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Tuesday, December 11th - Chromatin

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Pindyurin, A. V., Ilyin, A. A., Ivankin, A. V., Tselebrovsky, M. V., Nenasheva, V. V., Mikhaleva, E. A., Pagie, L., van Steensel, B. and Shevelyov, Y. Y. (2018). The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a. Epigenetics Chromatin 11(1): 65. PubMed ID: 30384843
Summary:
In most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin, as evidenced by microscopy observations and DamID mapping of lamina-associated domains (LADs) enriched in dimethylated Lys9 of histone H3 (H3K9me2). However, in Kc167 cell culture, the only Drosophilla cell type where LADs have previously been mapped, they are neither H3K9me2-enriched nor overlapped with the domains of heterochromatin protein 1a (HP1a). Using cell type-specific DamID this study mapped genome-wide LADs, HP1a and Polycomb (Pc) domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Strikingly, contrary to Kc167 cells of embryonic origin, in neurons and, to a lesser extent, in glia and the fat body, HP1a domains appear to overlap strongly with LADs in both the chromosome arms and pericentromeric regions. Accordingly, centromeres reside closer to the nuclear lamina in neurons than in Kc167 cells. As expected, active gene promoters are mostly not present in LADs, HP1a and Pc domains. These domains are occupied by silent or weakly expressed genes with genes residing in the HP1a-bound LADs expressed at the lowest level. In various differentiated Drosophila cell types, this study has discovered the existence of peripheral heterochromatin, similar to that observed in mammals. These findings support the model that peripheral heterochromatin matures enhancing the repression of unwanted genes as cells terminally differentiate.
Lee, H. and Oliver, B. (2018). Non-canonical Drosophila X chromosome dosage compensation and repressive topologically associated domains. Epigenetics Chromatin 11(1): 62. PubMed ID: 30355339
Summary:
In animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL) that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often derepressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then derepression could contribute to male dosage compensation. This study asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. These analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy; however, they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components. These results suggest that this non-canonical dosage compensation is due to the same transacting derepression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.
Scacchetti, A., Brueckner, L., Jain, D., Schauer, T., Zhang, X., Schnorrer, F., van Steensel, B., Straub, T. and Becker, P. B. (2018). CHRAC/ACF contribute to the repressive ground state of chromatin. Life Sci Alliance 1(1): e201800024. PubMed ID: 30456345
Summary:
The chromatin remodeling complexes chromatin accessibility complex and ATP-utilizing chromatin assembly and remodeling factor (ACF) combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression remains unclear. This study explored the influence of Drosophila melanogaster chromatin accessibility complex/ACF on transcription by using complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of an Acf knock-out allele showed that only lowly expressed genes are derepressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together, these results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.
Mashtalir, N., D'Avino, A. R., Michel, B. C., Luo, J., Pan, J., Otto, J. E., Zullow, H. J., McKenzie, Z. M., Kubiak, R. L., St Pierre, R., Valencia, A. M., Poynter, S. J., Cassel, S. H., Ranish, J. A. and Kadoch, C. (2018). Modular organization and assembly of SWI/SNF family chromatin remodeling complexes. Cell 175(5): 1272-1288. PubMed ID: 30343899
Summary:
Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. This study elucidates the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class were uncovered. Finally, human disease-associated mutations were mapped within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
Nazer, E., Dale, R. K., Palmer, C. and Lei, E. P. (2018). Argonaute2 attenuates active transcription by limiting RNA Polymerase II elongation in Drosophila melanogaster. Sci Rep 8(1): 15685. PubMed ID: 30356106
Summary:
Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. This study shows that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, differential ChIP-seq analysis was performed for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, specific accumulation of the elongating but not initiating form of Pol II was found after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
Raich, N., Mahmoudi, S., Emre, D. and Karess, R. E. (2018). Mad1 influences interphase nucleoplasm organization and chromatin regulation in Drosophila. Open Biol 8(10). PubMed ID: 30333236
Summary:
The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. This study found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

Monday, December 10th - Signaling

Pareek, G., Thomas, R. E., Vincow, E. S., Morris, D. R. and Pallanck, L. J. (2018). Lon protease inactivation in Drosophila causes unfolded protein stress and inhibition of mitochondrial translation. Cell Death Discov 5: 51. PubMed ID: 30374414
Summary:
Mitochondrial dysfunction is a frequent participant in common diseases and a principal suspect in aging. To combat mitochondrial dysfunction, eukaryotes have evolved a large repertoire of quality control mechanisms. One such mechanism involves the selective degradation of damaged or misfolded mitochondrial proteins by mitochondrial resident proteases, including proteases of the ATPase Associated with diverse cellular Activities (AAA(+)) family. The importance of the AAA(+) family of mitochondrial proteases is exemplified by the fact that mutations that impair their functions cause a variety of human diseases, yet knowledge of the cellular responses to their inactivation is limited. To address this matter, flies were created and characterized with complete or partial inactivation of the Drosophila matrix-localized AAA(+) protease Lon. A Lon null allele was found to confer early larval lethality and that severely reducing Lon expression using RNAi results in shortened lifespan, locomotor impairment, and respiratory defects specific to respiratory chain complexes that contain mitochondrially encoded subunits. The respiratory chain defects of Lon knockdown (Lon (KD) ) flies appeared to result from severely reduced translation of mitochondrially encoded genes. This translational defect was not a consequence of reduced mitochondrial transcription, as evidenced by the fact that mitochondrial transcripts were elevated in abundance in Lon (KD) flies. Rather, the translational defect of Lon (KD) flies appeared to be derived from sequestration of mitochondrially encoded transcripts in highly dense ribonucleoparticles. The translational defect of Lon (KD) flies was also accompanied by a substantial increase in unfolded mitochondrial proteins. Together, these findings suggest that the accumulation of unfolded mitochondrial proteins triggers a stress response that culminates in the inhibition of mitochondrial translation. This work provides a foundation to explore the underlying molecular mechanisms.
Palmer, W. H., Joosten, J., Overheul, G. J., Jansen, P. W., Vermeulen, M., Obbard, D. J. and Van Rij, R. P. (2018). Induction and suppression of NF-kappaB signalling by a DNA virus of Drosophila. J Virol. PubMed ID: 30404807
Summary:
Interactions between the insect immune system and RNA viruses have been extensively studied in Drosophila, where RNA interference, NF-kappaB and JAK-STAT pathways underlie antiviral immunity. In response to RNA interference, insect viruses have convergently evolved suppressors of this pathway that act by diverse mechanisms to permit viral replication. However, interactions between the insect immune system and DNA viruses have received less attention, primarily because few Drosophila-infecting DNA virus isolates are available. This study used a recently-isolated DNA virus of Drosophila melanogaster, Kallithea virus (family Nudiviridae), to probe known antiviral immune responses and virus evasion tactics in the context of DNA virus infection. Fly mutants for RNA interference and Immune deficiency (Imd), but not Toll, pathways are more susceptible to Kallithea virus infection. The Kallithea virus-encoded protein gp83 was identified as a potent inhibitor of Toll signalling, suggesting that Toll mediates antiviral defense against Kallithea virus infection, but that it is suppressed by the virus. This study found that Kallithea virus gp83 inhibits Toll signalling through the regulation of NF-kappaB transcription factors. Furthermore, gp83 of the closely related Drosophila innubila nudivirus (DiNV) suppresses D. melanogaster Toll signalling, suggesting an evolutionary conserved function of Toll in defense against DNA viruses. Together, these results provide a broad description of known antiviral pathways in the context of DNA virus infection and identify the first Toll pathway inhibitor in a Drosophila virus, extending the known diversity of insect virus-encoded immune inhibitors.
Khan, M., Shaukat, Z., Saint, R. and Gregory, S. L. (2018). Chromosomal instability causes sensitivity to protein folding stress and ATP depletion. Biol Open 7(10). PubMed ID: 30327366
Summary:
Aneuploidy -- having an unbalanced genome - is poorly tolerated at the cellular and organismal level. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift which makes these cells sensitive to internal and environmental stresses. Using Drosophila as a model, this study found that protein folding stress is exacerbated by redox stress that occurs in response to ongoing changes to ploidy (chromosomal instability, CIN). If de novo nucleotide synthesis is blocked, CIN cells are dependent on a high level of lysosome function to survive. Depletion of adenosine monophosphate (AMP) synthesis enzymes led to DNA damage in CIN cells, which showed elevated activity of the DNA repair enzyme activated poly(ADP ribose) polymerase (PARP). PARP activation causes depletion of its substrate, nicotinamide adenine dinucleotide (NAD+) and subsequent loss of Adenosine Tri-Phosphate (ATP), and this study found that adding ATP or nicotinamide (a precursor in the synthesis of NAD+) could rescue the observed phenotypes. These findings provide ways to interpret, target and exploit aneuploidy, which has the potential to offer tumour-specific therapies.
Kurshakova, M. M., Nabirochkina, E. N., Georgieva, S. G. and Kopytova, D. V. (2018). TRF4, the novel TBP-related protein of Drosophila melanogaster, is concentrated at the endoplasmic reticulum and copurifies with proteins participating in the processes associated with endoplasmic reticulum. J Cell Biochem. PubMed ID: 30426565
Summary:
Understanding the functions of TBP-related factors is essential for studying chromatin assembly and transcription regulation in higher eukaryotes. The novel TBP-related protein-coding gene, trf4, was described in Drosophila melanogaster. trf4 is found only in Drosophila and has likely originated in Drosophila common ancestor. TRF4 protein has a distant homology with TBP and TRF2 in the region of TBP-like domain and is evolutionarily conserved among distinct Drosophila species, which indicates its functional significance. TRF4 is widely expressed in D. melanogaster with high levels of its expression being observed in testes. Interestingly enough, TRF4 has become a cytoplasmic protein having lost nuclear localization signal sequence. TRF4 is concentrated at the endoplasmic reticulum (ER) and copurifies with the proteins participating in the ER-associated processes. It is suggested that trf4 gene is an example of homolog neofunctionalization by protein subcellular relocalization pathway, where the subcellular relocalization of gene product of duplicated gene leads to the new functions in ER-associated processes.
Ma, Z., Li, P., Hu, X. and Song, H. (2018). Polarity protein Canoe mediates overproliferation via modulation of JNK, Ras-MAPK and Hippo signalling. Cell Prolif: e12529. PubMed ID: 30328653
Summary:
Over the past decade an intriguing connection between cell polarity and tumorigenesis has emerged. Multiple core components of the junction complexes that help to form and maintain cell polarity display both pro- and anti-tumorigenic functions in a context-dependent manner, with the underlying mechanisms poorly understood. With transgenic fly lines that overexpress or knock down specific signalling components, this study performed genetic analysis to investigate the precise role of the polarity protein Canoe (Cno) in tumorigenesis and the downstream pathways. Overexpression of cno simultaneously activates JNK and Ras-MEK-ERK signalling, resulting in mixed phenotypes of both overproliferation and cell death in the Drosophila wing disc. Moderate alleviation of JNK activation eliminates the effect of Cno on cell death, leading to organ overgrowth and cell migration that mimic the formation and invasion of tumours. In addition, this study found that the Hippo pathway acts downstream of JNK and Ras signalling to mediate the effect of Cno on cell proliferation. This work reveals an oncogenic role of Cno and creates a new type of Drosophila tumour model for cancer research.
Negreiros, E., Herszterg, S., Hwa, K., Camara, A., Dias, W. B., Carneiro, K., Bier, E., Todeschini, A. and Araujo, H. (2018). N-linked glycosylation restricts the function of short gastrulation to bind and shuttle BMPs. Development. PubMed ID: 30355725
Summary:
Disorders of N-linked glycosylation are increasingly reported in the literature. However, targets responsible for the associated developmental and physiological defects are largely unknown. Bone Morphogenetic Proteins (BMPs) act as highly dynamic complexes to regulate several functions during development. The range and strength of BMP activity depend on interactions with glycosylated protein complexes in the extracellular milieu. This study investigated the role of glycosylation for the function of the conserved extracellular BMP antagonist Short gastrulation (Sog). Conserved N-glycosylated sites are identified, and the effect of mutating these residues on BMP pathway activity in Drosophila is described. Functional analysis reveals that loss of individual Sog glycosylation sites enhances BMP antagonism and/or increases the spatial range of Sog effects in the tissue. Mechanistically, evidence is provided that N-terminal and stem glycosylation controls extracellular Sog levels and distribution. The identification of similar residues in vertebrate Chordin proteins suggests that N-glycosylation may be an evolutionarily conserved process that adds complexity to the regulation of BMP activity.

Friday, December 7th - Transcriptional Regulation

Li, M., Ma, Z., Roy, S., Patel, S. K., Lane, D. C., Duffy, C. R. and Cai, H. N. (2018). Selective interactions between diverse STEs organize the ANT-C Hox cluster. Sci Rep 8(1): 15158. PubMed ID: 30310129
Summary:
This study used the SF1 insulator in the Drosophila Antennapedia homeotic gene complex (ANT-C) as a model to study the mechanism and regulation of chromatin looping events. Previously work has shown that SF1 tethers a transient chromatin loop in the early embryo that insulates the Hox gene Sex comb reduce from the neighbor non-Hox gene fushi tarazu for their independent regulation. To further probe the functional range and connectivity of SF1, high-resolution chromosomal conformation capture (3C) was used to search for SF1 looping partners across ANT-C. This study reports here the identification of three distal SF1 Tether Elements (STEs) located in the labial, Deformed and Antennapedia Hox gene regions, extending the range of SF1 looping network to the entire complex. These novel STEs are bound by four different combinations of insulator proteins and exhibit distinct behaviors in enhancer block, enhancer-bypass and boundary functions. Significantly, the six STEs identified so far map to all but one of the major boundaries between repressive and active histone domains, underlining the functional relevance of these long-range chromatin loops in organizing the Hox complex. Importantly, SF1 selectively captured with only 5 STEs out of ~20 sites that display similar insulator binding profiles, indicating that presence of insulator proteins alone is not sufficient to determine looping events. These findings suggest that selective interaction among diverse STE insulators organize the Drosophila Hox genes in the 3D nuclear space.
Hope, C. M., Webber, J. L., Tokamov, S. A. and Rebay, I. (2018). Tuned polymerization of the transcription factor Yan limits off-DNA sequestration to confer context-specific repression. Elife 7. PubMed ID: 30412049
Summary:
During development, transcriptional complexes at enhancers regulate gene expression in complex spatiotemporal patterns. To achieve robust expression without spurious activation, the affinity and specificity of transcription factor-DNA interactions must be precisely balanced. Protein-protein interactions among transcription factors are also critical, yet how their affinities impact enhancer output is not understood. The Drosophila transcription factor Yan provides a well-suited model to address this, as its function depends on the coordinated activities of two independent and essential domains: the DNA-binding ETS domain and the self-associating SAM domain. To explore how protein-protein affinity influences Yan function, mutants were engineered that increase SAM affinity over four orders of magnitude. This produced a dramatic subcellular redistribution of Yan into punctate structures, reduced repressive output and compromised survival. Cell-type specification and genetic interaction defects suggest distinct requirements for polymerization in different regulatory decisions. It is concluded that tuned protein-protein interactions enable the dynamic spectrum of complexes that are required for proper regulation.
Combs, P. A. and Fraser, H. B. (2018). Spatially varying cis-regulatory divergence in Drosophila embryos elucidates cis-regulatory logic. PLoS Genet 14(11): e1007631. PubMed ID: 30383747
Summary:
Spatial patterning of gene expression is a key process in development, yet how it evolves is still poorly understood. Both cis- and trans-acting changes could participate in complex interactions, so to isolate the cis-regulatory component of patterning evolution, allele-specific spatial gene expression patterns were measured in D. melanogaster x simulans hybrid embryos. RNA-seq of cryo-sectioned slices revealed 66 genes with strong spatially varying allele-specific expression. hunchback, a major regulator of developmental patterning, had reduced expression of the D. simulans allele specifically in the anterior tip of hybrid embryos. Mathematical modeling of hunchback cis-regulation suggested a candidate transcription factor binding site variant, which was verified as causal using CRISPR-Cas9 genome editing. In sum, even comparing morphologically near-identical species surprisingly extensive spatial variation was found in gene expression, suggesting not only that development is robust to many such changes, but also that natural selection may have ample raw material for evolving new body plans via changes in spatial patterning.
Wang, Y., Cho, D. Y., Lee, H., Fear, J., Oliver, B. and Przytycka, T. M. (2018). Reprogramming of regulatory network using expression uncovers sex-specific gene regulation in Drosophila. Nat Commun 9(1): 4061. PubMed ID: 30283019
Summary:
Gene regulatory networks (GRNs) describe regulatory relationships between transcription factors (TFs) and their target genes. Computational methods to infer GRNs typically combine evidence across different conditions to infer context-agnostic networks. A method, Network Reprogramming using EXpression (NetREX), has been developed that constructs a context-specific GRN given context-specific expression data and a context-agnostic prior network. NetREX remodels the prior network to obtain the topology that provides the best explanation for expression data. Because NetREX utilizes prior network topology, PriorBoost, a method that evaluates a prior network in terms of its consistency with the expression data was developed. NetREX and PriorBoost were validated using the "gold standard" E. coli GRN from the DREAM5 network inference challenge; they were applied to construct sex-specific Drosophila GRNs. NetREX constructed sex-specific Drosophila GRNs that, on all applied measures, outperform networks obtained from other methods indicating that NetREX is an important milestone toward building more accurate GRNs.
Jishage, M., Yu, X., Shi, Y., Ganesan, S. J., Chen, W. Y., Sali, A., Chait, B. T., Asturias, F. J. and Roeder, R. G. (2018). Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1. Nat Struct Mol Biol 25(9): 859-867. PubMed ID: 30190596
Summary:
Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. This study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function.
Nagy, O., Nuez, I., Savisaar, R., Peluffo, A. E., Yassin, A., Lang, M., Stern, D. L., Matute, D. R., David, J. R. and Courtier-Orgogozo, V. (2018). Correlated evolution of two copulatory organs via a single cis-regulatory nucleotide change. Curr Biol 28(21): 3450-3457.e3413. PubMed ID: 30344115
Summary:
Diverse traits often covary between species. The possibility that a single mutation could contribute to the evolution of several characters between species is rarely investigated as relatively few cases are dissected at the nucleotide level. Drosophila santomea has evolved additional sex comb sensory teeth on its legs and has lost two sensory bristles on its genitalia. Evidence is presented that a single nucleotide substitution in an enhancer of the scute gene contributes to both changes. The mutation alters a binding site for the Hox protein Abdominal-B in the developing genitalia, leading to bristle loss, and for another factor in the developing leg, leading to bristle gain. This study suggests that morphological evolution between species can occur through a single nucleotide change affecting several sexually dimorphic traits.

Thursday, December 6th - RNA

van den Beek, M., da Silva, B., Pouch, J., Ali Chaouche, M. E. A., Carre, C. and Antoniewski, C. (2018). Dual-layer transposon repression in heads of Drosophila melanogaster. RNA. PubMed ID: 30217866
Summary:
piRNA-mediated repression of transposable elements (TE) in the germline limits the accumulation of heritable mutations caused by their transposition in the genome. It is not clear whether the piRNA pathway plays a functional role in adult, non-gonadal tissues in Drosophila melanogaster. To address this question, the small RNA content of adult Drosophila melanogaster heads was analyzed. Varying amount of piRNA-sized, ping-pong positive molecules in heads correlates with contamination by gonadal tissue during RNA extraction, suggesting that most of piRNAs detected in head sequencing libraries originate from gonads. The heads of wild type and piwi mutants were sequenced to address whether piwi loss of function would affect the low amount of piRNA-sized, ping-pong negative molecules that are still detected in heads hand-checked to avoid gonadal contamination. Loss of piwi was found to not affect significantly these 24-28 RNA molecules. Instead, increased siRNA levels were found against the majority of Drosophila transposable element families. To determine the effect of this siRNA level change on transposon expression, the transcriptome of wild type, piwi, dicer-2 and piwi, dicer-2 double-mutant fly heads was sequenced. RNA expression levels of the majority of TE families in piwi or dicer-2 mutants remain unchanged and that TE transcript abundance increases significantly only in piwi, dicer-2 double-mutants. These results lead to the suggestion of a dual-layer model for TE repression in adult somatic tissues. Piwi-mediated transcriptional gene silencing (TGS) established during embryogenesis constitutes the first layer of TE repression whereas Dicer-2-dependent siRNA-mediated post-transcriptional gene silencing (PTGS) provide a backup mechanism to repress TEs that escape silencing by piwi-mediated TGS.
Kelleher, E. S., Jaweria, J., Akoma, U., Ortega, L. and Tang, W. (2018). QTL mapping of natural variation reveals that the developmental regulator bruno reduces tolerance to P-element transposition in the Drosophila female germline. PLoS Biol 16(10): e2006040. PubMed ID: 30376574
Summary:
Transposable elements (TEs) are obligate genetic parasites that propagate in host genomes by replicating in germline nuclei, thereby ensuring transmission to offspring. Resistance to TE propagation is enacted by germline-specific small-RNA-mediated silencing pathways, such as the Piwi-interacting RNA (piRNA) pathway, and is studied extensively. However, it remains entirely unknown whether host genomes may also evolve tolerance by desensitizing gametogenesis to the harmful effects of TEs. In part, the absence of research on tolerance reflects a lack of opportunity, as small-RNA-mediated silencing evolves rapidly after a new TE invades, thereby masking existing variation in tolerance. This study has exploited the recent historical invasion of the Drosophila melanogaster genome by P-element DNA transposons in order to study tolerance of TE activity. In the absence of piRNA-mediated silencing, the genotoxic stress imposed by P-elements disrupts oogenesis and, in extreme cases, leads to atrophied ovaries that completely lack germline cells. By performing quantitative trait locus (QTL) mapping on a panel of recombinant inbred lines (RILs) that lack piRNA-mediated silencing of P-elements, multiple QTL were uncovered that are associated with differences in tolerance of oogenesis to P-element transposition. The most significant QTL was localized to a small 230-kb euchromatic region, with the logarithm of the odds (LOD) peak occurring in the bruno locus, which codes for a critical and well-studied developmental regulator of oogenesis. Genetic, cytological, and expression analyses suggest that bruno dosage modulates germline stem cell (GSC) loss in the presence of P-element activity. These observations reveal segregating variation in TE tolerance for the first time, and implicate gametogenic regulators as a source of tolerant variants in natural populations.
Lee, M., Nguyen, T. M. T. and Kim, K. (2018). In-depth study of lin-28 suggests selectively conserved let-7 independent mechanism in Drosophila. Gene. PubMed ID: 30415010
Summary:
Lin-28 is a conserved RNA-binding protein that is involved in a wide range of developmental processes and pathogenesis. At the molecular level, Lin-28 blocks the maturation of let-7 and regulates translation of certain mRNA targets. In Drosophila, Lin-28 is reported to play a role in oogenesis, muscle formation, and the symmetric division of adult intestinal stem cells. This study characterized Drosophila Lin-28 through a detailed examination of its temporal and spatial expression. Lin-28 is specifically expressed in embryonic nervous and cardiac systems. However, loss or gain of lin-28 function does not cause any abnormality during embryonic development. Instead, the ubiquitous overexpression of Lin-28 leads to lethality from late larval stage to pupal stage, and eye-specific overexpression causes severe cell loss. The ectopic expression of human Lin28A has the same effect as Drosophila Lin-28, indicating functional conservation in Lin-28 orthologs. The effect of Lin-28 on let-7 biogenesis was examined through the mutant and overexpression analysis. Lin-28 does not block the production of let-7 in Drosophila, which suggests the let-7 independent pathway as a molecular mechanism of Lin-28.
Grobler, Y., Yun, C. Y., Kahler, D. J., Bergman, C. M., Lee, H., Oliver, B. and Lehmann, R. (2018). Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation. PLoS Pathog 14(11): e1007445. PubMed ID: 30422992
Summary:
Infection by the bacterium Wolbachia provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. Genome-wide RNAi screening was coupled with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. A diverse set of host gene networks was identified to regulate Wolbachia levels and unexpectedly revealed that perturbations of host translation components such as the ribosome and translation initiation factors results in increased Wolbachia levels both in vitro using RNAi and in vivo using mutants and a chemical-based translation inhibition assay. This work provides evidence for Wolbachia-host translation interaction and strengthens general understanding of the Wolbachia-host intracellular relationship.
Zhu, L., Kandasamy, S. K., Liao, S. E. and Fukunaga, R. (2018). LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes. Nat Commun 9(1): 4031. PubMed ID: 30279526
Summary:
Post-transcriptional regulation of gene expression plays an essential role during oocyte maturation. This study reports that Drosophila MARF1 (Meiosis Regulator And mRNA Stability Factor 1), which consists of one RNA-recognition motif and six tandem LOTUS domains with unknown molecular function, is essential for oocyte maturation. When tethered to a reporter mRNA, MARF1 post-transcriptionally silences reporter expression by shortening reporter mRNA poly-A tail length and thereby reducing reporter protein level. This activity is mediated by the MARF1 LOTUS domain, which binds the CCR4-NOT deadenylase complex. MARF1 binds cyclin A mRNA and shortens its poly-A tail to reduce Cyclin A protein level during oocyte maturation. This study identifies MARF1 as a regulator in oocyte maturation and defines the conserved LOTUS domain as a post-transcriptional effector domain that recruits CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress their translation.
Zhang, G., Tu, S., Yu, T., Zhang, X. O., Parhad, S. S., Weng, Z. and Theurkauf, W. E. (2018). Co-dependent assembly of Drosophila piRNA precursor complexes and piRNA cluster heterochromatin. Cell Rep 24(13): 3413-3422. PubMed ID: 30257203
Summary:
In Drosophila, the piRNAs that guide germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. Rhino is shown to promotes cluster transcript association with UAP56 and the THO complex, forming RNA-protein assemblies that are unique to piRNA precursors. UAP56 and THO are ubiquitous RNA-processing factors, and null alleles of uap56 and the THO subunit gene tho2 are lethal. However, uap56(sz15) and mutations in the THO subunit genes thoc5 and thoc7 are viable but sterile and disrupt piRNA biogenesis. The uap56(sz15) allele reduces UAP56 binding to THO, and the thoc5 and thoc7 mutations disrupt interactions among the remaining THO subunits and UAP56 binding to the core THO subunit Hpr1. These mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino at cluster heterochromatin.

Wednesday, December 5th - Behavior

Lam, V. H., Li, Y. H., Liu, X., Murphy, K. A., Diehl, J. S., Kwok, R. S. and Chiu, J. C. (2018). CK1alpha collaborates with DOUBLETIME to regulate PERIOD function in the Drosophila circadian clock. J Neurosci. PubMed ID: 30373768
Summary:
The animal circadian timing system interprets environmental time cues and internal metabolic status to orchestrate circadian rhythms of physiology, allowing animals to perform necessary tasks in a time-of-day dependent manner. In Drosophila, PERIOD (PER), TIMELESS (TIM), CLOCK (CLK), and CYCLE (CYC) are core clock proteins that function in a transcriptional-translational feedback mechanism to regulate the circadian transcriptome. Post-translational modifications of core clock proteins provide precise temporal control over when they are active as regulators of clock-controlled genes. In particular, phosphorylation is a key regulatory mechanism that dictates the subcellular localization, stability, and transcriptional activity of clock proteins. Previously, Casein kinase 1alpha (CK1alpha) was identified as a kinase that phosphorylates mammalian PER1 and modulates its stability, but the mechanisms by which it modulates PER protein stability is still unclear. Using Drosophila as a model, this study shows that CK1alpha has an overall function of speeding up PER metabolism, and is required to maintain the 24-hour period of circadian rhythms. The results indicate that CK1alpha collaborates with the key clock kinase DOUBLETIME (DBT) in both the cytoplasm and the nucleus to regulate the timing of PER-dependent repression of the circadian transcriptome. Specifically, it was observed that CK1alpha promotes PER nuclear localization by antagonizing the activity of DBT to inhibit PER nuclear translocation. Furthermore, CK1alpha enhances DBT-dependent PER phosphorylation and degradation once PER moves into the nucleus.
Grabowska, M. J., Steeves, J., Alpay, J., van de Poll, M., Ertekin, D. and van Swinderen, B. (2018). Innate visual preferences and behavioral flexibility in Drosophila. J Exp Biol. PubMed ID: 30322983
Summary:
Visual decision-making in animals is influenced by innate preferences as well as experience. Interaction between hard-wired responses and changing motivational states determines whether a visual stimulus is attractive, aversive, or neutral. It is however difficult to separate the relative contribution of nature versus nurture in experimental paradigms, especially for more complex visual parameters such as the shape of objects. Yhis study used a closed-loop virtual reality paradigm for walking Drosophila flies to uncover innate visual preferences for the shape and size of objects, in a recursive choice scenario allowing the flies to reveal their visual preferences over time. Drosophila flies were found to display a robust attraction / repulsion profile for a range of objects sizes in this paradigm, and that this visual preference profile remains evident under a variety of conditions and persists into old age. A level of flexibility in this behavior was also demonstrated: innate repulsion to certain objects could be transiently overridden if these were novel, although this effect was only evident in younger flies. Finally, a neuromodulatory circuit in the fly brain, Drosophila neuropeptide F (dNPF), can be recruited to guide visual decision-making. Optogenetic activation of dNPF-expressing neurons converted a visually repulsive object into a more attractive object. This suggests that dNPF activity in the Drosophila brain guides ongoing visual choices, to override innate preferences and thereby provide a necessary level of behavioral flexibility in visual decision-making.
Kutta, R. J., Archipowa, N. and Scrutton, N. S. (2018). The sacrificial inactivation of the blue-light photosensor cryptochrome from Drosophila melanogaster. Phys Chem Chem Phys. PubMed ID: 30417904
Summary:
Drosophila melanogaster Cryptochrome functions as the primary blue-light receptor that mediates circadian photo entrainment. Absorption of a photon leads to reduction of the protein-bound FAD via consecutive electron transfer along a conserved tryptophan tetrad resembling the signalling state required for conformational changes and induction of subsequent signalling cascades. However, how the initial photochemistry and subsequent dark processes leading to downstream signalling are linked to each other at the molecular level is still poorly understood. This study investigated in detail the initial photochemical events in DmCRY by time-resolved and stationary absorption spectroscopy combined with quantum chemical and molecular dynamics calculations. The early events along the conserved tryptophan tetrad and the final deprotonation of the terminal tryptophanyl radical cation were resolved. These initial events lead to conformational changes, such as the known C-terminal tail release, Trp decomposition, and finally FAD release providing evidence that DmCRY does not undergo a photocycle. It is proposed that light is a negative regulator of DmCRY stability even under in vitro conditions where the proteasomal machinery is missing, that is in line with its biological function, i.e. entrainment of the circadian clock.
Kirszenblat, L., Ertekin, D., Goodsell, J., Zhou, Y., Shaw, P. J. and van Swinderen, B. (2018). Sleep regulates visual selective attention in Drosophila. J Exp Biol. PubMed ID: 30355611
Summary:
Although sleep deprivation is known to impair attention in humans and other mammals, the underlying reasons are not well understood, and whether similar effects are present in non-mammalian species is not known. This study sought to investigate whether sleep is important for optimizing attention in an invertebrate species, the genetic model Drosophila melanogaster. A high-throughput paradigm was developed to measure visual attention in freely-walking Drosophila, using competing foreground/background visual stimuli. Whereas sleep-deprived flies could respond normally to either stimulus alone, they were more distracted by background cues in a visual competition task. Other stressful manipulations such as starvation, heat exposure, and mechanical stress had no effects on visual attention in this paradigm. In contrast to sleep deprivation, providing additional sleep using the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP) did not affect attention in wild-type flies, but specifically improved attention in the learning mutant dunce. These results reveal a key function of sleep in optimizing attention processes in Drosophila, and establish a behavioral paradigm that can be used to explore the molecular mechanisms involved.
Currier, T. A. and Nagel, K. I. (2018). Multisensory control of orientation in tethered flying Drosophila. Curr Biol. PubMed ID: 30393038
Summary:
A longstanding goal of systems neuroscience is to quantitatively describe how the brain integrates sensory cues over time. This study developed a closed-loop orienting paradigm in Drosophila to study the algorithms by which cues from two modalities are integrated during ongoing behavior. Flies exhibit two behaviors when presented simultaneously with an attractive visual stripe and aversive wind cue. First, flies perform a turn sequence where they initially turn away from the wind but later turn back toward the stripe, suggesting dynamic sensory processing. Second, turns toward the stripe are slowed by the presence of competing wind, suggesting summation of turning drives. A model is developed in which signals from each modality are filtered in space and time to generate turn commands and then summed to produce ongoing orienting behavior. This computational framework correctly predicts behavioral dynamics for a range of stimulus intensities and spatial arrangements.
Harbison, S. T., Kumar, S., Huang, W., McCoy, L. J., Smith, K. R. and Mackay, T. F. C. (2018). Genome-wide association study of circadian behavior in Drosophila melanogaster. Behav Genet. PubMed ID: 30341464
Summary:
Circadian rhythms influence physiological processes from sleep-wake cycles to body temperature and are controlled by highly conserved cycling molecules. Although the mechanistic basis of the circadian clock has been known for decades, the extent to which circadian rhythms vary in nature and the underlying genetic basis for that variation is not well understood. This study measured circadian period (T) and rhythmicity index in the Drosophila Genetic Reference Panel (DGRP) and observed extensive genetic variation in both. Seven DGRP lines had sexually dimorphic arrhythmicity and one line had an exceptionally long T. Genome-wide analyses identified 584 polymorphisms in 268 genes. Differences were observed among transcripts for nine genes predicted to interact among themselves and canonical clock genes in the long period line and a control. Mutations/RNAi knockdown targeting these genes also affected circadian behavior. These observations reveal that complex genetic interactions influence high levels of variation in circadian phenotypes.

Tuesday, December 2nd - CNS Development

Yin, J., Gibbs, M., Long, C., Rosenthal, J., Kim, H. S., Kim, A., Sheng, C., Ding, P., Javed, U. and Yuan, Q. (2018). Transcriptional regulation of lipophorin receptors supports neuronal adaptation to chronic elevations of activity. Cell Rep 25(5): 1181-1192. PubMed ID: 30380410
Summary:
Activity-dependent modifications strongly influence neural development. However, molecular programs underlying their context and circuit-specific effects are not well understood. To study global transcriptional changes associated with chronic elevation of synaptic activity, cell-type-specific transcriptome profiling was performed of Drosophila ventral lateral neurons (LNvs) in the developing visual circuit, and activity-modified transcripts were identified that are enriched in neuron morphogenesis, circadian regulation, and lipid metabolism and trafficking. Using bioinformatics and genetic analyses, activity-induced isoform-specific upregulation was validated of Drosophila lipophorin receptors LpR1 and LpR2, the homologs of mammalian low-density lipoprotein receptor (LDLR) family proteins. Furthermore, these morphological and physiological studies uncovered critical functions of neuronal lipophorin receptors (LpRs) in maintaining the structural and functional integrities in neurons challenged by chronic elevations of activity. Together, these findings identify LpRs as molecular targets for activity-dependent transcriptional regulation and reveal the functional significance of cell-type-specific regulation of neuronal lipid uptake in experience-dependent plasticity and adaptive responses.
Kwon, M. J., Han, M. H., Bagley, J. A., Hyeon, D. Y., Ko, B. S., Lee, Y. M., Cha, I. J., Kim, S. Y., Kim, D. Y., Kim, H. M., Hwang, D., Lee, S. B. and Jan, Y. N. (2018). Coiled-coil structure-dependent interactions between polyQ proteins and Foxo lead to dendrite pathology and behavioral defects. Proc Natl Acad Sci U S A 115(45): E10748-e10757. PubMed ID: 30348793
Summary:
Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. The molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. This study has demonstrated that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, three structural variants of SCA3 polyQ proteins were generated. Through comparative analysis of these variants, it was found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.
Chen, C. L., Hermans, L., Viswanathan, M. C., Fortun, D., Aymanns, F., Unser, M., Cammarato, A., Dickinson, M. H. and Ramdya, P. (2018). Imaging neural activity in the ventral nerve cord of behaving adult Drosophila. Nat Commun 9(1): 4390. PubMed ID: 30348941
Summary:
To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because limb premotor and motor circuits have been largely inaccessible for large-scale recordings in intact, moving animals-a constraint that is true for both vertebrate and invertebrate models. This study introduces a method for 2-photon functional imaging from the ventral nerve cord (VNC) of behaving adult Drosophila melanogaster. This method was used to reveal patterns of activity across nerve cord populations during grooming and walking and to uncover the functional encoding of moonwalker ascending neurons (MANs), moonwalker descending neurons (MDNs), and a previously uncharacterized class of locomotion-associated A1 descending neurons. Finally, a genetic reagent was developed to destroy the indirect flight muscles and to facilitate experimental access to the VNC. Taken together, these approaches enable the direct investigation of circuits associated with complex limb movements.
Berry, J. A., Phan, A. and Davis, R. L. (2018). Dopamine neurons mediate learning and forgetting through bidirectional modulation of a memory trace. Cell Rep 25(3): 651-662. PubMed ID: 30332645
Summary:
It remains unclear how memory engrams are altered by experience, such as new learning, to cause forgetting. This study reports that short-term aversive memory in Drosophila is encoded by and retrieved from the mushroom body output neuron MBOn-γ2α'1. Pairing an odor with aversive electric shock creates a robust depression in the calcium response of MBOn-gamma2alpha'1 and increases avoidance to the paired odor. Electric shock after learning, which activates the cognate dopamine neuron DAn-γ2α'1, restores the response properties of MBOn-γ2α'1 and causes behavioral forgetting. Conditioning with a second odor restores the responses of MBOn-γ2α'1 to a previously learned odor while depressing responses to the newly learned odor, showing that learning and forgetting can occur simultaneously. Moreover, optogenetic activation of DAn-γ2α'1 is sufficient for the bidirectional modulation of MBOn-γ2α'1 response properties. Thus, a single DAn can drive both learning and forgetting by bidirectionally modulating a cellular memory trace.
Heras, F. J. H., Vahasoyrinki, M. and Niven, J. E. (2018). Modulation of voltage-dependent K+ conductances in photoreceptors trades off investment in contrast gain for bandwidth. PLoS Comput Biol 14(11): e1006566. PubMed ID: 30399147
Summary:
Modulation is essential for adjusting neurons to prevailing conditions and differing demands. Yet understanding how modulators adjust neuronal properties to alter information processing remains unclear, as is the impact of neuromodulation on energy consumption. This study combine two computational models, one Hodgkin-Huxley type and the other analytic, to investigate the effects of neuromodulation upon Drosophila melanogaster photoreceptors. Voltage-dependent K+ conductances in these photoreceptors: (i) activate upon depolarisation to reduce membrane resistance and adjust bandwidth to functional requirements; (ii) produce negative feedback to increase bandwidth in an energy efficient way; (iii) produce shunt-peaking thereby increasing the membrane gain bandwidth product; and (iv) inactivate to amplify low frequencies. Through their effects on the voltage-dependent K+ conductances, three modulators, serotonin, calmodulin and PIP2, trade-off contrast gain against membrane bandwidth. Serotonin shifts the photoreceptor performance towards higher contrast gains and lower membrane bandwidths, whereas PIP2 and calmodulin shift performance towards lower contrast gains and higher membrane bandwidths. These neuromodulators have little effect upon the overall energy consumed by photoreceptors, instead they redistribute the energy invested in gain versus bandwidth. This demonstrates how modulators can shift neuronal information processing within the limitations of biophysics and energy consumption.
Horne, J. A., Langille, C., McLin, S., Wiederman, M., Lu, Z., Xu, C. S., Plaza, S. M., Scheffer, L. K., Hess, H. F. and Meinertzhagen, I. A. (2018). A resource for the Drosophila antennal lobe provided by the connectome of glomerulus VA1v. Elife 7. PubMed ID: 30382940
Summary:
Using focused ion beam-scanning electron microscope (FIB-SEM) this study reports the entire synaptic connectome of glomerulus VA1v of the right antennal lobe in Drosophila melanogaster. Within the glomerulus all neurons were densely reconstructed, including hitherto elusive local interneurons. The fruitless-positive, sexually dimorphic VA1v included >11,140 presynaptic sites with ~38,050 postsynaptic dendrites. These connected input olfactory receptor neurons (ORNs, 51 ipsilateral, 56 contralateral), output projection neurons (18 PNs), and local interneurons (56 of >150 previously reported LNs). ORNs are predominantly presynaptic and PNs predominantly postsynaptic; newly reported LN circuits are largely an equal mixture and confer extensive synaptic reciprocity, except the newly reported LN2V with input from ORNs and outputs mostly to monoglomerular PNs, however. PNs were more numerous than previously reported from genetic screens, suggesting that the latter failed to reach saturation. A matrix is reported of 192 bodies each having >50 connections; these form 88% of the glomerulus' pre/postsynaptic sites.

Monday, December 3rd. - Gonads

Sepil, I., Hopkins, B. R., Dean, R., Thezenas, M. L., Charles, P. D., Konietzny, R., Fischer, R., Kessler, B. and Wigby, S. (2018). Quantitative proteomics identification of seminal fluid proteins in male Drosophila melanogaster. Mol Cell Proteomics. PubMed ID: 30287546
Summary:
Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and particularly in insects, modifying female physiology and behaviour. This study presents a new and complementary method, which provides added sensitivity to Sfp identification. Label-free quantitative proteomics were applied to Drosophila melanogaster male reproductive tissue - where Sfps are unprocessed, and highly abundant - and Sfps quantified before and immediately after mating, to infer those transferred during copulation. Female reproductive tracts were also analyzed immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. These data were consistent with 125 previously reported Sfps. This study found nine high-confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. 42 more candidates were identified that are likely Sfps based on their abundance, known expression and predicted characteristics, and this study revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for these candidate Sfps were lower than for previously identified Sfps, supporting the idea that this technique provides a deeper analysis of the Sfp proteome than previous studies. These results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further the understanding of reproductive biology.
Vedelek, V., Bodai, L., Grezal, G., Kovacs, B., Boros, I. M., Laurinyecz, B. and Sinka, R. (2018). Analysis of Drosophila melanogaster testis transcriptome. BMC Genomics 19(1): 697. PubMed ID: 30249207
Summary:
The formation of matured and individual sperm involves a series of molecular and spectacular morphological changes of the developing cysts in Drosophila melanogaster testis. Recent advances in RNA Sequencing (RNA-Seq) technology help understanding the complexity of eukaryotic transcriptomes by dissecting different tissues and developmental stages of organisms. To gain a better understanding of cellular differentiation of spermatogenesis, RNA-Seq was applied to analyse the testis-specific transcriptome, including coding and non-coding genes. Three different parts of the wild-type testis were isolated by dissecting and cutting the different regions: 1.) the apical region, which contains stem cells and developing spermatocytes 2.) the middle region, with enrichment of meiotic cysts 3.) the basal region, which contains elongated post-meiotic cysts with spermatids. Total RNA was isolated from each region and analysed by next-generation sequencing. Data was collected from the annotated 17412 Drosophila genes and 5381 genes were identified with significant transcript accumulation differences between the regions, representing the main stages of spermatogenesis. The presence and region specific distribution is presented of 2061 lncRNAs in testis, with 203 significant differences. Using the available modENCODE RNA-Seq data, the tissue specificity indices was determined of Drosophila genes. Combining the indices with the current results, genes were identified with region-specific enrichment in testis. Searchable visualizations are presented that can facilitate the identification of new components that play role in the organisation and composition of different stages of spermatogenesis, including the less known, but complex regulation of post-meiotic stages.
Takahashi, K. H., Ishimori, M. and Iwata, H. (2018). HSP90 as a global genetic modifier for male genital morphology in Drosophila melanogaster. Evolution. PubMed ID: 30221481
Summary:
The molecular chaperone protein HSP90 has been proposed to modulate genotype-phenotype relationship in a broad range of organisms. This study explores the proposed genetic modifier effect of HSP90 through a genomewide analysis. HSP90 is shown to function as a genetic modifier of genital morphology in Drosophila melanogaster. A large number of single-nucleotide polymorphisms (SNPs) with an HSP90-dependent effect were identfied by using genome wide association analysis. The SNPs were clasified into the ones under capacitance effect (smaller allelic effect under HSP90 inhibition) or the ones under potentiation effect (larger allelic effect under HSP90 inhibition). Although the majority of SNPs are under capacitance, there are a large number of SNPs under potentiation. This observation provides support for a model in which Hsp90 is not described exclusively as a "genetic capacitor," but is described more broadly as a "genetic modifier." Because the majority of the candidate genes estimated from SNPs with an HSP90-dependent effect in the current study has never been reported to interact with HSP90 directly, the global genetic modifier effect of HSP90 may be exhibited through epistatic interactions in gene regulatory networks.
Eagle, W. V. I., Yeboah-Kordieh, D. K., Niepielko, M. G. and Gavis, E. R. (2018). Distinct cis-acting elements mediate targeting and clustering of Drosophila polar granule mRNAs. Development. PubMed ID: 30333216
Summary:
Specification and development of Drosophila germ cells depend on molecular determinants within the germ plasm, a specialized cytoplasmic domain at the posterior of the embryo. Localization of numerous mRNAs to the germ plasm occurs by their incorporation, as single-transcript ribonucleoprotein (RNP) particles, into complex RNP granules called polar granules. Incorporation of mRNAs into polar granules is followed by recruitment of additional like-transcripts to form discrete homotypic clusters. The cis-acting localization signals that target mRNAs to polar granules and promote homotypic clustering remain largely uncharacterized. This study shows that the polar granule component (pgc) and germ cell-less (gcl) 3' untranslated regions contain complex localization signals comprising multiple, independently weak and partially functionally redundant localization elements (LEs). Targeting of pgc to polar granules and self-assembly into homotypic clusters are functionally separable processes mediated by distinct classes of LEs. A sequence motif shared by other polar granule mRNAs was identifed that contributes to homotypic clustering. These results suggest that mRNA localization signal complexity may be a feature required by the targeting and self-recruitment mechanism that drives germ plasm mRNA localization.
Krishnakumar, P., Riemer, S., Perera, R., Lingner, T., Goloborodko, A., Khalifa, H., Bontems, F., Kaufholz, F., El-Brolosy, M. A. and Dosch, R. (2018). Functional equivalence of germ plasm organizers. PLoS Genet 14(11): e1007696. PubMed ID: 30399145
Summary:
The proteins Oskar (Osk) in Drosophila and Bucky ball (Buc) in zebrafish act as germ plasm organizers. Both proteins recapitulate germ plasm activities but seem to be unique to their animal groups. This study discovered that Osk and Buc show similar activities during germ cell specification. Drosophila Osk induces additional PGCs in zebrafish. Surprisingly, Osk and Buc do not show homologous protein motifs that would explain their related function. Nonetheless, both proteins contain stretches of intrinsically disordered regions (IDRs), which seem to be involved in protein aggregation. IDRs are known to rapidly change their sequence during evolution, which might obscure biochemical interaction motifs. Indeed, this study showed that Buc binds to the known Oskar interactors Vasa protein and nanos mRNA indicating conserved biochemical activities. These data provide a molecular framework for two proteins with unrelated sequence but with equivalent function to assemble a conserved core-complex nucleating germ plasm.
Singh, A., Buehner, N. A., Lin, H., Baranowski, K. J., Findlay, G. D. and Wolfner, M. F. (2018). Long-term interaction between Drosophila sperm and sex peptide is mediated by other seminal proteins that bind only transiently to sperm. Insect Biochem Mol Biol 102: 43-51. PubMed ID: 30217614
Summary:
Seminal fluid proteins elicit several post-mating physiological changes in mated Drosophila melanogaster females. Some of these changes persist for over a week after mating because the seminal protein that causes these changes, the Sex Peptide (SP), binds to sperm that are stored in the female reproductive tract. SP's sperm binding is mediated by a network of at least eight seminal proteins. Some of these network proteins (CG1656, CG1652, CG9997 and Antares) bind to sperm within 2h of mating, like SP. However, while SP remains bound to sperm at 4 days post-mating, none of the other network proteins are detectable at this time. It was also observed that the same network proteins are detectable at 2h post-mating in seminal receptacle tissue from which sperm have been removed, but are no longer detectable there by 4 days post-mating, suggesting short-term retention of these proteins in this female sperm storage organ. These results suggest that these network proteins act transiently to facilitate the conditions for SP's binding to sperm, perhaps by modifying SP or the sperm surface, but are not part of a long-acting complex that stably attaches SP to sperm.

Friday, November 30th - Signaling

Fernandez-Espartero, C. H., Rizzo, A., Fulford, A. D., Falo-Sanjuan, J., Goutte-Gattat, D. and Ribeiro, P. S. (2018). Prp8 regulates oncogene-induced hyperplastic growth in Drosophila. Development 145(22). PubMed ID: 30333215
Summary:
Although developmental signalling pathways control tumourigenic growth, the cellular mechanisms that abnormally proliferating cells rely on are still largely unknown. Drosophila melanogaster is a genetically tractable model that is used to study how specific genetic changes confer advantageous tumourigenic traits. Despite recent efforts, the role of deubiquitylating enzymes in cancer is particularly understudied. This study performed a Drosophila in vivo RNAi screen to identify deubiquitylating enzymes that modulate Ras(V12)-induced hyperplastic growth. The spliceosome core component Prp8 was identified as a crucial regulator of Ras-, EGFR-, Notch- or RET-driven hyperplasia. Loss of prp8 function alone decreased cell proliferation, increased cell death, and affected cell differentiation and polarity. In hyperplasia, Prp8 supported tissue overgrowth independently of caspase-dependent cell death. The depletion of prp8 efficiently blocked Ras-, EGFR- and Notch-driven tumours but, in contrast, enhanced tumours that were driven by oncogenic RET, suggesting a context-specific role in hyperplasia. These data show, for the first time, that Prp8 regulates hyperplasia, and extend recent observations on the potential role of the spliceosome in cancer. These findings suggest that targeting Prp8 could be beneficial in specific tumour types.
Gao, X., Xie, X. J., Hsu, F. N., Li, X., Liu, M., Hemba-Waduge, R. U., Xu, W. and Ji, J. Y. (2018). CDK8 mediates the dietary effects on developmental transition in Drosophila. Dev Biol. PubMed ID: 30352217
Summary:
The complex interplay between genetic and environmental factors, such as diet and lifestyle, defines the initiation and progression of multifactorial diseases, including cancer, cardiovascular and metabolic diseases, and neurological disorders. Given that most of the studies have been performed in controlled experimental settings to ensure the consistency and reproducibility, the impacts of environmental factors, such as dietary perturbation, on the development of animals with different genotypes and the pathogenesis of these diseases remain poorly understood. By analyzing the cdk8 and cyclin C (cycC) mutant larvae in Drosophila, it was previously reported that the CDK8-CycC complex coordinately regulates lipogenesis by repressing dSREBP (sterol regulatory element-binding protein)-activated transcription and developmental timing by activating EcR (ecdysone receptor)-dependent gene expression. This study reports that dietary nutrients, particularly proteins and carbohydrates, modulate the developmental timing through the CDK8/CycC/EcR pathway. cdk8 and cycC mutants are sensitive to the levels of dietary proteins and seven amino acids (arginine, glutamine, isoleucine, leucine, methionine, threonine, and valine). Those mutants are also sensitive to dietary carbohydrates, and they are more sensitive to monosaccharides than disaccharides. These results suggest that CDK8-CycC mediates the dietary effects on metabolism and developmental timing in Drosophila larvae.
Castro-Rodrigues, A. F., Zhao, Y., Fonseca, F., Gabant, G., Cadene, M., Robertson, G. A. and Morais-Cabral, J. H. (2018). The interaction between the Drosophila EAG potassium channel and the protein kinase CaMKII involves an extensive interface at the active site of the kinase. J Mol Biol. PubMed ID: 30381148
Summary:
The Drosophila EAG (dEAG) potassium channel is the founding member of the superfamily of KNCH channels, which are involved in cardiac repolarization, neuronal excitability and cellular proliferation. In flies, dEAG is involved in regulation of neuron firing and assembles with CaMKII to form a complex implicated in memory formation. This study has characterized the interaction between the kinase domain of CaMKII and a 53-residue fragment of the dEAG channel that includes a canonical CaMKII recognition sequence. Crystal structures together with biochemical/biophysical analysis show a substrate-kinase complex with an unusually tight and extensive interface that appears to be strengthened by phosphorylation of the channel fragment. Electrophysiological recordings show that catalytically active CaMKII is required to observe active dEAG channels. A previously identified phosphorylation site in the recognition sequence is not the substrate for this crucial kinase activity, but rather contributes importantly to the tight interaction of the kinase with the channel. The available data suggest that the dEAG channel is a docking platform for the kinase and that phosphorylation of the channel's kinase recognition sequence modulates the strength of the interaction between the channel and the kinase.
Crossman, S. H., Streichan, S. J. and Vincent, J. P. (2018). EGFR signaling coordinates patterning with cell survival during Drosophila epidermal development. PLoS Biol 16(10): e3000027. PubMed ID: 30379844
Summary:
Extensive apoptosis is often seen in patterning mutants, suggesting that tissues can detect and eliminate potentially harmful mis-specified cells. This study shows that the pattern of apoptosis in the embryonic epidermis of Drosophila is not a response to fate mis-specification but can instead be explained by the limiting availability of prosurvival signaling molecules released from locations determined by patterning information. In wild-type embryos, the segmentation cascade elicits the segmental production of several epidermal growth factor receptor (EGFR) ligands, including the transforming growth factor Spitz (TGFalpha), and the neuregulin, Vein. This leads to an undulating pattern of signaling activity, which prevents expression of the proapoptotic gene head involution defective (hid) throughout the epidermis. In segmentation mutants, where specific peaks of EGFR ligands fail to form, gaps in signaling activity appear, leading to coincident hid up-regulation and subsequent cell death. These data provide a mechanistic understanding of how cell survival, and thus appropriate tissue size, is made contingent on correct patterning.
Yao, W., Shan, Z., Gu, A., Fu, M., Shi, Z. and Wen, W. (2018). WW domain-mediated regulation and activation of E3 ubiquitin ligase Suppressor of Deltex. J Biol Chem. PubMed ID: 30213861
Summary:
The Nedd4 family E3 ligases Itch and WWP1/2 play crucial roles in the regulation of cell cycle progression and apoptosis, and are closely correlated with cancer development and metastasis. It has been recently shown that the ligase activities of Itch and WWP1/2 are tightly regulated with the HECT domain sequestered intramolecularly by a linker region connecting WW2 and WW3. This study shows that a similar autoinhibitory mechanism is utilized by the Drosophila ortholog of Itch and WWP1/2, Suppressor of Deltex [Su(dx)]. Su(dx) adopts an inactive steady state with the WW domain region interacting with the HECT domain. Both the linker and preceding WW2 are required for the efficient binding and regulation of Su(dx) HECT. Recruiting the multiple PY motif-containing adaptor dNdfip via WW domains relieves the inhibitory state of Su(dx) and leads to substrate (e.g., Notch) ubiquitination. This study demonstrates an evolutionarily conservative mechanism governing the regulation and activation of some Nedd4 family E3 ligases. These results also suggest a dual regulatory mechanism for specific Notch downregulation via dNdfip-Su(dx)-mediated Notch ubiquitination.
Hunter, M. V., Willoughby, P. M., Bruce, A. E. E. and Fernandez-Gonzalez, R. (2018). Oxidative stress orchestrates cell polarity to promote embryonic wound healing. Dev Cell 47(3): 377-387.e374. PubMed ID: 30399336
Summary:
Embryos have a striking ability to heal wounds rapidly and without scarring. Embryonic wound repair is a conserved process, driven by polarization of cell-cell junctions and the actomyosin cytoskeleton in the cells around the wound. However, the upstream signals that trigger cell polarization around wounds are unknown. This study used quantitative in vivo microscopy in Drosophila and zebrafish embryos to identify reactive oxygen species (ROS) as a critical signal that orchestrates cell polarity around wounds. ROS promote trafficking of adherens junctions and accumulation of actin and myosin at the wound edge and are necessary for wound closure. In Drosophila, ROS drive wound healing in part through an ortholog of Src kinase, Src42A, which is identified as a redox sensor that promotes polarization of junctions and the cytoskeleton around wounds. It is proposed that ROS are a reparative signal that drives rapid embryonic wound healing in vertebrate and invertebrate species.

Thursday November 29th - Synapses and Vesicles

Barber, K. R., Hruska, M., Bush, K. M., Martinez, J. A., Fei, H., Levitan, I. B., Dalva, M. B. and Wairkar, Y. P. (2018). Levels of Par-1 kinase determine the localization of Bruchpilot at the Drosophila neuromuscular junction synapses. Sci Rep 8(1): 16099. PubMed ID: 30382129
Summary:
Functional synaptic networks are compromised in many neurodevelopmental and neurodegenerative diseases. While the mechanisms of axonal transport and localization of synaptic vesicles and mitochondria are relatively well studied, little is known about the mechanisms that regulate the localization of proteins that localize to active zones. Recent finding suggests that mechanisms involved in transporting proteins destined to active zones are distinct from those that transport synaptic vesicles or mitochondria. This study reports that localization of BRP-an essential active zone scaffolding protein in Drosophila, depends on the precise balance of neuronal Par-1 kinase. Disruption of Par-1 levels leads to excess accumulation of BRP in axons at the expense of BRP at active zones. Temporal analyses demonstrate that accumulation of BRP within axons precedes the loss of synaptic function and its depletion from the active zones. Mechanistically, this study finds that Par-1 co-localizes with BRP and is present in the same molecular complex, raising the possibility of a novel mechanism for selective localization of BRP-like active zone scaffolding proteins. Taken together, these data suggest an intriguing possibility that mislocalization of active zone proteins like BRP might be one of the earliest signs of synapse perturbation and perhaps, synaptic networks that precede many neurological disorders.
Xing, X. and Wu, C. F. (2018). Inter-relationships among physical dimensions, distal-proximal rank orders, and basal GCaMP fluorescence levels in Ca(2+) imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions. J Neurogenet: 1-14. PubMed ID: 30322321
Summary:
GCaMP imaging is widely employed for investigating neuronal Ca(2+) dynamics. The Drosophila larval neuromuscular junction (NMJ) consists of three distinct types of motor terminals (type Ib, Is and II). This study investigated whether variability in synaptic bouton sizes and GCaMP expression levels confound interpretations of GCaMP readouts. Analysis of large data sets accumulated over years established the wide ranges of bouton sizes and GCaMP baseline fluorescence, with large overlaps among synaptic categories. Bouton size and GCaMP baseline fluorescence were not confounding factors in determining the characteristic frequency responses among type Ib, Is and II synapses. More importantly, the drastic phenotypes that hyperexcitability mutations manifest preferentially in particular synaptic categories, were not obscured by bouton heterogeneity in physical size and GCaMP expression level. The results illustrate the conditions that disrupt or enhance the distal-proximal gradients. For example, stimulus frequencies just above the threshold level produced the steepest gradient in low Ca(2+) (0.1 mM) saline, while supra-threshold stimulation flattened the gradient. Moreover, membrane hyperexcitability mutations (eag(1) Sh(120) and para(bss1)) and mitochondrial inhibition by dinitrophenol (DNP) disrupted the gradient. However, a novel distal-proximal gradient of decay kinetics appeared after long-term DNP incubation. Focal recording was performed to assess the failure rates in transmission at low Ca(2+) levels, which yielded indications of a mild distal-proximal gradient in release probability.
Yang, W. T., Li, Y. C., Lin, C. M., Tang, W. C., Chen, B. C., Suzuki, E. and Hsu, J. C. (2018). Adherens junction-associated pores mediate the intercellular transport of endosomes and cytoplasmic proteins. Biochem Biophys Res Commun. PubMed ID: 30286955
Summary:
Intercellular endosomes (IEs) are endocytosed vesicles shuttled through the adherens junctions (AJs) between two neighboring epidermal cells during Drosophila dorsal closure. The cell-to-cell transport of IEs requires DE-cadherin (DE-cad), microtubules (MTs) and kinesin. However, the mechanisms by which IEs can be transported through the AJs are unknown. This study demonstrate the presence of AJ-associated pores with MTs traversing through the pores. Live imaging allows direct visualization of IEs being transported through the AJ-associated pores. By using an optogenetic dimerization system, it was observe that the dimerized IE-kinesin complexes move across AJs into the neighboring cell. The AJ-associated pores also allow intercellular movement of soluble proteins. Importantly, most epidermal cells form dorsoventral-oriented two-cell syncytia. Together, this study presents a model in which an AJ-associated pore mediates the intercellular transport of IEs and proteins between two cells in direct contact.
Di Cara, F., Rachubinski, R. A. and Simmonds, A. J. (2018). Distinct roles for peroxisomal targeting signal receptors Pex5 and Pex7 in Drosophila. Genetics. PubMed ID: 30389805
Summary:
Peroxisomes are ubiquitous membrane-enclosed organelles involved in lipid processing and reactive oxygen detoxification. Mutations in human peroxisome biogenesis genes (Peroxin, PEX or Pex) cause developmental disabilities and often early death. Pex5 and Pex7 are receptors that recognize different peroxisomal targeting signals called PTS1 and PTS2, respectively, and traffic proteins to the peroxisomal matrix. This study characterized mutants of Drosophila melanogaster Pex5 and Pex7 and found that adult animals are affected in lipid processing. Pex5 mutants exhibited severe developmental defects in the embryonic nervous system and muscle similar to what is observed in humans with PEX5 mutations, while Pex7 fly mutants were weakly affected in brain development, suggesting different roles for fly Pex7 and human PEX7. Of note, although no PTS2-containing protein has been identified in Drosophila, Pex7 from Drosophila can function as a bona fide PTS2 receptor because it can rescue targeting of the PTS2-containing protein thiolase to peroxisomes in PEX7 mutant human fibroblasts.
Harris, N., Fetter, R. D., Brasier, D. J., Tong, A. and Davis, G. W. (2018). Molecular interface of neuronal innate immunity, synaptic vesicle stabilization, and presynaptic homeostatic plasticity. Neuron. PubMed ID: 30344041
Summary:
This study defines a homeostatic function for innate IMD immune signaling within neurons. A genetic analysis of the innate immune signaling genes IMD, IKKbeta, Tak1, and Relish demonstrates that each is essential for presynaptic homeostatic plasticity (PHP). Subsequent analyses define how the rapid induction of PHP (occurring in seconds) can be coordinated with the life-long maintenance of PHP, a time course that is conserved from invertebrates to mammals. This study defines a novel bifurcation of presynaptic innate immune signaling. Tak1 (Map3K) acts locally and is selective for rapid PHP induction. IMD, IKKbeta, and Relish are essential for long-term PHP maintenance. This study then define how Tak1 controls vesicle release. Tak1 stabilizes the docked vesicle state, which is essential for the homeostatic expansion of the readily releasable vesicle pool. This represents a mechanism for the control of vesicle release, and an interface of innate immune signaling with the vesicle fusion apparatus and homeostatic plasticity.
Huang, Y., Huang, S., Di Scala, C., Wang, Q., Wandall, H. H., Fantini, J. and Zhang, Y. Q. (2018). The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt. Elife 7. PubMed ID: 30355446
Summary:
Lipids are structural components of cellular membranes and signaling molecules that are widely involved in development and diseases, but the underlying molecular mechanisms are poorly understood, partly because of the vast variety of lipid species and complexity of synthetic and turnover pathways. From a genetic screen, this study identified that mannosyl glucosylceramide (MacCer), a species of glycosphingolipid (GSL), promotes synaptic bouton formation at the Drosophila neuromuscular junction (NMJ). Pharmacological and genetic analysis shows that the NMJ growth-promoting effect of MacCer depends on normal lipid rafts, which are known to be composed of sphingolipids, sterols and select proteins. MacCer positively regulates the synaptic level of Wnt1/Wingless (Wg) and facilitates presynaptic Wg signaling, whose activity is raft-dependent. Furthermore, a functional GSL-binding motif in Wg exhibiting a high affinity for MacCer is required for normal NMJ growth. These findings reveal a novel mechanism whereby the GSL MacCer promotes synaptic bouton formation via Wg signaling.

Thursday, November 29th - Evolution

Dupim, E. G., Goldstein, G., Vanderlinde, T., Vaz, S. C., Krsticevic, F., Bastos, A., Pinhao, T., Torres, M., David, J. R., Vilela, C. R. and Carvalho, A. B. (2018). An investigation of Y chromosome incorporations in 400 species of Drosophila and related genera. PLoS Genet 14(11): e1007770. PubMed ID: 30388103
Summary:
Y chromosomes are widely believed to evolve from a normal autosome through a process of massive gene loss (with preservation of some male genes), shaped by sex-antagonistic selection and complemented by occasional gains of male-related genes. The net result of these processes is a male-specialized chromosome. This might be expected to be an irreversible process, but it was found in 2005 that the Drosophila pseudoobscura Y chromosome was incorporated into an autosome. Y chromosome incorporations have important consequences: a formerly male-restricted chromosome reverts to autosomal inheritance, and the species may shift from an XY/XX to X0/XX sex-chromosome system. In order to assess the frequency and causes of this phenomenon Y chromosome incorporations was sought in 400 species from Drosophila and related genera. One additional large scale event of Y chromosome incorporation was found, affecting the whole montium subgroup (40 species in the sample); overall 13% of the sampled species (52/400) have Y incorporations. While previous data indicated that after the Y incorporation the ancestral Y disappeared as a free chromosome, the much larger data set analyzed here indicates that a copy of the Y survived as a free chromosome both in montium and pseudoobscura species, and that the current Y of the pseudoobscura lineage results from a fusion between this free Y and the neoY. The 400 species sample also showed that the previously suggested causal connection between X-autosome fusions and Y incorporations is, at best, weak: the new case of Y incorporation (montium) does not have X-autosome fusion, whereas nine independent cases of X-autosome fusions were not followed by Y incorporations. Y incorporation is an underappreciated mechanism affecting Y chromosome evolution; these results show that at least in Drosophila it plays a relevant role and highlight the need of similar studies in other groups.
Ebadi, H., Perry, M., Short, K., Klemm, K., Desplan, C., Stadler, P. F. and Mehta, A. (2018). Patterning the insect eye: From stochastic to deterministic mechanisms. PLoS Comput Biol 14(11): e1006363. PubMed ID: 30439954
Summary:
While most processes in biology are highly deterministic, stochastic mechanisms are sometimes used to increase cellular diversity. In human and Drosophila eyes, photoreceptors sensitive to different wavelengths of light are distributed in stochastic patterns, and one such patterning system has been analyzed in detail in the Drosophila retina. Interestingly, some species in the dipteran family Dolichopodidae (the "long legged" flies, or "Doli") instead exhibit highly orderly deterministic eye patterns. In these species, alternating columns of ommatidia (unit eyes) produce corneal lenses of different colors. Occasional perturbations in some individuals disrupt the regular columns in a way that suggests that patterning occurs via a posterior-to-anterior signaling relay during development, and that specification follows a local, cellular-automaton-like rule. It is hypothesized that the regulatory mechanisms that pattern the eye are largely conserved among flies and that the difference between unordered Drosophila and ordered dolichopodid eyes can be explained in terms of relative strengths of signaling interactions rather than a rewiring of the regulatory network itself. A simple stochastic model is presented that is capable of explaining both the stochastic Drosophila eye and the striped pattern of Dolichopodidae eyes and thereby characterize the least number of underlying developmental rules necessary to produce both stochastic and deterministic patterns. Only small changes to model parameters are needed to also reproduce intermediate, semi-random patterns observed in another Doli species, and quantification of ommatidial distributions in these eyes suggests that their patterning follows similar rules.
Craddock, E. M., Kambysellis, M. P., Franchi, L., Francisco, P., Grey, M., Hutchinson, A., Nanhoo, S. and Antar, S. (2018). Ultrastructural variation and adaptive evolution of the ovipositor in the endemic Hawaiian Drosophilidae. J Morphol. PubMed ID: 30397938
Summary:
Ecological diversification of the endemic Hawaiian Drosophilidae has been accompanied by striking divergence in egg morphology, and ovarian structure and function. To determine how these flies successfully oviposit in a variety of breeding substrates, Scanning Electron Microscopy was used to examine the ultrastructure of the ovipositor of a sample of 65 Drosophila species and five Scaptomyza species of this hyperdiverse monophyletic group. The Drosophila species analyzed included representatives of the fungus-breeding haleakalae group, the leaf-breeding antopocerus and modified tarsus groups, the modified mouthparts species group, the nudidrosophila, and the picture wing clade; the latter sample of 41 species from four species groups included stem- and bark-breeders, as well as tree sap flux-breeders. Ovipositor length was found to vary more than 12-fold among Hawaiian drosophilids, with the longest ovipositors observed in the bark-breeding species and the shortest among the Scaptomyza and fungus-breeders. More noteworthy is the striking variation in overall shape and proportions of the ovipositor, in the shape of the apical region, and in the pattern of sensory structures or ovisensilla. Ultrastructural observations of the pair of long subapical sensilla on the ventral side identify these as taste bristles. Ovipositor form correlates strongly with the oviposition substrate used by the species, being of a distinctive shape and size in each case. It is inferred that the observed morphological divergence in the ovipositor is adaptive and the product of natural selection for successful reproduction in alternate microhabitats.
Wensing, K. U. and Fricke, C. (2018). Divergence in sex peptide-mediated female post-mating responses in Drosophila melanogaster. Proc Biol Sci 285(1886). PubMed ID: 30209231
Summary:
Transfer and receipt of seminal fluid proteins crucially affect reproductive processes in animals. Evolution in these male ejaculatory proteins is explained with post-mating sexual selection, but a good understanding of the evolution of female post-mating responses (PMRs) to these proteins is lacking. Some of these proteins are expected to mediate sexually antagonistic coevolution generating the expectation that females evolve resistance. One candidate in Drosophila melanogaster is the sex peptide (SP) which confers cost of mating in females. This paper compared female SP-induced PMRs across three D. melanogaster wild-type populations after mating with SP-lacking versus control males including fitness measures. Surprisingly, no evidence was found for SP-mediated fitness costs in any of the populations. However, female lifetime reproductive success and lifespan were differently affected by SP receipt indicating that female PMRs diverged among populations. Injection of synthetic SP into virgin females further supported these findings and suggests that females from different populations require different amounts of SP to effectively initiate PMRs. Molecular analyses of the SP receptor suggest that genetic differences might explain the observed phenotypical divergence. The evolutionary processes that might have caused this divergence in female PMRs is discussed.
Filice, D. C. S. and Long, T. A. F. (2018). Genetic trade-offs between male reproductive traits in Drosophila melanogaster. Biol Lett 14(10). PubMed ID: 30333262
Summary:
In Drosophila melanogaster, males engage in both extensive pre- and post-copulatory competition for the opportunity to mate with females and subsequently sire offspring. The selection pressure for increased male reproductive success has resulted in the evolution of a wide diversity of sexual traits. However, despite strong selection, individuals often exhibit considerable phenotypic variation in the expression of these traits, and it is unclear if any of this variation is owing to underlying genetic trade-offs. Using hemiclonal flies this study examined how male reproductive success covaries with their ability to induce long-term stimulation of oogenesis and oviposition in their mates, and how this relationship may change over time. It was found that males from hemiclone lines with phenotypes that were more successful in a short-term reproductive 'scramble' environment were less effective at stimulating long-term fecundity in females. Furthermore, it was observed that males from hemiclone lines which showed the most improvement over a longer reproductive interaction period also tended to stimulate higher long-term fecundity in females. Together, these results indicate the presence of genetic trade-offs between different male reproductive traits and offer insights into the maintenance of their variation.
Castellano, D., James, J. and Eyre-Walker, A. (2018). Nearly neutral evolution across the Drosophila melanogaster genome. Mol Biol Evol 35(11): 2685-2694. PubMed ID: 30418639
Summary:
Under the nearly neutral theory of molecular evolution, the proportion of effectively neutral mutations is expected to depend upon the effective population size (Ne). This study investigate whether this is the case across the genome of Drosophila melanogaster using polymorphism data from North American and African lines. This study shows that the ratio of the number of nonsynonymous and synonymous polymorphisms is negatively correlated to the number of synonymous polymorphisms, even when the nonindependence is accounted for. The relationship is such that the proportion of effectively neutral nonsynonymous mutations increases by approximately 45% as Ne is halved. However, it was also shown that this relationship is steeper than expected from an independent estimate of the distribution of fitness effects from the site frequency spectrum. A number of potential explanations for this was investigated, and it was shown, using simulation, that this is consistent with a model of genetic hitchhiking: Genetic hitchhiking depresses diversity at neutral and weakly selected sites, but has little effect on the diversity of strongly selected sites.

Tuesday November 27th - Behavior

Zheng, Y., Xue, Y., Ren, X., Liu, M., Li, X., Jia, Y., Niu, Y., Ni, J. Q., Zhang, Y. and Ji, J. Y. (2018). The lysine demethylase dKDM2 is non-essential for viability, but regulates circadian rhythms in Drosophila. Front Genet 9: 354. PubMed ID: 30233643
Summary:
Post-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive depending on specific pathological contexts. However, the role of KDM2 proteins during development remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, two dKdm2 null alleles were generated using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately 1 h shorter than the control. Interestingly, lengthened circadian periods are observed when dKdm2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of dKDM2 and its orthologs in vertebrates regarding the regulation of circadian rhythms will advance understanding of the epigenetic regulations of circadian clocks.
Szczecinski, N. S., Bockemuhl, T., Chockley, A. S. and Buschges, A. (2018). Static stability predicts the continuum of interleg coordination patterns in Drosophila. J Exp Biol. PubMed ID: 30274987
Summary:
During walking, insects must coordinate the movements of their six legs for efficient locomotion. This interleg coordination is speed-dependent; fast walking in insects is associated with tripod coordination patterns, while slow walking is associated with more variable, tetrapod-like patterns. To date, however, there has been no comprehensive explanation as to why these speed-dependent shifts in interleg coordination should occur in insects. Tripod coordination would be sufficient at low walking speeds. The fact that insects use a different interleg coordination pattern at lower speeds suggests that it is more optimal or advantageous at these speeds. Furthermore, previous studies focused on discrete tripod and tetrapod coordination patterns. Experimental data, however, suggest that changes observed in interleg coordination are part of a speed-dependent spectrum. This study explored these issues in relation to static stability as an important aspect for interleg coordination in Drosophila. A model was created that uses basic experimentally measured parameters in fruit flies to find the interleg phase relationships that maximize stability for a given walking speed. The model predicted a continuum of interleg coordination patterns spanning the complete range of walking speeds as well as an anteriorly directed swing phase progression. Furthermore, for low walking speeds the model predicted tetrapod-like patterns to be most stable, while at high walking speeds tripod coordination emerged as most optimal. Finally, the basic assumption of a continuum of interleg coordination patterns was validated in a large set of experimental data from walking fruit flies and these data were compared with the model-based predictions.
Yurgel, M. E., Shah, K. D., Brown, E. B., Burns, C., Bennick, R. A., DiAngelo, J. R. and Keene, A. C. (2018). Ade2 functions in the Drosophila fat body to promote sleep. G3 (Bethesda). PubMed ID: 30249751
Summary:
Metabolic state is a potent modulator of sleep and circadian behavior and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, an RNAi screen selectively knocking down genes in the fat body. Knockdown was performed of Phosphoribosylformylglycinamidine synthase/Pfas(Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes.
Creamer, M. S., Mano, O. and Clark, D. A. (2018). Visual control of walking speed in Drosophila. Neuron. PubMed ID: 30415994
Summary:
An animal's self-motion generates optic flow across its retina, and it can use this visual signal to regulate its orientation and speed through the world. While orientation control has been studied extensively in Drosophila and other insects, much less is known about the visual cues and circuits that regulate translational speed. This study shows that flies regulate walking speed with an algorithm that is tuned to the speed of visual motion, causing them to slow when visual objects are nearby. This regulation does not depend strongly on the spatial structure or the direction of visual stimuli, making it algorithmically distinct from the classic computation that controls orientation. Despite the different algorithms, the visual circuits that regulate walking speed overlap with those that regulate orientation. Taken together, these findings suggest that walking speed is controlled by a hierarchical computation that combines multiple motion detectors with distinct tunings.
Duistermars, B. J., Pfeiffer, B. D., Hoopfer, E. D. and Anderson, D. J. (2018). A brain module for scalable control of complex, multi-motor threat displays. Neuron. PubMed ID: 30415997
Summary:
Threat displays are a universal feature of agonistic interactions. Whether threats are part of a continuum of aggressive behaviors or separately controlled remains unclear. Threats were analyzed in Drosophila; they are triggered by male cues and visual motion, and comprised of multiple motor elements that can be flexibly combined. A cluster of approximately 3 neurons was isolated whose activity is necessary for threat displays but not for other aggressive behaviors, and whose artificial activation suffices to evoke naturalistic threats in solitary flies, suggesting that the neural control of threats is modular with respect to other aggressive behaviors. Artificially evoked threats suffice to repel opponents from a resource in the absence of contact aggression. Depending on its level of artificial activation, this neural threat module can evoke different motor elements in a threshold-dependent manner. Such scalable modules may represent fundamental "building blocks" of neural circuits that mediate complex multi-motor behaviors.
Zacarias, R., Namiki, S., Card, G. M., Vasconcelos, M. L. and Moita, M. A. (2018). Speed dependent descending control of freezing behavior in Drosophila melanogaster. Nat Commun 9(1): 3697. PubMed ID: 30209268
Summary:
The most fundamental choice an animal has to make when it detects a threat is whether to freeze, reducing its chances of being noticed, or to flee to safety. This study shows that Drosophila melanogaster exposed to looming stimuli in a confined arena either freeze or flee. The probability of freezing versus fleeing is modulated by the fly's walking speed at the time of threat, demonstrating that freeze/flee decisions depend on behavioral state. A pair of descending neurons crucially implicated in freezing is described. Genetic silencing of DNp09 descending neurons disrupts freezing yet does not prevent fleeing. Optogenetic activation of both DNp09 neurons induces running and freezing in a state-dependent manner. These findings establish walking speed as a key factor in defensive response choices and reveal a pair of descending neurons as a critical component in the circuitry mediating selection and execution of freezing or fleeing behaviors.

Monday, December 26th - Stem Cells

Zhao, S., Fortier, T. M. and Baehrecke, E. H. (2018). Autophagy promotes tumor-like stem cell niche occupancy. Curr Biol 28(19): 3056-3064.e3053. PubMed ID: 30270184
Summary:
Adult stem cells usually reside in specialized niche microenvironments. Accumulating evidence indicates that competitive niche occupancy favors stem cells with oncogenic mutations, also known as tumor-like stem cells. However, the mechanisms that regulate tumor-like stem cell niche occupancy are largely unknown. This study used Drosophila ovarian germline stem cells as a model and use bam mutant cells as tumor-like stem cells. Interestingly, it was found that autophagy is low in wild-type stem cells but elevated in bam mutant stem cells. Significantly, autophagy is required for niche occupancy by bam mutant stem cells. Although loss of either atg6 or Fip200 alone in stem cells does not impact their competitiveness, loss of these conserved regulators of autophagy decreases bam mutant stem cell niche occupancy. In addition, starvation enhances the competition of bam mutant stem cells for niche occupancy in an autophagy-dependent manner. Of note, loss of autophagy slows the cell cycle of bam mutant stem cells and does not influence stem cell death. In contrast to canonical epithelial cell competition, loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influences the competition of bam mutant stem cells for niche occupancy. Additionally, autophagy promotes the tumor-like growth of bam mutant ovaries. Autophagy is known to be induced in a wide variety of tumors. Therefore, these results suggest that specifically targeting autophagy in tumor-like stem cells has potential as a therapeutic strategy.
Bodofsky, S., Liberatore, K., Pioppo, L., Lapadula, D., Thompson, L., Birnbaum, S., McClung, G., Kartik, A., Clever, S. and Wightman, B. (2018). A tissue-specific enhancer of the C. elegans nhr-67/tailless gene drives coordinated expression in uterine stem cells and the differentiated anchor cell. Gene Expr Patterns 30: 71-81. PubMed ID: 30404043
Summary:
The nhr-67 nuclear receptor gene of C. elegans encodes the ortholog of the Drosophila tailless genes. In C. elegans, nhr-67 plays multiple roles in the development of the uterus during L2 and L3 larval stages. Four pre-VU cells are born in the L2 stage and form the precursor complement for the ventral surface of the mature uterus. One of the four pre-VU cells becomes the anchor cell (AC), which exits the cell cycle and differentiates, while the remaining three VU cells serve as stem cells that populate the ventral uterus. The nhr-67 gene functions in the development of both VU cell lineages and AC differentiation. Hypomorphic mutations in nhr-67 identify a 276bp region of the distal promoter that is sufficient to activate nhr-67 expression in pre-VU cells and the AC. The 276bp region includes 8 conserved potential cis-acting sites, including two E boxes and a nuclear receptor binding site. Mutational analysis demonstrates that the two E boxes are required for expression of nhr-67 in uterine precursor cells. The E/daughterless ortholog HLH-2 binds these sites as a homodimer, thus playing a central role in activating nhr-67 expression in the uterine precursors. The organization of the nhr-67 uterine precursor enhancer is compared to similar conserved enhancers in the egl-43, lag-2, and lin-3 genes, which contain the same HLH-2-binding E boxes and are similarly expressed in both pre-VU cells and the AC. This basic regulatory module allows the coordinated expression of at least four genes. Expression of genes in different cells that must coordinate to form a mature organ is driven by a shared set of promoter elements, which integrate multiple transcription factor inputs.
Doupe, D. P., Marshall, O. J., Dayton, H., Brand, A. H. and Perrimon, N. (2018). Drosophila intestinal stem and progenitor cells are major sources and regulators of homeostatic niche signals. Proc Natl Acad Sci U S A. PubMed ID: 30404917
Summary:
Epithelial homeostasis requires the precise balance of epithelial stem/progenitor proliferation and differentiation. While many signaling pathways that regulate epithelial stem cells have been identified, it is probable that other regulators remain unidentified. This study uses gene-expression profiling by targeted DamID to identify the stem/progenitor-specific transcription and signaling factors in the Drosophila midgut. Many signaling pathway components, including ligands of most major pathways, exhibit stem/progenitor-specific expression and have regulatory regions bound by both intrinsic and extrinsic transcription factors. In addition to previously identified stem/progenitor-derived ligands, this study shows that both the insulin-like factor Ilp6 and TNF ligand eiger are specifically expressed in the stem/progenitors and regulate normal tissue homeostasis. It is proposed that intestinal stem cells not only integrate multiple signals but also contribute to and regulate the homeostatic signaling microenvironmental niche through the expression of autocrine and paracrine factors.
Mattila, J., Kokki, K., Hietakangas, V. and Boutros, M. (2018). Stem cell intrinsic hexosamine metabolism regulates intestinal adaptation to nutrient content. Dev Cell 47(1): 112-121.e113. PubMed ID: 30220570
Summary:
The intestine is an organ with an exceptionally high rate of cell turnover, and perturbations in this process can lead to severe diseases such as cancer or intestinal atrophy. Nutrition has a profound impact on intestinal volume and cellular architecture. However, how intestinal homeostasis is maintained in fluctuating dietary conditions remains insufficiently understood. By utilizing the Drosophila midgut model, this study reveals a novel stem cell intrinsic mechanism coupling cellular metabolism with stem cell extrinsic growth signal. The results show that intestinal stem cells (ISCs) employ the hexosamine biosynthesis pathway (HBP) to monitor nutritional status. Elevated activity of HBP promotes Warburg effect-like metabolic reprogramming required for adjusting the ISC division rate according to nutrient content. Furthermore, HBP activity is an essential facilitator for insulin signaling-induced ISC proliferation. In conclusion, ISC intrinsic hexosamine synthesis regulates metabolic pathway activities and defines the stem cell responsiveness to niche-derived growth signals.
Resende, L. P., Monteiro, A., Bras, R., Lopes, T. and Sunkel, C. E. (2018). Aneuploidy in intestinal stem cells promotes gut dysplasia in Drosophila. J Cell Biol. PubMed ID: 30282810
Summary:
Aneuploidy is associated with different human diseases including cancer. However, different cell types appear to respond differently to aneuploidy, either by promoting tumorigenesis or causing cell death. This study examined the behavior of adult Drosophila melanogaster intestinal stem cells (ISCs) after induction of chromosome missegregation either by abrogation of the spindle assembly checkpoint or through kinetochore disruption or centrosome amplification. These conditions induce moderate levels of aneuploidy in ISCs, and no evidence of apoptosis was found. Instead, a significant accumulation was found of ISCs associated with increased stem cell proliferation and an excess of enteroendocrine cells. Moreover, aneuploidy causes up-regulation of the JNK pathway throughout the posterior midgut, and specific inhibition of JNK signaling in ISCs is sufficient to prevent dysplasia. These findings highlight the importance of understanding the behavior of different stem cell populations to aneuploidy and how these can act as reservoirs for genomic alterations that can lead to tissue pathologies.
Tamirisa, S., Papagiannouli, F., Rempel, E., Ermakova, O., Trost, N., Zhou, J., Mundorf, J., Brunel, S., Ruhland, N., Boutros, M., Lohmann, J. U. and Lohmann, I. (2018). Decoding the regulatory logic of the Drosophila male stem cell system. Cell Rep 24(11): 3072-3086. PubMed ID: 30208329
Summary:
The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. This study elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Key roles were identified of nucleoporins and V-ATPase proton pumps, and their importance was demonstrated in controlling germline development from the support side. To make the dataset publicly available, an interactive analysis tool was generated, that uncovered conserved core genes of adult stem cells across species boundaries. The functional relevance of these genes was tested in the Drosophila testis and intestine, and a high frequency of stem cell defects was found. In summary, this dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types.

Friday, November 22nd - Chromatin

Shrestha, S., Oh, D. H., McKowen, J. K., Dassanayake, M. and Hart, C. M. (2018). 4C-seq characterization of Drosophila BEAF binding regions provides evidence for highly variable long-distance interactions between active chromatin. PLoS One 13(9): e0203843. PubMed ID: 30248133
Summary:
Chromatin organization is crucial for nuclear functions such as gene regulation, DNA replication and DNA repair. Insulator binding proteins, such as the Drosophila Boundary Element-Associated Factor (BEAF), are involved in chromatin organization. To further understand the role of BEAF, cis- and trans-interaction partners were detected of four BEAF binding regions (viewpoints) using 4C (circular chromosome conformation capture), and their association was analyzed with different genomic features. Previous genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes. These 4C data show the interaction partners of these viewpoints are highly variable and generally enriched for active chromatin marks. The most consistent association was with housekeeping genes, a feature in common with these viewpoints. Fluorescence in situ hybridization indicated that the long-distance interactions occur even in the absence of BEAF. These data are most consistent with a model in which BEAF is redundant with other factors found at active promoters. The results point to principles of long-distance interactions made by active chromatin, supporting a previously proposed model in which condensed chromatin is sticky and associates into topologically associating domains (TADs) separated by active chromatin. It is proposed that the highly variable long-distance interactions that were detected are driven by redundant factors that open chromatin to promote transcription, combined with active chromatin filling spaces between TADs while packing of TADs relative to each other varies from cell to cell.
Chang, C. H. and Larracuente, A. M. (2018). Heterochromatin-enriched assemblies reveal the sequence and organization of the Drosophila melanogaster Y chromosome. Genetics. PubMed ID: 30420487
Summary:
Heterochromatic regions of the genome are repeat-rich and poor in protein coding genes, and are therefore underrepresented in even the best genome assemblies. One of the most difficult regions of the genome to assemble are sex-limited chromosomes. The Drosophila melanogaster Y chromosome is entirely heterochromatic, yet has wide-ranging effects on male fertility, fitness, and genome-wide gene expression. The genetic basis of this phenotypic variation is difficult to study, in part because the detailed organization of the Y chromosome is not known. To study Y chromosome organization in D. melanogaster, an assembly strategy was developed involving the in silico enrichment of heterochromatic long single-molecule reads, and these reads were to create targeted de novo assemblies of heterochromatic sequences. Contigs were assigned to the Y chromosome using Illumina reads to identify male-specific sequences. This pipeline extends the D. melanogaster reference genome by 11.9 Mb, closes 43.8% of the gaps, and improves overall contiguity. The addition of 10.6 MB of Y-linked sequence permitted study of the organization of repeats and genes along the Y chromosome. A high rate of duplication was detected to the pericentric regions of the Y chromosome from other regions in the genome. Most of these duplicated genes exist in multiple copies. The evolutionary history of one sex-linked gene family-crystal-Stellate was detailed. While the Y chromosome does not undergo crossing over, high gene conversion rates were detected within and between members of the crystal-Stellate gene family, Su(Ste), and PCKR, compared to genome-wide estimates. These results suggest that gene conversion and gene duplication play an important role in the evolution of Y-linked genes.
Baldi, S., Jain, D. S., Harpprecht, L., Zabel, A., Scheibe, M., Butter, F., Straub, T. and Becker, P. B. (2018). Genome-wide rules of nucleosome phasing in Drosophila. Mol Cell 72(4): 661-672.e664. PubMed ID: 30392927
Summary:
Regular successions of positioned nucleosomes, or phased nucleosome arrays (PNAs), are predominantly known from transcriptional start sites (TSSs). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, spectral analysis was applied to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSSs and are strongly enriched for an uncharacterized sequence motif. Through genome-wide reconstitution of physiological chromatin in Drosophila embryo extracts, the molecular basis of PNA formation was uncovered. Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the motif, was uncovered. It also revealed how the global activity of the chromatin remodelers CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. This work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale in vitro.
Jankovics, F., Bence, M., Sinka, R., Farago, A., Bodai, L., Pettko-Szandtner, A., Ibrahim, K., Takacs, Z., Szarka-Kovacs, A. B. and Erdelyi, M. (2018). Drosophila small ovary gene is required for transposon silencing and heterochromatin organisation and ensures germline stem cell maintenance and differentiation. Development. PubMed ID: 30389853
Summary:
Self-renewal and differentiation of stem cells is one of the fundamental biological phenomena relying on proper chromatin organisation. This study describes a novel chromatin regulator encoded by the Drosophila small ovary (sov) gene. sov was shown to be required in both the germline stem cells (GSCs) and the surrounding somatic niche cells to ensure GSC survival and differentiation. Sov maintains niche integrity and function by repressing transposon mobility, not only in the germline, but also in the soma. Protein interactome analysis of Sov revealed an interaction between Sov and HP1a. In the germ cell nuclei, Sov co-localises with HP1a, suggesting that Sov affects transposon repression as a component of the heterochromatin. In a position effect variegation assay, a dominant genetic interaction was found between sov and HP1a, indicating their functional cooperation in promoting the spread of heterochromatin. An in vivo tethering assay and FRAP analysis revealed that Sov enhances heterochromatin formation by supporting the recruitment of HP1a to the chromatin. A model is proposed in which sov maintains GSC niche integrity by regulating transposon silencing and heterochromatin formation.
Anselm, E., Thomae, A. W., Jeyaprakash, A. A. and Heun, P. (2018). Oligomerization of Drosophila Nucleoplasmin-Like Protein is required for its centromere localization. Nucleic Acids Res. PubMed ID: 30357352
Summary:
The evolutionarily conserved nucleoplasmin family of histone chaperones has two paralogues in Drosophila, named Nucleoplasmin-Like Protein (NLP) and Nucleophosmin (NPH). NLP localizes to the centromere, yet molecular underpinnings of this localization are unknown. Moreover, similar to homologues in other organisms, NLP forms a pentamer in vitro, but the biological significance of its oligomerization has not been explored. This study characterize the oligomers formed by NLP and NPH in vivo and find that oligomerization of NLP is required for its localization at the centromere. It was further shown that oligomerization-deficient NLP is unable to bind the centromeric protein Hybrid Male Rescue (HMR), which in turn is required for targeting the NLP oligomer to the centromere. Finally, using super-resolution microscopy NLP and HMR were found to largely co-localize in domains that are immediately adjacent to, yet distinct from centromere domains defined by the centromeric histone dCENP-A.
Abed, J. A., Ghotbi, E., Ye, P., Frolov, A., Benes, J. and Jones, R. S. (2018). De novo recruitment of Polycomb-group proteins in Drosophila embryos. Development. PubMed ID: 30389849
Summary:
Polycomb-group (PcG)-mediated transcriptional repression of target genes can be delineated into two phases. First, following initial repression of target genes by gene-specific transcription factors, PcG proteins recognize the repressed state and assume control of the genes' repression. Once the silenced state is established, PcG proteins may maintain repression through an indefinite number of cell cycles. Little is understood about how PcG proteins initially recognize the repressed state of target genes and the steps leading to de novo establishment of PcG-mediated repression. This study describes a genetic system in which a Drosophila PcG target gene, giant (gt), is ubiquitously repressed during early embryogenesis by a maternally expressed transcription factor, and show the temporal recruitment of components of three PcG protein complexes, PhoRC, PRC1, and PRC2. De novo PcG recruitment follows a temporal hierarchy in which PhoRC stably localizes at the target gene at least one hour before stable recruitment of PRC2 and concurrent trimethylation of histone H3 at lysine 27 (H3K27me3). The presence of PRC2 and increased levels of H3K27me3 are found to precede stable binding by PRC1.

Wednesday, November 21st - Apoptosis and Autophagy

Baena-Lopez, L. A., Arthurton, L., Bischoff, M., Vincent, J. P., Alexandre, C. and McGregor, R. (2018). Novel initiator caspase reporters uncover unknown features of caspase-activating cells. Development. PubMed ID: 30413561
Summary:
The caspase-mediated regulation of many cellular processes, including apoptosis, justifies the substantial interest in understanding all of the biological features of these enzymes. To complement functional assays, it is critical to identify caspase-activating cells in live tissues. This work describes novel initiator caspase-reporters that, for the first time, provide direct information concerning the initial steps of the caspase activation cascade in Drosophila tissues. A caspase-sensor has capitalized on the rapid subcellular localization change of a fluorescent marker to uncover novel cellular apoptotic events. These refer to the actin-mediated positioning of the nucleus before cell delamination. The other construct has benefited from a caspase-induced nuclear translocation of a QF transcription factor. This feature enables the genetic manipulation of caspase-activating cells, whilst showing the spatio-temporal patterns of initiator caspase activity. Collectively, these sensors offer experimental opportunities not available by using previous reporters and therefore they have been proven useful to illuminate unknown aspects of caspase-dependent processes in apoptotic and non-apoptotic cellular scenarios.
Clark, S. G., Graybeal, L. L., Bhattacharjee, S., Thomas, C., Bhattacharya, S. and Cox, D. N. (2018). Basal autophagy is required for promoting dendritic terminal branching in Drosophila sensory neurons. PLoS One 13(11): e0206743. PubMed ID: 30395636
Summary:
Relatively little is known regarding the developmental role of basal autophagy in directing aspects of dendritic arborization. This study demonstrates that autophagy-related (Atg) genes are positively regulated by the homeodomain transcription factor Cut, and that basal autophagy functions as a downstream effector pathway for Cut-mediated dendritic terminal branching in Drosophila multidendritic (md) sensory neurons. Further, loss of function analyses implicate Atg genes in promoting cell type-specific dendritic arborization and terminal branching, while gain of function studies suggest that excessive autophagy leads to dramatic reductions in dendritic complexity. The Atg1 initiator kinase interacts with the dual leucine zipper kinase (DLK) pathway by negatively regulating the E3 ubiquitin ligase Highwire and positively regulating the MAPKKK Wallenda. Finally, autophagic induction partially rescues dendritic atrophy defects observed in a model of polyglutamine toxicity. Collectively, these studies implicate transcriptional control of basal autophagy in directing dendritic terminal branching and demonstrate the importance of homeostatic control of autophagic levels for dendritic arbor complexity under native or cellular stress conditions.
Singh, T., Lee, E. H., Hartman, T. R., Ruiz-Whalen, D. M. and O'Reilly, A. M. (2018). Opposing action of Hedgehog and insulin signaling balances proliferation and autophagy to determine follicle stem cell lifespan. Dev Cell 46(6): 720-734.e726. PubMed ID: 30197240
Summary:
Egg production declines with age in many species, a process linked with stem cell loss. Diet-dependent signaling has emerged as critical for stem cell maintenance during aging. Follicle stem cells (FSCs) in the Drosophila ovary are exquisitely responsive to diet-induced signals including Hedgehog (Hh) and insulin-IGF signaling (IIS), entering quiescence in the absence of nutrients and initiating proliferation rapidly upon feeding. Although highly proliferative FSCs generally exhibit an extended lifespan, this study found that constitutive Hh signaling drives FSC loss and premature sterility despite high proliferative rates. This occurs due to Hh-mediated induction of autophagy in FSCs via a Ptc-dependent, Smo-independent mechanism. Hh-dependent autophagy increases during aging, triggering FSC loss and consequent reproductive arrest. IIS is necessary and sufficient to suppress Hh-induced autophagy, promoting a stable proliferative state. These results suggest that opposing action of diet-responsive IIS and Hh signals determine reproductive lifespan by modulating the proliferation-autophagy balance in FSCs during aging.
Valsecchi, C. I. K., Basilicata, M. F., Semplicio, G., Georgiev, P., Gutierrez, N. M. and Akhtar, A. (2018). Facultative dosage compensation of developmental genes on autosomes in Drosophila and mouse embryonic stem cells. Nat Commun 9(1): 3626. PubMed ID: 30194291
Summary:
Haploinsufficiency and aneuploidy are two phenomena, where gene dosage alterations cause severe defects ultimately resulting in developmental failures and disease. One remarkable exception is the X chromosome, where copy number differences between sexes are buffered by dosage compensation systems. In Drosophila, the Male-Specific Lethal complex (MSLc) mediates upregulation of the single male X chromosome. The evolutionary origin and conservation of this process orchestrated by MSL2, the only male-specific protein within the fly MSLc, have remained unclear. This study reports that MSL2, in addition to regulating the X chromosome, targets autosomal genes involved in patterning and morphogenesis. Precise regulation of these genes by MSL2 is required for proper development. This set of dosage-sensitive genes maintains such regulation during evolution, as MSL2 binds and similarly regulates mouse orthologues via Histone H4 lysine 16 acetylation. It is proposed that this gene-by-gene dosage compensation mechanism was co-opted during evolution for chromosome-wide regulation of the Drosophila male X.

Tuesday, November 20th - Embryonic Development

Chandran, R. R., Scholl, A., Yang, Y. and Jiang, L. (2018). rebuff regulates apical luminal matrix to control tube size in Drosophila trachea. Biol Open 7(9). PubMed ID: 30185423
Summary:
The Drosophila embryonic tracheal network is an excellent model to study tube size. The chitin-based apical luminal matrix and cell polarity are well known to regulate tube size in Drosophila trachea. Defects in luminal matrix and cell polarity lead to tube overexpansion. This study addresses the novel function of the rebuff (reb) gene, which encodes an evolutionarily conserved Smad-like protein. In reb mutants, tracheal tubes are moderately over-elongated. Despite the establishment of normal cell polarity, significantly reduced apical luminal matrix was observed in rebf mutants. Among various luminal components, luminal Obstructor-A (ObstA) is drastically reduced. Interestingly, ObstA is localized in vesicle-like structures that are apically concentrated in reb mutants. To investigate the possibility that reb is involved in the endocytosis of ObstA, co-localization of ObstA and endocytic markers were colocalized in reb mutants. ObstA was found to be localized in late endosomes and recycling endosomes. This suggests that in reb mutant trachea, endocytosed ObstA is degraded or recycled back to the apical region. However, ObstA vesicles are retained in the apical region and are failed to be secreted to the lumen. Taken together, these results suggest one function of reb is regulating the endocytosis of luminal matrix components.
Altamirano-Torres, C., Salinas-Hernandez, J. E., Cardenas-Chavez, D. L., Rodriguez-Padilla, C. and Resendez-Perez, D. (2018). Transcription factor TFIIEbeta interacts with two exposed positions in helix 2 of the Antennapedia homeodomain to control homeotic function in Drosophila. PLoS One 13(10): e0205905. PubMed ID: 30321227
Summary:
Homeodomains (HDs) increase their DNA-binding specificity by interacting with additional cofactors outlining a Hox interactome with a multiplicity of protein-protein interactions. In Drosophila, the first link of functional contact with a general transcription factor (GTF) was found between Antennapedia (Antp) and BIP2 (TFIID complex). Hox proteins also interact with other components of Pol II machinery such as the subunit Med19 from Mediator (MED) complex, TFIIEbeta and transcription-pausing factor M1BP. This paper focused on the Antp-TFIIEbeta protein-protein interface to establish the specific contacts as well as its functional role. TFIIEbeta was found to interact with Antp through the HD independently of the YPWM motif and the direct physical interaction is at helix 2, specifically amino acidic positions I32 and H36 of Antp. These two positions in helix 2 are crucial for Antp homeotic function in head involution, and thoracic and antenna-to tarsus transformations. Interestingly, overexpression of Antp and TFIIEbeta in the antennal disc showed that this interaction is required for the antenna-to-tarsus transformation. These results open the possibility to more broadly analyze Antp-TFIIEbeta interaction on the transcriptional control for the activation and/or repression of target genes in the Hox interactome during Drosophila development.
Durrieu, L., Kirrmaier, D., Schneidt, T., Kats, I., Raghavan, S., Hufnagel, L., Saunders, T. E. and Knop, M. (2018). Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis. Mol Syst Biol 14(9): e8355. PubMed ID: 30181144
Summary:
Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. This study introduces introduce morphogen-age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, this study fljnd a gradient of increasing age of Bicoid along the anterior-posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis-diffusion-degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, the timer can detect transitions in the dynamics associated with syncytial cellularization. These results provide new insight into Bicoid gradient formation and demonstrate how morphogen-age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.
Sun, J. and Stathopoulos, A. (2018). FGF controls epithelial-mesenchymal transitions during gastrulation by regulating cell division and apicobasal polarity. Development. PubMed ID: 30190277
Summary:
To support tissue and organ development, cells transition between epithelial and mesenchymal states. This study investigated how mesoderm cells change state in Drosophila embryos and whether fibroblast growth factor (FGF) signaling plays a role. During gastrulation, presumptive mesoderm cells invaginate, undergo an epithelial-to-mesenchymal state transition (EMT) and migrate upon the ectoderm. The data show that EMT is a prolonged process in which adherens junctions progressively decrease in number throughout the mesoderm cells' migration. FGF influences adherens junction number and promotes mesoderm cell division, which is proposed to decrease cell-cell attachments to support slow EMT while retaining collective cell movement. It was also found that, at the completion of migration, cells form a monolayer and undergo a reverse mesenchymal-to-epithelial transition (MET). FGF activity leads to accumulation of beta-integrin Myospheroid basally and cell polarity factor Bazooka apically within mesoderm cells, thereby reestablishing apicobasal cell polarity in an epithelialized state in which cells express both E-Cadherin and N-Cadherin. In summary, FGF plays a dynamic role in supporting mesoderm cell development to ensure collective mesoderm cell movements as well as proper differentiation of mesoderm cell types.
Doubrovinski, K., Tchoufag, J. and Mandadapu, K. (2018). A simplified mechanism for anisotropic constriction in Drosophila mesoderm. Development. PubMed ID: 30401702
Summary:
Understanding how forces and material properties give rise to tissue shapes is a fundamental question in developmental biology. Although Drosophila gastrulation is a major system for investigating tissue morphogenesis, there does not exist a consensus mechanical model that explains all the key features of this process. One key feature of Drosophila gastrulation is its anisotropy - the mesoderm constricts much more along one axis than along the other. Previous explanations have involved graded stress, anisotropic stresses or material properties, or mechanosensitive feedback. This study shows that these mechanisms are not required to explain the anisotropy of constriction. Instead, constriction can be anisotropic if only two conditions are met: the tissue is elastic, as was demonstrated in a recent study, and the contractile domain is asymmetric. This conclusion is general and does not depend on the values of model parameters. This model can explain classical tissue grafting experiments and more recent laser ablation studies. Furthermore, this model may provide alternative explanations for experiments in other developmental systems, including C. elegans and zebrafish.
Vincent, B. J., Staller, M. V., Lopez-Rivera, F., Bragdon, M. D. J., Pym, E. C. G., Biette, K. M., Wunderlich, Z., Harden, T. T., Estrada, J. and DePace, A. H. (2018). Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos. PLoS Genet 14(9): e1007644. PubMed ID: 30192762
Summary:
Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. This study investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, this study found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. Evidence was found that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. These results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. How counter-repression may influence eve stripe 2 regulation and evolution is discussed.

Monday November 19th - Adult Development

Paul, M. S., Dutta, D., Singh, A., Mutsuddi, M. and Mukherjee, A. (2018). Regulation of Notch signaling in the developing Drosophila eye by a T-box containing transcription factor, Dorsocross. Genesis. PubMed ID: 30246928
Summary:
Owing to a multitude of functions, there is barely a tissue or a cellular process that is not being regulated by Notch signaling. To identify novel effectors of Notch signaling in Drosophila melanogaster, the whole transcriptome was analyzed of the wing and eye imaginal discs in which an activated form of Notch was overexpressed. Selected candidate genes from the transcriptome analysis were subjected to genetic interaction experiments with Notch pathway components. Among the candidate genes, T-box encoding gene, Dorsocross (Doc) showed strong genetic interaction with Notch ligand, Delta. Genetic interaction between them resulted in reduction of eye size, loss of cone cells, and cell death, which represent prominent Notch loss of function phenotypes. Immunocytochemical analysis in Df(3L)DocA/Dl(5f) trans-heterozygous eye discs showed accumulation of Notch at the membrane. This accumulation led to decreased Notch signaling activity as downregulation was found of Atonal, a Notch target and reduction in the rate of Notch-mediated cell proliferation. Doc mutant clones generated by FLP-FRT system showed depletion in the expression of Delta and subsequent reduction in the Notch signaling activity. Similarly, Doc overexpression in the eye discs led to modification of Delta expression, loss of Atonal expression and absence of eye structure in pharate adults. Taken together, these results suggest that Doc regulates the expression of Delta and influences the outcome of Notch signaling in the eye discs.
Yeung, K., Wang, F., Li, Y., Wang, K., Mardon, G. and Chen, R. (2018). Integrative genomic analysis reveals novel regulatory mechanisms of eyeless during Drosophila eye development. Nucleic Acids Res. PubMed ID: 30295802
Summary:
Eyeless (ey) is one of the most critical transcription factors for initiating the entire eye development in Drosophila. However, the molecular mechanisms through which Ey regulates target genes and pathways have not been characterized at the genomic level. Using ChIP-Seq, an endogenous Ey-binding profile was generated in Drosophila developing eyes. Ey binding occurred more frequently at promoter compared to non-promoter regions. Ey promoter binding was correlated with the active transcription of genes involved in development and transcription regulation. An integrative analysis revealed that Ey directly regulated a broad and highly connected genetic network, including many essential patterning pathways, and known and novel eye genes. Interestingly, it was observed that Ey could target multiple components of the same pathway, which might enhance its control of these pathways during eye development. In addition to protein-coding genes, it was discovered that Ey also targeted non-coding RNAs, representing a new regulatory mechanism employed by Ey. These findings suggest that Ey could use multiple molecular mechanisms to regulate target gene expression and pathway function, which might enable Ey to exhibit a greater flexibility in controlling different processes during eye development.
Du, L., Sohr, A., Yan, G. and Roy, S. (2018). Feedback regulation of cytoneme-mediated transport shapes a tissue-specific FGF morphogen gradient. Elife 7. PubMed ID: 30328809
Summary:
Gradients of signaling proteins are essential for inducing tissue morphogenesis. However, mechanisms of gradient formation remain controversial. This study characterized the distribution of fluorescently-tagged signaling proteins, FGF and FGFR, expressed at physiological levels from the genomic knock-in alleles in Drosophila. FGF produced in the larval wing imaginal-disc moves to the air-sac-primordium (ASP) through FGFR-containing cytonemes that extend from the ASP to contact the wing-disc source. The number of FGF-receiving cytonemes extended by ASP cells decreases gradually with increasing distance from the source, generating a recipient-specific FGF gradient. Acting as a morphogen in the ASP, FGF activates concentration-dependent gene expression, inducing pointed-P1 at higher and cut at lower levels. The transcription-factors Pointed-P1 and Cut antagonize each other and differentially regulate formation of FGFR-containing cytonemes, creating regions with higher-to-lower numbers of FGF-receiving cytonemes. These results reveal a robust mechanism where morphogens self-generate precise tissue-specific gradient contours through feedback regulation of cytoneme-mediated dispersion.
Yanku, Y., Bitman-Lotan, E., Zohar, Y., Kurant, E., Zilke, N., Eilers, M. and Orian, A. (2018). Drosophila HUWE1 ubiquitin ligase regulates endoreplication and antagonizes JNK signaling during salivary gland development. Cells 7(10). PubMed ID: 30261639
Summary:
The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. This study reports that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc protein expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development.
Aly, H., Akagi, K. and Ueda, H. (2018). Proteasome activity determines pupation timing through the degradation speed of timer molecule Blimp-1. Dev Growth Differ 60(8): 502-508. PubMed ID: 30368781
Summary:
The transcriptional repressor Blimp-1 is a labile protein. This characteristic is key for determining pupation timing because the timing of the disappearance of Blimp-1 affects pupation timing by regulating the expression of its target betaftz-f1. However, the molecular mechanisms that regulate the protein turnover of Blimp-1 are still unclear. This study demonstrates that Blimp-1 is regulated by the ubiquitin proteasome system. Blimp-1 degradation is inhibited by proteasome inhibitor MG132. Pupation timing was delayed in mutants of 26S proteasome subunits as well as FBXO11, which recruits target proteins to the 26S proteasome as a component of the SCF ubiquitin ligase complex by slowing down the degradation speed of Blimp-1. Delay in pupation timing in the FBXO11 mutant was suppressed by the induction of betaFTZ-F1. Furthermore, fat-body-specific knockdown of proteasomal activity was sufficient to induce a delay in pupation timing. These results suggest that Blimp-1 is degraded by the 26S proteasome and is recruited by FBXO11 in the fat body, which is important for determining pupation timing.
Pham, M. N., Schuweiler, M. and Ismat, A. (2018). The extracellular protease AdamTS-B inhibits vein formation in the Drosophila wing. Genesis. PubMed ID: 30296002
Summary:
Vein patterning in the Drosophila wing provides a powerful tool to study regulation of various signaling pathways. This study shows that the ADAMTS extracellular protease AdamTS-B (CG4096) is expressed in the embryonic wing imaginal disc precursor cells and the wing imaginal disc, and functions to inhibit wing vein formation. Knock-down of AdamTS-B displayed posterior crossveins (PCVs) with either extra branches or deltas, or wider PCVs, and a wandering distal tip of the L5 longitudinal vein. Conversely, over-expression of AdamTS-B resulted in a complete absence of the PCV, an incomplete anterior crossvein (ACV), and missing distal end of the L5 longitudinal vein. It is concluded that AdamTS-B inhibits wing vein formation through negative regulation of signaling pathways, possibly BMP as well as Egfr, displaying the complexity of roles for this family of extracellular proteases.

Friday, November 15th - Chromatin

Smolko, A. E., Shapiro-Kulnane, L. and Salz, H. K. (2018). The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells. Nat Commun 9(1): 4155. PubMed ID: 30297796
Summary:
The preservation of germ cell sexual identity is essential for gametogenesis. This study shows that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell specific loss of the H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, WDE, and HP1a, leads to ectopic expression of genes, many of which are normally expressed in testis. SETDB1 controls the accumulation of H3K9me3 over a subset of these genes without spreading into neighboring loci. At phf7, a regulator of male germ cell sexual fate, the H3K9me3 peak falls over the silenced testis-specific transcription start site. Furthermore, H3K9me3 recruitment to phf7 and repression of testis-specific transcription is dependent on the female sex determination gene Sxl. Thus, female identity is secured by an H3K9me3 epigenetic pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer, and phf7 is one of the critical SETDB1 target genes.
Varma, P. and Mishra, R. K. (2018). Little imaginal discs, a Trithorax group member, is a constituent of nuclear matrix of Drosophila melanogaster embryos. J Biosci 43(4): 621-633. PubMed ID: 30207309
Summary:
Attachment of chromatin to the Nuclear Matrix (NuMat) brings significant regulation of the transcriptional activity of particular genes. This study has identified Lid (Little imaginal discs) as one of the components of NuMat. It belongs to the TrxG group of proteins involved in activation of important developmental genes. However, unlike other activator proteins of TrxG, Lid is a Jumonji protein involved in H3K4me3 demethylation. This study reports the association of Lid and its various domains with NuMat which implicates its structural role in chromatin organization and epigenetic basis of cellular memory. Both N and C terminal regions of this protein were found to be capable of associating with NuMat. The association of individual domains was further mapped and it was found that, PHD, ARID and JmjC domains can associate with NuMat individually. Based on these findings, it is hypothesized that C terminal region of Lid which contains PHD fingers might be responsible for its NuMat association via protein-DNA interactions. However, for the N terminal region harboring both a PHD and an ARID finger, Lid anchors to the NuMat via both protein-protein and protein-DNA interactions. It is suggested that Lid, a demethylase, being part of NuMat might be involved in regulating the chromatin dynamics via its NuMat association.
Munden, A., Rong, Z., Sun, A., Gangula, R., Mallal, S. and Nordman, J. T. (2018). Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila. Elife 7. PubMed ID: 30277458
Summary:
Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR's SNF2 domain, an interaction between SUUR and Rif1 was identified. Rif1 has many roles in DNA metabolism and regulates the replication timing program. Repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in a partially SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. These findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.
Yao, Y., Li, X., Wang, W., Liu, Z., Chen, J., Ding, M. and Huang, X. (2018). MRT, functioning with NURF complex, regulates lipid droplet size. Cell Rep 24(11): 2972-2984. PubMed ID: 30208321
Summary:
Lipid droplets (LDs) are highly dynamic organelles that store neutral lipids. Through a gene overexpression screen in the Drosophila larval fat body, this study has identified that MRT, an Myb/switching-defective protein 3 (Swi3), Adaptor 2 (Ada2), Nuclear receptor co-repressor (N-CoR), Transcription factor (TF)IIIB (SANT)-like DNA-binding domain-containing protein, regulates LD size and lipid storage. MRT directly interacts with, and is functionally dependent on, the PZG and NURF chromatin-remodeling complex components. MRT binds to the promoter of plin1, the gene encoding the LD-resident protein perilipin, and inhibits the transcription of plin1. In vitro LD coalescence assays suggest that mrt overexpression or loss of plin1 function facilitates LD coalescence. These findings suggest that MRT functions together with chromatin-remodeling factors to regulate LD size, likely through the transcriptional repression of plin1.

Thursday, November 15th - Cell Cycle

Silva, R. D., Mirkovic, M., Guilgur, L. G., Rathore, O. S., Martinho, R. G. and Oliveira, R. A. (2018). Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion. Curr Biol. PubMed ID: 30122528
Summary:
The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin. Cleavage of cohesin marks anaphase onset. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC). To identify specific conditions capable of restoring defects associated with cohesion loss, a screen was carried out for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mps1, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.
Vaufrey, L., Balducci, C., Lafont, R., Prigent, C. and Le Bras, S. (2018). Size matters! Aurora A controls Drosophila larval development. Dev Biol. Pubmed ID: 29753017
Summary:
In metazoans, organisms arising from a fertilized egg, the embryo will develop through multiple series of cell divisions, both symmetric and asymmetric, leading to differentiation. Aurora A is a serine threonine kinase highly involved in such divisions. While intensively studied at the cell biology level, its function in the development of a whole organism has been neglected. This study investigated the pleiotropic effect of Aurora A loss-of-function in Drosophila larval early development. Aurora A was shown to be required for proper larval development timing control through direct and indirect means. In larval tissues, Aurora A is required for proper symmetric division rate and eventually development speed as was observed in central brain, wing disc and ring gland. Moreover, Aurora A inactivation induces a reduction of ecdysteroids levels and a pupariation delay as an indirect consequence of ring gland development deceleration. Finally, although central brain development is initially restricted, it was confirmed that brain lobe size eventually increases due to additive phenotypes: delayed pupariation and over-proliferation of cells with an intermediate cell-identity between neuroblast and ganglion mother cell resulting from defective asymmetric neuroblast cell division.
Song, F., Li, D., Wang, Y. and Bi, X. (2018). Drosophila Caliban mediates G1-S transition and ionizing radiation induced S phase checkpoint. Cell Cycle: 1-12. PubMed ID: 30231800
Summary:
Cell cycle progression is precisely regulated by diverse extrinsic and intrinsic cellular factors. Understanding the underlying mechanisms of cell cycle regulation is essential to address how normal development and tissue homeostasis are achieved. This study presents a novel cell cycle regulator Caliban (Clbn), the Drosophila ortholog of human Serologically defined colon cancer antigen 1 (SDCCAG1) gene. Ionizing radiation induces expression of clbn, and over-expression of clbn blocks G1-to-S cell cycle transition in Drosophila, while flies loss of clbn have defective S phase checkpoint in response to irradiation. Mechanistically, induced expression of clbn suppressed E2F1 activity and down-regulates the DNA replication and expression of its downstream target cyclin E, a key regulator of G1-to-S transition. Meanwhile, clbn over-expression leads to upregulation of the CDK inhibitor Dacapo (Dap), and upregulated Dap is decreased when e2f1 is over-expressed. Furthermore, expression of clbn is down-regulated in cells with e2f1 over-expression or rbf1 knockdown, indicating that Clbn and E2F1 act antagonistically in mediating G1-to-S transition. Thus this study provides genetic evidence that Clbn works together with E2F1 in regulating cell cycle progression, and Clbn is required for S phase cell cycle checkpoint in response to DNA damage.
Mehsen, H., Boudreau, V., Garrido, D., Bourouh, M., Larouche, M., Maddox, P. S., Swan, A. and Archambault, V. (2018). PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila. J Cell Biol. PubMed ID: 30309980
Summary:
As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. This study conducted a maternal-effect screen in Drosophila melanogaster to identify genes that function with PP2A-B55/Tws in the cell cycle. Eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often failrf development, concomitant with NE defects following meiosis and in syncytial mitoses. Mechanistic studies using Drosophila cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes Drosophila as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis.

Wednesday, November 14th - Adult Physiology

Wei, P., Guo, J., Xue, W., Zhao, Y., Yang, J. and Wang, J. (2018). RNF34 modulates the mitochondrial biogenesis and exercise capacity in muscle and lipid metabolism through ubiquitination of PGC-1 in Drosophila. Acta Biochim Biophys Sin (Shanghai) 50(10): 1038-1046. PubMed ID: 30247505
Summary:
The transcriptional co-activator PGC-1alpha is a key regulator of mitochondrial function and muscle fiber specification in the skeletal muscle. The E3 ubiquitin ligase RNF34 ubiquitinates PGC-1alpha and negatively regulates mammalian brown fat cell metabolism. However, the functional importance of RNF34 in the skeletal muscle and its impact on energy metabolism remain unknown. The Drosophila PGC-1 homolog dPGC-1 and its mammalian counterparts have conserved functions in mitochondria and insulin signaling. This study showed that the Drosophila RNF34 (dRNF34) ubiquitinates the Drosophila PGC-1alpha (dPGC-1) and promotes its degradation in HEK293T cells by immunoprecipitation and western blot analysis. This allows Drosophila to be used as a powerful model system to study the physiological role of RNF34 in mitochondrial function and metabolism. In the in vivo studies, by separately expressing two independent UAS-dRNF34 RNAi transgenes driven by the muscle-specific 24B-Gal4 driver, this study found that knockdown of dRNF34 specifically in muscle promotes mitochondrial biogenesis, improves negative geotaxis, extends climbing time to exhaustion in moderate aged flies and counteracts high-fat-diet-induced high triglyceride content. Furthermore, knockdown of dPGC-1 reversed the effects of the dRNF34 knockdown phenotypes described above. These results reveal that dRNF34 plays an important role in regulating mitochondrial biogenesis in muscle and lipid metabolism through dPGC-1. Thus, inhibition of RNF34 activity provides a potential novel therapeutic strategy for the treatment of age-related muscle dysfunction.
Yang, Z., Huang, R., Fu, X., Wang, G., Qi, W., Mao, D., Shi, Z., Shen, W. L. and Wang, L. (2018). A post-ingestive amino acid sensor promotes food consumption in Drosophila. Cell Res. PubMed ID: 30209352
Summary:
Adequate protein intake is crucial for the survival and well-being of animals. How animals assess prospective protein sources and ensure dietary amino acid intake plays a critical role in protein homeostasis. By using a quantitative feeding assay, this study shows that three amino acids, L-glutamate (L-Glu), L-alanine (L-Ala) and L-aspartate (L-Asp), but not their D-enantiomers or the other 17 natural L-amino acids combined, rapidly promote food consumption in the fruit fly Drosophila melanogaster. This feeding-promoting effect of dietary amino acids is independent of mating experience and internal nutritional status. In vivo and ex vivo calcium imagings show that six brain neurons expressing diuretic hormone 44 (DH44) can be rapidly and directly activated by these amino acids, suggesting that these neurons are an amino acid sensor. Genetic inactivation of DH44(+) neurons abolishes the increase in food consumption induced by dietary amino acids, whereas genetic activation of these neurons is sufficient to promote feeding, suggesting that DH44(+) neurons mediate the effect of dietary amino acids to promote food consumption. Single-cell transcriptome analysis and immunostaining reveal that a putative amino acid transporter, CG13248, is enriched in DH44(+) neurons. Knocking down CG13248 expression in DH44(+) neurons blocks the increase in food consumption and eliminates calcium responses induced by dietary amino acids. Therefore, these data identify DH44(+) neuron as a key sensor to detect amino acids and to enhance food intake via a putative transporter CG13248. These results shed critical light on the regulation of protein homeostasis at organismal levels by the nervous system.
Obniski, R., Sieber, M. and Spradling, A. C. (2018). Dietary lipids modulate Notch signaling and influence adult intestinal development and metabolism in Drosophila. Dev Cell 47(1): 98-111.e115. PubMed ID: 30220569
Summary:
Tissue homeostasis involves a complex balance of developmental signals and environmental cues that dictate stem cell function. This study found that dietary lipids control enteroendocrine cell production from Drosophila posterior midgut stem cells. Dietary cholesterol influences new intestinal cell differentiation in an Hr96-dependent manner by altering the level and duration of Notch signaling. Exogenous lipids modulate Delta ligand and Notch extracellular domain stability and alter their trafficking in endosomal vesicles. Lipid-modulated Notch signaling occurs in other nutrient-dependent tissues, suggesting that Delta trafficking in many cells is sensitive to cellular sterol levels. These diet-mediated alterations in young animals contribute to a metabolic program that persists after the diet changes. A low-sterol diet also slows the proliferation of enteroendocrine tumors initiated by Notch pathway disruption. Thus, a specific dietary nutrient can modify a key intercellular signaling pathway to shift stem cell differentiation and cause lasting changes in tissue structure and physiology.
Elya, C., Zhang, V., Ludington, W. B. and Eisen, M. B. (2016). Stable host gene expression in the gut of adult Drosophila melanogaster with different bacterial mono-associations. PLoS One 11(11): e0167357. PubMed ID: 27898741
Summary:
There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but how they accomplish this is still not understood. This study evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. It was observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, it was found whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. These data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body.
Prasad, N. and Hens, K. (2018). Sugar promotes feeding in flies via the serine protease homolog scarface. Cell Rep 24(12): 3194-3206.e3194. PubMed ID: 30232002
Summary:
A balanced diet of macronutrients is critical for animal health. A lack of specific elements can have profound effects on behavior, reproduction, and lifespan. This study used Drosophila to understand how the brain responds to carbohydrate deprivation. Serine protease homologs (SPHs) were enriched among genes that are transcriptionally regulated in flies deprived of carbohydrates. Stimulation of neurons expressing one of these SPHs, Scarface (Scaf), or overexpression of scaf positively regulates feeding on nutritious sugars, whereas inhibition of these neurons or knockdown of scaf reduces feeding. This modulation of food intake occurs only in sated flies while hunger-induced feeding is unaffected. Furthermore, scaf expression correlates with the presence of sugar in the food. As Scaf and Scaf neurons promote feeding independent of the hunger state, and the levels of scaf are positively regulated by the presence of sugar, it is concluded that scaf mediates the hedonic control of feeding.
Ugrankar, R., Theodoropoulos, P., Akdemir, F., Henne, W. M. and Graff, J. M. (2018). Circulating glucose levels inversely correlate with Drosophila larval feeding through insulin signaling and SLC5A11. Commun Biol 1: 110. PubMed ID: 30271990
Summary:
In mammals, blood glucose levels likely play a role in appetite regulation yet the mechanisms underlying this phenomenon remain opaque. Mechanisms can often be explored from Drosophila genetic approaches. To determine if circulating sugars might be involved in Drosophila feeding behaviors, hemolymph glucose and trehalose, and food ingestion were scored in larvae subjected to various diets, genetic mutations, or RNAi. Larvae with glucose elevations, hyperglycemia, were found to have an aversion to feeding; however, trehalose levels do not track with feeding behavior. It was further discovered that insulins and SLC5A11 may participate in glucose-regulated feeding. To see if food aversion might be an appropriate screening method for hyperglycemia candidates, a food aversion screen was developed to score larvae with abnormal feeding for glucose. It was found that many feeding defective larvae have glucose elevations. These findings highlight intriguing roles for glucose in fly biology as a potential cue and regulator of appetite.

Tuesday, November 13th - Signaling

White, K. A., Grillo-Hill, B. K., Esquivel, M., Peralta, J., Bui, V. N., Chire, I. and Barber, D. L. (2018). beta-Catenin is a pH sensor with decreased stability at higher intracellular pH. J Cell Biol. PubMed ID: 30315137
Summary:
beta-Catenin functions as an adherens junction protein for cell-cell adhesion and as a signaling protein. beta-catenin function is dependent on its stability, which is regulated by protein-protein interactions that stabilize beta-catenin or target it for proteasome-mediated degradation. This study shows that beta-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster. beta-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase beta-TrCP. While beta-catenin phosphorylation was pH independent, higher pHi induced increased beta-TrCP binding and decreased beta-catenin stability. An evolutionarily conserved histidine in beta-catenin (found in the beta-TrCP DSGIHS destruction motif) is required for pH-dependent binding to beta-TrCP. Expressing a cancer-associated H36R-beta-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic beta-catenin alleles. This study identified pHi dynamics as a previously unrecognized regulator of beta-catenin stability, functioning in coincidence with phosphorylation.
Wu, X., Chen, Z., Gao, Y., Wang, L., Sun, X., Jin, Y. and Liu, W. (2018). The Kruppel-like factor Dar1 restricts the proliferation of Drosophila intestinal stem cells. FEBS J. PubMed ID: 30188612
Summary:
The kruppel-like factors (KLFs) are a family of transcription factor proteins that regulate a wide range of biological processes. In an RNAi-based screening experiment, dendritic arbor reduction 1 (Dar1), which is a KLF member in Drosophila, inhibited the proliferation of intestinal stem cells (ISCs). Suppression of Dar1-activated ISC proliferation; as a consequence, the ISCs and the young differentiated cells were increased. On the other hand, overexpression (OE) of Dar1 inhibited ISC division and blocked the formation of ISC lineages. In order to explore the molecular mechanism of the Dar1 functions, the gene expression profiles of the Dar1 knockdown and Dar1 OE midguts was compared, using the deep RNA sequencing (RNA-Seq) technique. This experiment revealed that Dar1 negatively regulated the expression of several critical cell cycle genes. Evidence is provided that Dar1 has a function upstream of the JAK/STAT signaling pathway, suggesting Dar1 can regulate ISC proliferation through different mechanisms. Consistent with these findings, Dar1 was found to be downregulated in the wounded midguts, allowing increased ISC proliferation to promote intestinal repair. These data suggest that Dar1 is a functional homolog of the mammalian KLF4.
Li, P., Huang, P., Li, X., Yin, D., Ma, Z., Wang, H. and Song, H. (2018). Tankyrase mediates K63-linked ubiquitination of JNK to confer stress tolerance and influence lifespan in Drosophila. Cell Rep 25(2): 437-448. PubMed ID: 30304683
Summary:
Tankyrase (Tnks) transfers poly(ADP-ribose) on substrates. Whereas studies have highlighted the pivotal roles of Tnks in cancer, cherubism, systemic sclerosis, and viral infection, the requirement for Tnks under physiological contexts remains unclear. This study report that the loss of Tnks or its muscle-specific knockdown impairs lifespan, stress tolerance, and energy homeostasis in adult Drosophila. Tnks is a positive regulator in the JNK signaling pathway, and modest alterations in the activity of JNK signaling can strengthen or suppress the Tnks mutant phenotypes. JNK was identified as a direct substrate of Tnks. Although Tnks-dependent poly-ADP-ribosylation is tightly coupled to proteolysis in the proteasome, it was demonstrated that Tnks initiates degradation-independent ubiquitination on two lysine residues of JNK to promote its kinase activity and in vivo functions. This study uncovers a type of posttranslational modification of Tnks substrates and provides insights into Tnks-mediated physiological roles.
Ren, L., Mo, D., Li, Y., Liu, T., Yin, H., Jiang, N. and Zhang, J. (2018). A genetic mosaic screen identifies genes modulating Notch signaling in Drosophila. PLoS One 13(9): e0203781. PubMed ID: 30235233
Summary:
Notch signaling is conserved in most multicellular organisms and plays critical roles during animal development. The core components and major signal transduction mechanism of Notch signaling have been extensively studied. However, understanding of how Notch signaling activity is regulated in diverse developmental processes still remains incomplete. This study reports a genetic mosaic screen in Drosophila melanogaster that leads to identification of Notch signaling modulators during wing development. A group of genes was discovered required for the formation of the fly wing margin, a developmental process that is strictly dependent on the balanced Notch signaling activity. These genes encode transcription factors, protein phosphatases, vacuolar ATPases and factors required for RNA transport, stability, and translation. These data support the view that Notch signaling is controlled through a wide range of molecular processes. These results also provide foundations for further study by showing that Me31B and Wdr62 function as two novel modulators of Notch signaling activity.
Suzuki, T., Liu, C., Kato, S., Nishimura, K., Takechi, H., Yasugi, T., Takayama, R., Hakeda-Suzuki, S., Suzuki, T. and Sato, M. (2018). Netrin signaling defines the regional border in the Drosophila visual center. iScience 8: 148-160. PubMed ID: 30316037
Summary:
The brain consists of distinct domains defined by sharp borders. So far, the mechanisms of compartmentalization of developing tissues include cell adhesion, cell repulsion, and cortical tension. These mechanisms are tightly related to molecular machineries at the cell membrane. However, studies have demonstrated that Slit, a chemorepellent, is required to establish the borders in the fly brain. This study demonstrates that Netrin, a classic guidance molecule, is also involved in the compartmental subdivision in the fly brain. In Netrin mutants, many cells are intermingled with cells from the adjacent ganglia penetrating the ganglion borders, resulting in disorganized compartmental subdivisions. How do these guidance molecules regulate the compartmentalization? A mathematical model demonstrates that a simple combination of known guidance properties of Slit and Netrin is sufficient to explain their roles in boundary formation. These results suggest that Netrin indeed regulates boundary formation in combination with Slit in vivo.
Voolstra, O., Strauch, L., Mayer, M. and Huber, A. (2018). Functional characterization of the three Drosophila retinal degeneration C (RDGC) protein phosphatase isoforms. PLoS One 13(9): e0204933. PubMed ID: 30265717
Summary:
Drosophila retinal degeneration C (RDGC) is the founding member of the PPEF family of protein phosphatases. RDGC mediates dephosphorylation of the visual pigment rhodopsin and the TRP ion channel. From the rdgC locus, three protein isoforms, termed RDGC-S, -M, and -L, with different N-termini are generated. Due to fatty acylation, RDGC-M and -L are attached to the plasma membrane while RDGC-S is soluble. To assign physiological roles to these RDGC isoforms, flies were constructed that express various combinations of RDGC protein isoforms. Expression of the RDGC-L isoform alone did not fully prevent rhodopsin hyperphosphorylation and resulted in impaired photoreceptor physiology and in decelerated TRP dephosphorylation at Ser936. However, expression of RDGC-L alone as well as RDGC-S/M was sufficient to prevent degeneration of photoreceptor cells which is a hallmark of the rdgC null mutant. Membrane-attached RDGC-M displayed higher activity of TRP dephosphorylation than the soluble isoform RDGC-S. Taken together, in vivo activities of RDGC splice variants are controlled by their N-termini.

Monday, November 12th - Adult Neural Development and Function

Turrel, O., Goguel, V. and Preat, T. (2018). Amnesiac is required in the adult mushroom body for memory formation. J Neurosci. PubMed ID: 30201766
Summary:
It was proposed that the Drosophila amnesiac gene (amn) is required for consolidation of aversive memory in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the fly center for olfactory learning and memory. Yet, a previous conditional analysis suggested that amn might be involved in the development of brain structures that normally promote adult olfactory memory. To further investigate temporal and spatial requirements of Amnesiac in memory, RNA interference was used in combination with conditional drivers. The data show that acute modulation of amn expression in adult DPM neurons does not impact memory and that amn expression is required for normal development of DPM neurons. Detailed enhancer trap analyses suggest that an amn transcription unit contains two distinct enhancers, one specific of DPM neurons, and the other specific of alpha/beta MB neurons. This prompted an investigation of the role of Amnesiac in the adult MB. These results demonstrate that amn is acutely required in adult alpha/beta MB neurons for middle-term and long-term memory. The data thus establish that amn plays two distinct roles. Its expression is required in DPM neurons for their development, and in adult MB for olfactory memory.
Walcott, K. C. E., Mauthner, S. E., Tsubouchi, A., Robertson, J. and Tracey, W. D. (2018). The Drosophila small conductance calcium-activated potassium channel negatively regulates nociception. Cell Rep 24(12): 3125-3132. PubMed ID: 30231996
Summary:
Inhibition of nociceptor activity is important for the prevention of spontaneous pain and hyperalgesia. To identify the critical K(+) channels that regulate nociceptor excitability, a forward genetic screen was performed using a Drosophila larval nociception paradigm. Knockdown of three K(+) channel loci, the small conductance calcium-activated potassium channel (SK), seizure, and tiwaz, causes marked hypersensitive nociception behaviors. In more detailed studies of SK, this study found that hypersensitive phenotypes can be recapitulated with a genetically null allele. Optical recordings from nociceptive neurons showed a significant increase in mechanically activated Ca(2+) signals in SK mutant nociceptors. SK is expressed in peripheral neurons, including nociceptive neurons. Interestingly, SK proteins localize to axons of these neurons but are not detected in dendrites. These findings suggest a major role for SK channels in the regulation of nociceptor excitation and are inconsistent with the hypothesis that the important site of action is within dendrites.
Loyer, N. and Januschke, J. (2018). The last-born daughter cell contributes to division orientation of Drosophila larval neuroblasts. Nat Commun 9(1): 3745. PubMed ID: 30218051
Summary:
Controlling the orientation of cell division is important in the context of cell fate choices and tissue morphogenesis. However, the mechanisms providing the required positional information remain incompletely understood. This study used stem cells of the Drosophila larval brain that stably maintain their axis of polarity and division between cell cycles to identify cues that orient cell division. Using live cell imaging of cultured brains, laser ablation and genetics, this study reveals that division axis maintenance relies on their last-born daughter cell. It is proposed that, in addition to known intrinsic cues, stem cells in the developing fly brain are polarized by an extrinsic signal. It was further found that division axis maintenance allows neuroblasts to maximize their contact area with glial cells known to provide protective and proliferative signals to neuroblasts.
Richter, F. G., Fendl, S., Haag, J., Drews, M. S. and Borst, A. (2018) . Glutamate signaling in the fly visual system. iScience 7: 85-95. PubMed ID: 30267688
Summary:
For a proper understanding of neural circuit function, it is important to know which signals neurons relay to their downstream partners. Calcium imaging with genetically encoded calcium sensors like GCaMP has become the default approach for mapping these responses. How well such measurements represent the true neurotransmitter output of any given cell, however, remains unclear. This study demonstrates the viability of the glutamate sensor iGluSnFR for 2-photon in vivo imaging in Drosophila melanogaster and prove its usefulness for estimating spatiotemporal receptive fields in the visual system. The results obtained with iGluSnFR were compared with the ones obtained with GCaMP6f, and the spatial aspects of the receptive fields were shown to be preserved between indicators. In the temporal domain, however, measurements obtained with iGluSnFR reveal the underlying response properties to be much faster than those acquired with GCaMP6f. This approach thus offers a more accurate description of glutamatergic neurons in the fruit fly.
Mamiya, A., Gurung, P. and Tuthill, J. C. (2018). Neural coding of leg proprioception in Drosophila. Neuron. PubMed ID: 30293823
Summary:
Animals rely on an internal sense of body position and movement to effectively control motor behavior. This sense of proprioception is mediated by diverse populations of mechanosensory neurons distributed throughout the body. This study investigated neural coding of leg proprioception in Drosophila, using in vivo two-photon calcium imaging of proprioceptive sensory neurons during controlled movements of the fly tibia. The axons of leg proprioceptors are organized into distinct functional projections that contain topographic representations of specific kinematic features. Using subclass-specific genetic driver lines, this study shows that one group of axons encodes tibia position (flexion/extension), another encodes movement direction, and a third encodes bidirectional movement and vibration frequency. Overall, these findings reveal how proprioceptive stimuli from a single leg joint are encoded by a diverse population of sensory neurons, and provide a framework for understanding how proprioceptive feedback signals are used by motor circuits to coordinate the body.
Sanchez-Alcaniz, J. A., Silbering, A. F., Croset, V., Zappia, G., Sivasubramaniam, A. K., Abuin, L., Sahai, S. Y., Munch, D., Steck, K., Auer, T. O., Cruchet, S., Neagu-Maier, G. L., Sprecher, S. G., Ribeiro, C., Yapici, N. and Benton, R. (2018). An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing. Nat Commun 9(1): 4252. PubMed ID: 30315166
Summary:
Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, it was revealed that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. This study characterize IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. It was further demonstrated that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. This work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour.

Friday, November 9th - Behavior

Semaniuk, U. V., Gospodaryov, D. V., Feden'ko, K. M., Yurkevych, I. S., Vaiserman, A. M., Storey, K. B., Simpson, S. J. and Lushchak, O. (2018). Insulin-like peptides regulate feeding preference and metabolism in Drosophila. Front Physiol 9: 1083. PubMed ID: 30197596
Summary:
Fruit flies have eight identified Drosophila insulin-like peptides (DILPs) that are involved in the regulation of carbohydrate concentrations in hemolymph as well as in accumulation of storage metabolites. This study investigated diet-dependent roles of DILPs encoded by the genes dilp1-5, and dilp7 in the regulation of insect appetite, food choice, accumulation of triglycerides, glycogen, glucose, and trehalose in fruit fly bodies and carbohydrates in hemolymph. The wild type and the mutant lines demonstrate compensatory feeding for carbohydrates. However, mutants on dilp2, dilp3, dilp5, and dilp7 showed higher consumption of proteins on high yeast diets. High nutrient diets led to a moderate increase in concentration of glucose in hemolymph of the wild type flies. Mutations on dilp genes changed this pattern. The dilp2 mutation led to a drop in glycogen levels independently on diet, lack of dilp3 led to dramatic increase in circulating trehalose and glycogen levels, especially at low protein consumption. Lack of dilp5 led to decreased levels of glycogen and triglycerides on all diets, whereas knockout on dilp7 caused increase in glycogen levels and simultaneous decrease in triglyceride levels at low protein consumption. Fruit fly appetite was influenced by dilp3 and dilp7 genes. These data contribute to the understanding of Drosophila as a model for further studies of metabolic diseases.
Saurabh, S., Vanaphan, N., Wen, W. and Dauwalder, B. (2018). High functional conservation of takeout family members in a courtship model system. PLoS One 13(9): e0204615. PubMed ID: 30261021
Summary:
takeout (to) is one of the male-specific genes expressed in the fat body that regulate male courtship behavior, and has been shown to act as a secreted protein in conjunction with courtship circuits. There are 23 takeout family members in Drosophila melanogaster, and homologues of this family are distributed across insect species. Sequence conservation among family members is low. This study tested the functional conservation of takeout family members by examining whether they can rescue the takeout courtship defect. Despite their sequence divergence, takeout members from Aedes aegypti and Epiphas postvittana, as well as family members from D. melanogaster can substitute for takeout in courtship, demonstrating their functional conservation. Making use of the known E. postvittana Takeout structure, homology modeling and amphipathic helix analysis was used, and high overall structural conservation was found, including high conservation of the structure and amphipathic lining of an internal cavity that has been shown to accommodate hydrophobic ligands. Together these data suggest a high degree of structural conservation that likely underlies functional conservation in courtship. In addition, a role is identifed for a conserved exposed protein motif important for the protein's role in courtship.
Joseph, N. M., Elphick, N. Y., Mohammad, S. and Bauer, J. H. (2018). Altered pheromone biosynthesis is associated with sex-specific changes in life span and behavior in Drosophila melanogaster. Mech Ageing Dev. PubMed ID: 30312624
Summary:
Many insect behaviors, including foraging, aggression, mating or group behavior, are tightly regulated by pheromones. Recently, it has been shown that pheromones may influence extreme longevity in the honeybee Apis mellifera, while changes in pheromone profile have been observed during ageing in Drosophila melanogaster. These data suggest a potential link between the pheromone system, behavior and longevity in insects. This study investigated this potential link by examining changes in behavior and longevity in fruit flies with altered pheromone profiles. Oenocyte-specific reduction of desaturase activity was shown to be sufficient to dramatically alter the composition of the hydrocarbon mix displayed by the flies. In addition, flies with altered desaturase activity display changes in fecundity and stereotypical mating behavior, and, importantly, extended longevity. These data provide evidence for a potential link between hydrocarbon synthesis and life span, and suggest that longevity may be influenced by behavior.
Matsumoto, Y., Shimizu, K., Arahata, K., Suzuki, M., Shimizu, A., Takei, K., Yamauchi, J., Hakeda-Suzuki, S., Suzuki, T. and Morimoto, T. (2018). Prepulse inhibition in Drosophila melanogaster larvae. Biol Open 7(9). PubMed ID: 30262549
Summary:
The neural mechanisms of psychiatric diseases like autism spectrum disorder and schizophrenia have been intensively studied, and a number of candidate genes have been identified. However, the relationship between genes and neural system functioning remains unclear. Model organisms may serve as a powerful tool for addressing this question due to the availability of established genetic tools. This study reports prepulse inhibition (PPI) in Drosophila larvae for the first time. PPI is a neurological phenomenon found in humans and other organisms and is used in the diagnosis of schizophrenia and other psychiatric disorders. A weaker prestimulus (prepulse) inhibits the reaction to a subsequent strong, startling stimulus (pulse). Using the larval startle response to the buzz of a predator (wasp), PPI was examined in wild-type flies and two mutants: an fmr1 mutant, which is implicated in Fragile X syndrome, and a centaurin gamma 1A (CenG1A) mutant, which is associated with GTPase, PH, ArfGAP, and ANK domains and implicated in autism. Both mutants showed decreased PPI, whereas, interestingly, double mutants showed substantial PPI. The PPI phenomenon described here can provide a useful tool for the study of neural mechanisms of synaptic modification and psychiatric diseases.
Miwa, Y., Koganezawa, M. and Yamamoto, D. (2018). Antennae sense heat stress to inhibit mating and promote escaping in Drosophila females. J Neurogenet: 1-11. PubMed ID: 30231794
Summary:
Environmental stress is a major factor that affects courtship behavior and evolutionary fitness. Although mature virgin females of Drosophila melanogaster usually accept a courting male to mate, they may not mate under stressful conditions. Above the temperature optimal for mating (20-25 degrees C), copulation success of D. melanogaster declines with increasing temperature although vigorous courtship attempts were observed by males, and no copulation takes place at temperatures over 36 degrees C. Attempts were made to identify the sensory pathway for detecting heat threat that drives a female to escape rather than to engage in mating that detects hot temperature and suppresses courtship behavior. The artificial activation of warmth-sensitive neurons ('hot cells') in the antennal arista of females completely abrogates female copulation success even at permissive temperatures below 32 degrees C. Moreover, mutational loss of the GR28b.d thermoreceptor protein caused females to copulate even at 36 degrees C. These results indicate that antennal hot cells provide the input channel for detecting the high ambient temperature in the control of virgin female mating under stressful conditions.
Sun, J. S., Larter, N. K., Chahda, J. S., Rioux, D., Gumaste, A. and Carlson, J. R. (2018). Humidity response depends on the small soluble protein Obp59a in Drosophila. Elife 7. PubMed ID: 30230472
Summary:
Hygrosensation is an essential sensory modality that is used to find sources of moisture. Hygroreception allows animals to avoid desiccation, an existential threat that is increasing with climate change. Humidity response, however, remains poorly understood. This study found that humidity-detecting sensilla in the Drosophila antenna express and rely on a small protein, Obp59a. Mutants lacking this protein are defective in three hygrosensory behaviors, one operating over seconds, one over minutes, and one over hours. Remarkably, loss of Obp59a and humidity response leads to an increase in desiccation resistance. Obp59a is an exceptionally well-conserved, highly localized, and abundantly expressed member of a large family of secreted proteins. Antennal Obps have long been believed to transport hydrophobic odorants, and a role in hygroreception was unexpected. The results enhance understanding of hygroreception, Obp function, and desiccation resistance, a process that is critical to insect survival.

Thursday, November 8th - Synapse and Vesicles

Thomas, R. E., Vincow, E. S., Merrihew, G. E., MacCoss, M. J., Davis, M. Y. and Pallanck, L. J. (2018). Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles. PLoS Genet 14(9): e1007694. PubMed ID: 30256786
Summary:
Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. This study tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Striking changes were found in the turnover and abundance of proteins associated with extracellular vesicles (EVs), which have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, these findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles.
He, T., Nitabach, M. N. and Lnenicka, G. A. (2018). Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ. J Neurogenet: 1-12. PubMed ID: 30175644
Summary:
Presynaptic Ca(2+) appears to play multiple roles in synaptic development and physiology. This study examined the effect of buffering presynaptic Ca(2+) by expressing parvalbumin (PV) in Drosophila neurons, which do not normally express PV. The studies were performed on the identified Ib terminal that innervates muscle fiber 5. The volume-averaged, residual Ca(2+) resulting from single action potentials (APs) and AP trains was measured using the fluorescent Ca(2+) indicator, OGB-1. PV reduced the amplitude and decay time constant (tau) for single-AP Ca(2+) transients. For AP trains, there was a reduction in the rate of rise and decay of [Ca(2+)]i but the plateau [Ca(2+)]i was not affected. Electrophysiological recordings from muscle fiber 5 showed a reduction in paired-pulse facilitation, particularly the F1 component; this was likely due to the reduction in residual Ca(2+). These synapses also showed reduced synaptic enhancement during AP trains, presumably due to less buildup of synaptic facilitation. The transmitter release for single APs was increased for the PV-expressing terminals and this may have been a homeostatic response to the decrease in facilitation. Confocal microscopy was used to examine the structure of the motor terminals and PV expression resulted in smaller motor terminals with fewer synaptic boutons and active zones. This result supports earlier proposals that increased AP activity promotes motor terminal growth through increases in presynaptic [Ca(2+)]i.
Rosa-Ferreira, C., Sweeney, S. T. and Munro, S. (2018). The small G protein Arl8 contributes to lysosomal function and long-range axonal transport in Drosophila. Biol Open 7(9). PubMed ID: 30115618
Summary:
The small GTPase Arl8 has emerged as a major regulatory GTPase on lysosomes. Studies in mammalian cells have shown that it regulates both fusion with late endosomes and also lysosomal motility. In its active GTP-bound state, it recruits to lysosomes the HOPS (homotypic fusion and protein sorting) endosomal tethering complex and also proteins that link lysosomes to microtubule motors such as the kinesin adaptor PLEKHM2. To gain further insights into Arl8 biology, this study examined the single Drosophila ortholog. Drosophila Arl8 is essential for viability, and mitotic clones of mutant cells are able to continue to divide but show perturbation of the late endocytic pathway. Progeny-lacking Arl8 die as late larvae with movement-paralysis characteristic of defects in neuronal function. This phenotype was rescued by expression of Arl8 in motor neurons. Examination of these neurons in the mutant larvae revealed smaller synapses and axons with elevated levels of carriers containing synaptic components. Affinity chromatography revealed binding of Drosophila Arl8 to the HOPS complex, and to the Drosophila ortholog of RILP, a protein that, in mammals, recruits dynein to late endosomes, with dynein being known to be required for neuronal transport. Thus Drosophila Arl8 controls late endocytic function and transport via at least two distinct effectors.
Betancourt-Solis, M. A., Desai, T. and McNew, J. A. (2018). The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer. J Biol Chem. PubMed ID: 30287684
Summary:
The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. This study examined several Drosophila-human atlastin chimeras to identify functional domains of human atlastin-1 in vitro. Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro. It was also determined that co-reconstitution of atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, this study found that both human and Drosophila atlastin hydrophobic membrane anchors do not span the membrane, but rather forms two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.
Harris, K. P., Littleton, J. T. and Stewart, B. A. (2018). Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release. J Neurogenet: 1-9. PubMed ID: 30175640
Summary:
Signaling from the postsynaptic compartment regulates multiple aspects of synaptic development and function. Syntaxin 4 (Syx4) is a plasma membrane t-SNARE that promotes the growth and plasticity of Drosophila neuromuscular junctions (NMJs) by regulating the localization of key synaptic proteins in the postsynaptic compartment. This study describes electrophysiological analyses and reports that loss of Syx4 leads to enhanced neurotransmitter release, despite a decrease in the number of active zones. A requirement is described for postsynaptic Syx4 in regulating several presynaptic parameters, including Ca(2+) cooperativity and the abundance of the presynaptic calcium channel Cacophony (Cac) at active zones. These findings indicate Syx4 negatively regulates presynaptic neurotransmitter release through a retrograde signaling mechanism from the postsynaptic compartment.
Vukoja, A., Rey, U., Petzoldt, A. G., Ott, C., Vollweiter, D., Quentin, C., Puchkov, D., Reynolds, E., Lehmann, M., Hohensee, S., Rosa, S., Lipowsky, R., Sigrist, S. J. and Haucke, V. (2018). Presynaptic biogenesis requires axonal transport of lysosome-related vesicles. Neuron. PubMed ID: 30174114
Summary:
Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) supplying presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is unclear. Live imaging in Drosophila larvae and mouse hippocampal neurons provides evidence that presynaptic biogenesis depends on axonal co-transport of SV and AZ proteins in presynaptic lysosome-related vesicles (PLVs). Loss of the lysosomal kinesin adaptor Arl8 results in the accumulation of SV- and AZ-protein-containing vesicles in neuronal cell bodies and a corresponding depletion of SV and AZ components from presynaptic sites, leading to impaired neurotransmission. Conversely, presynaptic function is facilitated upon overexpression of Arl8. These data reveal an unexpected function for a lysosome-related organelle as an important building block for presynaptic biogenesis.

Wednesday, November 7th - Disease Models

Thomas, R. E., Vincow, E. S., Merrihew, G. E., MacCoss, M. J., Davis, M. Y. and Pallanck, L. J. (2018). Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles. PLoS Genet 14(9): e1007694. PubMed ID: 30256786
Summary:
Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. This study tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Likewise, Gba1b mutants did not have the marked impairment of mitochondrial protein turnover seen in mitophagy-deficient parkin mutants. Proteasome activity, microautophagy, and endocytic degradation also appeared unaffected in Gba1b mutants. However, striking changes were found in the turnover and abundance of proteins associated with extracellular vesicles (EVs), which have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. These changes were specific to Gba1b mutants and did not represent an acceleration of normal aging. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, these findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles.
He, T., Nitabach, M. N. and Lnenicka, G. A. (2018). Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ. J Neurogenet: 1-12. PubMed ID: 30175644
Summary:
Presynaptic Ca(2+) appears to play multiple roles in synaptic development and physiology. This study examined the effect of buffering presynaptic Ca(2+) by expressing parvalbumin (PV) in Drosophila neurons, which do not normally express PV. The studies were performed on the identified Ib terminal that innervates muscle fiber 5. The volume-averaged, residual Ca(2+) resulting from single action potentials (APs) and AP trains was measured using the fluorescent Ca(2+) indicator, OGB-1. PV reduced the amplitude and decay time constant (tau) for single-AP Ca(2+) transients. For AP trains, there was a reduction in the rate of rise and decay of [Ca(2+)]i but the plateau [Ca(2+)]i was not affected. Electrophysiological recordings from muscle fiber 5 showed a reduction in paired-pulse facilitation, particularly the F1 component; this was likely due to the reduction in residual Ca(2+). These synapses also showed reduced synaptic enhancement during AP trains, presumably due to less buildup of synaptic facilitation. The transmitter release for single APs was increased for the PV-expressing terminals and this may have been a homeostatic response to the decrease in facilitation. Confocal microscopy was used to examine the structure of the motor terminals and PV expression resulted in smaller motor terminals with fewer synaptic boutons and active zones. This result supports earlier proposals that increased AP activity promotes motor terminal growth through increases in presynaptic [Ca(2+)]i.
Rosa-Ferreira, C., Sweeney, S. T. and Munro, S. (2018). The small G protein Arl8 contributes to lysosomal function and long-range axonal transport in Drosophila. Biol Open 7(9). PubMed ID: 30115618
Summary:
The small GTPase Arl8 has emerged as a major regulatory GTPase on lysosomes. Studies in mammalian cells have shown that it regulates both fusion with late endosomes and also lysosomal motility. In its active GTP-bound state, it recruits to lysosomes the HOPS (homotypic fusion and protein sorting) endosomal tethering complex and also proteins that link lysosomes to microtubule motors such as the kinesin adaptor PLEKHM2. To gain further insights into Arl8 biology, this study examined the single Drosophila ortholog. Drosophila Arl8 is essential for viability, and mitotic clones of mutant cells are able to continue to divide but show perturbation of the late endocytic pathway. Progeny-lacking Arl8 die as late larvae with movement-paralysis characteristic of defects in neuronal function. This phenotype was rescued by expression of Arl8 in motor neurons. Examination of these neurons in the mutant larvae revealed smaller synapses and axons with elevated levels of carriers containing synaptic components. Affinity chromatography revealed binding of Drosophila Arl8 to the HOPS complex, and to the Drosophila ortholog of RILP, a protein that, in mammals, recruits dynein to late endosomes, with dynein being known to be required for neuronal transport. Thus Drosophila Arl8 controls late endocytic function and transport via at least two distinct effectors.
Betancourt-Solis, M. A., Desai, T. and McNew, J. A. (2018). The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer. J Biol Chem. PubMed ID: 30287684
Summary:
The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. This study examined several Drosophila-human atlastin chimeras to identify functional domains of human atlastin-1 in vitro. Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro. It was also determined that co-reconstitution of atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, this study found that both human and Drosophila atlastin hydrophobic membrane anchors do not span the membrane, but rather forms two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.
Harris, K. P., Littleton, J. T. and Stewart, B. A. (2018). Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release. J Neurogenet: 1-9. PubMed ID: 30175640
Summary:
Signaling from the postsynaptic compartment regulates multiple aspects of synaptic development and function. Syntaxin 4 (Syx4) is a plasma membrane t-SNARE that promotes the growth and plasticity of Drosophila neuromuscular junctions (NMJs) by regulating the localization of key synaptic proteins in the postsynaptic compartment. This study describes electrophysiological analyses and reports that loss of Syx4 leads to enhanced neurotransmitter release, despite a decrease in the number of active zones. A requirement is described for postsynaptic Syx4 in regulating several presynaptic parameters, including Ca(2+) cooperativity and the abundance of the presynaptic calcium channel Cacophony (Cac) at active zones. These findings indicate Syx4 negatively regulates presynaptic neurotransmitter release through a retrograde signaling mechanism from the postsynaptic compartment.
Vukoja, A., Rey, U., Petzoldt, A. G., Ott, C., Vollweiter, D., Quentin, C., Puchkov, D., Reynolds, E., Lehmann, M., Hohensee, S., Rosa, S., Lipowsky, R., Sigrist, S. J. and Haucke, V. (2018). Presynaptic biogenesis requires axonal transport of lysosome-related vesicles. Neuron. PubMed ID: 30174114
Summary:
Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) supplying presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is unclear. Live imaging in Drosophila larvae and mouse hippocampal neurons provides evidence that presynaptic biogenesis depends on axonal co-transport of SV and AZ proteins in presynaptic lysosome-related vesicles (PLVs). Loss of the lysosomal kinesin adaptor Arl8 results in the accumulation of SV- and AZ-protein-containing vesicles in neuronal cell bodies and a corresponding depletion of SV and AZ components from presynaptic sites, leading to impaired neurotransmission. Conversely, presynaptic function is facilitated upon overexpression of Arl8. These data reveal an unexpected function for a lysosome-related organelle as an important building block for presynaptic biogenesis.

Wednesday, November 7th

Sarkar, A., Gogia, N., Glenn, N., Singh, A., Jones, G., Powers, N., Srivastava, A., Kango-Singh, M. and Singh, A. (2018). A soy protein Lunasin can ameliorate amyloid-beta 42 mediated neurodegeneration in Drosophila eye. Sci Rep 8(1): 13545. PubMed ID: 30202077
Summary:
Alzheimer's disease (AD), a fatal progressive neurodegenerative disorder, also results from accumulation of amyloid-beta 42 (Abeta42) plaques. These Abeta42 plaques trigger oxidative stress, abnormal signaling, which results in neuronal death by unknown mechanism(s). This study misexpressed high levels of human Abeta42 in the differentiating retinal neurons of the Drosophila eye, which results in the Alzheimer's like neuropathology. Using s transgenic model, a soy-derived protein Lunasin (Lun) was tested for a possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer effect and reduces stress and inflammation. Misexpression of Lunasin by transgenic approach can rescue Abeta42 mediated neurodegeneration by blocking cell death in retinal neurons, and results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can prevent neuroprotective role of Lunasin in Abeta42 mediated neurodegeneration. This neuroprotective function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and is independent of Wingless and Decapentaplegic signaling pathways. Furthermore, Lunasin can significantly reduce mortality rate caused by misexpression of human Abeta42 in flies. These studies identified the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can ameliorate Abeta42 mediated neurodegeneration by downregulating JNK signaling.
Ran, D., Xie, B., Gan, Z., Sun, X., Gu, H. and Yang, J. (2018). Melatonin attenuates hLRRK2-induced long-term memory deficit in a Drosophila model of Parkinson's disease. Biomed Rep 9(3): 221-226. PubMed ID: 30271597
Summary:
As the most common genetic cause of Parkinson's disease (PD), the role of human leucine-rich repeat kinase 2 (hLRRK2) in the efficacy of PD treatment is a focus of study. A previous study demonstrated that mushroom body (MB) expression of hLRRK2 in Drosophila could recapitulate the clinical feature of sleep disturbances observed in PD patients, and melatonin (MT) treatment could attenuate the hLRRK2-induced sleep disorders and synaptic dysfunction, suggesting the therapeutic potential of MT in PD patients carrying hLRRK2 mutations; however, no further study into the impacts on memory deficit was conducted. Therefore, in the current paper, the study of the effects of MT on hLRRK2 flies was continued, to determine its potential role in the improvement of memory deficit in PD. To achieve this, the Drosophila learning and memory phases, including short- and long-term memory, were recorded; furthermore, the effect of MT on calcium channel activity during neurotransmission was detected using electrophysiology patch clamp recordings. It was demonstrated that MT treatment reversed hLRRK2-induced long-term memory deficits in Drosophila; furthermore, MT reduced MB calcium channel activities. These findings suggest that MT may exerts therapeutic effects on the long-term memory of PD patients via calcium channel modulation, thus providing indication of its potential to maintain cognitive function in PD patients.
Passarella, D. and Goedert, M. (2018). Beta-sheet assembly of Tau and neurodegeneration in Drosophila melanogaster. Neurobiol Aging 72: 98-105. PubMed ID: 30240946
Summary:
The assembly of Tau into abundant beta-sheet-rich filaments characterizes human tauopathies. A pathological pathway leading from monomeric to filamentous Tau is believed to be at the heart of these diseases. However, in Drosophila models of Tauopathy, neurodegeneration has been observed in the absence of abundant Tau filaments. This study investigated the role of Tau assembly into beta-sheets by expressing wild-type and Delta306-311 human Tau-383 in the retina and brain of Drosophila. Both lines were examined for eye abnormalities, brain vacuolization, Tau phosphorylation and assembly, as well as climbing activity and survival. Flies expressing wild-type Tau-383 showed MC-1 staining, Tau hyperphosphorylation, and neurodegeneration. By contrast, flies expressing Delta306-311 Tau-383 had less MC-1 staining, reduced Tau hyperphosphorylation, and no detectable neurodegeneration. Their climbing ability and lifespan were similar to those of nontransgenic flies. Fluorescence spectroscopy after addition of Thioflavin T, a dye that interacts with beta-sheets, showed no signal when Delta306-311 Tau-383 was incubated with heparin. These findings demonstrate that the assembly of Tau into beta-sheets is necessary for neurodegeneration.
Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B. and Voigt, A. (2018). Monitoring alpha-synuclein multimerization in vivo. Faseb j: fj201800148RRR. PubMed ID: 30252534
Summary:
The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of alpha-synuclein (alpha-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although alpha-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on alpha-Syn aggregation. This study used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor alpha-Syn accumulation by using a bimolecular fluorescence complementation assay. Both genetic and pharmacologic manipulations affected alpha-Syn accumulation. Interestingly, it was also found that alterations in the cellular protein degradation mechanisms strongly influenced alpha-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on alpha-Syn accumulation in vivo. Finally, this study showed that increasing phosphorylation of alpha-Syn at serine 129 enhances the accumulation and toxicity of alpha-Syn. Altogether, this study establishes a novel model to study alpha-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.
Qiao, H. H., Wang, F., Xu, R. G., Sun, J., Zhu, R., Mao, D., Ren, X., Wang, X., Jia, Y., Peng, P., Shen, D., Liu, L. P., Chang, Z., Wang, G., Li, S., Ji, J. Y., Liu, Q. and Ni, J. Q. (2018). An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila. Nat Commun 9(1): 4160. PubMed ID: 30297884
Summary:
Being relatively simple and practical, Drosophila transgenic RNAi is the technique of top priority choice to quickly study genes with pleiotropic functions. However, drawbacks have emerged over time, such as high level of false positive and negative results. To overcome these shortcomings and increase efficiency, specificity and versatility, a next generation transgenic RNAi system was developed. With this system, the leaky expression of the basal promoter is significantly reduced, as well as the heterozygous ratio of transgenic RNAi flies. In addition, it has been first achieved to precisely and efficiently modulate highly expressed genes. Furthermore, this study increased versatility which can simultaneously knock down multiple genes in one step. A case illustration is provided of how this system can be used to study the synthetic developmental effect of histone acetyltransferases. Finally, a collection of transgenic RNAi lines was generated for those genes that are highly homologous to human disease genes.
Roed, N. K., Viola, C. M., Kristensen, O., Schluckebier, G., Norrman, M., Sajid, W., Wade, J. D., Andersen, A. S., Kristensen, C., Ganderton, T. R., Turkenburg, J. P., De Meyts, P. and Brzozowski, A. M. (2018). Structures of insect Imp-L2 suggest an alternative strategy for regulating the bioavailability of insulin-like hormones. Nat Commun 9(1): 3860. PubMed ID: 30242155
Summary:
The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1-6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. This paper reports structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.

Tuesday, November 6th

Phillips, M. A., Rutledge, G. A., Kezos, J. N., Greenspan, Z. S., Talbott, A., Matty, S., Arain, H., Mueller, L. D., Rose, M. R. and Shahrestani, P. (2018). Effects of evolutionary history on genome wide and phenotypic convergence in Drosophila populations. BMC Genomics 19(1): 743. PubMed ID: 30305018
Summary:
Studies combining experimental evolution and next-generation sequencing have found that adaptation in sexually reproducing populations is primarily fueled by standing genetic variation. Consequently, the response to selection is rapid and highly repeatable across replicate populations. Some studies suggest that the response to selection is highly repeatable at both the phenotypic and genomic levels, and that evolutionary history has little impact. Other studies suggest that even when the response to selection is repeatable phenotypically, evolutionary history can have significant impacts at the genomic level. This study tests two hypotheses that may explain this discrepancy. Hypothesis 1: Past intense selection reduces evolutionary repeatability at the genomic and phenotypic levels when conditions change. Hypothesis 2: Previous intense selection does not reduce evolutionary repeatability, but other evolutionary mechanisms may. These hypotheses were tested using D. melanogaster populations that were subjected to 260 generations of intense selection for desiccation resistance and have since been under relaxed selection for the past 230 generations. It was found that, with the exception of longevity and to a lesser extent fecundity, 230 generations of relaxed selection has erased the extreme phenotypic differentiation previously found. No signs were found of genetic fixation, and only limited evidence of genetic differentiation between previously desiccation resistance selected populations and their controls. These findings suggest that evolution in this system is highly repeatable even when populations have been previously subjected to bouts of extreme selection. It is therefore concluded that evolutionary repeatability can overcome past bouts of extreme selection in Drosophila experimental evolution, provided experiments are sufficiently long and populations are not inbred.
Garlovsky, M. D. and Snook, R. R. (2018). Persistent postmating, prezygotic reproductive isolation between populations. Ecol Evol 8(17): 9062-9073. PubMed ID: 30271566
Summary:
Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate-female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. This study addressed these outstanding questions using multiple populations of Drosophila montana. A recurrent pattern of PMPZ isolation is shown, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. No evidence was found of variability in fertilization outcomes attributable to different female x male genotype interactions, and in combination with other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype x genotype interactions. These results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate-female reproductive tract interactions within species that may cause this PMPZ isolation.
Diaz, F., Allan, C. W. and Matzkin, L. M. (2018). Positive selection at sites of chemosensory genes is associated with the recent divergence and local ecological adaptation in cactophilic Drosophila. BMC Evol Biol 18(1): 144. PubMed ID: 30236055
Summary:
Adaptation to new hosts in phytophagous insects often involves mechanisms of host recognition by genes of sensory pathways. Most often the molecular evolution of sensory genes has been explained in the context of the birth-and-death model. The role of positive selection is less understood, especially associated with host adaptation and specialization. This study aimed to contribute evidence for this latter hypothesis by considering the case of Drosophila mojavensis, a species with an evolutionary history shaped by multiple host shifts in a relatively short time scale, and its generalist sister species, D. arizonae. A phylogenetic and population genetic analysis framework was used to test for positive selection in a subset of four chemoreceptor genes, one gustatory receptor (Gr) and three odorant receptors (Or), for which their expression has been previously associated with host shifts. Strong evidence was found of positive selection at several amino acid sites in all genes investigated, most of which exhibited changes predicted to cause functional effects in these transmembrane proteins. A significant portion of the sites identified as evolving positively were largely found in the cytoplasmic region, although a few were also present in the extracellular domains. The pattern of substitution observed suggests that some of these changes likely had an effect on signal transduction as well as odorant recognition and protein-protein interactions. These findings support the role of positive selection in shaping the pattern of variation at chemosensory receptors, both during the specialization onto one or a few related hosts, but as well as during the evolution and adaptation of generalist species into utilizing several hosts.
Comeault, A. A. and Matute, D. R. (2018). Genetic divergence and the number of hybridizing species affect the path to homoploid hybrid speciation. Proc Natl Acad Sci U S A 115(39): 9761-9766. PubMed ID: 30209213
Summary:
Hybridization is often maladaptive and in some instances has led to the loss of biodiversity. However, hybridization can also promote speciation, such as during homoploid hybrid speciation, thereby generating biodiversity. Despite examples of homoploid hybrid species, the importance of hybridization as a speciation mechanism is still widely debated, and a general understanding is lacking of the conditions most likely to generate homoploid hybrid species. This study shows that the level of genetic divergence between hybridizing species has a large effect on the probability that their hybrids evolve reproductive isolation. Populations of hybrids formed by parental species with intermediate levels of divergence were more likely to mate assortatively, and discriminate against their parental species, than those generated from weakly or strongly diverged parental species. Reproductive isolation was also found between hybrid populations, suggesting differential sorting of parental traits across populations. Finally, hybrid populations derived from three species were more likely to evolve reproductive isolation than those derived from two species, supporting arguments that hybridization-supplied genetic diversity can lead to the evolution of novel "adaptive systems" and promote speciation. These results illustrate when hybridization and admixture is expected to promote hybrid speciation. Whether homoploid hybrid speciation is a common speciation mechanism in general remains an outstanding empirical question.
Medina, P., Thornlow, B., Nielsen, R. and Corbett-Detig, R. (2018). Estimating the timing of multiple admixture pulses during local ancestry inference. Genetics. PubMed ID: 30206187
Summary:
Admixture, the mixing of genetically distinct populations, is increasingly recognized as a fundamental biological process. One major goal of admixture analyses is to estimate the timing of admixture events. Whereas most methods today can only detect the most recent admixture event, this study presents coalescent theory and associated software that can be used to estimate the timing of multiple admixture events in an admixed population. This approach was extensively validated and the conditions under which it can successfully distinguish one from two-pulse admixture models was validated. This approach was applied to real and simulated data of Drosophila melanogaster. Evidence was found of a single very recent pulse of cosmopolitan ancestry contributing to African populations as well as evidence for more ancient admixture among genetically differentiated populations in sub-Saharan Africa. These results suggest this method can quantify complex admixture histories involving genetic material introduced by multiple discrete admixture pulses. The new method facilitates the exploration of admixture and its contribution to adaptation, ecological divergence, and speciation.
Muto, L., Kamimura, Y., Tanaka, K. M. and Takahashi, A. (2018). An innovative ovipositor for niche exploitation impacts genital coevolution between sexes in a fruit-damaging Drosophila. Proc Biol Sci 285(1887). PubMed ID: 30257912
Summary:
Limited attention has been given to ecological factors influencing the coevolution of male and female genitalia. The innovative ovipositor of Drosophila suzukii, an invading fruit pest, represents an appealing case to document this phenomenon. The serrated saw-like ovipositor is used to pierce the hard skin of ripening fruits that are not used by other fruit flies that prefer soft decaying fruits. This study highlights another function of the ovipositor related to its involvement in genital coupling during copulation. The morphology and coupling of male and female genitalia in this species were compared to its sibling species, Drosophila subpulchrella, and to an outgroup species, Drosophila biarmipes. These comparisons and a surgical manipulation indicated that the shape of male genitalia in D. suzukii has had to be adjusted to ensure tight coupling, despite having to abandon the use of a hook-like structure, paramere, because of the more linearly elongated ovipositor. This phenomenon demonstrates that ecological niche exploitation can directly affect the mechanics of genital coupling and potentially cause incompatibility among divergent forms. This model case provides new insights towards elucidating the importance of the dual functions of ovipositors in other insect species that potentially induce genital coevolution and ecological speciation.

Monday, November 5th - Larval and Adult Development

Sawala, A. and Gould, A. P. (2018). Sex-lethal in neurons controls female body growth in Drosophila. Fly (Austin): 1-9. PubMed ID: 30126340
Summary:
Sexual size dimorphism (SSD), a sex difference in body size, is widespread throughout the animal kingdom, raising the question of how sex influences existing growth regulatory pathways to bring about SSD. In insects, somatic sexual differentiation has long been considered to be controlled strictly cell-autonomously. This paper discusses the surprising finding that in Drosophila larvae, the sex determination gene Sex-lethal (Sxl) functions in neurons to non-autonomously specify SSD. Sxl was found to be required in specific neuronal subsets to upregulate female body growth, including in the neurosecretory insulin producing cells, even though insulin-like peptides themselves appear not to be involved. SSD regulation by neuronal Sxl is also independent of its known splicing targets, transformer and msl-2, suggesting that it involves a new molecular mechanism. Interestingly, SSD control by neuronal Sxl is selective for larval, not imaginal tissue types, and operates in addition to cell-autonomous effects of Sxl and Tra, which are present in both larval and imaginal tissues. Overall, these findings add to a small but growing number of studies reporting non-autonomous, likely hormonal, control of sex differences in Drosophila, and suggest that the principles of sexual differentiation in insects and mammals may be more similar than previously thought.
Ho, E. C. Y., Malagon, J. N., Ahuja, A., Singh, R. and Larsen, E. (2018). Rotation of sex combs in Drosophila melanogaster requires precise and coordinated spatio-temporal dynamics from forces generated by epithelial cells. PLoS Comput Biol 14(10): e1006455. PubMed ID: 30303951
Summary:
The morphogenesis of sex combs (SCs), a male trait in many species of fruit flies, is an excellent system in which to study the cell biology, genetics and evolution of a trait. In Drosophila melanogaster, where the incipient SC rotates from horizontal to a vertical position, three signal comb properties have been documented: length, final angle and shape (linearity). During SC rotation, in which many cellular processes are occurring both spatially and temporally, it is difficult to distinguish which processes are crucial for which attributes of the comb. This study used a novel approach combining simulations and experiments to uncover the spatio-temporal dynamics underlying SC rotation. The results indicate that 1) the final SC shape is primarily controlled by the inhomogeneity of initial cell size in cells close to the immature comb, 2) the final angle is primarily controlled by later cell expansion and 3) a temporal sequence of cell expansion mitigates the malformations generally associated with longer rotated SCs. Overall, this work has linked together the morphological diversity of SCs and the cellular dynamics behind such diversity, thus providing important insights on how evolution may affect SC development via the behaviours of surrounding epithelial cells.
Cordoba, S. and Estella, C. (2018). The transcription factor Dysfusion promotes fold and joint morphogenesis through regulation of Rho1. PLoS Genet 14(8): e1007584. PubMed ID: 30080872
Summary:
The mechanisms that control tissue patterning and cell behavior are extensively studied separately, but much less is known about how these two processes are coordinated. This study shows that the Drosophila transcription factor Dysfusion (Dysf) directs leg epithelial folding and joint formation through the regulation of Rho1 activity. Dysf-induced Rho1 activity promotes apical constriction specifically in folding epithelial cells. This study shows that downregulation of Rho1 or its downstream effectors cause defects in fold and joint formation. In addition, Rho1 and its effectors are sufficient to induce the formation of epithelial folds when misexpressed in a flat epithelium. Furthermore, as apoptotic cells can actively control tissue remodeling, the role of cell death in the formation of tarsal folds and its relation to Rho1 activity was analyzed. Surprisingly, no defects were found in this process when apoptosis is inhibited. These results highlight the coordination between a patterning transcription factor and the cellular processes that cause the cell shape changes necessary to sculpt a flat epithelium into a three dimensional structure.
Ozturk-Colak, A., Stephan-Otto Attolini, C., Casanova, J. and Araujo, S. J. (2018). Blimp-1 mediates tracheal lumen maturation in Drosophila melanogaster. Genetics. PubMed ID: 30082278
Summary:
The specification of tissue identity during embryonic development requires precise spatio-temporal coordination of gene expression. Many transcription factors required for the development of organs have been identified and their expression patterns are known; however, the mechanisms through which they coordinate gene expression in time remain poorly understood. This study shows that hormone-induced transcription factor Blimp-1 participates in the temporal coordination of tubulogenesis in Drosophila melanogaster by regulating the expression of many genes involved in tube maturation. In particular, Blimp-1 was shown to regulates the expression of genes involved in chitin deposition and F-actin organization. Blimp-1 was shown to be involved in the temporal control of lumen maturation by regulating the beginning of chitin deposition. Blimp-1 represses a variety of genes involved in tracheal maturation. Kinase Btk29A was shown to serve as a link between Blimp-1 transcriptional repression and apical extra-cellular matrix organization.
Okamoto, N., Viswanatha, R., Bittar, R., Li, Z., Haga-Yamanaka, S., Perrimon, N. and Yamanaka, N. (2018). A membrane transporter is required for steroid hormone uptake in Drosophila. Dev Cell. PubMed ID: 30293839
Summary:
Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. This study challenges this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or Ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, these results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies.
Pan, Y., Alegot, H., Rauskolb, C. and Irvine, K. D. (2018). The dynamics of hippo signaling during Drosophila wing development. Development. PubMed ID: 30254143
Summary:
Tissue growth needs to be properly controlled for organs to reach their correct size and shape, but the mechanisms that control growth during normal development are not fully understood. This study reports that the activity of the Hippo signaling transcriptional activator Yorkie gradually decreases in the central region of the developing Drosophila wing disc. Spatial and temporal changes in Yorkie activity can be explained by changes in cytoskeletal tension and biomechanical regulators of Hippo signaling. These changes in cellular biomechanics correlate with changes in cell density, and experimental manipulations of cell density are sufficient to alter biomechanical Hippo signaling and Yorkie activity. The pattern of Yorkie activity in older discs was also related to patterns of cell proliferation. These results establish that spatial and temporal patterns of Hippo signaling occur during wing development, that these patterns depend upon cell-density modulated tissue mechanics, and that they contribute to the regulation of wing cell proliferation.

Friday, November 2nd - Testes and Oocytes

Valer, F. B., Machado, M. C. R., Silva-Junior, R. M. P. and Ramos, R. G. P. (2018). Expression of Hbs, Kirre and Rst during Drosophila ovarian development. Genesis. PubMed ID: 30114331
Summary:
The Irre Cell-Recognition Module (IRM) is a group of evolutionarily conserved and structurally related transmembrane glycoproteins of the immunoglobulin superfamily. In Drosophila melanogaster it comprises the products of the genes roughest (rst; also known as irreC-rst), kin-of-irre (kirre; also known as duf), stick-and-stones (sns) and hibris (hbs). In this model organism, the behavior of this group of proteins as a partly redundant functional unit mediating selective cell recognition was demonstrated in a variety of developmental contexts, but their possible involvement in ovarian development and oogenesis has not been investigated, notwithstanding the fact that some rst mutant alleles are also female sterile. This study shows that IRM genes are dynamically and, to some extent, coordinately transcribed in both pupal and adult ovaries. Additionally, the spatial distribution of Hbs, Kirre and Rst proteins indicates that they perform cooperative, although largely non-redundant, functions. Finally, phenotypical characterization of three different female sterile rst alleles uncovered two temporally separated and functionally distinct requirements for this locus in ovarian development: one in pupa, essential for the organization of peritoneal and epithelial sheaths that maintain the structural integrity of the adult organ and another, in mature ovarioles, needed for the progression of oogenesis beyond stage 10.
Lu, W., Lakonishok, M., Serpinskaya, A. S., Kirchenbuechler, D., Ling, S. C. and Gelfand, V. I. (2018). Ooplasmic flow cooperates with transport and anchorage in Drosophila oocyte posterior determination. J Cell Biol. PubMed ID: 30037924
Summary:
The posterior determination of the Drosophila melanogaster embryo is defined by the posterior localization of oskar (osk) mRNA in the oocyte. Defects of its localization result in a lack of germ cells and failure of abdomen specification. A microtubule motor kinesin-1 is essential for osk mRNA posterior localization. Because kinesin-1 is required for two essential functions in the oocyte-transport along microtubules and cytoplasmic streaming-it is unclear how individual kinesin-1 activities contribute to the posterior determination. Staufen, an RNA-binding protein that is colocalized with osk mRNA, was examined as a proxy of posterior determination, and mutants were used that either inhibit kinesin-driven transport along microtubules or cytoplasmic streaming. Late-stage streaming is partially redundant with early-stage transport along microtubules for Staufen posterior localization. Additionally, an actin motor, myosin V, is required for the Staufen anchoring to the actin cortex. A model is proposed whereby initial kinesin-driven transport, subsequent kinesin-driven streaming, and myosin V-based cortical retention cooperate in posterior determination.
Herrera, S. C. and Bach, E. A. (2018). JNK signaling triggers spermatogonial dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis. Elife 7. PubMed ID: 29985130
Summary:
Exhaustion of stem cells is a hallmark of aging. In the Drosophila testis, dedifferentiated germline stem cells (GSCs) derived from spermatogonia increases during lifespan, leading to the model that dedifferentiation counteracts the decline of GSCs in aged males. To test this, dedifferentiation was blocked by mis-expressing the differentiation factor bag of marbles (bam) in spermatogonia while lineage-labeling these cells. Strikingly, blocking bam-lineage dedifferentiation under normal conditions in virgin males has no impact on the GSC pool. However, in mated males or challenging conditions, inhibiting bam-lineage dedifferentiation markedly reduced the number of GSCs and their ability to proliferate and differentiate. bam-lineage derived GSCs have significantly higher proliferation rates than sibling GSCs in the same testis. Jun N-terminal kinase (JNK) activity is autonomously required for bam-lineage dedifferentiation. Overall, this study shows that dedifferentiation provides a mechanism to maintain the germline and ensure fertility under chronically stressful conditions.
McCarthy, A., Deiulio, A., Martin, E. T., Upadhyay, M. and Rangan, P. (2018). Tip60 complex promotes expression of a differentiation factor to regulate germline differentiation in female Drosophila. Mol Biol Cell: mbcE18060385. PubMed ID: 30230973
Summary:
Germline stem cells (GSCs) self-renew and differentiate to sustain a continuous production of gametes. In the female Drosophila germ line, two differentiation factors, bag of marbles (bam) and benign gonial cell neoplasm (bgcn), work in concert in the stem cell daughter to promote the generation. In GSCs, bam transcription is repressed by signaling from the niche and is activated in stem cell daughters. In contrast, bgcn is transcribed in both the GSCs and stem cell daughters, but little is known about how bgcn is transcriptionally modulated. This study finds that the conserved protein Nipped-A acts through the Tat interactive protein 60kDa (Tip60) Histone acetyl transferase (HAT) complex in the germ line to promote GSC daughter differentiation. Nipped-A is required for efficient exit from the gap phase 2 (G2) of cell cycle of the GSC daughter and for expression of a differentiation factor, bgcn. Loss of Nipped-A results in accumulation of GSC daughters . Forced expression of bgcn, in Nipped-A germline-depleted ovaries rescues this differentiation defect. Together, these results indicate that Tip60 complex coordinates cell cycle progression and expression of bgcn to help drive GSC daughters toward a differentiation program.
Greenspan, L. J. and Matunis, E. L. (2018). Retinoblastoma intrinsically regulates niche cell quiescence, identity, and niche number in the adult Drosophila testis. Cell Rep 24(13): 3466-3476.e3468. PubMed ID: 30257208
Summary:
Homeostasis in adult tissues depends on the precise regulation of stem cells and their surrounding microenvironments, or niches. This study shows that the cell cycle inhibitor and tumor suppressor Retinoblastoma (RB) is a critical regulator of niche cells in the Drosophila testis. The testis contains a single niche, composed of somatic hub cells, that signals to adjacent germline and somatic stem cells. Hub cells are normally quiescent, but knockdown of the RB homolog Rbf in these cells causes them to proliferate and convert to somatic stem cells. Over time, mutant hub cell clusters enlarge and split apart, forming ectopic hubs surrounded by active stem cells. Furthermore, this study shows that Rbf's ability to restrict niche number depends on the transcription factors E2F and Escargot and the adhesion molecule E-cadherin. Together this work reveals how precise modulation of niche cells, not only the stem cells they support, can drive regeneration and disease.
Kistler, K. E., Trcek, T., Hurd, T. R., Chen, R., Liang, F. X., Sall, J., Kato, M. and Lehmann, R. (2018). Phase transitioned nuclear Oskar promotes cell division of Drosophila primordial germ cells. Elife 7. PubMed ID: 30260314
Summary:
Germ granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. This study examined two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. Cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, Oskar was found to drive their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar's nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.

Thursday, November 1st - Cytoskeleton and Junctions

Oelz, D. B., Del Castillo, U., Gelfand, V. I. and Mogilner, A. (2018). Microtubule Dynamics, Kinesin-1 Sliding, and Dynein Action Drive Growth of Cell Processes. Biophys J. PubMed ID: 30268540
Summary:
Recent experimental studies of the role of microtubule sliding in neurite outgrowth suggested a qualitative model, according to which kinesin-1 motors push the minus-end-out microtubules against the cell membrane and generate the early cell processes. At the later stage, dynein takes over the sliding, expels the minus-end-out microtubules from the neurites, and pulls in the plus-end-out microtubules that continue to elongate the nascent axon. This model leaves unanswered a number of questions: why is dynein unable to generate the processes alone, whereas kinesin-1 can? What is the role of microtubule dynamics in process initiation and growth? Can the model correctly predict the rates of process growth in control and dynein-inhibited cases? What triggers the transition from kinesin-driven to dynein-driven sliding? To answer these questions, computational modeling of a network of elastic dynamic microtubules and kinesin-1 and dynein motors were combined with measurements of the process growth kinetics and pharmacological perturbations in Drosophila S2 cells. The results verify quantitatively the qualitative model of the microtubule polarity sorting and suggest that dynein-powered elongation is effective only when the processes are longer than a threshold length, which explains why kinesin-1 alone, but not dynein, is sufficient for the process growth. Furthermore, it was shown that the mechanism of process elongation depends critically on microtubule dynamic instability. Both modeling and experimental measurements show, surprisingly, that dynein inhibition accelerates the process extension. Implications of the model for the general problems of cell polarization, cytoskeletal polarity emergence, and cell process protrusion are discussed.
Jezzini, S. H., Merced, A. and Blagburn, J. M. (2018). Shaking-B misexpression increases the formation of gap junctions but not chemical synapses between auditory sensory neurons and the giant fiber of Drosophila melanogaster. PLoS One 13(8): e0198710. PubMed ID: 30118493
Summary:
The synapse between auditory Johnston's Organ neurons (JONs) and the giant fiber (GF) of Drosophila is structurally mixed, being composed of cholinergic chemical synapses and gap junctions, which consist of the innexin Shaking-B (ShakB). Misexpression of one ShakB isoform, ShakB(N+16), in a subset of JONs that do not normally form gap junctions results in their de novo dye coupling to the GF. Misexpression of the transcription factor Engrailed (En) in these neurons also has this effect, and in addition causes the formation of new chemical synapses. These results led to the hypothesis that ShakB would, like En, have an instructive effect on the distribution of mixed chemical/electrical contacts. To test this, it was first confirmed quantitatively that ShakB(N+16) misexpression increased the dye-coupling of JONs with the GF, indicating the formation of ectopic gap junctions. Immunocytochemistry of the ShakB protein showed that ShakB(N+16) increased gap junctional plaques in JON axons. To test the hypothesis, fluorescently-labeled presynaptic active zone protein (Brp) was expressed in JONs and the changes in its distribution on the GF dendrites was assayed with confocal microscopy in animals with misexpression of ShakB(N+16), ShakB(N) or, as a positive control, En. En misexpression increased the chemical synapses with the GF and the amount of GF medial dendrite branching. However, contrary to the hypothesis, misexpression of ShakB did not increase these parameters. It is concluded that both subsets of JON form chemical synapses onto the GF dendrites but only one population forms gap junctions, comprised of ShakB(N+16). Misexpression of this isoform in all JONs does not instruct the formation of new mixed chemical/electrical synapses, but results in the insertion of new gap junctions, presumably at the sites of existing chemical synaptic contacts with the GF.
Russell, S. L., Lemseffer, N. and Sullivan, W. T. (2018). Wolbachia and host germline components compete for kinesin-mediated transport to the posterior pole of the Drosophila oocyte. PLoS Pathog 14(8): e1007216. PubMed ID: 30110391
Summary:
Widespread success of the intracellular bacterium Wolbachia across insects and nematodes is due to efficient vertical transmission and reproductive manipulations. Many strains, including wMel from Drosophila melanogaster, exhibit a specific concentration to the germplasm at the posterior pole of the mature oocyte, thereby ensuring high fidelity of parent-offspring transmission. Transport of Wolbachia to the pole relies on microtubules and the plus-end directed motor kinesin heavy chain (KHC). However, the mechanisms mediating Wolbachia's association with KHC remain unknown. This study shows that reduced levels of the host canonical linker protein KLC results in dramatically increased levels of Wolbachia at the oocyte's posterior, suggesting that KLC and some key associated host cargos outcompete Wolbachia for association with a limited amount of KHC motor proteins. Consistent with this interpretation, over-expression of KHC causes similarly increased levels of posteriorly localized Wolbachia. However, excess KHC has no effect on levels of Vasa, a germplasm component that also requires KHC for posterior localization. Thus, Wolbachia transport is uniquely KHC-limited because these bacteria are likely outcompeted for binding to KHC by some host cargo/linker complexes. These results reveal a novel host-symbiont interaction that underscores the precise regulation required for an intracellular bacterium to co-opt, but not disrupt, vital host processes.
Manieu, C., Olivares, G. H., Vega-Macaya, F., Valdivia, M. and Olguin, P. (2018). Jitterbug/Filamin and Myosin-II form a complex in tendon cells required to maintain epithelial shape and polarity during musculoskeletal system development. Mech Dev. PubMed ID: 30213743
Summary:
During musculoskeletal system development, mechanical tension is generated between muscles and tendon-cells. This tension is required for muscle differentiation and is counterbalanced by tendon-cells avoiding tissue deformation. Both, Jbug/Filamin, an actin-meshwork organizing protein, and non-muscle Myosin-II (Myo-II) are required to maintain the shape and cell orientation of the Drosophila notum epithelium during flight muscle attachment to tendon cells. This study shows that halving the genetic dose of Rho kinase (Drok), the main activator of Myosin-II, enhances the epithelial deformation and bristle orientation defects associated with jbug/Filamin knockdown. Drok and activated Myo-II localize at the apical cell junctions, tendon processes and are associated to the myotendinous junction. Further, it was found that Jbug/Filamin co-distribute at tendon cells with activated Myo-II. Finally, it was found that Jbug/Filamin and Myo-II are in the same molecular complex and that the actin-binding domain of Jbug/Filamin is necessary for this interaction. These data together suggest that Jbug/Filamin and Myo-II proteins may act together in tendon cells to balance the tension generated during development of muscles-tendon interaction, maintaining the shape and polarity of the Drosophila notum epithelium.
Rome, P. and Ohkura, H. (2018). A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes. J Cell Biol. PubMed ID: 30087124
Summary:
The meiotic spindle in oocytes is assembled in the absence of centrosomes, the major microtubule nucleation sites in mitotic and male meiotic cells. A crucial, yet unresolved question in meiosis is how spindle microtubules are generated without centrosomes and only around chromosomes in the exceptionally large volume of oocytes. This study reports a novel oocyte-specific microtubule nucleation pathway that is essential for assembling most spindle microtubules complementarily with the Augmin pathway. This pathway is mediated by the kinesin-6 Subito/MKlp2, which recruits the gamma-tubulin complex to the spindle equator to nucleate microtubules in Drosophila oocytes. Away from chromosomes, Subito interaction with the gamma-tubulin complex is suppressed by its N-terminal region to prevent ectopic microtubule assembly in oocytes. It was further demonstrated in vitro that the Subito complex from ovaries can nucleate microtubules from pure tubulin dimers. Collectively, microtubule nucleation regulated by Subito drives spatially restricted spindle assembly in oocytes.
Ikawa, K. and Sugimura, K. (2018). AIP1 and cofilin ensure a resistance to tissue tension and promote directional cell rearrangement. Nat Commun 9(1): 3295. PubMed ID: 30202062
Summary:
In order to understand how tissue mechanics shapes animal body, it is critical to clarify how cells respond to and resist tissue stress when undergoing morphogenetic processes, such as cell rearrangement. This study addresses the question in the Drosophila wing epithelium, where anisotropic tissue tension orients cell rearrangements. Anisotropic tissue tension localizes actin interacting protein 1 (AIP1), a cofactor of cofilin, on the remodeling junction via cooperative binding of cofilin to F-actin. AIP1 and cofilin promote actin turnover and locally regulate the Canoe-mediated linkage between actomyosin and the junction. This mechanism is essential for cells to resist the mechanical load imposed on the remodeling junction perpendicular to the direction of tissue stretching. Thus, the present study delineates how AIP1 and cofilin achieve an optimal balance between resistance to tissue tension and morphogenesis.
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