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


Thursday January 31st, 2019 - Signaling

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Lo, P. K., Huang, Y. C., Corcoran, D., Jiao, R. and Deng, W. M. (2019). Drosophila chromatin assembly factor 1 p105 and p180 subunits are required for follicle cell proliferation via inhibiting Notch signaling. J Cell Sci. PubMed ID: 30630896
Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. This study reports another side of CAF1 wherein the subunits CAF1-p105 and CAF1-p180 inhibit expression of Notch target genes and shows this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt), leading to Cut downregulation and inhibition of follicle cell mitosis. These studies further show Notch is functionally responsible for these phenotypes observed in CAF1-p105/p180-deficient follicle cells. Moreover, this study reveals that CAF1-p105/p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative feedback regulatory loop between Cut and Hnt underlying CAF1-p105/p180 regulation, which is crucial for follicle cell differentiation. In conclusion, these studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner.
Moreno, E., Valon, L., Levillayer, F. and Levayer, R. (2019). Competition for space induces cell elimination through compaction-driven ERK downregulation. Curr Biol 29(1): 23-34. PubMed ID: 30554899
The plasticity of developing tissues relies on the adjustment of cell survival and growth rate to environmental cues. This includes the effect of mechanical cues on cell survival. Accordingly, compaction of an epithelium can lead to cell extrusion and cell death. This process was proposed to contribute to tissue homeostasis but also to facilitate the expansion of pretumoral cells through the compaction and elimination of the neighboring healthy cells. However, very little is known about the pathways that can trigger apoptosis upon tissue deformation, and the contribution of compaction-driven death to clone expansion has never been assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, it was shown first that tissue compaction induces cell elimination through the downregulation of epidermal growth factor receptor/extracellular signal regulated kinase (EGFR/ERK) pathway and the upregulation of the pro-apoptotic protein Hid. Those results suggest that the sensitivity of EGFR/ERK pathway to mechanics could play a more general role in the fine tuning of cell elimination during morphogenesis and tissue homeostasis. Second, the contribution of compaction-driven death to pretumoral cell expansion was assessed in vivo. The activation of the oncogene Ras in clones was found to downregulate ERK and activate apoptosis in the neighboring cells through their compaction, which eventually contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumor progression.
Hannaford, M., Loyer, N., Tonelli, F., Zoltner, M. and Januschke, J. (2019). A chemical-genetics approach to study the role of atypical protein kinase C in Drosophila. Development. PubMed ID: 30635282
Studying the function of proteins using genetics in cycling cells is complicated by the fact that there is often a delay between gene inactivation and the timepoint of phenotypic analysis. This is particularly true when studying kinases, that have pleiotropic functions and multiple substrates. Drosophila neuroblasts are rapidly dividing stem cells and an important model system to study cell polarity. Mutations in multiple kinases cause neuroblast polarity defects, but their precise functions at particular time points in the cell cycle are unknown. This study used chemical genetics and reports the generation of an analogue-sensitive (as) allele of Drosophila atypical protein kinase C (aPKC). The resulting mutant aPKC kinase can be specifically inhibited in vitro and in vivo. Acute inhibition of aPKC during neuroblast polarity establishment abolishes asymmetric localization of Miranda while its inhibition during NB polarity maintenance does not in the time frame of normal mitosis. However, aPKC contributes to sharpen the pattern of Miranda, by keeping it off the apical and lateral cortex after nuclear envelope breakdown.
Heigwer, F., Scheeder, C., Miersch, T., Schmitt, B., Blass, C., Pour Jamnani, M. V. and Boutros, M. (2018). Time-resolved mapping of genetic interactions to model rewiring of signaling pathways. Elife 7. PubMed ID: 30592458
Context-dependent changes in genetic interactions are an important feature of cellular pathways and their varying responses under different environmental conditions. However, methodological frameworks to investigate the plasticity of genetic interaction networks over time or in response to external stresses are largely lacking. To analyze the plasticity of genetic interactions, a combinatorial RNAi screen was performed in Drosophila cells at multiple time points and after pharmacological inhibition of Ras signaling activity. Using an image-based morphology assay to capture a broad range of phenotypes, the effect of 12768 pairwise RNAi perturbations was tested in six different conditions. Genetic interactions were found to form in different trajectories; an algorithm, termed MODIFI, was developed to analyze how genetic interactions rewire over time. Using this framework, more statistically significant interactions were identified compared to end-point assays, and several examples of context-dependent crosstalk between signaling pathways were further observed such as an interaction between Ras and Rel which is dependent on MEK activity.
Meschi, E., Leopold, P. and Delanoue, R. (2019). An EGF-responsive neural circuit couples insulin secretion with nutrition in Drosophila. Dev Cell 48(1): 76-86.e75. PubMed ID: 30555002
Developing organisms use fine-tuning mechanisms to adjust body growth to ever-changing nutritional conditions. In Drosophila, the secretory activity of insulin-producing cells (IPCs) is central to couple systemic growth with amino acids availability. This study identified a subpopulation of inhibitory neurons contacting the IPCs (IPC-connecting neurons or ICNs) that play a key role in this coupling. ICNs respond to growth-blocking peptides (GBPs), a family of fat-body-derived signals produced upon availability of dietary amino acids. GBPs are atypical ligands for the fly EGF receptor (EGFR). Upon activation of EGFR by adipose GBPs, ICN-mediated inhibition of IPC function is relieved, allowing insulin secretion. Tnis study reveals an unexpected role for EGF-like metabolic hormones and EGFR signaling as critical modulators of neural activity, coupling insulin secretion to the nutritional status.
Moreira, S., Osswald, M., Ventura, G., Goncalves, M., Sunkel, C. E. and Morais-de-Sa, E. (2019). PP1-mediated dephosphorylation of Lgl controls apical-basal polarity. Cell Rep 26(2): 293-301.e297. PubMed ID: 30625311
Apical-basal polarity is a common trait that underlies epithelial function. Although the asymmetric distribution of cortical polarity proteins works in a functioning equilibrium, it also retains plasticity to accommodate cell division, during which the basolateral determinant Lgl is released from the cortex. This study investigated how Lgl restores its cortical localization to maintain the integrity of dividing epithelia. Cytoplasmic Lgl is reloaded to the cortex at mitotic exit in Drosophila epithelia. Lgl cortical localization depends on protein phosphatase 1, which dephosphorylates Lgl on the serines phosphorylated by aPKC and Aurora A kinases through a mechanism that relies on the regulatory subunit Sds22 and a PP1-interacting RVxF motif of Lgl. This mechanism maintains epithelial polarity and is of particular importance at mitotic exit to couple Lgl cortical reloading with the polarization of the apical domain. Hence, PP1-mediated dephosphorylation of Lgl preserves the apical-basal organization of proliferative epithelia.

Wednesday, January 30th - Embryonic Development

Trebuchet, G., Cattenoz, P. B., Zsamboki, J., Mazaud, D., Siekhaus, D. E., Fanto, M. and Giangrande, A. (2018). The Repo homeodomain transcription factor suppresses hematopoiesis in Drosophila and preserves the glial fate. J Neurosci. PubMed ID: 30504274
Despite their different origins, Drosophila glia and hemocytes are related cell populations that provide an immune function. Drosophila hemocytes patrol the body cavity and act as macrophages outside the nervous system whereas glia originate from the neuroepithelium and provide the scavenger population of the nervous system. Drosophila glia are hence the functional orthologs of vertebrate microglia, even though the latter are cells of immune origin that subsequently move into the brain during development. Interestingly, the Drosophila immune cells within (glia) and outside the nervous system (hemocytes) require the same transcription factor Glide/Gcm for their development. This raises the issue of how do glia specifically differentiate in the nervous system and hemocytes in the procephalic mesoderm. The Repo homeodomain transcription factor and pan-glial direct target of Glide/Gcm is known to ensure glial terminal differentiation. This study shows that Repo also takes center stage in the process that discriminates between glia and hemocytes. First, Repo expression is repressed in the hemocyte anlagen by mesoderm-specific factors. Second, Repo ectopic activation in the procephalic mesoderm is sufficient to repress the expression of hemocyte-specific genes. Third, the lack of Repo triggers the expression of hemocyte markers in glia. Thus, a complex network of tissue-specific cues biases the potential of Glide/Gcm. These data allow revision of the concept of fate determinants and help to understand the bases of cell specification. Both sexes were analyzed.
Feng, R. et al. (2019). Dynamics expression of DmFKBP12/Calstabin during embryonic early development of Drosophila melanogaster. Cell Biosci 9: 8. PubMed ID: 30637096
Lack of calcium signaling protein FK506-binding protein 12 (FKBP12/Calstabin) is known to result in lethal cardiac dysfunction in mouse. This study identified both temporal and localization changes in expression of DmFKBP12, a translational and transcriptional regulator of Drosophila RyR and FKBP12, through embryonic development. DmFKBP12 is first expressed at the syncytial blastoderm stage and undergoes increased expression during the cellular blastoderm and early gastrulation stages. At late gastrulation, DmFKBP12 expression begins to decline until it reaches homeostasis, which it then maintains throughout the rest of development. DmFKBP12 mRNA remain stable, which indicates that protein dynamics are attributed to regulation at the mRNA to protein translation level. Although DmFKBP12 is distributed evenly between the anterior to posterior poles of the blastoderm egg, the protein is expressed more strongly in the cortex of the early Drosophila gastrula with the highest concentration found in the basement membrane of the cellular blastoderm. By late gastrulation, DmFKBP12 is no longer identified in the yolk or lumen of duct structures and has relocated to the future brain (suboesophageal and supraesophageal ganglions), ventral nervous system, and muscular system. Throughout these changes in distribution, in situ DmFKBP12 mRNA monitoring detected equal distribution of DmFKBP12 mRNA. As a critical regulator of the DmRyR-FKBP complex, DmFKBP12 expression in Drosophila fluctuates temporally and spatially with the formation of organ systems. These finding indicate that DmFKBP12 and RyR associated calcium signaling plays an essential role in the successful development of Drosophila melanogaster. Further study on the differences between mammalian RyR-FKBP12 and Drosophila DmRyR-FKBP12 can be exploited to develop safe pesticides.
Durney, C. H., Harris, T. J. C. and Feng, J. J. (2018). Dynamics of PAR proteins explain the oscillation and ratcheting mechanisms in dorsal closure. Biophys J 115(11): 2230-2241. PubMed ID: 30446158
This study presents a vertex-based model for Drosophila dorsal closure that predicts the mechanics of cell oscillation and contraction from the dynamics of the PAR proteins. Based on experimental observations of how aPKC, Par-6, and Bazooka translocate from the circumference of the apical surface to the medial domain, and how they interact with each other and ultimately regulate the apicomedial actomyosin, a system of differential equations was formulated that captures the key features of dorsal closure, including distinctive behaviors in its early, slow, and fast phases. The oscillation in cell area in the early phase of dorsal closure results from an intracellular negative feedback loop that involves myosin, an actomyosin regulator, aPKC, and Bazooka. In the slow phase, gradual sequestration of apicomedial aPKC by Bazooka clusters causes incomplete disassembly of the actomyosin network over each cycle of oscillation, thus producing a so-called ratchet. The fast phase of rapid cell and tissue contraction arises when medial myosin, no longer antagonized by aPKC, builds up in time and produces sustained contraction. Thus, a minimal set of rules governing the dynamics of the PAR proteins, extracted from experimental observations, can account for all major mechanical outcomes of dorsal closure, including the transitions between its three distinct phases.
Ishibashi, T., Hatori, R., Maeda, R., Nakamura, M., Taguchi, T., Matsuyama, Y. and Matsuno, K. (2019). E and ID proteins regulate cell chirality and left-right asymmetric development in Drosophila. Genes Cells. PubMed ID: 30624823
How left-right (LR) asymmetry forms in the animal body is a fundamental problem in Developmental Biology. While the mechanisms for LR asymmetry are well studied in some species, they are still poorly understood in invertebrates. It has been shown that the intrinsic LR asymmetry of cells (designated as cell chirality) drives LR asymmetric development in the Drosophila embryonic hindgut, although the machinery of the cell chirality formation remains elusive. This study found that the Drosophila homolog of the Id gene, extra macrochaetae (emc), is required for the normal LR asymmetric morphogenesis of this organ. Id proteins, including Emc, are known to interact with and inhibit E-box-binding proteins (E proteins), such as Drosophila Daughterless (Da). The suppression of da by wild-type emc was essential for cell chirality formation and for normal LR asymmetric development of the embryonic hindgut. MyosinID (MyoID), which encodes the Drosophila Myosin ID protein, is known to regulate cell chirality. It was further shown that Emc-Da regulates cell chirality formation, in which Emc functions upstream of or parallel to MyoID. Abnormal Id-E protein regulation is involved in various human diseases. These results suggest that defects in cell shape may contribute to the pathogenesis of such diseases.
Boos, A., Distler, J., Rudolf, H., Klingler, M. and El-Sherif, E. (2018). A re-inducible gap gene cascade patterns the anterior-posterior axis of insects in a threshold-free fashion. Elife 7. PubMed ID: 30570485
Gap genes mediate the division of the anterior-posterior axis of insects into different fates through regulating downstream hox genes. Decades of tinkering the segmentation gene network of Drosophila melanogaster led to the conclusion that gap genes are regulated (at least initially) through a threshold-based mechanism, guided by both anteriorly- and posteriorly-localized morphogen gradients. This paper shows that the response of the gap gene network in the beetle Tribolium castaneum upon perturbation is consistent with a threshold-free 'Speed Regulation' mechanism, in which the speed of a genetic cascade of gap genes is regulated by a posterior morphogen gradient. This is shown by re-inducing the leading gap gene (namely, hunchback) resulting in the re-induction of the gap gene cascade at arbitrary points in time. This demonstrates that the gap gene network is self-regulatory and is primarily under the control of a posterior regulator in Tribolium and possibly other short/intermediate-germ insects.
Itakura, Y., Inagaki, S., Wada, H. and Hayashi, S. (2018). Trynity controls epidermal barrier function and respiratory tube maturation in Drosophila by modulating apical extracellular matrix nano-patterning. PLoS One 13(12): e0209058. PubMed ID: 30576352
The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, the role of a conserved aECM protein, the ZP domain protein Trynity, was studied in Drosophila melanogaster. trynity was first identified as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small-molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. These findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.

Tuesday, January 29th - Gonads

Akishina, A. A., Vorontsova, J. E., Cherezov, R. O., Slezinger, M. S., Simonova, O. B. and Kuzin, B. A. (2018). NAP family CG5017 chaperone pleiotropically regulates human AHR target genes expression in Drosophila testis. Int J Mol Sci 20(1). PubMed ID: 30597983
To study the regulatory mechanism of the Aryl hydrocarbon receptor (AHR), target genes of transcription are necessary for understanding the normal developmental and pathological processes. This study examined the effects of human AHR ligands on male fecundity. To induce ectopic human AhR gene expression, Drosophila melanogaster was used that was transformed with human AhR under the control of a yeast UAS promoter element capable of activation in the two-component UAS-GAL4 system. Exogenous AHR ligands decrease the number of Drosophila gonadal Traffic jam-positive cells. Both an increase and decrease was observed of AHR target gene expression, including in genes that control homeostasis and testis development. This suggests that gonadal AHR activation may affect the expression of gene networks that control sperm production and could be critical for fertility not just in Drosophila but also in humans. Finally, the activation of the expression for some AHR target genes depends on the expression of testis-specific chaperone CG5017 in gonadal cells. Since CG5017 belongs to the nucleosome assembly protein (NAP) family and may participate in epigenetic regulation, it is proposed that this nucleotropic chaperone is essential to provide the human AHR with access to only the defined set of its target genes during spermatogenesis.
Inatomi, M., Shin, D., Lai, Y. T. and Matsuno, K. (2019). Proper direction of male genitalia is prerequisite for copulation in Drosophila, implying cooperative evolution between genitalia rotation and mating behavior. Sci Rep 9(1): 210. PubMed ID: 30659250
Animal morphology and behavior often appear to evolve cooperatively. However, it is difficult to assess how strictly these two traits depend on each other. The genitalia morphologies and courtship behaviors in insects, which vary widely, may be a good model for addressing this issue. In Diptera, phylogenetic analyses of mating positions suggested that the male-above position evolved from an end-to-end position. However, with this change in mating position, the dorsoventral direction of the male genitalia became upside down with respect to that of the female genitalia. It was proposed that to compensate for this incompatibility, the male genitalia rotated an additional 180 degrees during evolution, implying evolutionary cooperativity between the mating position and genitalia direction. According to this scenario, the proper direction of male genitalia is critical for successful mating. This study tested this hypothesis using a Drosophila Myosin31DF (Myo31DF) mutant, in which the rotation of the male genitalia terminates prematurely, resulting in various deviations in genitalia direction. The proper dorsoventral direction of the male genitalia was found to be a prerequisite for successful copulation, but it did not affect the other courtship behaviors. Therefore, these results suggested that the male genitalia rotation and mating position evolved cooperatively in Drosophila.
Baskar, R., Bakrhat, A. and Abdu, U. (2019). GFP-Forked, a genetic reporter for studying Drosophila oocyte polarity. Biol Open 8(1). PubMed ID: 30598482
The polarized organization of the Drosophila oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). This study used the gene forked, the putative Drosophila homologue of espin, to develop a unique genetic reporter for asymmetric oocyte organization. A null allele of the forked gene was generated using the CRISPR-Cas9 system, and forked was found not to be required for determining the axes of the Drosophila embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, it was demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal MT-organizing centers. These results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity.
Dubey, P., Kapoor, T., Gupta, S., Shirolikar, S. and Ray, K. (2019). Atypical septate junctions maintain the somatic enclosure around maturing spermatids and prevent premature sperm release in Drosophila testis. Biol Open. PubMed ID: 30635267
Tight junctions prevent paracellular flow and maintain cell polarity in an epithelium. These junctions are also required for maintaining the blood-testis-barrier, which is essential for sperm differentiation. Septate junctions in insects are orthologous to the tight junctions. In Drosophila testis, major septate junction components co-localize at the interface of germline and somatic cells initially and then condense between the two somatic cells in a cyst after germline meiosis. Their localization is extensively remodeled in subsequent stages. Characteristic septate junctions are formed between the somatic cyst cells at the elongated spermatid stage. Consistent with previous reports, knockdown of essential junctional components- Discs-large-1 and Neurexin-IV- during the early stages disrupted sperm differentiation beyond the spermatocyte stage. Knockdown of these proteins during the final stages of spermatid maturation caused premature release of spermatids inside the testes, resulting in partial loss of male fertility. These results indicate the importance of maintaining the integrity of the somatic enclosure during spermatid coiling and release in Drosophila testis. It also highlights the functional similarity with the tight junction proteins during mammalian spermatogenesis.
Augiere, C., Lapart, J. A., Duteyrat, J. L., Cortier, E., Maire, C., Thomas, J. and Durand, B. (2019). salto/CG13164 is required for sperm head morphogenesis in Drosophila. Mol Biol Cell: mbcE18070429. PubMed ID: 30601696
Producing mature spermatozoa is essential for sexual reproduction in metazoans. Spermiogenesis involves dramatic cell morphological changes going from sperm tail elongation, nuclear reshaping to cell membrane remodeling during sperm individualization and release. The sperm manchette, a temporary structure that assists in sperm elongation, plays a critical scaffolding function during nuclear remodeling by linking the nuclear lamina to the cytoskeleton. This study describes the role of an uncharacterized protein in Drosophila, Salto/CG13164, involved in nuclear shaping and spermatid individualization. Salto has a dynamic localization during spermatid differentiation, being progressively relocated from the sperm nuclear dense body, which is equivalent to the mammalian sperm manchette, to the centriolar adjunct and acrosomal cap during spermiogenesis. salto null male flies are sterile and exhibit complete spermatid individualization defects. salto deficient spermatids show coiled spermatid nuclei at late maturation stages and stalled individualization complexes. This work sheds light on a novel component involved in cytoskeleton based cell morphological changes during spermiogenesis.
Fic, W., Faria, C. and St Johnston, D. (2019). IMP regulates Kuzbanian to control the timing of Notch signalling in Drosophila follicle cells. Development 146(2). PubMed ID: 30635283
The timing of Drosophila egg chamber development is controlled by a germline Delta signal that activates Notch in the follicle cells to induce them to cease proliferation and differentiate. This report that follicle cells lacking the RNA-binding protein IGF-II mRNA-binding protein (IMP) go through one extra division owing to a delay in the Delta-dependent S2 cleavage of Notch. The timing of Notch activation has previously been shown to be controlled by cis-inhibition by Delta in the follicle cells, which is relieved when the miRNA pathway represses Delta expression. imp mutants are epistatic to Delta mutants and give an additive phenotype with belle and Dicer-1 mutants, indicating that IMP functions independently of both cis-inhibition and the miRNA pathway.The imp phenotype is rescued by overexpression of Kuzbanian, the metalloprotease that mediates the Notch S2 cleavage. Furthermore, Kuzbanian is not enriched at the apical membrane in imp mutants, accumulating instead in late endosomes. Thus, IMP regulates Notch signalling by controlling the localisation of Kuzbanian to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.

Monday, January 28th - Disease models

Held, A., Major, P., Sahin, A., Reenan, R., Lipscombe, D. and Wharton, K. A. (2019). Circuit dysfunction in SOD1-ALS model first detected in sensory feedback prior to motor neuron degeneration is alleviated by BMP signaling. J Neurosci. PubMed ID: 30659087
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose origin and underlying cellular defects are not fully understood. While motor neuron degeneration is the signature feature of ALS, it is not clear if motor neurons, or other cells of the motor circuit, are the site of disease initiation. To better understand the contribution of multiple cell types in ALS, use was made of a Drosophila Sod1(G85R) knock-in model, in which all cells harbor the disease allele. End-stage dSod1(G85R) animals of both sexes exhibit severe motor deficits with clear degeneration of motor neurons. Interestingly, earlier in dSod1(G85R) larvae, motor function is also compromised, but their motor neurons exhibit only subtle morphological and electrophysiological changes, that are unlikely to cause the observed decrease in locomotion. The intact motor circuit was analyzed, and a defect was identified in sensory feedback that likely accounts for the altered motor activity of dSod1(G85R). Cell-autonomous activation of BMP signaling in proprioceptor sensory neurons, critical for the relay of the contractile status of muscles back to the central nerve cord, is able to completely rescue early stage motor defects and partially rescue late stage motor function to extend lifespan. Identification of a defect in sensory feedback, as a potential initiating event in ALS motor dysfunction, coupled with the ability of modified proprioceptors to alleviate such motor deficits, underscores the critical role that non-motor neurons play in disease progression and highlights their potential as a site to identify early-stage ALS biomarkers and for therapeutic intervention.
Chaplot, K., Pimpale, L., Ramalingam, B., Deivasigamani, S., Kamat, S. S. and Ratnaparkhi, G. S. (2019). SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via ROS-induced proteasomal degradation in a Drosophila model of Amyotrophic Lateral Sclerosis. Dis Model Mech. PubMed ID: 30635270
Familial Amyotrophic Lateral Sclerosis (F-ALS) is an incurable, late onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated Protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase understanding of the disease and lead to potential therapeutics. A quantitative high-throughput, Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy was developed to score for genes involved in the modulation of aggregates of fly ortholog, VAP(P58S), fused with GFP. A targeted RNAi screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci SOD1, TDP43 and also genes belonging to the TOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the Drosophila larval brain was developed in order to validate the hits from the cell based screen. In the larval brain, it was found that reduction of SOD1 level or decreased TOR signalling reduces aggregation, presumably by increasing levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation which appears to be triggered by an increase in ROS. This study has thus uncovered an interesting interplay between SOD1, ROS and TOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to a better understanding of initiation and progression of ALS.
Portela, M., Segura-Collar, B., Argudo, I., Saiz, A., Gargini, R., Sanchez-Gomez, P. and Casas-Tinto, S. (2018). Oncogenic dependence of glioma cells on kish/TMEM167A regulation of vesicular trafficking. Glia. PubMed ID: 30506943
Genetic lesions in glioblastoma (GB) include constitutive activation of PI3K and EGFR pathways to drive cellular proliferation and tumor malignancy. An RNAi genetic screen, performed in Drosophila melanogaster to discover new modulators of GB development, identified a member of the secretory pathway: kish/TMEM167A. Downregulation of kish/TMEM167A impaired fly and human glioma formation and growth, with no effect on normal glia. Glioma cells increased the number of recycling endosomes, and reduced the number of lysosomes. In addition, EGFR vesicular localization was primed toward recycling in glioma cells. kish/TMEM167A downregulation in gliomas restored endosomal system to a physiological state and altered lysosomal function, fueling EGFR toward degradation by the proteasome. These endosomal effects mirrored the endo/lysosomal response of glioma cells to Brefeldin A (BFA), but not the Golgi disruption and the ER collapse, which are associated with the undesirable toxicity of BFA in other cancers. These results suggest that glioma growth depends on modifications of the vesicle transport system, reliant on kish/TMEM167A. Noncanonical genes in GB could be a key for future therapeutic strategies targeting EGFR-dependent gliomas.
Bardai, F. H., Ordonez, D. G., Bailey, R. M., Hamm, M., Lewis, J. and Feany, M. B. (2018). Lrrk promotes tau neurotoxicity through dysregulation of actin and mitochondrial dynamics. PLoS Biol 16(12): e2006265. PubMed ID: 30571694
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson disease. Genetics and neuropathology link Parkinson disease with the microtubule-binding protein tau, but the mechanism of action of LRRK2 mutations and the molecular connection between tau and Parkinson disease are unclear. This study investigated the interaction of LRRK and tau in Drosophila and mouse models of tauopathy. Either increasing or decreasing the level of fly Lrrk enhances tau neurotoxicity, which is further exacerbated by expressing Lrrk with dominantly acting Parkinson disease-associated mutations. At the cellular level, altering Lrrk expression promotes tau neurotoxicity via excess stabilization of filamentous actin (F-actin) and subsequent mislocalization of the critical mitochondrial fission protein dynamin-1-like protein (Drp1). Biochemically, monomeric LRRK2 exhibits actin-severing activity, which is reduced as increasing concentrations of wild-type LRRK2, or expression of mutant forms of LRRK2 promote oligomerization of the protein. Overall, these findings provide a potential mechanistic basis for a dominant negative mechanism in LRRK2-mediated Parkinson disease, suggest a common molecular pathway with other familial forms of Parkinson disease linked to abnormalities of mitochondrial dynamics and quality control, and raise the possibility of new therapeutic approaches to Parkinson disease and related disorders.
Coelho, D. S., Schwartz, S., Merino, M. M., Hauert, B., Topfel, B., Tieche, C., Rhiner, C. and Moreno, E. (2018). Culling less fit neurons protects against Amyloid-beta-induced brain damage and cognitive and motor decline. Cell Rep 25(13): 3661-3673. PubMed ID: 30590040
Alzheimer's disease (AD) is the most common form of dementia, impairing cognitive and motor functions. One of the pathological hallmarks of AD is neuronal loss, which is not reflected in mouse models of AD. Therefore, the role of neuronal death is still uncertain. This study used a Drosophila AD model expressing a secreted form of human amyloid-beta42 peptide and showed that it recapitulates key aspects of AD pathology, including neuronal death and impaired long-term memory. Neuronal apoptosis is mediated by cell fitness-driven neuronal culling, which selectively eliminates impaired neurons from brain circuits. Removal of less fit neurons delays beta-amyloid-induced brain damage and protects against cognitive and motor decline, suggesting that contrary to common knowledge, neuronal death may have a beneficial effect in AD.
Guida, M. C., Birse, R. T., Dall'Agnese, A., Toto, P. C., Diop, S. B., Mai, A., Adams, P. D., Puri, P. L. and Bodmer, R. (2019). Intergenerational inheritance of high fat diet-induced cardiac lipotoxicity in Drosophila. Nat Commun 10(1): 193. PubMed ID: 30643137
Obesity is strongly correlated with lipotoxic cardiomyopathy, heart failure and thus mortality. The incidence of obesity has reached alarming proportions worldwide, and increasing evidence suggests that the parents' nutritional status may predispose their offspring to lipotoxic cardiomyopathy. However, to date, mechanisms underlying intergenerational heart disease risks have yet to be elucidated. This study reports that cardiac dysfunction induced by high-fat-diet (HFD) persists for two subsequent generations in Drosophila and is associated with reduced expression of two key metabolic regulators, adipose triglyceride lipase (ATGL/bmm) and transcriptional cofactor PGC-1. Evidence is provided that targeted expression of ATGL/bmm in the offspring of HFD-fed parents protects them, and the subsequent generation, from cardio-lipotoxicity. Furthermore, it was found that intergenerational inheritance of lipotoxic cardiomyopathy correlates with elevated systemic H3K27 trimethylation. Lowering H3K27 trimethylation genetically or pharmacologically in the offspring of HFD-fed parents prevents cardiac pathology. This suggests that metabolic homeostasis is epigenetically regulated across generations.

Friday, January 25 - Chromatin and DNA Replication

Gonzalez de Cozar, J. M., Gerards, M., Teeri, E., George, J., Dufour, E., Jacobs, H. T. and Joers, P. (2019). RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA. J Biol Chem. PubMed ID: 30635398
Mitochondrial DNA (mtDNA) replication uses a simple core machinery similar to those of bacterial viruses and plasmids, but its components are challenging to unravel. This study found that, as in mammals, the single Drosophila gene for RNase H1 (rnh1) has alternative translational start sites, resulting in two polypeptides, targeted to either mitochondria or the nucleus. RNAi-mediated rnh1 knockdown did not influence growth or viability of S2 cells, but compromised mtDNA integrity and copy number. rnh1 knockdown in intact flies also produced a phenotype of impaired mitochondrial function, characterized by respiratory chain deficiency, locomotor dysfunction and decreased lifespan. Its over-expression in S2 cells resulted in cell-lethality after 5-9 days, attributable to the nuclear-localized isoform. rnh1 knockdown and over-expression produced opposite effects on mtDNA replication intermediates. The most pronounced effects were seen in genome regions beyond the major replication pauses, where the replication fork needs to progress through a gene cluster that is transcribed in the opposite direction. RNase H1 deficiency led to an accumulation of replication intermediates in these zones, abundant mtDNA molecules joined by 4-way junctions, and species consistent with fork regression from the origin. These findings indicate replication stalling due to the presence of unprocessed RNA/DNA heteroduplexes, potentially leading to the degradation of collapsed forks or to replication restart by a mechanism involving strand invasion. Both mitochondrial RNA and DNA syntheses were affected by rnh1 knockdown, suggesting that RNase H1 also plays a role in integrating or co-regulating these processes in Drosophila mitochondria.
Fedotova, A., Clendinen, C., Bonchuk, A., Mogila, V., Aoki, T., Georgiev, P. and Schedl, P. (2019). Functional dissection of the developmentally restricted BEN domain chromatin boundary factor Insensitive. Epigenetics Chromatin 12(1): 2. PubMed ID: 30602385
Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. This study used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to an ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7. It is concluded that the subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. These studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.
Li, K. L., Zhang, L., Yang, X. M., Fang, Q., Yin, X. F., Wei, H. M., Zhou, T., Li, Y. B., Chen, X. L., Tang, F., Li, Y. H., Chang, J. F., Li, W. and Sun, F. (2018). Histone acetyltransferase CBP-related H3K23 acetylation contributes to courtship learning in Drosophila. BMC Dev Biol 18(1): 20. PubMed ID: 30458702
Histone modifications are critical in regulating neuronal processes. However, the impacts of individual histone modifications on learning and memory are elusive. This study investigated the contributions of histone H3 lysine modifications to learning and memory in Drosophila by using histone lysine-to-alanine mutants. Behavioural analysis indicated that compared to the H3WT group, mutants overexpressing H3K23A displayed impaired courtship learning. Chromatin immunoprecipitation analysis of H3K23A mutants showed that H3K23 acetylation (H3K23ac) levels were decreased on learning-related genes. Knockdown of CREB-binding protein (CBP) decreased H3K23ac levels, attenuated the expression of learning-related genes, led to a courtship learning defect and altered development of the mushroom bodies. A decline in courtship learning ability was observed in both larvae and adult treatments with ICG-001. Furthermore, treatment of Drosophila overexpressing mutated H3K23A with a CBP inhibitor did not aggravate the learning defect. It is concluded that H3K23ac, catalysed by the acetyltransferases dCBP, contributes to Drosophila learning, likely by controlling the expression of specific genes. This is a novel epigenetic regulatory mechanism underlying neuronal behaviours.
Scacchetti, A., Brueckner, L., Jain, D., Schauer, T., Zhang, X., Schnorrer, F., van Steensel, B., Straub, T. and Becker, P. B. (2018). CHRAC/ACF contribute to the repressive ground state of chromatin. Life Sci Alliance 1(1): e201800024. PubMed ID: 30456345
The chromatin remodeling complexes chromatin accessibility complex and ATP-utilizing chromatin assembly and remodeling factor (ACF) combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression remains unclear. This study explored the influence of Drosophila melanogaster chromatin accessibility complex/ACF on transcription by using complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of an Acf knock-out allele showed that only lowly expressed genes are derepressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together, these results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.
Park, A. R., Liu, N., Neuenkirchen, N., Guo, Q. and Lin, H. (2018). The role of maternal HP1a in early Drosophila embryogenesis via regulation of maternal transcript production. Genetics. PubMed ID: 30442760
Heterochromatin protein 1a (HP1a) is a highly conserved and versatile epigenetic factor that can both silence and activate transcription. However, the function of HP1a in development has been under-investigated. This study reports the role of maternal HP1a in producing maternal transcripts that drive early Drosophila embryogenesis. Maternal HP1a up-regulates genes involved in translation, mRNA splicing, and cell division but down-regulates genes involved in neurogenesis, organogenesis, and germline development, which all occur later in development. This study reveals the earliest contribution of HP1a during oogenesis in regulating the production of maternal transcripts that drive early Drosophila embryogenesis.
De, I., Chittock, E. C., Grotsch, H., Miller, T. C. R., McCarthy, A. A. and Muller, C. W. (2018). Structural basis for the activation of the deubiquitinase Calypso by the Polycomb protein ASX. Structure. PubMed ID: 30639226
Ubiquitin C-terminal hydrolase deubiquitinase BAP1 is an essential tumor suppressor involved in cell growth control, DNA damage response, and transcriptional regulation. As part of the Polycomb repression machinery, BAP1 is activated by the deubiquitinase adaptor domain of ASXL1 mediating gene repression by cleaving ubiquitin (Ub) from histone H2A in nucleosomes. The molecular mechanism of BAP1 activation by ASXL1 remains elusive, as no structures are available for either BAP1 or ASXL1. This study presents the crystal structure of the BAP1 ortholog from Drosophila melanogaster, named Calypso, bound to its activator, ASX, homolog of ASXL1. Based on comparative structural and functional analysis, a model for Ub binding by Calypso/ASX, uncover decisive structural elements responsible for ASX-mediated Calypso activation, and characterize the interaction with ubiquitinated nucleosomes. The results give molecular insight into Calypso function and its regulation by ASX and provide the opportunity for the rational design of mechanism-based therapeutics to treat human BAP1/ASXL1-related tumors.

Thursday January 24th - RNA

Wang, C., Ge, L., Wu, J., Wang, X. and Yuan, L. (2018). MiR-219 represses expression of dFMR1 in Drosophila melanogaster. Life Sci. PubMed ID: 30528775
Fragile X mental retardation protein (FMRP) plays a vital role in mRNA trafficking and translation inhibition to regulate the synthesis of local proteins in neuronal axons and dendritic terminals. This study predicted and selected 11 miRNAs potentially acting on the Drosophila fragile X mental retardation 1 (dFMR1) transcript. These candidates were screened for modulation of dFMR1 transcript levels at the cellular level using a dual luciferase reporter system. In addition, a transgenic Drosophila model was constructed overexpressing miR-219 in the nervous system, and dFMRP was quantified by western blotting. The neuromuscular junction phenotype in the model was studied by immunofluorescence staining. Among the 11 miRNAs screened, miR-219 and miR-960 reduced luciferase gene activity by binding to the 3'-UTR of the dFMR1 transcript. Mutation of the miR-219 or miR-960 binding sites on the transcript resulted in complete or partial elimination of the miRNA-induced repression. Western blots revealed that dFMRP expression was decreased in the miR-219 overexpression model (Elav>miR-219). Drosophila larvae overexpressing miR-219 showed morphological abnormalities at the neuromuscular junction (increased synaptic boutons and synaptic branches). These results suggest that miR-219 regulates dFMR1 expression in Drosophila and is involved in fragile X syndrome pathogenesis.
Flora, P., Wong-Deyrup, S. W., Martin, E. T., Palumbo, R. J., Nasrallah, M., Oligney, A., Blatt, P., Patel, D., Fuchs, G. and Rangan, P. (2018). Sequential regulation of maternal mRNAs through a conserved cis-acting element in their 3' UTRs . Cell Rep 25(13): 3828-3843.e3829. PubMed ID: 30590052
Maternal mRNAs synthesized during oogenesis initiate the development of future generations. Some maternal mRNAs are either somatic or germline determinants and must be translationally repressed until embryogenesis. However, the translational repressors themselves are temporally regulated. This study used polar granule component (pgc), a Drosophila maternal mRNA, to ask how maternal transcripts are repressed while the regulatory landscape is shifting. pgc, a germline determinant, is translationally regulated throughout oogenesis. Different conserved RNA-binding proteins bind a 10-nt sequence in the 3' UTR of pgc mRNA to continuously repress translation at different stages of oogenesis. Pumilio binds to this sequence in undifferentiated and early-differentiating oocytes to block Pgc translation. After differentiation, Bruno levels increase, allowing Bruno to bind the same sequence and take over translational repression of pgc mRNA. This study has identified a class of maternal mRNAs that are regulated similarly, including zelda, the activator of the zygotic genome.
Xu, M., Xiang, Y., Liu, X., Bai, B., Chen, R., Liu, L. and Li, M. (2018). Long noncoding RNA SMRG regulates Drosophila macrochaetes by antagonizing scute through E(spl)mbeta. RNA Biol. PubMed ID: 30526271
It is obvious that the majority of cellular transcripts are long noncoding RNAs (lncRNAs). Although studies suggested that lncRNAs participate in many biological processes through diverse mechanisms, however, little is known about their effects on epidermal mechanoreceptors. This study identified one novel Drosophila lncRNA, Scutellar Macrochaetes Regulatory Gene (SMRG), which regulates scutellar macrochaetes that act as mechanoreceptors by antagonizing the proneural gene scute (sc), through the repressor Enhancer-of-split mbeta (E(spl)mbeta). SMRG deficiency induced supernumerary scutellar macrochaetes and simultaneously a high sc RNA level in the adult thorax. Genetically, sc overexpression enhanced this supernumerary phenotype, while heterozygous sc mutant rescued this phenotype, both of which were mediated by E(spl)mbeta. At the molecular level, SMRG recruited E(spl)mbeta to the sc promoter region, which in turn suppressed sc expression. This work presents a novel function of lncRNA and offers insights into the molecular mechanism underlying mechanoreceptor development.
Shakhmantsir, I., Nayak, S., Grant, G. R. and Sehgal, A. (2018). Spliceosome factors target timeless (tim) mRNA to control clock protein accumulation and circadian behavior in Drosophila. Elife 7. PubMed ID: 30516472
Transcription-translation feedback loops that comprise eukaryotic circadian clocks rely upon temporal delays that separate the phase of active transcription of clock genes, such as Drosophila period (per) and timeless (tim), from negative feedback by the two proteins. However, understanding of the mechanisms involved is incomplete. Through an RNA interference screen, this study found that pre-mRNA processing 4 (PRP4) kinase, a component of the U4/U5.U6 triple small nuclear ribonucleoprotein (tri-snRNP) spliceosome, and other tri-snRNP components regulate cycling of the molecular clock as well as rest:activity rhythms. Unbiased RNA-Sequencing uncovered an alternatively spliced intron in tim whose increased retention upon prp4 downregulation leads to decreased TIM levels. The splicing of tim is rhythmic with a phase that parallels delayed accumulation of the protein in a 24 hr cycle. It is proposed that alternative splicing constitutes an important clock mechanism for delaying the daily accumulation of clock proteins, and thereby negative feedback by them.
Chen, D., Brovkina, M., Matzat, L. H. and Lei, E. P. (2019). Shep RNA-binding capacity is required for antagonism of gypsy chromatin insulator activity. G3 (Bethesda). PubMed ID: 30630880
Chromatin insulators are DNA-protein complexes that regulate chromatin structure and gene expression in a wide range of organisms. These complexes also harbor enhancer blocking and barrier activities. Increasing evidence suggests that RNA molecules are integral components of insulator complexes. However, how these RNA molecules are involved in insulator function remains unclear. The Drosophila RNA-binding protein Shep associates with the gypsy insulator complex and inhibits insulator activities. By mutating key residues in the RRM domains, a Shep mutant protein incapable of RNA-binding was generated, and this mutant lost the ability to inhibit barrier activity. In addition, one of many wildtype Shep isoforms but not RRM mutant Shep was sufficient to repress enhancer blocking activities. Finally, wildtype Shep rescued synthetic lethality of shep, mod(mdg4) double-mutants and developmental defects of shep mutant neurons, whereas mutant Shep failed to do so. These results indicate that the RNA-binding ability of Shep is essential for its ability to antagonize insulator activities and promote neuronal maturation. The findings suggest that regulation of insulator function by RNA-binding proteins relies on RNA-mediated interactions.
Van den Brande, S., Gijbels, M., Wynant, N., Santos, D., Mingels, L., Gansemans, Y., Van Nieuwerburgh, F. and Vanden Broeck, J. (2018). The presence of extracellular microRNAs in the media of cultured Drosophila cells. Sci Rep 8(1): 17312. PubMed ID: 30470777
While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. This study demonstrates the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), the presence was analyzed of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed the qRT-PCR results and provided evidence for selective miRNA secretion in EVs. This is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals.

Wednesday, January 23rd - Adult Neural Function

Damulewicz, M., Swiatek, M., Loboda, A., Dulak, J., Bilska, B., Przewlocki, R. and Pyza, E. (2018). Daily regulation of phototransduction, circadian clock, DNA repair, and immune gene expression by Heme Oxygenase in the retina of Drosophila. Genes (Basel) 10(1). PubMed ID: 30583479
The daily expression of genes and the changes in gene expression after silencing the heme oxygenase (ho) gene were examined in the retina of Drosophila using microarray and SybrGreen qPCR (quantitative polymerase chain reaction) methods. The HO decrease in the morning upregulated 83 genes and downregulated 57 genes. At night, 80 genes were upregulated and 22 were downregulated. The top 20 genes downregulated after ho silencing in the morning modulate phototransduction, immune responses, autophagy, phagocytosis, apoptosis, the carbon monoxide (CO) response, the oxidative stress/UV response, and translation. In turn, the genes that upregulated at night were involved in translation-the response to oxidative stress, DNA damage, and phototransduction. Among the top 20 genes downregulated at night were genes involved in phototransduction, immune responses, and autophagy. For some genes, a low level of HO had an opposite effect in the morning compared to those at night. Silencing ho also changed the expression of circadian clock genes, while the HO decrease during the night enhanced the expression of immune system genes. The results showed that the cyclic expression of HO is important for controlling several processes in the retina, including neuroprotection and those involved in the innate immune system.
Avetisyan, A. and Salzberg, A. (2019). Accurate elimination of superfluous attachment cells is critical for the construction of functional multicellular proprioceptors in Drosophila. Cell Death Differ. PubMed ID: 30622305
This study shows that developmental cell death plays a critical role in the morphogenesis of multicellular proprioceptors in Drosophila. The most prominent multicellular proprioceptive organ in the fly larva, the pentascolopidial (LCh5) organ, consists of a cluster of five stretch-responsive sensory organs that are anchored to the cuticle via specialized attachment cells. Stable attachment of the organ to the cuticle is critical for its ability to perceive mechanical stimuli arising from muscle contractions and the resulting displacement of its attachment sites. This study shows that five attachment cells are born within the LCh5 lineage, but three of them are rapidly eliminated, normally, by apoptosis. Strong genetic evidence attests to the existence of an autophagic gene-dependent safeguard mechanism that guarantees elimination of the unwanted cells upon perturbation of the apoptotic pathway prior to caspase liberation. The removal of the three superfluous cells guarantees the right ratio between the number of sensory organs and the number of attachment cells that anchor them to the cuticle. This accurate matching seems imperative for the attachment of cell growth and functionality and is thus vital for normal morphogenesis and functionality of the sensory organ.
Budelli, G., Ni, L., Berciu, C., van Giesen, L., Knecht, Z. A., Chang, E. C., Kaminski, B., Silbering, A. F., Samuel, A., Klein, M., Benton, R., Nicastro, D. and Garrity, P. A. (2019). Ionotropic receptors specify the morphogenesis of phasic sensors controlling rapid thermal preference in Drosophila. Neuron. PubMed ID: 30654923
Thermosensation is critical for avoiding thermal extremes and regulating body temperature. While thermosensors activated by noxious temperatures respond to hot or cold, many innocuous thermosensors exhibit robust baseline activity and lack discrete temperature thresholds, suggesting they are not simply warm and cool detectors. This study investigated how the aristal Cold Cells encode innocuous temperatures in Drosophila. They are not cold sensors but cooling-activated and warming-inhibited phasic thermosensors that operate similarly at warm and cool temperatures; it is proposed renaming them "Cooling Cells." Unexpectedly, Cooling Cell thermosensing does not require the previously reported Brivido Transient Receptor Potential (TRP) channels. Instead, three Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, specify both the unique structure of Cooling Cell cilia endings and their thermosensitivity. Behaviorally, Cooling Cells promote both warm and cool avoidance. These findings reveal a morphogenetic role for IRs and demonstrate the central role of phasic thermosensing in innocuous thermosensation.
Yanagawa, A., Couto, A., Sandoz, J. C., Hata, T., Mitra, A., Ali Agha, M. and Marion-Poll, F. (2018). LPS perception through taste-induced reflex in Drosophila melanogaster. J Insect Physiol. PubMed ID: 30528842
In flies, grooming serves several purposes, including protection against pathogens and parasites. Previous work has shown that Escherichia coli or lipopolysaccharides (LPS) can induce grooming behavior via activation of contact chemoreceptors on Drosophila wing. This suggested that specific taste receptors may contribute to this detection. This study examined the perception of commercially available LPS on Drosophila wing chemoreceptors in grooming reflex. Behavioral tests conducted with bitter, sweet and salty gustation such as caffeine, sucrose and salt, using flies carrying a defect in one of their taste receptors related to the detection of bitter molecules (Gr66a, Gr33a), sugars (Gr5a, Gr64f), or salt (IR76b). LPS and tastants of each category were applied to wing sensilla of these taste defective flies and to wild-type Canton Special (CS) flies. The results indicate that the grooming reflex induced by LPS requires a wide range of gustatory genes, and the inactivation of any of tested genes expressing cells causes a significant reduction of the behavior. This suggests that, while the grooming reflex is strongly regulated by cues perceived as aversive, other sapid cues traditionally related to sweet and salty tastes are also contributing to this behavior.
Gao, R., Asano, S. M., Upadhyayula, S., Pisarev, I., Milkie, D. E., Liu, T. L., Singh, V., Graves, A., Huynh, G. H., Zhao, Y., Bogovic, J., Colonell, J., Ott, C. M., Zugates, C., Tappan, S., Rodriguez, A., Mosaliganti, K. R., Sheu, S. H., Pasolli, H. A., Pang, S., Xu, C. S., Megason, S. G., Hess, H., Lippincott-Schwartz, J., Hantman, A., Rubin, G. M., Kirchhausen, T., Saalfeld, S., Aso, Y., Boyden, E. S. and Betzig, E. (2019). Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science 363(6424). PubMed ID: 30655415
Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. This study combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.
Alejevski, F., Saint-Charles, A., Michard-Vanhee, C., Martin, B., Galant, S., Vasiliauskas, D. and Rouyer, F. (2019). The HisCl1 histamine receptor acts in photoreceptors to synchronize Drosophila behavioral rhythms with light-dark cycles. Nat Commun 10(1): 252. PubMed ID: 30651542
In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. This study shows that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. These results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina.

Tuesday, January 22nd - Signaling

Alegot, H., Markosian, C., Rauskolb, C., Yang, J., Kirichenko, E., Wang, Y. C. and Irvine, K. D. (2019). Recruitment of Jub by alpha-catenin promotes Yki activity and Drosophila wing growth. J Cell Sci. PubMed ID: 30659113
The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions. This study investigated the mechanism by which the Drosophila Ajuba family protein, Jub, is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. Alpha-catenin as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of alpha-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. Increased Jub recruitment to alpha-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. These observations establish alpha-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to alpha-catenin as a key contributor to biomechanical regulation of Hippo signaling.
Zhang, Z., Ahmed-Braimah, Y., Goldberg, M. L. and Wolfner, M. F. (2018). Calcineurin dependent protein phosphorylation changes during egg activation in Drosophila melanogaster. Mol Cell Proteomics. PubMed ID: 30478224
In almost all animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transitions into an actively developing embryo, initiates with an increase in Ca2+ in the oocyte's cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepares the oocyte for embryogenesis. Calcineurin is a highly conserved phosphatase that is activated by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus, ascidians, and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. This study investigated the regulatory role of calcineurin during egg activation in Drosophila melanogaster. Using mass spectrometry, the phosphoproteomic and proteomic changes were quantified that occur during egg activation, and how these events are affected when calcineurin function is perturbed in female germ cells was examined. The results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. Calcineurin-dependent changes in cell cycle regulators including Fizzy (Fzy), Greatwall (Gwl) and Endosulfine (Endos); in protein translation modulators including PNG, NAT, eIF4G, and eIF4B; and in important components of signaling pathways including GSK3beta and Akt1. These results help elucidate the events that occur during the transition from oocyte to embryo.
Dutta, D., Mutsuddi, M. and Mukherjee, A. (2018). Synergistic interaction of Deltex and Hrp48 leads to JNK activation. Cell Biol Int. PubMed ID: 30597717
The communication among the cells plays a seminal role in metazoan development by coordinating multiple cellular processes that, in turn, helps in the maintenance of biological homeostasis. A previous study demonstrated that Dx and Hrp48 together downregulate Notch signaling and induce cell death in Drosophila. To understand the signaling events behind the Dx and Hrp48-induced cell death in a greater detail, a set of genetic experiments was performed followed by immunocytochemical analyses. The data revealed that Dx along with Hrp48 induced JNK activation and consequently cell death in the eye tissue. Additionally, using genetic and molecular approaches, the domain of Dx protein responsible for its synergistic activity with Hrp48 was identified. Altogether, these analyses suggest that coexpression of Dx and Hrp48 activates JNK pathway to induce cell death in eye disc of Drosophila melanogaster.
Ye, F., Huang, Y., Li, J., Ma, Y., Xie, C., Liu, Z., Deng, X., Wan, J., Xue, T., Liu, W. and Zhang, M. (2018). An unexpected INAD PDZ tandem-mediated PLCbeta binding in Drosophila photo receptors. Elife 7. PubMed ID: 30526850
INAD assembles key enzymes of Drosophila compound eye photo-transduction pathway into a supramolecular complex, supporting efficient and fast light signaling. However, the molecular mechanism governing the interaction between INAD and NORPA (phospholipase Cbeta, PLCbeta), a key step for the fast kinetics of the light signaling, is not known. This study shows that the NORPA C-terminal coiled-coil domain and PDZ-binding motif (CC-PBM) synergistically bind to INAD PDZ45 tandem with an unexpected mode and unprecedentedly high affinity. Guided by the INAD/NORPA complex structure, this study discovers that INADL is likely a mammalian counterpart of INAD. The INADL PDZ89 tandem specifically binds to PLCbeta4 with a strikingly similar mode to that of the INAD/NORPA complex as revealed by the structure of the INADL PDZ89/PLCbeta4 CC-PBM complex. Therefore, this study suggests that the highly specific PDZ tandem/PLCbeta interactions are an evolutionarily conserved mechanism in PLCbeta signaling of the animal kingdom.
Baillon, L., Germani, F., Rockel, C., Hilchenbach, J. and Basler, K. (2018). Xrp1 is a transcription factor required for cell competition-driven elimination of loser cells. Sci Rep 8(1): 17712. PubMed ID: 30531963
The elimination of unfit cells from a tissue is a process known in Drosophila and mammals as cell competition. In a well-studied paradigm "loser" cells that are heterozygous mutant for a haploinsufficient ribosomal protein gene are eliminated from developing tissues via apoptosis when surrounded by fitter wild-type cells, referred to as "winner" cells. However, the mechanisms underlying the induction of this phenomenon are not fully understood. This paper reports that a CCAAT-Enhancer-Binding Protein (C/EBP), Xrp1, which is known to help maintaining genomic stability after genotoxic stress, is necessary for the elimination of loser clones in cell competition. In loser cells, Xrp1 is transcriptionally upregulated by an autoregulatory loop and is able to trigger apoptosis - driving cell elimination. Xrp1 acts in the nucleus to regulate the transcription of several genes that have been previously involved in cell competition. It is therefore speculated that Xrp1 might play a fundamental role as a molecular caretaker of the genomic integrity of tissues.
Yang, G., Humphrey, S. J., Murashige, D. S., Francis, D., Wang, Q. P., Cooke, K. C., Neely, G. and James, D. E. (2018). RagC phosphorylation autoregulates mTOR complex 1. EMBO J. PubMed ID: 30552228
The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb and an amino acid-responsive pathway mediated via the Rag GTPases. This study identified and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation-induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, mTORC1 was identified as the upstream kinase of RagC on S21. These data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity.

Monday, January 21st - Evolution

Atallah, J. and Lott, S. E. (2018). Evolution of maternal and zygotic mRNA complements in the early Drosophila embryo. PLoS Genet 14(12): e1007838. PubMed ID: 30557299
The earliest stages of animal development are controlled by maternally deposited mRNA transcripts and proteins. Once the zygote is able to transcribe its own genome, maternal transcripts are degraded, in a tightly regulated process known as the maternal to zygotic transition (MZT). While this process has been well-studied within model species, there is little knowledge of how the pools of maternal and zygotic transcripts evolve. To characterize the evolutionary dynamics and functional constraints on early embryonic expression, a transcriptomic dataset was created for 14 Drosophila species spanning over 50 million years of evolution, at developmental stages before and after the MZT, and the results were compared with a previously published Aedes aegypti developmental time course. Deep conservation was found over 250 million years of a core set of genes transcribed only by the zygote. This select group is highly enriched in transcription factors that play critical roles in early development. However, a surprisingly high level of change was also identifed in the transcripts represented at both stages over the phylogeny. While mRNA levels of genes with maternally deposited transcripts are more highly conserved than zygotic genes, those maternal transcripts that are completely degraded at the MZT vary dramatically between species. It was also shown that hundreds of genes have different isoform usage between the maternal and zygotic genomes. This work suggests that maternal transcript deposition and early zygotic transcription are remarkably dynamic over evolutionary time, despite the widespread conservation of early developmental processes.
Fraisse, C., Puixeu Sala, G. and Vicoso, B. (2018). Pleiotropy modulates the efficacy of selection in Drosophila melanogaster. Mol Biol Evol. PubMed ID: 30590559
Pleiotropy is the well-established idea that a single mutation affects multiple phenotypes. If a mutation has opposite effects on fitness when expressed in different contexts, then genetic conflict arises. Pleiotropic conflict is expected to reduce the efficacy of selection by limiting the fixation of beneficial mutations through adaptation, and the removal of deleterious mutations through purifying selection. While this has been widely discussed, in particular in the context of a putative "gender load", it has yet to be systematically quantified. This work empirically estimates to which extent different pleiotropic regimes impede the efficacy of selection in Drosophila melanogaster. Whole-genome polymorphism data from a single African population and divergence data from D. simulans were used to estimate the fraction of adaptive fixations (alpha), the rate of adaptation (omegaA) and the direction of selection (DoS). After controlling for confounding covariates, it was found that the different pleiotropic regimes have a relatively small, but significant, effect on selection efficacy. Specifically, the results suggest that pleiotropic sexual antagonism may restrict the efficacy of selection, but that this conflict can be resolved by limiting the expression of genes to the sex where they are beneficial. Intermediate levels of pleiotropy across tissues and life stages can also lead to maladaptation in D. melanogaster, due to inefficient purifying selection combined with low frequency of mutations that confer a selective advantage. Thus, this study highlights the need to consider the efficacy of selection in the context of antagonistic pleiotropy, and of genetic conflict in general.
Combs, P. A., Krupp, J. J., Khosla, N. M., Bua, D., Petrov, D. A., Levine, J. D. and Fraser, H. B. (2018). Tissue-specific cis-regulatory divergence implicates eloF in inhibiting interspecies mating in Drosophila. Curr Biol. PubMed ID: 30503619
Reproductive isolation is a key component of speciation. In many insects, a major driver of this isolation is cuticular hydrocarbon pheromones, which help to identify potential intraspecific mates. When the distributions of related species overlap, there may be strong selection on mate choice for intraspecific partners because interspecific hybridization carries significant fitness costs. Drosophila has been a key model for the investigation of reproductive isolation; although both male and female mate choices have been extensively investigated, the genes underlying species recognition remain largely unknown. To explore the molecular mechanisms underlying Drosophila speciation, tissue-specific cis-regulatory divergence was identifed using RNA sequencing (RNA-seq) in D. simulans x D. sechellia hybrids. By focusing on cis-regulatory changes specific to female oenocytes, the tissue that produces cuticular hydrocarbons, this study identified a small number of candidate genes were identified. One of these, the fatty acid elongase eloF, broadly affects the hydrocarbons present on D. sechellia and D. melanogaster females, as well as the propensity of D. simulans males to mate with them. Therefore, cis-regulatory changes in eloF may be a major driver in the sexual isolation of D. simulans from multiple other species. The RNA-seq approach proved to be far more efficient than quantitative trait locus (QTL) mapping in identifying candidate genes; the same framework can be used to pinpoint candidate drivers of cis-regulatory divergence in traits differing between any interfertile species.
Mageeney, C. M., Kearse, M. G., Gershman, B. W., Pritchard, C. E., Colquhoun, J. M. and Ware, V. C. (2018). Functional interplay between ribosomal protein paralogues in the eRpL22 family in Drosophila melanogaster. Fly (Austin): 1-21. PubMed ID: 30465696
Duplicated ribosomal protein (RP) genes in the Drosophila melanogaster eRpL22 family encode structurally-divergent and differentially-expressed rRNA-binding RPs. eRpL22 is expressed ubiquitously and eRpL22-like expression is tissue-restricted with highest levels in the adult male germline. Paralogue functional equivalence was explored using the GAL4-UAS system for paralogue knockdown or overexpression and a conditional eRpL22-like knockout in a heat- shock flippase/FRT line. Ubiquitous eRpL22 knockdown with Actin-GAL4 resulted in embryonic lethality, confirming eRpL22 essentiality. eRpL22-like knockdown (60%) was insufficient to cause lethality; yet, conditional eRpL22-like knockout at one hour following egg deposition caused lethality within each developmental stage. Therefore, each paralogue is essential. Variation in timing of heat-shock-induced eRpL22-like knockout highlighted early embryogenesis as the critical period where eRpL22-like expression (not compensated for by eRpL22) is required for normal development of several organ systems, including testis development and subsequent sperm production. To determine if eRpL22-like can substitute for eRpL22, Actin-GAL4 for ubiquitous eRpL22 knockdown and eRpL22-like-FLAG (or FLAG-eRpL22: control) overexpression. Emergence of adults demonstrated that ubiquitous eRpL22-like-FLAG or FLAG-eRpL22 expression eliminates embryonic lethality resulting from eRpL22 depletion. Adults rescued by eRpL22-like-FLAG (but not by FLAG-eRpL22) overexpression had reduced fertility and longevity. It is concluded that eRpL22 paralogue roles are not completely interchangeable and include functionally-diverse roles in development and spermatogenesis.
Meiklejohn, C. D., Landeen, E. L., Gordon, K. E., Rzatkiewicz, T., Kingan, S. B., Geneva, A. J., Vedanayagam, J. P., Muirhead, C. A., Garrigan, D., Stern, D. L. and Presgraves, D. C. (2018). Gene flow mediates the role of sex chromosome meiotic drive during complex speciation. Elife 7. PubMed ID: 30543325
During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build-up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, this study combined high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, Drosophila mauritiana and D. simulans. The findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation.
Amei, A. and Zhou, S. (2019). Inferring the distribution of selective effects from a time inhomogeneous model. PLoS One 14(1): e0194709. PubMed ID: 30657757
A Poisson random field model has been developed for estimating the distribution of selective effects of newly arisen nonsynonymous mutations that could be observed as polymorphism or divergence in samples of two related species under the assumption that the two species populations are not at mutation-selection-drift equilibrium. The model is applied to 91 Drosophila genes by comparing levels of polymorphism in an African population of D. melanogaster with divergence to a reference strain of D. simulans. Based on the difference of gene expression level between testes and ovaries, the 91 genes were classified as 33 male-biased, 28 female-biased, and 30 sex-unbiased genes. Under a Bayesian framework, Markov chain Monte Carlo simulations are implemented to the model in which the distribution of selective effects is assumed to be Gaussian with a mean that may differ from one gene to the other to sample key parameters. Based on estimates, the majority of newly-arisen nonsynonymous mutations that could contribute to polymorphism or divergence in Drosophila species are mildly deleterious with a mean scaled selection coefficient of -2.81, while almost 86% of the fixed differences between species are driven by positive selection. There are only 16.6% of the nonsynonymous mutations observed in sex-unbiased genes that are under positive selection in comparison to 30% of male-biased and 46% of female-biased genes that are beneficial. It was also estimated that D. melanogaster and D. simulans may have diverged 1.72 million years ago.

Friday, January 18th - CNS Development

Mishra, A. K., Bernardo-Garcia, F. J., Fritsch, C., Humberg, T. H., Egger, B. and Sprecher, S. G. (2018). Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode. PLoS Genet 14(4): e1007353. PubMed ID: 29677185
The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. This study characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define photoreceptor subtype identity. This model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning.
Sun, B., Tu, J., Liang, Q., Cheng, X., Fan, X., Li, Y., Wallbank, R. W. R. and Yang, M. (2018). Expression of mammalian ASH1 and ASH4 in Drosophila reveals opposing functional roles in neurogenesis. Gene 688: 132-139. PubMed ID: 30529096
To investigate whether the members of the mammalian Achaete-Scute Complex homologue (ASH) gene family have evolved functional differences, the patterning of bristles was used as a phenotypic marker. Drosophila uses a single genetic locus - the Achaete-Scute Complex - to demarcate the regions of the body where bristles can form. 4-5 Achaete-Scute Complex homologue genes (ASH) are found in the mammalian genome, which are homologous with scute in Drosophila. Although ASH2 and ASH3 have gained new functions during evolution, the function of ASH4 and its evolutionary changes are still unclear. In this study, mouse and human ASH1 and ASH4 were overexpressed in the Drosophila notum respectively. The results show that both the protein sequence and cis-regulatory elements of mammalian ASH1 have conserved an ancient proneural function during evolution. However, mouse ASH4 has lost proneural function partly due to truncation of a C-terminal amino acid domain. Interestingly, instead of a similar loss of proneural function, human ASH4 can actually inhibit Drosophila bristle development, implying that human ASH4 may be a potential factor relating to skin development in human being. These results demonstrate gene duplication of the ASH family may have led to a novel function during evolution.
Shih, M. M., Davis, F. P., Henry, G. L. and Dubnau, J. (2018). Nuclear transcriptomes of the seven neuronal cell types that constitute the Drosophila mushroom bodies. G3 (Bethesda). PubMed ID: 30397017
The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The Drosophila melanogaster MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes (the γ, α'/β' and α/β) and 7 cell subtypes (γd, γm, α''/β'ap, α''/β'm, α'/βp, α'/βs and α/βc) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. This study used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. 350 MB class- or subtype-specific genes were identified, including the widely used α/β class marker Fas2 and the α'/β' class marker trio. Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, the data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the Drosophila MB provides a valuable resource for the fly neuroscience community.
Olesnicky, E. C., Antonacci, S., Popitsch, N., Lybecker, M. C., Titus, M. B., Valadez, R., Derkach, P. G., Marean, A., Miller, K., Mathai, S. K. and Killian, D. J. (2018). Shep interacts with posttranscriptional regulators to control dendrite morphogenesis in sensory neurons. Dev Biol. PubMed ID: 30352216
RNA binding proteins (RBPs) mediate posttranscriptional gene regulatory events throughout development. During neurogenesis, many RBPs are required for proper dendrite morphogenesis within Drosophila sensory neurons. Despite their fundamental role in neuronal morphogenesis, little is known about the molecular mechanisms in which most RBPs participate during neurogenesis. In Drosophila, alan shepard (shep) encodes a highly conserved RBP that regulates dendrite morphogenesis in sensory neurons. Moreover, the C. elegans ortholog sup-26 has also been implicated in sensory neuron dendrite morphogenesis. Nonetheless, the molecular mechanism by which Shep/SUP-26 regulate dendrite development is not understood. This study shows that Shep interacts with the RBPs Trailer Hitch (Tral), Ypsilon schachtel (Yps), Belle (Bel), and Poly(A)-Binding Protein (PABP), to direct dendrite morphogenesis in Drosophila sensory neurons. Moreover, a conserved set of Shep/SUP-26 target RNAs was identified that include regulators of cell signaling, posttranscriptional gene regulators, and known regulators of dendrite development.
Averbukh, I., Lai, S. L., Doe, C. Q. and Barkai, N. (2018). A repressor-decay timer for robust temporal patterning in embryonic Drosophila neuroblast lineages. Elife 7. PubMed ID: 30526852
Biological timers synchronize patterning processes during embryonic development. In the Drosophila embryo, neural progenitors (neuroblasts; NBs) produce a sequence of unique neurons whose identities depend on the sequential expression of temporal transcription factors (TTFs). The stereotypy and precision of NB lineages indicate reproducible TTF timer progression. This study combines theory and experiments to define the timer mechanism. The TTF timer is commonly described as a relay of activators, but its regulatory circuit is also consistent with a repressor-decay timer, where TTF expression begins when its repressor decays. Theory shows that repressor-decay timers are more robust to parameter variations than activator-relay timers. This motivated an experimental comparison of the relative importance of the relay and decay interactions in-vivo. Comparing WT and mutant NBs at high temporal resolution, this study show that the TTF sequence progresses primarily by repressor-decay. It is suggested that need for robust performance shapes the evolutionary-selected designs of biological circuits.
Schilling, T., Ali, A. H., Leonhardt, A., Borst, A. and Pujol-Marti, J. (2019). Transcriptional control of morphological properties of direction-selective T4/T5 neurons in Drosophila. Development. PubMed ID: 30642835
In the Drosophila visual system, T4/T5 neurons represent the first stage in which the direction of visual motion is computed. T4 and T5 neurons exist in four subtypes, each responding to motion in one of the four cardinal directions and projecting axons into one of the four lobula plate layers. However, all T4/T5 neurons share properties essential for sensing motion. How T4/T5 neurons acquire their properties during development is poorly understood. This study reveals that SoxN and Sox102F transcription factors control the acquisition of properties common to all T4/T5 neuron subtypes, i.e. the layer specificity of dendrites and axons. Accordingly, adult flies are motion blind after disrupting SoxN or Sox102F in maturing T4/T5 neurons. It was further foud that the transcription factors Ato and Dac are redundantly required in T4/T5 neuron progenitors for SoxN and Sox102F expression in T4/T5 neurons, linking the transcriptional programs specifying progenitor identity to those regulating the acquisition of morphological properties in neurons. This work will help to link structure, function and development in a neuronal type performing a computation conserved across vertebrate and invertebrate visual systems.

Thursday, January 17th - Adult Physiology

Trinh, I., Gluscencova, O. B. and Boulianne, G. L. (2018). An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion. Mol Metab. PubMed ID: 30389349
Obesity is a complex disorder involving many genetic and environmental factors that are required to maintain energy homeostasis. While studies in human populations have led to significant progress in the generation of an obesity gene map and broadened understanding of the genetic basis of common obesity, there is still a large portion of heritability and etiology that remains unknown. This study used the genetically tractable fruit fly, Drosophila melanogaster, to identify genes/pathways that function in the nervous system to regulate energy balance. An in vivo RNAi screen was performed in Drosophila neurons. and obese or lean phenotypes were assayed by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects. 24 genes were identified including 20 genes that have not been previously associated with energy homeostasis. One identified hit, Diacylglycerol kinase (Dgk), has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. Downregulation of neuronal Diacyl glycerol kinase (Dgk) levels increases TAG and carbohydrate levels and these phenotypes can be recapitulated by reducing Dgk levels specifically within the insulin-producing cells that secrete Drosophila insulin-like peptides (dILPs). Conversely, overexpression of kinase-dead Dgk, but not wild-type, decreased circulating dILP2 and dILP5 levels resulting in lower insulin signalling activity. Despite having higher circulating dILP levels, Dgk RNAi flies have decreased pathway activity suggesting that they are insulin-resistant. Altogether, this study has identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila.
Zandawala, M., Yurgel, M. E., Texada, M. J., Liao, S., Rewitz, K. F., Keene, A. C. and Nassel, D. R. (2018). Modulation of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin. PLoS Genet 14(11): e1007767. PubMed ID: 30457986
Behavior and physiology are orchestrated by neuropeptides acting as central neuromodulators and circulating hormones. An outstanding question is how these neuropeptides function to coordinate complex and competing behaviors. In Drosophila, the neuropeptide leucokinin (LK) modulates diverse functions, but mechanisms underlying these complex interactions remain poorly understood. As a first step towards understanding these mechanisms, LK circuitry that governs various aspects of post-feeding physiology and behavior was delimited. Processes were found that impaired LK signaling in Lk and Lk receptor (Lkr) mutants affects diverse but coordinated processes, including regulation of stress, water homeostasis, feeding, locomotor activity, and metabolic rate. Next, attempts were made to define the populations of LK neurons that contribute to the different aspects of this physiology. The calcium activity in abdominal ganglia LK neurons (ABLKs), but not in the two sets of brain neurons, increases specifically following water consumption, suggesting that ABLKs regulate water homeostasis and its associated physiology. To identify targets of LK peptide, the distribution of Lkr expression was mapped, a brain single-cell transcriptome dataset was mined for genes coexpressed with Lkr, and synaptic partners of LK neurons were identified. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) and transcripts in IPCs and increased starvation resistance. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, these data suggest that the three sets of LK neurons have different targets, but modulate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating feeding-related neuroendocrine regulation of vital behavior and physiology.
Iatsenko, I., Boquete, J. P. and Lemaitre, B. (2018). Microbiota-derived lactate activates production of reactive oxygen species by the intestinal NADPH Oxidase Nox and shortens Drosophila lifespan. Immunity 49(5): 929-942.e925. PubMed ID: 30446385
Commensal microbes colonize the gut epithelia of virtually all animals and provide several benefits to their hosts. Changes in commensal populations can lead to dysbiosis, which is associated with numerous pathologies and decreased lifespan. Peptidoglycan recognition proteins (PGRPs) are important regulators of the commensal microbiota and intestinal homeostasis. This study found that a null mutation in Drosophila PGRP-SD was associated with overgrowth of Lactobacillus plantarum in the fly gut and a shortened lifespan. L. plantarum-derived lactic acid triggered the activation of the intestinal NADPH oxidase Nox and the generation of reactive oxygen species (ROS). In turn, ROS production promoted intestinal damage, increased proliferation of intestinal stem cells, and dysplasia. Nox-mediated ROS production required lactate oxidation by the host intestinal lactate dehydrogenase, revealing a host-commensal metabolic crosstalk that is probably broadly conserved. These findings outline a mechanism whereby host immune dysfunction leads to commensal dysbiosis that in turn promotes age-related pathologies.
Wang, W., Xin, J., Yang, X., Lam, S. M., Shui, G., Wang, Y. and Huang, X. (2018). Lipid-gene regulatory network reveals coregulations of triacylglycerol with phosphatidylinositol/lysophosphatidylinositol and with hexosyl-ceramide. Biochim Biophys Acta Mol Cell Biol Lipids 1864(2): 168-180. PubMed ID: 30521938
Lipid homeostasis is important for executing normal cellular functions and maintaining physiological conditions. The biophysical properties and intricate metabolic network of lipids underlie the coordinated regulation of different lipid species in lipid homeostasis. To reveal the homeostatic response among different lipids, this study systematically knocked down 40 lipid metabolism genes in Drosophila S2 cells by RNAi and profiled the lipidomic changes. Clustering analyses of lipids reveal that many pairs of genes acting in a sequential fashion or sharing the same substrate are tightly clustered. Through a lipid-gene regulatory network analysis, it was further found that a reduction of triacylglycerol (TAG) is associated with an increase of phosphatidylinositol (PI) and lysophosphatidylinositol (LPI) or a reduction of hexosyl-ceramide (HexCer) and hydroxylated hexosyl-ceramide (OH-HexCer). Importantly, negative coregulation between TAG and LPI/PI, and positive coregulation between TAG and HexCer, were also found in human Hela cells. Together, these results reveal coregulations of TAG with PI/LPI and with HexCer in lipid homeostasis.
Meschi, E., Leopold, P. and Delanoue, R. (2019). An EGF-Responsive Neural Circuit Couples Insulin Secretion with Nutrition in Drosophila. Dev Cell 48(1): 76-86 e75. PubMed ID: 30555002
Developing organisms use fine-tuning mechanisms to adjust body growth to ever-changing nutritional conditions. In Drosophila, the secretory activity of insulin-producing cells (IPCs) is central to couple systemic growth with amino acids availability. This study identified a subpopulation of inhibitory neurons contacting the IPCs (IPC-connecting neurons or ICNs) that play a key role in this coupling. ICNs respond to growth-blocking peptides (GBPs), a family of fat-body-derived signals produced upon availability of dietary amino acids. GBPs are atypical ligands for the fly EGF receptor (EGFR). Upon activation of EGFR by adipose GBPs, ICN-mediated inhibition of IPC function is relieved, allowing insulin secretion. This study reveals an unexpected role for EGF-like metabolic hormones and EGFR signaling as critical modulators of neural activity, coupling insulin secretion to the nutritional status.
Post, S., Liao, S., Yamamoto, R., Veenstra, J. A., Nassel, D. R. and Tatar, M. (2018). Drosophila insulin-like peptide dilp1 increases lifespan and glucagon-like Akh expression epistatic to dilp2. Aging Cell: e12863. PubMed ID: 30511458
The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. This study finds that dilp1 is highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Dilp1 and dilp2 single and double mutants were used to describe interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 - dilp2 double mutants. dilp1 and dilp2 interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. Repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. The data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide.

Wednesday, January 16th - Adult Neural Function

Omoto, J. J., Nguyen, B. M., Kandimalla, P., Lovick, J. K., Donlea, J. M. and Hartenstein, V. (2018). Neuronal constituents and putative interactions within the Drosophila ellipsoid body neuropil. Front Neural Circuits 12: 103. PubMed ID: 30546298
The central complex (CX) is a midline-situated collection of neuropil compartments in the arthropod central brain, implicated in higher-order processes such as goal-directed navigation. This study provides a systematic genetic-neuroanatomical analysis of the ellipsoid body (EB), a compartment which represents a major afferent portal of the Drosophila CX. The neuropil volume of the EB, along with its prominent input compartment, called the bulb, is subdivided into precisely tessellated domains, distinguishable based on intensity of the global marker DN-cadherin. EB tangential elements (so-called ring neurons), most of which are derived from the DALv2 neuroblast lineage, predominantly interconnect the bulb and EB domains in a topographically organized fashion. Using the DN-cadherin domains as a framework, this connectivity was first characterized by Gal4 driver lines expressed in different DALv2 ring neuron (R-neuron) subclasses. 11 subclasses were identified, 6 of which correspond to previously described projection patterns, and 5 novel patterns. These subclasses both spatially (based on EB innervation pattern) and numerically (cell counts) summate to the total EB volume and R-neuron cell number, suggesting that this compilation of R-neuron subclasses approaches completion. EB columnar elements, as well as non-DALv2 derived extrinsic ring neurons (ExR-neurons), were also incorporated into this anatomical framework. Finally, the connectivity between R-neurons and their targets was addressed, using the anterograde trans-synaptic labeling method, trans-Tango.
Razetti, A., Medioni, C., Malandain, G., Besse, F. and Descombes, X. (2018). A stochastic framework to model axon interactions within growing neuronal populations. PLoS Comput Biol 14(12): e1006627. PubMed ID: 30507939
The confined and crowded environment of developing brains imposes spatial constraints on neuronal cells that have evolved individual and collective strategies to optimize their growth. These include organizing neurons into populations extending their axons to common target territories. How individual axons interact with each other within such populations to optimize innervation is currently unclear and difficult to analyze experimentally in vivo. This study developed a stochastic model of 3D axon growth that takes into account spatial environmental constraints, physical interactions between neighboring axons, and branch formation. This general, predictive and robust model, when fed with parameters estimated on real neurons from the Drosophila brain, enabled the study of the mechanistic principles underlying the growth of axonal populations. First, it provided a novel explanation for the diversity of growth and branching patterns observed in vivo within populations of genetically identical neurons. Second, it uncovered that axon branching could be a strategy optimizing the overall growth of axons competing with others in contexts of high axonal density. The flexibility of this framework will make it possible to investigate the rules underlying axon growth and regeneration in the context of various neuronal populations.
Pirez, N., Bernabei-Cornejo, S. G., Fernandez-Acosta, M., Duhart, J. M. and Ceriani, M. F. (2018). Contribution of non-circadian neurons to the temporal organization of locomotor activity. Biol Open. PubMed ID: 30530810
In the fruit fly, Drosophila melanogaster, the daily cycle of rest and activity is a rhythmic behavior that relies on the activity of a small number of neurons. The small Lateral Neurons ventral (sLNvs) are considered key in the control of locomotor rhythmicity. Previous work has shown that these neurons undergo structural remodeling on its axonal projections on a daily basis. Such remodeling endows sLNvs with the possibility to make synaptic contacts with different partners at different times along the day as has been previously described. By using different genetic tools to alter membrane excitability of the sLNv putative postsynaptic partners, their functional role on the control of locomotor activity was tested. Optical imaging was used to test the functionality of these contacts. These different neuronal groups affect the consolidation of rhythmic activity, suggesting that non-circadian cells are part of the circuit that controls locomotor activity. The results suggest that new neuronal groups, in addition to the well-characterized clock neurons, contribute to the operations of the circadian network that controls locomotor activity in Drosophila melanogaster.
Poe, A. R., Wang, B., Sapar, M. L., Ji, H., Li, K., Onabajo, T., Fazliyeva, R., Gibbs, M., Qiu, Y., Hu, Y. and Han, C. (2018). Robust CRISPR/Cas9-mediated tissue specific mutagenesis reveals gene redundancy and perdurance in Drosophila. Genetics. PubMed ID: 30504366
Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. This study presents a simple yet highly efficient CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously-expressed multi-guide RNA (gRNA) transgene. Convenient toolkits are described for making enhancer-driven Cas9 lines and multi-gRNAs that are optimized for mutagenizing somatic cells. Indels in coding sequences were found to more reliably cause somatic mutations than DNA excisions induced by two gRNAs. It was further shown that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasked redundant SNARE gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage revealed the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISRPR tools can be applied to analyze tissue-specific gene function in many biological processes.
Xu, S., Xiao, Q., Cosmanescu, F., Sergeeva, A. P., Yoo, J., Lin, Y., Katsamba, P. S., Ahlsen, G., Kaufman, J., Linaval, N. T., Lee, P. T., Bellen, H. J., Shapiro, L., Honig, B., Tan, L. and Zipursky, S. L. (2018). Interactions between the Ig-superfamily proteins DIP-alpha and Dpr6/10 regulate assembly of neural circuits. Neuron. PubMed ID: 30467079
Drosophila Dpr (21 paralogs) and DIP proteins (11 paralogs) are cell recognition molecules of the immunoglobulin superfamily (IgSF) that form a complex protein interaction network. DIP and Dpr proteins are expressed in a synaptic layer-specific fashion in the visual system. How interactions between these proteins regulate layer-specific synaptic circuitry is not known. This study establishes that DIP-alpha and its interacting partners Dpr6 and Dpr10 regulate multiple processes, including arborization within layers, synapse number, layer specificity, and cell survival. Heterophilic binding between Dpr6/10 and DIP-alpha and homophilic binding between DIP-alpha proteins promote interactions between processes in vivo. Knockin mutants disrupting the DIP/Dpr binding interface reveal a role for these proteins during normal development, while ectopic expression studies support an instructive role for interactions between DIPs and Dprs in circuit development. These studies support an important role for the DIP/Dpr protein interaction network in regulating cell-type-specific connectivity patterns.
Salazar-Gatzimas, E., Agrochao, M., Fitzgerald, J. E. and Clark, D. A. (2018). The neuronal basis of an illusory motion percept is explained by decorrelation of parallel motion pathways. Curr Biol 28(23): 3748-3762.e3748. PubMed ID: 30471993
Both vertebrates and invertebrates perceive illusory motion, known as "reverse-phi," in visual stimuli that contain sequential luminance increments and decrements. However, increment (ON) and decrement (OFF) signals are initially processed by separate visual neurons, and parallel elementary motion detectors downstream respond selectively to the motion of light or dark edges, often termed ON- and OFF-edges. It remains unknown how and where ON and OFF signals combine to generate reverse-phi motion signals. This study shows that each of Drosophila's elementary motion detectors encodes motion by combining both ON and OFF signals. Their pattern of responses reflects combinations of increments and decrements that co-occur in natural motion, serving to decorrelate their outputs. These results suggest that the general principle of signal decorrelation drives the functional specialization of parallel motion detection channels, including their selectivity for moving light or dark edges.

Tuesday, January 15th - Signaling

Nagy, A., Kovacs, L., Lipinszki, Z., Pal, M. and Deak, P. (2018). Developmental and tissue specific changes of ubiquitin forms in Drosophila melanogaster. PLoS One 13(12): e0209080. PubMed ID: 30543682
In most Eukaryotes, ubiquitin either exists as free monoubiquitin or as a molecule that is covalently linked to other proteins. These two forms cycle between each other and due to the concerted antagonistic activity of ubiquitylating and deubiquitylating enzymes, an intracellular ubiquitin equilibrium is maintained that is essential for normal biological function. However, measuring the level and ratio of these forms of ubiquitin has been difficult and time consuming. This paper has adapted a simple immunoblotting technique to monitor ubiquitin content and equilibrium dynamics in different developmental stages and tissues of Drosophila. The data show that the level of total ubiquitin is distinct in different developmental stages, lowest at the larval-pupal transition and in three days old adult males, and highest in first instar larvae. Interestingly, the ratio of free mono-ubiquitin remains within 30-50% range of the total throughout larval development, but peaks to 70-80% at the larval-pupal and the pupal-adult transitions. It stays within the 70-80% range in adults. In developmentally and physiologically active tissues, the ratio of free ubiquitin is similarly high, most likely reflecting a high demand for ubiquitin availability. This method was used to demonstrate the disruption of the finely tuned ubiquitin equilibrium by the abolition of proteasome function or the housekeeping deubiquitylase, Usp5. These data support the notion that the ubiquitin equilibrium is regulated by tissue- and developmental stage-specific mechanisms.
Matsui, Y., Zhang, Y., Paulson, R. F. and Lai, Z. C. (2018). Dual role of a C-terminally truncated isoform of large tumor suppressor kinase 1 in the regulation of Hippo signaling and tissue growth. DNA Cell Biol. PubMed ID: 30461308
The considerable amount of experimental evidence has defined the Hippo pathway as a tumor suppressive pathway and increased expression and/or activity of its oncogenic effectors is frequently observed in cancer. However, clinical studies have failed to attribute cancer development and progression to mutations in the pathway. In explaining this conundrum, this study investigated the expression and functions of a C-terminally truncated isoform of large tumor suppressor kinase 1 (LATS1) called short LATS1 (sLATS1) in human cell lines and Drosophila. Intriguingly, through overexpression of sLATS1, it was demonstrated that sLATS1 either activates or suppresses the activity of Yes-associated protein (YAP; see Drosophila Yorkie), one of the effectors of the Hippo pathway, in a cell type-specific manner. The activation is mediated through inhibition of full-length LATS1, whereas suppression of YAP is accomplished through sLATS1-YAP interaction. In HEK293T cells, the former mechanism may affect the cellular response more dominantly, whereas in U2OS cells and developing tissues in Drosophila, the latter mechanism may be solely carried out. Finally, to find the clinical relevance of this molecule, the expression of sLATS1 was examined in breast cancer patients. The transcriptome analysis showed that the ratio of sLATS1 to LATS1 was increased in tumor tissues comparing to their adjacent normal tissues.
Li, Y., Liu, T. and Zhang, J. (2018). The ATPase TER94 regulates Notch signaling during Drosophila wing development. Biol Open. PubMed ID: 30530809
The evolutionarily conserved Notch signaling pathway plays crucial roles in various developmental contexts. Multiple mechanisms are involved in regulation of the Notch pathway activity. Identified through a genetic mosaic screen, this study shows that the ATPase TER94 acts as a positive regulator of Notch signaling during Drosophila wing development. Depletion of TER94 causes marginal notches in the adult wing and reduction of Notch target genes wingless and cut during wing margin formation. Evidence is provided that TER94 is likely required for proper Notch protein localization and activation. Furthermore, knockdown of the TER94 adaptor dNpl4 leads to similar Notch signaling defects. Although the TER94 complex is implicated in various cellular processes, its role in the regulation of Notch pathways was previously uncharacterized. This study demonstrates that TER94 positively regulates Notch signaling, thus reveals a novel role of TER94 in development.
Kanoh, H., Kato, H., Suda, Y., Hori, A., Kurata, S. and Kuraishi, T. (2018). Dual comprehensive approach to decipher the Drosophila Toll pathway, ex vivo RNAi screenings and immunoprecipitation-mass spectrometry. Biochem Biophys Res Commun. PubMed ID: 30497778
The Drosophila Toll pathway is involved in embryonic development, innate immunity, and cell-cell interactions. However, compared to the mammalian Toll-like receptor innate immune pathway, its intracellular signaling mechanisms are not fully understood. A series of ex vivo genome-wide RNAi screenings was performed to identify genes required for the activation of the Toll pathway. This study has conducted an additional genome-wide RNAi screening using the overexpression of Tube, an adapter molecule in the Toll pathway, and has performed a co-immunoprecipitation assay to identify components present in the dMyd88-Tube complex. Based on the results of these assays, a bioinformatic analysis was performed, and describe candidate molecules and post-translational modifications that could be involved in Drosophila Toll signaling.
Poon, C. L. C., Liu, W., Song, Y., Gomez, M., Kulaberoglu, Y., Zhang, X., Xu, W., Veraksa, A., Hergovich, A., Ghabrial, A. and Harvey, K. F. (2018). A Hippo-like signaling pathway controls tracheal morphogenesis in Drosophila melanogaster. Dev Cell 47(5): 564-575.e565. PubMed ID: 30458981
Hippo-like pathways are ancient signaling modules first identified in yeasts. The best-defined metazoan module forms the core of the Hippo pathway, which regulates organ size and cell fate. Hippo-like kinase modules consist of a Sterile 20-like kinase, an NDR kinase, and non-catalytic protein scaffolds. In the Hippo pathway, the upstream kinase Hippo can be activated by another kinase, Tao-1. This study delineate a related Hippo-like signaling module that Tao-1 regulates to control tracheal morphogenesis in Drosophila melanogaster. Tao-1 activates the Sterile 20-like kinase GckIII by phosphorylating its activation loop, a mode of regulation that is conserved in humans. Tao-1 and GckIII act upstream of the NDR kinase Tricornered to ensure proper tube formation in trachea. This study reveals that Tao-1 activates two related kinase modules to control both growth and morphogenesis. The Hippo-like signaling pathway delineated by this study has a potential role in the human vascular disease cerebral cavernous malformation.
Vuong, L. T., Iomini, C., Balmer, S., Esposito, D., Aaronson, S. A. and Mlodzik, M. (2018). Kinesin-2 and IFT-A act as a complex promoting nuclear localization of beta-catenin during Wnt signalling. Nat Commun 9(1): 5304. PubMed ID: 30546012
Wnt/Wg-signalling is critical signalling in all metazoans. Recent studies suggest that IFT-A proteins and Kinesin-2 modulate canonical Wnt/Wg-signalling independently of their ciliary role. Whether they function together in Wnt-signalling and their mechanistic role in the pathway remained unresolved. This study demonstrated that Kinesin-2 and IFT-A proteins act as a complex during Drosophila Wg-signalling, affecting pathway activity in the same manner, interacting genetically and physically, and co-localizing with beta-catenin, the mediator of Wnt/Wg-signalling on microtubules. Following pathway activation, Kinesin-2/IFT-A mutant cells exhibit high cytoplasmic beta-catenin levels, yet fail to activate Wg-targets. In mutant tissues in both, Drosophila and mouse/MEFs, nuclear localization of beta-catenin is markedly reduced. A conserved, motor-domain dependent function of the Kinesin-2/IFT-A complex is demonstrated in promoting nuclear translocation of beta-catenin. This is mediated by protecting beta-catenin from a conserved cytoplasmic retention process, thus identifying a mechanism for Kinesin-2/IFT-A in Wnt-signalling that is independent of their ciliary role.

Monday, January 14th - Transcriptional Regulation

Vuilleumier, R., Lian, T., Flibotte, S., Khan, Z. N., Fuchs, A., Pyrowolakis, G. and Allan, D. W. (2018). Retrograde BMP signaling activates neuronal gene expression through widespread deployment of a conserved BMP-responsive cis-regulatory activation element. Nucleic Acids Res. PubMed ID: 30476189
Retrograde Bone Morphogenetic Protein (BMP) signaling in neurons is essential for the differentiation and synaptic function of many neuronal subtypes. BMP signaling regulates these processes via Smad transcription factor activity, yet the scope and nature of Smad-dependent gene regulation in neurons are mostly unknown. This study applied a computational approach to predict Smad-binding cis-regulatory BMP-Activating Elements (BMP-AEs) in Drosophila, followed by transgenic in vivo reporter analysis to test their neuronal subtype enhancer activity in the larval central nervous system (CNS). 34 BMP-AE-containing genomic fragments were identifed that are responsive to BMP signaling in neurons; the embedded BMP-AEs are required for this activity. RNA-seq analysis identified BMP-responsive genes in the CNS and revealed that BMP-AEs selectively enrich near BMP-activated genes. These data suggest that functional BMP-AEs control nearby BMP-activated genes, which were validated experimentally. Finally, it was demonstrated that the BMP-AE motif mediates a conserved Smad-responsive function in the Drosophila and vertebrate CNS. The results provide evidence that BMP signaling controls neuronal function by directly coordinating the expression of a battery of genes through widespread deployment of a conserved Smad-responsive cis-regulatory motif.
Vizcaya-Molina, E., Klein, C. C., Serras, F., Mishra, R. K., Guigo, R. and Corominas, M. (2018). Damage-responsive elements in Drosophila regeneration. Genome Res 28(12): 1852-1866. PubMed ID: 30459214
One of the most important questions in regenerative biology is to unveil how and when genes change expression and trigger regeneration programs. The resetting of gene expression patterns during response to injury is governed by coordinated actions of genomic regions that control the activity of multiple sequence-specific DNA binding proteins. Using genome-wide approaches to interrogate chromatin function, this study identified the elements that regulate tissue recovery in Drosophila imaginal discs, which show a high regenerative capacity after genetically induced cell death. These findings indicate there is global coregulation of gene expression as well as a regeneration program driven by different types of regulatory elements. Novel enhancers acting exclusively within damaged tissue cooperate with enhancers co-opted from other tissues and other developmental stages, as well as with endogenous enhancers that show increased activity after injury. Together, these enhancers host binding sites for regulatory proteins that include a core set of conserved transcription factors that control regeneration across metazoans.
Albig, C., Tikhonova, E., Krause, S., Maksimenko, O., Regnard, C. and Becker, P. B. (2018). Factor cooperation for chromosome discrimination in Drosophila. Nucleic Acids Res. PubMed ID: 30541149
Transcription regulators select their genomic binding sites from a large pool of similar, non-functional sequences. Although general principles that allow such discrimination are known, the complexity of DNA elements often precludes a prediction of functional sites. The process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex (DCC) selectively binds to some 300 X chromosomal 'High Affinity Sites' (HAS) containing GA-rich 'MSL recognition elements' (MREs), but disregards thousands of other MRE sequences in the genome. The DNA-binding subunit MSL2 alone identifies a subset of MREs, but fails to recognize most MREs within HAS. The 'Chromatin-linked adaptor for MSL proteins' (CLAMP) also interacts with many MREs genome-wide and promotes DCC binding to HAS. Using genome-wide DNA-immunoprecipitation extensive cooperativity id described between both factors, depending on the nature of the binding sites. These are explained by physical interaction between MSL2 and CLAMP. In vivo, both factors cooperate to compete with nucleosome formation at HAS. The male-specific MSL2 thus synergises with a ubiquitous GA-repeat binding protein for refined X/autosome discrimination.
Dufourt, J., Trullo, A., Hunter, J., Fernandez, C., Lazaro, J., Dejean, M., Morales, L., Nait-Amer, S., Schulz, K. N., Harrison, M. M., Favard, C., Radulescu, O. and Lagha, M. (2018). Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs. Nat Commun 9(1): 5194. PubMed ID: 30518940
Pioneer transcription factors can engage nucleosomal DNA, which leads to local chromatin remodeling and to the establishment of transcriptional competence. However, the impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains unclear. This study employs quantitative live imaging methods and mathematical modeling to test the effect of the pioneer factor Zelda on transcriptional dynamics and memory in Drosophila embryos. Increasing the number of Zelda binding sites accelerates the kinetics of nuclei transcriptional activation regardless of their transcriptional past. Despite its known pioneering activities, Zelda does not remain detectably associated with mitotic chromosomes and is neither necessary nor sufficient to foster memory.It was further revealed that Zelda forms sub-nuclear dynamic hubs where Zelda binding events are transient. It is proposed that Zelda facilitates transcriptional activation by accumulating in microenvironments where it could accelerate the duration of multiple pre-initiation steps.
Zoller, B., Little, S. C. and Gregor, T. (2018). Diverse spatial expression patterns emerge from unified kinetics of transcriptional bursting. Cell 175(3): 835-847. PubMed ID: 30340044
How transcriptional bursting relates to gene regulation is a central question that has persisted for more than a decade. This study measured nascent transcriptional activity in early Drosophila embryos and characterize the variability in absolute activity levels across expression boundaries. Boundary formation was demonstrated to follow a common transcription principle: a single control parameter determines the distribution of transcriptional activity, regardless of gene identity, boundary position, or enhancer-promoter architecture. The underlying bursting kinetics were inferred and the key regulatory parameter was identified as the fraction of time a gene is in a transcriptionally active state. Unexpectedly, both the rate of polymerase initiation and the switching rates for bcd, hb, Kr, kni and gt and are tightly constrained across all expression levels, predicting synchronous patterning outcomes at all positions in the embryo. These results point to a shared simplicity underlying the apparently complex transcriptional processes of early embryonic patterning and indicate a path to general rules in transcriptional regulation.

Friday, January 11th - Disease Models

Pons, M., Prieto, S., Miguel, L., Frebourg, T., Campion, D., Sune, C. and Lecourtois, M. (2018). Identification of TCERG1 as a new genetic modulator of TDP-43 production in Drosophila. Acta Neuropathol Commun 6(1): 138. PubMed ID: 30541625
TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed DNA-/RNA-binding protein that has been linked to numerous aspects of the mRNA life cycle. Similar to many RNA-binding proteins, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Cell function and survival depend on the strict control of TDP-43 protein levels. TDP-43 has been identified as the major constituent of ubiquitin-positive inclusions in patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several observations argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulation of the cycle of TDP-43 production might therefore provide a new therapeutic strategy. Using a Drosophila model mimicking key features of the TDP-43 autoregulatory feedback loop, this study identified CG42724 as a genetic modulator of TDP-43 production in vivo. CG42724 protein was found to influence qualitatively and quantitatively the TDP-43 mRNA transcript pattern. CG42724 overexpression promotes the production of transcripts that can be efficiently released into the cytoplasm for protein translation. Importantly, TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, these data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.
Maor, G., Rapaport, D., Horowitz, M. (2019).. The effect of mutant GBA1 on accumulation and aggregation of alpha-synuclein. Hum Mol Genet. [Epub ahead of print]. PubMed ID: 30615125
Gaucher disease (GD) patients and carriers of GD mutations have a higher propensity to develop Parkinson disease (PD) in comparison to the non-GD population. This implies that mutant GBA1 allele is a predisposing factor for the development of PD. One of the major characteristics of PD is the presence of oligomeric α-synuclein-positive inclusions known as Lewy bodies in the dopaminergic neurons localized to the substantia nigra pars compacta. This study tested whether presence of human mutant GCase leads to accumulation and aggregation of α-synuclein in two models: in SHSY5Y neuroblastoma cells endogenously expressing α-synuclein and stably transfected with human GCase variants, and in Drosophila melanogaster co-expressing normal human α-synuclein and mutant human GCase. The results showed that heterologous expression of mutant, but not WT, human GCase in SHSY5Y cells, led to a significant stabilization of α-synuclein and to its aggregation. In parallel, there was also a significant stabilization of mutant, but not WT, GCase. Co-expression of human α-synuclein and human mutant GCase in the dopaminergic cells of flies initiated α-synuclein aggregation, earlier death of these cells and significantly shorter life span, compared to flies expressing α-synuclein or mutant GCase alone. Taken together, these results strongly indicate that human mutant GCase contributes to accumulation and aggregation of α-synuclein. In the fly, this aggregation leads to development of more severe parkinsonian signs in comparison to flies expressing either mutant GCase or α-synuclein alone.
Flintham, E. O., Yoshida, T., Smith, S., Pavlou, H. J., Goodwin, S. F., Carazo, P. and Wigby, S. (2018). Interactions between the sexual identity of the nervous system and the social environment mediate lifespan in Drosophila melanogaster. Proc Biol Sci 285(1892). PubMed ID: 30487307
Sex differences in lifespan are ubiquitous, but the underlying causal factors remain poorly understood. Inter- and intrasexual social interactions are well known to influence lifespan in many taxa, but it has proved challenging to separate the role of sex-specific behaviours from wider physiological differences between the sexes. To address this problem, the sexual identity of the nervous system - and hence sexual behaviour - was genetically manipulated in Drosophila melanogaster, and lifespan was measured under varying social conditions. Consistent with previous studies, masculinization of the nervous system in females induced male-specific courtship behaviour and aggression, while nervous system feminization in males induced male-male courtship and reduced aggression. Control females outlived males, but masculinized female groups displayed male-like lifespans and male-like costs of group living. By varying the mixture of control and masculinized females within social groups, male-specific behaviours were shown to be costly to recipients, even when received from females. However, consistent with recent findings, the data suggest courtship expression to be surprisingly low cost. Overall, this study indicates that nervous system-mediated expression of sex-specific behaviour per se-independent of wider physiological differences between the sexes, or the receipt of aggression or courtship-plays a limited role in mediating sex differences in lifespan.
Halperin, D. et al. (2018). SEC31A mutation affects ER homeostasis, causing neurological syndrome. J Med Genet. PubMed ID: 30464055
Consanguineous kindred presented with an autosomal recessive syndrome of intrauterine growth retardation, marked developmental delay, spastic quadriplegia with profound contractures, pseudobulbar palsy with recurrent aspirations, epilepsy, dysmorphism, neurosensory deafness and optic nerve atrophy with no eye fixation. This study aimed at elucidating the molecular basis of this disease. Genome-wide linkage analysis combined with whole exome sequencing were performed to identify disease-causing variants. Functional consequences were investigated in fruit flies null mutant for the Drosophila SEC31A orthologue. SEC31A knockout SH-SY5Y and HEK293T cell-lines were generated using CRISPR/Cas9 and studied through qRT-PCR, immunoblotting and viability assays. Through genetic studies, a disease-associated homozygous nonsense mutation in SEC31A was identified. SEC31A was shown to be ubiquitously expressed, and the mutation triggers nonsense-mediated decay of its transcript, comprising a practical null mutation. Similar to the human disease phenotype, knockdown SEC31A flies had defective brains and early lethality. Moreover, in line with SEC31A encoding one of the two coating layers comprising the Coat protein complex II (COP-II) complex, trafficking newly synthesised proteins from the endoplasmic reticulum (ER) to the Golgi, CRISPR/Cas9-mediated SEC31A null mutant cells demonstrated reduced viability through upregulation of ER-stress pathways. This study demonstrated through human and Drosophila genetic and in vitro molecular studies, that a severe neurological syndrome is caused by a null mutation in SEC31A, reducing cell viability through enhanced ER-stress response, in line with SEC31A's role in the COP-II complex.
Kadas, D., Papanikolopoulou, K., Xirou, S., Consoulas, C. and Skoulakis, E. M. C. (2018). Human Tau isoform-specific presynaptic deficits in a Drosophila central nervous system circuit. Neurobiol Dis 124: 311-321. PubMed ID: 30529489
Accumulation of normal or mutant human Tau isoforms (see Drosophila Tau) in Central Nervous System (CNS) neurons of vertebrate and invertebrate models underlies pathologies ranging from behavioral deficits to neurodegeneration that broadly recapitulate human Tauopathies. Although some functional differences have begun to emerge, it is still largely unclear whether normal and mutant Tau isoforms induce differential effects on the synaptic physiology of CNS neurons. This study used the oligosynaptic Giant Fiber System in the adult Drosophila CNS to address this question and revealed that 3R and 4R isoforms affect distinct synaptic parameters. Whereas 0N3R increased failure rate upon high frequency stimulation, 0N4R compromised stimulus conduction and response speed at a specific cholinergic synapse in an age-dependent manner. In contrast, accumulation of the R406W mutant of 0N4R induced mild, age-dependent conduction velocity defects. Because 0N4R and its mutant isoform are expressed equivalently, this demonstrates that the defects are not merely consequent of exogenous human Tau accumulation and suggests distinct functional properties of 3R and 4R isoforms in cholinergic presynapses.
Petruccelli, E., Feyder, M., Ledru, N., Jaques, Y., Anderson, E. and Kaun, K. R. (2018). Alcohol activates Scabrous-Notch to influence associated memories. Neuron 100(5): 1209-1223 PubMed ID: 30482693
Drugs of abuse, like alcohol, modulate gene expression in reward circuits and consequently alter behavior. However, the in vivo cellular mechanisms through which alcohol induces lasting transcriptional changes are unclear. This study shows that Drosophila Notch/Su(H) signaling and the secreted fibrinogen-related protein Scabrous in mushroom body (MB) memory circuitry are important for the enduring preference of cues associated with alcohol's rewarding properties. Alcohol exposure affects Notch responsivity in the adult MB and alters Su(H) targeting at the dopamine-2-like receptor (Dop2R). Alcohol cue training also caused lasting changes to the MB nuclear transcriptome, including changes in the alternative splicing of Dop2R and newly implicated transcripts like Stat92E. Together, these data suggest that alcohol-induced activation of the highly conserved Notch pathway and accompanying transcriptional responses in memory circuitry contribute to addiction. Ultimately, this provides mechanistic insight into the etiology and pathophysiology of alcohol use disorder.

Thursday, January 1Oth - Behavior

Busch, C., Borst, A. and Mauss, A. S. (2018). Bi-directional control of walking behavior by horizontal optic flow sensors. Curr Biol. PubMed ID: 30528583
Moving animals experience constant sensory feedback, such as panoramic image shifts on the retina, termed optic flow. Underlying neuronal signals are thought to be important for exploratory behavior by signaling unintended course deviations and by providing spatial information about the environment. Particularly in insects, the encoding of self-motion-related optic flow is well understood. However, a gap remains in understanding how the associated neuronal activity controls locomotor trajectories. In flies, visual projection neurons belonging to two groups encode panoramic horizontal motion: horizontal system (HS) cells respond with depolarization to front-to-back motion and hyperpolarization to the opposite direction, and other neurons have the mirror-symmetrical response profile. With primarily monocular sensitivity, the neurons' responses are ambiguous for different rotational and translational self-movement components. Such ambiguities can be greatly reduced by combining signals from both eyes to determine turning and movement speed. This study explores the underlying functional logic by optogenetic HS cell manipulation in tethered walking Drosophila. De- and hyperpolarization were shown to evoke opposite turning behavior, indicating that both direction-selective signals are transmitted to descending pathways for course control. Further experiments reveal a negative effect of bilaterally symmetric de- and hyperpolarization on walking velocity. The results are therefore consistent with a functional architecture in which the HS cells' membrane potential influences walking behavior bi-directionally via two decelerating pathways.
Brockmann, A., Basu, P., Shakeel, M., Murata, S., Murashima, N., Boyapati, R. K., Prabhu, N. G., Herman, J. J. and Tanimura, T. (2018). Sugar intake elicits intelligent searching behavior in flies and honey bees. Front Behav Neurosci 12: 280. PubMed ID: 30546299
This study presents a comparison of the sugar-elicited search behavior in Drosophila melanogaster and Apis mellifera. In both species, intake of sugar-water elicits a complex of searching responses. The most obvious response was an increase in turning frequency. However, it was also found that flies and honey bees returned to the location of the sugar drop. They even returned to the food location when they were prevented from using visual and chemosensory cues. Analyses of the recorded trajectories indicated that flies and bees use two mechanisms, a locomotor pattern involving an increased turning frequency and path integration to increase the probability to stay close or even return to the sugar drop location. However, evidence for the use of path integration in honey bees was less clear. In general, walking trajectories of honey bees showed a higher degree of curvature and were more spacious; two characters which likely masked evidence for the use of path integration in these experiments. Visual cues, i.e., a black dot, presented underneath the sugar drop made flies and honey bees stay closer to the starting point of the search. In honey bees, vertical black columns close to the sugar drop increased the probability to visit similar cues in the vicinity. An additional one trial learning experiment suggested that the intake of sugar-water likely has the potential to initiate an associative learning process. Together, these experiments indicate that the sugar-elicited local search is more complex than previously assumed. Most importantly, this local search behavior appeared to exhibit major behavioral capabilities of large-scale navigation. Thus, it is proposed that sugar-elicited search behavior has the potential to become a fruitful behavioral paradigm to identify neural and molecular mechanisms involved in general mechanisms of navigation.
van Breugel, F., Huda, A. and Dickinson, M. H. (2018). Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila. Nature. PubMed ID: 30464346
Carbon dioxide is produced by many organic processes and is a convenient volatile cue for insects that are searching for blood hosts, flowers, communal nests, fruit and wildfires. Although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments suggest that walking flies avoid CO2. This study resolved this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when they are in an active state associated with foraging. Their aversion to CO2 at low-activity levels may be an adaptation to avoid parasites that seek CO2, or to avoid succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature. In contrast to CO2, flies are attracted to ethanol in all behavioural states, and invest twice the time searching near ethanol compared to CO2. These behavioural differences reflect the fact that ethanol is a unique signature of yeast fermentation, whereas CO2 is generated by many natural processes. Using genetic tools, it was determined that the evolutionarily conserved ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved in this attraction. This study lays the foundation for future research to determine the neural circuits that underlie both state- and odorant-dependent decision-making in Drosophila.
Mansourian, S., Enjin, A., Jirle, E. V., Ramesh, V., Rehermann, G., Becher, P. G., Pool, J. E. and Stensmyr, M. C. (2018). Wild african Drosophila melanogaster are seasonal specialists on marula fruit. Curr Biol. PubMed ID: 30528579
Although the vinegar fly Drosophila melanogaster is arguably the most studied organism on the planet, fundamental aspects of this species' natural ecology have remained enigmatic. This study has investigated a wild population of D. melanogaster from a mopane forest in Zimbabwe. These flies are closely associated with marula fruit (Sclerocarya birrea), and it is proposed that this seasonally abundant and predominantly Southern African fruit is a key ancestral host of D. melanogaster. Moreover, when fruiting, marula is nearly exclusively used by D. melanogaster, suggesting that these forest-dwelling D. melanogaster are seasonal specialists, in a similar manner to, e.g., Drosophila erecta on screw pine cones. It was further demonstrated that the main chemicals released by marula activate odorant receptors that mediate species-specific host choice (Or22a) and oviposition site selection (Or19a). The Or22a-expressing neurons-ab3A-respond strongly to the marula ester ethyl isovalerate, a volatile rarely encountered in high amounts in other fruit. Or22a differs among African populations sampled from a wide range of habitats, in line with a function associated with host fruit usage. Flies from Southern Africa, most of which carry a distinct allele at the Or22a/Or22b locus, have ab3A neurons that are more sensitive to ethyl isovalerate than, e.g., European flies. Finally, the possibility is discussed that marula, which is also a culturally and nutritionally important resource to humans, may have helped the transition to commensalism in D. melanogaster.
Artiushin, G., Zhang, S. L., Tricoire, H. and Sehgal, A. (2018). Endocytosis at the Drosophila blood-brain barrier as a function for sleep. Elife 7. PubMed ID: 30475209
Glia are important modulators of neural activity, yet few studies link glia to sleep regulation. This study found that blocking activity of the endocytosis protein, dynamin, in adult Drosophila glia increases sleep and enhances sleep need, manifest as resistance to sleep deprivation. Surface glia comprising the fly equivalent of the blood-brain barrier (BBB) mediate the effect of dynamin on sleep. Blocking dynamin in the surface glia causes ultrastructural changes, albeit without compromising the integrity of the barrier. Supporting a role for endocytic trafficking in sleep, a screen of Rab GTPases identifies sleep-modulating effects of the recycling endosome Rab11 in surface glia. It was also found that endocytosis is increased in BBB glia during sleep and reflects sleep need. It is proposed that endocytic trafficking through the BBB represents a function of sleep.
Tastekin, I., Khandelwal, A., Tadres, D., Fessner, N. D., Truman, J. W., Zlatic, M., Cardona, A. and Louis, M. (2018). Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva. Elife 7. PubMed ID: 30465650
Sensory navigation results from coordinated transitions between distinct behavioral programs. During chemotaxis in the Drosophila melanogaster larva, the detection of positive odor gradients extends runs while negative gradients promote stops and turns. This algorithm represents a foundation for the control of sensory navigation across phyla. The present work identified an olfactory descending neuron, PDM-DN, which plays a pivotal role in the organization of stops and turns in response to the detection of graded changes in odor concentrations. Artificial activation of this descending neuron induces deterministic stops followed by the initiation of turning maneuvers through head casts. Using electron microscopy, the main pathway was reconstructed that connects the PDM-DN neuron to the peripheral olfactory system and to the pre-motor circuit responsible for the actuation of forward peristalsis. The results set the stage for a detailed mechanistic analysis of the sensorimotor conversion of graded olfactory inputs into action selection to perform goal-oriented navigation.

Wednesday, January 9th - RNA and Transposons

Kirsch, R., Seemann, S. E., Ruzzo, W. L., Cohen, S. M., Stadler, P. F. and Gorodkin, J. (2018). Identification and characterization of novel conserved RNA structures in Drosophila. BMC Genomics 19(1): 899. PubMed ID: 30537930
Comparative genomics approaches have facilitated the discovery of many novel non-coding and structured RNAs (ncRNAs). Earlier work identifying ncRNAs in Drosophila melanogaster made use of sequence-based alignments and employed a sliding window approach, inevitably biasing identification toward RNAs encoded in the more conserved parts of the genome. To search for conserved RNA structures (CRSs) that may not be highly conserved in sequence and to assess the expression of CRSs, a genome-wide structural alignment screen of 27 insect genomes including D. melanogaster was conducted, and this was integrated with an extensive set of tiling array data. The structural alignment screen revealed approximately 30,000 novel candidate CRSs at an estimated false discovery rate of less than 10%. With more than one quarter of all individual CRS motifs showing sequence identities below 60%, the predicted CRSs largely complement the findings of sliding window approaches applied previously. While a sixth of the CRSs were ubiquitously expressed, most were expressed in specific developmental stages or cell lines. Notably, most statistically significant enrichment of CRSs were observed in pupae, mainly in exons of untranslated regions, promotors, enhancers, and long ncRNAs. Interestingly, cell lines were found to express a different set of CRSs than were found in vivo. Only a small fraction of intergenic CRSs were co-expressed with the adjacent protein coding genes, which suggests that most intergenic CRSs are independent genetic units. This study provides a more comprehensive view of the ncRNA transcriptome in fly as well as evidence for differential expression of CRSs during development and in cell lines.
Lerat, E., Goubert, C., Guirao-Rico, S., Merenciano, M., Dufour, A. B., Vieira, C. and Gonzalez, J. (2018). Population specific dynamics and selection patterns of transposable element insertions in European natural populations. Mol Ecol. PubMed ID: 30506554
Transposable elements (TEs) are ubiquitous sequences in genomes of virtually all species. While TEs have been investigated for several decades, only recently has the opportunity appeared to study their genome-wide population dynamics. Most of the studies so far have been restricted either to the analysis of the insertions annotated in the reference genome or to the analysis of a limited number of populations. Taking advantage of the European Drosophila population genomics consortium (DrosEU) sequencing dataset, this study has identified and measured the dynamics of TEs in a large sample of European Drosophila melanogaster natural populations. The mobilome landscape is population specific and highly diverse depending on the TE family. In contrast with previous studies based on SNP variants, no geographical structure was observed for TE abundance or TE divergence in European populations. De novo individual insertions were also identified using two available programs and, as expected, most of the insertions were present at low frequencies. Nevertheless, a subset of TEs present at high frequencies was identified and located in genomic regions with a high recombination rate. These TEs are candidates for being the target of positive selection, although neutral processes should be discarded before reaching any conclusion on the type of selection acting on them. Finally, parallel patterns of association between the frequency of TE insertions and several geographical and temporal variables were found between European and North American populations, suggesting that TEs can be potentially implicated in the adaptation of populations across continents.
Mikhaleva, E. A., Leinsoo, T. A., Ishizu, H., Gvozdev, V. A. and Klenov, M. S. (2018). The nucleolar transcriptome regulates Piwi shuttling between the nucleolus and the nucleoplasm. Chromosome Res. PubMed ID: 30539407
The nucleolus contains a lot of proteins unrelated to ribosome biogenesis. Some of these proteins shuttle between the nucleolus and the nucleoplasm regulating the cell cycle and stress response. The piRNA binding protein Piwi is involved in silencing of transposable elements (TEs) in the Drosophila gonads. This study used cultured ovarian somatic cells (OSC) to characterize Piwi as a visitor to the nucleolus. Dynamic Piwi localization was shown to vary from its uniform distribution between the nucleoplasm and the nucleolus to pronounced nucleolar immobilization. This localization behavior was intriguing, and nascent nucleolar transcripts recruit Piwi for nucleolar retention were revealed. Piwi eviction from the nucleolus was observed upon RNase treatment and after RNA polymerase (Pol) I inhibition, but not after Pol II inactivation. On the contrary, heat shock caused drastic Piwi redistribution from the nucleoplasm to the nucleolus, which occurred only in the presence of Pol I-mediated transcription. These results led to a hypothesis that specific stress-induced transcripts made by Pol I promote the nucleolar sequestration of proteins in Drosophila, similar to previous observations in mammalian cells. It was also found that in OSC, Piwi partially restricts expression of the rDNA copies containing R1 and R2 retrotransposon insertions especially upon heat shock-induced activation of these copies. Therefore, it is suggested that Piwi intranuclear shuttling may have a functional role in ensuring a balance between silencing of rDNA-specific TEs under stress and the canonical Piwi function in non-nucleolar TE repression.
Kim, M., Faucillion, M. L. and Larsson, J. (2018). RNA-on-X 1 and 2 in Drosophila melanogaster fulfill separate functions in dosage compensation. PLoS Genet 14(12): e1007842. PubMed ID: 30532158
In Drosophila melanogaster, the male-specific lethal (MSL) complex plays a key role in dosage compensation by stimulating expression of male X-chromosome genes. It consists of MSL proteins and two long noncoding RNAs, roX1 and roX2, that are required for spreading of the complex on the chromosome and are redundant in the sense that loss of either does not affect male viability. However, despite rapid evolution, both roX species are present in diverse Drosophilidae species, raising doubts about their full functional redundancy. Thus, this study investigated consequences of deleting roX1 and/or roX2 to probe their specific roles and redundancies in D. melanogaster. Anew mutant allele of roX2 was created, and roX1 and roX2 were shown to have partly separable functions in dosage compensation. In larvae, roX1 is the most abundant variant and the only variant present in the MSL complex when the complex is transmitted (physically associated with the X-chromosome) in mitosis. Loss of roX1 results in reduced expression of the genes on the X-chromosome, while loss of roX2 leads to MSL-independent upregulation of genes with male-biased testis-specific transcription. In roX1 roX2 mutant, gene expression is strongly reduced in a manner that is not related to proximity to high-affinity sites. These results suggest that high tolerance of mis-expression of the X-chromosome has evolved. It is proposed that this may be a common property of sex-chromosomes, that dosage compensation is a stochastic process and its precision for each individual gene is regulated by the density of high-affinity sites in the locus.
Kobayashi, H., Shoji, K., Kiyokawa, K., Negishi, L. and Tomari, Y. (2018). Iruka eliminates dysfunctional Argonaute by selective ubiquitination of its empty state. Mol Cell. PubMed ID: 30503771
MicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear. This study discovered a RING-type E3 ubiquitin ligase named Iruka (Iru), which selectively ubiquitinates the empty form of Drosophila Ago1 to trigger its degradation. Iru preferentially binds empty Ago1 and ubiquitinates Lys514 in the L2 linker, which is predicted to be inaccessible in the miRNA-loaded state. Depletion of Iru results in global impairment of miRNA-mediated silencing of target genes and in the accumulation of aberrant Ago1 that is dysfunctional for canonical protein-protein interactions and miRNA loading. These findings reveal a sophisticated mechanism for the selective degradation of empty AGO that underlies a quality control process to ensure AGO function.
Pellacani, C., Bucciarelli, E., Renda, F., Hayward, D., Palena, A., Chen, J., Bonaccorsi, S., Wakefield, J. G., Gatti, M. and Somma, M. P. (2018). Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation. Elife 7. PubMed ID: 30475206
Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. This study shows that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. Both SFs were demonstrated to bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells.

Tuesday, January 8th - Synapses and Vesicles

Ou, M., Wang, S., Sun, M., An, J., Lv, H., Zeng, X., Hou, S. X. and Xie, W. (2018).. The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction. Exp Cell Res. PubMed ID: 30553967
Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. The study analyzed the role of Gef26 in synaptic development and function. Significant decreases were identified in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, the data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a betaPS integrin-mediated signaling pathway. Taken together, these findings define a novel role of Gef26 in regulating NMJ development and function.
Kerr, C. H., Dalwadi, U., Scott, N. E., Yip, C. K., Foster, L. J. and Jan, E. (2018). Transmission of Cricket paralysis virus via exosome-like vesicles during infection of Drosophila cells. Sci Rep 8(1): 17353. PubMed ID: 30478341
Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped viruses (e.g. hepatitis A virus) can acquire an envelope during infection by hijacking host cellular pathways. This study examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket paralysis virus (CrPV). Utilizing quantitative proteomics, it was demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the diverse strategies of nonenveloped viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission.
Lembke, K. M., Law, A. D., Ahrar, J. and Morton, D. B. (2018). Deletion of a specific exon in the voltage-gated calcium channel, cacophony, causes disrupted locomotion in Drosophila larvae. J Exp Biol. PubMed ID: 30397173
Tar DNA binding protein 43 (TDP-43) is an RNA binding protein that regulates transcription, translation, and alternative splicing of mRNA. Null mutations of the Drosophila orthologue, Tar DNA-binding homologue (tbph), has been shown to cause severe locomotion defects in larvae that are mediated by a reduction in the expression of the type II voltage-gated calcium channel, cacophony (cac). TDP-43 also regulates the inclusion of alternatively spliced exons of cacophony; tbph mutants showed significantly increased expression of cacophony isoforms lacking exon 7, a particularly notable finding as only one out of the 15 predicted isoforms lacks exon 7. To investigate the function of exon 7, Drosophila mutant lines were generated with a deletion that eliminates exon 7. This deletion phenocopies many defects in tbph mutants: a reduction in Cacophony protein expression, locomotion defects in male and female third instar larvae, disrupted larval motor output, and also reduced activity levels in adult male flies. All these defects were rescued by expression of cacophony transcripts containing exon 7. By contrast, expression of a cacophony cDNA lacking exon 7 resulted in reduced Cacophony protein levels and failed to rescue larval locomotion.
Phan, A., Thomas, C. I., Chakraborty, M., Berry, J. A., Kamasawa, N. and Davis, R. L. (2018). Stromalin constrains memory acquisition by developmentally limiting synaptic vesicle pool size. Neuron. PubMed ID: 30503644
Stromalin, a cohesin complex protein, was recently identified as a novel memory suppressor gene, but its mechanism remained unknown. This study shows that Stromalin functions as a negative regulator of synaptic vesicle (SV) pool size in Drosophila neurons. Stromalin knockdown in dopamine neurons during a critical developmental period enhances learning and increases SV pool size without altering the number of dopamine neurons, their axons, or synapses. The developmental effect of Stromalin knockdown persists into adulthood, leading to strengthened synaptic connections and enhanced olfactory memory acquisition in adult flies. Correcting the SV content in dopamine neuron axon terminals by impairing anterograde SV trafficking motor protein Unc104/KIF1A rescues the enhanced-learning phenotype in Stromalin knockdown flies. These results identify a new mechanism for memory suppression and reveal that the size of the SV pool is controlled genetically and independent from other aspects of neuron structure and function through Stromalin.
Saadin, A. and Starz-Gaiano, M. (2018). Cytokine exocytosis and JAK/STAT activation in the Drosophila ovary requires the vesicle trafficking regulator alpha-Snap. J Cell Sci. PubMed ID: 30404830
How vesicle trafficking components actively contribute to regulation of paracrine signaling is unclear. This study has genetically uncovered a requirement for alpha-Soluble NSF Attachment Protein (alpha-Snap) in the activation of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway during Drosophila egg development. alpha-Snap, a well-conserved vesicle trafficking regulator, mediates association of N-ethylmaleimide-Sensitive Factor (NSF) and SNAREs to promote vesicle fusion. Depletion of alpha-Snap or the SNARE family member Syntaxin1A in epithelia blocks polar cells maintenance and prevents specification of motile border cells. Blocking apoptosis rescues polar cell maintenance in alpha-Snap-depleted egg chambers, indicating that the lack of border cells in mutants is due to impaired signaling. Genetic experiments implicate alpha-Snap and NSF in secretion of a STAT-activating cytokine. Live imaging suggests that changes in intracellular calcium may be linked to this event. These data suggest a cell-type specific requirement for particular vesicle trafficking components in regulated exocytosis during development. Given the central role for STAT signaling in immunity, this work may shed light on regulation of cytokine release in humans.
Oswald, M. C. W., Brooks, P. S., Zwart, M. F., Mukherjee, A., West, R. J. H., Giachello, C. N. G., Morarach, K., Baines, R. A., Sweeney, S. T. and Landgraf, M. (2018). Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila. Elife 7. PubMed ID: 30540251
Reactive oxygen species (ROS) have been extensively studied as damaging agents associated with ageing and neurodegenerative conditions. Their role in the nervous system under non-pathological conditions has remained poorly understood. Working with the Drosophila larval locomotor network, this study showed that in neurons ROS act as obligate signals required for neuronal activity-dependent structural plasticity, of both pre- and postsynaptic terminals. ROS signaling is also necessary for maintaining evoked synaptic transmission at the neuromuscular junction, and for activity-regulated homeostatic adjustment of motor network output, as measured by larval crawling behavior. The highly conserved Parkinson's disease-linked protein DJ-1ss was identified as a redox sensor in neurons where it regulates structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. This study provides a new conceptual framework of neuronal ROS as second messengers required for neuronal plasticity and for network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction.
Bollinger, W. L., Sial, N. and Dawson-Scully, K. (2018). BK channels and a cGMP-dependent protein kinase (PKG) function through independent mechanisms to regulate the tolerance of synaptic transmission to acute oxidative stress at the Drosophila larval neuromuscular junction. J Neurogenet 32(3): 246-255. PubMed ID: 30484391
A cGMP-dependent protein kinase (PKG) has previously been shown to regulate synaptic transmission at the Drosophila neuromuscular junction (NMJ) during acute oxidative stress, potentially through modulation of downstream K(+) channel kinetics; however, the specific K(+) channels through which PKG functions remains unclear. It was hypothesized that PKG may be acting on calcium-activated large-conductance Slo K(+) channels, or BK channels. Genetic elimination and pharmacological inhibition of BK channel conductance increases synaptic transmission tolerance to acute H2O2-induced oxidative stress. Furthermore, it was discovered that activation of PKG in BK channel loss-of-function (Slo4) mutants significantly decreases time to stimulus-induced synaptic failure, providing the first evidence of PKG and BK channels functioning independently to control synaptic transmission tolerance to acute oxidative stress.
Prince, E., Kroeger, B., Gligorov, D., Wilson, C., Eaton, S., Karch, F., Brankatschk, M. and Maeda, R. K. (2018). Rab-mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity. Traffic. PubMed ID: 30426623
The male seminal fluid contains factors that affect female post-mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, their molecular organization was investigated, particularly with respect to the intracellular membrane transport machinery. Large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long-term post-mating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, this study has created a searchable, online, open-access imaging resource to display complete findings regarding Rab localization in the accessory gland.

Tuesday, December 7th - Cytoskeleton and Junctions

Banerjee, S. and Riordan, M. (2018). Coordinated regulation of axonal microtubule organization and transport by Drosophila Neurexin and BMP pathway. Sci Rep 8(1): 17337. PubMed ID: 30478335
Neurexins are well known trans-synaptic cell adhesion molecules that are required for proper synaptic development and function across species. Beyond synapse organization and function, little is known about other roles Neurexins might have in the nervous system. This study reports novel phenotypic consequences of mutations in Drosophila neurexin (dnrx), which alters axonal microtubule organization and transport. dnrx mutants display phenotypic similarities with the BMP receptor wishful thinking (wit) and one of the downstream effectors, futsch, which is a known regulator of microtubule organization and stability. dnrx has genetic interactions with wit and futsch. Loss of Dnrx also results in reduced levels of other downstream effectors of BMP signaling, phosphorylated-Mad and Trio. Interestingly, postsynaptic overexpression of the BMP ligand, Glass bottom boat, in dnrx mutants partially rescues the axonal transport defects but not the synapse undergrowth at the neuromuscular junctions. These data suggest that Dnrx and BMP signaling are involved in many diverse functions and that regulation of axonal MT organization and transport might be distinct from regulation of synaptic growth in dnrx mutants. Together, this work uncovers a novel function of Drosophila Neurexin and may provide insights into functions of Neurexins in vertebrates.
Olivares-Castineira, I. and Llimargas, M. (2018). Anisotropic Crb accumulation, modulated by Src42A, is coupled to polarised epithelial tube growth in Drosophila. PLoS Genet 14(11): e1007824. PubMed ID: 30475799
Tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. This study investigated these mechanisms using the Drosophila tracheal system. The apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. Evidence is provided indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. This study finds that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. The results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, these results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process.
Edzuka, T. and Goshima, G. (2018). Drosophila kinesin-8 stabilizes the kinetochore-microtubule interaction. J Cell Biol. PubMed ID: 30538142
Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. This study finds that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end-stabilizing and -destabilizing activity, in addition to kinesin-8's commonly observed MT plus end-directed motility and tubulin-binding activity in vitro. This study further shows that Klp67A is required for stable kinetochore-MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an "end-on" fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end-stabilizing factor CLASP, but not by artificial shortening of MTs. Human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore-MT attachment.
Babatz, F., Naffin, E. and Klambt, C. (2018). The Drosophila blood-brain barrier adapts to cell growth by unfolding of pre-existing septate junctions. Dev Cell. PubMed ID: 30482667
]The blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. This study used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. Septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish.
Krueger, D., Tardivo, P., Nguyen, C. and De Renzis, S. (2018). Downregulation of basal myosin-II is required for cell shape changes and tissue invagination. EMBO J 37(23). PubMed ID: 30442834
Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin-mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. This study used optogenetics to quantitatively control myosin-II levels at the basal surface of invaginating cells during Drosophila gastrulation. While basal myosin-II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre-invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube-like structure. Collectively, these data demonstrate the requirement of myosin-II polarization and basal relaxation throughout the entire invagination process.
Kwiatkowski, S., Seliga, A. K., Vertommen, D., Terreri, M., Ishikawa, T., Grabowska, I., Tiebe, M., Teleman, A. A., Jagielski, A. K., Veiga-da-Cunha, M. and Drozak, J. (2018). SETD3 protein is the actin-specific histidine N-methyltransferase. Elife 7. PubMed ID: 30526847
Protein histidine methylation is a rare post-translational modification of unknown biochemical importance. In vertebrates, only a few methylhistidine-containing proteins have been reported, including beta-actin as an essential example. The evolutionary conserved methylation of beta-actin H73 is catalyzed by an as yet unknown histidine N-methyltransferase. This study reports that the protein SETD3 is the actin-specific histidine N-methyltransferase. In vitro, recombinant rat and human SETD3 methylated beta-actin at H73. Knocking-out SETD3 in both human HAP1 cells and in Drosophila melanogaster resulted in the absence of methylation at beta-actin H73 in vivo, whereas beta-actin from wildtype cells or flies was > 90% methylated. As a consequence, it was shown that Setd3-deficient HAP1 cells have less cellular F-actin and an increased glycolytic phenotype. In conclusion, by identifying SETD3 as the actin-specific histidine N-methyltransferase, this work pioneers new research into the possible role of this modification in health and disease and questions the substrate specificity of SET-domain-containing enzymes.

Friday, January 4th - Adult CNS Function

Kimura, K. I., Urushizaki, A., Sato, C. and Yamamoto, D. (2018). A novel sex difference in Drosophila contact chemosensory neurons unveiled using single cell labeling. J Neurogenet: 1-9. PubMed ID: 30457022
Among the sensory modalities involved in controlling mating behavior in Drosophila melanogaster, contact sex pheromones play a primary role. The key receptor neurons for contact sex pheromones are located on the forelegs, which are activated in males upon touching the female abdomen during tapping events in courtship actions. A fruitless (fru)-positive (fru [+]) male-pheromone sensing cell (M-cell) and a fru [+] female-pheromone sensing cell (F-cell) are paired in a sensory bristle on the legs, and some fru [+] chemoreceptor axons project across the midline in the thoracic neuromere in males but not in females. However, the receptor cells that form sexually dimorphic axon terminals in the thoracic ganglia remain unknown. By generating labeled single-cell clones, this study shows that only a specific subset of fru [+] chemosensory neurons have axons that cross the midline in males. It was further demonstrated that there exist two male-specific bristles, each harboring two chemosensory neurons; neither of which exhibits midline crossing, a masculine characteristic. This study reveals hitherto unrecognized sex differences in chemosensory neurons, imposing a reinvestigation of the pheromone input pathways that impinge on the central courtship circuit.
Keles, M. F., Mongeau, J. M. and Frye, M. A. (2018). Object features and T4/T5 motion detectors modulate the dynamics of bar tracking by Drosophila. J Exp Biol. PubMed ID: 30446539
Visual objects can be discriminated by static spatial features such as luminance or dynamic features such as relative movement. Flies track a solid dark vertical bar moving on a bright background, a behavioral reaction so strong that for a rigidly tethered fly, the steering trajectory is phase advanced relative to the moving bar, apparently in anticipation of its future position. By contrast, flickering bars that generate no coherent motion, or whose surface texture moves in the direction opposite to the bar generate steering responses that lag behind the stimulus. It remains unclear how the spatial properties of a bar influence behavioral response dynamics. A dark bar defined by its luminance contrast to the uniform background drives a co-directional steering response that is phase-advanced relative to the response to a textured bar defined only by its motion relative to a stationary textured background. The textured bar drives an initial contra-directional turn and phase-locked tracking. The qualitatively distinct response dynamics could indicate parallel visual processing of a luminance versus motion-defined object. Calcium imaging shows that T4/T5 motion detecting neurons are more responsive to a solid dark bar than a motion defined bar. Genetically blocking T4/T5 neurons eliminates the phase-advanced co-directional response to the luminance-defined bar, leaving the orientation response largely intact. It is concluded that T4/T5 neurons mediate a co-directional optomotor response to a luminance defined bar, thereby driving phase-advanced wing kinematics, whereas separate unknown visual pathways elicit the contra-directional orientation response.
Miyashita, T., Kikuchi, E., Horiuchi, J. and Saitoe, M. (2018). Long-term memory engram cells are established by c-Fos/CREB transcriptional cycling. Cell Rep 25(10): 2716-2728.e2713. PubMed ID: 30517860
=Training-dependent increases in c-fos have been used to identify engram cells encoding long-term memories (LTMs). However, the interaction between transcription factors required for LTM, including CREB and c-Fos, and activating kinases such as phosphorylated ERK (pERK) in the establishment of memory engrams has been unclear. Formation of LTM of an aversive olfactory association in flies requires repeated training trials with rest intervals between trainings. This study finds that prolonged rest interval-dependent increases in pERK induce transcriptional cycling between c-Fos and CREB in a subset of KCs in the mushroom bodies, where olfactory associations are made and stored. Preexisting CREB is required for initial c-fos induction, while c-Fos is required later to increase CREB expression. Blocking or activating c-fos-positive engram neurons inhibits memory recall or induces memory-associated behaviors. These results suggest that c-Fos/CREB cycling defines LTM engram cells required for LTM.
Miroschnikow, A., Schlegel, P., Schoofs, A., Hueckesfeld, S., Li, F., Schneider-Mizell, C. M., Fetter, R. D., Truman, J. W., Cardona, A. and Pankratz, M. J. (2018). Convergence of monosynaptic and polysynaptic sensory paths onto common motor outputs in a Drosophila feeding connectome. Elife 7. PubMed ID: 30526854
This study reconstructed, from a whole CNS EM volume, the synaptic map of input and output neurons that underlie food intake behavior of Drosophila larvae. Input neurons originate from enteric, pharyngeal and external sensory organs and converge onto seven distinct sensory synaptic compartments within the CNS. Output neurons consist of feeding motor, serotonergic modulatory and neuroendocrine neurons. Monosynaptic connections from a set of sensory synaptic compartments cover the motor, modulatory and neuroendocrine targets in overlapping domains. Polysynaptic routes are superimposed on top of monosynaptic connections, resulting in divergent sensory paths that converge on common outputs. A completely different set of sensory compartments is connected to the mushroom body calyx. The mushroom body output neurons are connected to interneurons that directly target the feeding output neurons. These results illustrate a circuit architecture in which monosynaptic and multisynaptic connections from sensory inputs traverse onto output neurons via a series of converging paths.
Hamid, R., Hajirnis, N., Kushwaha, S., Saleem, S., Kumar, V. and Mishra, R. K. (2018). Drosophila Choline transporter non-canonically regulates pupal eclosion and NMJ integrity through a neuronal subset of mushroom body. Dev Biol. PubMed ID: 30529058
Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. To date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. This study reports a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. Knockdown of ChT in a sub-type of MB neurons called alpha/beta core (alpha/betac) and Upsilon neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in alpha/betac and Upsilon neurons nor by knockdown of ChT in cholinergic neurons. Thus, this study provides insights into non-canonical roles of ChT in MB.
Kacsoh, B. Z., Barton, S., Jiang, Y., Zhou, N., Mooney, S. D., Friedberg, I., Radivojac, P., Greene, C. S. and Bosco, G. (2018). New Drosophila long-term memory genes revealed by assessing computational function prediction methods. G3 (Bethesda). PubMed ID: 30463884
A major bottleneck to understanding of the genetic and molecular foundation of life lies in the ability to assign function to a gene and, subsequently, a protein. Traditional molecular and genetic experiments can provide the most reliable forms of identification, but are generally low-throughput, making such discovery and assignment a daunting task. The bottleneck has led to an increasing role for computational approaches. The Critical Assessment of Functional Annotation (CAFA) effort seeks to measure the performance of computational methods. In CAFA3, selected screens were performed, including an effort focused on long-term memory.Homology and previous CAFA predictions were used to identify 29 key Drosophila genes, which were tested via a long-term memory screen. 11 novel genes were found that are involved in long-term memory formation and show a high level of connectivity with previously identified learning and memory genes. This study provides first higher-order behavioral assay and organism screen used for CAFA assessments and revealed previously uncharacterized roles of multiple genes as possible regulators of neuronal plasticity at the boundary of information acquisition and memory formation.

Thursday, January 3rd - Adult Development

Meserve, J. H. and Duronio, R. J. (2018). Fate mapping during regeneration: cells that undergo compensatory proliferation in damaged Drosophila eye imaginal discs differentiate into multiple retinal accessory cell types. Dev Biol. PubMed ID: 30347187
Regeneration of tissues that have been damaged by cell loss requires new growth, often via proliferation of precursor cells followed by differentiation to replace loss of specific cell types. When regeneration occurs after normal differentiation of the tissue is complete, developmental pathways driving differentiation must be re-activated. How proliferation and differentiation are induced and balanced during regeneration is not well understood. To investigate these processes, a paradigm was used for tissue damage and regeneration in the developing Drosophila melanogaster eye. Previous studies have demonstrated that tissue damage resulting from extensive cell death stimulates quiescent, undifferentiated cells in the developing larval eye to re-enter the cell cycle and proliferate. Whether these cells are restricted to certain fates or can contribute to all retinal cell types and thus potentially be fully regenerative is not known. This study found by fate mapping experiments that these cells are competent to differentiate into all accessory cell types in the retina but do not differentiate into photoreceptors, likely because cell cycle re-entry in response to damage occurs after photoreceptor differentiation has completed. It is concluded that the ability to re-enter the cell cycle in response to tissue damage in the developing Drosophila eye is not restricted to precursors of a specific cell type and that cell cycle re-entry following damage does not disrupt developmental programs that control differentiation.
Tran, H. T., Cho, E., Jeong, S., Jeong, E. B., Lee, H. S., Jeong, S. Y., Hwang, J. S. and Kim, E. Y. (2018). Makorin 1 regulates developmental timing in Drosophila. Mol Cells. PubMed ID: 30396233
The central mechanisms coordinating growth and sexual maturation are well conserved across invertebrates and vertebrates. Although mutations in the gene encoding makorin RING finger protein 3 (mkrn3) are associated with central precocious puberty in humans, a causal relationship has not been elucidated. This study examined the role of the Drosophila ortholog of mammalian makorin genes, in the regulation of developmental timing. Loss of MKRN1 in mkrn1exS prolonged the 3rd instar stage and delayed the onset of pupariation, resulting in bigger size pupae. MKRN1 was expressed in the prothoracic gland, where the steroid hormone ecdysone is produced. Furthermore, mkrn1exS larvae exhibited reduced mRNA levels of phantom, which encodes ecdysone-synthesizing enzyme and E74, which is a downstream target of ecdysone. Collectively, these results indicate that MKRN1 fine-tunes developmental timing and sexual maturation by affecting ecdysone synthesis in Drosophila. Moreover, this study supports the notion that malfunction of makorin gene family member, mkrn3 dysregulates the timing of puberty in mammals.
Bruce, L., Singkornrat, D., Wilson, K., Hausman, W., Robbins, K., Huang, L., Foss, K. and Binninger, D. (2018). In vivo effects of Methionine Sulfoxide Reductase deficiency in Drosophila melanogaster. Antioxidants (Basel) 7(11). PubMed ID: 30388828
The deleterious alteration of protein structure and function due to the oxidation of methionine residues has been studied extensively in age-associated neurodegenerative disorders such as Alzheimer's and Parkinson's Disease. Methionine sulfoxide reductases (MSR) have three well-characterized biological functions. The most commonly studied function is the reduction of oxidized methionine residues back into functional methionine thus, often restoring biological function to proteins. Previous studies have successfully overexpressed and silenced MSR activity in numerous model organisms correlating its activity to longevity and oxidative stress. The present study characterized in vivo effects of MSR deficiency in Drosophila. Interestingly, no significant phenotype was found in animals lacking either methionine sulfoxide reductase A (MSRA) or methionine sulfoxide reductase B (MSRB). However, Drosophila lacking any known MSR activity exhibited a prolonged larval third instar development and a shortened lifespan. These data suggest an essential role of MSR in key biological processes.
Sui, L., Alt, S., Weigert, M., Dye, N., Eaton, S., Jug, F., Myers, E. W., Julicher, F., Salbreux, G. and Dahmann, C. (2018). Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms. Nat Commun 9(1): 4620. PubMed ID: 30397306
Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. This study shows that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. This combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.
Mangione, F. and Martin-Blanco, E. (2018). The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen. Cell Rep 25(10): 2836-2850.e2834. PubMed ID: 30517870
The achievement of the final form of an individual requires not only the control of cell size and differentiation but also integrative directional cues to instruct cell movements, positions, and orientations. In Drosophila, the adult epidermis of the abdomen is created de novo by histoblasts. As these expand and fuse, they uniformly orient along the anteroposterior axis. The Dachsous/Fat/Four-jointed (Ds/Ft/Fj) pathway is key for their alignment. The refinement of the tissue-wide expression of the atypical cadherins Ds and Ft result in their polarization and directional adhesiveness. Mechanistically, the axially oriented changes in histoblasts respond to the redesign of the epithelial field. It is suggested that the role of Ds/Ft/Fj in long-range oriented cell alignment is a general function and that the regulation of the expression of its components will be crucial in other morphogenetic models or during tissue repair.
Tanaka, R., Miyata, S., Yamaguchi, M. and Yoshida, H. (2018). Role of the smallish gene during Drosophila eye development. Gene 684: 10-19. PubMed ID: 30359736
Smallish (Smash; CG43427), the Drosophila homologue of human LIM domain only 7 (LMO7), is a key regulator of Drosophila embryogenesis associated with planer cell polarity and actomyosin contractility at the zonula adherence. Although smash mRNA is expressed in several tissues during Drosophila development, only Smash function at the adherence junction in the embryonic epithelial cells has been reported. This study demonstrated that the knockdown of smash in eye imaginal discs induced morphological aberrations in adult compound eyes that were associated with increased apoptosis. Furthermore, immunohistochemical analyses revealed that Smash localized to the nucleus in several tissues, including eye imaginal discs. The knockdown of smash in eye imaginal discs down-regulated the expression of the otefin and bocksbeutel genes as well as the Drosophila homologue of the emerin gene, which is a target of LMO7. Collectively, these results indicate that Smash functions in proper Drosophila eye development mediated by the regulation of ote and bocks gene expression.

Wednesday, January 2nd - Signaling

Delgado, R., Delgado, M. G., Bastin-Heline, L., Glavic, A., O'Day, P. M. and Bacigalupo, J. (2018). Light-induced opening of the TRP channel in isolated membrane patches excised from photosensitive microvilli from Drosophila photoreceptors. Neuroscience 396: 66-72. PubMed ID: 30458219
Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H(+). This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. This study reports that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-gamma-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.
Graze, R. M., Tzeng, R. Y., Howard, T. S. and Arbeitman, M. N. (2018). Perturbation of IIS/TOR signaling alters the landscape of sex-differential gene expression in Drosophila. BMC Genomics 19(1): 893. PubMed ID: 30526477
The core functions of the insulin/insulin-like signaling and target of rapamycin (IIS/TOR) pathway are nutrient sensing, energy homeostasis, growth, and regulation of stress responses. This pathway is also known to interact directly and indirectly with the sex determination regulatory hierarchy. The IIS/TOR pathway plays a role in directing sexually dimorphic traits, including dimorphism of growth, metabolism, stress and behavior. To understand the degree to which the environmentally responsive insulin signaling pathway contributes to sexual dimorphism of gene expression, the effect of perturbation of the pathway on gene expression was examined in male and female Drosophila heads. The data reveal a large effect of insulin signaling on gene expression, with greater than 50% of genes examined changing expression. Males and females have a shared gene expression response to knock-down of InR function, with significant enrichment for pathways involved in metabolism. Perturbation of insulin signaling has a greater impact on gene expression in males, with more genes changing expression and with gene expression differences of larger magnitude. Primarily as a consequence of the response in males, this study found that reduced insulin signaling results in a striking increase in sex-differential expression. This includes sex-differences in expression of immune, defense and stress response genes, genes involved in modulating reproductive behavior, genes linking insulin signaling and ageing, and in the insulin signaling pathway itself. These results demonstrate that perturbation of insulin signaling results in thousands of genes displaying sex differences in expression that are not differentially expressed in control conditions. Thus, insulin signaling may play a role in variability of somatic, sex-differential expression. The finding that perturbation of the IIS/TOR pathway results in an altered landscape of sex-differential expression suggests a role of insulin signaling in the physiological underpinnings of trade-offs, sexual conflict and sex differences in expression variability.
Hwang, S. H., Bang, S., Kang, K. S., Kang, D. and Chung, J. (2018). ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled. Biochem Biophys Res Commun. PubMed ID: 30497781
Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Among the core components of Wnt signaling pathway, this study discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/beta-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, it was found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, it was further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, the results suggested that Dsh forms an SDS-resistant high molecular weight complex with beta-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, it is proposed that ULK1 plays a pivotal role in the regulation of Wnt/beta-catenin signaling pathway by phosphorylating Dsh.
Contreras, E. G., Egger, B., Gold, K. S. and Brand, A. H. (2018). Dynamic Notch signalling regulates neural stem cell state progression in the Drosophila optic lobe. Neural Dev 13(1): 25. PubMed ID: 30466475
Neural stem cells generate all of the neurons and glial cells in the central nervous system, both during development and in the adult to maintain homeostasis. In the Drosophila optic lobe, neuroepithelial cells progress through two transient progenitor states, PI and PII, before transforming into neuroblasts. This study analysed the role of Notch signalling in the transition from neuroepithelial cells to neuroblasts.Dynamic regulation of Notch signalling was observed: strong activity in PI progenitors, low signalling in PII progenitors, and increased activity after neuroblast transformation. Ectopic expression of the Notch ligand Delta induced the formation of ectopic PI progenitors. Interestingly, the E3 ubiquitin ligase, Neuralized, regulates Delta levels and Notch signalling activity at the transition zone. The proneural transcription factor, Lethal of scute, is essential to induce expression of Neuralized and promote the transition from the PI progenitor to the PII progenitor state. These results show dynamic regulation of Notch signalling activity in the transition from neuroepithelial cells to neuroblasts. A model is proposed in which Lethal of scute activates Notch signalling in a non-cell autonomous manner by regulating the expression of Neuralized, thereby promoting the progression between different neural stem cell states
Giordano, C., Ruel, L., Poux, C. and Therond, P. (2018). Protein association changes in the Hedgehog signaling complex mediate differential signaling strength. Development 145(24). PubMed ID: 30541874
Hedgehog (Hh) is a conserved morphogen that controls cell differentiation and tissue patterning in metazoans. In Drosophila, the Hh signal is transduced from the G protein-coupled receptor Smoothened (Smo) to the cytoplasmic Hh signaling complex (HSC). How activated Smo is translated into a graded activation of the downstream pathway is still not well understood. This study shows that the last amino acids of the cytoplasmic tail of Smo, in combination with G protein-coupled receptor kinase 2 (Gprk2), bind to the regulatory domain of Fused (Fu) and highly activate its kinase activity. This binding induces changes in the association of Fu protein with the HSC and increases the proximity of the Fu catalytic domain to its substrate, the Costal2 kinesin. A new model is proposed in which, depending on the magnitude of Hh signaling, Smo and Gprk2 modulate protein association and conformational changes in the HSC, which are responsible for the differential activation of the pathway.
Hall, E. T., Hoesing, E., Sinkovics, E. and Verheyen, E. M. (2018). Actomyosin contractility modulates Wnt signaling through adherens junction stability. Mol Biol Cell: mbcE18060345. PubMed ID: 30540525
Actomyosin contractility can influence the canonical Wnt signaling pathway in processes like mesoderm differentiation and tissue stiffness during tumorigenesis. This study found that increased non-muscle myosin II activation and cellular contraction inhibited Wnt target gene transcription in developing Drosophila imaginal discs. This effect is due to myosin-induced accumulation of cortical F-actin resulting in clustering and accumulation of E-cadherin to the adherens junctions. This results in E-cadherin titrating any available beta-catenin, the Wnt pathway transcriptional co-activator, to the adherens junctions in order to maintain cell-cell adhesion under contraction. Decreased levels of cytoplasmic beta-catenin result in insufficient nuclear translocation for full Wnt target gene transcription. This study shows the consequences of modulating myosin phosphatase. This work elucidates a mechanism in which the dynamic promotion of actomyosin contractility refines patterning of Wnt transcription during development and maintenance of epithelial tissue in organisms.
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