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


Friday May 29th, 2020 - Chromatin

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Gingell, L. F. and McLean, J. R. (2020). A Protamine Knockdown Mimics the Function of Sd in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32321837
Segregation Distorter (SD) is an autosomal meiotic drive system found worldwide in natural populations of Drosophila melanogaster This gene complex induces the preferential and nearly exclusive transmission of the SD chromosome in SD/SD(+) males. This selfish propagation occurs through the interplay of the Sd locus, its enhancers and the Rsp(s) locus during spermatid development. The key distorter locus, Sd, encodes a truncated but enzymatically active RanGAP (RanGTPase-activating protein), a key nuclear transport factor in the Ran signaling pathway. When encoded by Sd, RanGAP is mislocalized to the nucleus interior, which then traps Ran inside the nucleus and disrupts nuclear import. As a result of this aberrant nuclear transport, a process known as the histone-to-protamine transition that is required for proper spermatid condensation fails to occur in SD/SD (+) males. In this process, sperm-specific protamine proteins enter the spermatid nucleus and replace the formerly chromatin-complexed histones. Previous work has shown that mutations affecting nuclear import and export can enhance distortion in an SD background, thus verifying that a defect in nuclear transport is responsible for the unequal transmission of chromosomes. This study shows that specifically reducing protamines induces distortion in an SD background, verifying that protamines are transported via the RanGAP/GEF pathway and indicating that E(SD) plays a significant and unique role in the process of distortion.
Brown, E. J., Nguyen, A. H. and Bachtrog, D. (2020). The Drosophila Y chromosome affects heterochromatin integrity genome-wide. Mol Biol Evol. PubMed ID: 32211857
The Y influences expression of hundreds of genes genome-wide, possibly by sequestering key components of the heterochromatin machinery away from other positions in the genome. To test the influence of the Y chromosome on the genome-wide chromatin landscape, the genomic distribution was assayed of histone modifications associated with gene activation (H3K4me3) or heterochromatin (H3K9me2 and H3K9me3) in fruit flies with varying sex chromosome complements (X0, XY and XYY males; XX and XXY females). Consistent with the general deficiency of active chromatin modifications on the Y, this study found that Y gene dose has little influence on the genomic distribution of H3K4me3. In contrast, both the presence and the number of Y-chromosomes strongly influence genome-wide enrichment patterns of repressive chromatin modifications. Highly repetitive regions such as the pericentromeres, the dot, and the Y chromosome (if present) are enriched for heterochromatic modifications in wildtype males and females, and even more strongly in X0 flies. In contrast, the additional Y chromosome in XYY males and XXY females diminishes the heterochromatic signal in these normally silenced, repeat-rich regions, which is accompanied by an increase in expression of Y-linked repeats. Hundreds of genes were found that are expressed differentially between individuals with aberrant sex chromosome karyotypes, many of which also show sex-biased expression in wildtype Drosophila. Thus, differences in the chromatin landscape of males and females may also contribute to sex-biased gene expression and sexual dimorphisms.
Brown, E. J., Nguyen, A. H. and Bachtrog, D. (2020). The Y chromosome may contribute to sex-specific ageing in Drosophila. Nat Ecol Evol. PubMed ID: 32313175
Heterochromatin suppresses repetitive DNA, and a loss of heterochromatin has been observed in aged cells of several species, including humans and Drosophila. Males often contain substantially more heterochromatic DNA than females, due to the presence of a large, repeat-rich Y chromosome, and male flies generally have a shorter average lifespan than females. This study shows that repetitive DNA becomes de-repressed more rapidly in old male flies relative to females, and repeats on the Y chromosome are disproportionally mis-expressed during ageing. This is associated with a loss of heterochromatin at repetitive elements during ageing in male flies, and a general loss of repressive chromatin in aged males away from pericentromeric regions and the Y. By generating flies with different sex chromosome karyotypes (XXY females and X0 and XYY males), this study shows that repeat de-repression and average lifespan is correlated with the number of Y chromosomes. This suggests that sex-specific chromatin differences may contribute to sex-specific ageing in flies.
Khoroshko, V. A., Pokholkova, G. V., Levitsky, V. G., Zykova, T. Y., Antonenko, O. V., Belyaeva, E. S. and Zhimulev, I. F. (2020). Genes Containing Long Introns Occupy Series of Bands and Interbands In Drosophila melanogaster polytene Chromosomes. Genes (Basel) 11(4). PubMed ID: 32290448
Despite of the long-term studies available on genetic organization of polytene chromosome bands and interbands, little is known regarding long gene location on chromosomes. To analyze it, bioinformatic approaches were used, and genome-wide distribution of introns in gene bodies and in different chromatin states was characterized, and using fluorescent in situ hybridization they were juxtaposed with the chromosome structures. Short introns up to 2 kb in length are located in the bodies of housekeeping genes (grey bands or lazurite chromatin). In the group of 70 longest genes in the Drosophila genome, 95% of total gene length accrues to introns. The mapping of the 15 long genes showed that they could occupy extended sections of polytene chromosomes containing band and interband series, with promoters located in the interband fragments (aquamarine chromatin). Introns (malachite and ruby chromatin) in polytene chromosomes form independent bands, which can contain either both introns and exons or intron material only. Thus, a novel type of the gene arrangement in polytene chromosomes was discovered; peculiarities of such genetic organization are discussed.
Hassan, A., Araguas Rodriguez, P., Heidmann, S. K., Walmsley, E. L., Aughey, G. N. and Southall, T. D. (2020). Condensin I subunit Cap-G is essential for proper gene expression during the maturation of post-mitotic neurons. Elife 9. PubMed ID: 32255428
Condensin complexes are essential for mitotic chromosome assembly and segregation during cell divisions, however, little is known about their functions in post-mitotic cells. This study reports a role for the condensin I subunit Cap-G in Drosophila neurons. Despite not requiring condensin for mitotic chromosome compaction, post-mitotic neurons express Cap-G. Knockdown of Cap-G specifically in neurons (from their birth onwards) results in developmental arrest, behavioural defects, and dramatic gene expression changes, including reduced expression of a subset of neuronal genes and aberrant expression of genes that are not normally expressed in the developing brain. Knockdown of Cap-G in mature neurons results in similar phenotypes but to a lesser degree. Furthermore, dynamic binding of Cap-G is seen at distinct loci in progenitor cells and differentiated neurons. Therefore, Cap-G is essential for proper gene expression in neurons and plays an important role during the early stages of neuronal development.
Kyrchanova, O., Maksimenko, O., Ibragimov, A., Sokolov, V., Postika, N., Lukyanova, M., Schedl, P. and Georgiev, P. (2020). The insulator functions of the Drosophila polydactyl C2H2 zinc finger protein CTCF: Necessity versus sufficiency. Sci Adv 6(13): eaaz3152. PubMed ID: 32232161
In mammals, a C2H2 zinc finger (C2H2) protein, CTCF, acts as the master regulator of chromosomal architecture and of the expression of Hox gene clusters. Like mammalian CTCF, the Drosophila homolog, dCTCF, localizes to boundaries in the bithorax complex (BX-C). This study has determined the minimal requirements for the assembly of a functional boundary by dCTCF and two other C2H2 zinc finger proteins, Pita and Su(Hw). Although binding sites for these proteins are essential for the insulator activity of BX-C boundaries, these binding sites alone are insufficient to create a functional boundary. dCTCF cannot effectively bind to a single recognition sequence in chromatin or generate a functional insulator without the help of additional proteins. In addition, for boundary elements in BX-C at least four binding sites for dCTCF or the presence of additional DNA binding factors is required to generate a functional insulator.

Thursday, May 28th - Enhancers and gene regulation

Bravo Gonzalez-Blas, C., Quan, X. J., Duran-Romana, R., Taskiran, II, Koldere, D., Davie, K., Christiaens, V., Makhzami, S., Hulselmans, G., de Waegeneer, M., Mauduit, D., Poovathingal, S., Aibar, S. and Aerts, S. (2020). Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics. Mol Syst Biol 16(5): e9438. PubMed ID: 32431014
Single-cell technologies allow measuring chromatin accessibility and gene expression in each cell, but jointly utilizing both layers to map bona fide gene regulatory networks and enhancers remains challenging. This study generate independent single-cell RNA-seq and single-cell ATAC-seq atlases of the Drosophila eye-antennal disc and spatially integrate the data into a virtual latent space that mimics the organization of the 2D tissue using ScoMAP (Single-Cell Omics Mapping into spatial Axes using Pseudotime ordering). To validate spatially predicted enhancers, a large collection of enhancer-reporter lines were used, and ~ 85% of enhancers were identifed in which chromatin accessibility and enhancer activity are coupled. Next, enhancer-to-gene relationships in the virtual space were inferred, finding that genes are mostly regulated by multiple, often redundant, enhancers. Exploiting cell type-specific enhancers, cell type-specific effects of bulk-derived chromatin accessibility QTLs were deconvoluted. Finally, it was discovered that Prospero drives neuronal differentiation through the binding of a GGG motif. In summary, a comprehensive spatial characterization of gene regulation is provided in a 2D tissue.
Jorgensen, V., Chen, J., Vander Wende, H., Harris, D., McCarthy, A., Breznak, S., Wong-Deyrup, S. W., Chen, Y., Rangan, P., Brar, G. A., Sawyer, E. M., Chan, L. Y. and Unal, E. (2020). Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32213532
Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in Alcohol dehydrogenase (Adh) gene in Drosophila is regulated by two promoters. When the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. This study employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae and that the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. It is concluded that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.
Kim, M. K., Tranvo, A., Hurlburt, A. M., Verma, N., Phan, P., Luo, J., Ranish, J. and Stumph, W. E. (2020). Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA gene promoter in Drosophila melanogaster. Mol Cell Biol. PubMed ID: 32253345
U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB, and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previous work found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. This study shows that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and a region of Bdp1 was identified that is sufficient for TBP recruitment. Moreover, it was found that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed the build of a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.
Hoermann, N., Schilling, T., Haji Ali, A., Serbe, E., Mayer, C., Borst, A. and Pujol-Marti, J. (2020). A combinatorial code of transcription factors specifies subtypes of visual motion-sensing neurons in Drosophila. Development. PubMed ID: 32238425
Direction-selective T4/T5 neurons exist in four subtypes, each tuned to visual motion along one of the four cardinal directions. Along with their directional tuning, neurons of each T4/T5 subtype orient their dendrites and project their axons in a subtype-specific manner. Directional tuning, thus, appears strictly linked to morphology in T4/T5 neurons. How the four T4/T5 subtypes acquire their distinct morphologies during development remains largely unknown. This study investigated when and how the dendrites of the four T4/T5 subtypes acquire their specific orientations, and profiled the transcriptomes of all T4/T5 neurons during this process. This revealed a simple and stable combinatorial code of transcription factors defining the four T4/T5 subtypes during their development. Changing the combination of transcription factors of specific T4/T5 subtypes resulted in predictable and complete conversions of subtype-specific properties, i.e. dendrite orientation and matching axon projection pattern. Therefore, a combinatorial code of transcription factors coordinates the development of dendrite and axon morphologies to generate anatomical specializations differentiating subtypes of T4/T5 motion-sensing neurons.
Yokoshi, M., Segawa, K. and Fukaya, T. (2020). Visualizing the role of boundary elements in enhancer-promoter communication. Mol Cell. PubMed ID: 32109364
Formation of self-associating loop domains is a fundamental organizational feature of metazoan genomes. This study employed quantitative live-imaging methods to visualize impacts of higher-order chromosome topology on enhancer-promoter communication in developing Drosophila embryos. Evidence is provided that distal enhancers effectively produce transcriptional bursting from target promoters over distances when they are flanked with boundary elements. Importantly, neither inversion nor deletion of a boundary element abrogates this "enhancer-assisting activity," suggesting that they can facilitate intra-domain enhancer-promoter interaction and production of transcriptional bursting independently of topologically associating domain (TAD) formation. In contrast, domain-skipping activity of distal enhancers was lost after disruption of topological domains. This observation raises a possibility that intra-domain and inter-domain enhancer-promoter interactions are differentially regulated by chromosome topology.
Kuang, Y., Golan, O., Preusse, K., Cain, B., Christensen, C. J., Salomone, J., Campbell, I., Okwubido-Williams, F. V., Hass, M. R., Yuan, Z., Eafergan, N., Moberg, K. H., Kovall, R. A., Kopan, R., Sprinzak, D. and Gebelein, B. (2020). Enhancer architecture sensitizes cell-specific responses to Notch gene dose via a bind and discard mechanism. Elife 9. PubMed ID: 32297857
Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, there is only a limited mechanistic understanding of phenotype variability due to gene dosage. This study unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, it was shown that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. Mathematical modeling was combined with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.

Wednesday, May 27th - Embryonic Development

Ruiz-Sobrino, A., Martin-Blanco, C. A., Navarro, T., Almudi, I., Masiero, G., Jimenez-Caballero, M., Buchwalter, D. B., Funk, D. H., Gattolliat, J. L., Lemos, M. C., Jimenez, F. and Casares, F. (2020). Space colonization by branching trachea explains the morphospace of a simple respiratory organ. Dev Biol. PubMed ID: 32109442
Branching morphogenesis helps increase the efficiency of gas and liquid transport in many animal organs. Studies in several model organisms have highlighted the molecular and cellular complexity behind branching morphogenesis. To understand this complexity, computational models have been developed with the goal of identifying the "major rules" that globally explain the branching patterns. These models also guide further experimental exploration of the biological processes that execute and maintain these rules. This paper introduces the tracheal gills of mayfly (Ephemeroptera) larvae as a model system to study the generation of branched respiratory patterns. First, the gills of the mayfly Cloeon dipterum are described, and the geometry of its branching trachea is quantitatively characterized. This characterization was extended to those of related species to generate the morphospace of branching patterns. Then, it was shown how an algorithm based on the "space colonization" concept (SCA) can generate this branching morphospace via growth towards a hypothetical attractor molecule (M). SCA differs from other branch-generating algorithms in that the geometry generated depends to a great extent on its perception of the "external" space available for branching, uses few rules and, importantly, can be easily translated into a realistic "biological patterning algorithm". A gene was identified in the C. dipterum genome (Cd-bnl) that is orthologous to the fibroblast growth factor branchless (bnl), which stimulates growth and branching of embryonic trachea in Drosophila. In C. dipterum, this gene is expressed in the gill margins and areas of finer tracheolar branching from thicker trachea. Thus, Cd-bnl may perform the function of M in the model. Finally, this general mechanism is discussed in the context of other branching pattern-generating algorithms.
Al Asafen, H., Bandodkar, P. U., Carrell-Noel, S., Schloop, A. E., Friedman, J. and Reeves, G. T. (2020). Robustness of the Dorsal morphogen gradient with respect to morphogen dosage. PLoS Comput Biol 16(4): e1007750. PubMed ID: 32251432
In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. This work investigated the robustness of the Dorsal/NF-kappaB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and an empirical description of the Dorsal gradient was found to be highly sensitive to maternal dorsal dosage. In contrast, it was found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, a mechanistic model of the Dorsal signaling module was analyzed, and Cactus, a cytoplasmic inhibitor for Dorsal, had to be be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. This work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.
Ko, C. S., Kalakuntla, P. and Martin, A. C. (2020). Apical constriction reversal upon mitotic entry underlies different morphogenetic outcomes of cell division. Mol Biol Cell: mbcE19120673. PubMed ID: 32129704
During development, coordinated cell shape changes and cell divisions sculpt tissues. While these individual cell behaviors have been extensively studied, how cell shape changes and cell divisions that occur concurrently in epithelia influence tissue shape is less understood. This question was addressed in two contexts of the early Drosophila embryo: premature cell division during mesoderm invagination, and native ectodermal cell divisions with ectopic activation of apical contractility. Using quantitative live-cell imaging, it was demonstrated that mitotic entry reverses apical contractility by interfering with medioapical RhoA signaling. While premature mitotic entry inhibits mesoderm invagination, which relies on apical constriction, mitotic entry in an artificially contractile ectoderm induced ectopic tissue invaginations. Ectopic invaginations resulted from medioapical myosin loss in neighboring mitotic cells. This myosin loss enabled non-mitotic cells to apically constrict through mitotic cell stretching. Thus, the spatial pattern of mitotic entry can differentially regulate tissue shape through signal interference between apical contractility and mitosis.
Konigsmann, T., Parfentev, I., Urlaub, H., Riedel, D. and Schuh, R. (2020). The bicistronic gene wurmchen encodes two essential components for epithelial development in Drosophila. Dev Biol. PubMed ID: 32361005
Epithelial tissues are fundamental for the establishment and maintenance of different body compartments in multicellular animals. To achieve this specific task epithelial sheets secrete an apical extracellular matrix for tissue strength and protection and they organize a transepithelial barrier function, which is mediated by tight junctions in vertebrates or septate junctions in invertebrates. This study shows that the bicistronic gene wurmchen (CG43780) is functionally expressed in epithelial tissues. CRISPR/Cas9-mediated mutations in both coding sequences reveal two essential polypeptides, Wurmchen1 and Wurmchen2, which are both necessary for normal epithelial tissue development. Wurmchen1 represents a genuine septate junction core component. It is required during embryogenesis for septate junction organization, the establishment of a transepithelial barrier function, distinct cellular transport processes and tracheal system morphogenesis. Wurmchen2 is localized in the apical membrane region of epithelial tissues and in a central core of the tracheal lumen during embryogenesis. It is essential during the later larval development.
Johnson, H. E. and Toettcher, J. E. (2019). Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo. Dev Cell 48(3): 361-370 e363. PubMed ID: 30753836
The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? This study combined optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. Erk activity was found to be sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. The endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.
Dutta, S., Djabrayan, N. J., Smits, C. M., Rowley, C. W. and Shvartsman, S. Y. (2020). Excess dNTPs Trigger Oscillatory Surface Flow in the Early Drosophila Embryo. Biophys J. PubMed ID: 32247330
During the first 2 hours of Drosophila development, precisely orchestrated nuclear cleavages, cytoskeletal rearrangements, and directed membrane growth lead to the formation of an epithelial sheet around the yolk. The newly formed epithelium remains relatively quiescent during the next hour as it is patterned by maternal inductive signals and zygotic gene products. It was discovered that this mechanically quiet period is disrupted in embryos with high levels of dNTPs, which have been recently shown to cause abnormally fast nuclear cleavages and interfere with zygotic transcription. High levels of dNTPs are associated with robust onset of oscillatory two-dimensional flows during the third hour of development. Tissue cartography, particle image velocimetry, and dimensionality reduction techniques reveal that these oscillatory flows are low dimensional and are characterized by the presence of spiral vortices. It is speculated that these aberrant flows emerge through an instability triggered by deregulated mechanical coupling between the nascent epithelium and three-dimensional yolk. These results highlight an unexplored connection between a core metabolic process and large-scale mechanics in a rapidly developing embryo.

Tuesday May 26 - Adult neural development and function

Delestro, F., Scheunemann, L., Pedrazzani, M., Tchenio, P., Preat, T. and Genovesio, A. (2020). In vivo large-scale analysis of Drosophila neuronal calcium traces by automated tracking of single somata. Sci Rep 10(1): 7153. PubMed ID: 32346011
How does the concerted activity of neuronal populations shape behavior? In Drosophila melanogaster, the mushroom body (MB) represents an excellent model to analyze sensory coding and memory plasticity. This work presents an experimental setup coupled with a dedicated computational method that provides in vivo measurements of the activity of hundreds of densely packed somata uniformly spread in the MB. This study exploited spinning-disk confocal 3D imaging over time of the whole MB cell body layer in vivo while it is exposed to olfactory stimulation. Importantly, to derive individual signal from densely packed somata, a fully automated image analysis procedure was developed that takes advantage of the specificities of the data. After anisotropy correction, this approach operates a dedicated spot detection and registration over the entire time sequence to transform trajectories to identifiable clusters. This enabled discarding spurious detections and reconstruct missing ones in a robust way. It was demonstrated that this approach outperformed existing methods in this specific context and made possible high-throughput analysis of approximately 500 single somata uniformly spread over the MB in various conditions. Applying this approach, it was found that learned experiences change the population code of odor representations in the MB. After long-term memory (LTM) formation,an increase in responsive somata count and a stable single neuron signal were quantified. It is predicted that this method, which should further enable studying the population pattern of neuronal activity, has the potential to uncover fine details of sensory processing and memory plasticity.
Hunter, A. C., Petley-Ragan, L. M., Das, M. and Auld, V. J. (2020). Basigin Associates with Integrin in Order to Regulate Perineurial Glia and Drosophila Nervous System Morphology. J Neurosci 40(17): 3360-3373. PubMed ID: 32265259
The Drosophila nervous system is ensheathed by a layer of outer glial cells, the perineurial glia, and a specialized extracellular matrix, the neural lamella. The function of perineurial glial cells and how they interact with the extracellular matrix are just beginning to be elucidated. Integrin-based focal adhesion complexes link the glial membrane to the extracellular matrix, but little is known about integrin's regulators in the glia. The transmembrane Ig domain protein Basigin/CD147/EMMPRIN is highly expressed in the perineurial glia surrounding the Drosophila larval nervous system. This study shows that Basigin associates with integrin at the focal adhesions to uphold the structure of the glia-extracellular matrix sheath. Knockdown of Basigin in perineurial glia using RNAi results in significant shortening of the ventral nerve cord, compression of the glia and extracellular matrix in the peripheral nerves, and reduction in larval locomotion. It was determined that Basigin is expressed in close proximity to integrin at the glial membrane, and that expression of the extracellular integrin-binding domain of Basigin is sufficient to rescue peripheral glial compression. It was also found that a reduction in expression of integrin at the membrane rescues the ventral nerve cord shortening, peripheral glial compression, and locomotor phenotypes, and that reduction in the integrin-binding protein Talin can partially rescue glial compression. These results identify Basigin as a potential negative regulator of integrin in the glia, supporting proper glial and extracellular matrix ensheathment of the nervous system.
Ishii, K., Wohl, M., DeSouza, A. and Asahina, K. (2020). Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons. Elife 9. PubMed ID: 32314964
For successful mating, a male animal must execute effective courtship behaviors toward a receptive target sex, which is female. Whether the courtship execution capability and upregulation of courtship toward females are specified through separable sex-determining genetic pathways remains uncharacterized. This study found that one of the two Drosophila sex-determining genes, doublesex (dsx), specifies a male-specific neuronal component that serves as an execution mechanism for courtship behavior, whereas fruitless (fru) is required for enhancement of courtship behavior toward females. The dsx-dependent courtship execution mechanism includes a specific subclass within a neuronal cluster that co-express dsx and fru. This cluster contains at least another subclass that is specified cooperatively by both dsx and fru. Although these neuronal populations can also promote aggressive behavior toward male flies, this capacity requires fru-dependent mechanisms. These results uncover how sex-determining genes specify execution capability and female-specific enhancement of courtship behavior through separable yet cooperative neurogenetic mechanisms.
Kim, J. H., Ki, Y., Lee, H., Hur, M. S., Baik, B., Hur, J. H., Nam, D. and Lim, C. (2020). The voltage-gated potassium channel Shaker promotes sleep via thermosensitive GABA transmission. Commun Biol 3(1): 174. PubMed ID: 32296133
Genes and neural circuits coordinately regulate animal sleep. However, it remains elusive how these endogenous factors shape sleep upon environmental changes. This study demonstrates that Shaker (Sh)-expressing GABAergic neurons projecting onto dorsal fan-shaped body (dFSB) regulate temperature-adaptive sleep behaviors in Drosophila. Loss of Sh function suppressed sleep at low temperature whereas light and high temperature cooperatively gated Sh effects on sleep. Sh depletion in GABAergic neurons partially phenocopied Sh mutants. Furthermore, the ionotropic GABA receptor, Resistant to dieldrin (Rdl), in dFSB neurons acted downstream of Sh and antagonized its sleep-promoting effects. In fact, Rdl inhibited the intracellular cAMP signaling of constitutively active dopaminergic synapses onto dFSB at low temperature. High temperature silenced GABAergic synapses onto dFSB, thereby potentiating the wake-promoting dopamine transmission. It is proposed that temperature-dependent switching between these two synaptic transmission modalities may adaptively tune the neural property of dFSB neurons to temperature shifts and reorganize sleep architecture for animal fitness.
Eschbach, C., Fushiki, A., Winding, M., Schneider-Mizell, C. M., Shao, M., Arruda, R., Eichler, K., Valdes-Aleman, J., Ohyama, T., Thum, A. S., Gerber, B., Fetter, R. D., Truman, J. W., Litwin-Kumar, A., Cardona, A. and Zlatic, M. (2020). Recurrent architecture for adaptive regulation of learning in the insect brain. Nat Neurosci 23(4): 544-555. PubMed ID: 32203499
Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. This study provides a synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mushroom body of Drosophila larva. Afferent sensory pathways and a large population of neurons were discovered that provide feedback from mushroom body output neurons and link distinct memory systems (aversive and appetitive). This was combined with functional studies of DANs and their presynaptic partners and with comprehensive circuit modeling. It was found that DANs compare convergent feedback from aversive and appetitive systems, which enables the computation of integrated predictions that may improve future learning. Computational modeling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. This study provides the most detailed view to date of biological circuit motifs that support associative learning.
Jacob, P. F. and Waddell, S. (2020). Spaced Training Forms Complementary Long-Term Memories of Opposite Valence in Drosophila. Neuron. PubMed ID: 32289250
Forming long-term memory (LTM) often requires repetitive experience spread over time. Studies in Drosophila suggest aversive olfactory LTM is optimal after spaced training, multiple trials of differential odor conditioning with rest intervals. Memory after spaced training is frequently compared to that after the same number of trials without intervals. This study shows that, after spaced training, flies acquire additional information and form an aversive memory for the shock-paired odor and a slowly emerging and more persistent "safety-memory" for the explicitly unpaired odor. Safety-memory acquisition requires repetition, order, and spacing of the training trials and relies on triggering specific rewarding dopaminergic neurons. Co-existence of aversive and safety memories is evident as depression of odor-specific responses at different combinations of junctions in the mushroom body output network; combining two outputs appears to signal relative safety. Having complementary aversive and safety memories augments LTM performance after spaced training by making the odor preference more certain.

Friday, May 22nd - Disease Models

Gogia, N., Sarkar, A., Mehta, A. S., Ramesh, N., Deshpande, P., Kango-Singh, M., Pandey, U. B. and Singh, A. (2020). Inactivation of Hippo and cJun-N-terminal Kinase (JNK) signaling mitigate FUS mediated neurodegeneration in vivo. Neurobiol Dis 140: 104837. PubMed ID: 32199908
Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. A screen was performed for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly defined deficiencies. hippo (hpo), a component of the evolutionarily conserved Hippo growth regulatory pathway, was identified as a genetic modifier of FUS mediated neurodegeneration. Gain-of-function of hpo triggers cell death whereas its loss-of-function promotes cell proliferation. Downregulation of the Hippo signaling pathway, using mutants of Hippo signaling, exhibit rescue of FUS-mediated neurodegeneration in the Drosophila eye, as evident from reduction in the number of TUNEL positive nuclei as well as rescue of axonal targeting from the retina to the brain. The Hippo pathway activates c-Jun amino-terminal (NH2) Kinase (JNK) mediated cell death. Downregulation of JNK signaling is sufficient to rescue FUS-mediated neurodegeneration in the Drosophila eye. This study elucidates that Hippo signaling and JNK signaling are activated in response to FUS accumulation to induce neurodegeneration. These studies will shed light on the genetic mechanism involved in neurodegeneration observed in ALS and other associated disorders.
Irwin, M., Tare, M., Singh, A., Puli, O. R., Gogia, N., Riccetti, M., Deshpande, P., Kango-Singh, M. and Singh, A. (2020). A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration. Front Cell Dev Biol 8: 117. PubMed ID: 32232042
Alzheimer's disease is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Abeta42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. A Drosophila eye model of AD was developed by targeted misexpression of human Abeta42 in the differentiating retinal neurons, where an accumulation of Abeta42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, a molecularly defined deficiency was identified that suppresses Abeta42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. Hippo signaling is activated in Abeta42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Abeta42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Abeta42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. This study found that activation of JNK signaling enhances the Abeta42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Abeta42-mediated neurodegeneration. The nature of interactions between Hippo signaling and JNK signaling was tested in Abeta42-mediated neurodegeneration using genetic epistasis approach. The data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Abeta42 plaques to trigger cell death. These studies demonstrate a novel role of Hippo signaling pathway in Abeta42-mediated neurodegeneration.
Chung, H. L., Mao, X., Wang, H., Park, Y. J., Marcogliese, P. C., Rosenfeld, J. A., Burrage, L. C., Liu, P., Murdock, D. R., Yamamoto, S., Wangler, M. F., Chao, H. T., Long, H., Feng, L., Bacino, C. A., Bellen, H. J. and Xiao, B. (2020). De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy. Am J Hum Genet. PubMed ID: 32330417
Three unrelated individuals were identified with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, these findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.
Dutta, D., Briere, L. C., Kanca, O., Marcogliese, P. C., Walker, M. A., High, F. A., Vanderver, A., Krier, J., Carmichael, N., Callahan, C., Taft, R. J., Simons, C., Helman, G., Network, U. D., Wangler, M. F., Yamamoto, S., Sweetser, D. A. and Bellen, H. J. (2020). De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment. Hum Mol Genet. PubMed ID: 32356556
The Translocase of Outer Mitochondrial Membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. This study reports two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyperreflexia, ataxia, dystonia, and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1, was found to have a p.Thr607Ile variant while individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, the Drosophila Tom70 coding region was replaced with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4 driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knock-down of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, these data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals.
Huang, Y., Mao, X., van Jaarsveld, R. H., Shu, L., Terhal, P. A., Jia, Z., Xi, H., Peng, Y., Yan, H., Yuan, S., Li, Q., Wang, H. and Bellen, H. J. (2020). Variants in CAPZA2, a member of a F-actin capping complex, cause intellectual disability and developmental delay. Hum Mol Genet. PubMed ID: 32338762
The actin cytoskeleton is regulated by many proteins including capping proteins that stabilize actin filaments (F-actin) by inhibiting actin polymerization and depolymerization. This study report two pediatric probands who carry damaging heterozygous de novo mutations in CAPZA2 (HGNC: 1490) and exhibit neurological symptoms with shared phenotypes including global motor development delay, speech delay, intellectual disability, hypotonia and a history of seizures. CAPZA2 encodes a subunit of an F-actin-capping protein complex (CapZ). CapZ is an obligate heterodimer consisting of alpha and beta heterodimer conserved from yeast to human. Vertebrate genomes contain three alpha subunits encoded by three different genes and CAPZA2 encodes the alpha2 subunit. The single orthologue of CAPZA genes in Drosophila is cpa. Loss of cpa leads to lethality in early development and expression of the human reference CAPZA2 rescues this lethality. However, the two CAPZA2 variants identified in the probands rescue this lethality at lower efficiency than the reference. Moreover, expression of the CAPZA2 variants affects bristle morphogenesis, a process that requires extensive actin polymerization and bundling during development. Taken together, these findings suggest that variants in CAPZA2 lead to a non-syndromic neurodevelopmental disorder in children.
Hope, K. A., Johnson, D., Miller, P. W., Lopez-Ferrer, D., Kakhniashvili, D. and Reiter, L. T. (2020). Transcriptomic and proteomic profiling of glial versus neuronal Dube3a overexpression reveals common molecular changes in gliopathic epilepsies. Neurobiol Dis: 104879. PubMed ID: 32344153
Epilepsy affects millions of individuals worldwide and many cases are pharmacoresistant. Duplication 15q syndrome (Dup15q) is a genetic disorder caused by duplications of the 15q11.2-q13.1 region. Phenotypes include in a high rate of pharmacoresistant epilepsy. This study developed a Dup15q model in Drosophila melanogaster that recapitulates seizures in Dup15q by over-expressing fly Dube3a or human UBE3A in glial cells, but not neurons, implicating glia in the Dup15q epilepsy phenotype. Dube3a overexpression in glia (repo>Dube3a) versus neurons (elav>Dube3a) using transcriptomics and proteomics of whole fly head extracts. 851 transcripts differentially regulated in repo>Dube3a were identified, including an upregulation glutathione S-transferase (GST) genes that occurred cell autonomously within glial cells. Approximately 2,500 proteins were reliably measured by proteomics, most of which were also quantified at the transcript level. Combined transcriptomic and proteomic analysis revealed an enrichment of 21 synaptic transmission genes downregulated at the transcript and protein in repo>Dube3a indicating synaptic proteins change in a cell non-autonomous manner in repo>Dube3a flies. Six additional glia originating bang-sensitive seizure lines were identified, and upregulation of GSTs in 4 out of these 6 lines was identified. These data suggest GST upregulation is common among gliopathic seizures and may ultimately provide insight for treating epilepsy.

Thursday, May 21st - Behavior

Jantrapirom, S., Enomoto, Y., Karinchai, J., Yamaguchi, M., Yoshida, H., Fukusaki, E., Shimma, S. and Yamaguchi, M. (2020). The depletion of ubiquilin in Drosophila melanogaster disturbs neurochemical regulation to drive activity and behavioral deficits. Sci Rep 10(1): 5689. PubMed ID: 32231214
Drosophila melanogaster is a useful and highly tractable model organism for understanding the molecular mechanisms of human diseases. Previously work characterized a new dUbqn knockdown model that induces learning-memory and locomotive deficits mediated by impaired proteostasis. Although proteinopathies are the main causes of neurodegenerative diseases, limited information is currently available on the relationship between proteostasis and neurodegenerative-related behavioral perturbations, such as locomotion, wakefulness, and sexual activities. Thus, this study aimed to elucidate the mechanisms by which dUbqn depletion which is known to cause proteinopathies, affects neurodegenerative-related behavioral perturbations. Pan-neuronal dUbqn-depleted flies showed significantly reduced evening activity along with altered pre- and postsynaptic structural NMJ's proteins by attenuating signals of Bruchpilot puncta and GluRIIA clustering. In addition, the neurochemical profiles of GABA, glutamate, dopamine, and serotonin were disturbed and these changes also affected courtship behaviors in dUbqn-depleted flies. Collectively, these results extend understanding on how dUbqn depletion affects neurochemical regulation to drive behavioral disturbances that are generally found in the early stage of neurodegenerative diseases. Moreover, the present study may contribute a novel finding to the design of new agents that prevent disease progression or even treat diseases related to neurodegeneration.
Zhang, N., Guo, L. and Simpson, J. H. (2020). Spatial comparisons of mechanosensory information govern the grooming sequence in Drosophila. Curr Biol. PubMed ID: 32142695
Animals integrate information from different sensory modalities, body parts, and time points to inform behavioral choice, but the relevant sensory comparisons and the underlying neural circuits are still largely unknown. This study used the grooming behavior of Drosophila melanogaster as a model to investigate the sensory comparisons that govern a motor sequence. Flies perform grooming movements spontaneously, but when covered with dust, they clean their bodies following an anterior-to-posterior sequence. After investigating different sensory modalities that could detect dust, focus was placed on mechanosensory bristle neurons, whose optogenetic activation induces a similar sequence. Computational modeling predicts that higher sensory input strength to the head will cause anterior grooming to occur first. This prediction was tested using an optogenetic competition assay whereby two targeted light beams independently activate mechanosensory bristle neurons on different body parts. It was found that the initial choice of grooming movement is determined by the ratio of sensory inputs to different body parts. In dust-covered flies, sensory inputs change as a result of successful cleaning movements. Simulations from this model suggest that this change results in sequence progression. One possibility is that flies perform frequent comparisons between anterior and posterior sensory inputs, and the changing ratios drive different behavior choices. Alternatively, flies may track the temporal change in sensory input to a given body part to measure cleaning effectiveness. The first hypothesis is supported by the optogenetic competition experiments: iterative spatial comparisons of sensory inputs between body parts is essential for organizing grooming movements in sequence.
Versace, E., Caffini, M., Werkhoven, Z. and de Bivort, B. L. (2020). Individual, but not population asymmetries, are modulated by social environment and genotype in Drosophila melanogaster. Sci Rep 10(1): 4480. PubMed ID: 32161330
Theory predicts that social interactions can induce an alignment of behavioral asymmetries between individuals (i.e., population-level lateralization), but evidence for this effect is mixed. To understand how interaction with other individuals affects behavioral asymmetries, this study systematically manipulated the social environment of Drosophila melanogaster, testing individual flies and dyads (female-male, female-female and male-male pairs). In these social contexts individual and population asymmetries in individual behaviors (circling asymmetry, wing use) and dyadic behaviors (relative position and orientation between two flies) were measured in five different genotypes. It was reasoned that if coordination between individuals drives alignment of behavioral asymmetries, greater alignment at the population-level should be observed in social contexts compared to solitary individuals. It was observed that the presence of other individuals influenced the behavior and position of flies but had unexpected effects on individual and population asymmetries: individual-level asymmetries were strong and modulated by the social context but population-level asymmetries were mild or absent. Moreover, the strength of individual-level asymmetries differed between strains, but this was not the case for population-level asymmetries. These findings suggest that the degree of social interaction found in Drosophila is insufficient to drive population-level behavioral asymmetries.
Williams, M. J., Akram, M., Barkauskaite, D., Patil, S., Kotsidou, E., Kheder, S., Vitale, G., Filaferro, M., Blemings, S. W., Maestri, G., Hazim, N., Vergoni, A. V. and Schioth, H. B. (2020). CCAP regulates feeding behavior via the NPF pathway in Drosophila adults. Proc Natl Acad Sci U S A. PubMed ID: 32179671
The intake of macronutrients is crucial for the fitness of any animal and is mainly regulated by peripheral signals to the brain. How the brain receives and translates these peripheral signals or how these interactions lead to changes in feeding behavior is not well-understood. This study discovered that 2 crustacean cardioactive peptide (CCAP)-expressing neurons in Drosophila adults regulate feeding behavior and metabolism. Notably, loss of CCAP, or knocking down the CCAP receptor (CCAP-R) in 2 dorsal median neurons, inhibits the release of neuropeptide F (NPF), which regulates feeding behavior. Furthermore, under starvation conditions, flies normally have an increased sensitivity to sugar; however, loss of CCAP, or CCAP-R in 2 dorsal median NPF neurons, inhibited sugar sensitivity in satiated and starved flies. Separate from its regulation of NPF signaling, the CCAP peptide also regulates triglyceride levels. Additionally, genetic and optogenetic studies demonstrate that CCAP signaling is necessary and sufficient to stimulate a reflexive feeding behavior, the proboscis extension reflex (PER), elicited when external food cues are interpreted as palatable. Dopaminergic signaling was also sufficient to induce a PER. On the other hand, although necessary, NPF neurons were not able to induce a PER. These data illustrate that the CCAP peptide is a central regulator of feeding behavior and metabolism in adult flies, and that NPF neurons have an important regulatory role within this system.
Bath, E., Biscocho, E. R., Easton-Calabria, A. and Wigby, S. (2020). Temporal and genetic variation in female aggression after mating. PLoS One 15(4): e0229633. PubMed ID: 32348317
Aggression between individuals of the same sex is almost ubiquitous across the animal kingdom. Winners of intrasexual contests often garner considerable fitness benefits, through greater access to mates, food, or social dominance. In females, aggression is often tightly linked to reproduction, with females displaying increases in aggressive behavior when mated, gestating or lactating, or when protecting dependent offspring. In the fruit fly, Drosophila melanogaster, females spend twice as long fighting over food after mating as when they are virgins. However, it is unknown when this increase in aggression begins or whether it is consistent across genotypes. This study shows that aggression in females increases between 2 to 4 hours after mating and remains elevated for at least a week after a single mating. In addition, this increase in aggression 24 hours after mating is consistent across three diverse genotypes, suggesting this may be a universal response to mating in the species. This study also reports the first use of automated tracking and classification software to study female aggression in Drosophila and assess its accuracy for this behavior. Dissecting the genetic diversity and temporal patterns of female aggression assists in better understanding its generality and adaptive function, and will facilitate the identification of its underlying mechanisms.
Chowdhury, T., Calhoun, R. M., Bruch, K. and Moehring, A. J. (2020). The fruitless gene affects female receptivity and species isolation. Proc Biol Sci 287(1923): 20192765. PubMed ID: 32208837
Female mate rejection acts as a major selective force within species, and can serve as a reproductive barrier between species. In spite of its critical role in fitness and reproduction, surprisingly little is known about the genetic or neural basis of variation in female mate choice. This study identified fruitless as a gene affecting female receptivity within Drosophila melanogaster, as well as female Drosophila simulans rejection of male D. melanogaster. Of the multiple transcripts this gene produces, by far the most widely studied is the sex-specifically spliced transcript involved in the sex determination pathway. However, this study found that female rejection behaviour is affected by a non-sex-specifically spliced fruitless transcript. This is the first implication of fruitless in female behaviour, and the first behavioural role identified for a fruitless non-sex-specifically spliced transcript. It was found that this locus does not influence preferences via a single sensory modality, examining courtship song, antennal pheromone perception, or perception of substrate vibrations, and it is concluded that fruitless influences mate choice via the integration of multiple signals or through another sensory modality.

Wednesday May 20th - RNA and Transposons

Warsinger-Pepe, N., Li, D. and Yamashita, Y. M. (2020). Regulation of Nucleolar Dominance in Drosophila melanogaster. Genetics. PubMed ID: 32122935
In eukaryotic genomes, ribosomal RNA (rRNA) genes exist as tandemly repeated clusters, forming ribosomal DNA (rDNA) loci. Each rDNA locus typically contains hundreds of rRNA genes to meet the high demand of ribosome biogenesis. Nucleolar dominance is a phenomenon, whereby individual rDNA loci are entirely silenced or transcribed, and is believed to be a mechanism to control rRNA dosage. Nucleolar dominance was originally noted to occur in interspecies hybrids, and has been shown to occur within a species (i.e. non-hybrid context). However, studying nucleolar dominance within a species has been challenging due to the highly homogenous sequence across rDNA loci. By utilizing single nucleotide polymorphisms (SNPs) between X rDNA vs. Y rDNA loci in males, as well as sequence variations between two X rDNA loci in females, this study conducted a thorough characterization of nucleolar dominance throughout development of D. melanogaster. Nucleolar dominance is a developmentally-regulated program that occurs in non-hybrid, wild type D. melanogaster, where Y rDNA dominance is established during male embryogenesis, whereas females normally do not exhibit dominance between two X rDNA loci. By utilizing various chromosomal complements (e.g. X/Y, X/X, X/X/Y) and a chromosome rearrangement, this study shows that the short arm of the Y chromosome including the Y rDNA likely contains information that instructs the state of nucleolar dominance. This study begins to reveal the mechanisms underlying the selection of rDNA loci for activation/silencing in nucleolar dominance in the context of non-hybrid D. melanogaster.
Yuan, L., Ren, X., Zheng, Y., Qian, J., Xu, L. and Sun, M. (2020). MiR-315 is required for neural development and represses the expression of dFMR1 in Drosophila melanogaster. Biochem Biophys Res Commun. PubMed ID: 32107003
The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered. This study detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups. This study determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, miR-315 was found to regulate synaptic structure and transmission by targeting dfmr1. These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression.
Buddika, K., Ariyapala, I. S., Hazuga, M. A., Riffert, D. and Sokol, N. S. (2020). Canonical nucleators are dispensable for stress granule assembly in intestinal progenitors. J Cell Sci. PubMed ID: 32265270
Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remain poorly characterized. This study reports that Drosophila orthologs of mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP, LIN-28, PABP, and TIAR are enriched in adult intestinal progenitor cells where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs). Treatment with sodium arsenite or rapamycin reorganized these mRNPs into large cytoplasmic granules. Formation of these intestinal progenitor stress granules (IPSGs) depended on polysome disassembly, led to translational downregulation, and was reversible. While canonical SG nucleators ATX2 and G3BP were sufficient for IPSG formation in the absence of stress, neither of them, nor TIAR, either individually or collectively, were required for stress-induced IPSG formation. This work therefore finds that IPSGs do not assemble via a canonical mechanism, raising the possibility that other stem cell populations employ a similar stress-response mechanism.
Wu, Y. C., Chawla, G. and Sokol, N. (2020). let-7-complex microRNAs regulate Broad-Z3, which together with Chinmo maintains adult lineage neurons in an immature state. G3 (Bethesda). PubMed ID: 32071070
During Drosophila melanogaster metamorphosis, arrested immature neurons born during larval development differentiate into their functional adult form. This differentiation coincides with the downregulation of two zinc-finger transcription factors, Chronologically Inappropriate Morphogenesis (Chinmo) and the Z3 isoform of Broad (Br-Z3). This study shows that br-Z3 is regulated by two microRNAs, let-7 and miR-125, that are activated at the larval-to-pupal transition and are known to also regulate chinmo The br-Z3 3'UTR contains functional binding sites for both let-7 and miR-125 that confers sensitivity to both of these microRNAs, as determined by deletion analysis in reporter assays. Forced expression of let-7 and miR-125 miRNAs leads to early silencing of Br-Z3 and Chinmo and is associated with inappropriate neuronal sprouting and outgrowth. Similar phenotypes were observed by the combined but not separate depletion of br-Z3 and chinmo. Because persistent Br-Z3 was not detected in let-7-C mutants, this work suggests a model in which let-7 and miR-125 activation at the onset of metamorphosis may act as a failsafe mechanism that ensures the coordinated silencing of both br-Z3 and chinmo needed for the timely outgrowth of neurons arrested during larval development. The let-7 andmiR-125 binding site sequences are conserved across Drosophila species and possibly other insects as well, suggesting that this functional relationship is evolutionarily conserved.
Deng, P., Khan, A., Jacobson, D., Sambrani, N., McGurk, L., Li, X., Jayasree, A., Hejatko, J., Shohat-Ophir, G., O'Connell, M. A., Li, J. B. and Keegan, L. P. (2020). Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila. Nat Commun 11(1): 1580. PubMed ID: 32221286
ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. A Drosophila Adar(E374A) mutant strain encoding a catalytically inactive Adar was generated with CRISPR/Cas9. Adar adenosine deamination activity was shown to be necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. This work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.
Fant, C. B., Levandowski, C. B., Gupta, K., Maas, Z. L., Moir, J., Rubin, J. D., Sawyer, A., Esbin, M. N., Rimel, J. K., Luyties, O., Marr, M. T., Berger, I., Dowell, R. D. and Taatjes, D. J. (2020). TFIID Enables RNA Polymerase II Promoter-Proximal Pausing. Mol Cell. PubMed ID: 32229306
RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, human RNAPII promoter-proximal pausing was reconstituted in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.

Tuesday, May 19th - Synapse and Vesicles

McNeill, E. M., Warinner, C., Alkins, S., Taylor, A., Heggeness, H., DeLuca, T. F., Fulga, T. A., Wall, D. P., Griffith, L. C. and Van Vactor, D. (2020). The conserved microRNA miR-34 regulates synaptogenesis via coordination of distinct mechanisms in presynaptic and postsynaptic cells. Nat Commun 11(1): 1092. PubMed ID: 32107390
Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, however, relatively few specific miRNAs have been identified that control synapse formation. Using a conditional transgenic toolkit for competitive inhibition of miRNA function in Drosophila, an unbiased screen was performed for novel regulators of synapse morphogenesis at the larval neuromuscular junction (NMJ). From a set of ten new validated regulators of NMJ growth, miR-34 mutants were discovered to display synaptic phenotypes and cell type-specific functions suggesting distinct downstream mechanisms in the presynaptic and postsynaptic compartments. A search for conserved downstream targets for miR-34 identified the junctional receptor CNTNAP4/Neurexin-IV (Nrx-IV) and the membrane cytoskeletal effector Adducin/Hu-li tai shao (Hts) as proteins whose synaptic expression is restricted by miR-34. Manipulation of miR-34, Nrx-IV or Hts-M function in motor neurons or muscle supports a model where presynaptic miR-34 inhibits Nrx-IV to influence active zone formation, whereas, postsynaptic miR-34 inhibits Hts to regulate the initiation of bouton formation from presynaptic terminals.
McNeill, E. M., Thompson, C., Berke, B., Chou, V. T., Rusch, J., Duckworth, A., DeProto, J., Taylor, A., Gates, J., Gertler, F., Keshishian, H. and Van Vactor, D. (2020). Drosophila enabled promotes synapse morphogenesis and regulates active zone form and function. Neural Dev 15(1): 4. PubMed ID: 32183907
Recent studies of synapse form and function highlight the importance of the actin cytoskeleton in regulating multiple aspects of morphogenesis, neurotransmission, and neural plasticity. The conserved actin-associated protein Enabled (Ena) is known to regulate development of the Drosophila larval neuromuscular junction through a postsynaptic mechanism. However, the functions and regulation of Ena within the presynaptic terminal has not been determined. This study used a conditional genetic approach to address a presynaptic role for Ena on presynaptic morphology and ultrastructure, and also examine the pathway in which Ena functions through epistasis experiments. Ena was found to is required to promote the morphogenesis of presynaptic boutons and branches, in contrast to its inhibitory role in muscle. Moreover, while postsynaptic Ena is regulated by microRNA-mediated mechanisms, presynaptic Ena relays the output of the highly conserved receptor protein tyrosine phosphatase Dlar and associated proteins including the heparan sulfate proteoglycan Syndecan, and the non-receptor Abelson tyrosine kinase to regulate addition of presynaptic varicosities. Interestingly, Ena also influences active zones, where it restricts active zone size, regulates the recruitment of synaptic vesicles, and controls the amplitude and frequency of spontaneous glutamate release. This study thus shows that Ena, under control of the Dlar pathway, is required for presynaptic terminal morphogenesis and bouton addition and that Ena has active zone and neurotransmission phenotypes. Notably, in contrast to Dlar, Ena appears to integrate multiple pathways that regulate synapse form and function.
Kiral, F. R., Linneweber, G. A., Mathejczyk, T., Georgiev, S. V., Wernet, M. F., Hassan, B. A., von Kleist, M. and Hiesinger, P. R. (2020). Autophagy-dependent filopodial kinetics restrict synaptic partner choice during Drosophila brain wiring. Nat Commun 11(1): 1325. PubMed ID: 32165611
Brain wiring is remarkably precise, yet most neurons readily form synapses with incorrect partners when given the opportunity. Dynamic axon-dendritic positioning can restrict synaptogenic encounters, but the spatiotemporal interaction kinetics and their regulation remain essentially unknown inside developing brains. This study shows that the kinetics of axonal filopodia restrict synapse formation and partner choice for neurons that are not otherwise prevented from making incorrect synapses. Using 4D imaging in developing Drosophila brains, this study shows that filopodial kinetics are regulated by autophagy, a prevalent degradation mechanism whose role in brain development remains poorly understood. With surprising specificity, autophagosomes form in synaptogenic filopodia, followed by filopodial collapse. Altered autophagic degradation of synaptic building material quantitatively regulates synapse formation as shown by computational modeling and genetic experiments. Increased filopodial stability enables incorrect synaptic partnerships. Hence, filopodial autophagy restricts inappropriate partner choice through a process of kinetic exclusion that critically contributes to wiring specificity.
Kobbersmed, J. R. L., Grasskamp, A. T., Jusyte, M., Bohme, M. A., Ditlevsen, S., Sorensen, J. B. and Walter, A. M. (2020). Rapid regulation of vesicle priming explains synaptic facilitation despite heterogeneous vesicle:Ca(2+) channel distances. Elife 9. PubMed ID: 32077852
Chemical synaptic transmission relies on the Ca(2+)-induced fusion of transmitter-laden vesicles whose coupling distance to Ca(2+)-channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Using electron- and super-resolution microscopy at the Drosophila neuromuscular junction this study quantitatively mapped vesicle:Ca(2+)-channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. It was shown that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca(2+)-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. Activity-dependent inhibition of vesicle un-priming or release site activation are proposed as novel facilitation mechanisms.
Zhang, Z., Bai, M., Barbosa, G. O., Chen, A., Wei, Y., Luo, S., Wang, X., Wang, B., Tsukui, T., Li, H., Sheppard, D., Kornberg, T. B. and Ma, D. K. (2020). Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking. Sci Adv 6(7): eaay7667. PubMed ID: 32095531
Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, this study identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. Amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. Evidence is provided that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.
Du, G., Xiong, L., Li, X., Zhuo, Z., Zhuang, X., Yu, Z., Wu, L., Xiao, D., Liu, Z., Jie, M., Liu, X., Luo, G., Guo, Z. and Chen, H. (2020). Peroxisome Elevation Induces Stem Cell Differentiation and Intestinal Epithelial Repair. Dev Cell 53(2): 169-184. PubMed ID: 32243783
Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. This study found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, it was found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients.

Monday, May 18th - Cytoskeleton and Junctions

Deng, H., Yang, L., Wen, P., Lei, H., Blount, P. and Pan, D. (2020). Spectrin couples cell shape, cortical tension, and Hippo signaling in retinal epithelial morphogenesis. J Cell Biol 219(4). PubMed ID: 32328630
Although extracellular force has a profound effect on cell shape, cytoskeleton tension, and cell proliferation through the Hippo signaling effector Yki/YAP/TAZ, how intracellular force regulates these processes remains poorly understood. This study reports an essential role for spectrin in specifying cell shape by transmitting intracellular actomyosin force to cell membrane. While activation of myosin II in Drosophila melanogaster pupal retina leads to increased cortical tension, apical constriction, and Yki-mediated hyperplasia, spectrin mutant cells, despite showing myosin II activation and Yki-mediated hyperplasia, paradoxically display decreased cortical tension and expanded apical area. Mechanistically, this study shows that spectrin is required for tethering cortical F-actin to cell membrane domains outside the adherens junctions (AJs). Thus, in the absence of spectrin, the weakened attachment of cortical F-actin to plasma membrane results in a failure to transmit actomyosin force to cell membrane, causing an expansion of apical surfaces. These results uncover an essential mechanism that couples cell shape, cortical tension, and Hippo signaling and highlight the importance of non-AJ membrane domains in dictating cell shape in tissue morphogenesis.
Drechsler, M., Lang, L. F., Al-Khatib, L., Dirks, H., Burger, M., Schonlieb, C. B. and Palacios, I. M. (2020). Optical flow analysis reveals that Kinesin-mediated advection impacts on the orientation of microtubules in the Drosophila oocyte. Mol Biol Cell: mbcE19080440. PubMed ID: 32267197
The orientation of microtubule networks is exploited by motors to deliver cargoes to specific intracellular destinations, and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of microtubule filaments. In cells however, microtubules are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. This study implemented variational optical flow analysis as a new approach to analyze the polarity of microtubules in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing microtubule orientation from confocal movies, it was found that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, it was found that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of microtubule plus ends is also altered. These findings lead to a proposal that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, a possible mechanism is proposed for a specialised cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds; to counteract the recruitment of Kinesin to microtubules.
Blackie, L., Walther, R. F., Staddon, M. F., Banerjee, S. and Pichaud, F. (2020). Cell-type specific mechanical response and myosin dynamics during retinal lens development in Drosophila. Mol Biol Cell: mbcE19090523. PubMed ID: 32320320
During organogenesis, different cell types need to work together to induce functional multicellular structures. To study this process, use was made of the genetically tractable fly retina, with a focus on the mechanisms that coordinate morphogenesis between the different epithelial cell types that make up the optical lens. This work shows that these epithelial cells present contractile apical-medial MyosinII meshworks, which control the apical area and junctional geometry of these cells during lens development. This study also suggests that MyosinII meshworks drive cell shape changes in response to external forces, and thus they mediate part of the biomechanical coupling that takes place between these cells. Importantly, this work, including mathematical modelling of forces and material stiffness during lens development, raises the possibility that increased cell stiffness acts as a mechanism for limiting this mechanical coupling. It is proposed that this might be required in complex tissues, where different cell types undergo concurrent morphogenesis and where averaging out of forces across cells could compromise individual cell apical geometry and thereby organ function.
Dey, B. and Rikhy, R. (2020). DE-cadherin and Myosin II balance regulates furrow length for onset of polygon shape in syncytial Drosophila embryos. J Cell Sci. PubMed ID: 32265269
Cell shape morphogenesis from spherical to polygonal occurs in epithelial cell formation in metazoan embryogenesis. In syncytial Drosophila embryos, the plasma membrane incompletely surrounds each nucleus and is organized as a polygonal epithelial-like array. Each cortical syncytial division cycle shows circular to polygonal plasma membrane transition along with furrow extension between adjacent nuclei from interphase to metaphase. This study assessed the relative contribution of DE-cadherin and Myosin II at the furrow for polygonal shape transition. Polygonality initiates during each cortical syncytial division cycle when the furrow extends from 4.75 to 5.75 microm. Polygon plasma membrane organization correlates with increased junctional tension, increased DE-cadherin and decreased Myosin II mobility. DE-cadherin regulates furrow length and polygonality. Decreased Myosin II activity allows for polygonality to occur at a lower length than controls. Increased Myosin II activity leads to loss of lateral furrow formation and complete disruption of polygonal shape transition. These studies show that DE-cadherin-Myosin II balance regulates an optimal lateral membrane length during each syncytial cycle for polygonal shape transition.
Chougule, A., Lapraz, F., Foldi, I., Cerezo, D., Mihaly, J. and Noselli, S. (2020). The Drosophila actin nucleator DAAM is essential for left-right asymmetry. PLoS Genet 16(4): e1008758. PubMed ID: 32324733
Left-Right (LR) asymmetry is essential for organ positioning, shape and function. Myosin 1D (Myo1D) has emerged as an evolutionary conserved chirality determinant in both Drosophila and vertebrates. However, the molecular interplay between Myo1D and the actin cytoskeleton underlying symmetry breaking remains poorly understood. To address this question, a dual genetic screen was performed to identify new cytoskeletal factors involved in LR asymmetry. The conserved actin nucleator DAAM was identified as an essential factor required for both dextral and sinistral development. In the absence of DAAM, organs lose their LR asymmetry, while its overexpression enhances Myo1D-induced de novo LR asymmetry. These results show that DAAM is a limiting, LR-specific actin nucleator connecting up Myo1D with a dedicated F-actin network important for symmetry breaking.
Chen, H. J., Li, Q., Nirala, N. K. and Ip, Y. T. (2020). The Snakeskin-Mesh Complex of Smooth Septate Junction Restricts Yorkie to Regulate Intestinal Homeostasis in Drosophila. Stem Cell Reports. PubMed ID: 32330445
Tight junctions in mammals and septate junctions in insects are essential for epithelial integrity. This study shows that, in the Drosophila intestine, smooth septate junction proteins provide barrier and signaling functions. During an RNAi screen for genes that regulate adult midgut tissue growth, it was found that loss of two smooth septate junction components, Snakeskin and Mesh, caused a hyperproliferation phenotype. By examining epitope-tagged endogenous Snakeskin and Mesh, it was demonstrated that the two proteins are present in the cytoplasm of differentiating enteroblasts and in cytoplasm and septate junctions of mature enterocytes. In both enteroblasts and enterocytes, loss of Snakeskin and Mesh causes Yorkie-dependent expression of the JAK-STAT pathway ligand Upd3, which in turn promotes proliferation of intestinal stem cells. Snakeskin and Mesh form a complex with each other, with other septate junction proteins and with Yorkie. Therefore, the Snakeskin-Mesh complex has both barrier and signaling function to maintain stem cell-mediated tissue homeostasis.

Friday May 15th - Adult neural development and function

Cattaneo, V., San Martin, A., Lew, S. E., Gelb, B. D. and Pagani, M. R. (2020). Repeating or Spacing learning sessions are strategies for memory improvement with shared molecular and neuronal components. Neurobiol Learn Mem: 107233. PubMed ID: 32360730
Intellectual disability is a common feature in genetic disorders with enhanced RAS-ERK1/2 signaling, including neurofibromatosis type 1 (NF1) and Noonan syndrome (NS). Additional training trials and additional spacing between trials, respectively, restores memory deficits in animal models of NF1 and NS. However, the relationship between the underlying mechanisms in these strategies remain obscure. This study developed an approach to examine the effect of adding training trials or spacing to a weak training protocol and used genetic and behavioral manipulations in Drosophila to explore such question. Repetition and spacing effects are highly related, being equally effective to improve memory in control flies and sharing mechanistic bases, including the requirement of RAS activity in mushroom body neurons and protein synthesis dependence. After spacing or repeating learning trials, memory improvement depends on the formation of long-term memory (LTM). Moreover, a disease-related gain-of-function RasV152G allele impaired LTM. Using minimal training protocols, this study established that both learning strategies were also equally effective for memory rescue in the RasV152G mutant and showed non-additive interaction of the spacing and repetition effects. Memory improvement was never detected after Ras inhibition. It is concluded that memory improvement by spacing or repeating training trials are two ways of using the same molecular resources, including RAS-ERK1/2-dependent signaling. This evidence supports the concept that learning problems in RAS-related disorders depend on the impaired ability to exploit the repetition and the spacing effect required for long-term memory induction.
de Azevedo, R. V. M., Hansen, C., Chen, K. F., Rosato, E. and Kyriacou, C. P. (2020). Disrupted Glutamate Signaling in Drosophila Generates Locomotor Rhythms in Constant Light. Front Physiol 11: 145. PubMed ID: 32210832
The Cambridge Protein Trap resource (CPTI) was used to screen for flies whose locomotor rhythms are rhythmic in constant light (LL) as a means of identifying circadian photoreception genes. From the screen of approximately 150 CPTI lines, seven hits were obtained, two of which targeted the glutamate pathway, Got1 (Glutamate oxaloacetate transaminase 1) and Gs2 (Glutamine synthetase 2). The genetic background was important with a strong interaction observed with the common and naturally occurring timeless (tim) polymorphisms, ls-tim and s-tim. The less circadian photosensitive ls-tim allele generated high levels of LL rhythmicity in combination with Got1 or Gs2, even though ls-tim and s-tim alleles do not, by themselves, generate the LL phenotype. The use of dsRNAi for both genes as well as for Gad (Glutamic acid decarboxylase) and the metabotropic glutamate receptor DmGluRA driven by clock gene promoters also revealed high levels of LL rhythmicity compared to controls. It is clear that the glutamate pathway is heavily implicated in circadian photoreception. TIM levels in Got1 and Gs2 mutants cycled and were more abundant than in controls under LL. Got1 but not Gs2 mutants showed diminished phase shifts to 10 min light pulses. Neurogenetic dissection of the LL rhythmic phenotype using the gal4/gal80 UAS bipartite system suggested that the more dorsal CRY-negative clock neurons, DNs and LNds were responsible for the LL phenotype. Immunocytochemistry using the CPTI YFP tagged insertions for the two genes revealed that the DN1s but not the DN2 and DN3s expressed Got1 and Gs2, but expression was also observed in the lateral neurons, the LNds and s-LNvs. These results suggest a model whereby the DN1s and possibly CRY-negative LNds use glutamate signaling to suppress the pacemaker s-LNvs in LL.
DeAngelis, B. D., Zavatone-Veth, J. A., Gonzalez-Suarez, A. D. and Clark, D. A. (2020). Spatiotemporally precise optogenetic activation of sensory neurons in freely walking Drosophila. Elife 9. PubMed ID: 32319425
Previous work has characterized how walking Drosophila coordinate the movements of individual limbs. To understand the circuit basis of this coordination, one must characterize how sensory feedback from each limb affects walking behavior. However, it has remained difficult to manipulate neural activity in individual limbs of freely moving animals. This study demonstrates a simple method for optogenetic stimulation with body side-, body segment-, and limb-specificity that does not require real-time tracking. Instead, precise locations were activated at random in time and space and use post hoc analysis to determine behavioral responses to specific activations. Using this method, this study has characterized limb coordination and walking behavior in response to transient activation of mechanosensitive bristle neurons and sweet-sensing chemoreceptor neurons. these findings reveal that activating these neurons has opposite effects on turning, and that activations in different limbs and body regions produce distinct behaviors.
Choi, J., Yu, S., Choi, M. S., Jang, S., Han, I. J., Maier, G. L., Sprecher, S. G. and Kwon, J. Y. (2020). Cellular Basis of Bitter-Driven Aversive Behaviors in Drosophila Larva. eNeuro 7(2). PubMed ID: 32220859
Feeding, a critical behavior for survival, consists of a complex series of behavioral steps. In Drosophila larvae, the initial steps of feeding are food choice, during which the quality of a potential food source is judged, and ingestion, during which the selected food source is ingested into the digestive tract. It remains unclear whether these steps employ different mechanisms of neural perception. This study provides insight into the two initial steps of feeding in Drosophila larva. Substrate choice and ingestion were found to be determined by independent circuits at the cellular level. First, 22 candidate bitter compounds were taken, and their influence on choice preference and ingestion behavior was examined. Interestingly, certain bitter tastants caused different responses in choice and ingestion, suggesting distinct mechanisms of perception. Evidence is further provided that certain gustatory receptor neurons (GRNs) in the external terminal organ (TO) are involved in determining choice preference, and a pair of larval pharyngeal GRNs is involved in mediating both avoidance and suppression of ingestion. These results show that feeding behavior is coordinated by a multistep regulatory process employing relatively independent neural elements. These findings are consistent with a model in which distinct sensory pathways act as modulatory circuits controlling distinct subprograms during feeding.
Chakravarti Dilley, L., Szuperak, M., Gong, N. N., Williams, C. E., Saldana, R. L., Garbe, D. S., Syed, M. H., Jain, R. and Kayser, M. S. (2020). Identification of a molecular basis for the juvenile sleep state. Elife 9. PubMed ID: 32202500
Across species, sleep in young animals is critical for normal brain maturation. The molecular determinants of early life sleep remain unknown. Through an RNAi-based screen, this study identified a gene, pdm3, required for sleep maturation in Drosophila. Pdm3, a transcription factor, coordinates an early developmental program that prepares the brain to later execute high levels of juvenile adult sleep. PDM3 controls the wiring of wake-promoting dopaminergic (DA) neurites to a sleep-promoting region, and loss of PDM3 prematurely increases DA inhibition of the sleep center, abolishing the juvenile sleep state. RNA-Seq/ChIP-Seq and a subsequent modifier screen reveal that pdm3 represses expression of the synaptogenesis gene Msp300 to establish the appropriate window for DA innervation. These studies define the molecular cues governing sleep behavioral and circuit development, and suggest sleep disorders may be of neurodevelopmental origin.
Bilz, F., Geurten, B. R. H., Hancock, C. E., Widmann, A. and Fiala, A. (2020). Visualization of a Distributed Synaptic Memory Code in the Drosophila Brain. Neuron. PubMed ID: 32268119
During associative conditioning, animals learn which sensory cues are predictive for positive or negative conditions. Because sensory cues are encoded by distributed neurons, one has to monitor plasticity across many synapses to capture how learned information is encoded. This study analyzed synaptic boutons of Kenyon cells of the Drosophila mushroom body gamma lobe, a brain structure that mediates olfactory learning. A fluorescent Ca(2+) sensor was expressed in single Kenyon cells so that axonal boutons could be assigned to distinct cells and Ca(2+) could be measured across many animals. Learning induced directed synaptic plasticity in specific compartments along the axons. Moreover, it was shown that odor-evoked Ca(2+) dynamics across boutons decorrelate as a result of associative learning. Information theory indicates that learning renders the stimulus representation more distinct compared with naive stimuli. These data reveal that synaptic boutons rather than cells act as individually modifiable units, and coherence among them is a memory-encoding parameter.

Thursday, May 14th - Signaling

Cheong, H. S. J., Nona, M., Guerra, S. B. and VanBerkum, M. F. (2020). The first quarter of the C-terminal domain of Abelson regulates the WAVE regulatory complex and Enabled in axon guidance. Neural Dev 15(1): 7. PubMed ID: 32359359
Abelson tyrosine kinase (Abl) plays a key role in axon guidance in linking guidance receptors to actin dynamics. The long C-terminal domain (CTD) of Drosophila Abl is important for this role, and previous work identified the 'first quarter' (1Q) of the CTD as essential. This study links the physical interactions of 1Q binding partners to Abl's function in axon guidance. Protein binding partners of 1Q were identified by GST pulldown and mass spectrometry and validated using axon guidance assays in the embryonic nerve cord and motoneurons. The role of 1Q was assessed genetically, utilizing a battery of Abl transgenes in combination with mutation or overexpression of the genes of pulled down proteins, and their partners in actin dynamics. The set of Abl transgenes had the following regions deleted: all of 1Q, each half of 1Q ('eighths', 1E and 2E) or a PxxP motif in 2E, which may bind SH3 domains. GST pulldown identified Hem and Sra-1 as binding partners of 1Q, and genetic analyses show that both proteins function with Abl in axon guidance, with Sra-1 likely interacting with 1Q. As Hem and Sra-1 are part of the actin-polymerizing WAVE regulatory complex (WRC), the analyses was extended to Abi and Trio, which interact with Abl and WRC members. Overall, the 1Q region (and especially 2E and its PxxP motif) are important for Abl's ability to work with WRC in axon guidance. These areas are also important for Abl's ability to function with the actin regulator Enabled. In comparison, 1E contributes to Abl function with the WRC at the midline, but less so with Enabled. It is concluded that the 1Q region, and especially the 2E region with its PxxP motif, links Abl with the WRC, its regulators Trio and Abi, and the actin regulator Ena. Removing 1E has specific effects suggesting it may help modulate Abl's interaction with the WRC or Ena. Thus, the 1Q region of Abl plays a key role in regulating actin dynamics during axon guidance.
Cairns, L., Patterson, A., Weingartner, K. A., Koehler, T. J., DeAngelis, D. R., Tripp, K. W., Bothner, B. and Kavran, J. M. (2020). Biophysical characterization of SARAH domain-mediated multimerization of Hippo pathway complexes in Drosophila. J Biol Chem 295(18): 6202-6213. PubMed ID: 32213597
Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain-mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain-mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, this study biophysically characterized the entire suite of SARAH domain-mediated complexes. The three SARAH domains were purified from Drosophila melanogaster, and an unbiased pulldown assay was performed to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, it was found that the Salvador SARAH domain homodimerizes and demonstrated that this interaction is conserved in Salvador's mammalian homolog. Using native MS, each of these complexes were shown to be dimeric in solution. The stability of each SARAH domain complex was measured, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized in this study comprehensively reveal the nature of SARAH domain-mediated complex formation and provide mechanistic insights into how SARAH domain-mediated interactions influence Hippo pathway activity.
Catinozzi, M., Mallik, M., Frickenhaus, M., Been, M., Sijlmans, C., Kulshrestha, D., Alexopoulos, I., Weitkunat, M., Schnorrer, F. and Storkebaum, E. (2020). The Drosophila FUS ortholog cabeza promotes adult founder myoblast selection by Xrp1-dependent regulation of FGF signaling. PLoS Genet 16(4): e1008731. PubMed ID: 32302304
The number of adult myofibers in Drosophila is determined by the number of founder myoblasts selected from a myoblast pool, a process governed by fibroblast growth factor (FGF) signaling. This study shows that loss of cabeza (caz) function results in a reduced number of adult founder myoblasts, leading to a reduced number and misorientation of adult dorsal abdominal muscles. Genetic experiments revealed that loss of caz function in both adult myoblasts and neurons contributes to caz mutant muscle phenotypes. Selective overexpression of the FGF receptor Htl or the FGF receptor-specific signaling molecule Stumps in adult myoblasts partially rescued caz mutant muscle phenotypes, and Stumps levels were reduced in caz mutant founder myoblasts, indicating FGF pathway deregulation. In both adult myoblasts and neurons, caz mutant muscle phenotypes were mediated by increased expression levels of Xrp1, a DNA-binding protein involved in gene expression regulation. Xrp1-induced phenotypes were dependent on the DNA-binding capacity of its AT-hook motif, and increased Xrp1 levels in founder myoblasts reduced Stumps expression. Thus, control of Xrp1 expression by Caz is required for regulation of Stumps expression in founder myoblasts, resulting in correct founder myoblast selection.
Chen, L., Zhang, T., Ge, M., Liu, Y., Xing, Y., Liu, L., Li, F. and Cheng, L. (2020). The Nrf2-Keap1 pathway: A secret weapon against pesticide persecution in Drosophila Kc cells. Pestic Biochem Physiol 164: 47-57. PubMed ID: 32284136
Nrf2-Keap1 pathway defends organisms against the detrimental effects of oxidative stress, and play pivotal roles in preventing xenobiotic-related toxicity. Experiments were designed to explore and verify its role and function under deltamethrin (DM) stress. In experiments, DM was selected as the inducer, and Drosophila Kc cells were treated as the objects. The result showed the oxidative stress of cells proliferated in a very short time after DM treatment, reaching the maximum after one hour of treatment. The experimental data showed Nrf2 could be up-regulated and activated by DM which were manifested by the increase of Nrf2 mRNA, Nrf2 protein in the nucleus and the expression of detoxification enzyme genes. The activity of all groups was further tested, and the survival rate of cells was found to be basically proportional to the expression of Nrf2. Based on the above experimental results, Keap1 overexpression (K+), Nrf2 overexpression (N+) or interference (N-) cells were used to verified the relationship between Nrf2, cell survival and detoxification enzymes expression. It was found the cell survival rate of N+ group was significantly higher than that of other groups and the expression of detoxification enzymes were increased compared to the control group. These results demonstrated that Nrf2 is related to cell detoxification and associated with the tolerance to DM. These evidence suggested Nrf2 is a potential therapeutic target for oxidative stress and a potential molecular target gene of resistance control.
Bunnag, N., Tan, Q. H., Kaur, P., Ramamoorthy, A., Sung, I. C. H., Lusk, J. and Tolwinski, N. S. (2020). An Optogenetic Method to Study Signal Transduction in Intestinal Stem Cell Homeostasis. J Mol Biol. PubMed ID: 32201167
Homeostasis in adult organs involves replacement of cells from a stem cell pool maintained in specialized niches regulated by extracellular signals. This cell-to-cell communication employs signal transduction pathways allowing cells to respond with a variety of behaviors. To study these cellular behaviors, signaling must be perturbed within tissues in precise patterns, a technique recently made possible by the development of optogenetic tools. Tools have been developed to study signal transduction in vivo in an adult fly midgut stem cell model where signaling was regulated by the application of light. Activation was achieved by clustering of membrane receptors EGFR and Toll, while inactivation was achieved by clustering the downstream activators ERK/Rolled and NFkappaB/Dorsal in the cytoplasm, preventing nuclear translocation and transcriptional activation. Both pathways contribute to stem and transit amplifying cell numbers and affect the lifespan of adult flies. This study further presents new approaches to overcome overexpression phenotypes and novel methods for the integration of optogenetics into the already-established genetic toolkit of Drosophila.
Boukhatmi, H., Martins, T., Pillidge, Z., Kamenova, T. and Bray, S. (2020). Notch mediates inter-tissue communication to promote tumorigenesis. Curr Biol. PubMed ID: 32275875
Disease progression in many tumor types involves the interaction of genetically abnormal cancer cells with normal stromal cells. Neoplastic transformation in a Drosophila genetic model of Epidermal growth factor receptor (EGFR)-driven tumorigenesis similarly relies on the interaction between epithelial and mesenchymal cells, providing a simple system to investigate mechanisms used for the cross-talk. Using the Drosophila model, this study shows that the transformed epithelium hijacks the mesenchymal cells through Notch signaling, which prevents their differentiation and promotes proliferation. A key downstream target in the mesenchyme is Zfh1/ZEB. When Notch or zfh1 are depleted in the mesenchymal cells, tumor growth is compromised. The ligand Delta is highly upregulated in the epithelial cells where it is found on long cellular processes. By using a live transcription assay in cultured cells and by depleting actin-rich processes in the tumor epithelium, this study provides evidence that signaling can be mediated by cytonemes from Delta-expressing cells. It is thus proposed that high Notch activity in the unmodified mesenchymal cells is driven by ligands produced by the cancerous epithelial. This long-range Notch signaling integrates the two tissues to promote tumorigenesis, by co-opting a normal regulatory mechanism that prevents the mesenchymal cells from differentiating.

Wednesday, May 13 - Disease Models

Huang, W., Campbell, T., Carbone, M. A., Jones, W. E., Unselt, D., Anholt, R. R. H. and Mackay, T. F. C. (2020). Context-dependent genetic architecture of Drosophila life span. PLoS Biol 18(3): e3000645. PubMed ID: 32134916
Understanding the genetic basis of variation in life span is a major challenge that is difficult to address in human populations. Evolutionary theory predicts that alleles affecting natural variation in life span will have properties that enable them to persist in populations at intermediate frequencies, such as late-life-specific deleterious effects, antagonistic pleiotropic effects on early and late-age fitness components, and/or sex- and environment-specific or antagonistic effects. This study quantified variation in life span in males and females reared in 3 thermal environments for the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and an advanced intercross outbred population derived from a subset of DGRP lines. Quantitative genetic analyses of life span and the micro-environmental variance of life span in the DGRP revealed significant genetic variance for both traits within each sex and environment, as well as significant genotype-by-sex interaction (GSI) and genotype-by-environment interaction (GEI). Genome-wide association (GWA) mapping in both populations implicates over 2,000 candidate genes with sex- and environment-specific or antagonistic pleiotropic allelic effects. Over 1,000 of these genes are associated with variation in life span in other D. melanogaster populations. The effects of 15 candidate genes were functionally assessed using RNA interference (RNAi): all affected life span and/or micro-environmental variance of life span in at least one sex and environment and exhibited sex-and environment-specific effects. These results implicate novel candidate genes affecting life span and suggest that variation for life span may be maintained by variable allelic effects in heterogeneous environments.
Cassar, M., Law, A. D., Chow, E. S., Giebultowicz, J. M. and Kretzschmar, D. (2020). Disease-Associated Mutant Tau Prevents Circadian Changes in the Cytoskeleton of Central Pacemaker Neurons. Front Neurosci 14: 232. PubMed ID: 32292325
A hallmark feature of Alzheimer's disease (AD) and other Tauopathies, like Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17), is the accumulation of neurofibrillary tangles composed of the microtubule-associated protein Tau. As in AD, symptoms of FTDP-17 include cognitive decline, neuronal degeneration, and disruptions of sleep patterns. However, mechanisms by which Tau may lead to these disturbances in sleep and activity patterns are unknown. To identify such mechanisms, this study has generated novel Drosophila Tauopathy models by replacing endogenous fly dTau with normal human Tau (hTau) or the FTDP-17 causing hTau(V337M) mutation. This mutation is localized in one of the microtubule-binding domains of hTau and has a dominant effect. Analyzing heterozygous flies, this study found that aged hTau(V337M) flies show neuronal degeneration and locomotion deficits when compared to wild type or hTau(WT) flies. Furthermore, hTau(V337M) flies are hyperactive and they show a fragmented sleep pattern. These changes in the sleep/activity pattern are accompanied by morphological changes in the projection pattern of the central pacemaker neurons. These neurons show daily fluctuations in their connectivity, whereby synapses are increased during the day and reduced during sleep. Synapse formation requires cytoskeletal changes that can be detected by the accumulation of the end-binding protein 1 (EB1) at the site of synapse formation. Whereas, hTau(WT) flies show the normal day/night changes in EB1 accumulation, hTau(V337M) flies do not show this fluctuation. This suggests that hTau(V337M) disrupts sleep patterns by interfering with the cytoskeletal changes that are required for the synaptic homeostasis of central pacemaker neurons.
Xu, Y., Xie, M., Xue, J., Xiang, L., Li, Y., Xiao, J., Xiao, G. and Wang, H. L. (2020). EGCG ameliorates neuronal and behavioral defects by remodeling gut microbiota and TotM expression in Drosophila models of Parkinson's disease. Faseb j. PubMed ID: 32157731
Parkinson's disease (PD) is the second most common neurodegenerative disease. Eigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is known to exert a beneficial effect on PD patients. Although some mechanisms were suggested to underlie this intervention, it remains unknown if the EGCG-mediated protection was achieved by remodeling gut microbiota. In the present study, 0.1 mM or 0.5 mM EGCG was administered to the Drosophila melanogaster with PINK1 (PTEN induced putative kinase 1) mutations, a prototype PD model, and their behavioral performances, as well as neuronal/mitochondrial morphology (only for 0.5 mM EGCG treatment) were determined. According to the results, the mutant PINK1(B9) flies exhibited dopaminergic, survival, and behavioral deficits, which were rescued by EGCG supplementation. Meanwhile, EGCG resulted in profound changes in gut microbial compositions in PINK1(B9) flies, restoring the abundance of a set of bacteria. Notably, EGCG protection was blunted when gut microbiota was disrupted by antibiotics. Four bacterial strains were isolated from fly guts and the supplementation of individual Lactobacillus plantarum or Acetobacter pomorum strain exacerbated the neuronal and behavioral dysfunction of PD flies, which could not be rescued by EGCG. Transcriptomic analysis identified TotM as the central gene responding to EGCG or microbial manipulations. Genetic ablation of TotM blocked the recovery activity of EGCG, suggesting that EGCG-mediated protection warrants TotM. Apart from familial form, EGCG was also potent in improving sporadic PD symptoms induced by rotenone treatment, wherein gut microbiota shared regulatory roles. Together, these results suggest the relevance of the gut microbiota-TotM pathway in EGCG-mediated neuroprotection, providing insight into indirect mechanisms underlying nutritional intervention of Parkinson's disease.
Pirooznia, S. K., Yuan, C., Khan, M. R., Karuppagounder, S. S., Wang, L., Xiong, Y., Kang, S. U., Lee, Y., Dawson, V. L. and Dawson, T. M. (2020). PARIS induced defects in mitochondrial biogenesis drive dopamine neuron loss under conditions of parkin or PINK1 deficiency. Mol Neurodegener 15(1): 17. PubMed ID: 32138754
Mutations in PINK1 and parkin cause autosomal recessive Parkinson's disease (PD). Evidence placing PINK1 and parkin in common pathways regulating multiple aspects of mitochondrial quality control is burgeoning. However, compelling evidence to causatively link specific PINK1/parkin dependent mitochondrial pathways to dopamine neuron degeneration in PD is lacking. This study examined how PINK1/parkin mediated regulation of the pathogenic substrate PARIS impacts dopaminergic mitochondrial network homeostasis and neuronal survival in Drosophila. The UAS-Gal4 system was employed for cell-type specific expression of the various transgenes. Effects on dopamine neuronal survival and function were assessed by anti-TH immunostaining and negative geotaxis assays. Defects in mitochondrial biogenesis were shown to drive adult onset progressive loss of dopamine neurons and motor deficits in Drosophila models of PINK1 or parkin insufficiency. Such defects result from PARIS dependent repression of dopaminergic PGC-1alpha and its downstream transcription factors NRF1 and TFAM that cooperatively promote mitochondrial biogenesis. Dopaminergic accumulation of human or Drosophila PARIS recapitulates these neurodegenerative phenotypes that are effectively reversed by PINK1, parkin or PGC-1alpha overexpression in vivo. PARIS is the only co-substrate of PINK1 and parkin to specifically accumulate in the DA neurons and cause neurodegeneration and locomotor defects stemming from disrupted dopamine signaling. These findings identify a highly conserved role for PINK1 and parkin in regulating mitochondrial biogenesis and promoting mitochondrial health via the PARIS/ PGC-1alpha axis. The Drosophila models described in this study effectively recapitulate the cardinal PD phenotypes and thus will facilitate identification of novel regulators of mitochondrial biogenesis for physiologically relevant therapeutic interventions.
Miguel, L., Frebourg, T., Campion, D. and Lecourtois, M. (2020). Moderate Overexpression of Tau in Drosophila Exacerbates Amyloid-beta-Induced Neuronal Phenotypes and Correlates with Tau Oligomerization. J Alzheimers Dis. PubMed ID: 32065789
Alzheimer's disease (AD) is neuropathologically defined by two key hallmarks: extracellular senile plaques composed primarily of amyloid-beta (Abeta) peptide and intraneuronal neurofibrillary tangles, containing abnormally hyperphosphorylated tau protein. The tau protein is encoded by the MAPT gene. Recently, the H1 and H2 haplotypes of the MAPT gene were associated with AD risk. The minor MAPT H2 haplotype has been linked with a decreased risk of developing late-onset AD (LOAD). MAPT haplotypes show different levels of MAPT/Tau expression with H1 being approximately 1.5-fold more expressed than H2, suggesting that MAPT expression level could be related to LOAD risk. This study investigated whether this moderate difference in MAPT/Tau expression could influence Abeta-induced toxicity in vivo. It was shown that modest overexpression of tau protein in Drosophila exacerbates neuronal phenotypes in AbetaPP/BACE1 (amyloid-beta protein precursor/Beta-Secretase 1) flies. The exacerbation of neuronal defects correlates with the accumulation of insoluble dTau oligomers, suggesting that the moderate difference in level of tau expression observed between H1 and H2 haplotypes could influence Abeta toxicity through the production of oligomeric Tau insoluble species.
Thackray, A. M., Lam, B., Shahira Binti Ab Razak, A., Yeo, G. and Bujdoso, R. (2020). Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease. Biochem J 477(4): 833-852. PubMed ID: 32108870
Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. This study used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, these data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology.

Tuesday, May 12th - Signaling

Bakopoulos, D., Forbes-Beadle, L., Esposito, K. M., Mirth, C. K., Warr, C. G. and Johnson, T. K. (2020). Insulin-Like Signalling Influences the Coordination of Larval Hemocyte Number with Body Size in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32341056
Blood cells, known as hemocytes in invertebrates, play important and conserved roles in immunity, wound healing and tissue remodelling. The control of hemocyte number is therefore critical to ensure these functions are not compromised, and studies using Drosophila melanogaster are proving useful for understanding how this occurs. Recently, the embryonic patterning gene, torso-like (tsl), was identified as being required both for normal hemocyte development and for providing immunity against certain pathogens. This study reports that Tsl is required specifically during the larval phase of hematopoiesis, and that tsl mutant larvae likely have reduced hemocyte numbers due to a reduced larval growth rate and compromised insulin signalling. Consistent with this, impairing insulin-mediated growth, either by nutrient deprivation or genetically, results in fewer hemocytes. This is likely the result of impaired insulin-like signalling in the hemocytes themselves, since modulation of Insulin-like Receptor (InR) activity specifically in hemocytes causes concomitant changes to their population size in developing larvae. Taken together, this work reveals the strong relationship that exists between body size and hemocyte number, and suggests that insulin-like signalling contributes to, but is not solely responsible for, keeping these tightly aligned during larval development.
Vissers, J. H. A., Dent, L. G., House, C. M., Kondo, S. and Harvey, K. F. (2020). Pits and CtBP Control Tissue Growth in Drosophila melanogaster with the Hippo Pathway Transcription Repressor, Tgi. Genetics. PubMed ID: 32122936
The Hippo pathway is an evolutionary conserved signalling network that regulates organ size, cell fate control and tumorigenesis. The central Hippo signalling effector is the transcriptional co-activator Yorkie, which controls gene expression in partnership with different transcription factors, most notably Scalloped. When it is not activated by Yorkie, Scalloped can act as a repressor of transcription, at least in part due to its interaction with the corepressor protein Tgi. The mechanism by which Tgi represses transcription is incompletely understood and therefore this study sought to identify proteins that potentially operate together with it. Using an affinity purification and mass-spectrometry approach this study identified Pits and CtBP as Tgi-interacting proteins, both of which have been linked to transcriptional repression. Both Pits and CtBP were required for Tgi to suppress the growth of the Drosophila melanogaster eye and CtBP loss suppressed the undergrowth of yorkie mutant eye tissue. Furthermore, as reported previously for Tgi, overexpression of Pits repressed transcription of Hippo pathway target genes. These findings suggest that Tgi might operate together with Pits and CtBP to repress transcription of genes that normally promote tissue growth. The human orthologues of Tgi, CtBP and Pits (VGLL4, CTBP2 and IRF2BP2) have previously been shown to physically and functionally interact to control transcription, implying that the mechanism by which these proteins control transcriptional repression is conserved throughout evolution (Vissers, 2020).
Vega-Cuesta, P., Ruiz-Gomez, A., Molnar, C., Organista, M. F., Resnik-Docampo, M., Falo-Sanjuan, J., Lopez-Varea, A. and de Celis, J. F. (2020). Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability. Dev Biol. PubMed ID: 32061885
Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. This study finds that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.
Yeom, E., Kwon, D. W., Lee, J., Kim, S. H., Lee, J. H., Min, K. J., Lee, K. S. and Yu, K. (2020). Asparaginyl-tRNA Synthetase, a Novel Component of Hippo Signaling, Binds to Salvador and Enhances Yorkie-Mediated Tumorigenesis. Front Cell Dev Biol 8: 32. PubMed ID: 32117966
Aminoacyl-tRNA synthetases (ARSs), which are essential for protein translation, were recently shown to have non-translational functions in various pathological conditions including cancer. However, the molecular mechanism underlying the role of ARSs in cancer remains unknown. This study demonstrates that asparaginyl-tRNA synthetase (NRS) regulates Yorkie-mediated tumorigenesis by binding to the Hippo pathway component Salvador. NRS-RNAi and the NRS inhibitor tirandamycin B (TirB) suppressed Yorkie-mediated tumor phenotypes in Drosophila. Genetic analysis showed that NRS interacted with Salvador, and NRS activated Hippo target genes by regulating Yorkie phosphorylation. Biochemical analyses showed that NRS blocked Salvador-Hippo binding by interacting directly with Salvador, and TirB treatment inhibited NRS-Salvador binding. YAP target genes were upregulated in a mammalian cancer cell line with high expression of NRS, whereas TirB treatment suppressed cancer cell proliferation. These results indicate that NRS regulates tumor growth by interacting with Salvador in the Hippo signaling pathway.
Blanco-Obregon, D., Katz, M. J., Durrieu, L., Gandara, L. and Wappner, P. (2020). Context-specific functions of Notch in Drosophila blood cell progenitors. Dev Biol. PubMed ID: 32243888
Drosophila Larval hematopoiesis takes place at the lymph gland, where myeloid-like progenitors differentiate into Plasmatocytes and Crystal Cells, under regulation of conserved signaling pathways. It has been established that the Notch pathway plays a specific role in Crystal Cell differentiation and maintenance. In mammalian hematopoiesis, the Notch pathway has been proposed to fulfill broader functions, including Hematopoietic Stem Cell maintenance and cell fate decision in progenitors. This work describes different roles that Notch plays in the lymph gland. Notch, activated by its ligand Serrate, is expressed at the Posterior Signaling Center is required to restrain Core Progenitor differentiation. A novel population of blood cell progenitors is defined that was named Distal Progenitors, where Notch, activated by Serrate expressed in Lineage Specifying Cells at the Medullary Zone/Cortical Zone boundary, regulates a binary decision between Plasmatocyte and Crystal Cell fates. Thus, Notch plays context-specific functions in different blood cell progenitor populations of the Drosophila lymph gland.
Weiner, A. T., Seebold, D. Y., Torres-Gutierrez, P., Folker, C.... Axelrod, J. D. and Rolls, M. M. (2020). Endosomal Wnt signaling proteins control microtubule nucleation in dendrites. PLoS Biol 18(3): e3000647. PubMed ID: 32163403
Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, this study found that canonical Wnt signaling proteins regulate localization of the core nucleation protein gammaTubulin (gammaTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Igamma, G proteins, and Axin reduced gammaTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic gammaTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with Fz, Dsh, Axin, and Rab5. It is proposed that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.

Monday, May 11th - Cytoskeleton and Junctions

Manhart, A., Azevedo, M., Baylies, M. and Mogilner, A. (2020). Reverse engineering forces responsible for dynamic clustering and spreading of multiple nuclei in developing muscle cells. Mol Biol Cell: mbcE19120711. PubMed ID: 32129712
How cells position organelles is a fundamental biological question. During Drosophila embryonic muscle development, multiple nuclei transition from being clustered together, to splitting into two smaller clusters, to spreading along the myotube's length. Perturbations of microtubules and motor proteins disrupt this sequence of events. These perturbations do not allow intuiting which molecular forces govern the nuclear positioning; therefore computational screening was used to reverse engineer and identify these forces. The screen reveals three models: two suggest that the initial clustering is due to the nuclear repulsion from the cell poles, while the third, most robust, model poses that this clustering is due to a short-ranged internuclear attraction. All three models suggest that the nuclear spreading is due to the long-ranged internuclear repulsion. The robust model was quantitatively tested by comparing it to data from perturbed muscle cells. The model was also tested by using agent-based simulations with elastic dynamic microtubules and molecular motors. The model predicts that, in longer mammalian myotubes with a great number of nuclei, the spreading stage would be preceded with segregation of the nuclei into a large number of clusters, proportional to the myotube length, with a small average number of nuclei per cluster.
Beyenbach, K. W., Schoene, F., Breitsprecher, L. F., Tiburcy, F., Furuse, M., Izumi, Y., Meyer, H., Jonusaite, S., Rodan, A. R. and Paululat, A. (2020). The Septate Junction Protein Tetraspanin 2A is critical to the Structure and Function of Malpighian tubules in Drosophila melanogaster. Am J Physiol Cell Physiol. PubMed ID: 32267718
Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, this study used the GAL4/UAS system to selectively knockdown Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh and Discs Large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels in Malpighian tubules together with diminished V-type H(+)-ATPase activity is consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifest an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.
Cerqueira Campos, F., Dennis, C., Alegot, H., Fritsch, C., Isabella, A., Pouchin, P., Bardot, O., Horne-Badovinac, S. and Mirouse, V. (2020). Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation. Development. PubMed ID: 32156755
How extracellular matrix participates to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. This study found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell autonomous control of F-actin fibers orientation relies on the previous BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a critical organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation.
Zheng, Y., Buchwalter, R. A., Zheng, C., Wight, E. M., Chen, J. V. and Megraw, T. L. (2020). A perinuclear microtubule-organizing centre controls nuclear positioning and basement membrane secretion. Nat Cell Biol 22(3): 297-309. PubMed ID: 32066907
Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. This study reports a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor gamma-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.
Galletta, B. J., Ortega, J. M., Smith, S. L., Fagerstrom, C. J., Fear, J. M., Mahadevaraju, S., Oliver, B. and Rusan, N. M. (2020). Sperm Head-Tail Linkage Requires Restriction of Pericentriolar Material to the Proximal Centriole End. Dev Cell. PubMed ID: 32169161
The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, this study identified a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage.
Yaniv, S. P., Meltzer, H., Alyagor, I. and Schuldiner, O. (2020). Developmental axon regrowth and primary neuron sprouting utilize distinct actin elongation factors. J Cell Biol 219(5). PubMed ID: 32191286
Intrinsic neurite growth potential is a key determinant of neuronal regeneration efficiency following injury. The stereotypical remodeling of Drosophila gamma-neurons includes developmental regrowth of pruned axons to form adult specific connections, thereby offering a unique system to uncover growth potential regulators. Motivated by the dynamic expression in remodeling gamma-neurons, this study focused on the role of actin elongation factors as potential regulators of developmental axon regrowth. Regrowth in vivo was found to require the actin elongation factors Ena and profilin, but not the formins that are expressed in gamma-neurons. In contrast, primary gamma-neuron sprouting in vitro requires profilin and the formin DAAM, but not Ena. Furthermore, this study demonstrates that DAAM can compensate for the loss of Ena in vivo. Similarly, DAAM mutants express invariably high levels of Ena in vitro. Thus, this study shows that different linear actin elongation factors function in distinct contexts even within the same cell type and that they can partially compensate for each other.

Friday, May 8th - Adult neural development

Sudarsanam, S., Yaniv, S., Meltzer, H. and Schuldiner, O. (2020). Cofilin regulates axon growth and branching of Drosophila gamma neurons. J Cell Sci. PubMed ID: 32152181
The mechanisms that control intrinsic axon growth potential, and thus axon regeneration following injury, are not well understood. Developmental axon regrowth of Drosophila mushroom body gamma neurons during neuronal remodeling offers a unique opportunity to study the molecular mechanisms controlling intrinsic growth potential. Motivated by the recently uncovered developmental expression atlas of gamma neurons, this study focused on the role of the actin severing protein cofilin during axon regrowth. Twinstar (Tsr), the fly cofilin, is a crucial regulator of both axon growth and branching during developmental remodeling of gamma neurons. tsr mutant axons demonstrate growth defects both in vivo and in vitro and also exhibit actin rich filopodial-like structures at failed branch points in vivo. These data is inconsistent with Tsr being important for increasing G-actin availability. Furthermore, analysis of microtubule localization suggests that Tsr is required for microtubule infiltration into the axon tips and branch points. Taken together, this study shows that Tsr promotes axon growth and branching, likely by clearing F-actin to facilitate microtubules protrusion.
Warth Perez Arias, C. C., Frosch, P., Fiala, A. and Riemensperger, T. D. (2020). Stochastic and arbitrarily generated input patterns to the mushroom bodies can serve as conditioned stimuli in Drosophila. Front Physiol 11: 53. PubMed ID: 32116764
Single neurons in the brains of insects often have individual genetic identities and can be unambiguously identified between animals. The overall neuronal connectivity is also genetically determined and hard-wired to a large degree. Experience-dependent structural and functional plasticity is believed to be superimposed onto this more-or-less fixed connectome. However, in Drosophila, it has been shown that the connectivity between the olfactory projection neurons (OPNs) and Kenyon cells, the intrinsic neurons of the mushroom body, is highly stochastic and idiosyncratic between individuals. Ensembles of distinctly and sparsely activated Kenyon cells represent information about the identity of the olfactory input, and behavioral relevance can be assigned to this representation in the course of associative olfactory learning. This study has tested the hypothesis that the mushroom body can learn any stochastic neuronal input pattern as behaviorally relevant, independent of its exact origin. Fruit flies can learn thermogenetically generated, stochastic activity patterns of OPNs as conditioned stimuli, irrespective of glomerular identity, the innate valence that the projection neurons carry, or inter-hemispheric symmetry.
Sherer, L. M., Catudio Garrett, E., Morgan, H. R., Brewer, E. D., Sirrs, L. A., Shearin, H. K., Williams, J. L., McCabe, B. D., Stowers, R. S. and Certel, S. J. (2020). Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons. PLoS Genet 16(2): e1008609. PubMed ID: 32097408
Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. This study establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. These findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network.
Zhang, T., Cheng, D., Wu, C., Wang, X., Ke, Q., Lou, H., Zhu, L., Wang, X. D., Duan, S. and Liu, Y. J. (2020). Lysosomal hydrolase Cathepsin D non-proteolytically modulates dendritic morphology in Drosophila. Neurosci Bull. PubMed ID: 32170568
The main lysosomal protease cathepsin D (cathD) is essential for maintaining tissue homeostasis via its degradative function, and its loss leads to ceroid accumulation in the mammalian nervous system, which results in progressive neurodegeneration. Increasing evidence implies non-proteolytic roles of cathD in regulating various biological processes such as apoptosis, cell proliferation, and migration. Along these lines, this study shows that cathD is required for modulating dendritic architecture in the nervous system independent of its traditional degradative function. Upon cathD depletion, class I and class III arborization (da) neurons in Drosophila larvae exhibited aberrant dendritic morphology, including over-branching, aberrant turning, and elongation defects. Re-introduction of wild-type cathD or its proteolytically-inactive mutant dramatically abolished these morphological defects. Moreover, cathD knockdown also led to dendritic defects in the adult mushroom bodies, suggesting that cathD-mediated processes are required in both the peripheral and central nervous systems. Taken together, these results demonstrate a critical role of cathD in shaping dendritic architecture independent of its proteolytic function.
Sun, H., Nishioka, T., Hiramatsu, S., Kondo, S., Amano, M., Kaibuchi, K., Ichinose, T. and Tanimoto, H. (2020). Dopamine receptor Dop1R2 stabilizes appetitive olfactory memory through the Raf/MAPK pathway in Drosophila. J Neurosci. PubMed ID: 32102921
In Drosophila, dopamine signaling to the mushroom body intrinsic neurons, Kenyon cells (KCs), is critical to stabilize olfactory memory. Little is known about the downstream intracellular molecular signaling underlying memory stabilization. This study addresses this question in the context of sugar-rewarded olfactory long-term memory (LTM). Associative conditioning increases the phosphorylation of MAPK in KCs, via Dop1R2 signaling. Consistently, the attenuation of Dop1R2, Raf or MAPK expression in KCs selectively impairs LTM but not short-term memory. Moreover, this study shows that the LTM deficit caused by the knockdown of Dop1R2 can be rescued by expressing active Raf in KCs. Thus, the Dop1R2/Raf/MAPK pathway is a pivotal downstream effector of dopamine signaling for stabilizing appetitive olfactory memory.
Allen, A. M., Neville, M. C., Birtles, S., Croset, V., Treiber, C. D., Waddell, S. and Goodwin, S. F. (2020). A single-cell transcriptomic atlas of the adult Drosophila ventral nerve cord. Elife 9. PubMed ID: 32314735
The Drosophila ventral nerve cord (VNC) receives and processes descending signals from the brain to produce a variety of coordinated locomotor outputs. It also integrates sensory information from the periphery and sends ascending signals to the brain. This study used single-cell transcriptomics to generate an unbiased classification of cellular diversity in the VNC of five-day old adult flies. An atlas was produced of 26,000 high-quality cells, representing more than 100 transcriptionally distinct cell types. The predominant gene signatures defining neuronal cell types reflect shared developmental histories based on the neuroblast from which cells were derived, as well as their birth order. The relative position of cells along the anterior-posterior axis could also be assigned using adult Hox gene expression. This single-cell transcriptional atlas of the adult fly VNC will be a valuable resource for future studies of neurodevelopment and behavior.

Thursday, May 7th - Chromatin

Kurihara, M., Komatsu, K., Awane, R. and Inoue, Y. H. (2020). Loss of Histone Locus Bodies in the Mature Hemocytes of Larval Lymph Gland Result in Hyperplasia of the Tissue in mxc Mutants of Drosophila. Int J Mol Sci 21(5). PubMed ID: 32111032
Mutations in the multi sex combs (mxc) gene in Drosophila results in ma lignant hyperplasia in larval hematopoietic tissues, called lymph glands (LG). mxc encodes a component of the histone locus body (HLB) that is essential for cell cycle-dependent transcription and processing of histone mRNAs. The mammalian nuclear protein ataxia-telangiectasia (NPAT) gene, encoded by the responsible gene for ataxia telangiectasia, is a functional Mxc orthologue. However, their roles in tumorigenesis are unclear. Genetic analyses of the mxc mutants and larvae having LG-specific depletion revealed that a reduced activity of the gene resulted in the hyperplasia, which is caused by hyper-proliferation of immature LG cells. The depletion of mxc in mature hemocytes of the LG resulted in the hyperplasia. Furthermore, the inhibition of HLB formation was required for LG hyperplasia. In the mutant larvae, the total mRNA levels of the five canonical histones decreased, and abnormal forms of polyadenylated histone mRNAs, detected rarely in normal larvae, were generated. The ectopic expression of the polyadenylated mRNAs was sufficient for the reproduction of the hyperplasia. The loss of HLB function, especially 3-end processing of histone mRNAs, is critical for malignant LG hyperplasia in this leukemia model in Drosophila. It is proposed that mxc is involved in the activation to induce adenosine deaminase-related growth factor A (Adgf-A), which suppresses immature cell proliferation in LG.
Kursel, L. E., Welsh, F. C. and Malik, H. S. (2020). Ancient co-retention of paralogs of Cid centromeric histones and Cal1 chaperones in mosquito species. Mol Biol Evol. PubMed ID: 32125433
Four instances have been identified of gene duplication and specialization of Cid, which encodes for the centromeric histone in Drosophila. It was hypothesized that retention of specialized Cid paralogs could be selectively advantageous to resolve the intralocus conflict that occurs on essential genes like Cid, which are subject to divergent selective pressures to perform multiple functions. If this were the case, finding other instances of co-retention and specialization of centromeric proteins during animal evolution would be expected. Consistent with this hypothesis, this study found that most mosquito species encode two CenH3 (mosqCid) genes, mosqCid1 and mosqCid2, which have been co-retained for over 150 million years. In addition, Aedes species encode a third mosqCid3 gene, which arose from an independent gene duplication of mosqCid1. Like Drosophila Cid paralogs, mosqCid paralogs evolve under different selective constraints and show tissue-specific expression patterns. Analysis of mosqCid N-terminal protein motifs further supports the model that mosqCid paralogs have functionally diverged. Extending this survey to other centromeric proteins, it was found that all Anopheles mosquitos encode two CAL1 paralogs, which are the chaperones that deposit CenH3 proteins at centromeres in Diptera, but a single CENP-C paralog. The ancient co-retention of paralogs of centromeric proteins adds further support to the hypothesis that intralocus conflict can drive their co-retention and functional specialization.
Medina-Pritchard, B., Lazou, V., Zou, J., Byron, O., Abad, M. A., Rappsilber, J., Heun, P. and Jeyaprakash, A. A. (2020). Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1. EMBO J: e103234. PubMed ID: 32134144
Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X-ray crystallography, this study shows how Drosophila CAL1, an evolutionarily distinct CENP-A histone chaperone, binds both CENP-A and the centromere receptor CENP-C without the requirement for the Mis18 complex. While an N-terminal CAL1 fragment wraps around CENP-A/H4 through multiple physical contacts, a C-terminal CAL1 fragment directly binds a CENP-C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP-A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP-C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP-C dimer. Overall, by demonstrating how CAL1 binds CENP-A/H4 and CENP-C, this study provides key insights into the minimalistic principles underlying centromere maintenance.
Torres-Campana, D., Kimura, S., Orsi, G. A., Horard, B., Benoit, G. and Loppin, B. (2020). The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition. PLoS Genet 16(3): e1008543. PubMed ID: 32134927
Following fertilization of a mature oocyte, the formation of a diploid zygote involves a series of coordinated cellular events that ends with the first embryonic mitosis. In animals, this complex developmental transition is almost entirely controlled by maternal gene products. How such a crucial transcriptional program is established during oogenesis remains poorly understood. This study performed an shRNA-based genetic screen in Drosophila to identify genes required to form a diploid zygote. The Lid/KDM5 histone demethylase and its partner, the Sin3A-HDAC1 deacetylase complex, are necessary for sperm nuclear decompaction and karyogamy. Surprisingly, transcriptomic analyses revealed that these histone modifiers are required for the massive transcriptional activation of deadhead (dhd), which encodes a maternal thioredoxin involved in sperm chromatin remodeling. Unexpectedly, while lid knock-down tends to slightly favor the accumulation of its target, H3K4me3, on the genome, this mark was lost at the dhd locus. It is proposed that Lid/KDM5 and Sin3A cooperate to establish a local chromatin environment facilitating the unusually high expression of dhd, a key effector of the oocyte-to-zygote transition.
Santana, J. F., Parida, M., Long, A., Wankum, J., Lilienthal, A. J., Nukala, K. M. and Manak, J. R. (2020). The Dm-Myb Oncoprotein Contributes to Insulator Function and Stabilizes Repressive H3K27me3 PcG Domains. Cell Rep 30(10): 3218-3228. PubMed ID: 32160531
Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. This study further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. This study now reveals a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation.
Alecki, C., Chiwara, V., Sanz, L. A., Grau, D., Arias Perez, O., Boulier, E. L., Armache, K. J., Chedin, F. and Francis, N. J. (2020). RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2. Nat Commun 11(1): 1781. PubMed ID: 32286294
Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. This study shows that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, this study found that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. These results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.

Wednesday, May 6th - Adult physiology

Ahmed, M. A. I. and Vogel, C. F. A. (2020). Hazardous effects of octopamine receptor agonists on altering metabolism-related genes and behavior of Drosophila melanogaster. Chemosphere 253: 126629. PubMed ID: 32283422
Recent reports demonstrate that octopamine receptor (OR) agonists such as formamidine pesticides cause reproductive and developmental toxicity through endocrine disrupting effects in both humans and animals. The effects of different sublethal concentrations of OR agonists, Amitraz and Chlordimeform, on growth, development, and reproduction of D. melanogaster were studied from a genotype perspective view. As a result, the sublethal concentrations for both OR agonists delayed the developmental time including pupation and eclosion. It significantly reduced the lifespan, eclosion rate, and production of eggs. The mRNA expression of genes relevant for development and metabolism was significantly changed after exposure to sublethal concentrations of both OR agonists. Octopamine receptor in mushroom bodies (Oamb), trehalase enzyme (Treh), hemocyte proliferation (RyR), and immune response (IM4) genes were upregulated whereas, trehalose sugar (Tret1-1), mixed function oxidase enzyme (Cyp9f2), lifespan (Atg7), male mating behavior (Ple), female fertility (Ddc), and lipid metabolism (Sxe2) genes were downregulated. These results support the conclusion that OR agonists activate the octopamine receptor in D. melanogaster leading to an increase of trehalase enzyme activity and degradation of trehalose sugar into free glucose which results in rapid energy exhaustion, hyperexcitation, and disturbing of the octopaminergic system in D. melanogaster.
Sejour, R., Sanguino, R. A., Mikolajczak, M., Ahmadi, W. and Villa-Cuesta, E. (2020). Sirt4 modulates oxidative metabolism and sensitivity to rapamycin through species-dependent phenotypes in Drosophila mtDNA haplotypes. G3 (Bethesda). PubMed ID: 32152006
The endosymbiotic theory proposes that eukaryotes evolved from the symbiotic relationship between anaerobic (host) and aerobic prokaryotes. Through iterative genetic transfers, the mitochondrial and nuclear genomes coevolved, establishing the mitochondria as the hub of oxidative metabolism. To study this coevolution, this study disrupt mitochondrial-nuclear epistatic interactions by using strains that have mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from evolutionarily divergent species. A multifaceted approach was undertaken generating introgressed Drosophila strains containing D. simulans mtDNA and D. melanogaster nDNA with Sirtuin 4 (Sirt4)-knockouts. Sirt4 is a nuclear-encoded enzyme that functions, exclusively within the mitochondria, as a master regulator of oxidative metabolism. Flies were exposed to the drug rapamycin in order to eliminate TOR signaling, thereby compromising the cytoplasmic crosstalk between the mitochondria and nucleus. The results indicate that D. simulans and D. melanogaster mtDNA haplotypes display opposite Sirt4-mediated phenotypes in the regulation of whole-fly oxygen consumption. Moreover, the data reflect that the deletion of Sirt4 rescued the metabolic response to rapamycin among the introgressed strains. It is proposed that Sirt4 is a suitable candidate for studying the properties of mitochondrial-nuclear epistasis in modulating mitochondrial metabolism.
Scialo, F., Sriram, A., Stefanatos, R., Spriggs, R. V., Loh, S. H. Y., Martins, L. M. and Sanz, A. (2020). Mitochondrial complex I derived ROS regulate stress adaptation in Drosophila melanogaster. Redox Biol: 101450. PubMed ID: 32146156
Reactive Oxygen Species (ROS) are essential cellular messengers required for cellular homeostasis and regulate the lifespan of several animal species. The main site of ROS production is the mitochondrion, and within it, respiratory complex I (CI) is the main ROS generator. ROS produced by CI trigger several physiological responses that are essential for the survival of neurons, cardiomyocytes and macrophages. This study show that CI produces ROS when electrons flow in either the forward (Forward Electron Transport, FET) or reverse direction (Reverse Electron Transport, RET). ROS production via RET (ROS-RET) is activated under thermal stress conditions, and interruption of ROS-RET production, through ectopic expression of the alternative oxidase AOX, attenuates the activation of pro-survival pathways in response to stress. Accordingly, this study found that both suppressing ROS-RET signalling or decreasing levels of mitochondrial H2O2 by overexpressing mitochondrial catalase (mtCAT), reduces survival dramatically in flies under stress. These results uncover a specific ROS signalling pathway where hydrogen peroxide (H2O2) generated by CI via RET is required to activate adaptive mechanisms, maximising survival under stress conditions.
Tsuboyama, K., Osaki, T., Matsuura-Suzuki, E., Kozuka-Hata, H., Okada, Y., Oyama, M., Ikeuchi, Y., Iwasaki, S. and Tomari, Y. (2020). A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation. PLoS Biol 18(3): e3000632. PubMed ID: 32163402
Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. This study reports that heat-resistant obscure (Hero; see CG17931, CG12384, or CG11444) proteins, which remain soluble after boiling at 95 degrees C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. This study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.
van Dam, E., van Leeuwen, L. A. G., Dos Santos, E., James, J., Best, L., Lennicke, C., Vincent, A. J., Marinos, G., Foley, A., Buricova, M., Mokochinski, J. B., Kramer, H. B., Lieb, W., Laudes, M., Franke, A., Kaleta, C. and Cocheme, H. M. (2020). Sugar-Induced Obesity and Insulin Resistance Are Uncoupled from Shortened Survival in Drosophila. Cell Metab. PubMed ID: 32197072
High-sugar diets cause thirst, obesity, and metabolic dysregulation, leading to diseases including type 2 diabetes and shortened lifespan. However, the impact of obesity and water imbalance on health and survival is complex and difficult to disentangle. This study shows that high sugar induces dehydration in adult Drosophila, and water supplementation fully rescues their lifespan. Conversely, the metabolic defects are water-independent, showing uncoupling between sugar-induced obesity and insulin resistance with reduced survival in vivo. High-sugar diets promote accumulation of uric acid, an end-product of purine catabolism, and the formation of renal stones, a process aggravated by dehydration and physiological acidification. Importantly, regulating uric acid production impacts on lifespan in a water-dependent manner. Furthermore, metabolomics analysis in a human cohort reveals that dietary sugar intake strongly predicts circulating purine levels. This model explains the pathophysiology of high-sugar diets independently of obesity and insulin resistance and highlights purine metabolism as a pro-longevity target.
Wu, Q., Yu, G., Cheng, X., Gao, Y., Fan, X., Yang, D., Xie, M., Wang, T., Piper, M. D. W. and Yang, M. (2020). Sexual dimorphism in the nutritional requirement for adult lifespan in Drosophila melanogaster. Aging Cell 19(3): e13120. PubMed ID: 32069521
The nutritional requirements of Drosophila have mostly been studied for development and reproduction, but the minimal requirements for adult male and female flies for lifespan have not been established. Following development on a complete diet, this study found substantial sex difference in the basic nutritional requirement of adult flies for full length of life. Relative to females, males require less of each nutrient, and for some nutrients that are essential for development, adult males have no requirement at all for lifespan. The most extreme (and surprising) sex differences were that chronic cholesterol and vitamin deficiencies had no effect on the lifespan of adult males, but they greatly decreased lifespan in females. Female oogenesis rather than chromosomal karyotype and mating status is the key cause of this gender difference in life-sustaining nutritional requirements. These data are important to the way the mechanisms are understood by which diet modifies lifespan.

Tuesday, May 5th- RNA and transposions

Torri, A., Mongelli, V., Mondotte, J. A. and Saleh, M. C. (2020). Viral Infection and Stress Affect Protein Levels of Dicer 2 and Argonaute 2 in Drosophila melanogaster. Front Immunol 11: 362. PubMed ID: 32194567
The small interfering RNA (siRNA) pathway of Drosophila melanogaster, mainly characterized by the activity of the enzymes Dicer 2 (Dcr-2) and Argonaute 2 (Ago-2), has been described as the major antiviral immune response. Several lines of evidence demonstrated its pivotal role in conferring resistance against viral infections at cellular and systemic level. However, only few studies have addressed the regulation and induction of this system upon infection and knowledge on stability and turnover of the siRNA pathway core components transcripts and proteins remains scarce. This work, explores whether the siRNA pathway is regulated following viral infection in D. melanogaster. After infecting different fly strains with two different viruses and modes of infection, changes were observed in Dcr-2 and Ago-2 protein concentrations that were not related with changes in gene expression. This response was observed either upon viral infection or upon stress-related experimental procedure, indicating a bivalent function of the siRNA system operating as a general gene regulation rather than a specific antiviral system.
Sproul, J. S., Khost, D. E., Eickbush, D. G., Negm, S., Wei, X., Wong, I. and Larracuente, A. M. (2020). Dynamic evolution of euchromatic satellites on the X chromosome in Drosophila melanogaster and the simulans clade. Mol Biol Evol. PubMed ID: 32191304
This study use high-quality genome assemblies to study the evolutionary dynamics of two complex satellite DNAs (satDNAs), Rsp-like and 1.688 gm/cm3, in Drosophila melanogaster and its three nearest relatives in the simulans clade. Large blocks of these repeats are highly dynamic in the heterochromatin, where their genomic location varies across species. Small blocks of satDNA were discovered that are abundant in X chromosome euchromatin are similarly dynamic, with repeats changing in abundance, location, and composition among species. The proliferation of a rare satellite (Rsp-like) across the X chromosome was detailed in D. simulans and D. mauritiana. Rsp-like spread by inserting into existing clusters of the older, more abundant 1.688 satellite, in events likely facilitated by microhomology-mediated repair pathways. Rsp-like is abundant on extrachromosomal circular DNA in D. simulans, which may have contributed to its dynamic evolution. Intralocus satDNA expansions via unequal exchange and the movement of higher-order repeats also contribute to the fluidity of the repeat landscape. Evidence was found that euchromatic satDNA repeats experience cycles of proliferation and diversification somewhat analogous to bursts of transposable element proliferation. This study lays a foundation for mechanistic studies of satDNA proliferation and the functional and evolutionary consequences of satDNA movement.
Venkei, Z. G., Choi, C., Feng, S., Chen, C., Jacobsen, S. E., Kim, J. K. and Yamashita, Y. M. (2020). A kinesin Klp10A mediates cell cycle-dependent shuttling of Piwi between nucleus and nuage. PLoS Genet 16(3): e1008648. PubMed ID: 32168327
The piRNA pathway protects germline genomes from selfish genetic elements (e.g. transposons) through their transcript cleavage in the cytoplasm and/or their transcriptional silencing in the nucleus. This study describes a mechanism by which the nuclear and cytoplasmic arms of the piRNA pathway are linked. During mitosis of Drosophila spermatogonia, nuclear Piwi interacts with nuage, the compartment that mediates the cytoplasmic arm of the piRNA pathway. At the end of mitosis, Piwi leaves nuage to return to the nucleus. Dissociation of Piwi from nuage occurs at the depolymerizing microtubules of the central spindle, mediated by a microtubule-depolymerizing kinesin, Klp10A. Depletion of klp10A delays the return of Piwi to the nucleus and affects piRNA production, suggesting the role of nuclear-cytoplasmic communication in piRNA biogenesis. It is proposed that cell cycle-dependent communication between the nuclear and cytoplasmic arms of the piRNA pathway may play a previously unappreciated role in piRNA regulation.
Soule, S., Mellottee, L., Arab, A., Chen, C. and Martin, J. R. (2020). Jouvence a small nucleolar RNA required in the gut extends lifespan in Drosophila. Nat Commun 11(1): 987. PubMed ID: 32080190
Longevity is influenced by genetic and environmental factors, but the underlying mechanisms remain elusive. This study functionally characterise a Drosophila small nucleolar RNA (snoRNA), named jouvence whose loss of function reduces lifespan. The genomic region of jouvence rescues the longevity in mutant, while its overexpression in wild-type increases lifespan. Jouvence is required in enterocytes. In mutants, the epithelium of the gut presents more hyperplasia, while the overexpression of jouvence prevents it. Molecularly, the mutant lack pseudouridylation on 18S and 28S-rRNA, a function rescued by targeted expression of jouvence in the gut. A transcriptomic analysis performed from the gut reveals that several genes are either up- or down-regulated, while restoring the mRNA level of two genes (ninaD or CG6296) rescue the longevity. Since snoRNAs are structurally and functionally well conserved throughout evolution, this study identified a putative jouvence orthologue in mammals including humans, suggesting that its function in longevity could be conserved.
Salces-Ortiz, J., Vargas-Chavez, C., Guio, L., Rech, G. E. and Gonzalez, J. (2020). Transposable elements contribute to the genomic response to insecticides in Drosophila melanogaster. Philos Trans R Soc Lond B Biol Sci 375(1795): 20190341. PubMed ID: 32075557
Most of the genotype-phenotype analyses to date have largely centered attention on single nucleotide polymorphisms. However, transposable element (TE) insertions have arisen as a plausible addition to the study of the genotypic-phenotypic link because of to their role in genome function and evolution. This work investigated the contribution of TE insertions to the regulation of gene expression in response to insecticides. Four Drosophila melanogaster strains were exposed to malathion, a commonly used organophosphate insecticide. By combining information from different approaches, including RNA-seq and ATAC-seq, it was found that TEs can contribute to the regulation of gene expression under insecticide exposure by rewiring cis-regulatory networks. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.
Machyna, M., Kiefer, L. and Simon, M. D. (2020). Enhanced nucleotide chemistry and toehold nanotechnology reveals lncRNA spreading on chromatin. Nat Struct Mol Biol 27(3): 297-304. PubMed ID: 32157249
Understanding the targeting and spreading patterns of long non-coding RNAs (lncRNAs) on chromatin requires a technique that can detect both high-intensity binding sites and reveal genome-wide changes in spreading patterns with high precision and confidence. This study determine lncRNA localization using biotinylated locked nucleic acid (LNA)-containing oligonucleotides with toehold architecture capable of hybridizing to target RNA through strand-exchange reaction. During hybridization, a protecting strand competitively displaces contaminating species, leading to highly specific RNA capture of individual RNAs. Analysis of Drosophila roX2 lncRNA using this approach revealed that heat shock, unlike the unfolded protein response, leads to reduced spreading of roX2 on the X chromosome, but surprisingly also to relocalization to sites on autosomes. These results demonstrate that this improved hybridization capture approach can reveal previously uncharacterized changes in the targeting and spreading of lncRNAs on chromatin.

Monday, May 4th - Signal Transduction

Schaefer, K. N., Pronobis, M., Williams, C. E., Zhang, S., Bauer, L., Goldfarb, D., Yan, F., Major, M. B. and Peifer, M. (2020). Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex. Mol Biol Cell: mbcE19110647. PubMed ID: 32129710
Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector beta-catenin. The multiprotein destruction complex binds and phosphorylates beta-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing beta-catenin. Recent work clarified underlying mechanisms, but important questions remain. This study explored beta-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. This study found that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin's RGS domain plays an important role. Elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When Dishevelled levels were elevated, it forms its own cytoplasmic puncta, but these do not recruit Axin. Superresolution imaging in mammalian cells raises the possibility that this may result by promoting Dishevelled:Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.
Shin, M., Cha, N., Koranteng, F., Cho, B. and Shim, J. (2020). Subpopulation of Macrophage-Like Plasmatocytes Attenuates Systemic Growth via JAK/STAT in the Drosophila Fat Body. Front Immunol 11: 63. PubMed ID: 32082322
Drosophila hemocytes, like those of mammals, are given rise from two distinctive phases during both the embryonic and larval hematopoiesis. Embryonically derived hemocytes, mostly composed of macrophage-like plasmatocytes, are largely identified by genetic markers. However, the cellular diversity and distinct functions of possible subpopulations within plasmatocytes have not been explored in Drosophila larvae. This study shows that larval plasmatocytes exhibit differential expressions of Hemolectin (Hml) and Peroxidasin (Pxn) during development. Moreover, removal of plasmatocytes by overexpressing pro-apoptotic genes, hid and reaper in Hml-positive plasmatocytes, feeding high sucrose diet, or wasp infestation results in increased circulating hemocytes that are Hml-negative. Interestingly these Hml-negative plasmatocytes retain Pxn expression, and animals expressing Hml-negative and Pxn-positive subtype largely attenuate growth and abrogate metabolism. Furthermore, elevated levels of a cytokine, Unpaired 3, are detected when Hml-positive hemocytes are ablated, which in turn activates JAK/STAT activity in several tissues including the fat body. Finally, it was observed that insulin signaling is inhibited in this background, which can be recovered by concurrent loss of upd3. Overall, this study highlights heterogeneity in Drosophila plasmatocytes and a functional plasticity of each subtype, which reaffirms extension of their role beyond immunity into metabolic regulation for cooperatively maintaining internal homeostatic balance.
Matsumura, T., Uryu, O., Matsuhisa, F., Tajiri, K., Matsumoto, H. and Hayakawa, Y. (2020). N-acetyl-l-tyrosine is an intrinsic triggering factor of mitohormesis in stressed animals. EMBO Rep: e49211. PubMed ID: 32118349
Under stress conditions, mitochondria release low levels of reactive oxygen species (ROS), which triggers a cytoprotective response, called "mitohormesis". It still remains unclear how mitochondria respond to stress-derived stimuli and release a low level of ROS. This study shows that N-acetyl-l-tyrosine (NAT) functions as a plausible intrinsic factor responsible for these tasks in stressed animals. NAT is present in the blood or hemolymph of healthy animals, and its concentrations increase in response to heat stress. Pretreatment with NAT significantly increases the stress tolerance of tested insects and mice. Analyses using Drosophila larvae and cultured cells demonstrate that the hormetic effects are triggered by transient NAT-induced perturbation of mitochondria, which causes a small increase in ROS production and leads to sequential retrograde responses: NAT-dependent FoxO activation increases in the gene expression of antioxidant enzymes and Keap1. Moreover, NAT represses tumor growth, possibly via the activation of Keap1. In sum, it is proposed that NAT is a vital endogenous molecule that could serve as a triggering factor for mitohormesis.
Strilbytska, O. M., Storey, K. B. and Lushchak, O. V. (2020). TOR signaling inhibition in intestinal stem and progenitor cells affects physiology and metabolism in Drosophila. Comp Biochem Physiol B Biochem Mol Biol 243-244: 110424. PubMed ID: 32088257
In all eukaryotic organisms, the control of growth, metabolism, reproduction, and lifespan is realized by interactions of genetic and environmental signals. An important player in the regulatory network is the target of rapamycin (TOR) signaling pathway, which is triggered by nutritional cues. Given the pivotal role of TOR in regulating multiple processes in organisms, this study inhibited TOR by inducible expression of specific RNAi in Drosophila intestinal stem and progenitor cells or progenitor cells alone. TOR inhibition in stem and progenitor cells shortened the lifespan on both regular diet and under malnutrition. Moreover, flies became more short-lived under starvation or oxidative stress conditions if TOR was inhibited. TOR-RNAi expression resulted in a decrease in body glycogen and TAG levels. All these physiological and metabolic changes might be partially explained by significant changes in mRNA levels for genes encoding the Drosophila insulin-like peptides (dilp2, dilp3 and dilp5) with subsequent effects on insulin signaling to modulate gene expression in peripheral tissues (e.g. tobi and pepck transcripts). In the gut, a strong increase in transcript levels of cytokines upd2, upd3 and downstream target socs36e of the JAK/STAT signaling pathway in the gut indicate an important role for this signaling pathway when TOR is inhibited.
Sui, L. and Dahmann, C. (2020). Wingless counteracts epithelial folding by increasing mechanical tension at basal cell edges in Drosophila. Development 147(5). PubMed ID: 32161062
The modulation of mechanical tension is important for sculpturing tissues during animal development, yet how mechanical tension is controlled remains poorly understood. In Drosophila wing discs, the local reduction of mechanical tension at basal cell edges results in basal relaxation and the formation of an epithelial fold. This study shows that Wingless, which is expressed next to this fold, promotes basal cell edge tension to suppress the formation of this fold. Ectopic expression of Wingless blocks fold formation, whereas the depletion of Wingless increases fold depth. Moreover, local depletion of Wingless in a region where Wingless signal transduction is normally high results in ectopic fold formation. The depletion of Wingless also results in decreased basal cell edge tension and basal cell area relaxation. Conversely, the activation of Wingless signal transduction leads to increased basal cell edge tension and basal cell area constriction. These results identify the Wingless signal transduction pathway as a crucial modulator of mechanical tension that is important for proper wing disc morphogenesis.
Sun, J., Zhang, J., Wang, D. and Shen, J. (2020). The transcription factor spalt and human homologue SALL4 induce cell invasion via the dMyc-JNK pathway in Drosophila. Biol Open. PubMed ID: 32098783
Cancer cell metastasis is a leading cause of mortality in cancer patients. Therefore, revealing the molecular mechanism of cancer cell invasion is of great significance for the treatment of cancer. In human patients, the hyperactivity of transcription factor Spalt-like 4 (SALL4) is sufficient to induce malignant tumorigenesis and metastasis. This study found that when ectopically expressing the Drosophila homologue spalt (sal) or human SALL4 in Drosophila, epithelial cells delaminated basally with penetration of the basal lamina and degradation of the extracellular matrix, which are essential properties of cell invasion. Further assay found that sal/SALL4 promoted cell invasion via dMyc-JNK signaling. Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway through suppressing matrix metalloprotease 1 or basket can achieve suppression of cell invasion. Moreover, expression of dMyc, a suppressor of JNK signaling, dramatically blocked cell invasion induced by sal/SALL4 in the wing disc. These findings reveal a conserved role of sal/SALL4 in invasive cell movement and link the crucial mediator of tumor invasion, the JNK pathway, to SALL4-mediated cancer progression.

Friday, May 1st - Evolution

Chandler, C. H., Mammel, A. and Dworkin, I. (2020). Sexual Selection Does Not Increase the Rate of Compensatory Adaptation to a Mutation Influencing a Secondary Sexual Trait in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32122961
Theoretical work predicts that sexual selection can enhance natural selection, increasing the rate of adaptation to new environments and helping purge harmful mutations. While some experiments support these predictions, remarkably little work has addressed the role of sexual selection on compensatory adaptation-populations' ability to compensate for the costs of deleterious alleles that are already present. This study tested whether sexual selection, as well as the degree of standing genetic variation, affect the rate of compensatory evolution via phenotypic suppression in experimental populations of Drosophila melanogaster. These populations were fixed for a spontaneous mutation causing mild abnormalities in the male sex comb, a structure important for mating success.This mutation was mapped to an ~85 kb region on the X chromosome containing three candidate genes, showed that the mutation is deleterious, and that its phenotypic expression and penetrance vary by genetic background. Experimental evolution was then performed, including a treatment where opportunity for mate choice was limited by experimentally enforced monogamy. Although evolved populations did show some phenotypic suppression of the morphological abnormalities in the sex comb, the amount of suppression did not depend on the opportunity for sexual selection. Sexual selection, therefore, may not always enhance natural selection; instead, the interaction between these two forces may depend on additional factors.

Han, X., Guo, J., Pang, E., Song, H. and Lin, K. (2020). Ab initio construction and evolutionary analysis of protein-coding gene families with partially homologous relationships: Closely related Drosophila genomes as a case study. Genome Biol Evol. PubMed ID: 32108239
How have genes evolved within a well-known genome phylogeny? Many protein-coding genes should have evolved as a whole at the gene level, and some should have evolved partly through fragments at the subgene level. To comprehensively explore such complex homologous relationships and better understand gene family evolution, in this study, with de novo-identified modules, the subgene units which could consecutively cover proteins within a set of closely related species, a new phylogeny-based approach was applied that considers evolutionary models with partial homology to classify all protein-coding genes in nine Drosophila genomes. Compared with two other popular methods for gene family construction, this approach improved practical gene family classifications with a more reasonable view of homology and provided a much more complete landscape of gene family evolution at the gene and subgene levels. This study found that most expanded gene families might have evolved mainly through module rearrangements rather than gene duplications and mainly generated single-module genes through partial gene duplication, suggesting that there might be pervasive subgene rearrangement in the evolution of protein-coding gene families. The use of a phylogeny-based approach with partial homology to classify and analyze protein-coding gene families may provide a more comprehensive landscape depicting how genes evolve within a well-known genome phylogeny.
Johri, P., Charlesworth, B. and Jensen, J. D. (2020). Towards an Evolutionarily Appropriate Null Model: Jointly Inferring Demography and Purifying Selection. Genetics. PubMed ID: 32152045
The question of the relative evolutionary roles of adaptive and non-adaptive processes has been a central debate in population genetics for nearly a century. While advances have been made in the theoretical development of the underlying models, and statistical methods for estimating their parameters from large-scale genomic data, a framework for an appropriate null model remains elusive. A model incorporating evolutionary processes known known to be in constant operation - genetic drift (as modulated by the demographic history of the population) and purifying selection - is lacking. Without such a null model, the role of adaptive processes in shaping within- and between-population variation may not be accurately assessed. This study investigated how population size changes and the strength of purifying selection affect patterns of variation at neutral sites near functional genomic components. A novel statistical framework is proposed for jointly inferring the contribution of the relevant selective and demographic parameters. By means of extensive performance analyses, the utility of the approach was quantified, the most important statistics for parameter estimation were identified, and the results with existing methods were compared. Finally, genome-wide population-level data was re-analyzed from a Zambian population of Drosophila melanogaster, and it was found that the population has experienced a much slower rate of population growth than was inferred when the effects of purifying selection were neglected. This approach represents an appropriate null model, against which the effects of positive selection can be assessed.
Morrow, J. L., Schneider, D. I., Klasson, L., Janitz, C., Miller, W. J. and Riegler, M. (2020). Parallel sequencing of Wolbachia wCer2 from donor and novel hosts reveals multiple incompatibility factors and genome stability after host transfers. Genome Biol Evol. PubMed ID: 32163151
The application of Wolbachia in insect pest and vector control requires the establishment of genotypically stable host associations. The cytoplasmic incompatibility (CI) inducing Wolbachia strain wCer2 naturally occurs in the cherry fruit fly Rhagoletis cerasi as co-infection with other strains and was transferred to other fruit flies by embryonic microinjections. wCer2 genome data was obtained from its native and three novel hosts, Drosophila simulans, Drosophila melanogaster and Ceratitis capitata and assessed its genome stability, characteristics, and CI factor (cif) genes. De novo assembly was successful from Wolbachia cell-enriched singly infected D. simulans embryos, with minimal host and other bacterial genome traces. The low yield of Wolbachia sequence reads from total genomic extracts of one multiply infected R. cerasi pupa and one singly infected C. capitata adult limited de novo assemblies but was sufficient for comparative analyses. Across hosts wCer2 was stable in genome synteny and content. Polymorphic nucleotide sites were found in wCer2 of each host, however only one nucleotide was different between R. cerasi and C. capitata, and none between replicated D. simulans lines. The wCer2 genome is highly similar to wAu (D. simulans), wMel (D. melanogaster) and wRec (Drosophila recens). In contrast to wMel and wRec (each with one cif gene pair) and wAu (without any cif genes), wCer2 has three pairs of Type I cif genes and one Type IV cifB gene without a cifA complement. This may explain previously reported CI patterns of wCer2, including incomplete rescue of its own CI modification in three novel host species.
Strausfeld, N. J., Wolff, G. H. and Sayre, M. E. (2020). Mushroom body evolution demonstrates homology and divergence across Pancrustacea. Elife 9. PubMed ID: 32124731
Descriptions of crustacean brains have focused mainly on three highly derived lineages of malacostracans: the reptantian infraorders represented by spiny lobsters, lobsters, and crayfish. Those descriptions advocate the view that dome- or cap-like neuropils, referred to as 'hemiellipsoid bodies,' are the ground pattern organization of centers that are comparable to insect mushroom bodies in processing olfactory information. This study challenges the doctrine that hemiellipsoid bodies are a derived trait of crustaceans, whereas mushroom bodies are a derived trait of hexapods. It was demonstrated that mushroom bodies typify lineages that arose before Reptantia and exist in Reptantia thereby indicating that the mushroom body, not the hemiellipsoid body, provides the ground pattern for both crustaceans and hexapods. Evolved variations of the mushroom body ground pattern are, in some lineages, defined by extreme diminution or loss and, in others, by the incorporation of mushroom body circuits into lobeless centers. Such transformations are ascribed to modifications of the columnar organization of mushroom body lobes that, as shown in Drosophila and other hexapods, contain networks essential for learning and memory.
Chen, Y., Han, H., Seo, G., Vargas, R. E., Yang, B., Chuc, K., Zhao, H. and Wang, W. (2020). Systematic analysis of the Hippo pathway organization and oncogenic alteration in evolution. Sci Rep 10(1): 3173. PubMed ID: 32081887
The Hippo pathway is a central regulator of organ size and a key tumor suppressor via coordinating cell proliferation and death. Initially discovered in Drosophila, the Hippo pathway has been implicated as an evolutionarily conserved pathway in mammals; however, how this pathway was evolved to be functional from its origin is still largely unknown. This study traced the Hippo pathway in premetazoan species, characterized the intrinsic functions of its ancestor components, and unveiled the evolutionary history of this key signaling pathway from its unicellular origin. In addition, the paralogous gene history for the mammalian Hippo pathway components was elucidated and their cancer-derived somatic mutations were characterized from an evolutionary perspective. Taken together, these findings not only traced the conserved function of the Hippo pathway to its unicellular ancestor components, but also provided novel evolutionary insights into the Hippo pathway organization and oncogenic alteration.
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