Monday, August 20th - Drosophila Disease Models

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Jablonska, J., Dubinska-Magiera, M., Jagla, T., Jagla, K. and Daczewska, M. (2018). Drosophila Hsp67Bc hot-spot variants alter muscle structure and function. Cell Mol Life Sci. PubMed ID: 30032358
The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. This study reports the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, a different impact was observed on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant.
Martin-Pena, A., Rincon-Limas, D. E. and Fernandez-Funez, P. (2018). Engineered Hsp70 chaperones prevent Abeta42-induced memory impairments in a Drosophila model of Alzheimer's disease. Sci Rep 8(1): 9915. PubMed ID: 29967544
Proteinopathies constitute a group of diseases in which certain proteins are abnormally folded leading to aggregation and eventual cell failure. Most neurodegenerative diseases belong to protein misfolding disorders and, among them, Alzheimer's disease (AD) is the most prevalent. AD is characterized by accumulation of the amyloid-beta42 (Abeta42) peptide in the extracellular space. Hence, this study genetically engineered a molecular chaperone that was selectively delivered to this cellular location. It has been reported that the heat shock protein 70 (Hsp70) binds Abeta42 (see Drosophila Appl) preventing self-aggregation. This study employed two isoforms of the Hsp70, cytosolic and extracellular, to evaluate their potential protective effect against the memory decline triggered by extracellular deposition of Abeta42. Both Hsp70 isoforms significantly improved memory performance of flies expressing Abeta42, irrespective of their age or the level of Abeta42 load. Using olfactory classical conditioning, a Drosophila model of AD was established based on Abeta42 neurotoxicity and memory decline was monitored through aging. The onset of the memory impairment observed was proportional to the cumulative level of Abeta42 in the Drosophila brain. These data support the use of this Drosophila model of AD to further investigate molecules with a protective activity against Abeta42-induced memory loss, contributing to the development of palliative therapies for AD.
Chen, Z. S., Li, L., Peng, S., Chen, F. M., Zhang, Q., An, Y., Lin, X., Li, W., Koon, A. C., Chan, T. F., Lau, K. F., Ngo, J. C. K., Wong, W. T., Kwan, K. M. and Chan, H. Y. E. (2018). Planar cell polarity gene Fuz triggers apoptosis in neurodegenerative disease models. EMBO Rep. PubMed ID: 30026307
Planar cell polarity (PCP) describes a cell-cell communication process through which individual cells coordinate and align within the plane of a tissue. This study shows that overexpression of Fuz, a PCP gene, triggers neuronal apoptosis via the Dishevelled/Rac1 GTPase/MEKK1/JNK/caspase signalling axis. Consistent with this finding, endogenous Fuz expression is upregulated in models of polyglutamine (polyQ) diseases and in fibroblasts from spinocerebellar ataxia type 3 (SCA3) patients. The disruption of this upregulation mitigates polyQ-induced neurodegeneration in Drosophila. The transcriptional regulator Yin Yang 1 (YY1) associates with the Fuz promoter. Overexpression of YY1 promotes the hypermethylation of Fuz promoter, causing transcriptional repression of Fuz. Remarkably, YY1 protein is recruited to ATXN3-Q84 aggregates, which reduces the level of functional, soluble YY1, resulting in Fuz transcriptional derepression and induction of neuronal apoptosis. Furthermore, Fuz transcript level is elevated in amyloid beta-peptide, Tau and alpha-synuclein models, implicating its potential involvement in other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Taken together, this study unveils a generic Fuz-mediated apoptotic cell death pathway in neurodegenerative disorders.
Iyer, J., Singh, M. D., Jensen, M., Patel, P., Pizzo, L., Huber, E., Koerselman, H., Weiner, A. T., Lepanto, P., Vadodaria, K., Kubina, A., Wang, Q., Talbert, A., Yennawar, S., Badano, J., Manak, J. R., Rolls, M. M., Krishnan, A. and Girirajan, S. (2018). Pervasive genetic interactions modulate neurodevelopmental defects of the autism-associated 16p11.2 deletion in Drosophila melanogaster. Nat Commun 9(1): 2548. PubMed ID: 29959322
As opposed to syndromic copy number variants (CNVs) caused by single genes, extensive phenotypic heterogeneity in variably-expressive CNVs complicates disease gene discovery and functional evaluation. This study proposes a complex interaction model for pathogenicity of the autism-associated 16p11.2 deletion, where CNV genes interact with each other in conserved pathways to modulate expression of the phenotype. Using multiple quantitative methods in Drosophila RNAi lines, a range of neurodevelopmental phenotypes was identified for knockdown of individual 16p11.2 homologs in different tissues. 565 pairwise knockdowns were tested in the developing eye, and 24 interactions were identified between pairs of 16p11.2 homologs and 46 interactions between 16p11.2 homologs and neurodevelopmental genes that suppress or enhance cell proliferation phenotypes compared to one-hit knockdowns. These interactions within cell proliferation pathways are also enriched in a human brain-specific network, providing translational relevance in humans. This study indicates a role for pervasive genetic interactions within CNVs towards cellular and developmental phenotypes.

Friday, August 17th - Behavior

Mandel, S. J., Shoaf, M. L., Braco, J. T., Silver, W. L. and Johnson, E. C. (2018). Behavioral aversion to AITC requires both Painless and dTRPA1 in Drosophila. Front Neural Circuits 12: 45. PubMed ID: 30018539
There has been disagreement over the functional roles of the painless gene product in the detection and subsequent behavioral aversion to the active ingredient in wasabi, allyl isothiocyanate (AITC). Originally, painless was reported to eliminate the behavioral aversion to AITC, although subsequent reports suggested that another trpA homolog, dTRPA1, was responsible for AITC aversion. This study re-evaluated the role of the painless gene in the detection of AITC. Using the proboscis extension reflex (PER) assay, it was observed that AITC did not reduce PER frequencies in painless or dTRPA1 mutants but did in wild-type genotypes. Quantification of food intake showed a significant decline in food consumption in the presence of AITC in wild-type, but not painless mutants. An oviposition choice assay was adapted and it was found wild-type oviposit on substrates lacking AITC, in contrast to painless and dTRPA1 mutants. Lastly, tracking individual flies relative to a point source of AITC, showed a consistent clustering of wild-type animals away from the point source, which was absent in painless mutants. Expression patterns were evaluated of both dTRPA1 and painless, which showed expression in distinct central and peripheral populations. The transmitter phenotypes of subsets of painless and dTRPA1 neurons were evaluated, and similar neuropeptides were found as those expressed by mammalian trpA expressing neurons. Using a calcium reporter, it was observed AITC-evoked responses in both painless and dTRPA1 expressing neurons. Collectively, these results reaffirm the necessity of painless in nociceptive behaviors and suggest experiments to further resolve the molecular basis of aversion.
Park, J., Kondo, S., Tanimoto, H., Kohsaka, H. and Nose, A. (2018). Data-driven analysis of motor activity implicates 5-HT2A neurons in backward locomotion of larval Drosophila. Sci Rep 8(1): 10307. PubMed ID: 29985473
Rhythmic animal behaviors are regulated in part by neural circuits called the central pattern generators (CPGs). Classifying neural population activities correlated with body movements and identifying the associated component neurons are critical steps in understanding CPGs. Previous methods that classify neural dynamics obtained by dimension reduction algorithms often require manual optimization which could be laborious and preparation-specific. This study presents a simpler and more flexible method that is based on the pre-trained convolutional neural network model VGG-16 and unsupervised learning, and successfully classifies the fictive motor patterns in Drosophila larvae under various imaging conditions. Voxel-wise correlation mapping was also used to identify neurons associated with motor patterns. By applying these methods to neurons targeted by 5-HT2A-GAL4, which was generated by the CRISPR/Cas9-system, two classes of interneurons were identified, termed Seta and Leta, which are specifically active during backward but not forward fictive locomotion. Optogenetic activation of Seta and Leta neurons increased backward locomotion. Conversely, thermogenetic inhibition of 5-HT2A-GAL4 neurons or application of a 5-HT2 antagonist decreased backward locomotion induced by noxious light stimuli. This study establishes an accelerated pipeline for activity profiling and cell identification in larval Drosophila and implicates the serotonergic system in the modulation of backward locomotion.
de Andres-Bragado, L., Mazza, C., Senn, W. and Sprecher, S. G. (2018). Statistical modelling of navigational decisions based on intensity versus directionality in Drosophila larval phototaxis. Sci Rep 8(1): 11272. PubMed ID: 30050066
The fruit fly larva stands as a powerful model to study decision-making processes that underlie directed navigation. This study has quantitatively measured phototaxis in response to well-defined sensory inputs. Subsequently, a statistical stochastic model based on biased Markov chains was formulated to characterize the behavioural basis of negative phototaxis. These experiments show that larvae make navigational decisions depending on two independent physical variables: light intensity and its spatial gradient. Furthermore, the statistical model quantifies how larvae balance two potentially-contradictory factors: minimizing exposure to light intensity and at the same time maximizing their distance to the light source. The response to the light field is manifestly non-linear, and saturates above an intensity threshold. The model has been validated against experimental biological data yielding insight into the strategy that larvae use to achieve their goal with respect to the navigational cue of light, an important piece of information for future work to study the role of the different neuronal components in larval phototaxis.
Kacsoh, B. Z., Bozler, J. and Bosco, G. (2018). Drosophila species learn dialects through communal living. PLoS Genet 14(7): e1007430. PubMed ID: 30024883
Many species are able to share information about their environment by communicating through auditory, visual, and olfactory cues. In Drosophila melanogaster, exposure to parasitoid wasps leads to a decline in egg laying, and exposed females communicate this threat to naive flies, which also depress egg laying. This study found that species across the genus Drosophila respond to wasps by egg laying reduction, activate cleaved caspase in oocytes, and communicate the presence of wasps to naive individuals. Communication within a species and between closely related species is efficient, while more distantly related species exhibit partial communication. Remarkably, partial communication between some species is enhanced after a cohabitation period that requires exchange of visual and olfactory signals. This interspecies "dialect learning" requires neuronal cAMP signaling in the mushroom body, suggesting neuronal plasticity facilitates dialect learning and memory. These observations establish Drosophila as genetic models for interspecies social communication and evolution of dialects.
Jois, S., Chan, Y. B., Fernandez, M. P. and Leung, A. K. (2018). Characterization of the sexually dimorphic fruitless neurons that regulate copulation duration. Front Physiol 9: 780. PubMed ID: 29988589
Male courtship in Drosophila melanogaster is a sexually dimorphic innate behavior that is hardwired in the nervous system. Understanding the neural mechanism of courtship behavior requires the anatomical and functional characterization of all the neurons involved. Courtship involves a series of distinctive behavioral patterns, culminating in the final copulation step, where sperms from the male are transferred to the female. The duration of this process is tightly controlled by multiple genes. The fruitless (fru) gene is one of the factors that regulate the duration of copulation. Using several intersectional genetic combinations to restrict the labeling of GAL4 lines, this study found that a subset of a serotonergic cluster of fru neurons co-express the dopamine-synthesizing enzyme, tyrosine hydroxylase, and provide behavioral and immunological evidence that these neurons are involved in the regulation of copulation duration.
Hill, V. M., O'Connor, R. M., Sissoko, G. B., Irobunda, I. S., Leong, S., Canman, J. C., Stavropoulos, N. and Shirasu-Hiza, M. (2018). A bidirectional relationship between sleep and oxidative stress in Drosophila. PLoS Biol 16(7): e2005206. PubMed ID: 30001323
Although sleep appears to be broadly conserved in animals, the physiological functions of sleep remain unclear. This study sought to identify a physiological defect common to a diverse group of short-sleeping Drosophila mutants, which might provide insight into the function and regulation of sleep. These short-sleeping mutants share a common phenotype of sensitivity to acute oxidative stress, exhibiting shorter survival times than controls. It was further shown that increasing sleep in wild-type flies using genetic or pharmacological approaches increases survival after oxidative challenge. Moreover, reducing oxidative stress in the neurons of wild-type flies by overexpression of antioxidant genes reduces the amount of sleep. Together, these results support the hypothesis that a key function of sleep is to defend against oxidative stress and also point to a reciprocal role for reactive oxygen species (ROS) in neurons in the regulation of sleep.

Thursday, August 16th - RNA

Kane, N. S., Vora, M., Padgett, R. W. and Li, Y. (2018). bantam microRNA is a negative regulator of the Drosophila decapentaplegic pathway. Fly (Austin). PubMed ID: 30015555
Decapentaplegic (Dpp), the Drosophila homolog of the vertebrate bone morphogenetic protein (BMP2/4), is crucial for patterning and growth in many developmental contexts. The Dpp pathway is regulated at many different levels to exquisitely control its activity. This study shows that bantam (ban), a microRNA, modulates Dpp signaling activity. Overexpression of ban decreases phosphorylated Mothers against decapentaplegic (Mad) levels and negatively affects Dpp pathway transcriptional target genes, while null mutant clones of ban upregulate the pathway. Evidence is provided that dpp upregulates ban in the wing imaginal disc, and attenuation of Dpp signaling results in a reduction of ban expression, showing that they function in a feedback loop. Furthermore, this feedback loop is shown to be important for maintaining anterior-posterior compartment boundary stability in the wing disc through regulation of optomotor blind (omb), a known target of the pathway. These results support a model that ban functions with dpp in a negative feedback loop.
Yu, B., Lin, Y. A., Parhad, S. S., Jin, Z., Ma, J., Theurkauf, W. E., Zhang, Z. Z. and Huang, Y. (2018). Structural insights into Rhino-Deadlock complex for germline piRNA cluster specification. EMBO Rep. PubMed ID: 29858487
PIWI-interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species-specific manner and so defines the piRNA-producing loci in the Drosophila genome. This study determined the crystal structures of Rhino-Deadlock complex in Drosophila melanogaster and simulans. In both species, one Rhino binds the N-terminal helix-hairpin-helix motif of one Deadlock protein through a novel interface formed by the beta-sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. These structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross-species incompatibility between the sibling species. By determining the molecular architecture of this piRNA-producing machinery, a novel HP1-partner interacting mode was discovered that is crucial to piRNA biogenesis and transposon silencing. This study thus explains the cross-species incompatibility of two sibling species at the molecular level.
Wang, Q., Abruzzi, K. C., Rosbash, M. and Rio, D. C. (2018). Striking circadian neuron diversity and cycling of Drosophila alternative splicing. Elife 7. PubMed ID: 29863472
Although alternative pre-mRNA splicing (AS) significantly diversifies the neuronal proteome, the extent of AS is still unknown due in part to the large number of diverse cell types in the brain. To address this complexity issue, this study used an annotation-free computational method to analyze and compare the AS profiles between small specific groups of Drosophila circadian neurons. The method, the Junction Usage Model (JUM), allows the comprehensive profiling of both known and novel AS events from specific RNA-seq libraries. The results show that many diverse and novel pre-mRNA isoforms are preferentially expressed in one class of clock neuron and also absent from the more standard Drosophila head RNA preparation. These AS events are enriched in potassium channels important for neuronal firing, and there are also cycling isoforms with no detectable underlying transcriptional oscillations. The results suggest massive AS regulation in the brain that is also likely important for circadian regulation.
Weber, G., DeKoster, G. T., Holton, N., Hall, K. B. and Wahl, M. C. (2018). Molecular principles underlying dual RNA specificity in the Drosophila SNF protein. Nat Commun 9(1): 2220. PubMed ID: 29880797
The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A' protein. This study investigated its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A' or U2 stem-loop IV and U2A', SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. Different loop-closing base pairs and a nucleotide exchange at the tips of the loops were found to contribute to differential SNF affinity for the RNAs. U2A' immobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. These findings show how U2A' can modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts.

Wednesday, August 15th - Chromatin

Ogiyama, Y., Schuettengruber, B., Papadopoulos, G. L., Chang, J. M. and Cavalli, G. (2018). Polycomb-dependent chromatin looping contributes to gene silencing during Drosophila development. Mol Cell 71(1): 73-88.e75. PubMed ID: 30008320
Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, this study analyzed higher order 3D chromatin organization during Drosophila embryogenesis and identified active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development.
Gambetta, M. C. and Furlong, E. E. M. (2018). The insulator protein CTCF is required for correct hox gene expression, but not for embryonic development in Drosophila. Genetics. PubMed ID: 30021792
Among Drosophila insulator binding proteins (IBPs) only CCCTC-binding factor (CTCF) has an obvious ortholog in Mammals. CTCF is essential for mammalian cell viability and is an important regulator of genome architecture. In flies, CTCF is both maternally deposited and zygotically expressed. Flies lacking zygotic CTCF die as young adults with homeotic defects, suggesting that specific Hox genes are misexpressed in inappropriate body segments. The lack of any major embryonic defects was assumed to be due to the maternal supply of CTCF protein, as maternally contributed factors are often sufficient to progress through much of embryogenesis. This study determined the requirement of CTCF for developmental progression in Drosophila. Animals were generated that completely lack both maternal and zygotic CTCF and it was found that, contrary to expectation, these mutants progress through embryogenesis and larval life. They develop to pharate adults, which fail to eclose from their pupal case. These mutants show exacerbated homeotic defects compared to zygotic mutants, misexpressing the Hox gene Abdominal-B outside of its normal expression domain early in development. These results indicate that loss of Drosophila CTCF is not accompanied by widespread effects on gene expression, which may be due to redundant functions with other IBPs. Rather, CTCF is required for correct Hox gene expression patterns and for the viability of adult Drosophila.
Giaimo, B. D., Ferrante, F., Vallejo, D. M., Hein, K., Gutierrez-Perez, I., Nist, A., Stiewe, T., Mittler, G., Herold, S., Zimmermann, T., Bartkuhn, M., Schwarz, P., Oswald, F., Dominguez, M. and Borggrefe, T. (2018). Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response. Nucleic Acids Res. PubMed ID: 29986055
A fundamental as yet incompletely understood feature of Notch signal transduction is a transcriptional shift from repression to activation that depends on chromatin regulation mediated by transcription factor RBP-J and associated cofactors. Incorporation of histone variants alter the functional properties of chromatin and are implicated in the regulation of gene expression. This study shows that depletion of histone variant H2A.Z leads to upregulation of canonical Notch target genes and that the H2A.Z-chaperone TRRAP/p400/Tip60 complex physically associates with RBP-J at Notch-dependent enhancers. When targeted to RBP-J-bound enhancers, the acetyltransferase Tip60 acetylates H2A.Z and upregulates Notch target gene expression. Importantly, the Drosophila homologs of Tip60, p400 and H2A.Z modulate Notch signaling response and growth in vivo. Together, these data reveal that loading and acetylation of H2A.Z are required to assure tight control of canonical Notch activation.
Hu, J., Gu, L., Ye, Y., Zheng, M., Xu, Z., Lin, J., Du, Y., Tian, M., Luo, L., Wang, B., Zhang, X., Weng, Z. and Jiang, C. (2018). Dynamic placement of the linker histone H1 associated with nucleosome arrangement and gene transcription in early Drosophila embryonic development. Cell Death Dis 9(7): 765. PubMed ID: 29988149
The linker histone H1 is critical to maintenance of higher-order chromatin structures and to gene expression regulation. However, H1 dynamics and its functions in embryonic development remain unresolved. This study has profiled gene expression, nucleosome positions, and H1 locations in early Drosophila embryos. The results show that H1 binding is positively correlated with the stability of beads-on-a-string nucleosome organization likely through stabilizing nucleosome positioning and maintaining nucleosome spacing. Strikingly, nucleosomes with H1 placement deviating to the left or the right relative to the dyad shift to the left or the right, respectively, during early Drosophila embryonic development. H1 occupancy on genic nucleosomes is inversely correlated with nucleosome distance to the transcription start sites. This inverse correlation reduces as gene transcription levels decrease. Additionally, H1 occupancy is lower at the 5' border of genic nucleosomes than that at the 3' border. This asymmetrical pattern of H1 occupancy on genic nucleosomes diminishes as gene transcription levels decrease. These findings shed new lights into how H1 placement dynamics correlates with nucleosome positioning and gene transcription during early Drosophila embryonic development.

Tuesday, August 14th - Adult Neural Function

Hall, H., Ma, J., Shekhar, S., Leon-Salas, W. D. and Weake, V. M. (2018). Blue light induces a neuroprotective gene expression program in Drosophila photoreceptors. BMC Neurosci 19(1): 43. PubMed ID: 30029619
Light exposure induces oxidative stress, which contributes to ocular diseases of aging. In contrast to mature adults, which undergo retinal degeneration when exposed to prolonged blue light, newly-eclosed flies are resistant to blue light-induced retinal degeneration. This study sought to characterize the gene expression programs induced by blue light in flies of different ages by profiling the nuclear transcriptome of Drosophila photoreceptors. Flies were exposed to 3 h blue light, which increases levels of reactive oxygen species but does not cause retinal degeneration. Substantial gene expression changes were identified in response to blue light only in six-day-old flies. In six-day-old flies, blue light induced a neuroprotective gene expression program that included upregulation of stress response pathways and downregulation of genes involved in light response, calcium influx and ion transport. An intact phototransduction pathway and calcium influx were required for upregulation, but not downregulation, of genes in response to blue light, suggesting that distinct pathways mediate the blue light-associated transcriptional response. These data demonstrate that under phototoxic conditions, Drosophila photoreceptors upregulate stress response pathways and simultaneously, downregulate expression of phototransduction components, ion transporters, and calcium channels. Together, this gene expression program both counteracts the calcium influx resulting from prolonged light exposure, and ameliorates the oxidative stress resulting from this calcium influx.Developmental transitions during the first week of adult Drosophila life lead to an altered gene expression program in photoreceptors that includes reduced expression of genes that maintain redox and calcium homeostasis, reducing the capacity of six-day-old flies to cope with longer periods (8 h) of light exposure. Together, these data provide insight into the neuroprotective gene regulatory mechanisms that enable photoreceptors to withstand light-induced oxidative stress.
Aboudhiaf, S., Alves, G., Parrot, S., Amri, M., Simonnet, M. M., Grosjean, Y., Maniere, G. and Seugnet, L. (2018). LAT1-like transporters regulate dopaminergic transmission and sleep in Drosophila. Sleep. PubMed ID: 30016498
Amino-acid transporters are involved in functions reportedly linked to the sleep/wake cycle: neurotransmitter synthesis and recycling, the regulation of synaptic strength, protein synthesis and energy metabolism. In addition, the existence of bidirectional relationships between extracellular content, transport systems and sleep/wake states is receiving emerging support. Nevertheless, the connection between amino-acid transport and sleep/wake regulation remains elusive. To address this question, this study used Drosophila melanogaster and investigated the role of LAT1 (Large neutral Amino-acid Transporter 1) transporters. This study shows that the two Drosophila LAT1-like transporters: JhI-21 and minidiscs (Mnd) are required in dopaminergic neurons for sleep/wake regulation. Down-regulating either gene in dopaminergic neurons resulted in higher daily sleep and longer sleep bout duration during the night, suggesting a defect in dopaminergic transmission. Since LAT1 transporters can mediate in mammals the uptake of L-DOPA, a precursor of dopamine, amino-acid transport efficiency was assessed by L-DOPA feeding. Downregulation of JhI-21, but not Mnd, reduced the sensitivity to L-DOPA as measured by sleep loss. JhI-21 downregulation also attenuated the sleep loss induced by continuous activation of dopaminergic neurons. Since LAT1 transporters are known to regulate TOR (Target Of Rapamycin) signaling, the role of this amino-acid sensing pathway in dopaminergic neurons was investigated. Consistently, it is reported that TOR activity in dopaminergic neurons modulates sleep/wake states. Altogether, this study provides evidence that LAT1 mediated amino-acid transport in dopaminergic neurons, is playing a significant role in sleep/wake regulation, and is providing several entry points to elucidate the role of nutrients such as amino-acids in sleep/wake regulation.
Gouzi, J. Y., Bouraimi, M., Roussou, I. G., Moressis, A. and Skoulakis, E. M. C. (2018). The Drosophila Receptor Tyrosine Kinase Alk constrains long-term memory formation. J Neurosci. PubMed ID: 30030398
In addition to mechanisms promoting protein-synthesis dependent long-term memory (PSD-LTM), the process appears to also be specifically constrained. This study presents evidence that the highly conserved Receptor Tyrosine Kinase dAlk is a novel PSD-LTM attenuator in Drosophila. Reduction of dAlk levels in adult α/β mushroom body (MB) neurons during conditioning elevates LTM, whereas its overexpression impairs it. Unlike other memory suppressor proteins and miRNAs, dAlk within the MBs constrains PSD-LTM specifically, but constrains learning outside the MBs as previously shown. Dendritic dAlk levels rise rapidly in MB neurons upon conditioning, a process apparently controlled by the 3'UTR of its mRNA and interruption of the 3'UTR leads to enhanced LTM. Because its activating ligand Jeb is dispensable for LTM attenuation, it is proposed that post-conditioning elevation of dAlk within α/β dendrites results in its auto-activation and constrains formation of the energy costly PSD-LTM, acting as a novel memory filter.
Cusumano, P., Biscontin, A., Sandrelli, F., Mazzotta, G. M., Tregnago, C., De Pitta, C. and Costa, R. (2018). Modulation of miR-210 alters phasing of circadian locomotor activity and impairs projections of PDF clock neurons in Drosophila melanogaster. PLoS Genet 14(7): e1007500. PubMed ID: 30011269
This study investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.

Monday, August 13th - Signaling

Eusebio, N., Tavares, L. and Pereira, P. S. (2018). CtBP represses Dpp-dependent Mad activation during Drosophila eye development. Dev Biol. PubMed ID: 30031756
Complex networks of signaling pathways maintain the correct balance between positive and negative growth signals, ensuring that tissues achieve proper sizes and differentiation pattern during development. In Drosophila, Dpp, a member of the TGFbeta family, plays two main roles during larval eye development. In the early eye primordium, Dpp promotes growth and cell survival, but later on, it switches its function to induce a developmentally-regulated cell cycle arrest in the G1 phase and neuronal photoreceptor differentiation. To advance in the identification and characterization of regulators and targets of Dpp signaling required for retinal development, an in vivo eye-targeted double-RNAi screen was carried out to identify punt (Type II TGFbeta receptor) interactors. Using a set of 251 genes associated with eye development, CtBP, Dad, Ago and Brk were identified as punt genetic interactors. This study shows that downregulation of Ago, or conditions causing increased tissue growth including overexpression of Myc or CyclinD-Cdk4 are sufficient to partially rescue punt-dependent growth and photoreceptor differentiation. Interestingly, a novel role is shown for the transcriptional co-repressor CtBP in inhibiting Dpp-dependent Mad activation by phosphorylation, downstream or in parallel to Dad, the inhibitory Smad. Furthermore, CtBP downregulation activates JNK signaling pathway, implying a complex regulation of signaling pathways by CtBP during eye development.
Ding, L., Yang, X., Tian, H., Liang, J., Zhang, F., Wang, G., Wang, Y., Ding, M., Shui, G. and Huang, X. (2018). Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism. Embo J. PubMed ID: 30049710
Seipin, the gene that causes Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), is important for adipocyte differentiation and lipid homeostasis. Previous studies in Drosophila revealed that Seipin promotes ER calcium homeostasis through the Ca(2+)-ATPase SERCA, but little is known about the events downstream of perturbed ER calcium homeostasis that lead to decreased lipid storage in Drosophila dSeipin mutants. This study shows that glycolytic metabolites accumulate and the downstream mitochondrial TCA cycle is impaired in dSeipin mutants. The impaired TCA cycle further leads to a decreased level of citrate, a critical component of lipogenesis. Mechanistically, Seipin/SERCA-mediated ER calcium homeostasis is important for maintaining mitochondrial calcium homeostasis. Reduced mitochondrial calcium in dSeipin mutants affects the TCA cycle and mitochondrial function. The lipid storage defects in dSeipin mutant fat cells can be rescued by replenishing mitochondrial calcium or by restoring the level of citrate through genetic manipulations or supplementation with exogenous metabolites. Together, these results reveal that Seipin promotes adipose tissue lipid storage via calcium-dependent mitochondrial metabolism.
Green, H. J., Griffiths, A. G. M., Ylanne, J. and Brown, N. H. (2018). Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila. Elife 7. PubMed ID: 30028294
This study used the myotendinous junction of Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere.
Gupta, A., Fabian, L. and Brill, J. A. (2018). Phosphatidylinositol 4,5-bisphosphate regulates cilium transition zone maturation in Drosophila melanogaster. J Cell Sci. PubMed ID: 30054387
Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. This study reports a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in TZ maturation in the Drosophila melanogaster male germline. Reduction of cellular PIP2 levels by ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. It is also reported that the onion rings (onr) allele of Drosophila exo84 decouples TZ hyperelongation from loss of cilium-plasma membrane tethering. These results reveal a requirement for PIP2 in supporting ciliogenesis by promoting proper TZ maturation.

Friday, August 10th - Gonads

Sharma, A., Halder, S., Felix, M., Nisaa, K., Deshpande, G. and Prasad, M. (2018). Insulin signaling modulates border cell movement in Drosophila oogenesis. Development. PubMed ID: 29950391
As collective cell migration is intimately involved in different aspects of metazoan development, molecular mechanisms underlying this process are being explored in a variety of developmental contexts. Border cell (BC) migration during Drosophila oogenesis has emerged as an excellent genetic model for studying collective cell migration. BCs are of epithelial origin but acquire partial mesenchymal characteristics before migrating as a group towards the oocyte. This study reports that insulin signalling modulates collective BC movement during Drosophila oogenesis. Supporting the involvement of Insulin pathway, it was demonstrated that compromising Insulin-like Receptor (dInR) levels in BCs, inhibits their migration. Furthermore, it was shown that canonical Insulin signalling pathway components participate in this process. Interestingly, visualization of dInR-depleted BC clusters, using time-lapse imaging, revealed a delay in detachment of BC clusters from the surrounding anterior follicle cells and altered protrusion dynamics. Lastly, based on genetic interactions between dInR, the polarity determinant, dPAR-1 and a regulatory subunit of Drosophila Myosin, (Spaghetti squash) it is proposed that Insulin signalling likely influences dPAR-1 activity to engineer border cell detachment and subsequent movement via Drosophila Myosin.
Li, W., Young, J. F. and Sun, J. (2018). NADPH oxidase-generated reactive oxygen species in mature follicles are essential for Drosophila ovulation. Proc Natl Acad Sci U S A 115(30): 7765-7770. PubMed ID: 29987037
Ovarian reactive oxygen species (ROS) are believed to regulate ovulation in mammals, but the details of ROS production in follicles and the role of ROS in ovulation in other species remain underexplored. In Drosophila ovulation, matrix metalloproteinase 2 (MMP2) is required for follicle rupture by degradation of posterior follicle cells surrounding a mature oocyte. MMP2 activation and follicle rupture are regulated by the neuronal hormone octopamine (OA) and the octopamine receptor in mushroom body (OAMB). This study investigated the role of the superoxide-generating enzyme NADPH oxidase (NOX) in Drosophila ovulation. Nox is highly enriched in mature follicle cells, and Nox knockdown in these cells leads to a reduction in superoxide and to defective ovulation. Similar to MMP2 activation, NOX enzymatic activity is also controlled by the OA/OAMB-Ca(2+) signaling pathway. In addition, this study reports that extracellular superoxide dismutase 3 (SOD3) is required to convert superoxide to hydrogen peroxide, which acts as the key signaling molecule for follicle rupture, independent of MMP2 activation. Given that Nox homologs are expressed in mammalian follicles, the NOX-dependent hydrogen peroxide signaling pathway that is described in this study could play a conserved role in regulating ovulation in other species.
Weaver, L. N. and Drummond-Barbosa, D. (2018). Maintenance of proper germline stem cell number requires adipocyte collagen in adult Drosophila females. Genetics. PubMed ID: 29884747
Stem cells reside in specialized niches and are regulated by a variety of physiological inputs. Adipocytes influence whole-body physiology and stem cell lineages; however, the molecular mechanisms linking adipocytes to stem cells are poorly understood. This study reports that collagen IV produced in adipocytes is transported to the ovary to maintain proper germline stem cell (GSC) number in adult Drosophila females. Adipocyte-derived collagen IV acts through beta-integrin signaling to maintain normal levels of E-cadherin at the niche, thereby ensuring proper adhesion to GSCs. These findings demonstrate that extracellular matrix components produced in adipocytes can be transported to and incorporated into an established adult tissue to influence stem cell number.
Collins, C. M., Malacrida, B., Burke, C., Kiely, P. A. and Dunleavy, E. M. (2018). ATP synthase F1 subunits recruited to centromeres by CENP-A are required for male meiosis. Nat Commun 9(1): 2702. PubMed ID: 30006572
The histone H3 variant CENP-A epigenetically defines the centromere and is critical for chromosome segregation. This study reports an interaction between CENP-A and subunits of the mitochondrial ATP synthase complex in the germline of male Drosophila. Furthermore, knockdown of CENP-A, as well as subunits ATPsyn-alpha, -betalike (a testis-specific paralogue of ATPsyn-beta) and -gamma disrupts sister centromere cohesion in meiotic prophase I. This disruption is likely independent of reduced ATP levels. ATPsyn-alpha and -betalike were found to localise to meiotic centromeres; and this localisation is dependent on the presence of CENP-A. ATPsyn-alpha directly interacts with the N-terminus of CENP-A in vitro, and truncation of its N terminus perturbs sister centromere cohesion in prophase I. It is proposed that the CENP-A N-terminus recruits ATPsyn-alpha and -betalike to centromeres to promote sister centromere cohesion in a nuclear function that is independent of oxidative phosphorylation.
Gupta, S., Varshney, B., Chatterjee, S. and Ray, K. (2018). Somatic ERK activation during transit amplification is essential for maintaining the synchrony of germline divisions in Drosophila testis. Open Biol 8(7). PubMed ID: 30045884
Transit amplification (TA) of progenitor cells maintains tissue homeostasis by balancing proliferation and differentiation. In Drosophila testis, the germline proliferation is tightly regulated by factors present in both the germline and the neighbouring somatic cyst cells (SCCs). Although the exact mechanism is unclear, the epidermal growth factor receptor (EGFR) activation in SCCs has been reported to control spermatogonial divisions within a cyst, through downstream activations of Rac1-dependent pathways. This study reports that somatic activation of the mitogen-activated protein kinase (Rolled/ERK) downstream of EGFR is required to synchronize the mitotic divisions and regulate the transition to meiosis. The process operates independently of the Bag-of-marble activity in the germline. Also, the integrity of the somatic cyst enclosure is inessential for this purpose. Together, these results suggest that synchronization of germ-cell divisions through somatic activation of distinct ERK-downstream targets independently regulates TA and subsequent differentiation of neighbouring germline cells.
Ghiglione, C., Jouandin, P., Cerezo, D. and Noselli, S. (2018). The Drosophila insulin pathway controls Profilin expression and dynamic actin-rich protrusions during collective cell migration. Development. PubMed ID: 29980565
Understanding how different cell types acquire their motile behaviour is central to many normal and pathological processes. Drosophila border cells represent a powerful model to address this question and to specifically decipher the mechanisms controlling collective cell migration. This study has identified the Drosophila Insulin/Insulin-like growth factor Signalling (IIS) pathway as a key regulator controlling actin dynamics in border cells, independently of its function in growth control. Loss of IIS activity blocks the formation of actin-rich long cellular extensions that are important for the delamination and the migration of the invasive cluster. IIS specifically activates the expression of the actin regulator chickadee, the Drosophila homolog of Profilin, essential for promoting the formation of actin extensions and migration through the egg chamber. In this process, the transcription factor dFoxO acts as a repressor of chickadee expression. Altogether, these results show that local activation of IIS controls collective cell migration through regulation of actin homeostasis and protrusion dynamics.

Thursday, August 9th - Cytoskeleton

Strunov, A., Boldyreva, L. V., Andreyeva, E. N., Pavlova, G. A., Popova, J. V., Razuvaeva, A. V., Anders, A. F., Renda, F., Pindyurin, A. V., Gatti, M. and Kiseleva, E. (2018). Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors. BMC Biol 16(1): 68. PubMed ID: 29907103
This study provides a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM). A random sample of 144 cells undergoing mitosis was analyzed by TEM, focusing on intracellular membrane and microtubule (MT) behaviors. S2 cells were found to exhibit a behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. A peculiar metaphase spindle organization was observed. Kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. This study has discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.
Malerod, L., Le Borgne, R., Lie-Jensen, A., Eikenes, A. H., Brech, A., Liestol, K., Stenmark, H. and Haglund, K. (2018). Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics. Embo J. PubMed ID: 29858227
The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. This study reports a novel role for the multifunctional adaptor protein ALG-2-interacting protein X (ALIX) in regulating MS orientation in addition to its well-established role in cytokinesis. ALIX is recruited to the pericentriolar material (PCM) of the centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of gamma-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, these results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division.
Ye, A. A., Verma, V. and Maresca, T. J. (2018). NOD is a plus end-directed motor that binds EB1 via a new microtubule tip localization sequence. J Cell Biol. PubMed ID: 29899040
Chromosome congression, the process of positioning chromosomes in the midspindle, promotes the stable transmission of the genome to daughter cells during cell division. Congression is typically facilitated by DNA-associated, microtubule (MT) plus end-directed motors called chromokinesins. The Drosophila melanogaster chromokinesin NOD contributes to congression, but the means by which it does so are unknown in large part because NOD has been classified as a nonmotile, orphan kinesin. It has been postulated that NOD promotes congression, not by conventional plus end-directed motility, but by harnessing polymerization forces by end-tracking on growing MT plus ends via a mechanism that is also uncertain. This study demonstrates that NOD possesses MT plus end-directed motility. Furthermore, NOD directly binds EB1 through unconventional EB1-interaction motifs that are similar to a newly characterized MT tip localization sequence. It is proposed that NOD produces congression forces by MT plus end-directed motility and tip-tracking on polymerizing MT plus ends via association with EB1.
Kovacs, L., Chao-Chu, J., Schneider, S., Gottardo, M., Tzolovsky, G., Dzhindzhev, N. S., Riparbelli, M. G., Callaini, G. and Glover, D. M. (2018). Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles. Nat Genet. PubMed ID: 29892014
A Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. The separation of centriole and Golgi functions of Drosophila Gorab were demonstrated in two ways: first, Gorab variants were created that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies; second, it was shown that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. These findings suggest that during animal evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.

Wednesday, August 8th

Schwarz, B., Hollfelder, D., Scharf, K., Hartmann, L. and Reim, I. (2018). Diversification of heart progenitor cells by EGF signaling and differential modulation of ETS protein activity. Elife 7. PubMed ID: 29869981
For coordinated circulation, vertebrate and invertebrate hearts require stereotyped arrangements of diverse cell populations. This study explores the process of cardiac cell diversification in the Drosophila heart, focusing on the two major cardioblast subpopulations: generic working myocardial cells and inflow valve-forming ostial cardioblasts. By screening a large collection of randomly induced mutants several genes involved in cardiac patterning were identified. Further analysis revealed an unexpected, specific requirement of EGF signaling for the specification of generic cardioblasts and a subset of pericardial cells. The Tbx20 ortholog Midline acts as a direct target of the EGFR effector Pointed to repress ostial fates. Furthermore, Edl/Mae, an antagonist of the ETS factor Pointed, was identified as a novel cardiac regulator crucial for ostial cardioblast specification. Combining these findings a regulatory model is proposed in which the balance between activation of Pointed and its inhibition by Edl controls cardioblast subtype-specific gene expression.
Baker, L. R., Weasner, B. M., Nagel, A., Neuman, S. D., Bashirullah, A. and Kumar, J. P. (2018). Eyeless/Pax6 initiates eye formation non-autonomously from the peripodial epithelium. Development. PubMed ID: 29980566
The Pax6 transcription factor is considered the master control gene for eye formation because (1) it is present within the genomes and retina/lens in all seeing animals; (2) severe retinal defects accompany its loss; (2) Pax6 genes have the ability to substitute for one another across the animal kingdom; and (4) Pax6 genes are capable of inducing ectopic eye/lens in flies and mammals. Many roles of Pax6 were first elucidated in Drosophila through studies of the gene eyeless (ey), which controls both growth of the entire eye-antennal imaginal disc and fate specification of the eye. This study shows that Ey also plays a surprising role within cells of the peripodial epithelium to control pattern formation. It regulates the expression of decapentaplegic (dpp), which is required for the initiation of the morphogenetic furrow in the eye itself. Loss of Ey within the peripodial epithelium leads to the loss of dpp expression within the eye, the failure of the furrow to initiate, and the abrogation of retinal development. These findings reveal an unexpected mechanism for how Pax6 controls eye development in Drosophila.
Bazzi, W., Cattenoz, P. B., Delaporte, C., Dasari, V., Sakr, R., Yuasa, Y. and Giangrande, A. (2018). Embryonic hematopoiesis modulates the inflammatory response and larval hematopoiesis in Drosophila. Elife 7. PubMed ID: 29992900
Recent lineage tracing analyses have significantly improved understanding of immune system development and highlighted the importance of the different hematopoietic waves. The current challenge is to understand whether these waves interact and whether this affects the function of the immune system. This study reports a molecular pathway regulating the immune response and involving the communication between embryonic and larval hematopoietic waves in Drosophila. Down-regulating the transcription factor Gcm specific to embryonic hematopoiesis enhances the larval phenotypes induced by over-expressing the pro-inflammatory Jak/Stat pathway or by wasp infestation. Gcm works by modulating the transduction of the Upd cytokines to the site of larval hematopoiesis and hence the response to chronic (Jak/Stat over-expression) and acute (wasp infestation) immune challenges. Thus, homeostatic interactions control the function of the immune system in physiology and pathology. The data also indicate that a transiently expressed developmental pathway has a long-lasting effect on the immune response.
Diaz-de-la-Loza, M. D., Ray, R. P., Ganguly, P. S., Alt, S., Davis, J. R., Hoppe, A., Tapon, N., Salbreux, G. and Thompson, B. J. (2018). Apical and basal matrix remodeling control epithelial morphogenesis. Dev Cell 46(1): 23-39.e25. PubMed ID: 29974861
Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. This study shows that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions.

Tuesday, August 7th - Signaling

Baldeosingh, R., Gao, H., Wu, X. and Fossett, N. (2018). Hedgehog signaling from the Posterior Signaling Center maintains U-shaped expression and a prohemocyte population in Drosophila. Dev Biol. PubMed ID: 29966604
The hematopoetic lymph gland is zonally arranged, with progenitors located in medullary zone, differentiating cells in the cortical zone, and the stem cell niche or Posterior Signaling Center (PSC) residing at the base of the medullary zone (MZ). This arrangement facilitates investigations into how signaling from the microenvironment controls progenitor choice. The Drosophila Friend of GATA transcriptional regulator, U-shaped, is a conserved hematopoietic regulator. To identify additional novel intrinsic and extrinsic regulators that interface with U-shaped to control hematopoiesis, this study conducted an in vivo screen for factors that genetically interact with u-shaped. Smoothened, a downstream effector of Hedgehog signaling, was one of the factors identified in the screen. This study reports studies that characterized the relationship between Smoothened and U-shaped. The PSC and Hedgehog signaling are required for U-shaped expression, and U-shaped is an important intrinsic progenitor regulator. These observations identify a potential link between the progenitor regulatory machinery and extrinsic signals from the PSC. Furthermore, both Hedgehog signaling and the PSC were shown to be required to maintain a subpopulation of progenitors. This led to a delineation of PSC-dependent versus PSC-independent progenitors and provided further evidence that the MZ progenitor population is heterogeneous. Overall, a connection has been identified between a conserved hematopoietic master regulator and a putative stem cell niche, which adds to understanding of how signals from the microenvironment regulate progenitor multipotency.
Qi, X., Schmiege, P., Coutavas, E., Wang, J. and Li, X. (2018). Structures of human Patched and its complex with native palmitoylated sonic hedgehog. Nature 560(7716): 128-132. PubMed ID: 29995851
Hedgehog (HH) signalling governs embryogenesis and adult tissue homeostasis in mammals and other multicellular organisms. Whereas deficient HH signalling leads to birth defects, unrestrained HH signalling is implicated in human cancers. N-terminally palmitoylated HH releases the repression of Patched to the oncoprotein Smoothened (SMO); however, the mechanism by which HH recognizes Patched is unclear. This study reports cryo-electron microscopy structures of human patched 1 (PTCH1) alone and in complex with the N-terminal domain of 'native' sonic hedgehog (native SHH-N has both a C-terminal cholesterol and an N-terminal fatty-acid modification), at resolutions of 3.5 A and 3.8 A, respectively. The structure of PTCH1 has internal two-fold pseudosymmetry in the transmembrane core, which features a sterol-sensing domain and two homologous extracellular domains, resembling the architecture of Niemann-Pick C1 (NPC1) protein. The palmitoylated N terminus of SHH-N inserts into a cavity between the extracellular domains of PTCH1 and dominates the PTCH1-SHH-N interface, which is distinct from that reported for SHH-N co-receptors. These biochemical assays show that SHH-N may use another interface, one that is required for its co-receptor binding, to recruit PTCH1 in the absence of a covalently attached palmitate. This work provides atomic insights into the recognition of the N-terminal domain of HH (HH-N) by PTCH1, offers a structural basis for cooperative binding of HH-N to various receptors and serves as a molecular framework for HH signalling and its malfunction in disease.
Chandra, V., Fetter-Pruneda, I., Oxley, P. R., Ritgerm A. L., McKenzie, S. K., Libbrecht, R. and Kronauer, D. J. C. (2018). Social regulation of insulin signaling and the evolution of eusociality in ants. Science 361(6400): 398-402. PubMed ID: 30049879
Queens and workers of eusocial Hymenoptera are considered homologous to the reproductive and brood care phases of an ancestral subsocial life cycle. However, the molecular mechanisms underlying the evolution of reproductive division of labor remain obscure. Using a brain transcriptomics screen, this study identified a single gene, insulin-like peptide 2 (ilp2), which is always up-regulated in ant reproductives, likely because they are better nourished than their nonreproductive nestmates. In clonal raider ants (Ooceraea biroi), larval signals inhibit adult reproduction by suppressing ilp2, thus producing a colony reproductive cycle reminiscent of ancestral subsociality. However, increasing ILP2 peptide levels overrides larval suppression, thereby breaking the colony cycle and inducing a stable division of labor. These findings suggest a simple model for the origin of ant eusociality via nutritionally determined reproductive asymmetries potentially amplified by larval signals.
Balakrishnan, S. S., Basu, U., Shinde, D., Thakur, R., Jaiswal, M. and Raghu, P. (2018). Regulation of PI4P levels by PI4KIIIalpha during G-protein coupled PLC signaling in Drosophila photoreceptors. J Cell Sci. PubMed ID: 29980590
The activation of phospholipase C (PLC) is a conserved mechanism of receptor activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and continued signaling requires the resynthesis and availability of PI(4,5)P2 at the plasma membrane. PI(4,5)P2 is synthesized by the phosphorylation of phosphatidylinositol-4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. This study shows that in Drosophila photoreceptors, PI4KIIIalpha activity is required to support signaling during G-protein coupled PLC activation. Depletion of PI4KIIIalpha results in impaired electrical responses to light and reduced plasma membrane levels of PI4P and PI(4,5)P2. Depletion of conserved proteins Efr3 and TTC7 that assemble PI4KIIIalpha at the plasma membrane also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P2 levels. Thus PI4KIIIalpha activity at the plasma membrane generates PI4P and supports PI(4,5)P2 levels during receptor activated PLC signaling.

Monday, August 6th - Behavior

Sonn, J. Y., Lee, J., Sung, M. K., Ri, H., Choi, J. K., Lim, C. and Choe, J. (2018). Serine metabolism in the brain regulates starvation-induced sleep suppression in Drosophila melanogaster. Proc Natl Acad Sci U S A. PubMed ID: 29915051
Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. This study identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. It is proposed that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs.
Classen, G. and Scholz, H. (2018). Octopamine shifts the behavioral response from indecision to approach or aversion in Drosophila melanogaster. Front Behav Neurosci 12: 131. PubMed ID: 30018540
To analyze whether the activation of the octopaminergic neurotransmitter system can shift bias the behavioral response towards approach or aversion, different sets of octopaminergic neurons using optogenetics were activated in Drosophila and the choice of the flies was analyzed using a binary odor trap assay. The release of octopamine from a set of neurons and not acetylcholine acts as positive reinforcer for one food odor source resulting in attraction. The activation of a subset of these neurons causes the opposite behavior and results in aversion. This aversion is due to octopamine release and not tyramine, since in Tyramine-beta-hydroxylase mutants (Tbetah) lacking octopamine, the aversion is suppressed. When given the choice between two different attractive food odor sources the activation of the octopaminergic neurotransmitter system switches the attraction for ethanol-containing food odor to a less attractive food odor. Consistent with the requirement for octopamine in biasing the behavioral outcome, Tbetah mutants fail to switch their attraction. The execution of attraction does not require octopamine but rather initiation of the behavior or a switch of the behavioral response. The attraction to ethanol also depends on octopamine. Pharmacological increases in octopamine signaling in Tbetah mutants increase ethanol attraction and blocking octopamine receptor function reduces ethanol attraction. Taken together, octopamine in the central brain orchestrates behavioral outcomes by biasing the decision of the animal towards food odors. This finding might uncover a basic principle of how octopamine gates behavioral outcomes in the brain.
Ostrowski, D., Salari, A., Zars, M. and Zars, T. (2018). A biphasic locomotor response to acute unsignaled high temperature exposure in Drosophila. PLoS One 13(6): e0198702. PubMed ID: 29883493
Unsignaled stress can have profound effects on animal behavior. While most investigation of stress-effects on behavior follows chronic exposures, less is understood about acute exposures and potential after-effects. This study examined walking activity in Drosophila following acute exposure to high temperature or electric shock. Compared to initial walking activity, flies first increase walking with exposure to high temperatures then have a strong reduction in activity. These effects are related to the intensity of the high temperature and number of exposures. The reduction in walking activity following high temperature and electric shock exposures survives context changes and lasts at least five hours. Reduction in the function of the biogenic amines octopamine / tyramine and serotonin both strongly blunt the increase in locomotor activity with high temperature exposure. However, neither set of biogenic amines alter the long lasting depression in walking activity after exposure.
Chandra, V., Fetter-Pruneda, I., Oxley, P. R., Ritger, A. L., McKenzie, S. K., Libbrecht, R. and Kronauer, D. J. C. (2018). Social regulation of insulin signaling and the evolution of eusociality in ants. Science 361(6400): 398-402. PubMed ID: 30049879
Queens and workers of eusocial Hymenoptera are considered homologous to the reproductive and brood care phases of an ancestral subsocial life cycle. However, the molecular mechanisms underlying the evolution of reproductive division of labor remain obscure. Using a brain transcriptomics screen, this study identified a single gene, insulin-like peptide 2 (ilp2), which is always up-regulated in ant reproductives, likely because they are better nourished than their nonreproductive nestmates. In clonal raider ants (Ooceraea biroi), larval signals inhibit adult reproduction by suppressing ilp2, thus producing a colony reproductive cycle reminiscent of ancestral subsociality. However, increasing ILP2 peptide levels overrides larval suppression, thereby breaking the colony cycle and inducing a stable division of labor. These findings suggest a simple model for the origin of ant eusociality via nutritionally determined reproductive asymmetries potentially amplified by larval signals.

Friday, August 3rd

Deshpande, N. and Meller, V. H. (2018). Chromatin that guides dosage compensation is modulated by the siRNA pathway in Drosophila melanogaster. Genetics. PubMed ID: 29921620
Many heterogametic organisms adjust sex chromosome expression to accommodate differences in gene dosage. This requires selective recruitment of regulatory factors to the modulated chromosome. How these factors are localized to a chromosome with requisite accuracy is poorly understood. Drosophila melanogaster males increase expression from their single X chromosome. Identification of this chromosome involves cooperation between different classes of X-identity elements. The Chromatin Entry Sites (CES) recruit a chromatin-modifying complex that spreads into nearby genes and increases expression. In addition, a family of satellite repeats that is enriched on the X chromosome, the 1.688(X) repeats, promotes recruitment of the complex to nearby genes. The 1.688(X) repeats and CES are dissimilar, and appear to operate through different mechanisms. Interestingly, the siRNA pathway and siRNA from a 1.688(X) repeat also promote X recognition. This study postulatex that siRNA-dependent modification of 1.688(X) chromatin contributes to recognition of nearby genes. In accord with this, enrichment of the siRNA effector Argonaute2 (Ago2) was found at some 1.688(X) repeats. Mutations in several proteins that physically interact with Ago2, including the histone methyltransferase Su(var)3-9, enhance the lethality of males with defective X recognition. Su(var)3-9 deposits H3K9me2 on some 1.688(X) repeats, and this mark is disrupted upon ectopic expression of 1.688(X) siRNA. Furthermore, integration of 1.688(X) DNA on an autosome induces local H3K9me2 deposition, but enhances expression of nearby genes in a siRNA-dependent manner. These findings are consistent with a model in which siRNA-directed modification of 1.688(X) chromatin contributes to recognition of the male X chromosome for dosage compensation.
Lu, D., Li, Z., Li, L., Yang, L., Chen, G., Yang, D., Zhang, Y., Singh, V., Smith, S., Xiao, Y., Wang, E., Ye, Y., Zhang, W., Zhou, L., Rong, Y. and Zhou, J. (2018). The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila. PLoS One 13(6): e0199353. PubMed ID: 29928011
Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. The chromatin barrier activity of Fab-8 was studied and how it is affected by the zygosity of the transgene. Fab-8 was able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments were performed on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.
La Fortezza, M., Grigolon, G., Cosolo, A., Pinduyrin, A., Breimann, L., Blum, H., van Steensel, B. and Classen, A. K. (2018). DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis. Epigenetics Chromatin 11(1): 27. PubMed ID: 29871666
Tracking dynamic protein-chromatin interactions in vivo is key to unravel transcriptional and epigenetic transitions in development and disease. However, limited availability and heterogeneous tissue composition of in vivo source material impose challenges on many experimental approaches. This study has adapted cell-type-specific DamID-seq profiling for use in Drosophila imaginal discs and make FLP/FRT-based induction accessible to GAL driver-mediated targeting of specific cell lineages. In a proof-of-principle approach, ubiquitous DamID expression was used to describe dynamic transitions of Polycomb-binding sites during wing imaginal disc development and in a scrib tumorigenesis model. Atf3 and Ets21C as novel Polycomb target genes involved in scrib tumorigenesis and suggest that target gene regulation by Atf3 and AP-1 transcription factors, as well as modulation of insulator function, plays crucial roles in dynamic Polycomb-binding at target sites. These findings by DamID-seq analysis of wing imaginal disc samples derived from 10 larvae. This study opens avenues for robust profiling of small cell population in imaginal discs in vivo and provides insights into epigenetic changes underlying transcriptional responses to tumorigenic transformation.
Dardalhon-Cumenal, D., Deraze, J., Dupont, C. A., Ribeiro, V., Coleno-Costes, A., Pouch, J., Le Crom, S., Thomassin, H., Debat, V., Randsholt, N. B. and Peronnet, F. (2018). Cyclin G and the Polycomb Repressive complexes PRC1 and PR-DUB cooperate for developmental stability. PLoS Genet 14(7): e1007498. PubMed ID: 29995890
In Drosophila, ubiquitous expression of a short Cyclin G isoform generates extreme developmental noise estimated by fluctuating asymmetry (FA), providing a model to tackle developmental stability. This transcriptional cyclin interacts with chromatin regulators of the Enhancer of Trithorax and Polycomb (ETP; see Corto) and Polycomb families. This led to an investigation of the importance of these interactions in developmental stability. Deregulation of Cyclin G highlights an organ intrinsic control of developmental noise, linked to the ETP-interacting domain, and enhanced by mutations in genes encoding members of the Polycomb Repressive complexes PRC1 and PR-DUB. Deep-sequencing of wing imaginal discs deregulating CycG reveals that high developmental noise correlates with up-regulation of genes involved in translation and down-regulation of genes involved in energy production. Most Cyclin G direct transcriptional targets are also direct targets of PRC1 and RNAPolII in the developing wing. Altogether, these results suggest that Cyclin G, PRC1 and PR-DUB cooperate for developmental stability.

Thursday, August 2nd - Transcriptional Regulation

Collins, A. P. and Anderson, P. C. (2018). Complete coupled binding-folding pathway of the intrinsically disordered transcription factor protein brinker revealed by molecular dynamics simulations and Markov state modeling. Biochemistry. PubMed ID: 29990433
Intrinsically disordered proteins (IDPs) make up a large class of proteins that lack stable structures in solution, existing instead as dynamic conformational ensembles. To perform their biological functions, many IDPs bind to other proteins or nucleic acids. Although IDPs are unstructured in solution, when they interact with binding partners, they fold into defined three-dimensional structures via coupled binding-folding processes. Because they frequently underlie IDP function, the mechanisms of this coupled binding-folding process are of great interest. However, given the flexibility inherent to IDPs and the sparse populations of intermediate states, it is difficult to reveal binding-folding pathways at atomic resolution using experimental methods. Computer simulations are another tool for studying these pathways at high resolution. Accordingly, this study has applied 40 mμs of unbiased molecular dynamics simulations and Markov state modeling to map the complete binding-folding pathway of a model IDP, the 59-residue C-terminal portion of the DNA binding domain of Drosophila melanogaster transcription factor Brinker (BrkDBD). The modeling indicates that BrkDBD binds to its cognate DNA and folds in approximately 50 mμs by an induced fit mechanism, acquiring most of its stable secondary and tertiary structure only after it reaches the final binding site on the DNA. The protein follows numerous pathways en route to its bound and folded conformation, occasionally becoming stuck in kinetic traps. Each binding-folding pathway involves weakly bound, increasingly folded intermediate states located at different sites on the DNA surface. These findings agree with experimental data and provide additional insight into the BrkDBD folding mechanism and kinetics.
Kim, S., Kim, T., Jeong, Y., Choi, S., Yamaguchi, M. and Lee, I. S. (2018). The Drosophila histone methyltransferase NSD is positively regulated by the DRE/DREF system. Genes Genomics 40(5): 475-484. PubMed ID: 29892958
The Drosophila nuclear receptor-binding SET domain protein (NSD) gene encodes the Drosophila ortholog of mammalian NSD family members that are important in many aspects of development and disease in humans. This study observed that overexpression of Drosophila NSD in imaginal discs induces organ atrophy. Thus, to gain an understanding of the transcriptional regulation of the gene, the NSD promoter region was analyzed. First, the presence was identified of three putative DNA replication-related element (DRE) sequences in its promoter region, where DRE-binding factor (DREF) could bind for transcriptional activation. In the experiments with the fly GAL4-UAS system, it was demonstrated that overexpressed DREF increased the endogenous NSD transcription. To confirm the role of DREF as a transcriptional activator on the NSD expression, a series of luciferase reporter gene constructs were generated containing deleted portions of the 5'-flanking regions as well as point mutations in the putative DRE sites. When transiently transfected into S2 cells, the deletion construct containing no DRE sites showed dramatic decrease in the NSD promoter activity, but only two sites near the transcriptional start site were important. Furthermore, the direct interaction of DREF with the two positively cis-acting sequences on the NSD promoter was demonstrated by chromatin immunoprecipitation assay. Taken together, these results demonstrated that NSD is one of the downstream targets of the DRE/DREF pathway that is associated with various cellular processes in Drosophila, indicating that the findings may contribute to the understanding of molecular mechanisms in complex disorders associated with NSD family members in humans.
Bothma, J. P., Norstad, M. R., Alamos, S. and Garcia, H. G. (2018). LlamaTags: A versatile tool to image transcription factor dynamics in live embryos. Cell 173(7):1810-1822. PubMed ID: 29754814
Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. This study pioneered a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, stochastic bursts in the concentration of transcription factors were discovered that are correlated with bursts in transcription. LlamaTags were used to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; it is speculated that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.
Chen, H., Levo, M., Barinov, L., Fujioka, M., Jaynes, J. B. and Gregor, T. (2018). Dynamic interplay between enhancer-promoter topology and gene activity. Nat Genet. PubMed ID: 30038397
A long-standing question in gene regulation is how remote enhancers communicate with their target promoters, and specifically how chromatin topology dynamically relates to gene activation. This study combined genome editing and multi-color live imaging to simultaneously visualize physical enhancer-promoter interaction and transcription at the single-cell level in Drosophila embryos. By examining transcriptional activation of a reporter by the endogenous even-skipped enhancers, which are located 150 kb away, three distinct topological conformation states were identified and their transition kinetics were measured. Sustained proximity of the enhancer to its target is required for activation. Transcription in turn affects the three-dimensional topology as it enhances the temporal stability of the proximal conformation and is associated with further spatial compaction. Furthermore, the facilitated long-range activation results in transcriptional competition at the locus, causing corresponding developmental defects. This approach offers quantitative insight into the spatial and temporal determinants of long-range gene regulation and their implications for cellular fates (Chen, 2018).
Verma, N., Hurlburt, A. M., Wolfe, A., Kim, M. K., Kang, Y. S., Kang, J. J. and Stumph, W. E. (2018). Bdp1 interacts with SNAPc bound to a U6, but not U1, snRNA gene promoter element to establish a stable protein-DNA complex. FEBS Lett. PubMed ID: 29932462
In metazoans, U6 small nuclear RNA (snRNA) gene promoters utilize a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). SNAPc interacts with the transcription factor TFIIIB, which consists of the subunits TBP, Brf1 (Brf2 in vertebrates), and Bdp1. This study show that in Drosophila melanogaster, DmSNAPc directly recruits Bdp1 to the U6 promoter, and an 87-residue region of Bdp1 involved in this interaction was identified. Importantly, Bdp1 recruitment requires that DmSNAPc be bound to a U6 PSE rather than a U1 PSE. This is consistent with the concept that DmSNAPc adopts different conformations on U6 and U1 PSEs, which leads to the subsequent recruitment of distinct general transcription factors and RNA polymerases for U6 and U1 gene transcription.
Ross, J., Kuzin, A., Brody, T. and Odenwald, W. F. (2018). Mutational analysis of a Drosophila neuroblast enhancer governing nubbin expression during CNS development. Genesis. PubMed ID: 30005136
While developmental studies of Drosophila neural stem cell lineages have identified transcription factors (TFs) important to cell identity decisions, currently only an incomplete understanding exists of the cis-regulatory elements that control the dynamic expression of these TFs. Previous studies have identified multiple enhancers that regulate the POU-domain TF paralogs nubbin and pdm-2 genes. Evolutionary comparative analysis of these enhancers reveals that they each contain multiple conserved sequence blocks (CSBs) that span TF DNA-binding sites for known regulators of neuroblast (NB) gene expression in addition to novel sequences. This study functionally analyzes the conserved DNA sequence elements within a NB enhancer located within the nubbin gene and highlights a high level of complexity underlying enhancer structure. Mutational analysis has revealed CSBs that are important for enhancer activation and silencing in the developing CNS. Adjusting the number and relative positions of the TF binding sites within these CSBs alters enhancer function.

Wednesday, August 1st - Drosophila as a model for human diseases

Xu, S., Pany, S., Benny, K., Tarique, K., Al-Hatem, O., Gajewski, K., Leasure, J. L., Das, J. and Roman, G. (2018). Ethanol regulates presynaptic activity and sedation through presynaptic Unc13 uroteins in Drosophila. eNeuro 5(3). PubMed ID: 29911175
One possible presynaptic effector for ethanol is the Munc13-1 protein. This study shows that ethanol binding to the rat Munc13-1 C1 domain, at concentrations consistent with binge exposure, reduces diacylglycerol (DAG) binding. The inhibition of DAG binding is predicted to reduce the activity of Munc13-1 and presynaptic release. In Drosophila, this study shows that sedating concentrations of ethanol significantly reduce synaptic vesicle release in olfactory sensory neurons (OSNs), while having no significant impact on membrane depolarization and Ca(2+) influx into the presynaptic compartment. These data indicate that ethanol targets the active zone in reducing synaptic vesicle exocytosis. Drosophila, haploinsufficent for the Munc13-1 ortholog Dunc13, are more resistant to the effect of ethanol on presynaptic inhibition. Genetically reducing the activity of Dunc13 through mutation or expression of RNAi transgenes also leads to a significant resistance to the sedative effects of ethanol. The neuronal expression of Munc13-1 in heterozygotes for a Dunc13 loss-of-function mutation can largely rescue the ethanol sedation resistance phenotype, indicating a conservation of function between Munc13-1 and Dunc13 in ethanol sedation. Hence, reducing Dunc13 activity leads to naive physiological and behavioral resistance to sedating concentrations of ethanol. It is propose that reducing Dunc13 activity, genetically or pharmacologically by ethanol binding to the C1 domain of Munc13-1/Dunc13, promotes a homeostatic response that leads to ethanol tolerance.
Valadas, J. S., Esposito, G., Vandekerkhove, D., Miskiewicz, K., Deaulmerie, L., Raitano, S., Seibler, P., Klein, C. and Verstreken, P. (2018). ER lipid defects in neuropeptidergic neurons impair sleep patterns in Parkinson's disease. Neuron 98(6): 1155-1169.e1156. PubMed ID: 29887339
Parkinson's disease patients report disturbed sleep patterns long before motor dysfunction. In parkin and pink1 models, this study has identified circadian rhythm and sleep pattern defects and has mapped these to specific neuropeptidergic neurons in fly models and in hypothalamic neurons differentiated from patient induced pluripotent stem cells (iPSCs). Parkin and Pink1 control the clearance of mitochondria by protein ubiquitination. Although major defects were not observed in mitochondria of mutant neuropeptidergic neurons, excess of endoplasmic reticulum-mitochondrial contacts was found. These excessive contact sites cause abnormal lipid trafficking that depletes phosphatidylserine from the endoplasmic reticulum (ER) and disrupts the production of neuropeptide-containing vesicles. Feeding mutant animals phosphatidylserine rescues neuropeptidergic vesicle production and acutely restores normal sleep patterns in mutant animals. Hence, sleep patterns and circadian disturbances in Parkinson's disease models are explained by excessive ER-mitochondrial contacts, and blocking their formation or increasing phosphatidylserine levels rescues the defects in vivo.
Younan, N. D., Chen, K. F., Rose, R. S., Crowther, D. C. and Viles, J. H. (2018). Prion protein stabilizes amyloid-beta (Abeta) oligomers and enhances Abeta neurotoxicity in a Drosophila model of Alzheimer disease. J Biol Chem. PubMed ID: 29887525
The cellular prion protein (PrPC) can act as a cell-surface receptor for amyloid-beta (Abeta) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. This study has expressed a range of Abeta isoforms and PrPC in the Drosophila brain. Co-expression of Abeta and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Abeta deposition in the fly brain. In contrast, under the same conditions, expression of Abeta or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Abeta as oligomeric assemblies and fragment-preformed Abeta fibers. The ability of membrane-anchored PrPC to trap Abeta as cytotoxic oligomers at the membrane surface and fragment inert Abeta fibers, suggests a mechanism by which PrPC exacerbates Abeta deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Abeta toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Abeta and PrPC represents a potential therapeutic strategy.
Bogaert, E., Boeynaems, S., Kato, M., Guo, L., Caulfield, T. R., Steyaert, J., Scheveneels, W., Wilmans, N., Haeck, W., Hersmus, N., Schymkowitz, J., Rousseau, F., Shorter, J., Callaerts, P., Robberecht, W., Van Damme, P. and Van Den Bosch, L. (2018). Molecular dissection of FUS points at synergistic effect of low-complexity domains in toxicity. Cell Rep 24(3): 529-537.e524. PubMed ID: 30021151
RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, this study investigated FUS, which is mutated and aggregated in both ALS and FTLD. This study generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, this study found that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins.
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