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Current papers in developmental biology and gene function


Thursday February 28th, 2019 - Apoptosis and Autophagy

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Guo, T., Nan, Z., Miao, C., Jin, X., Yang, W., Wang, Z., Tu, Y., Bao, H., Lyu, J., Zheng, H., Deng, Q., Guo, P., Xi, Y., Yang, X. and Ge, W. (2019). The autophagy-related gene Atg101 in Drosophila regulates both neuron and midgut homeostasis. J Biol Chem. PubMed ID: 30760524
Atg101 is an autophagy-related gene identified in worms, flies, mice, and mammals and encodes a protein that functions in autophagosome formation by associating with the ULK1-Atg13-Fip200 complex. Atg101's physiological role both during development and in adulthood remains poorly understood. This study describes the generation and characterization of an Atg101 loss-of-function mutant in Drosophila and reports on the roles of Atg101 in maintaining tissue homeostasis in both adult brains and midguts. Homozygous or hemizygous Atg101 mutants were semi-lethal, with only some of them surviving as adults. Both developmental and starvation-induced autophagy processes were defective in the Atg101 mutant animals, and Atg101 mutant adult flies had a significantly shorter lifespan and displayed a mobility defect. Moreover, accumulation of ubiquitin-positive aggregates were observed in Atg101 mutant brains, indicating a neuronal defect. Interestingly, Atg101 mutant adult midguts were shorter and thicker and exhibited abnormal morphology with enlarged enterocytes. Detailed analysis also revealed that the differentiation from intestinal stem cells to enterocytes was impaired in these midguts. Cell type-specific rescue experiments disclosed that Atg101 had a function in enterocytes and limited their growth. In summary, the results of this study indicate that Drosophila Atg101 is essential for tissue homeostasis in both adult brains and midguts. It is proposed that Atg101 may have a role in age-related processes.
Bourouis, M., Mondin, M., Dussert, A. and Leopold, P. (2019). Control of basal autophagy rate by vacuolar peduncle. PLoS One 14(2): e0209759. PubMed ID: 30735514
Basal autophagy is as a compressive catabolic mechanism engaged in the breakdown of damaged macromolecules and organelles leading to the recycling of elementary nutrients. Thought essential to cellular refreshing, little is known about the origin of a constitutional rate of basal autophagy. This study found that loss of Drosophila vacuolar peduncle (vap), a presumed GAP enzyme, is associated with enhanced basal autophagy rate and physiological alterations resulting in a wasteful cell energy balance, a hallmark of overactive autophagy. By contrast, starvation-induced autophagy was disrupted in vap mutant conditions, leading to a block of maturation into autolysosomes. This phenotype stem for exacerbated biogenesis of PI(3)P-dependent endomembranes, including autophagosome membranes and ectopic fusions of vesicles. These findings shed new light on the neurodegenerative phenotype found associated to mutant vap adult brains in a former study. A partner of Vap, Sprint (Spri), acting as an endocytic GEF for Rab5, had the converse effect of leading to a reduction in PI(3)P-dependent endomembrane formation in mutants. Spri was conditional to normal basal autophagy and instrumental to the starvation-sensitivity phenotype specific of vap. Rab5 activity itself was essential for PI(3)P and for pre-autophagosome structures formation. It is proposed that Vap/Spri complexes promote a cell surface-derived flow of endocytic Rab5-containing vesicles, the traffic of which is crucial for the implementation of a basal autophagy rate.
Denton, D., Xu, T., Dayan, S., Nicolson, S. and Kumar, S. (2019). Crosstalk between Dpp and Tor signaling coordinates autophagy-dependent midgut degradation. Cell Death Dis 10(2): 111. PubMed ID: 30737370
The majority of developmentally programmed cell death (PCD) is mediated by caspase-dependent apoptosis; however, additional modalities, including autophagy-dependent cell death, have important spatiotemporally restricted functions. Autophagy involves the engulfment of cytoplasmic components in a double membrane vesicle for delivery to the lysosome. An established model for autophagy-dependent PCD is Drosophila larval midgut removal during metamorphosis. Previous work demonstrated that growth arrest is required to initiate autophagy-dependent midgut degradation and Target of rapamycin (Tor) limits autophagy induction. This study uncovered a role for Decapentaplegic (Dpp) in coordinating midgut degradation. This study provides new data to show that Dpp interacts with Tor during midgut degradation. Inhibiting Tor rescued the block in midgut degradation due to Dpp signaling. It is proposed that Dpp is upstream of Tor and down-regulation promotes growth arrest and autophagy-dependent midgut degradation. These findings underscore a relationship between Dpp and Tor signaling in the regulation of cell growth and tissue removal.
Fujisawa, Y., Kosakamoto, H., Chihara, T. and Miura, M. (2019). Non-apoptotic function of Drosophila caspase activation in epithelial thorax closure and wound healing. Development 146(4). PubMed ID: 30770378
Non-apoptotic caspase activation involves multiple cellular events. However, the link between visible non-apoptotic caspase activation and its function in living organisms has not yet been revealed. This study visualized sub-lethal activation of apoptotic signaling with the combination of a sensitive indicator for caspase 3 activation and in vivo live-imaging analysis of Drosophila. During thorax closure in pupal development, caspase 3 activation was specifically observed at the leading edge cells, with no signs of apoptosis. Inhibition of caspase activation led to an increase in thorax closing speed, which suggests a role of non-apoptotic caspase activity in cell motility. Importantly, sub-lethal activation of caspase 3 was also observed during wound closure at the fusion sites at which thorax closure had previously taken place. Further genetic analysis revealed that the activation of the initiator caspase Dronc is coupled with the generation of reactive oxygen species. The activation of Dronc also regulates myosin levels and delays wound healing. These findings suggest a possible function for non-apoptotic caspase activation in the fine-tuning of cell migratory behavior during epithelial closure.

Wednesday, February 27th - RNA and Transposons

Yang, W. R., Ardeljan, D., Pacyna, C. N., Payer, L. M. and Burns, K. H. (2019). SQuIRE reveals locus-specific regulation of interspersed repeat expression. Nucleic Acids Res. PubMed ID: 30624635
Transposable elements (TEs) are interspersed repeat sequences that make up much of the human genome. Their expression has been implicated in development and disease. However, TE-derived RNA-seq reads are difficult to quantify. Past approaches have excluded these reads or aggregated RNA expression to subfamilies shared by similar TE copies, sacrificing quantitative accuracy or the genomic context necessary to understand the basis of TE transcription. As a result, the effects of TEs on gene expression and associated phenotypes are not well understood. This study presents Software for Quantifying Interspersed Repeat Expression (SQuIRE), the first RNA-seq analysis pipeline that provides a quantitative and locus-specific picture of TE expression. SQuIRE is an accurate and user-friendly tool that can be used for a variety of species. SQuIRE was applied to RNA-seq from normal mouse tissues and a Drosophila model of amyotrophic lateral sclerosis. In both model organisms, previously reported TE subfamily expression levels were recapitulated and locus-specific TE expression was revealed. Differences were identified in TE transcription patterns relating to transcript type, gene expression and RNA splicing that would be lost with other approaches using subfamily-level analyses. Altogether, these findings illustrate the importance of studying TE transcription with locus-level resolution.
Wakisaka, K. T., et al. (2018). Novel roles of Drosophila FUS and Aub responsible for piRNA biogenesis in neuronal disorders. Brain Res. PubMed ID: 30578769
This study examined the possible roles of Aubergine (Aub), a Piwi-family protein (PIWI) responsible for piRNAs biogenesis, in the neuronal disorders, using the Cabeza (Caz) knockdown Drosophila. Caz is a Drosophila homologue of FUS, which is one of the genes causing amyotrophic lateral sclerosis (ALS). Aub overexpression enhanced the mobility defects accompanied by anatomical defects in motoneurons at neuromuscular junctions induced by the neuron-specific knockdown of Caz. In order to elucidate the underlying mechanisms, this study examined pre-piRNA and mature-size piRNA levels under these conditions. qRT-PCR and RNA-seq analyses revealed that the Caz knockdown increased pre-piRNA levels, but reduced mature-size piRNA levels in the central nervous system (CNS), suggesting a role in the pre-piRNAs production. Aub overexpression did not increase mature-size piRNA levels. These results suggest that the accumulated pre-piRNAs are abnormal abortive pre-piRNAs that cannot be further processed by slicers, including Aub. A relationship between Caz and pre-piRNAs in the CNS was demonstrated by RNA immunoprecipitation. Aub overexpression induced the abnormal cytoplasmic localization of Caz. Based on these results, a model is proposed in which Caz knockdown-induced abnormal pre-piRNAs associate with Caz, then translocate and accumulate in the cytoplasm, a process that may be mediated by Aub.
Sandler, J. E., Irizarry, J., Stepanik, V., Dunipace, L., Amrhein, H. and Stathopoulos, A. (2018). A developmental program truncates long transcripts to temporally regulate cell signaling. Dev Cell 47(6): 773-784.e776. PubMed ID: 30562515
Rapid mitotic divisions and a fixed transcription rate limit the maximal length of transcripts in early Drosophila embryos. Previous studies suggested that transcription of long genes is initiated but aborted, as early nuclear divisions have short interphases. This study identified long genes that are expressed during short nuclear cycles as truncated transcripts. The RNA binding protein Sex-lethal physically associates with transcripts for these genes and is required to support early termination to specify shorter transcript isoforms in early embryos of both sexes. In addition, one truncated transcript for the gene short-gastrulation encodes a product in embryos that functionally relates to a previously characterized dominant-negative form, which maintains TGF-beta signaling in the off-state. In summary, these results reveal a developmental program of short transcripts functioning to help temporally regulate Drosophila embryonic development, keeping cell signaling at early stages to a minimum in order to support its proper initiation at cellularization.
Valanne, S., Salminen, T. S., Jarvela-Stolting, M., Vesala, L. and Ramet, M. (2019). Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster. PLoS Pathog 15(1): e1007504. PubMed ID: 30633769
Non-coding RNAs have important roles in regulating physiology, including immunity. Transcriptome profiling of immune-responsive genes in Drosophila melanogaster was performed during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. Upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-kappaB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism.
Fablet, M., Jacquet, A., Rebollo, R., Haudry, A., Rey, C., Salces-Ortiz, J., Bajad, P., Burlet, N., Jantsch, M. F., Garcia Guerreiro, M. P. and Vieira, C. (2019). Dynamic Interactions between the genome and an endogenous retrovirus: Tirant in Drosophila simulans wild-type strains. G3 (Bethesda). PubMed ID: 30658967
All genomes contain repeated sequences that are known as transposable elements (TEs). Among these are endogenous retroviruses (ERVs), which are sequences similar to retroviruses and are transmitted across generations from parent to progeny. These sequences are controlled in genomes through epigenetic mechanisms. At the center of the epigenetic control of TEs are small interfering RNAs of the piRNA class, which trigger heterochromatinization of TE sequences. The tirant ERV of Drosophila simulans displays intra-specific variability in copy numbers, insertion sites, and transcription levels, providing a well-suited model to study the dynamic relationship between a TE family and the host genome through epigenetic mechanisms. tirant transcript amounts and piRNA amounts are positively correlated in ovaries in normal conditions, unlike what was previously described following divergent crosses. In addition, tirant insertion polymorphism in the genomes of three D. simulans wild-type strains is described, revealing a limited number of insertions that may be associated with gene transcript level changes through heterochromatin spreading having phenotypic impacts. Taken together, these results participate in the understanding of the equilibrium between the host genome and its TEs.
Duarte Junior, F. F., Bueno, P. S. A., Pedersen, S. L., Rando, F. D. S., Pattaro Junior, J. R., Caligari, D., Ramos, A. C., Polizelli, L. G., Lima, A., de Lima Neto, Q. A., Krude, T., Seixas, F. A. V. and Fernandez, M. A. (2019). Identification and characterization of stem-bulge RNAs in Drosophila melanogaster. RNA Biol 16(3): 330-339. PubMed ID: 30666901
Non-coding Y RNAs and stem-bulge RNAs are homologous small RNAs in vertebrates and nematodes, respectively. They share a conserved function in the replication of chromosomal DNA in these two groups of organisms. However, functional homologues have not been found in insects, despite their common early evolutionary history. This study describes the identification and functional characterization of two sbRNAs in Drosophila melanogaster, termed Dm1 and Dm2. The genes coding for these two RNAs were identified by a computational search in the genome of D. melanogaster for conserved sequence motifs present in nematode sbRNAs. The predicted secondary structures of Dm1 and Dm2 partially resemble nematode sbRNAs and show stability in molecular dynamics simulations. Both RNAs are phylogenetically closer related to nematode sbRNAs than to vertebrate Y RNAs. Dm1, but not Dm2 sbRNA is abundantly expressed in D. melanogaster S2 cells and adult flies. Only Dm1, but not Dm2 sbRNA can functionally replace Y RNAs in a human cell-free DNA replication initiation system. Therefore, Dm1 is the first functional sbRNA described in insects, allowing future investigations into the physiological roles of sbRNAs in the genetically tractable model organism D. melanogaster.

Tuesday, February 26th - Chromatin

Berlandi, J., Chaouch, A., De Jay, N., Tegeder, I., Thiel, K., Shirinian, M., Kleinman, C. L., Jeibmann, A., Lasko, P., Jabado, N. and Hasselblatt, M. (2019). Identification of genes functionally involved in the detrimental effects of mutant histone H3.3-K27M in Drosophila melanogaster. Neuro Oncol. PubMed ID: 30715493
Recurrent specific mutations in evolutionarily conserved Histone 3 (H3) variants drive pediatric high-grade gliomas (HGG), but little is known about their downstream effects. The aim of this study was to identify genes involved in the detrimental effects of mutant H3.3-K27M, the main genetic driver in lethal midline HGG, in a transgenic Drosophila model. Mutant and wild-type histone H3.3 expressing flies were generated using a phiC31-based integration system. Genetic modifier screens were performed by crossing H3.3-K27M expressing driver strains and 194 fly lines expressing shRNA targeting genes selected based on their potential role in the detrimental effects of mutant H3. Expression of the human orthologues of genes with functional relevance in the fly model was validated in H3-K27M mutant HGG. Ubiquitous and midline glia-specific expression of H3.3-K27M but not wild-type H3.3 caused pupal lethality, morphological alterations and decreased H3K27me3. Knockdown of 17 candidate genes shifted the lethal phenotype to later stages of development. These included histone modifying and chromatin remodeling genes as well as genes regulating cell differentiation and proliferation. Notably, several of these genes were over-expressed in mutant H3-K27M mutated HGG. It is concluded that rapid screening, identification and validation of relevant targets in "oncohistone" mediated pathogenesis has proven a challenge and a barrier to providing novel therapies. These results provide further evidence on the role of chromatin modifiers in the genesis of H3.3-K27M. Notably, they validate Drosophila as a model system for rapid identification of relevant genes functionally involved in the detrimental effects of H3.3-K27M mutagenesis.
Armstrong, R. L., Penke, T. J. R., Chao, S. K., Gentile, G. M., Strahl, B. D., Matera, A. G., McKay, D. J. and Duronio, R. J. (2019). H3K9 Promotes under-replication of pericentromeric heterochromatin in Drosophila salivary gland Polytene Chromosomes. Genes (Basel) 10(2). PubMed ID: 30700014
Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. This study employed a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9), but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.
Bag, I., Dale, R. K., Palmer, C. and Lei, E. P. (2019). The zinc-finger protein CLAMP promotes gypsy chromatin insulator function in Drosophila. J Cell Sci. PubMed ID: 30718365
Chromatin insulators are DNA-protein complexes that establish independent higher order DNA domains to influence transcription. Insulators are functionally defined by two different properties: they can block communication between an enhancer and a promoter and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. This study identified a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is depleted, gypsy-dependent enhancer blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, but depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. These results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity.
Zhang, W., Zhang, X., Xue, Z., Li, Y., Ma, Q., Ren, X., Zhang, J., Yang, S., Yang, L., Wu, M., Ren, M., Xi, R., Wu, Z., Liu, J. L., Matunis, E., Dai, J. and Gao, G. (2018). Probing the function of metazoan histones with a systematic library of H3 and H4 mutants. Dev Cell. PubMed ID: 30595536
Replication-dependent histone genes often reside in tandemly arrayed gene clusters, hindering systematic loss-of-function analyses. This study used CRISPR/Cas9 and the attP/attB double-integration system to alter numbers and sequences of histone genes in their original genomic context in Drosophila melanogaster. As few as 8 copies of the histone gene unit supported embryo development and adult viability, whereas flies with 20 copies were indistinguishable from wild-types. By hierarchical assembly, 40 alanine-substitution mutations (covering all known modified residues in histones H3 and H4) were introduced and characterized. Mutations at multiple residues compromised viability, fertility, and DNA-damage responses. In particular, H4K16 was necessary for expression of male X-linked genes, male viability, and maintenance of ovarian germline stem cells, whereas H3K27 was essential for late embryogenesis. Simplified mosaic analysis showed that H3R26 is required for H3K27 trimethylation. This study has developed a powerful strategy and valuable reagents to systematically probe histone functions in D. melanogaster.
Chathoth, K. T. and Zabet, N. R. (2019). Chromatin architecture reorganisation during neuronal cell differentiation in Drosophila genome. Genome Res. PubMed ID: 30709849
The organization of the genome into topologically associating domains (TADs) was shown to have a regulatory role in development and cellular functioning, but the mechanism involved in TAD establishment is still unclear. This study presented the first high-resolution contact map of Drosophila neuronal cells (BG3) and identified different classes of TADs by comparing this to genome organization in embryonic cells (Kc167). Only some TADs were found to be conserved in both cell lines, whereas the rest are cell-specific TADs. This is supported by a change in the enrichment of architectural proteins at TAD borders, with BEAF-32 present in embryonic cells and CTCF in neuronal cells. Furthermore, strong divergent transcription was observed, together with RNA Polymerase II occupancy, and an increase in DNA accessibility at the TAD borders. TAD borders that are specific to neuronal cells are enriched in enhancers controlled by neuronal-specific transcription factors. These results suggest that TADs are dynamic across developmental stages and reflect the interplay between insulators, transcriptional states and enhancer activities.
De, S., Cheng, Y., Sun, M. A., Gehred, N. D. and Kassis, J. A. (2019). Structure and function of an ectopic Polycomb chromatin domain. Sci Adv 5(1): eaau9739. PubMed ID: 30662949
Polycomb group proteins (PcGs) drive target gene repression and form large chromatin domains. In Drosophila, DNA elements known as Polycomb group response elements (PREs) recruit PcGs to the DNA. This study shows that, within the invected-engrailed (inv-en) Polycomb domain, strong, constitutive PREs are dispensable for Polycomb domain structure and function. It is suggested that the endogenous chromosomal location imparts stability to this Polycomb domain. To test this possibility, a 79-kb en transgene was inserted into other chromosomal locations. This transgene is functional and forms a Polycomb domain. The spreading of the H3K27me3 repressive mark, characteristic of PcG domains, varies depending on the chromatin context of the transgene. Unlike at the endogenous locus, deletion of the strong, constitutive PREs from the transgene leads to both loss- and gain-of function phenotypes, demonstrating the important role of these regulatory elements. These data show that chromatin context plays an important role in Polycomb domain structure and function.

Monday, February 25th - Behavior

Brown, E. B., Rayens, E. and Rollmann, S. M. (2019). The gene CG6767 affects olfactory behavior in Drosophila melanogaster. Behav Genet. PubMed ID: 30710192
Chemosensory systems mediate some of the most vital animal behaviors. However, knowledge of the genetic mechanisms that underlie behavioral responses to olfactory cues remains fragmented. Genome-wide association mapping has greatly advanced the ability to identify candidate loci associated with variation in olfactory behavior, but functional validation of these candidates remain a necessary next step in understanding the mechanisms by which these genes influence chemoreception. In previous genome-wide association analyses, a genomic region that spans multiple polymorphic loci on the left arm of the third chromosome was found to be significantly associated with variation in olfactory behavioral responses to the odorant 2,3-butanedione, a volatile compound present in fermenting fruit. In this study, behavioral analysis of flies possessing either the major or minor haplotype for this region confirmed the association between polymorphisms in the region and variation in olfactory behavior. Moreover, functional dissection of the genes within this region using P-element insertional mutagenesis together with targeted RNAi experiments revealed that the gene CG6767, a gene of previously unknown function but predicted to encode an enzyme responsible for the synthesis and metabolism of nucleic acids, affects olfactory behavioral responses to 2,3-butanedione. Specifically, RNAi mediated knockdown of CG6767 expression in different neuroanatomical populations of the olfactory system suggests that this gene functions in local interneurons of the antennal lobe. These results reveal a new role for CG6767 and its importance in olfactory behavior.
Churchill, E. R., Dytham, C. and Thom, M. D. F. (2019). Differing effects of age and starvation on reproductive performance in Drosophila melanogaster. Sci Rep 9(1): 2167. PubMed ID: 30770855
Successful reproduction requires the completion of many, often condition-dependent, stages, from mate searching and courtship through to sperm transfer, fertilisation and offspring production. Animals can plastically adjust their investment in each stage according to the physical and social environment, their own condition, their future reproductive potential, and the condition of their partner. This study manipulate age and condition, through a nutritional challenge early or late in life, of both male and female Drosophila melanogaster and measure the effects on courtship, mating, and fitness when paired with a standardized (unmanipulated) partner. Older males were slower to start courting and mating, and courted at a slower rate, but males were indifferent to female age or condition despite older females laying and hatching fewer eggs. Female condition had a substantial effect on mating acceptance rate, which dropped dramatically after starvation, and particularly recent starvation experience. In contrast, male condition had little effect on any of the components of reproductive performance measured. Intriguingly, no evidence was found for additive or multiplicative effects of ageing and starvation: the only significant interaction between these variables was on male latency to initiate courtship - older males were slower to start courting unless they had experienced starvation early in life. These results indicate that the immediate costs of mating differ between males and females, and that the sexes differ in their perception of the opportunity cost sustained by refusing a mating opportunity. These results support the idea that ageing has more wide-ranging impact on reproductive behaviours than does nutritional challenge.
Soto-Yeber, L., Soto-Ortiz, J., Godoy, P. and Godoy-Herrera, R. (2018). The behavior of adult Drosophila in the wild. PLoS One 13(12): e0209917. PubMed ID: 30596767
Little is known about how Drosophila adults behave in the wild, including mating, allocation of food and space, and escape from predators. This lack of information has negative implications for the ability to understand the capabilities of the nervous system to integrate sensory cues necessary for the adaptation of organisms in natural conditions. This study characterized a set of behavioral routines of D. melanogaster and D. simulans adults in three ecologically different orchards: grape, apple and prickly pear. How the flies identify conspecifics and aliens in the wild were also investigated to better understand relationships between group formation and adaptation of Drosophila to breeding sites. The locations were characterized by recording in each orchard humidity, temperature, illumination conditions, pH of fruits, the presence/absence of other Drosophila species and the predator ant Linepithema humile. The findings suggest that the home range of these species of Drosophila includes decaying fruits and, principally, a variety of microhabitats that surround the fruits. The ecological heterogeneity of the orchards and odors emitted by adult D. melanogaster and D. simulans influence perch preferences, cluster formation, court and mating, egg-laying site selection, and use of space. This is one of the first large examinations of the association between changing, complex environments and a set of adult behaviors of Drosophila. Therefore, these results have implications for understanding the genetic differentiation and evolution of populations of species in the genus Drosophila.
Eriksson, A., Anand, P., Gorson, J., Grijuc, C., Hadelia, E., Stewart, J. C., Holford, M. and Claridge-Chang, A. (2018). Using Drosophila behavioral assays to characterize terebrid venom-peptide bioactivity. Sci Rep 8(1): 15276. PubMed ID: 30323294
The number of newly discovered peptides from the transcriptomes and proteomes of animal venom arsenals is rapidly increasing, resulting in an abundance of uncharacterized peptides. There is a pressing need for a systematic, cost effective, and scalable approach to identify physiological effects of venom peptides. To address this discovery-to-function gap, a sequence driven:activity-based hybrid approach was developed for screening venom peptides that is amenable to large-venom peptide libraries with minimal amounts of peptide. Using this approach, the physiological and behavioral phenotypes of two peptides were characterized from the venom of predatory terebrid marine snails, teretoxins Tv1 from Terebra variegata and Tsu1.1 from Terebra subulata. The results indicate that Tv1 and Tsu1.1 have distinct bioactivity. Tv1 (100 microM) had an antinociceptive effect in adult Drosophila using a thermal nociception assay to measure heat avoidance. Alternatively, Tsu1.1 (100 microM) increased food intake. These findings describe the first functional bioactivity of terebrid venom peptides in relation to pain and diet and indicate that Tv1 and Tsu1.1 may, respectively, act as antinociceptive and orexigenic agents. Tv1 and Tsu1.1 are distinct from previously identified venom peptides, expanding the toolkit of peptides that can potentially be used to investigate the physiological mechanisms of pain and diet.
Sato, K., Ito, H., Yokoyama, A., Toba, G. and Yamamoto, D. (2019). Partial proteasomal degradation of Lola triggers the male-to-female switch of a dimorphic courtship circuit. Nat Commun 10(1): 166. PubMed ID: 30635583
In Drosophila, some neurons develop sex-specific neurites that contribute to dimorphic circuits for sex-specific behavior. As opposed to the idea that the sexual dichotomy in transcriptional profiles produced by a sex-specific factor underlies such sex differences, the sex-specific cleavage was found to confer the activity as a sexual-fate inducer on the pleiotropic transcription factor Longitudinals lacking (Lola). Surprisingly, Fruitless, another transcription factor with a master regulator role for courtship circuitry formation, directly binds to Lola to protect its cleavage in males. Lola cleavage involves E3 ubiquitin ligase Cullin1 and 26S proteasome. This work adds a new dimension to the study of sex-specific behavior and its circuit basis by unveiling a mechanistic link between proteolysis and the sexually dimorphic patterning of circuits. These findings may also provide new insights into potential causes of the sex-biased incidence of some neuropsychiatric diseases and inspire novel therapeutic approaches to such disorders.
Zhao, W., Zhou, P., Gong, C., Ouyang, Z., Wang, J., Zheng, N. and Gong, Z. (2019). A disinhibitory mechanism biases Drosophila innate light preference. Nat Commun 10(1): 124. PubMed ID: 30631066
Innate preference toward environmental conditions is crucial for animal survival. Although much is known about the neural processing of sensory information, how the aversive or attractive sensory stimulus is transformed through central brain neurons into avoidance or approaching behavior is largely unclear. This study shows that Drosophila larval light preference behavior is regulated by a disinhibitory mechanism. In the disinhibitory circuit, a pair of GABAergic neurons exerts tonic inhibition on one pair of contralateral projecting neurons that control larval reorientation behavior. When a larva enters the light area, the reorientation-controlling neurons are disinhibited to allow reorientation to occur as the upstream inhibitory neurons are repressed by light. When the larva exits the light area, the inhibition on the downstream neurons is restored to repress further reorientation and thus prevents the larva from re-entering the light area. It is suggested that disinhibition may serve as a common neural mechanism for animal innate preference behavior.

Friday, February 22nd - - Adult neural development and function

Wang, Z., Tacchelly-Benites, O., Noble, G. P., Johnson, M. K., Gagne, J. P., Poirier, G. G. and Ahmed, Y. (2018). A context-dependent role for the RNF146 ubiquitin ligase in Wingless/Wnt signaling in Drosophila. Genetics. PubMed ID: 30593492
Aberrant activation of the Wnt signal transduction pathway triggers the development of colorectal cancer. The ADP-ribose polymerase Tankyrase (TNKS) mediates proteolysis of Axin, a negative regulator of Wnt signaling, and provides a promising therapeutic target for Wnt-driven diseases. Proteolysis of TNKS substrates is mediated through their ubiquitination by the poly-ADP-ribose (pADPr)-dependent RING-domain E3 ubiquitin ligase RNF146/Iduna. Like TNKS, RNF146 promotes Axin proteolysis and Wnt pathway activation in some cultured cell lines, but in contrast with TNKS, RNF146 is dispensable for Axin degradation in colorectal carcinoma cells. Thus the contexts in which RNF146 is essential for TNKS-mediated Axin destabilization and Wnt signaling remain uncertain. This study tested the requirement for RNF146 in TNKS-mediated Axin proteolysis and Wnt pathway activation in a range of in vivo settings. Using null mutants in Drosophila, genetic and biochemical evidence is provided that Rnf146 and Tnks function in the same proteolysis pathway in vivo. Furthermore, like Tnks, Drosophila Rnf146 promotes Wingless signaling in multiple developmental contexts by buffering Axin levels to ensure they remain below the threshold at which Wingless signaling is inhibited. However, in contrast with Tnks, Rnf146 is dispensable for Wingless target gene activation and the Wingless-dependent control of intestinal stem cell proliferation in the adult midgut during homeostasis. Together, these findings demonstrate that the requirement for Rnf146 in Tnks-mediated Axin proteolysis and Wingless pathway activation is dependent on physiological context, and suggest that in some cell types, functionally redundant pADPr-dependent E3 ligases or other compensatory mechanisms promote the Tnks-dependent proteolysis of Axin in both mammalian and Drosophila cells.
Brodskiy, P. A., Wu, Q., Soundarrajan, D. K., Huizar, F. J., Chen, J., Liang, P., Narciso, C., Levis, M. K., Arredondo-Walsh, N., Chen, D. Z. and Zartman, J. J. (2019). Decoding calcium signaling dynamics during Drosophila wing disc development. Biophys J. PubMed ID: 30704858
The robust specification of organ development depends on coordinated cell-cell communication. This process requires signal integration among multiple pathways, relying on second messengers such as calcium ions. Calcium signaling encodes a significant portion of the cellular state by regulating transcription factors, enzymes, and cytoskeletal proteins. However, the relationships between the inputs specifying cell and organ development, calcium signaling dynamics, and final organ morphology are poorly understood. In this study a quantitative image-analysis pipeline was designed for decoding organ-level calcium signaling. With this pipeline, spatiotemporal features were extracted of calcium signaling dynamics during the development of the Drosophila larval wing disc, a genetic model for organogenesis. Specific classes of wing phenotypes were identified that resulted from calcium signaling pathway perturbations, including defects in gross morphology, vein differentiation, and overall size. Four qualitative classes of calcium signaling activity were. These classes can be ordered based on agonist stimulation strength Galphaq-mediated signaling. In vivo calcium signaling dynamics depend on both receptor tyrosine kinase/phospholipase C gamma and G protein-coupled receptor/phospholipase C beta activities. Spatially patterned calcium dynamics were found to correlate with known differential growth rates between anterior and posterior compartments. Integrated calcium signaling activity decreases with increasing tissue size, and it responds to morphogenetic perturbations that impact organ growth. Together, these findings define how calcium signaling dynamics integrate upstream inputs to mediate multiple response outputs in developing epithelial organs.
Zhou, Y., Popadowski, S. E., Deustchman, E. and Halfon, M. S. (2019). Distinct roles and requirements for Ras pathway signaling in visceral versus somatic muscle founder specification. Development 146(2). PubMed ID: 30630823
Pleiotropic signaling pathways must somehow engender specific cellular responses. In the Drosophila mesoderm, Ras pathway signaling specifies muscle founder cells from among the broader population of myoblasts. For somatic muscles, this is an inductive process mediated by the ETS-domain downstream Ras effectors Pointed and Aop (Yan). For the circular visceral muscles, despite superficial similarities, a significantly different specification mechanism is at work. Not only is visceral founder cell specification not dependent on Pointed or Aop, but Ras pathway signaling in its entirety can be bypassed. These results show that de-repression, not activation, is the predominant role of Ras signaling in the visceral mesoderm and that, accordingly, Ras signaling is not required in the absence of repression. The key repressor acts downstream of the transcription factor Lame duck and is likely a member of the ETS transcription factor family. These findings fit with a growing body of data that point to a complex interplay between the Ras pathway, ETS transcription factors, and enhancer binding as a crucial mechanism for determining unique responses to Ras signaling.
Zahoor, M. K., Poidevin, M., Lecerf, C., Garrido, D. and Montagne, J. (2019). A Drosophila genetic screen for suppressors of S6kinase-dependent growth identifies the F-box subunit Archipelago/FBXW7. Mol Genet Genomics. PubMed ID: 30656413
This study was designed to identify novel negative regulators of the Drosophila S6kinase (dS6K). S6K is a downstream effector of the mTORC1 pathway. Nutrients activate mTORC1, which in turn induces the phosphorylation of S6K to promote cell growth, whereas fasting represses mTORC1 activity. This study screened 11,000 RNA-interfering (RNAi) lines and retained those that enhanced a dS6K-dependent growth phenotype. Since RNAi induces gene knockdown, enhanced tissue growth supports the idea that the targeted gene acts as a growth suppressor. To validate the resulting candidate genes, dS6K phosphorylation and protein levels were monitored in double-stranded RNAi-treated S2 cells. Archipelago (Ago), an E3-ubiquitin-ligase subunit that loads ubiquitin units onto target substrates for proteasome-mediated degradation, was identified. Loss-of-ago/fbxw7 in larvae resulted in an increase in dS6K protein levels, but no change in the levels of phosphorylated dS6K or dS6K transcripts, suggesting that Ago/FBXW7 indirectly controls dS6K translation or stability. Through the identification of novel negative regulators of the downstream target, dS6K, this study may help deciphering the underlying mechanisms driving deregulations of mTORC1, which underlies several human diseases.
Ashe, S. and Yadav, S. (2019). Maintenance of Rhodopsin levels in Drosophila photoreceptor and phototransduction requires Protein Kinase D. Fly (Austin): 1-10. PubMed ID: 30663936
During Drosophila phototransduction, the G protein coupled receptor (GPCR) Rhodopsin (Rh1) transduces photon absorption into electrical signal via G-protein coupled activation of phospholipase C (PLC). Rh1 levels in the plasma membrane are critical for normal sensitivity to light. This paper reports that Protein Kinase D (dPKD) regulates Rh1 homeostasis in adult photoreceptors. Although eye development and retinal structure are unaffected in the dPKD hypomorph (dPKD(H)), it exhibited elevated levels of Rh1. Surprisingly, despite having elevated levels of Rh1, no defect was observed in the electrical response to light in these flies. By contrast the levels of another transmembrane protein of the photoreceptor plasma membrane, Transient receptor potential (TRP) was not altered in dPKD(H). These results indicate that dPKD is dispensable for eye development but is required for maintaining Rh1 levels in adult photoreceptors.
Bertran, M. T., Mouilleron, S., Zhou, Y., Bajaj, R., Uliana, F., Kumar, G. S., van Drogen, A., Lee, R., Banerjee, J. J., Hauri, S., O'Reilly, N., Gstaiger, M., Page, R., Peti, W. and Tapon, N. (2019). ASPP proteins discriminate between PP1 catalytic subunits through their SH3 domain and the PP1 C-tail. Nat Commun 10(1): 771. PubMed ID: 30770806
Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. This study shows that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. The crystal structure of the human ASPP2/PP1 complex was solved; ASPP2 was shown to recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1 (Bertran, 2019).

Thursday, February 21st

Batchelor, A. V. and Wilson, R. I. (2019). Sound localization behavior in Drosophila melanogaster depends on inter-antenna vibration amplitude comparisons. J Exp Biol 222(Pt 3). PubMed ID: 30733260
Drosophila melanogaster hear with their antennae: sound evokes vibration of the distal antennal segment, and this vibration is transduced by specialized mechanoreceptor cells. The left and right antennae vibrate preferentially in response to sounds arising from different azimuthal angles. Therefore, by comparing signals from the two antennae, it should be possible to obtain information about the azimuthal angle of a sound source. However, behavioral evidence of sound localization has not been reported in Drosophila. This study shows that walking D. melanogaster do indeed turn in response to lateralized sounds. This behavior is evoked by vibrations of the distal antennal segment. The rule for turning is different for sounds arriving from different locations: flies turn toward sounds in their front hemifield, but they turn away from sounds in their rear hemifield, and they do not turn at all in response to sounds from 90 or -90 deg. All of these findings can be explained by a simple rule: the fly steers away from the antenna with the larger vibration amplitude. Finally, this study shows that these behaviors generalize to sound stimuli with diverse spectro-temporal features, and that these behaviors are found in both sexes. These findings demonstrate the behavioral relevance of the antenna's directional tuning properties. They also pave the way for investigating the neural implementation of sound localization, as well as the potential roles of sound-guided steering in courtship and exploration.
Andrade, I. V., Riebli, N., Nguyen, B. M., Omoto, J. J., Cardona, A. and Hartenstein, V. (2019). Developmentally arrested precursors of pontine neurons establish an embryonic blueprint of the Drosophila central complex. Curr Biol 29(3): 412-425.e413. PubMed ID: 30661802
Serial electron microscopic analysis shows that the Drosophila brain at hatching possesses a large fraction of developmentally arrested neurons with a small soma, heterochromatin-rich nucleus, and unbranched axon lacking synapses. All 802 "small undifferentiated" (SU) neurons were digitally reconstructed and assigned to the known brain lineages. By establishing the coordinates and reconstructing trajectories of the SU neuron tracts, a framework is provided of landmarks for the ongoing analyses of the L1 brain circuitry. To address the later fate of SU neurons, focus was placed on the 54 SU neurons belonging to the DM1-DM4 lineages, which generate all columnar neurons of the central complex. Regarding their topologically ordered projection pattern, these neurons form an embryonic nucleus of the fan-shaped body ("FB pioneers"). Fan-shaped body pioneers survive into the adult stage, where they develop into a specific class of bi-columnar elements, the pontine neurons. Later born, unicolumnar DM1-DM4 neurons fasciculate with the fan-shaped body pioneers. Selective ablation of the fan-shaped body pioneers altered the architecture of the larval fan-shaped body primordium but did not result in gross abnormalities of the trajectories of unicolumnar neurons, indicating that axonal pathfinding of the two systems may be controlled independently. This comprehensive spatial and developmental analysis of the SU neurons adds to understanding of the establishment of neuronal circuitry.
Semelidou, O., Acevedo, S. F. and Skoulakis, E. M. (2018). Temporally specific engagement of distinct neuronal circuits regulating olfactory habituation in Drosophila. Elife 7. PubMed ID: 30576281
Habituation is the process that enables salience filtering, precipitating perceptual changes that alter the value of environmental stimuli. To discern the neuronal circuits underlying habituation to brief inconsequential stimuli, a novel olfactory habituation paradigm was developed, identifying two distinct phases of the response that engage distinct neuronal circuits. Responsiveness to the continuous odor stimulus is maintained initially, a phase termed habituation latency; it requires Rutabaga Adenylyl-Cyclase-depended neurotransmission from GABAergic Antennal Lobe Interneurons and activation of excitatory Projection Neurons (PNs) and the Mushroom Bodies. In contrast, habituation depends on the inhibitory PNs of the middle Antenno-Cerebral Track, requires inner Antenno-Cerebral Track PN activation and defines a temporally distinct phase. Collectively, these data support the involvement of Lateral Horn excitatory and inhibitory stimulation in habituation. These results provide essential cellular substrates for future analyses of the molecular mechanisms that govern the duration and transition between these distinct temporal habituation phases.
Shinomiya, K., Huang, G., Lu, Z., Parag, T., Xu, C. S., Aniceto, R., Ansari, N., Cheatham, N., Lauchie, S., Neace, E., Ogundeyi, O., Ordish, C., Peel, D., Shinomiya, A., Smith, C., Takemura, S., Talebi, I., Rivlin, P. K., Nern, A., Scheffer, L. K., Plaza, S. M. and Meinertzhagen, I. A. (2019). Comparisons between the ON- and OFF-edge motion pathways in the Drosophila brain. Elife 8. PubMed ID: 30624205
Understanding the circuit mechanisms behind motion detection is a long-standing question in visual neuroscience. In Drosophila melanogaster, recently discovered synapse-level connectomes in the optic lobe, particularly in ON-pathway (T4) receptive-field circuits, in concert with physiological studies, suggest a motion model that is increasingly intricate when compared with the ubiquitous Hassenstein-Reichardt model. By contrast, knowledge of OFF-pathway (T5) has been incomplete. This study presents a conclusive and comprehensive connectome that, for the first time, integrates detailed connectivity information for inputs to both the T4 and T5 pathways in a single EM dataset covering the entire optic lobe. With novel reconstruction methods using automated synapse prediction suited to such a large connectome, previous findings in the T4 pathway were successfully corroborate, and inputs and receptive fields for T5 were comprehensively identified. Although the two pathways are probably evolutionarily linked and exhibit many similarities, interesting differences and interactions were uncovered that may underlie their distinct functional properties.
Akin, O., Bajar, B. T., Keles, M. F., Frye, M. A. and Zipursky, S. L. (2019). Cell-type-specific patterned stimulus-independent neuronal activity in the Drosophila visual system during synapse formation. Neuron. PubMed ID: 30711355
Stereotyped synaptic connections define the neural circuits of the brain. In vertebrates, stimulus-independent activity contributes to neural circuit formation. It is unknown whether this type of activity is a general feature of nervous system development. This study reports patterned, stimulus-independent neural activity in the Drosophila visual system during synaptogenesis. Using in vivo calcium, voltage, and glutamate imaging, it was found that all neurons participate in this spontaneous activity, which is characterized by brain-wide periodic active and silent phases. Glia are active in a complementary pattern. Each of the 15 of over 100 specific neuron types in the fly visual system examined exhibited a unique activity signature. The activity of neurons that are synaptic partners in the adult was highly correlated during development. It is proposed that this cell-type-specific activity coordinates the development of the functional circuitry of the adult brain.
Aimon, S., Katsuki, T., Jia, T., Grosenick, L., Broxton, M., Deisseroth, K., Sejnowski, T. J. and Greenspan, R. J. (2019). Fast near-whole-brain imaging in adult Drosophila during responses to stimuli and behavior. PLoS Biol 17(2): e2006732. PubMed ID: 30768592
Whole-brain recordings give a global perspective of the brain in action. This study describes a method using light field microscopy to record near-whole brain calcium and voltage activity at high speed in behaving adult flies. Global activity maps were first obtained for various stimuli and behaviors. Notably, brain activity was found to increase on a global scale when the fly walked but not when it groomed. This global increase with walking was particularly strong in dopamine neurons, which showed little activity otherwise. Second, maps were extracted of spatially distinct sources of activity as well as their time series using principal component analysis and independent component analysis. The characteristic shapes in the maps matched the anatomy of subneuropil regions and, in some cases, a specific neuron type. Brain structures that responded to light and odor were consistent with previous reports, confirming the new technique's validity. Previously uncharacterized behavior-related activity wee also observed as well as patterns of spontaneous voltage activity.

Wednesday, February 20th - Evolution

Liu, Y., Huang, A., Booth, R. M., Mendes, G. G., Merchant, Z., Matthews, K. S. and Bondos, S. E. (2018). Evolution of the activation domain in a Hox transcription factor. Int J Dev Biol 62(11-12): 745-753. PubMed ID: 30604844
Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved.
Rane, R. V., Pearce, S. L., Li, F., Coppin, C., Schiffer, M., Shirriffs, J., Sgro, C. M., Griffin, P. C., Zhang, G., Lee, S. F., Hoffmann, A. A. and Oakeshott, J. G. (2019). Genomic changes associated with adaptation to arid environments in cactophilic Drosophila species. BMC Genomics 20(1): 52. PubMed ID: 30651071
Insights into the genetic capacities of species to adapt to future climate change can be gained by using comparative genomic and transcriptomic data to reconstruct the genetic changes associated with such adaptations in the past. This study investigated the genetic changes associated with adaptation to arid environments, specifically climatic extremes and new cactus hosts, through such an analysis of five repleta group Drosophila species. Disproportionately high rates of gene gains were found in internal branches in the species' phylogeny where cactus use and subsequently cactus specialisation and high heat and desiccation tolerance evolved. The terminal branch leading to the most heat and desiccation resistant species, Drosophila aldrichi, also shows disproportionately high rates of both gene gains and positive selection. Several Gene Ontology terms related to metabolism were enriched in gene gain events in lineages where cactus use was evolving, while some regulatory and developmental genes were strongly selected in the Drosophila aldrichi branch. Transcriptomic analysis of flies subjected to sublethal heat shocks showed many more downregulation responses to the stress in a heat sensitive versus heat resistant species, confirming the existence of widespread regulatory as well as structural changes in the species' differing adaptations. Gene Ontology terms related to metabolism were enriched in the differentially expressed genes in the resistant species while terms related to stress response were over-represented in the sensitive one. It is concluded that daptations to new cactus hosts and hot desiccating environments were associated with periods of accelerated evolutionary change in diverse biochemistries. The hundreds of genes involved suggest adaptations of this sort would be difficult to achieve in the timeframes projected for anthropogenic climate change.
Xu, M. and Shaw, K. L. (2019). The genetics of mating song evolution underlying speciation: Linking quantitative variation to candidate genes for behavioral isolation. Genetics. PubMed ID: 30647070
Differences in mating behaviors evolve early during speciation, eventually contributing to reproductive barriers between species. Knowledge of the genetic and genomic bases of these behaviors is therefore integral to a causal understanding of speciation. Acoustic behaviors are often part of the mating ritual in animal species. The temporal rhythms of mating songs are notably species-specific in many vertebrates and arthropods and often underlie assortative mating. Despite discoveries of mutations that disrupt the temporal rhythm of these songs, little is known about genes affecting naturally occurring variation in the temporal pattern of singing behavior. In the rapidly speciating Hawaiian cricket genus Laupala, the striking species variation in song rhythms constitutes a behavioral barrier to reproduction between species. This study mapped the largest-effect locus underlying interspecific variation in song rhythm between two Laupala species to a narrow genomic region, wherein no known candidate genes were found affecting song temporal rhythm in Drosophila. Whole genome sequencing, gene prediction and functional annotation of this region reveal an exciting and promising candidate gene, the putative cyclic nucleotide-gated ion channel-like gene, for natural variation in mating behavior. Identification and molecular characterization of the candidate gene reveals a non-synonymous mutation in a conserved binding domain, suggesting the hypothesis that ion channels are important targets of selection of rhythmic signaling during establishment of behavioral isolation and rapid speciation.
Ali, S., Signor, S. A., Kozlov, K. and Nuzhdin, S. V. (2019). Novel approach to quantitative spatial gene expression uncovers genetic stochasticity in the developing Drosophila eye. Evol Dev. PubMed ID: 30756455
Robustness in development allows for the accumulation of genetically based variation in expression. However, this variation is usually examined in response to large perturbations, and examination of this variation has been limited to being spatial, or quantitative, but because of technical restrictions not both. This study bridged these gaps by investigating replicated quantitative spatial gene expression using rigorous statistical models, in different genotypes, sexes, and species (Drosophila melanogaster and D. simulans). Using this type of quantitative approach with molecular developmental data allows for comparison among conditions, such as different genetic backgrounds. This approach was applied to the morphogenetic furrow, a wave of differentiation that patterns the developing eye disc. Within the morphogenetic furrow, focus was placed on four genes, hairy, atonal, hedgehog, and Delta. Hybridization chain reaction quantitatively measures spatial gene expression, co-staining for all four genes simultaneously. Considerable variation was found in the spatial expression pattern of these genes in the eye between species, genotypes, and sexes. It was also found that there has been evolution of the regulatory relationship between these genes and that their spatial interrelationships have evolved between species. This variation has no phenotypic effect, and could be buffered by network thresholds or compensation from other genes. Both of these mechanisms could potentially be contributing to long term developmental systems drift.
Morimoto, J., McDonald, G. C., Smith, E., Smith, D. T., Perry, J. C., Chapman, T., Pizzari, T. and Wigby, S. (2019). Sex peptide receptor-regulated polyandry modulates the balance of pre- and post-copulatory sexual selection in Drosophila. Nat Commun 10(1): 283. PubMed ID: 30655522
Polyandry prolongs sexual selection on males by forcing ejaculates to compete for fertilisation. Recent theory predicts that increasing polyandry may weaken pre-copulatory sexual selection on males and increase the relative importance of post-copulatory sexual selection, but experimental tests of this prediction are lacking. This study manipulated the polyandry levels in groups of Drosophila melanogaster by deletion of the female sex peptide receptor. Groups in which the sex-peptide-receptor is absent in females (SPR-) have higher polyandry, and - as a result - weaker pre-copulatory sexual selection on male mating success, compared to controls. Post-copulatory selection on male paternity share is relatively more important in SPR- groups, where males gain additional paternity by mating repeatedly with the same females. These results provide experimental evidence that elevated polyandry weakens pre-copulatory sexual selection on males, shifts selection to post-copulatory events, and that the sex peptide pathway can play a key role in modulating this process in Drosophila.
Wang, S. P. and Althoff, D. M. (2019). Phenotypic plasticity facilitates initial colonization of a novel environment. Evolution. PubMed ID: 30618131
Phenotypic plasticity can allow organisms to respond to environmental changes by producing better matching phenotypes without any genetic change. Because of this, plasticity is predicted to be a major mechanism by which a population can survive the initial stage of colonizing a novel environment. This prediction was tested by challenging wild Drosophila melanogaster with increasingly extreme larval environments and then examining expression of alcohol dehydrogenase (ADH) and its relationship to larval survival in the first generation of encountering a novel environment. Most families responded in the adaptive direction of increased ADH activity in higher alcohol environments and families with higher plasticity were also more likely to survive in the highest alcohol environment. Thus, plasticity of ADH activity was positively selected in the most extreme environment and was a key trait influencing fitness. Furthermore, there was significant heritability of ADH plasticity that can allow plasticity to evolve in subsequent generations after initial colonization. The adaptive value of plasticity, however, was only evident in the most extreme environment and had little impact on fitness in less extreme environments. The results provide one of the first direct tests of the adaptive role of phenotypic plasticity in colonizing a novel environment.

Tuesday, January 19th - Larval and Adult Development

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 was identified 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. It was also shown that 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.
Srivastava, M., James, A., Varma, V., Sharma, V. K. and Sheeba, V. (2018). Environmental cycles regulate development time via circadian clock mediated gating of adult emergence. BMC Dev Biol 18(1): 21. PubMed ID: 30577765
The contribution of circadian clocks in determining time taken for pre-adult development has remained unclear. This study presents results of studies aimed to understand this influence by examining populations of fruit flies carrying three different alleles of the period gene and hence having different free-running periods. Attempts were made to achieve similarity of genetic background among the three strains while also ensuring that they harbored sufficient variation on loci other than period gene. Under constant conditions, flies with long period were found to have slower development whereas in presence of light-dark cycles (LD) of various lengths, the speed of development for each genotype is influenced by whether their eclosion rhythms can entrain to them. Under LD 12:12 (T24), where all three strains entrain, they do not show any difference in time taken for emergence, whereas under LD 10:10 (T20) where long period flies do not entrain and LD 14:14 (T28) where short period flies do not entrain, they have slower and faster pre-adult development, respectively, compared to the controls. A prior stage in development, namely pupation, is not rhythmic, though time taken for pupation is determined by both the environmental cycle and period allele.
Setiawan, L., Pan, X., Woods, A. L., O'Connor, M. B. and Hariharan, I. K. (2018). The BMP2/4 ortholog Dpp can function as an inter-organ signal that regulates developmental timing. Life Sci Alliance 1(6): e201800216. PubMed ID: 30515478
Developmental transitions are often triggered by a neuroendocrine axis and can be contingent upon multiple organs achieving sufficient growth and maturation. How the neurodendocrine axis senses the size and maturity of peripheral organs is not known. In Drosophila larvae, metamorphosis is triggered by a sharp increase in the level of the steroid hormone ecdysone, secreted by the prothoracic gland (PG). This study shows that the BMP2/4 ortholog Dpp can function as a systemic signal to regulate developmental timing. Dpp from peripheral tissues, mostly imaginal discs, can reach the PG and inhibit ecdysone biosynthesis. As the discs grow, reduced Dpp signaling in the PG is observed, consistent with the possibility that Dpp functions in a checkpoint mechanism that prevents metamorphosis when growth is insufficient. Indeed, upon starvation early in the third larval instar, reducing Dpp signaling in the PG abrogates the critical-weight checkpoint which normally prevents pupariation under these conditions. It is suggested that increased local trapping of morphogen within tissues as they grow would reduce circulating levels and hence provide a systemic readout of their growth status.
Skouloudaki, K., Papadopoulos, D. K., Tomancak, P. and Knust, E. (2019). The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation. PLoS Genet 15(1): e1007852. PubMed ID: 30645584
Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. This study has identified apnoia (apn; CG15887), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. It is concluded that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.
Storelli, G., Nam, H. J., Simcox, J., Villanueva, C. J. and Thummel, C. S. (2018). Drosophila HNF4 directs a switch in lipid metabolism that supports the transition to adulthood. Dev Cell. PubMed ID: 30554999
Animals must adjust their metabolism as they progress through development in order to meet the needs of each stage in the life cycle. This study shows that the dHNF4 nuclear receptor acts at the onset of Drosophila adulthood to direct an essential switch in lipid metabolism. Lipid stores are consumed shortly after metamorphosis but contribute little to energy metabolism. Rather, dHNF4 directs their conversion to very long chain fatty acids and hydrocarbons, which waterproof the animal to preserve fluid homeostasis. Similarly, HNF4alpha is required in mouse hepatocytes for the expression of fatty acid elongases that contribute to a waterproof epidermis, suggesting that this pathway is conserved through evolution. This developmental switch in Drosophila lipid metabolism promotes lifespan and desiccation resistance in adults and suppresses hallmarks of diabetes, including elevated glucose levels and intolerance to dietary sugars. These studies establish dHNF4 as a regulator of the adult metabolic state.
Zappia, M. P., Rogers, A., Islam, A. and Frolov, M. V. (2019). Rbf activates the myogenic transcriptional program to promote skeletal muscle differentiation. Cell Rep 26(3): 702-719. PubMed ID: 30650361
The importance of the retinoblastoma tumor suppressor protein pRB in cell cycle control is well established. However, less is known about its role in differentiation during animal development. This study investigated the role of Rbf, the Drosophila pRB homolog, in adult skeletal muscles. Depletion of Rbf severely reduced muscle growth and altered myofibrillogenesis but only minimally affected myoblast proliferation. An Rbf-dependent transcriptional program in late muscle development was identified that is distinct from the canonical role of Rbf in cell cycle control. Unexpectedly, Rbf acts as a transcriptional activator of the myogenic and metabolic genes in the growing muscles. The genomic regions bound by Rbf contained the binding sites of several factors that genetically interacted with Rbf by modulating Rbf-dependent phenotype. Thus, these results reveal a distinctive role for Rbf as a direct activator of the myogenic transcriptional program that drives late muscle differentiation.

Friday, February 17th - Apoptosis and Autophagy

Nandy, A., Lin, L., Velentzas, P. D., Wu, L. P., Baehrecke, E. H. and Silverman, N. (2018). The NF-kappaB factor Relish regulates Atg1 expression and controls autophagy. Cell Rep 25(8): 2110-2120.e2113. PubMed ID: 30463009
Macroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. This study shows that the nuclear factor kappaB (NF-kappaB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation. Surprisingly, of the classic Imd pathway components, only Relish and the PGRP receptors were involved in salivary gland degradation. Significantly, Relish controls salivary gland degradation by regulating autophagy but not caspases. In addition, expression of either Relish or PGRP-LC causes premature autophagy induction and subsequent gland degradation. Relish controls autophagy by regulating the expression of Atg1, a core component and activator of the autophagy pathway. Together these findings demonstrate that a NF-kappaB pathway regulates autophagy during developmentally programmed cell death.
Lee, G., Sehgal, R., Wang, Z. and Park, J. H. (2019). Ultraspiracle-independent anti-apoptotic function of ecdysone receptors is required for the survival of larval peptidergic neurons via suppression of grim expression in Drosophila melanogaster. Apoptosis. PubMed ID: 30637539
Crustacean cardioactive peptide (CCAP)-producing neurons in the CNS are developmentally programmed to die shortly after adult emergence. Disruption of endogenous EcR function by ectopic expression of dominant negative forms of EcRs (EcR(DN)) causes premature death of larval CCAP neurons. This event is rescued by co-expression of individual EcR isoforms. Furthermore, larval CCAP neurons are largely normal in ecr mutants lacking either EcR-A or EcR-B isoforms, suggesting that EcR isoforms redundantly function to protect larval CCAP neurons. Ultraspiracle (Usp) is dispensable in the protection of CCAP neurons, whereas both EcR and Usp are required for inducing metamorphoptosis of vCrz neurons shortly after prepupal formation. grim is an essential cell death gene for the EcR(DN)-mediated CCAP neuronal death. These results suggest that Usp-independent EcR actions protect CCAP neurons from their premature death by repressing grim expression until their normally scheduled apoptosis at post-emergence. These studies highlight two opposite roles played by EcR function for apotosis of two different peptidergic neuronal groups, proapoptotic (vCrz) versus antiapoptotic (CCAP).
Robin, M., Issa, A. R., Santos, C. C., Napoletano, F., Petitgas, C., Chatelain, G., Ruby, M., Walter, L., Birman, S., Domingos, P. M., Calvi, B. R. and Mollereau, B. (2018). Drosophila p53 integrates the antagonism between autophagy and apoptosis in response to stress. Autophagy. PubMed ID: 30563404
The tumor suppressor TP53/p53 is a known regulator of apoptosis and macroautophagy/autophagy. However, the molecular mechanism by which TP53 regulates 2 apparently incompatible processes remains unknown. This study found that Drosophila lacking p53 displayed impaired autophagic flux, higher caspase activation and mortality in response to oxidative stress compared with wild-type flies. Moreover, autophagy and apoptosis were differentially regulated by the p53 (p53B) and DeltaNp53 (p53A) isoforms: while the former induced autophagy in differentiated neurons, which protected against cell death, the latter inhibited autophagy by activating the caspases Dronc, Drice, and Dcp-1. These results demonstrate that the differential use of p53 isoforms combined with the antagonism between apoptosis and autophagy ensures the generation of an appropriate p53 biological response to stress.
Ariss, M. M., Islam, A., Critcher, M., Zappia, M. P. and Frolov, M. V. (2018). Single cell RNA-sequencing identifies a metabolic aspect of apoptosis in Rbf mutant. Nat Commun 9(1): 5024. PubMed ID: 30479347
The function of Retinoblastoma tumor suppressor (pRB) is greatly influenced by the cellular context, therefore the consequences of pRB inactivation are cell-type-specific. This study employed single cell RNA-sequencing (scRNA-seq) to profile the impact of an Rbf mutation during Drosophila eye development. First, a catalogue was built of 11,500 wild type eye disc cells containing major known cell types. A transcriptional switch was found occurring in differentiating photoreceptors at the time of axonogenesis. Next, a cell landscape of Rbf mutant was mapped, and a mutant-specific cell population was identifgied that shows intracellular acidification due to increase in glycolytic activity. Genetic experiments demonstrate that such metabolic changes, restricted to this unique Rbf mutant population, sensitize cells to apoptosis and define the pattern of cell death in Rbf mutant eye disc. Thus, these results illustrate how scRNA-seq can be applied to dissect mutant phenotypes.

Thursday, February 14th - Adult Physiology

Brown, E. B., Slocumb, M. E., Szuperak, M., Kerbs, A., Gibbs, A. G., Kayser, M. S. and Keene, A. C. (2019). Starvation resistance is associated with developmentally specified changes in sleep, feeding and metabolic rate. J Exp Biol. PubMed ID: 30606795
Food shortage represents a primary challenge to survival, and animals have adapted diverse developmental, physiological, and behavioral strategies to survive when food becomes unavailable. Starvation resistance is strongly influenced by ecological and evolutionary history, yet the genetic basis for the evolution of starvation resistance remains poorly understood. The fruit fly, Drosophila melanogaster, provides a powerful model for leveraging experimental evolution to investigate traits associated with starvation resistance. While control populations only live a few days without food, selection for starvation resistance results in populations that can survive weeks. Previous work has shown that selection for starvation resistance results in increased sleep and reduced feeding in adult flies. This study investigated the ontogeny of starvation resistance-associated behavioral and metabolic phenotypes in these experimentally selected flies. Selection for starvation resistance was found to result in delayed development and a reduction in metabolic rate in larvae that persists into adulthood, suggesting that these traits may allow for the accumulation of energy stores and an increase in body size within these selected populations. In addition, larval sleep was found to be largely unaffected by starvation selection and feeding increases during the late larval stages, suggesting that experimental evolution for starvation resistance produces developmentally specified changes in behavioral regulation. Together, these findings reveal a critical role for development in the evolution of starvation resistance and indicate that selection can selectively influence behavior during defined developmental timepoints.
Towarnicki, S. G. and Ballard, J. W. O. (2018). Mitotype interacts With diet to influence longevity, fitness, and mitochondrial functions in adult female Drosophila. Front Genet 9: 593. PubMed ID: 30555517
Mitochondrial DNA (mtDNA) and the dietary macronutrient ratio are known to influence a wide range of phenotypic traits including longevity, fitness and energy production. Commonly mtDNA mutations are posited to be selectively neutral or reduce fitness and, to date, no selectively advantageous mtDNA mutations have been experimentally demonstrated in adult female Drosophila. This study proposes that a ND V161L mutation interacted with diets differing in their macronutrient ratios to influence organismal physiology and mitochondrial traits, but further studies are required to definitively show no linked mtDNA mutations are functionally significant. Two mtDNA types (mitotypes) fed either a 1:2 Protein: Carbohydrate (P:C) or 1:16 P:C diet were used. When fed the former diet, Dahomey females harboring the V161L mitotype lived longer than those with the Alstonville mitotype and had higher climbing, basal reactive oxygen species (ROS) and elevated glutathione S-transferase E1 expression. The short lived Alstonville females ate more, had higher walking speed and elevated mitochondrial functions as suggested by respiratory control ratio (RCR), mtDNA copy number and expression of mitochondrial transcription termination factor 3. In contrast, Dahomey females fed 1:16 P:C were shorter lived, had higher fecundity, walking speed and mitochondrial functions. They had reduced climbing. This result suggests that mtDNA cannot be assumed to be a strictly neutral evolutionary marker when the dietary macronutrient ratio of a species varies over time and space and supports the hypothesis that mtDNA diversity may reflect the amount of time since the last selective sweep rather than strictly demographic processes.
Xiao, G., Liu, Z. H., Zhao, M., Wang, H. L. and Zhou, B. (2019). Transferrin 1 functions in iron trafficking and genetically interacts with Ferritin in Drosophila melanogaster. Cell Rep 26(3): 748-758. PubMed ID: 30650364
Iron metabolism is an essential process that when dysregulated causes disease. Mammalian serum transferrin (TF) plays a primary role in delivering iron to cells. To improve understanding of the conservation of iron metabolism between species, the function of the TF homolog in Drosophila melanogaster, transferrin 1 (Tsf1) has been investigated. Tsf1 knockdown results in iron accumulation in the gut and iron deficiency in the fat body (which is analogous to the mammalian liver). Fat body-derived Tsf1 localizes to the gut surface, suggesting that Tsf1 functions in trafficking iron between the gut and the fat body, similar to TF in mammals. Moreover, Tsf1 knockdown strongly suppresses the phenotypic effects of ferritin (Fer1HCH) RNAi, an established iron trafficker in Drosophila. It is proposed that Tsf1 and ferritin compete for iron in the Drosophila intestine, and the value of using Drosophila for investigating iron trafficking and the evolution of systemic iron regulation is demonstrated.

Wednesday, February 13th - Chromatin

Janssen, A., Colmenares, S. U., Lee, T. and Karpen, G. H. (2019). Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A. Genes Dev 33(1-2): 103-115. PubMed ID: 30578303
Repair of DNA double-strand breaks (DSBs) must be orchestrated properly within diverse chromatin domains in order to maintain genetic stability. Euchromatin and heterochromatin domains display major differences in histone modifications, biophysical properties, and spatiotemporal dynamics of DSB repair. However, it is unclear whether differential histone-modifying activities are required for DSB repair in these distinct domains. Previous work has shown that the Drosophila melanogaster KDM4A (dKDM4A) histone demethylase is required for heterochromatic DSB mobility. This study used locus-specific DSB induction in Drosophila animal tissues and cultured cells to more deeply interrogate the mpact of dKDM4A on chromatin changes, temporal progression, and pathway utilization during DSB repair. dKDM4A was found to promote the demethylation of heterochromatin-associated histone marks at DSBs in heterochromatin but not euchromatin. Most importantly, it was demonstrated that dKDM4A is required to complete DSB repair in a timely manner and regulate the relative utilization of homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways but exclusively for heterochromatic DSBs. It is concluded that the temporal kinetics and pathway utilization during heterochromatic DSB repair depend on dKDM4A-dependent demethylation of heterochromatic histone marks. Thus, distinct pre-existing chromatin states require specialized epigenetic alterations to ensure proper DSB repair.
Shindo, Y. and Amodeo, A. A. (2019). Dynamics of free and chromatin-bound Histone H3 during early embryogenesis. Curr Biol. PubMed ID: 30639105
During zygotic genome activation (ZGA), the chromatin environment undergoes profound changes, including the formation of topologically associated domains, refinements in nucleosome positioning on promoters, and the emergence of heterochromatin. In many organisms, including Drosophila, ZGA is associated with the end of a period of extremely rapid, exponential cleavage divisions that are facilitated by large maternally provided pools of nuclear components. It is therefore imperative to understand how the supply of chromatin components relative to the exponentially increasing demand affects nuclear and chromatin composition during early embryogenesis. This study examined the nuclear trafficking and chromatin dynamics of histones during the cleavage divisions in Drosophila using a photo-switchable H3-Dendra2 reporter. Total H3-Dendra2 in the nucleus decreases with each cleavage cycle. This change in nuclear composition is due to depletion of large pools (>50%) of free protein that are present in the early cycles. The per nucleus import rate halves with each cycle, and a mathematical model was constructed in which increasing histone demand determines the dynamics of nuclear H3 supply. Finally, it was shown that these changes in H3 availability correspond to a large (~ 40%) reduction in global H3 occupancy on the chromatin, which is compensated by the increased incorporation of H3.3. The observed changes in free nuclear H3 and chromatin composition may contribute to the cell-cycle slowing, changes in chromatin structure, and the onset of transcription associated with this developmental stage.
Lee, D. H., Ryu, H. W., Kim, G. W. and Kwon, S. H. (2019). Comparison of three heterochromatin protein 1 homologs of Drosophila. J Cell Sci. PubMed ID: 30659116
Heterochromatin protein 1 (HP1) is an epigenetic regulator of chromatin structure and genome function in eukaryotes. Despite shared features, most eukaryotes have minimum three HP1 homologs with differential localization patterns and functions. Most studies focus on Drosophila HP1a, and little is known about the properties of HP1b and HP1c. To determine the features of the three HP1 homologs,the first comprehensive comparative analysis was performed of Drosophila HP1 homologs. HP1 differentially homodimerizes and heterodimerizes in vivo, and in vitro HP1b and HP1c, but not HP1a, are localized to both the nucleus and cytoplasm. The C-terminal extension region (CTE) targets HP1c and HP1b to the cytoplasm. Biochemical approaches show that HP1 binds to various interacting partners with a differential binding affinity. Each HP1 associates differently with RNA polymerase II; a gene reporter assay revealed that HP1a and HP1b, but not HP1c, inhibit transcriptional activity, suggesting that HP1c serves as a positive regulator in transcription. Thus, these studies provide the basic clues pertaining to the molecular mechanism by which HP1 might control cellular processes in a homolog-specific manner.
Lake, C. M., Nielsen, R. J., Bonner, A. M., Eche, S., White-Brown, S., McKim, K. S. and Hawley, R. S. (2019). Narya, a RING finger domain-containing protein, is required for meiotic DNA double-strand break formation and crossover maturation in Drosophila melanogaster. PLoS Genet 15(1): e1007886. PubMed ID: 30615609
Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. This study investigated the roles of two paralogous genes, CG12200 and CG31053, which have been named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. narya recently evolved from nenya by a gene duplication event, and these two RING finger domain-containing proteins were shown to be functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. Data is provided on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over.
Yang, F., Quan, Z., Huang, H., He, M., Liu, X., Cai, T. and Xi, R. (2019). Ovaries absent links dLsd1 to HP1a for local H3K4 demethylation required for heterochromatic gene silencing. Elife 8. PubMed ID: 30648969
Heterochromatin Protein 1 (HP1) is a conserved chromosomal protein in eukaryotic cells that has a major role in directing heterochromatin formation, a process that requires co-transcriptional gene silencing mediated by small RNAs and their associated argonaute proteins. Heterochromatin formation requires erasing the active epigenetic mark, such as H3K4me2, but the molecular link between HP1 and H3K4 demethylation remains unclear. In a fertility screen in female Drosophila, this study identified ovaries absent (ova/CG5694), which functions in the stem cell niche, downstream of Piwi, to support germline stem cell differentiation. Moreover, ova acts as a suppressor of position effect variegation, and is required for silencing telomeric transposons in the germline. Biochemically, Ova acts to link the H3K4 demethylase dLsd1 to HP1a for local histone modifications. Therefore, this study provides a molecular connection between HP1a and local H3K4 demethylation during HP1a-mediated gene silencing that is required for ovary development, transposon silencing, and heterochromatin formation.
Torres-Zelada, E. F., Stephenson, R. E., Alpsoy, A., Anderson, B. D., Swanson, S. K., Florens, L., Dykhuizen, E. C., Washburn, M. P. and Weake, V. M. (2019). The Drosophila Dbf4 ortholog Chiffon forms a complex with Gcn5 that is necessary for histone acetylation and viability. J Cell Sci 132(2). PubMed ID: 30559249
Metazoans contain two homologs of the Gcn5-binding protein Ada2, Ada2a and Ada2b, which nucleate formation of the ATAC and SAGA complexes, respectively. In Drosophila melanogaster, there are two splice isoforms of Ada2b: Ada2b-PA and Ada2b-PB. This study shows that only the Ada2b-PB isoform is in SAGA; in contrast, Ada2b-PA associates with Gcn5, Ada3, Sgf29 and Chiffon, forming the Chiffon histone acetyltransferase (CHAT) complex. Chiffon is the Drosophila ortholog of Dbf4, which binds and activates the cell cycle kinase Cdc7 to initiate DNA replication. In flies, Chiffon and Cdc7 are required in ovary follicle cells for gene amplification, a specialized form of DNA re-replication. Although chiffon was previously reported to be dispensable for viability, this study finds that Chiffon is required for both histone acetylation and viability in flies. Surprisingly, chiffon is a dicistronic gene that encodes distinct Cdc7- and CHAT-binding polypeptides. Although the Cdc7-binding domain of Chiffon is not required for viability in flies, the CHAT-binding domain is essential for viability, but is not required for gene amplification, arguing against a role in DNA replication.

Tuesday, February 12th - Cytoskeleton and Junctions

Harker, A. J., Katkar, H. H., Bidone, T. C., Aydin, F., Voth, G. A., Applewhite, D. A. and Kovar, D. R. (2019). Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia crosslinker fascin. Mol Biol Cell: mbcE18080500. PubMed ID: 30601697
Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different crosslinking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (alpha-actinin). Previous work has show that Ena takes approximately 3-fold longer processive runs on trailing barbed ends of fascin-bundled F-actin. This study used single-molecule TIRFM and developed a kinetic model to further dissect Ena/VASP's processive mechanism on bundled filaments. Ena's enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or alpha-actinin bundles. Notably, Ena/VASP's processive run length increases with the number of both fascin-bundled filaments and Ena 'arms', revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologs, including human VASP and C. elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, a novel regulatory process was discovered whereby bundle size and bundling protein specificity control activities of a processive assembly factor.
Rosen, J. N., Azevedo, M., Soffar, D. B., Boyko, V. P., Brendel, M. B., Schulman, V. K. and Baylies, M. K. (2019). The Drosophila Ninein homologue Bsg25D cooperates with Ensconsin in myonuclear positioning. J Cell Biol. PubMed ID: 30626718
Skeletal muscle consists of multinucleated cells in which the myonuclei are evenly spaced throughout the cell. In Drosophila, this pattern is established in embryonic myotubes, where myonuclei move via microtubules (MTs) and the MT-associated protein Ensconsin (Ens)/MAP7, to achieve their distribution. Ens regulates multiple aspects of MT biology, but little is known about how Ens itself is regulated. This study finds that Ens physically interacts and colocalizes with Blastoderm-specific gene 25D (Bsg25D), the Drosophila homologue of the centrosomal protein Ninein. Bsg25D loss enhances myonuclear positioning defects in embryos sensitized by partial Ens loss. Bsg25D overexpression causes severe positioning defects in immature myotubes and fully differentiated myofibers, where it forms ectopic MT organizing centers, disrupts perinuclear MT arrays, reduces muscle stiffness, and decreases larval crawling velocity. These studies define a novel relationship between Ens and Bsg25D. At endogenous levels, Bsg25D positively regulates Ens activity during myonuclear positioning, but excess Bsg25D disrupts Ens localization and MT organization, with disastrous consequences for myonuclear positioning and muscle function.
Hudson, A. M., Mannix, K. M., Gerdes, J. A., Kottemann, M. C. and Cooley, L. (2019). Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion. Development 146(1). PubMed ID: 30559276
During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3(Kelch)) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. This study used tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3(Kelch) ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. Genetic evidence is presented consistent with HtsRC being the CRL3(Kelch) substrate; biochemical evidence indicates that HtsRC is ubiquitylated and degraded by the proteasome. Finally, a short sequence motif was identified in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.
Sumi, A., Hayes, P., D'Angelo, A., Colombelli, J., Salbreux, G., Dierkes, K. and Solon, J. (2018). Adherens junction length during tissue contraction is controlled by the mechanosensitive activity of actomyosin and junctional recycling. Dev Cell 47(4): 453-463.e453. PubMed ID: 30458138
During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. This study shows that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. This study reveals that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis.
Scarpa, E., Finet, C., Blanchard, G. B. and Sanson, B. (2018). Actomyosin-driven tension at compartmental boundaries orients cell division independently of cell geometry in vivo. Dev Cell. PubMed ID: 30503752
Cell shape is known to influence the plane of cell division. In vitro, mechanical constraints can also orient mitoses; however, in vivo it is not clear whether tension can orient the mitotic spindle directly, because tissue-scale forces can change cell shape. During segmentation of the Drosophila embryo, actomyosin is enriched along compartment boundaries forming supracellular cables that keep cells segregated into distinct compartments. This study shows that these actomyosin cables orient the planar division of boundary cells perpendicular to the boundaries. This bias overrides the influence of cell shape, when cells are mildly elongated. By decreasing actomyosin cable tension with laser ablation or, conversely, ectopically increasing tension with laser wounding, this study demonstrates that local tension is necessary and sufficient to orient mitoses in vivo. This involves capture of the spindle pole by the actomyosin cortex. These findings highlight the importance of actomyosin-mediated tension in spindle orientation in vivo.
Wu, L., Dong, A., Dong, L., Wang, S. Q. and Li, Y. (2019). PARIS, an optogenetic method for functionally mapping gap junctions. Elife 8. PubMed ID: 30638447
Cell-cell communication via gap junctions (see Optic ganglion reduced & Innexin 2) regulates a wide range of physiological processes by enabling the direct intercellular electrical and chemical coupling. However, the in vivo distribution and function of gap junctions remain poorly understood, partly due to the lack of non-invasive tools with both cell-type specificity and high spatiotemporal resolution. This study developed PARIS (pairing actuators and receivers to optically isolate gap junctions), a new fully genetically encoded tool for measuring the cell-specific gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC in several cultured cell lines under physiologically relevant conditions and in distinct genetically defined neurons in Drosophila brain, with ~10-sec temporal resolution and sub-cellular spatial resolution. These results demonstrate that PARIS is a robust, highly sensitive tool for mapping functional gap junctions and study their regulation in both health and disease.

Monday, February 11th - Signaling

Reynolds, E. R., Himmelwright, R., Sanginiti, C. and Pfaffmann, J. O. (2019). An agent-based model of the Notch signaling pathway elucidates three levels of complexity in the determination of developmental patterning. BMC Syst Biol 13(1): 7. PubMed ID: 30642357
The Notch signaling pathway is involved in cell fate decision and developmental patterning in diverse organisms. A receptor molecule, Notch (N), and a ligand molecule (in this case Delta or Dl) are the central molecules in this pathway. In early Drosophila embryos, these molecules determine neural vs. skin fates in a reproducible rosette pattern. This study has created an agent-based model (ABM) that simulates the molecular components for this signaling pathway as agents acting within a spatial representation of a cell. The model captures the changing levels of these components, their transition from one state to another, and their movement from the nucleus to the cell membrane and back to the nucleus again. The model introduces stochastic variation into the system using a random generator within the Netlogo programming environment. The model uses these representations to understand the biological systems at three levels: individual cell fate, the interactions between cells, and the formation of pattern across the system. Using a set of assessment tools, the current model was shown to accurately reproduce the rosette pattern of neurons and skin cells in the system over a wide set of parameters. Oscillations in the level of the N agent eventually stabilize cell fate into this pattern. The dynamic timing and the availability of the N and Dl agents in neighboring cells are central to the formation of a correct and stable pattern. A feedback loop to the production of both components is necessary for a correct and stable pattern. The signaling pathways within and between cells in this model interact in real time to create a spatially correct field of neurons and skin cells. This model predicts that cells with high N and low Dl drive the formation of the pattern. This model also be used to elucidate general rules of biological self-patterning and decision-making.
Smylla, T. K., Meier, M., Preiss, A. and Maier, D. (2019). The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments. Dev Genes Evol. PubMed ID: 30612166
During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. This study analysed three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins H(FA), H(LLAA) and H(WA). For in vivo analysis, UAS-lines H(FA), H(LLAA) and H(WA) were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the H(LD) control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. It is concluded that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.
Wang, X., Zhang, Y. and Blair, S. S. (2019). Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination. Proc Natl Acad Sci U S A. PubMed ID: 30606799
The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. Previous work has identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat's regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. This study characterizes a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, Dlish was found to binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. It was further shown that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex.
Suisse, A. and Treisman, J. E. (2019). Reduced SERCA function preferentially affects Wnt signaling by retaining E-Cadherin in the endoplasmic reticulum. Cell Rep 26(2): 322-329.e323. PubMed ID: 30625314
Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. This study shows that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/beta-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed definition of three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics.
Tsuzuki, K., Itoh, Y., Inoue, Y. and Hayashi, H. (2018). TRB1 negatively regulates gluconeogenesis by suppressing the transcriptional activity of FOXO1. FEBS Lett. PubMed ID: 30556236
Tribbles related homolog 1 is the mammalian ortholog of Tribbles, which controls cell division and migration during development in Drosophila. TRB1 is a pseudokinase and functions as a scaffold protein. Recent findings suggest that TRB1 plays important roles in hepatic lipid metabolism and participates in insulin resistance. However, the underlying mechanisms have not yet been elucidated. This study demonstrates that TRB1 suppresses FOXO1 (see Drosophila Foxo) transcriptional activity to downregulate the expression of G6Pase and PEPCK, which encode gluconeogenic rate-limiting enzymes. TRB1 knockdown enhances FOXO1 binding to the gluconeogenic gene promoters. It also increases FOXO1 acetylation and recruits CBP to the binding sequence of FOXO1. These results suggest that TRB1 suppresses the expression of G6Pase and PEPCK by attenuating FOXO1 transcriptional activity and negatively regulates gluconeogenesis.
Ryu, T. H., Yeom, E., Subramanian, M., Lee, K. S. and Yu, K. (2018). Prominin-like regulates longevity and glucose metabolism via insulin signaling in Drosophila. J Gerontol A Biol Sci Med Sci. PubMed ID: 30590420
CD133, also called Prominin-1, is a biomarker for mammalian stem cells. It is involved in cell growth, development, and tumor biology. However, the function of CD133 at the organismal level has not been investigated. This study found that prominin-like (promL) loss-of-function mutant flies show an extended lifespan and metabolic defects such as increased circulating carbohydrates, lipid storage, and starvation resistance. The mRNA expression levels of Drosophila insulin-like peptides (Dilps) were reduced in loss-of-function promL mutants. Furthermore, the level of phosphorylated AKT, a downstream component of insulin signaling, was lower in promL loss-of-function mutants than in the w- control flies. Importantly, the PromL protein is predominantly expressed in the pars intercerebralis region with insulin producing cells (IPCs) of the adult brain. When promL was inhibited in IPCs, these flies showed an extended lifespan, metabolic defects, and reduced insulin signaling. These results indicate that the promL gene regulates longevity and glucose metabolism by controlling insulin signaling in Drosophila.

Friday, February 8th - Cell Cycle

Bonner, A. M. and Hawley, R. S. (2018). Functional consequences of the evolution of matrimony, a meiosis-specific inhibitor of Polo kinase. Mol Biol Evol. PubMed ID: 30351378
Meiosis is a defining characteristic of eukaryotes, believed to have evolved only once, over one billion years ago. While the general progression of meiotic events is conserved across multiple diverse organisms, the specific pathways and proteins involved can be highly divergent, even within species from the same genus. This study investigate the rapid evolution of Matrimony (Mtrm), a female meiosis-specific regulator of Polo kinase (Polo) in Drosophila. Mtrm physically interacts with Polo and is required to restrict the activity of Polo during meiosis. Despite Mtrm's critical role in meiosis, sequence conservation within the genus Drosophila is poor. To explore the functional significance of this rapid divergence, Mtrm proteins from 12 different Drosophila species were expressed in the D. melanogaster female germline. Distantly related Mtrm homologs are able to both physically interact with D. melanogaster Polo and rescue the meiotic defects seen in mtrm mutants. However, these distant homologs are not properly degraded after the completion of meiosis. Rather, they continue to inhibit Polo function in the early embryo, resulting in dominant maternal-effect lethality. The ability of Mtrm to be properly degraded, and thus release Polo, is partially due to residues or motifs found within Mtrm's least-conserved regions. It is hypothesized that, while Mtrm regions critical for its meiotic function are under strong purifying selection, changes that occurred in its unconserved regions may have been advantageous, potentially by affecting the timing or duration of meiosis and/or the early embryonic divisions.
Merigliano, C., Mascolo, E., Cesta, A., Saggio, I. and Verni, F. (2019). A new role for Drosophila Aurora-A in maintaining chromosome integrity. Chromosoma. PubMed ID: 30612150
Aurora-A is a conserved mitotic kinase overexpressed in many types of cancer. Growing evidence shows that Aurora-A plays a crucial role in DNA damage response (DDR) although this aspect has been less characterized. A new aur-A mutation, named aur-A949, was isolated in Drosophila, and it was shown to causes chromosome aberrations (CABs). In addition, aur-A949 mutants were sensitive to X-ray treatment and showed impaired gamma-H2Av foci dissolution kinetics. To identify the pathway in which Aur-A works, an epistasis analysis was carried out by evaluating CAB frequencies in double mutants carrying aur-A(949) mutation combined to mutations in genes related to DNA damage response (DDR). Mutations in tefu (ATM) and in the histone variant H2Av were epistatic over aur-A949 indicating that Aur-A works in DDR and that it is required for gamma-H2Av foci dissolution. More interestingly, it was found that a mutation in lig4, a gene belonging to the non-homologous end joining (NHEJ) repair pathway, was epistatic over aur-A949. Based on studies in other systems, which show that phosphorylation is important to target Lig4 for degradation, it was hypothesized that in aur-A949 mutant cells, there is a persistence of Lig4 that could be, in the end, responsible for CABs. Finally, a synergistic interaction was observed between Aur-A and the homologous recombination (HR) repair system component Rad 51 in the process that converts chromatid deletions into isochromatid deletions. Altogether, these data indicate that Aur-A depletion can elicit chromosome damage. This conclusion should be taken into consideration, since some anticancer therapies are aimed at reducing Aurora-A expression.
Stormo, B. M. and Fox, D. T. (2018). Interphase cohesin regulation ensures mitotic fidelity after genome reduplication. Mol Biol Cell: mbcE17100582. PubMed ID: 30462577
To ensure faithful genome propagation, mitotic cells alternate one round of chromosome duplication with one round of chromosome separation. Chromosome separation failure thus causes genome reduplication, which alters mitotic chromosome structure. Such structural alterations are well-documented to impair mitotic fidelity following aberrant genome re-duplication, including in diseased states. In contrast, recent work has shown that naturally occurring genome re-duplication does not alter mitotic chromosome structure in Drosophila papillar cells. This discovery raised the question of how a cell undergoing genome reduplication might regulate chromosome structure to prevent mitotic errors. This study shows that papillar cells ensure mitotic fidelity through interphase cohesin regulation. A requirement is demonstrated for cohesins during programmed rounds of papillar genome reduplication known as endocycles. This interphase cohesin regulation relies on cohesin release but not cohesin cleavage, and depends on the conserved cohesin regulator Pds5. These data suggest that a distinct form of interphase cohesin regulation ensures mitotic fidelity after genome reduplication.
Ramani, A., Mariappan, A., Gottardo, M., Mandad, S., Urlaub, H., Avidor-Reiss, T., Riparbelli, M., Callaini, G., Debec, A., Feederle, R. and Gopalakrishnan, J. (2018). Plk1/Polo phosphorylates Sas-4 at the onset of mitosis for an efficient recruitment of pericentriolar material to centrosomes. Cell Rep 25(13): 3618-3630.e3616. PubMed ID: 30590037
Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. This study identified that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4 localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and gamma-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and gamma-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.
Li, N., Liu, Q., Xiong, Y. and Yu, J. (2019). Headcase and Unkempt regulate tissue growth and cell cycle progression in response to nutrient restriction. Cell Rep 26(3): 733-747. PubMed ID: 30650363
Nutrient restriction (NR) decreases the incidence and growth of many types of tumors, yet the underlying mechanisms are not fully understood. This study identified Headcase (Hdc) and Unkempt (Unk) as two NR-specific tumor suppressor proteins that form a complex to restrict cell cycle progression and tissue growth in response to NR in Drosophila. Loss of Hdc or Unk does not confer apparent growth advantage under normal nutrient conditions but leads to accelerated cell cycle progression and tissue overgrowth under NR. Hdc and Unk bind to the TORC1 component Raptor and preferentially regulate S6 phosphorylation in a TORC1-dependent manner. It was further shown that HECA and UNK, the human counterparts of Drosophila Hdc and Unk, respectively, have a conserved function in regulating S6 phosphorylation and tissue growth. The identification of Hdc and Unk as two NR-specific tumor suppressors provides insight into molecular mechanisms underlying the anti-tumorigenic effects of NR.
Sollazzo, M., Genchi, C., Paglia, S., Di Giacomo, S., Pession, A., de Biase, D. and Grifoni, D. (2018). High MYC levels favour multifocal carcinogenesis. Front Genet 9: 612. PubMed ID: 30619451
The term "field cancerisation" describes the formation of tissue sub-areas highly susceptible to multifocal tumourigenesis. This behaviour recalls cell competition, a process based on a reciprocal fitness comparison: when cells with a growth advantage arise in a tissue, they are able to commit wild-type neighbours to death and to proliferate at their expense. It is known that cells expressing high MYC levels behave as super-competitors, able to kill and replace less performant adjacent cells; given MYC upregulation in most human cancers, MYC-mediated cell competition is likely to pioneer field cancerisation. This study shows that MYC overexpression in a sub-territory of the larval wing epithelium of Drosophila is sufficient to trigger a number of cellular responses specific to mammalian pre-malignant tissues. Moreover, following induction of different second mutations, high MYC-expressing epithelia were found to be susceptible to multifocal growth, a hallmark of mammalian pre-cancerous fields. In summary, this study identified an early molecular alteration implicated in field cancerisation and established a genetically amenable model which may help study the molecular basis of early carcinogenesis.

Thursday, February 7th - Adult Neural Function

Schiemann, R., Lammers, K., Janz, M., Lohmann, J., Paululat, A. and Meyer, H. (2018). Identification and in vivo characterisation of cardioactive peptides in Drosophila melanogaster. Int J Mol Sci 20(1). PubMed ID: 30577424
Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. The present study analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. Numerous peptides were identifed that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, the in vivo relevance of the respective peptidergic regulation was assessed. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans.
Kennedy, T. and Broadie, K. (2018). Newly identified electrically coupled neurons support development of the Drosophila giant fiber model circuit. eNeuro 5(6). PubMed ID: 30627638
The Drosophila giant fiber (GF) escape circuit is an extensively studied model for neuron connectivity and function. Researchers have long taken advantage of the simple linear neuronal pathway, which begins at peripheral sensory modalities, travels through the central GF interneuron (GFI) to motor neurons, and terminates on wing/leg muscles. This circuit is more complex than it seems, however, as there exists a complex web of coupled neurons connected to the GFI that widely innervates the thoracic ganglion. This study defines four new neuron clusters dye coupled to the central GFI, which were named GF coupled (GFC) 1-4. New transgenic Gal4 drivers were identified that express specifically in these neurons, and both neuronal architecture and synaptic polarity were mapped. GFC1-4 share a central site of GFI connectivity, the inframedial bridge, where the neurons each form electrical synapses. Targeted apoptotic ablation of GFC1 reveals a key role for the proper development of the GF circuit, including the maintenance of GFI connectivity with upstream and downstream synaptic partners. GFC1 ablation frequently results in the loss of one GFI, which is always compensated for by contralateral innervation from a branch of the persisting GFI axon. Overall, this work reveals extensively coupled interconnectivity within the GF circuit, and the requirement of coupled neurons for circuit development. Identification of this large population of electrically coupled neurons in this classic model, and the ability to genetically manipulate these electrically synapsed neurons, expands the GF system capabilities for the nuanced, sophisticated circuit dissection necessary for deeper investigations into brain formation.
Mecklenburg, K. L., Weghorst, F. P., Freed, S. A. and O'Tousa, J. E. (2018). Discordant responses to MAPK pathway stimulation include axonal growths in adult Drosophila photoreceptors. Front Mol Neurosci 11: 441. PubMed ID: 30564098
Wallenda (WND) is the Drosophila member of a conserved family of dual leucine-zipper kinases (DLK) active in both neuronal regeneration and degeneration. This study examined the role of WND over-expression on sensory neuron morphology by driving WND in multiple subtypes of Drosophila photoreceptors. WND overexpression under control of the pan-retinal GAL4 driver GMR causes multiple photoreceptor defects including cell death, rhabdomere degeneration, and axonal sprouting. Individual photoreceptor subtypes were assayed using GAL4 drivers specific for each photoreceptor class. Many R7 and R8 cells exhibit axonal sprouting while some show cell degeneration. Delaying the onset of WND overexpression until 20 days of age showed that older adult R7 cells retain the ability to initiate new axon growth. R1-6 photoreceptor cells degenerate in response to WND expression and exhibit rhodopsin loss and rhabdomere degeneration. RNAi knockdown of the MAPK signaling components Kayak (KAY) and Hemipterous (HEP) attenuates the WND-induced loss of Rh1 rhodopsin. UAS-induced HEP expression is similar to WND expression, causing degeneration in R1-6 photoreceptors and axonal sprouting in R7 photoreceptors. These results demonstrate that WND in adult Drosophila photoreceptor cells acts through MAPK signaling activity with both regenerative and degenerative responses. These photoreceptors provide a tractable experimental model to reveal cellular mechanisms driving contradictory WND signaling responses.
Humberg, T. H. and Sprecher, S. G. (2018). Two pairs of Drosophila central brain neurons mediate larval navigational strategies based on temporal light information processing. Front Behav Neurosci 12: 305. PubMed ID: 30568583
Some animals are attracted by sun light, others are highly repulsed by it. Especially for slowly moving animals, such as Drosophila larvae, direct sunlight may be perceived as noxious stimulus as it increases the risk of desiccation, DNA-damaging by UV-light and exposure to predators. For several reasons, model organisms like Drosophila larvae are well-suited for investigating how light cues are translated into an appropriate behavioral output. First, many of the genetic tools, which were created for use in adult fruit flies, work also in larvae. Second, the lower number of cells in Drosophila larvae compared to adults makes this system adequate for reconstructing neural circuits. Third, the relatively simple behavioral repertoire of larvae facilitates the study of basic functions like navigation with regards to light. Larvae navigate robustly away from a light source by the use of several sophisticated behavioral strategies which are based on temporal or spatial information processing. Two central brain neurons, the NP394-neurons, are highly important for larval light avoidance. It was even reported that these cells seem to play a functional role in a putative larval light preference switch right before pupation. However, the exact function of the NP394-neurons in light navigation remains unknown. This study shows that the functional role of NP394-neurons in larval light navigation is restricted to behaviors based on temporal information processing, but not for spatial navigation.
Schlichting, M., Menegazzi, P., Rosbash, M. and Helfrich-Forster, C. (2019). A distinct visual pathway mediates high light intensity adaptation of the circadian clock in Drosophila. J Neurosci. PubMed ID: 30606757
In order to provide organisms a fitness advantage, circadian clocks have to react appropriately to changes in their environment. High light intensities (HI) play an essential role in the adaptation to hot summer days, which especially endanger insects of desiccation or prey visibility. This study shows that solely increasing light intensity leads to an increased midday siesta in Drosophila behavior. Interestingly, this change is independent of the fly's circadian photoreceptor cryptochrome (CRY), and solely caused by a small visual organ, the Hofbauer-Buchner (HB) eyelets. Using receptor knockdowns, immunostaining, as well as recently developed calcium tools, the eyelets were shown to activate key core clock neurons, namely the s-LNvs, at HI. This activation delays the decrease of PER in the middle of the day and propagates to downstream target clock neurons that prolong the siesta. Together a new pathway is shown for integrating light intensity information into the clock network, suggesting new network properties and surprising parallels between Drosophila and the mammalian system.
Manjila, S. B., Kuruvilla, M., Ferveur, J. F., Sane, S. P. and Hasan, G. (2018). Extended flight bouts require disinhibition from GABAergic mushroom body neurons. Curr Biol. PubMed ID: 30612904
Insect flight is a complex behavior that requires the integration of multiple sensory inputs with flight motor output. Although previous genetic studies identified central brain monoaminergic neurons that modulate Drosophila flight, neuro-modulatory circuits underlying sustained flight bouts remain unexplored. Certain classes of dopaminergic and octopaminergic neurons that project to the mushroom body, a higher integrating center in the insect brain, are known to modify neuronal output based on contextual cues and thereby organismal behavior. This study focuses on how monoaminergic modulation of mushroom body GABAergic output neurons (MBONs) regulates the duration of flight bouts. Octopaminergic neurons in the sub-esophageal zone stimulate central dopaminergic neurons (protocerebral anterior medial, PAM) that project to GABAergic MBONs. Either inhibition of octopaminergic and dopaminergic neurons or activation of GABAergic MBONs reduces the duration of flight bouts. Moreover, activity in the PAM neurons inhibits the GABAergic MBONs. These data suggest that disinhibition of the identified neural circuit very likely occurs after flight initiation and is required to maintain the "flight state" when searching for distant sites, possibly related to food sources, mating partners, or a suitable egg-laying site.

Wednesday, February 6th - Behavior

Narasimha, S., Nagornov, K. O., Menin, L., Mucciolo, A., Rohwedder, A., Humbel, B. M., Stevens, M., Thum, A. S., Tsybin, Y. O. and Vijendravarma, R. K. (2019). Drosophila melanogaster cloak their eggs with pheromones, which prevents cannibalism. PLoS Biol 17(1): e2006012. PubMed ID: 30629594
Oviparous animals across many taxa have evolved diverse strategies that deter egg predation, providing valuable tests of how natural selection mitigates direct fitness loss. Communal egg laying in nonsocial species minimizes egg predation. However, in cannibalistic species, this very behavior facilitates egg predation by conspecifics (cannibalism). Similarly, toxins and aposematic signaling that deter egg predators are often inefficient against resistant conspecifics. Egg cannibalism can be adaptive, wherein cannibals may benefit through reduced competition and added nutrition, but since it reduces Darwinian fitness, the evolution of anticannibalistic strategies is rife. However, such strategies are likely to be nontoxic because deploying toxins against related individuals would reduce inclusive fitness. This study reports how D. melanogaster use specific hydrocarbons to chemically mask their eggs from cannibal larvae. Using an integrative approach combining behavioral, sensory, and mass spectrometry methods, this study demonstrates that maternally provisioned pheromone 7,11-heptacosadiene (7,11-HD) in the eggshell's wax layer deters egg cannibalism. Furthermore, 7,11-HD is shown to be nontoxic, can mask underlying substrates (for example, yeast) when coated upon them, and its detection requires pickpocket 23 (ppk23) gene function. Finally, using light and electron microscopy, it was demonstrated how maternal pheromones leak-proof the egg, consequently concealing it from conspecific larvae. These data suggest that semiochemicals possibly subserve in deceptive functions across taxa, especially when predators rely on chemical cues to forage and stimulate further research on deceptive strategies mediated through nonvisual sensory modules. This study thus highlights how integrative approaches can illuminate understanding of the adaptive significance of deceptive defenses and the mechanisms through which they operate.
Boomgarden, A. C., Sagewalker, G. D., Shah, A. C., Haider, S. D., Patel, P., Wheeler, H. E., Dubowy, C. M. and Cavanaugh, D. J. (2019). Chronic circadian misalignment results in reduced longevity and large-scale changes in gene expression in Drosophila. BMC Genomics 20(1): 14. PubMed ID: 30616504
Circadian clocks ensure that behavioral and physiological processes occur at optimal times of day and in the correct temporal order. It is becoming increasingly clear that chronic circadian misalignment (CCM), such as occurs in shift workers, has profound metabolic and cognitive consequences, but the proximate mechanisms connecting CCM with reduced organismal health are unknown. This study investigated the consequences of CCM in the powerful model system of the fruit fly, Drosophila melanogaster. Flies were subjected to daily 4-h phase delays in the light-dark schedule, and the Drosophila Activity Monitoring (DAM) system was used to continuously track locomotor activity and sleep while simultaneously monitoring fly lifespan. Consistent with previous results, exposing flies to CCM leads to a ~ 15% reduction in median lifespan in both male and female flies. Importantly, it was demonstrated that the reduced longevity occurs independent of changes in overall sleep or activity. To uncover potential molecular mechanisms of CCM-induced reduction in lifespan, whole body RNA-sequencing was conducted to assess differences in gene transcription between control and misaligned flies. CCM caused progressive, large-scale changes in gene expression characterized by upregulation of genes involved in response to toxic substances, aging and oxidative stress, and downregulation of genes involved in regulation of development and differentiation, gene expression and biosynthesis. Many of these gene expression changes mimic those that occur during natural aging, consistent with the idea that CCM results in premature organismal decline, however, genes involved in lipid metabolism are overrepresented among those that are differentially regulated by CCM and aging. This category of genes is also among the earliest to exhibit CCM-induced changes in expression, thus highlighting altered lipid metabolism as a potentially important mediator of the negative health consequences of CCM.
Verschut, T. A., Inouye, B. D. and Hamback, P. A. (2018). Sensory deficiencies affect resource selection and associational effects at two spatial scales. Ecol Evol 8(21): 10569-10577. PubMed ID: 30464828
Many insect species have limited sensory abilities and may not be able to perceive the quality of different resource types while approaching patchily distributed resources. These restrictions may lead to differences in selection rates between separate patches and between different resource types within a patch, which may have consequences for associational effects between resources. This study used an oviposition assay containing different frequencies of apple and banana substrates divided over two patches to compare resource selection rates of wild-type Drosophila melanogaster at the between- and within-patch scales. Next, the wild-type behavior was compared with that of the olfactory-deficient strain Orco (2) and the gustatory-deficient strain Poxn (DeltaM22-B5), and comparable responses were found to patch heterogeneity and similarly strong selection rates for apple at both scales for the wild-type and olfactory-deficient flies. Their oviposition behavior translated into associational susceptibility for apple and associational resistance for banana. The gustatory-deficient flies, on the other hand, no longer had a strong selection rate for apple, strongly differed in between- and within-patch selection rates from the wild-type flies, and caused no associational effects between the resources. This study suggests that differences in sensory capabilities can affect resource selection at different search behavior scales in different ways and in turn underlie associational effects between resources at different spatial scales.
Clark, D. A., Odell, S. R., Armstrong, J. M., Turcotte, M., Kohler, D., Mathis, A., Schmidt, D. R. and Mathew, D. (2018). Behavior responses to chemical and optogenetic stimuli in Drosophila larvae. Front Behav Neurosci 12: 324. PubMed ID: 30622461
An animal's ability to navigate an olfactory environment is critically dependent on the activities of its first-order olfactory receptor neurons (ORNs). While considerable research has focused on ORN responses to odorants, the mechanisms by which olfactory information is encoded in the activities of ORNs and translated into navigational behavior remain poorly understood. This study sought to determine the contributions of Drosophila larval ORNs to navigational behavior. Using odorants to activate ORNs and a larval tracking assay to measure the corresponding behavioral response, larval ORN activators were observed cluster into four groups based on the behavior responses elicited from larvae. This is significant because it provides new insights into the functional relationship between ORN activity and behavioral response. Subsequent optogenetic analyses of a subset of ORNs revealed previously undescribed properties of larval ORNs. Furthermore, the results indicated that different temporal patterns of ORN activation elicit different behavioral outputs: some ORNs respond to stimulus increments while others respond to stimulus decrements. These results suggest that the ability of ORNs to encode temporal patterns of stimulation increases the coding capacity of the olfactory circuit. Moreover, the ability of ORNs to sense stimulus increments and decrements facilitates instantaneous evaluations of concentration changes in the environment. Together, these ORN properties enable larvae to efficiently navigate a complex olfactory environment.
Diaz, M. M., Schlichting, M., Abruzzi, K. C., Long, X. and Rosbash, M. (2019). Allatostatin-C/AstC-R2 is a novel pathway to modulate the circadian activity pattern in Drosophila. Curr Biol 29(1): 13-22.e13. PubMed ID: 30554904
Seven neuropeptides are expressed within the Drosophila brain circadian network. Previous mRNA profiling suggested that Allatostatin-C (AstC) is an eighth neuropeptide and specifically expressed in dorsal clock neurons (DN1s). The results of this study show that AstC is, indeed, expressed in DN1s, where it oscillates. AstC is also expressed in two less well-characterized circadian neuronal clusters, the DN3s and lateral-posterior neurons (LPNs). Behavioral experiments indicate that clock-neuron-derived AstC is required to mediate evening locomotor activity under short (winter-like) and long (summer-like) photoperiods. The AstC-Receptor 2 (AstC-R2) is expressed in LNds, the clock neurons that drive evening locomotor activity, and AstC-R2 is required in these neurons to modulate the same short photoperiod evening phenotype. Ex vivo calcium imaging indicates that AstC directly inhibits a single LNd. The results suggest that a novel AstC/AstC-R2 signaling pathway, from dorsal circadian neurons to an LNd, regulates the evening phase in Drosophila.
Ramin, M., Li, Y., Chang, W. T., Shaw, H. and Rao, Y. (2019). The peacefulness gene promotes aggression in Drosophila. Mol Brain 12(1): 1. PubMed ID: 30606245
Natural aggressiveness is commonly observed in all animal species, and is displayed frequently when animals compete for food, territory and mating. Aggression is an innate behaviour, and is influenced by both environmental and genetic factors. However, the genetics of aggression remains largely unclear. This study identified the peacefulness (pfs) gene as a novel player in the control of male-male aggression in Drosophila. Mutations in pfs decreased intermale aggressiveness, but did not affect locomotor activity, olfactory avoidance response and sexual behaviours. pfs encodes for the evolutionarily conserved molybdenum cofactor (MoCo) synthesis 1 protein (Mocs1), which catalyzes the first step in the MoCo biosynthesis pathway. Neuronal-specific knockdown of pfs decreased aggressiveness. By contrast, overexpression of pfs greatly increased aggressiveness. Knocking down Cinnamon (Cin) catalyzing the final step in the MoCo synthesis pathway, caused a pfs-like aggression phenotype. In humans, inhibition of MoCo-dependent enzymes displays anti-aggressive effects. Thus, the control of aggression by Pfs-dependent MoCo pathways may be conserved throughout evolution.

Tuesday, February 5th - Transcriptional Regulation

Cambon, M. and Sanchez, O. (2019). Analysis of the transcriptional logic governing differential spatial expression in Hh target genes. PLoS One 14(1): e0209349. PubMed ID: 30615641
This work provides theoretical tools to analyse the transcriptional effects of certain biochemical mechanisms (i.e. affinity and cooperativity) that have been proposed in previous literature to explain the proper spatial expression of Hedgehog target genes involved in Drosophila development. Specifically this study has focused on the expression of decapentaplegic, wingless, stripe and patched. The transcription of these genes is believed to be controlled by enhancer modules able to interpret opposing gradients of the activator and repressor forms of the transcription factor Cubitus interruptus (Ci). This study is based on a thermodynamic approach, which provides expression rates for these genes. These expression rates are controlled by transcription factors which are competing and cooperating for common binding sites. Mathematical representations have been made of the different expression rates which depend on multiple factors and variables. The expressions obtained with the model have been refined to produce simpler equivalent formulae which allow for their mathematical analysis. Thanks to this, the correlation between the different interactions involved in transcription and the biological features observed at tissue level can be evaluated. These mathematical models can be applied to other morphogenes to help understand the complex transcriptional logic of opposing activator and repressor gradients.
Papadopoulos, D. K., Skouloudaki, K., Engstrom, Y., Terenius, L., Rigler, R., Zechner, C., Vukojevic, V. and Tomancak, P. (2019). Control of Hox transcription factor concentration and cell-to-cell variability by an auto-regulatory switch. Development. PubMed ID: 30642837
The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, since this requires the use of quantitative methods with single-molecule sensitivity. Using Fluorescence Correlation Spectroscopy (FCS), this study characterized the concentration and variability of 14 endogenously tagged TFs in live Drosophila imaginal discs. For the Hox TF Antennapedia this study investigated whether protein variability results from random stochastic events or is developmentally regulated. Antennapedia was found to transition from low concentration/high variability early, to high concentration/low variability later, in development. FCS and temporally resolved genetic studies uncovered that Antennapedia itself is necessary and sufficient to drive a developmental regulatory switch from auto-activation to auto-repression, thereby reducing variability. This switch is controlled by progressive changes in relative concentrations of preferentially activating and repressing Antennapedia isoforms, which bind chromatin with different affinities. Mathematical modelling demonstrated that the experimentally supported auto-regulatory circuit can explain the increase of Antennapedia concentration and suppression of variability over time.
Zandvakili, A., Uhl, J. D., Campbell, I., Salomone, J., Song, Y. C. and Gebelein, B. (2018). The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila. Dev Biol. PubMed ID: 30468713
During development diverse transcription factor inputs are integrated by cis-regulatory modules (CRMs) to yield cell-specific gene expression. Defining how CRMs recruit the appropriate combinations of factors to either activate or repress gene expression remains a challenge. This study compares and contrasts the ability of two CRMs within the Drosophila embryo to recruit functional Hox transcription factor complexes. The Distal-less DCRE CRM recruits Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox complexes that include the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the Distal-less leg selector gene, whereas the RhoA CRM selectively recruits Abd-A/Exd/Hth complexes to activate rhomboid and stimulate Epidermal Growth Factor secretion in sensory cell precursors. By swapping binding sites between these elements, it was found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE. It was further shown that the orientation and spacing of Hox sites relative to additional binding sites within the RhoA and DCRE is critical to mediate cell- and segment-specific output. These results indicate that the configuration of Exd, Hth, and Hox site within RhoA is neither Abd-A specific nor activation specific. Instead Hox specific output is largely dependent upon the presence of appropriately spaced and oriented binding sites for additional TF inputs. Taken together, these studies provide insight into the cis-regulatory logic used to generate cell-specific outputs via recruiting Hox transcription factor complexes.
Arbel, H., Basu, S., Fisher, W. W., Hammonds, A. S., Wan, K. H., Park, S., Weiszmann, R., Booth, B. W., Keranen, S. V., Henriquez, C., Shams Solari, O., Bickel, P. J., Biggin, M. D., Celniker, S. E. and Brown, J. B. (2019). Exploiting regulatory heterogeneity to systematically identify enhancers with high accuracy. Proc Natl Acad Sci U S A 116(3): 900-908. PubMed ID: 30598455
Identifying functional enhancer elements in metazoan systems is a major challenge. Large-scale validation of enhancers predicted by ENCODE reveal false-positive rates of at least 70%. This study used the pregrastrula-patterning network of Drosophila melanogaster to demonstrate that loss in accuracy in held-out data results from heterogeneity of functional signatures in enhancer elements. At least two classes of enhancers are active during early Drosophila embryogenesis and by focusing on a single, relatively homogeneous class of elements, greater than 98% prediction accuracy can be achieved in a balanced, completely held-out test set. The class of well-predicted elements is composed predominantly of enhancers driving multistage segmentation patterns, which were designated segmentation driving enhancers (SDE). Prediction is driven by the DNA occupancy of early developmental transcription factors, with almost no additional power derived from histone modifications. Improved accuracy is not a property of a particular prediction method: after conditioning on the SDE set, naive Bayes and logistic regression perform as well as more sophisticated tools. Applying this method to a genome-wide scan, 1,640 SDEs are predicted that cover 1.6% of the genome. An analysis of 32 SDEs using whole-mount embryonic imaging of stably integrated reporter constructs chosen throughout a prediction rank-list showed >90% drove expression patterns. 86.7% precision was achieved on a genome-wide scan, with an estimated recall of at least 98%, indicating high accuracy and completeness in annotating this class of functional elements.
Rinaldi, L., Saurin, A. J. and Graba, Y. (2018). Fattening the perspective of Hox protein specificity through SLiMming. Int J Dev Biol 62(11-12): 755-766. PubMed ID: 30604845
The functional identification and dissection of protein domains has been a successful approach towards the understanding of Hox protein specificity. However, only a few functional protein domains have been identified; this has been a major limitation in deciphering the molecular modalities of Hox protein action. This study explored, by in silico survey of short linear motifs (SLiMs) in Hox proteins, the contribution of SLiMs to Hox proteins, focusing on the mouse, chick and Drosophila Hox complement. The findings reveal a widespread and uniform distribution of SLiMs along Hox protein sequences and identify the most apparent features of Hox associated SLiMs. While few motifs have been associated with Hox proteins so far, this work suggests that many more contribute to Hox protein functions. The potential and difficulties to apprehend the full contribution of SLiMs in controlling Hox protein functions are discussed.
Mir, M., Stadler, M. R., Ortiz, S. A., Hannon, C. E., Harrison, M. M., Darzacq, X. and Eisen, M. B. (2018). Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos. Elife 7. PubMed ID: 30589412
The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. This study used lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, this study shows that both factors interact with DNA with surprisingly high off-rates. Both factors form dynamic subnuclear hubs, and Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on these observations, it is hypothesized that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.

Monday, February 4th - Disease Models

Hong, H., Koon, A. C., Chen, Z. S., Wei, Y., An, Y., Li, W., Lau, M. H. Y., Lau, K. F., Ngo, J. C. K., Wong, C. H., Au-Yeung, H. Y., Zimmerman, S. C. and Chan, H. Y. E. (2018). AQAMAN, a bisamidine-based inhibitor of toxic protein inclusions in neurons, ameliorates cytotoxicity in polyglutamine disease models. J Biol Chem. PubMed ID: 30593503
Polyglutamine (polyQ) diseases are a group of dominantly inherited neurodegenerative disorders caused by the expansion of an unstable CAG repeat in the coding region of the affected genes. Hallmarks of polyQ diseases include the accumulation of misfolded protein aggregates, leading to neuronal degeneration and cell death. PolyQ diseases are currently incurable, highlighting the urgent need for approaches that inhibit the formation of or disaggregate cytotoxic polyQ protein inclusions. This study screened for bisamidine-based inhibitors that can inhibit neuronal polyQ protein inclusions. One inhibitor, AQAMAN, prevents polyQ protein aggregation and promotes deaggregation of self-assembled polyQ proteins in several models of polyQ diseases. Using immunocytochemistry, AQAMAN was found to significantly reduce polyQ protein aggregation and specifically suppresses polyQ protein-induced cell death. Using a recombinant and purified polyQ protein (Trx-Huntingtin-Q46), it was further demonstrated that AQAMAN interferes with polyQ self-assembly, preventing polyQ aggregation, and dissociates preformed polyQ aggregates in a cell-free system. Remarkably, AQAMAN feeding of Drosophila expressing expanded polyQ disease protein suppresses polyQ-induced neurodegeneration in vivo. In addition, using inhibitors and activators of the autophagy pathway, it was demonstrated that AQAMAN's cytoprotective effect against polyQ toxicity is autophagy-dependent. In summary, this study has identified AQAMAN as a potential therapeutic for combating polyQ protein toxicity in polyQ diseases. These findings further highlight the importance of the autophagy pathway in clearing harmful polyQ proteins.
Huang, K., Chen, W., Zhu, F., Li, P. W., Kapahi, P. and Bai, H. (2019). RiboTag translatomic profiling of Drosophila oenocytes under aging and induced oxidative stress. BMC Genomics 20(1): 50. PubMed ID: 30651069
Aging is accompanied with loss of tissue homeostasis and accumulation of cellular damages. As one of the important metabolic centers, liver shows age-related dysregulation of lipid metabolism, impaired detoxification pathway, increased inflammation and oxidative stress response. However, the mechanisms for these age-related changes still remain unclear. In the fruit fly, Drosophila melanogaster, liver-like functions are controlled by two distinct tissues, fat body and oenocytes. Compared to fat body, little is known about how oenocytes age and what are their roles in aging regulation. To characterize age- and stress-regulated gene expression in oenocytes, cell-type-specific ribosome profiling (RiboTag) was performed to examine the impacts of aging and oxidative stress on oenocyte translatome in Drosophila. Aging and oxidant paraquat significantly increased the levels of reactive oxygen species (ROS) in adult oenocytes of Drosophila, and aged oenocytes exhibited reduced sensitivity to paraquat treatment. Through RiboTag sequencing, 3324 and 949 differentially expressed genes were identified in oenocytes under aging and paraquat treatment, respectively. Aging and paraquat exhibit both shared and distinct regulations on oenocyte translatome. Among all age-regulated genes, oxidative phosphorylation, ribosome, proteasome, fatty acid metabolism, and cytochrome P450 pathways were down-regulated, whereas DNA replication and immune response pathways were up-regulated. In addition, most of the peroxisomal genes were down-regulated in aged oenocytes, including genes involved in peroxisomal biogenesis factors and fatty acid beta-oxidation. Many age-related mRNA translational changes in oenocytes are similar to aged mammalian liver, such as up-regulation of innate immune response and Ras/MAPK signaling pathway and down-regulation of peroxisome and fatty acid metabolism. Furthermore, oenocytes highly expressed genes involving in liver-like processes (e.g., ketogenesis). Yhis translatome analysis provides important genomic resource for future dissection of oenocyte function and its role in lipid metabolism, stress response and aging regulation.
Nagy, S., Maurer, G. W., Hentze, J. L., Rose, M., Werge, T. M. and Rewitz, K. (2018). AMPK signaling linked to the schizophrenia-associated 1q21.1 deletion is required for neuronal and sleep maintenance. PLoS Genet 14(12): e1007623. PubMed ID: 30566533
The human 1q21.1 deletion of ten genes is associated with increased risk of schizophrenia. This deletion involves the beta-subunit of the AMP-activated protein kinase (AMPK) complex, a key energy sensor in the cell. Although neurons have a high demand for energy and low capacity to store nutrients, the role of AMPK in neuronal physiology is poorly defined. This study shows that AMPK is important in the nervous system for maintaining neuronal integrity and for stress survival and longevity in Drosophila. To understand the impact of this signaling system on behavior and its potential contribution to the 1q21.1 deletion syndrome, this study focused on sleep, an important role of which is proposed to be the reestablishment of neuronal energy levels that are diminished during energy-demanding wakefulness. Sleep disturbances are one of the most common problems affecting individuals with psychiatric disorders. This study shows that AMPK is required for maintenance of proper sleep architecture and for sleep recovery following sleep deprivation. Neuronal AMPKbeta loss specifically leads to sleep fragmentation and causes dysregulation of genes believed to play a role in sleep homeostasis. These data also suggest that AMPKbeta loss may contribute to the increased risk of developing mental disorders and sleep disturbances associated with the human 1q21.1 deletion.
Pacifico, R., MacMullen, C. M., Walkinshaw, E., Zhang, X. and Davis, R. L. (2018). Brain transcriptome changes in the aging Drosophila melanogaster accompany olfactory memory performance deficits. PLoS One 13(12): e0209405. PubMed ID: 30576353
Cognitive decline is a common occurrence of the natural aging process in animals and studying age-related changes in gene expression in the brain might shed light on disrupted molecular pathways that play a role in this decline. The fruit fly is a useful neurobiological model for studying aging due to its short generational time and relatively small brain size. This study investigated age-dependent changes in the Drosophila melanogaster whole-brain transcriptome by comparing 5-, 20-, 30- and 40-day-old flies of both sexes. RNA-sequencing of dissected brain samples followed by differential expression, temporal clustering, co-expression network and gene ontology enrichment analyses were performed. An overall decline was observed in expression of genes from the mitochondrial oxidative phosphorylation pathway that occurred as part of aging. In females, a pattern of continuously declining expression was detected for many neuronal function genes, which was unexpectedly reversed later in life. This group of genes was highly enriched in memory-impairing genes previously identified through an RNAi screen. Deficits in short-term olfactory memory performance was observed in older flies of both sexes, some of which matched the timing of certain changes in the brain transcriptome. This study provides the first transcriptome profile of aging brains from fruit flies of both sexes, and it will serve as an important resource for those who study aging and cognitive decline in this model.
Huang, Y., Wan, Z., Wang, Z. and Zhou, B. (2019). Insulin signaling in Drosophila melanogaster mediates Abeta toxicity. Commun Biol 2: 13. PubMed ID: 30652125
Alzheimer's disease (AD) and diabetes are clinically positively correlated. However, the connection between them is not clarified. Using Drosophila as a model system, this study shows that reducing insulin signaling can effectively suppress the toxicity from Abeta (Amyloid beta 42) expression. On the other hand, Abeta accumulation led to the elevation of fly insulin-like peptides (ILPs) and activation of insulin signaling in the brain. Mechanistically, these observations are attributed to a reciprocal competition between Drosophila insulin-like peptides and Abeta for the activity of insulin-degrading enzyme (IDE). Intriguingly, peripheral insulin signaling is decreased despite its heightened activity in the brain. While many upstream factors may modify Abeta toxicity, the results suggest that insulin signaling is the main downstream executor of Abeta damage, and thus may serve as a promising target for Alzheimer's treatment in non-diabetes patients. This study explains why more Alzheimer's cases are found in diabetes patients.
Kopp, Z. and Park, Y. (2019). Longer lifespan in the Rpd3 and Loco signaling results from the reduced catabolism in young age with noncoding RNA. Aging (Albany NY) 11(1): 230-239. PubMed ID: 30620723
Downregulation of Rpd3 (histone deacetylase) or Loco (regulator of G-protein signaling protein) extends Drosophila lifespan with higher stress resistance. rpd3-downregulated long-lived flies genetically interact with loco-upregulated short-lived flies in stress resistance and lifespan. Gene expression profiles revealed that they regulate common target genes in metabolic enzymes and signaling pathways. Functional analyses of more significantly changed genes indicated that the activities of catabolic enzymes and uptake/storage proteins are reduced in long-lived flies with Rpd3 downregulation. This reduced catabolism exhibited from a young age is considered to be necessary for the resultant longer lifespan of the Rpd3- and Loco-downregulated old flies, which mimics the dietary restriction (DR) effect that extends lifespan in the several species. Inversely, those catabolic activities that break down carbohydrates, lipids, and peptides were high in the short lifespan of Loco-upregulated flies. Long noncoding gene, dntRL (CR45923), was also found as a putative target modulated by Rpd3 and Loco for the longevity. Interestingly, this dntRL could affect stress resistance and lifespan, suggesting that the dntRL lncRNA may be involved in the metabolic mechanism of Rpd3 and Loco signaling.

Friday, February 1st - RNA

Menzel, P., McCorkindale, A. L., Stefanov, S. R., Zinzen, R. P. and Meyer, I. M. (2018). Transcriptional dynamics of microRNAs and their targets during Drosophila neurogenesis. RNA Biol. PubMed ID: 30582411
During Drosophila melanogaster embryogenesis, tight regulation of gene expression in time and space is required for the orderly emergence of specific cell types. While the general importance of microRNAs in regulating eukaryotic gene expression has been well-established, their role in early neurogenesis remains to be addressed. This survey investigated the transcriptional dynamics of microRNAs and their target transcripts during neurogenesis of Drosophila melanogaster. To this end, the recently developed DIV-MARIS protocol, a method for enriching specific cell types from the Drosophila embryo in vivo, was used to sequence cell-type-specific transcriptomes. Dedicated small and total RNA-seq libraries were generated for neuroblasts, neurons and glia cells at early (6-8 h after egg laying (AEL)) and late (18-22 h AEL) stage. This allowed direct comparison of these transcriptomes and investigation of the potential functional roles of individual microRNAs with spatio-temporal resolution genome-wide, which is beyond the capabilities of existing in-situ hybridization studies. Overall, 74 microRNAs were identified that are significantly differentially expressed between the three cell types and the two developmental stages. In all cell types, predicted target genes of down-regulated microRNAs show a significant enrichment of their target genes related to neurogenesis. How microRNAs regulate the transcriptome was also investigated by targeting transcription factors; many candidate microRNAs were found with putative roles in neurogenesis. This survey highlights the roles of miRNAs as regulators of differentiation and glioneurognesis in the fruit fly and provides distinct starting points for dedicated functional follow-up studies.
Matera, A. G., Raimer, A. C., Schmidt, C. A., Kelly, J. A., Droby, G. N., Baillat, D., Ten Have, S., Lamond, A. I., Wagner, E. J. and Gray, K. M. (2018). Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 30563832
Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, this study investigated its composition in Drosophila melanogaster. Using transgenic flies that exclusively express Flag-tagged SMN from its native promoter, it was previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. This study show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D. melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.
Lv, M., Yao, Y., Li, F., Xu, L., Yang, L., Gong, Q., Xu, Y. Z., Shi, Y., Fan, Y. J. and Tang, Y. (2019). Structural insights reveal the specific recognition of roX RNA by the dsRNA-binding domains of the RNA helicase MLE and its indispensable role in dosage compensation in Drosophila. Nucleic Acids Res. PubMed ID: 30649456
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). This study reports a 2.90A crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, this research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
Joosten, J., Miesen, P., Taskopru, E., Pennings, B., Jansen, P. W., Huynen, M. A., Vermeulen, M. and Van Rij, R. P. (2018). The Tudor protein Veneno assembles the ping-pong amplification complex that produces viral piRNAs in Aedes mosquitoes. Nucleic Acids Res. PubMed ID: 30566680
PIWI-interacting RNAs (piRNAs) comprise a class of small RNAs best known for suppressing transposable elements in germline tissues. The vector mosquito Aedes aegypti encodes seven PIWI genes, four of which are somatically expressed. This somatic piRNA pathway generates piRNAs from viral RNA during infection with cytoplasmic RNA viruses through ping-pong amplification by the PIWI proteins Ago3 and Piwi5. Yet, additional insights into the molecular mechanisms mediating non-canonical piRNA production are lacking. TUDOR-domain containing (Tudor) proteins facilitate piRNA biogenesis in Drosophila melanogaster and other model organisms. It was thus hypothesized that Tudor proteins are required for viral piRNA production, and a knockdown screen was performed targeting all A. aegypti Tudor genes. Knockdown of the Tudor genes AAEL012437, Vreteno, Yb, SMN and AAEL008101-RB resulted in significantly reduced viral piRNA levels, with AAEL012437-depletion having the strongest effect. This protein, which was named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts with Piwi5. It is proposed that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from viral RNA.
Lim, D. H., Lee, S., Han, J. Y., Choi, M. S., Hong, J. S. and Lee, Y. S. (2018). MicroRNA miR-252 targets mbt to control the developmental growth of Drosophila. Insect Mol Biol. PubMed ID: 30582233
Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The ecdysone-responsive miRNA, miR-252, is normally upregulated during the pupal and adult stages of Drosophila development. This study found that overexpression of miR-252 in the larval fat body decreased total tissue mass through a reduction in both cell size and cell number, causing a concomitant decrease in larval size. Furthermore, miR-252 overexpression led to a delayed larval-to-pupal transition with defective anterior spiracle eversion, as well as a decrease in adult size and mass. Conversely, adult flies lacking miR-252 showed an increase in mass compared with control flies. miR-252 directly targeted mbt, encoding a p21-activated kinase, to repress its expression. Notably, co-overexpression of mbt rescued the developmental and growth defects associated with miR-252 overexpression, indicating that mbtis a biologically relevant target of miR-252. Overall, the data support a role for the ecdysone/miR-252/mbt regulatory axis in growth control during Drosophila development.
Moschall, R., Rass, M., Rossbach, O., Lehmann, G., Kullmann, L., Eichner, N., Strauss, D., Meister, G., Schneuwly, S., Krahn, M. P. and Medenbach, J. (2018). Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing. Nucleic Acids Res. PubMed ID: 30590805
In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. This study has identified the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern.
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