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Current papers in developmental biology and gene function


Thursday May 31st, 2018 - Adult nervous system development and function

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Sun, J., Xu, A. Q., Giraud, J., Poppinga, H., Riemensperger, T., Fiala, A. and Birman, S. (2018). Neural control of startle-induced locomotion by the mushroom bodies and associated neurons in Drosophila. Front Syst Neurosci 12: 6. PubMed ID: 29643770
Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. This study combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. The activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). Previous observations were confirmed that activating the MB alpha'beta', but not alphabeta, KCs decreased the SING response, and further MB neurons implicated in SING control were identified, including KCs of the gamma lobe and two subtypes of MB output neurons (MBONs). It was also observed that co-activating the alphabeta KCs antagonizes alpha'beta' and gamma KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.
Patella, P. and Wilson, R. I. (2018). Functional maps of mechanosensory features in the Drosophila brain. Curr Biol 28(8): 1189-1203. PubMed ID: 29657118
Johnston's organ is the largest mechanosensory organ in Drosophila. It contributes to hearing, touch, vestibular sensing, proprioception, and wind sensing. This study used in vivo 2-photon calcium imaging and unsupervised image segmentation to map the tuning properties of Johnston's organ neurons (JONs) at the site where their axons enter the brain. The same methodology was then applied to study two key brain regions that process signals from JONs: the antennal mechanosensory and motor center (AMMC) and the wedge, which is downstream of the AMMC. First, a diversity of JON response types was identified that tile frequency space and form a rough tonotopic map. Some JON response types are direction selective; others are specialized to encode amplitude modulations over a specific range (dynamic range fractionation). Next, it was discovered that both the AMMC and the wedge contain a tonotopic map, with a significant increase in tonotopy-and a narrowing of frequency tuning-at the level of the wedge. Whereas the AMMC tonotopic map is unilateral, the wedge tonotopic map is bilateral. Finally, a subregion of the AMMC/wedge was identfied that responds preferentially to the coherent rotation of the two mechanical organs in the same angular direction, indicative of oriented steady air flow (directional wind). Together, these maps reveal the broad organization of the primary and secondary mechanosensory regions of the brain. They provide a framework for future efforts to identify the specific cell types and mechanisms that underlie the hierarchical re-mapping of mechanosensory information in this system.
Liao, C. P., Li, H., Lee, H. H., Chien, C. T. and Pan, C. L. (2018). Cell-autonomous regulation of dendrite self-avoidance by the Wnt secretory factor MIG-14/Wntless. Neuron 98(2): 320-334 PubMed ID: 29673481
Self-avoidance allows sister dendrites from the same neuron to form non-redundant coverage of the sensory territory and is important for neural circuitry functions. This study reports an unexpected, cell-autonomous role of the Wnt-secretory factor MIG-14/Wntless in mediating dendrite self-avoidance in the C. elegans multidendritic PVD neurons. Similar findings in Drosophila suggest that this novel function of Wntless is conserved. The mig-14 mutant shows defects in dendrite self-avoidance, and ectopic MIG-14 expression triggers dendrite repulsion. Functions of dendrite self-avoidance and Wnt secretion could be mapped to distinct MIG-14 domains, indicating that these two functions of MIG-14 are genetically separable, consistent with lack of self-avoidance defects in the Wnt mutants. It was further demonstrated that MIG-14 engages Wiskott-Aldrich syndrome protein (WASP)-dependent actin assembly to regulate dendrite self-avoidance. This work expands the repertoire of self-avoidance molecules and uncovers a previously unknown, Wnt-independent function of MIG-14/Wntless.
Pinto-Teixeira, F., Koo, C., Rossi, A. M., Neriec, N., Bertet, C., Li, X., Del-Valle-Rodriguez, A. and Desplan, C. (2018). Development of concurrent retinotopic maps in the fly motion detection circuit. Cell 173(2): 485-498.e411. PubMed ID: 29576455
Understanding how complex brain wiring is produced during development is a daunting challenge. In Drosophila, information from 800 retinal ommatidia is processed in distinct brain neuropiles, each subdivided into 800 matching retinotopic columns. The lobula plate comprises four T4 and four T5 neuronal subtypes. T4 neurons respond to bright edge motion, whereas T5 neurons respond to dark edge motion. Each is tuned to motion in one of the four cardinal directions, effectively establishing eight concurrent retinotopic maps to support wide-field motion. A mode of neurogenesis was discovered where two sequential Notch-dependent divisions of either a horizontal or a vertical progenitor produce matching sets of two T4 and two T5 neurons retinotopically coincident with pairwise opposite direction selectivity. Retinotopy is an emergent characteristic of this neurogenic program and derives directly from neuronal birth order. This work illustrates how simple developmental rules can implement complex neural organization.
Pu, Y., Palombo, M. M. M. and Shen, P. (2018). Contribution of DA signaling to appetitive odor perception in a Drosophila model. Sci Rep 8(1): 5978. PubMed ID: 29654277
>Understanding cognitive processes that translate chemically diverse olfactory stimuli to specific appetitive drives remains challenging. Food-related odors arouse impulsive-like feeding of food media that are palatable and readily accessible in well-nourished Drosophila larvae. This study provides evidence that two assemblies of four dopamine (DA) neurons, one per brain hemisphere, contribute to perceptual processing of the qualitative and quantitative attributes of food scents. These DA neurons receive neural representations of chemically diverse food-related odors, and their combined neuronal activities become increasingly important as the chemical complexity of an appetizing odor stimulus increases. Furthermore, in each assembly of DA neurons, integrated odor signals are transformed to one-dimensional DA outputs that have no intrinsic reward values. Finally, a genetic analysis has revealed a D1-type DA receptor (Dop1R1)-gated mechanism in neuropeptide Y-like neurons that assigns appetitive significance to selected DA outputs. These findings suggest that fly larvae provide a useful platform for elucidation of molecular and circuit mechanisms underlying cognitive processing of olfactory and possibly other sensory cues.
Scheunemann, L., Placais, P. Y., Dromard, Y., Schwarzel, M. and Preat, T. (2018). Dunce phosphodiesterase acts as a checkpoint for Drosophila long-term memory in a pair of serotonergic neurons. Neuron 98(2): 350-365.e355. PubMed ID: 29673482
A key function of the brain is to filter essential information and store it in the form of stable, long-term memory (LTM). The Dunce (Dnc) phosphodiesterase, an important enzyme that degrades cAMP, acts as a molecular switch that controls LTM formation in Drosophila. During LTM formation, Dnc is inhibited in the SPN, a pair of newly characterized serotonergic projection neurons, which stimulates the cAMP/PKA pathway. As a consequence, the SPN activates downstream dopaminergic neurons, opening the gate for LTM formation in the olfactory memory center, the mushroom body. Strikingly, transient inhibition of Dnc in the SPN by RNAi was sufficient to induce LTM formation with a training protocol that normally generates only short-lived memory. Thus, Dnc activity in the SPN acts as a memory checkpoint to guarantee that only the most relevant learned experiences are consolidated into stable memory.

Wednesday, May 30th - Behavior

Warren, T. L., Weir, P. T. and Dickinson, M. H. (2018). Flying Drosophila maintain arbitrary but stable headings relative to the angle of polarized light. J Exp Biol. PubMed ID: 29593084
Animals must use external cues to maintain a straight course over long distances. This study investigated how the fruit fly, Drosophila melanogaster, selects and maintains a flight heading relative to the axis of linearly polarized light, a visual cue produced by the atmospheric scattering of sunlight. To track flies' headings over extended periods, a flight simulator was used that coupled the angular velocity of dorsally presented polarized light to the stroke amplitude difference of the animal's wings. In the simulator, most flies actively maintained a stable heading relative to the axis of polarized light for the duration of 15 minute flights. Individuals selected arbitrary, unpredictable headings relative to the polarization axis, which demonstrates that Drosophila can perform proportional navigation using a polarized light pattern. When flies flew in two consecutive bouts separated by a 5 minute gap, the two flight headings were correlated, suggesting individuals retain a memory of their chosen heading. Adding a polarized light pattern to a light intensity gradient was found to enhance flies' orientation ability, suggesting Drosophila use a combination of cues to navigate. For both polarized light and intensity cues, flies' capacity to maintain a stable heading gradually increased over several minutes from the onset of flight. These findings are consistent with a model in which each individual initially orients haphazardly but then settles on a heading which is maintained via a self-reinforcing process. This may be a general dispersal strategy for animals with no target destination.
Bentzur, A., Shmueli, A., Omesi, L., Ryvkin, J., Knapp, J. M., Parnas, M., Davis, F. P. and Shohat-Ophir, G. (2018). Odorant binding protein 69a connects social interaction to modulation of social responsiveness in Drosophila. PLoS Genet 14(4): e1007328. PubMed ID: 29630598
Living in a social environment requires the ability to respond to specific social stimuli and to incorporate information obtained from prior interactions into future ones. One of the mechanisms that facilitates social interaction is pheromone-based communication. In Drosophila melanogaster, the male-specific pheromone cis-vaccenyl acetate (cVA) elicits different responses in male and female flies, and functions to modulate behavior in a context and experience-dependent manner. This study explored the functional link between social interaction and pheromone-based communication and discovered an odorant binding protein that links social interaction to sex specific changes in cVA related responses. Odorant binding protein 69a (Obp69a) is expressed in auxiliary cells and secreted into the olfactory sensilla. Its expression is inversely regulated in male and female flies by social interactions: cVA exposure reduces its levels in male flies and increases its levels in female flies. Increasing or decreasing Obp69a levels by genetic means establishes a functional link between Obp69a levels and the extent of male aggression and female receptivity. Activation of cVA-sensing neurons was shown to be sufficient to regulate Obp69a levels in the absence of cVA, and requires active neurotransmission between the sensory neuron to the second order olfactory neuron. The cross-talk between sensory neurons and non-neuronal auxiliary cells at the olfactory sensilla, represents an additional component in the machinery that promotes behavioral plasticity to the same sensory stimuli in male and female flies.
Bai, L., Lee, Y., Hsu, C. T., Williams, J. A., Cavanaugh, D., Zheng, X., Stein, C., Haynes, P., Wang, H., Gutmann, D. H. and Sehgal, A. (2018). A conserved circadian function for the Neurofibromatosis 1 gene. Cell Rep 22(13): 3416-3426. PubMed ID: 29590612
Loss of the Neurofibromatosis 1 (Nf1) protein, neurofibromin, in Drosophila disrupts circadian rhythms of locomotor activity without impairing central clock function, suggesting effects downstream of the clock. However, the relevant cellular mechanisms are not known. Leveraging the discovery of output circuits for locomotor rhythms, cellular actions of neurofibromin in recently identified substrates were identified. This study shows that neurofibromin affects the levels and cycling of calcium in multiple circadian peptidergic neurons. A prominent site of action is the pars intercerebralis (PI), the fly equivalent of the hypothalamus, with cell-autonomous effects of Nf1 in PI cells that secrete DH44. Nf1 interacts genetically with peptide signaling to affect circadian behavior. These studies were extended to mammals to demonstrate that mouse astrocytes exhibit a 24-hr rhythm of calcium levels, which is also attenuated by lack of neurofibromin. These findings establish a conserved role for neurofibromin in intracellular signaling rhythms within the nervous system.
Landayan, D., Feldman, D. S. and Wolf, F. W. (2018). Satiation state-dependent dopaminergic control of foraging in Drosophila. Sci Rep 8(1): 5777. PubMed ID: 29636522
Hunger evokes stereotypic behaviors that favor the discovery of nutrients. The neural pathways that coordinate internal and external cues to motivate foraging behaviors are only partly known. Drosophila that are food deprived increase locomotor activity, are more efficient in locating a discrete source of nutrition, and are willing to overcome adversity to obtain food. A simple open field assay was developed that allows flies to freely perform multiple steps of the foraging sequence, and it was shown that two distinct dopaminergic neural circuits regulate measures of foraging behaviors. One group, the PAM neurons, functions in food deprived flies while the other functions in well fed flies, and both promote foraging. These satiation state-dependent circuits converge on dopamine D1 receptor-expressing Kenyon cells of the mushroom body, where neural activity promotes foraging independent of satiation state. These findings provide evidence for active foraging in well-fed flies that is separable from hunger-driven foraging.
Schultzhaus, J. N., Bennett, C. J., Iftikhar, H., Yew, J. Y., Mallett, J. and Carney, G. E. (2018). High fat diet alters Drosophila melanogaster sexual behavior and traits: decreased attractiveness and changes in pheromone profiles. Sci Rep 8(1): 5387. PubMed ID: 29599496
Sexual traits convey information about individual quality to potential mates. Environmental and genetic factors affect sexual trait expression and perception via effects on animal condition and health. High fat diet (HFD) is one environmental factor that adversely affects Drosophila melanogaster health, and its effects on animal health are mediated through conserved metabolic signaling pathways. HFD decreases female attractiveness, resulting in reduced male mating behaviors toward HFD females. HFD also affects the ability of males to judge mate attractiveness and likely alters fly condition and sexual traits to impact mating behavior. This study shows that HFD affects both visual (body size) and non-visual (pheromone profiles) sexual traits, which likely contribute to decreased fly attractiveness. It was also demonstrated that adult-specific HFD effects on male mate preference can be rescued by changing metabolic signaling. These results demonstrate that HFD alters Drosophila sexual cues to reflect concurrent effects on condition and that less severe behavioral defects can be reversed by genetic manipulations that rescue fly health. This work expands on current knowledge of the role that metabolic signaling pathways play in linking animal health, sexual traits, and mating behavior, and provides a robust assay in a genetically tractable system to continue examining these processes.
Snellings, Y., Herrera, B., Wildemann, B., Beelen, M., Zwarts, L., Wenseleers, T. and Callaerts, P. (2018). The role of cuticular hydrocarbons in mate recognition in Drosophila suzukii. Sci Rep 8(1): 4996. PubMed ID: 29567945
Cuticular hydrocarbons (CHCs) play a central role in the chemical communication of many insects. In Drosophila suzukii, an economically important pest insect, very little is known about chemical communication and the possible role of CHCs. This study identified 60 CHCs of Drosophila suzukii and studied their changes in function of age (maturation), sex and interactions with the opposite sex. Age (maturation) was identified is the key factor driving changes in the CHC profiles. The effect on courtship behaviour and mating of six CHCs, five of which were positively associated with maturation and one negatively. The results of these experiments demonstrate that four of the major CHC peaks with a chain length of 23 carbons, namely 9-tricosene (9-C23:1), 7-tricosene (7-C23:1), 5-tricosene (5-C23:1) and tricosane (n-C23), negatively regulated courtship and mating, even though all these compounds were characteristic for sexually mature flies. The study then goes on to show that this effect on courtship and mating is likely due to the disruption of the natural ratios in which these hydrocarbons occur in Drosophila suzukii. Overall, these results provide key insights into the cuticular hydrocarbon signals that play a role in D. suzukii mate recognition.

Tuesday, May 29th - RNA biology

Kittelmann, S., Buffry, A. D., Franke, F. A., Almudi, I., Yoth, M., Sabaris, G., Couso, J. P., Nunes, M. D. S., Frankel, N., Gomez-Skarmeta, J. L., Pueyo-Marques, J., Arif, S. and McGregor, A. P. (2018). Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution. PLoS Genet 14(5): e1007375. PubMed ID: 29723190
Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary 'hotspots' are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the 'naked valley' on T2 femurs in Drosophila melanogaster is caused by reduced microRNA-92a (miR-92a) expression rather than changes in svb. The expression and function of components between the larval and leg trichome GRNs were compared to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. Key differences were found between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. This work shows that variability in GRNs across different developmental contexts, as well as whether a morphological feature is lost versus gained, influence the nodes at which a GRN evolves to cause morphological change. Therefore, these findings have important implications for understanding the pathways and predictability of evolution.
Leggio, L., Guarino, F., Magri, A., Accardi-Gheit, R., Reina, S., Specchia, V., Damiano, F., Tomasello, M. F., Tommasino, M. and Messina, A. (2018). Mechanism of translation control of the alternative Drosophila melanogaster Voltage Dependent Anion-selective Channel 1 mRNAs. Sci Rep 8(1): 5347. PubMed ID: 29593233
The eukaryotic porin, also called the Voltage Dependent Anion-selective Channel (VDAC), is the main pore-forming protein of the outer mitochondrial membrane. In Drosophila melanogaster, a cluster of genes evolutionarily linked to VDAC is present on chromosome 2L. The main VDAC isoform, called VDAC1 (Porin1), is expressed from the first gene of the cluster. The porin1 gene produces two splice variants, 1A-VDAC and 1B-VDAC, with the same coding sequence but different 5' untranslated regions (UTRs). The influence of the two 5' UTRs, 1A-5' UTR and 1B-5' UTR, was studied on transcription and translation of VDAC1 mRNAs. In porin-less yeast cells, transformation with a construct carrying 1A-VDAC results in the expression of the corresponding protein and in complementation of a defective cell phenotype, whereas the 1B-VDAC sequence actively represses VDAC expression. Identical results were obtained using constructs containing the two 5' UTRs upstream of the GFP reporter. A short region of 15 nucleotides in the 1B-5' UTR should be able to pair with an exposed helix of 18S ribosomal RNA (rRNA), and this interaction could be involved in the translational repression. These data suggest that contacts between the 5' UTR and 18S rRNA sequences could modulate the translation of Drosophila 1B-VDAC mRNA. The evolutionary significance of this finding is discussed.
Szostak, E., Garcia-Beyaert, M., Guitart, T., Graindorge, A., Coll, O. and Gebauer, F. (2018). Hrp48 and eIF3d contribute to msl-2 mRNA translational repression. Nucleic Acids Res 46(8):4099-4113. PubMed ID: 29635389
Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. This study identified Heterogeneous nuclear ribonucleoprotein at 27C as a SXL co-factor. Hrp48 binds to the 3' UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5' UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. These results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.
Wharton, T. H., Nomie, K. J. and Wharton, R. P. (2018). No significant regulation of bicoid mRNA by Pumilio or Nanos in the early Drosophila embryo. PLoS One 13(3): e0194865. PubMed ID: 29601592
Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3'-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal hunchback (hb) mRNA, takes place exclusively in the posterior. An exceptional case of Nos-independent regulation by Pum has been described-repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3'-UTR of the mRNA. This study re-investigated regulation of bcd in the early embryo; the experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, it was found that Pum and Nos control the accumulation of bcd mRNA in testes.
Landskron, L., Steinmann, V., Bonnay, F., Burkard, T. R., Steinmann, J., Reichardt, I., Harzer, H., Laurenson, A. S., Reichert, H. and Knoblich, J. A. (2018). The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells. Elife 7. PubMed ID: 29580384
Tumor cells display features that are not found in healthy cells. How they become immortal and how their specific features can be exploited to combat tumorigenesis are key questions in tumor biology.This study describes the long non-coding RNA cherub (long non-coding RNA:CR43283) that is critically required for the development of brain tumors in Drosophila but is dispensable for normal development. In mitotic Drosophila neural stem cells, cherub localizes to the cell periphery and segregates into the differentiating daughter cell. During tumorigenesis, de-differentiation of cherub-high cells leads to the formation of tumorigenic stem cells that accumulate abnormally high cherub levels. cherub establishes a molecular link between the RNA-binding proteins Staufen and Syncrip. As Syncrip is part of the molecular machinery specifying temporal identity in neural stem cells, it is proposed that tumor cells proliferate indefinitely, because cherub accumulation no longer allows them to complete their temporal neurogenesis program.
Joseph, B., Kondo, S. and Lai, E. C. (2018). Short cryptic exons mediate recursive splicing in Drosophila. Nat Struct Mol Biol. PubMed ID: 29632374
Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect 'zero-nucleotide exons', whose sequential processing subdivides removal of long host introns. This study used CRISPR-Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40-80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.

Friday, May 25th - Transcriptional regulation

Eksi, S. E., Barmina, O., McCallough, C. L., Kopp, A. and Orenic, T. V. (2018). A Distalless-responsive enhancer of the Hox gene Sex combs reduced is required for segment- and sex-specific sensory organ development in Drosophila. PLoS Genet 14(4): e1007320. PubMed ID: 29634724
Hox genes are involved in the patterning of animal body parts at multiple levels of regulatory hierarchies. Early expression of Hox genes in different domains along the embryonic anterior-posterior (A/P) axis in insects, vertebrates, and other animals establishes segmental or regional identity. However, Hox gene function is also required later in development for the patterning and morphogenesis of limbs and other organs. In Drosophila, spatiotemporal modulation of Sex combs reduced (Scr) expression within the first thoracic (T1) leg underlies the generation of segment- and sex-specific sense organ patterns. High Scr expression in defined domains of the T1 leg is required for the development of T1-specific transverse bristle rows in both sexes and sex combs in males, implying that the patterning of segment-specific sense organs involves incorporation of Scr into the leg development and sex determination gene networks. This study sought to gain insight into this process by identifying the cis-and trans-regulatory factors that direct Scr expression during leg development. Two cis-regulatory elements were identified that control spatially modulated Scr expression within T1 legs. One of these enhancers directs sexually dimorphic expression and is required for the formation of T1-specific bristle patterns. The Distalless and Engrailed homeodomain transcription factors act through sequences in this enhancer to establish elevated Scr expression in spatially defined domains. This enhancer functions to integrate Scr into the intrasegmental gene regulatory network, such that Scr serves as a link between leg patterning, sex determination, and sensory organ development.
Sarkar, A., Gogia, N., Farley, K., Payton, L. and Singh, A. (2018). Characterization of a morphogenetic furrow specific Gal4 driver in the developing Drosophila eye. PLoS One 13(4): e0196365. PubMed ID: 29702674
During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. There are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. GMR lines harboring regulatory regions of dpp fused with Gal4 coding region were screened to identify MF specific enhancer of dpp using a GFP reporter gene. Immuno-histochemical approaches were used to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. Two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 were identified that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. Misexpression phenotypes were compared of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using the newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in these studies.
Abidi, S. N. F. and Smith-Bolton, R. K. (2018). Cell fate changes induced by a Distal-less enhancer-trap transgene in the Drosophila antennal imaginal disc. Sci Rep 8(1): 4950. PubMed ID: 29563503
The imaginal discs of the genetically tractable model organism Drosophila melanogaster have been used to study cell-fate specification and plasticity, including homeotic changes and regeneration-induced transdetermination. The identity of the reprogramming mechanisms that induce plasticity has been of great interest in the field. This study identified a change from antennal fate to eye fate induced by a Distal-less-GAL4 (DllGAL4) P-element insertion that is a mutant allele of Dll and expresses GAL4 in the antennal imaginal disc. While this fate change is not induced by tissue damage, it appears to be a hybrid of transdetermination and homeosis as the GAL4 expression causes upregulation of Wingless, and the Dll mutation is required for the fate change. Neither GAL4 expression nor a Dll mutation on its own is able to induce antenna-to-eye fate changes. This plasticity appears to be unique to the DllGAL4 line, possibly due to cellular stress induced by the high GAL4 expression combined with the severity of the Dll mutation. Thus, it is proposed that even in the absence of tissue damage, other forms of cellular stress caused by high GAL4 expression can induce determined cell fates to change, and selector gene mutations can sensitize the tissue to these transformations.
Chakraborty, M., Sellier, C., Ney, M., Pascal, V., Charlet-Berguerand, N., Artero, R. and Llamusi, B. (2018). Daunorubicin reduces MBNL1 sequestration caused by CUG-repeat expansion and rescues cardiac dysfunctions in a Drosophila model of myotonic dystrophy. Dis Model Mech 11(4). PubMed ID: 29592894
Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder caused by expression of mutant myotonin-protein kinase (DMPK) transcripts containing expanded CUG repeats. Pathogenic DMPK RNA sequesters the muscleblind-like (MBNL) proteins, causing alterations in metabolism of various RNAs. Cardiac dysfunction represents the second most common cause of death in DM type 1 (DM1) patients. However, the contribution of MBNL sequestration in DM1 cardiac dysfunction is unclear. This study overexpressed Muscleblind (Mbl), the Drosophila MBNL orthologue, in cardiomyocytes of DM1 model flies and observed a rescue of heart dysfunctions, which are characteristic of these model flies and resemble cardiac defects observed in patients. A drug - daunorubicin hydrochloride - was identified that directly binds to CUG repeats and alleviates Mbl sequestration in Drosophila DM1 cardiomyocytes, resulting in mis-splicing rescue and cardiac function recovery. These results demonstrate the relevance of Mbl sequestration caused by expanded-CUG-repeat RNA in cardiac dysfunctions in DM1, and highlight the potential of strategies aimed at inhibiting this protein-RNA interaction to recover normal cardiac function.
Kaye, E. G., Booker, M., Kurland, J. V., Conicella, A. E., Fawzi, N. L., Bulyk, M. L., Tolstorukov, M. Y. and Larschan, E. (2018). Differential occupancy of two GA-binding proteins promotes targeting of the Drosophila dosage compensation complex to the male X chromosome. Cell Rep 22(12): 3227-3239. PubMed ID: 29562179
Little is known about how variation in sequence composition alters transcription factor occupancy to precisely recruit large transcription complexes. A key model for understanding how transcription complexes are targeted is the Drosophila dosage compensation system in which the male-specific lethal (MSL) transcription complex specifically identifies and regulates the male X chromosome. The chromatin-linked adaptor for MSL proteins (CLAMP) zinc-finger protein targets MSL to the X chromosome but also binds to GA-rich sequence elements throughout the genome. Furthermore, the GAGA-associated factor (GAF) transcription factor also recognizes GA-rich sequences but does not associate with the MSL complex. This study demonstrated that MSL complex recruitment sites are optimal CLAMP targets. Specificity for CLAMP binding versus GAF binding is driven by variability in sequence composition within similar GA-rich motifs. Therefore, variation within seemingly similar cis elements drives the context-specific targeting of a large transcription complex.
Rennie, S., Dalby, M., Lloret-Llinares, M., Bakoulis, S., Dalager Vaagenso, C., Heick Jensen, T. and Andersson, R. (2018). Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities. Nucleic Acids Res. PubMed ID: 29659982
Mammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. This study investigated the transcription initiation landscape and its associated RNA decay in Drosophila melanogaster. The majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. Quantitative relationships were observed between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The seemingly separable layer of regulation by gene promoters with housekeeping enhancer potential is also indicated by chromatin interaction data. These results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

Thursday, May 24th - Gonadogenesis

Coux, R. X., Teixeira, F. K. and Lehmann, R. (2018). L(3)mbt and the LINT complex safeguard cellular identity in the Drosophila ovary. Development. PubMed ID: 29511022
Maintenance of cellular identity is essential for tissue development and homeostasis. At the molecular level, cell identity is determined by the coordinated activation and repression of defined sets of genes. The tumor suppressor L(3)mbt was shown to secure cellular identity in Drosophila larval brains by repressing germline-specific genes. This study interrogates the temporal and spatial requirements for L(3)mbt in the Drosophila ovary, and show that it safeguards the integrity of both somatic and germline tissues. L(3)mbt mutant ovaries exhibit multiple developmental defects, which were found to be largely caused by the inappropriate expression of a single gene, nanos, a key regulator of germline fate, in the somatic ovarian cells. In the female germline, L(3)mbt was found to repress testis-specific and neuronal genes. Molecularly, L(3)mbt function in the ovary was shown to be mediated through its cofactor Lint1 but independent of the dREAM complex. Together, this work uncovers a more complex role for L(3)mbt than previously understood and demonstrates that L(3)mbt secures tissue identity by preventing the simultaneous expression of original identity markers and tissue-specific misexpression signatures.
Su, Y. H., Rastegri, E., Kao, S. H., Lai, C. M., Lin, K. Y., Liao, H. Y., Wang, M. H. and Hsu, H. J. (2018). Diet regulates membrane extension and survival of niche escort cells for germline homeostasis via insulin signaling. Development. PubMed ID: 29549109
Diet is an important regulator of stem cell homeostasis, however, the underlying mechanisms of this regulation are not fully known. This study reports that insulin signaling mediates dietary maintenance of Drosophila ovarian germline stem cells (GSCs) by promoting the extension of niche escort cell (EC) membranes to wrap around GSCs. This wrapping may facilitate the delivery of BMP stemness factors from ECs in the niche to GSCs. In addition to the effects on GSCs, insulin signaling-mediated regulation of EC number and protrusions controls the division and growth of GSC progeny. The effects of insulin signaling on EC membrane extension are, at least in part, driven by enhanced translation of Failed axon connections (Fax) via Ribosomal protein S6 kinase. Fax is a membrane protein that may participate in Abl-regulated cytoskeletal dynamics and is known to be involved in axon bundle formation. Therefore, it is concluded that dietary cues stimulate insulin signaling in the niche to regulate EC cellular structure, probably via Fax-dependent cytoskeleton remodeling. This mechanism enhances intercellular contact and facilitates homeostatic interactions between somatic and germline cells in response to diet.
Reina, J., Gottardo, M., Riparbelli, M. G., Llamazares, S., Callaini, G. and Gonzalez, C. (2018). Centrobin is essential for C-tubule assembly and flagellum development in Drosophila melanogaster spermatogenesis. J Cell Biol. PubMed ID: 29712734
Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila) inhibits basal body function. These data open the question of CNB's role in spermatocytes, where daughter and mother centrioles become basal bodies. This study reports that in these cells, CNB localizes equally to mother and daughter centrioles and is essential for C-tubules to attain the right position and remain attached to B-tubules as well as for centrioles to grow in length. CNB appears to be dispensable for meiosis, but flagellum development is severely compromised in Cnb mutant males. Remarkably, three N-terminal POLO phosphorylation sites that are critical for CNB function in neuroblasts are dispensable for spermatogenesis. These results underpin the multifunctional nature of CNB that plays different roles in different cell types in Drosophila, and they identify CNB as an essential component for C-tubule assembly and flagellum development in Drosophila spermatogenesis.
Alegot, H., Pouchin, P., Bardot, O. and Mirouse, V. (2018). Jak-Stat pathway induces Drosophila follicle elongation by a gradient of apical contractility. Elife 7. PubMed ID: 29420170
Tissue elongation and its control by spatiotemporal signals is a major developmental question. Currently, it is thought that Drosophila ovarian follicular epithelium elongation requires the planar polarization of the basal domain cytoskeleton and of the extra-cellular matrix, associated with a dynamic process of rotation around the anteroposterior axis. This study shows, by careful kinetic analysis of fat2 mutants, that neither basal planar polarization nor rotation is required during a first phase of follicle elongation. Conversely, a JAK-STAT signaling gradient from each follicle pole orients early elongation. JAK-STAT controls apical pulsatile contractions, and Myosin II activity inhibition affects both pulses and early elongation. Early elongation is associated with apical constriction at the poles and with oriented cell rearrangements, but without any visible planar cell polarization of the apical domain. Thus, a morphogen gradient can trigger tissue elongation through a control of cell pulsing and without a planar cell polarity requirement.
Allbee, A. W., Rincon-Limas, D. E. and Biteau, B. (2018). Lmx1a is required for the development of the ovarian stem cell niche in Drosophila. Development 145(8). PubMed ID: 29615466
The Drosophila ovary serves as a model for pioneering studies of stem cell niches, with defined cell types and signaling pathways supporting both germline and somatic stem cells. The establishment of the niche units begins during larval stages with the formation of terminal filament-cap structures; however, the genetics underlying their development remains largely unknown. This study shows that the transcription factor Lmx1a is required for ovary morphogenesis. Lmx1a is expressed in early ovarian somatic lineages and becomes progressively restricted to terminal filaments and cap cells. Lmx1a is required for the formation of terminal filaments, during the larval-pupal transition. Finally, the data demonstrate that Lmx1a functions genetically downstream of Bric-a-Brac, and is crucial for the expression of key components of several conserved pathways essential to ovarian stem cell niche development. Importantly, expression of chicken Lmx1b is sufficient to rescue the null Lmx1a phenotype, indicating functional conservation across the animal kingdom. These results significantly expand our understanding of the mechanisms controlling stem cell niche development in the fly ovary.
Kairamkonda, S. and Nongthomba, U. (2018). Beadex, a Drosophila LIM domain only protein, function in follicle cells is essential for egg development and fertility. Exp Cell Res 367(1): 97-103. PubMed ID: 29580687
LIM domain, constituted by two tandem C2H2 zinc finger motif, proteins regulate several biological processes. They are usually found associated with various functional domains like Homeodomain, kinase domain and other protein binding domains. LIM proteins that are devoid of other domains are called LIM only proteins (LMO). LMO proteins were first identified in humans and are implicated in development and oncogenesis. They regulate various cell specifications by regulating the activity of respective transcriptional complexes. The Drosophila LMO protein (dLMO), Beadex (Bx), regulates various developmental processes like wing margin development and bristle development. It also regulates Drosophila behavior in response to cocaine and ethanol. Analysis of Bx null flies has shown Bx essential function in neurons for multiple aspects of female reproduction. However, it was not known whether Bx affects reproduction through its independent function in ovaries. This paper shows that female flies null for Bx lay eggs with multiple defects. Further, through knock down studies the function of Bx in follicle cells was shown to be required for normal egg development. Function of Bx is particularly required in border cells for Drosophila fertility.

Wednesday, May 23 - Signal transduction

Langerak, S., Kim, M. J., Lamberg, H., Godinez, M., Main, M., Winslow, L., O'Connor, M. B. and Zhu, C. C. (2018). The Drosophila TGF-beta/Activin-like ligands Dawdle and Myoglianin appear to modulate adult lifespan through regulation of 26S proteasome function in adult muscle. Biol Open 7(4). PubMed ID: 29615416
The Drosophila Activin signaling pathway employs at least three separate ligands - Activin-β (Actβ), Dawdle (Daw), and Myoglianin (Myo) - to regulate several general aspects of fruit fly larval development, including cell proliferation, neuronal remodeling, and metabolism. This study provides experimental evidence indicating that both Daw and Myo are anti-ageing factors in adult fruit flies. Knockdown of Myo or Daw reduced mean lifespan, while overexpression of either ligand in adult muscle enhanced mean lifespan. An examination of ubiquitinated protein aggregates in adult muscles revealed a strong inverse correlation between Myo- or Daw-initiated Activin signaling and the amount of ubiquitinated protein aggregates, demonstrating that the lifespan extension effect caused by overexpression of wild-type Daw or Myo in adult muscle tissues can be completely abrogated by knockdown of a 26S proteasome regulatory subunit Rpn1 in adult fly muscle, and that the prolonged lifespan caused by overexpression of Daw or Myo in adult muscle could be due to enhanced protein levels of the key subunits of 26S proteasome.
Liu, C. H., Bollepalli, M. K., Long, S. V., Asteriti, S., Tan, J., Brill, J. A. and Hardie, R. C. (2018). Genetic dissection of the phosphoinositide cycle in Drosophila photoreceptors. J Cell Sci 131(8). PubMed ID: 29567856
Phototransduction in Drosophila is mediated by phospholipase C-dependent hydrolysis of PIP2-, and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in Drosophila photoreceptors. This study used transgenic flies expressing fluorescently tagged probes (P4M and Tb(R332H)), which allow in vivo quantitative measurements of PI4P and PIP2 dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, Drosophila PI4KIIIalpha (CG10260) was identified as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. These results also indicate that PI4KIIIalpha activity requires rbo (the Drosophila orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIalpha activity in yeast and mammals. However, the evidence indicates that the recently reported central role of dPIP5K59B (CG3682) in PIP2 synthesis in the rhabdomeres should be re-evaluated; although PIP2 resynthesis was suppressed by RNAi directed against dPIP5K59B, little or no defect was detected in a reportedly null mutant (dPIP5K(18)).
Lin, C., Top, D., Manahan, C. C., Young, M. W. and Crane, B. R. (2018). Circadian clock activity of cryptochrome relies on tryptophan-mediated photoreduction. Proc Natl Acad Sci U S A 115(15): 3822-3827. PubMed ID: 29581265
Cryptochromes (CRYs) entrain the circadian clocks of plants and animals to light. Irradiation of the Drosophila cryptochrome (dCRY) causes reduction of an oxidized flavin cofactor by a chain of conserved tryptophan (Trp) residues. However, it is unclear how redox chemistry within the Trp chain couples to dCRY-mediated signaling. This study shows that substitutions of four key Trp residues to redox-active tyrosine and redox-inactive phenylalanine tune the light sensitivity of dCRY photoreduction, conformational activation, cellular stability, and targeted degradation of the clock protein Timeless (TIM). An essential surface Trp gates electron flow into the flavin cofactor, but can be relocated for enhanced photoactivation. Differential effects of Trp-mediated flavin photoreduction on cellular turnover of TIM and dCRY indicate that these activities are separated in time and space. Overall, the dCRY Trp chain has evolutionary importance for light sensing, and its manipulation has implications for optogenetic applications of CRYs.
Malzer, E., Dominicus, C. S., Chambers, J. E., Dickens, J. A., Mookerjee, S. and Marciniak, S. J. (2018). The integrated stress response regulates BMP signalling through effects on translation. BMC Biol 16(1): 34. PubMed ID: 29609607
Developmental pathways must be responsive to the environment. Phosphorylation of eIF2alpha enables a family of stress-sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone morphogenetic proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. This study shows in Drosophila that GCN2 antagonises BMP signalling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2alpha phosphatase dPPP1R15 impairs developmental BMP signalling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signalling. Moreover, loss of d4E-BP, a target of crc, augments BMP signalling in vitro and rescues tissue development in vivo. These results identify a novel mechanism by which the ISR modulates BMP signalling during development.
Nowak, K., Gupta, A. and Stocker, H. (2018). FoxO restricts growth and differentiation of cells with elevated TORC1 activity under nutrient restriction. PLoS Genet 14(4): e1007347. PubMed ID: 29677182
TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In Drosophila, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of PTEN mutant tissue under NR. This study demonstrates that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of Rheb is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, Tsc1 mutant cells show decreased PKB activity, and the extent of Tsc1 mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from Tsc1 mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of Tsc1 mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1-in contrast to PTEN-rarely become malignant.
Paul, M. S., Singh, A., Dutta, D., Mutsuddi, M. and Mukherjee, A. (2018). Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling. BMC Res Notes 11(1): 247. PubMed ID: 29661224
Oncogenic potential of Notch signaling and its cooperation with other factors to affect proliferation are widely established. Notch exhibits a cooperative effect with loss of a cell polarity gene, scribble to induce neoplastic overgrowth. Oncogenic Ras also show cooperative effect with loss of cell polarity genes such as scribble, lethal giant larvae (lgl) and discs large to induce neoplastic overgrowth and invasion. This study aims at assessing the cooperation of activated Notch with loss of function of lgl in tumor overgrowth, and the mode of JNK signaling activation in this context. Drosophila was used as an in vivo model to show the synergy between activated Notch (Nact) and loss of function of lgl (lgl-IR) in tumor progression. Coexpression of Nact and lgl-IR results in massive tumor overgrowth and displays hallmarks of cancer, such as MMP1 upregulation and loss of epithelial integrity. Activation of JNK signaling and upregulation of its receptor, Grindelwald in Nact /lgl-IR tumor. In contrast to previously described Nact/scrib-/- tumor, these experiments in Nact/lgl-IR tumor showed the presence of dying cells along with tumorous overgrowth.

Tuesday, May 22 - Stem Cells

Pyo, J. H., Jeon, H. J., Park, J. S., Lee, J. S., Chung, H. Y. and Yoo, M. A. (2018). Drosophila PEBP1 inhibits intestinal stem cell aging via suppression of ERK pathway. Oncotarget 9(26): 17980-17993. PubMed ID: 29719584
The intestine is a high cellular turnover tissue largely dependent on the regenerative function of stem cell throughout life, and a signaling center for the health and viability of organisms. Therefore, better understanding of the mechanisms underlying the regulation of intestinal stem cell (ISC) regenerative potential is essential for the possible intervention of aging process and age-related diseases. Drosophila midgut is a well-established model system for studying the mechanisms underlying ISC regenerative potential during aging. This study reporta the requirement of Drosophila phosphatidylethanolamine binding protein 1 (PEBP1) in ISC regenerative potential. PEBP1 was strongly expressed in enterocytes (ECs) of guts and its decrease with age and oxidative stress. Furthermore, the downregulation of PEBP1 in ECs accelerates ISC aging, as evidenced by ISC hyper-proliferation, gammaH2AX accumulation, and centrosome amplification, and intestinal hyperplasia. The decrease in PEBP1 expression was associated with increased extracellular signal-regulated kinase (ERK) activity in ECs. All these phenotypes by EC-specific depletion of PEBP1 were rescued by the concomitant inhibition of ERK signaling. These findings evidence that the age-related downregulation of PEBP1 in ECs is a novel cause accelerating ISC aging and that PEBP1 is an EC-intrinsic suppressor of epidermal growth factor receptor (EGFR)/ERK signaling. This study provides molecular insights into the tight regulation of EGFR/ERK signaling in niches for stem cell regenerative potential.
Reilein, A., Melamed, D., Tavare, S. and Kalderon, D. (2018). Division-independent differentiation mandates proliferative competition among stem cells. Proc Natl Acad Sci U S A. PubMed ID: 29555768
Cancer-initiating gatekeeper mutations that arise in stem cells would be especially potent if they stabilize and expand an affected stem cell lineage. It is therefore important to understand how different stem cell organization strategies promote or prevent variant stem cell amplification in response to different types of mutation, including those that activate proliferation. Stem cell numbers can be maintained constant while producing differentiated products through individually asymmetrical division outcomes or by population asymmetry strategies in which individual stem cell lineages necessarily compete for niche space. This study considers alternative mechanisms underlying population asymmetry and used quantitative modeling to predict starkly different consequences of altering proliferation rate: A variant, faster proliferating mutant stem cell should compete better only when stem cell division and differentiation are independent processes. For most types of stem cells, it has not been possible to ascertain experimentally whether division and differentiation are coupled. However, Drosophila follicle stem cells (FSCs) provided a favorable system with which to investigate population asymmetry mechanisms and also for measuring the impact of altered proliferation on competition. Detailed cell lineage studies that division and differentiation of an individual FSC were found to be uncoupled. FSC representation, reflecting maintenance and amplification, was highly responsive to genetic changes that altered only the rate of FSC proliferation. The FSC paradigm therefore provides definitive experimental evidence for the general principle that relative proliferation rate will always be a major determinant of competition among stem cells specifically when stem cell division and differentiation are independent.
Park, J. S., Jeon, H. J., Pyo, J. H., Kim, Y. S. and Yoo, M. A. (2018). Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila. Aging (Albany NY). PubMed ID: 29514136
Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. This study showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. Flies were generated with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. These data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. These findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.
Mora, N., Oliva, C., Fiers, M., Ejsmont, R., Soldano, A., Zhang, T. T., Yan, J., Claeys, A., De Geest, N. and Hassan, B. A. (2018). A temporal transcriptional switch governs stem cell division, neuronal numbers, and maintenance of differentiation. Dev Cell 45(1): 53-66.e55. PubMed ID: 29576424
The importance of producing the correct numbers of neurons during development is illustrated by both evolutionary enhancement of cognitive capacities in larger brains, and developmental disorders of brain size. In humans, increased neuronal numbers during development is speculated to partly derive from a unique subtype of neural stem cells (NSCs) that undergo a phase of expansion through symmetric self-amplifying divisions before generating neurons. Symmetric amplification also appears to underlie adult neural stem maintenance in the mouse. However, the mechanisms regulating this behavior are unclear. This study reports the discovery of self-amplifying NSCs in Drosophila and shows that they arise by a spatiotemporal conversion of classical self-renewing NSCs. This conversion is regulated by a temporal transition in the expression of proneural transcription factors prior to cell division. A causal link as found between stem cell self-amplification and increased neuronal numbers. It was further shown that the temporal transcriptional switch controls both stem cell division and subsequent neuronal differentiation.
Obata, F., Tsuda-Sakurai, K., Yamazaki, T., Nishio, R., Nishimura, K., Kimura, M., Funakoshi, M. and Miura, M. (2018). Nutritional control of stem cell division through S-Adenosylmethionine in Drosophila intestine. Dev Cell 44(6): 741-751.e743. PubMed ID: 29587144
The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, S-adenosylmethionine (SAM) was identified as a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. This study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response.
Nagy, P., Sandor, G. O. and Juhasz, G. (2018). Autophagy maintains stem cells and intestinal homeostasis in Drosophila. Sci Rep 8(1): 4644. PubMed ID: 29545557
Intestinal homeostasis is maintained by tightly controlled proliferation and differentiation of tissue-resident multipotent stem cells during aging and regeneration, which ensures organismal adaptation. This study shows that autophagy is required in Drosophila intestinal stem cells to sustain proliferation, and preserves the stem cell pool. Autophagy-deficient stem cells show elevated DNA damage and cell cycle arrest during aging, and are frequently eliminated via JNK-mediated apoptosis. Interestingly, loss of Chk2, a DNA damage-activated kinase that arrests the cell cycle and promotes DNA repair and apoptosis, leads to uncontrolled proliferation of intestinal stem cells regardless of their autophagy status. Chk2 accumulates in the nuclei of autophagy-deficient stem cells, raising the possibility that its activation may contribute to the effects of autophagy inhibition in intestinal stem cells. This study reveals the crucial role of autophagy in preserving proper stem cell function for the continuous renewal of the intestinal epithelium in Drosophila.
Ote, M. and Yamamoto, D. (2018). Enhancing Nanos expression via the bacterial TomO protein is a conserved strategy used by the symbiont Wolbachia to fuel germ stem cell maintenance in infected Drosophila females. Arch Insect Biochem Physiol: e21471. PubMed ID: 29701280
The toxic manipulator of oogenesis (TomO) protein has been identified in the wMel strain of Wolbachia that symbioses with the vinegar fly Drosophila melanogaster, as a protein that affects host reproduction. TomO protects germ stem cells (GSCs) from degeneration, which otherwise occurs in ovaries of host females that are mutant for the gene Sex-lethal (Sxl). TomO homologs were isolated from wPip, a Wolbachia strain from the mosquito Culex quinquefasciatus. One of the homologs, TomOwPip 1, exerted the GSC rescue activity in fly Sxl mutants when lacking its hydrophobic stretches. The GSC-rescuing action of the TomOwPip 1 variant was ascribable to its abilities to associate with Nanos (nos) mRNA and to enhance Nos protein expression. The analysis of structure-activity relationships with TomO homologs and TomO deletion variants revealed distinct modules in the protein that are each dedicated to different functions, i.e., subcellular localization, nos mRNA binding or Nos expression enhancement. It is proposed that modular reshuffling is the basis for structural and functional diversification of TomO protein members.
Otsuki, L. and Brand, A. H. (2018). Cell cycle heterogeneity directs the timing of neural stem cell activation from quiescence. Science 360(6384): 99-102. PubMed ID: 29622651
Quiescent stem cells in adult tissues can be activated for homeostasis or repair. Neural stem cells (NSCs) in Drosophila are reactivated from quiescence in response to nutrition by the insulin signaling pathway. It is widely accepted that quiescent stem cells are arrested in G0. In this study, however, it was demonstrated that quiescent NSCs (qNSCs) are arrested in either G2 or G0 G2-G0 heterogeneity directs NSC behavior: G2 qNSCs reactivate before G0 qNSCs. In addition, it was shown that the evolutionarily conserved pseudokinase Tribbles (Trbl) induces G2 NSCs to enter quiescence by promoting degradation of Cdc25(String) and that it subsequently maintains quiescence by inhibiting Akt activation. Insulin signaling overrides repression of Akt and silences trbl transcription, allowing NSCs to exit quiescence. These results have implications for identifying and manipulating quiescent stem cells for regenerative purposes.

Monday, May 21st - Chromatin

Kolesnikova, T. D., Goncharov, F. P. and Zhimulev, I. F. (2018). Similarity in replication timing between polytene and diploid cells is associated with the organization of the Drosophila genome. PLoS One 13(4): e0195207. PubMed ID: 29659604
Morphologically, polytene chromosomes of Drosophila melanogaster consist of compact "black" bands alternating with less compact "grey" bands and interbands. This study developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, the replication timetable was obtained for all the bands mapped. The results allowed comparison of replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the Drosophila genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in Drosophila.
Lo Piccolo, L., Bonaccorso, R., Attardi, A., Li Greci, L., Romano, G., Sollazzo, M., Giurato, G., Ingrassia, A. M. R., Feiguin, F., Corona, D. F. V. and Onorati, M. C. (2018). Loss of ISWI function in Drosophila nuclear bodies drives cytoplasmic redistribution of Drosophila TDP-43. Int J Mol Sci 19(4). PubMed ID: 29617352
Over the past decade, evidence has identified a link between protein aggregation, RNA biology, and a subset of degenerative diseases. An important feature of these disorders is the cytoplasmic or nuclear aggregation of RNA-binding proteins (RBPs). Redistribution of RBPs, such as the human TAR DNA-binding 43 protein (TDP-43) from the nucleus to cytoplasmic inclusions is a pathological feature of several diseases. Indeed, sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration share as hallmarks ubiquitin-positive inclusions. Recently, the wide spectrum of neurodegenerative diseases characterized by RBPs functions' alteration and loss was collectively named proteinopathies. This study shows that TBPH (TAR DNA-binding protein-43 homolog), the Drosophila ortholog of human TDP-43 TAR DNA-binding protein-43, interacts with the 'architectural RNA' (arcRNA) hsromega and with hsromega-associated hnRNPs. Additionally, it was found that the loss of the omega speckles remodeler ISWI (Imitation SWI) changes the TBPH sub-cellular localization to drive a TBPH cytoplasmic accumulation. These results, hence, identify TBPH as a new component of omega speckles and highlight a role of chromatin remodelers in hnRNPs nuclear compartmentalization.
Rybina, O. Y., Rozovsky, Y. M., Veselkina, E. R. and Pasyukova, E. G. (2018). Polycomb/Trithorax group-dependent regulation of the neuronal gene Lim3 involved in Drosophila lifespan control. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 29555581
Molecular mechanisms governing gene expression and defining complex phenotypes are central to understanding the basics of development and aging. This study demonstrates that naturally occurring polymorphisms of the Lim3 regulatory region that are associated with variation in gene expression and Drosophila lifespan control are located exclusively in the Polycomb response element (PRE). This study found that the Polycomb group (PcG) protein Polycomb (PC) is bound to the PRE only in embryos where Lim3 is present in both repressed and active states. In contrast, the Trithorax group (TrxG) protein Absent, small, or homeotic discs 1 (ASH1) is bound downstream of the PRE, to a region adjacent to the Lim3 transcription start site in embryos and adult flies, in which Lim3 is in an active state. Furthermore, mutations in Pc and ash1 genes affect Lim3 expression depending on the structural integrity of the Lim3 PRE, thus confirming functional interactions between these proteins and Lim3 regulatory region. In addition, this study demonstrated that the evolutionary conserved Lim3 core promoter provides basic Lim3 expression, whereas structural changes in the Lim3 PRE of distal promoter provide stage-, and tissue-specific Lim3 expression. Therefore, it is hypothesized that PcG/TrxG proteins, which are directly involved in Lim3 transcription regulation, participate in lifespan control.
Szabo, Q., Jost, D., Chang, J. M., Cattoni, D. I., Papadopoulos, G. L., Bonev, B., Sexton, T., Gurgo, J., Jacquier, C., Nollmann, M., Bantignies, F. and Cavalli, G. (2018). TADs are 3D structural units of higher-order chromosome organization in Drosophila. Sci Adv 4(2): eaar8082. PubMed ID: 29503869
Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, this study provides an integrative view of chromatin folding in Drosophila. Repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.
Lim, B., Heist, T., Levine, M. and Fukaya, T. (2018). Visualization of transvection in living Drosophila embryos. Mol Cell 70(2): 287-296. PubMed ID: 29606591
How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. This study exploits the properties of transvection, the interaction between an allele on one chromosome and the corresponding allele on the homologous chromosome, to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. The activation of an MS2-tagged reporter gene was successfully visualized by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability-but not the frequency-of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, it was found that a shared enhancer co-activates separate PP7 and MS2 reporter genes in cis and in trans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. It is proposed that transvecting alleles form a trans-homolog "hub," which serves as a scaffold for the accumulation of transcription complexes.
Huang, Y., Zhao, W., Wang, C., Zhu, Y., Liu, M., Tong, H., Xia, Y., Jiang, Q. and Qin, J. (2018). Combinatorial control of recruitment of a variant PRC1.6 complex in embryonic stem cells. Cell Rep 22(11): 3032-3043. PubMed ID: 29539429
Evolutionary Homolog Study

Though genetic data suggest that Polycomb group proteins (PcGs) are central chromatin modifiers and repressors that have been implicated in control of embryonic stem cell (ESC) pluripotency, the precise mechanism of PcG complex recruitment remains elusive, especially in mammals. This study reports that the first and second MBT repeats of L3mbtl2 (see Drosophila L(3)mbt) are important structural and functional features that are necessary and sufficient for L3mbtl2-mediated recruitment of PRC1.6 complex to target promoters. Interestingly, this region of L3mbtl2 harbors the evolutionarily conserved Pho-binding pocket also present in Drosophila Sfmbt, and mutation of the critical residues within this pocket completely abolishes its interaction with target promoters. Additionally, decreased PRC1.6 chromatin occupancy was observed following loss of individual components (L3mbtl2, Pcgf6, and Max) of the complex. These findings suggest that the recruitment of noncanonical PRC1.6 complex in ESCs might be the result of L3mbtl2's interaction with multiple components of the complex.

Friday, May 18th - Signaling

Cruz-Becerra, G., Valerio-Cabrera, S., Juarez, M., Bucio-Mendez, A. and Zurita, M. (2018). TFIIH is highly dynamic during zygotic genome activation in Drosophila and its depletion causes catastrophic mitosis. J Cell Sci [Epub ahead of print]. PubMed ID: 29643118
In Drosophila zygotic genome activation occurs in pre-blastoderm embryos during rapid mitotic divisions. How the transcription machinery is coordinated to achieve this goal in a very brief time span is still poorly understood. TFIIH is fundamental for transcription initiation by RNA polymerase II (RNAPII). This study showed the in vivo dynamics of TFIIH at the onset of transcription in Drosophila embryos. TFIIH shows an oscillatory behaviour between the nucleus and cytoplasm. TFIIH foci are observed from interphase to metaphase, and co-localize with the RNAPII phosphorylated at serine 5 (RNAPIIS5P) at prophase, suggesting that transcription overlaps with the first mitotic phases. Furthermore, embryos with defects in subunits of either the CAK or the core subcomplexes of TFIIH show catastrophic mitosis. Although, transcriptome analyses show altered expression of several maternal genes that participate in mitosis, the global level of RNAPIIS5P in TFIIH mutant embryos is similar to wild type, therefore, a direct role for TFIIH in mitosis cannot be ruled out. These results provide important insights regarding the role of a basal transcription machinery component when the zygotic genome is activated.
Zeng, Q., et al. (2018). The bHLH protein Nulp1 is essential for femur development via acting as a cofactor in Wnt signaling in Drosophila. Curr Mol Med 17(7): 509-517. PubMed ID: 29437009
Nuclear localized protein-1 (Nulp1) might act as a novel bHLH transcriptional factor to mediate cellular functions. However, its role in development in vivo remains unknown. Drosophila Nulp1 (dNulp1) mutants are generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal. Expression of Wg target genes are analyzed by qRT-PCR. The Top-Flash luciferase reporter was used assay to response to Wg signaling. Drosophila Nulp1 (dNulp1) mutants, generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal, are partially homozygous lethal and the rare escapers have bent femurs, which are similar to the major manifestation of congenital bent-bone dysplasia in human Stuve- Weidemann syndrome. The fly phenotype can be rescued by dNulp1 over-expression, indicating that dNulp1 is essential for fly femur development and survival. Moreover, dNulp1 overexpression suppresses the notch wing phenotype caused by the overexpression of sgg/GSK3beta, an inhibitor of the canonical Wnt cascade. Furthermore, qRT-PCR analyses show that seven target genes positively regulated by Wg signaling pathway are down-regulated in response to dNulp1 knockout, while two negatively regulated Wg targets are up-regulated in dNulp1 mutants. Finally, dNulp1 overexpression significantly activates the Top-Flash Wnt signaling reporter. It is concluded that bHLH protein dNulp1 is essential for femur development and survival in Drosophila by acting as a positive cofactor in Wnt/Wingless signaling.
Course, M. M., Scott, A. I., Schoor, C., Hsieh, C. H., Papakyrikos, A. M., Winter, D., Cowan, T. M. and Wang, X. (2018). Phosphorylation of MCAD selectively rescues PINK1 deficiencies in behavior and metabolism. Mol Biol Cell [Epub ahead of print]. PubMed ID: 29563254
PINK1 is a mitochondria-targeted kinase, whose mutations are a cause of Parkinson's disease. This study set out to better understand PINK1's effects on mitochondrial proteins in vivo. Using an unbiased phosphoproteomic screen in Drosophila, it was found that PINK1 mediates the phosphorylation of MCAD, a mitochondrial matrix protein critical to fatty acid metabolism. By mimicking phosphorylation of this protein in a PINK1 null background, PINK1 null's climbing, flight, thorax, and wing deficiencies were restored. Due to MCAD's role in fatty acid metabolism, the metabolic profile of PINK1 null flies were examined; significant disruptions were uncovered in both acylcarnitines and amino acids. Some of these disruptions were rescued by phosphorylation of MCAD, consistent with MCAD's rescue of PINK1 null's organismal phenotypes. This work validates and extends the current knowledge of PINK1, identifies a novel function of MCAD, and illuminates the need for and effectiveness of metabolic profiling in models of neurodegenerative disease.
Yu, J. and Pan, D. (2018). Validating upstream regulators of Yorkie activity in Hippo signaling through scalloped-based genetic epistasis. Development 145(4). PubMed ID: 29467233
Genetic studies in Drosophila have been instrumental in characterizing the Hippo pathway, which converges on the co-activator Yorkie to regulate target gene transcription. A routinely used strategy to interrogate upstream regulators of Yorkie involves the examination of selected Hippo target genes upon loss or gain of function of a suspected pathway regulator. A caveat with this strategy is that aberrant expression of a given Hippo target per se does not distinguish whether it is caused by changes in Yorkie or Yorkie-independent inputs converging on the same target gene. Building on previous findings that the DNA-binding transcription factor Scalloped mediates both Yorkie overexpression and loss-of-function phenotypes yet is itself dispensable for normal eye development, this study describes a simple strategy to distinguish these possibilities by analyzing double-mutant clones of scalloped and a suspected Yorkie regulator. Proof of principle is provided that this strategy can be used effectively to validate canonical Yorkie regulators and to exclude proteins that impact target expression independent of Yorkie. The described methodology and reagents should facilitate efforts to assess the expanding repertoire of proteins implicated in regulation of Yorkie activity.
Gerlach, S. U., Eichenlaub, T. and Herranz, H. (2018). Yorkie and JNK control tumorigenesis in Drosophila cells with cytokinesis failure. Cell Rep 23(5): 1491-1503. PubMed ID: 29719260
Cytokinesis failure may result in the formation of polyploid cells, and subsequent mitosis can lead to aneuploidy and tumor formation. Tumor suppressor mechanisms limiting the oncogenic potential of these cells have been described. However, the universal applicability of these tumor-suppressive barriers remains controversial. This study used Drosophila epithelial cells to investigate the consequences of cytokinesis failure in vivo. Cleavage defects trigger the activation of the JNK pathway, leading to downregulation of the inhibitor of apoptosis DIAP1 and programmed cell death. Yorkie overcomes the tumor-suppressive role of JNK and induces neoplasia. Yorkie regulates the cell cycle phosphatase Cdc25/string, which drives tumorigenesis in a context of cytokinesis failure. These results highlight the functional significance of the JNK pathway in epithelial cells with defective cytokinesis and elucidate a mechanism used by emerging tumor cells to bypass this tumor-suppressive barrier and develop into tumors.
Katanaev, V. L., Egger-Adam, D. and Tomlinson, A. (2018). Antagonistic PCP signaling pathways in the developing Drosophila eye. Sci Rep 8(1): 5741. PubMed ID: 29636485
In Planar cell polarity (PCP), cells coordinately polarize their cytoskeletons within the plane of the epithelium in which they lie. In most insect epithelia this is indicated by the coordinated projections of the hairs secreted by the ectodermal cells. PCP of this form has been effectively studied in Drosophila, but it has proven difficult to achieve an integrated description of the roles played by the various proteins. In the insect eye, PCP is not evident as the polarization of individual cells, but as the asymmetric arrangements of the cells of the ommatidia. This different form of PCP allows different studies to be performed, and using this system the action of two antagonistic signaling pathways (one that promotes the R3 fate and the other that promotes R4) was detected. Even though antagonistic, the two pathways synergize and cooperate to ensure that the correct arrangement of the cells is achieved. The cooperative use of antagonistic signaling pathways occurs in the polarization of chemotacting cells, and the possibility is discussed that a similar molecular principle may underlie PCP.

Thursday, May 17th - Adult CNS development and function

Gruber, L., Rybak, J., Hansson, B. S. and Cantera, R. (2018). Synaptic spinules in the olfactory circuit of Drosophila melanogaster. Front Cell Neurosci 12: 86. PubMed ID: 29636666
his study reports on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. It is proposed that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity.
Humberg, T. H., Bruegger, P., Afonso, B., Zlatic, M., Truman, J. W., Gershow, M., Samuel, A. and Sprecher, S. G. (2018). Dedicated photoreceptor pathways in Drosophila larvae mediate navigation by processing either spatial or temporal cues. Nat Commun 9(1): 1260. PubMed ID: 29593252
To integrate changing environmental cues with high spatial and temporal resolution is critical for animals to orient themselves. Drosophila larvae show an effective motor program to navigate away from light sources. How the larval visual circuit processes light stimuli to control navigational decision remains unknown. The larval visual system is composed of two sensory input channels, Rhodopsin5 (Rh5) and Rhodopsin6 (Rh6) expressing photoreceptors (PRs). This study characterized how spatial and temporal information are used to control navigation. Rh6-PRs are required to perceive temporal changes of light intensity during head casts, while Rh5-PRs are required to control behaviors that allow navigation in response to spatial cues. This study characterized how distinct behaviors are modulated and identify parallel acting and converging features of the visual circuit. Functional features of the larval visual circuit highlight the principle of how early in a sensory circuit distinct behaviors may be computed by partly overlapping sensory pathways.
Croset, V., Treiber, C. D. and Waddell, S. (2018). Cellular diversity in the Drosophila midbrain revealed by single-cell transcriptomics. Elife 7. PubMed ID: 29671739
To understand the brain, molecular details need to be overlaid onto neural wiring diagrams so that synaptic mode, neuromodulation and critical signaling operations can be considered. Single-cell transcriptomics provide a unique opportunity to collect this information. This study presents an initial analysis of thousands of individual cells from Drosophila midbrain, that were acquired using Drop-Seq. A number of approaches permitted the assignment of transcriptional profiles to several major brain regions and cell-types. Expression of biosynthetic enzymes and reuptake mechanisms allows all the neurons to be typed according to the neurotransmitter or neuromodulator that they produce and presumably release. Some neuropeptides are preferentially co-expressed in neurons using a particular fast-acting transmitter, or monoamine. Neuromodulatory and neurotransmitter receptor subunit expression illustrates the potential of these molecules in generating complexity in neural circuit function. This cell atlas dataset provides an important resource to link molecular operations to brain regions and complex neural processes.
Johnson, P. W., Doe, C. Q. and Lai, S. L. (2018). Drosophila nucleostemin 3 is required to maintain larval neuroblast proliferation. Dev Biol. [Epub ahead of print] PubMed ID: 29679561
Stem cells must maintain proliferation during tissue development, repair and homeostasis, yet avoid tumor formation. In Drosophila, neural stem cells (neuroblasts) maintain proliferation during embryonic and larval development and terminate cell cycle during metamorphosis. An important question for understanding how tissues are generated and maintained is: what regulates stem cell proliferation versus differentiation? A genetic screen was performed that identified nucleostemin 3 (ns3) as a gene required to maintain neuroblast proliferation. ns3 is evolutionarily conserved with yeast and human Lsg1, which encode putative GTPases and are essential for organism growth and viability. NS3 is cytoplasmic and it is required to retain the cell cycle repressor Prospero in neuroblast cytoplasm via a Ran-independent pathway. NS3 is also required for proper neuroblast cell polarity and asymmetric cell division. Structure-function analysis further shows that the GTP-binding domain and acidic domain are required for NS3 function in neuroblast proliferation. It is concluded NS3 has novel roles in regulating neuroblast cell polarity and proliferation.
Hsu, I. U., Linsley, J. W., Varineau, J. E., Shafer, O. T. and Kuwada, J. Y. (2018). Dstac is required for normal circadian activity rhythms in Drosophila. Chronobiol Int: 1-11. PubMed ID: 29621409
The genetic, molecular and neuronal mechanism underlying circadian activity rhythms is well characterized in the brain of Drosophila. The small ventrolateral neurons (s-LNVs) and pigment dispersing factor (PDF) expressed by them are especially important for regulating circadian locomotion. This study describes a novel gene, Dstac, which is similar to the stac genes found in vertebrates that encode adaptor proteins, which bind and regulate L-type voltage-gated Ca(2+) channels (CaChs). Dstac is coexpressed with PDF by the s-LNVs and regulates circadian activity. Furthermore, the L-type CaCh, Dmca1D, appears to be expressed by the s-LNVs. Since vertebrate Stac3 regulates an L-type CaCh it is hypothesized that Dstac regulates Dmca1D in s-LNVs and circadian activity.
Groschner, L. N., Chan Wah Hak, L., Bogacz, R., DasGupta, S. and Miesenbock, G. (2018). Dendritic integration of sensory evidence in perceptual decision-making. Cell 173(4): 894-905.e813. PubMed ID: 29706545
Perceptual decisions require the accumulation of sensory information to a response criterion. Most accounts of how the brain performs this process of temporal integration have focused on evolving patterns of spiking activity. This study reports that subthreshold changes in membrane voltage can represent accumulating evidence before a choice. αβ core Kenyon cells (αβc KCs) in the mushroom bodies of fruit flies integrate odor-evoked synaptic inputs to action potential threshold at timescales matching the speed of olfactory discrimination. The forkhead box P transcription factor (FoxP) sets neuronal integration and behavioral decision times by controlling the abundance of the voltage-gated potassium channel Shal (KV4) in αβc KC dendrites. αβc KCs thus tailor, through a particular constellation of biophysical properties, the generic process of synaptic integration to the demands of sequential sampling.

Wednesday, May 16th - Synapses and Vesicles

Xing, X. and Wu, C. F. (2018). Unraveling synaptic GCaMP Signals: Differential excitability and clearance mechanisms underlying distinct Ca(2+) dynamics in tonic and phasic excitatory, and aminergic modulatory motor terminals in Drosophila. eNeuro 5(1). PubMed ID: 29464198
GCaMP is an optogenetic Ca(2+) sensor widely used for monitoring neuronal activities but the precise physiological implications of GCaMP signals remain to be further delineated among functionally distinct synapses. The Drosophila neuromuscular junction (NMJ), a powerful genetic system for studying synaptic function and plasticity, consists of tonic and phasic glutamatergic and modulatory aminergic motor terminals of distinct properties. This study reports a first simultaneous imaging and electric recording study to directly contrast the frequency characteristics of GCaMP signals of the three synapses for physiological implications. Distinct mutational and drug effects on GCaMP signals indicate differential roles of Na(+) and K(+) channels, encoded by genes including paralytic (para), Shaker (Sh), Shab, and ether-a-go-go (eag), in excitability control of different motor terminals. Moreover, the Ca(2+) handling properties reflected by the characteristic frequency dependence of the synaptic GCaMP signals were determined to a large extent by differential capacity of mitochondria-powered Ca(2+) clearance mechanisms. Simultaneous focal recordings of synaptic activities further revealed that GCaMPs were ineffective in tracking the rapid dynamics of Ca(2+) influx that triggers transmitter release, especially during low-frequency activities, but more adequately reflected cytosolic residual Ca(2+) accumulation, a major factor governing activity-dependent synaptic plasticity. These results highlight the vast range of GCaMP response patterns in functionally distinct synaptic types and provide relevant information for establishing basic guidelines for the physiological interpretations of presynaptic GCaMP signals from in situ imaging studies.
Hur, J. H., Lee, S. H., Kim, A. Y. and Koh, Y. H. (2018). Regulation of synaptic architecture and synaptic vesicle pools by Nervous wreck at Drosophila Type 1b glutamatergic synapses. Exp Mol Med 50(3): e462. PubMed ID: 29568072
Nervous wreck (Nwk), a protein that is present at Type 1 glutamatergic synapses that contains an SH3 domain and an FCH motif, is a Drosophila homolog of the human srGAP3/MEGAP protein, which is associated with mental retardation. Confocal microscopy revealed that circles in Nwk reticulum enclosed T-shaped active zones (T-AZs) and partially colocalized with synaptic vesicle (SV) markers and both exocytosis and endocytosis components. Results from an electron microscopic (EM) analysis showed that Nwk proteins localized at synaptic edges and in SV pools. Both the synaptic areas and the number of SVs in the readily releasable (RRPs) and reserve (RPs) SV pools in nwk2 were significantly reduced. Synergistic, morphological phenotypes observed from eag1;;nwk2 neuromuscular junctions suggested that Nwk may regulate synaptic plasticity differently from activity-dependent Hebbian plasticity. Although the synaptic areas in eag1;;nwk2 boutons were not significantly different from those of nwk2, the number of SVs in the RRPs was similar to those of Canton-S. In addition, three-dimensional, high-voltage EM tomographic analysis demonstrated that significantly fewer enlarged SVs were present in nwk2 RRPs. Furthermore, Nwk formed protein complexes with Drosophila Synapsin and Synaptotagmin 1 (DSypt1). Taken together, these findings suggest that Nwk is able to maintain synaptic architecture and both SV size and distribution at T-AZs by interacting with Synapsin and DSypt1.
Fulterer, A., Andlauer, T. F. M., Ender, A., Maglione, M., Eyring, K., Woitkuhn, J., Lehmann, M., Matkovic-Rachid, T., Geiger, J. R. P., Walter, A. M., Nagel, K. I. and Sigrist, S. J. (2018). Active zone scaffold protein ratios tune functional diversity across brain synapses. Cell Rep 23(5): 1259-1274. PubMed ID: 29719243
High-throughput electron microscopy has started to reveal synaptic connectivity maps of single circuits and whole brain regions, for example, in the Drosophila olfactory system. However, efficacy, timing, and frequency tuning of synaptic vesicle release are also highly diversified across brain synapses. These features critically depend on the nanometer-scale coupling distance between voltage-gated Ca(2+) channels (VGCCs) and the synaptic vesicle release machinery. Combining light super resolution microscopy with in vivo electrophysiology, this study shows that two orthogonal scaffold proteins (ELKS family Bruchpilot, BRP, and Syd-1) cluster-specific (M)Unc13 release factor isoforms either close (BRP/Unc13A) or further away (Syd-1/Unc13B) from VGCCs across synapses of the Drosophila olfactory system, resulting in different synapse-characteristic forms of short-term plasticity. Moreover, BRP/Unc13A versus Syd-1/Unc13B ratios were different between synapse types. Thus, variation in tightly versus loosely coupled scaffold protein/(M)Unc13 modules can tune synapse-type-specific release features, and "nanoscopic molecular fingerprints" might identify synapses with specific temporal features.
Xing, G., Li, M., Sun, Y., Rui, M., Zhuang, Y., Lv, H., Han, J., Jia, Z. and Xie, W. (2018). Neurexin-Neuroligin 1 regulates synaptic morphology and function via the WAVE regulatory complex in Drosophila neuromuscular junction. Elife 7. PubMed ID: 29537369
Neuroligins are postsynaptic adhesion molecules that are essential for postsynaptic specialization and synaptic function. But the underlying molecular mechanisms of Neuroligin functions remain unclear. This study found that Drosophila Neuroligin1 (DNlg1) regulates synaptic structure and function through WAVE regulatory complex (WRC)-mediated postsynaptic actin reorganization. The disruption of DNlg1, DNlg2, or their presynaptic partner Neurexin (DNrx) led to a dramatic decrease in the amount of F-actin. Further study showed that DNlg1, but not DNlg2 or DNlg3, directly interacts with the WRC via its C-terminal interacting receptor sequence. That interaction is required to recruit WRC to the postsynaptic membrane to promote F-actin assembly. Furthermore, the interaction between DNlg1 and the WRC is essential for DNlg1 to rescue the morphological and electrophysiological defects in dnlg1 knockout mutants. The results reveal a novel mechanism by which the DNrx-DNlg1 trans-synaptic interaction coordinates structural and functional properties at the neuromuscular junction.
Segal, D., Zaritsky, A., Schejter, E. D. and Shilo, B. Z. (2018). Feedback inhibition of actin on Rho mediates content release from large secretory vesicles. J Cell Biol [Epub ahead of print]. PubMed ID: 29496739
Secretion of adhesive glycoproteins to the lumen of Drosophila melanogaster larval salivary glands is performed by contraction of an actomyosin network assembled around large secretory vesicles, after their fusion to the apical membranes. This study has identified a cycle of actin coat nucleation and disassembly that is independent of myosin. Recruitment of active Rho1 to the fused vesicle triggers activation of the formin Diaphanous and actin nucleation. This leads to actin-dependent localization of a RhoGAP protein that locally shuts off Rho1, promoting disassembly of the actin coat. When contraction of vesicles is blocked, the strict temporal order of the recruited elements generates repeated oscillations of actin coat formation and disassembly. Interestingly, different blocks to actin coat disassembly arrested vesicle contraction, indicating that actin turnover is an integral part of the actomyosin contraction cycle. The capacity of F-actin to trigger a negative feedback on its own production may be widely used to coordinate a succession of morphogenetic events or maintain homeostasis.
Li, X., Goel, P., Chen, C., Angajala, V., Chen, X. and Dickman, D. K. (2018). Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation. Elife 7. PubMed ID: 29620520
Postsynaptic compartments can be specifically modulated during various forms of synaptic plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic homeostatic plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular junction, where a retrograde enhancement of presynaptic neurotransmitter release compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP induction and expression, a genetic manipulation was developed to reduce postsynaptic receptor expression at one of the two muscles innervated by a single motor neuron. PHP can be induced and expressed at a subset of synapses, over both acute and chronic time scales, without influencing transmission at adjacent release sites. Further, homeostatic modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and expression mechanisms are locally transmitted and restricted to specific synaptic compartments.

Tuesday, May 15th - Immune Response

Yadav, S., Frazer, J., Banga, A., Pruitt, K., Harsh, S., Jaenike, J. and Eleftherianos, I. (2018). Endosymbiont-based immunity in Drosophila melanogaster against parasitic nematode infection. PLoS One 13(2): e0192183. PubMed ID: 29466376
Associations between endosymbiotic bacteria and their hosts represent a complex ecosystem within organisms ranging from humans to protozoa. Drosophila species are known to naturally harbor Wolbachia and Spiroplasma endosymbionts, which play a protective role against certain microbial infections. Thhis study investigated whether the presence or absence of endosymbionts affects the immune response of Drosophila melanogaster larvae to infection by Steinernema carpocapsae nematodes carrying or lacking their mutualistic Gram-negative bacteria Xenorhabdus nematophila (symbiotic or axenic nematodes, respectively).The presence of Wolbachia alone or together with Spiroplasma was found to promote the survival of larvae in response to infection with S. carpocapsae symbiotic nematodes, but not against axenic nematodes. Wolbachia numbers are reduced in Spiroplasma-free larvae infected with axenic compared to symbiotic nematodes, and they are also reduced in Spiroplasma-containing compared to Spiroplasma-free larvae infected with axenic nematodes. It was further shown that S. carpocapsae axenic nematode infection induces the Toll pathway in the absence of Wolbachia, and that symbiotic nematode infection leads to increased phenoloxidase activity in D. melanogaster larvae devoid of endosymbionts. Finally, infection with either type of nematode alters the metabolic status and the fat body lipid droplet size in D. melanogaster larvae containing only Wolbachia or both endosymbionts. These results suggest an interaction between Wolbachia endosymbionts with the immune response of D. melanogaster against infection with the entomopathogenic nematodes S. carpocapsae. Results from this study indicate a complex interplay between insect hosts, endosymbiotic microbes and pathogenic organisms.
Sigle, L. T. and Hillyer, J. F. (2018). Eater and draper are involved in the periostial haemocyte immune response in the mosquito Anopheles gambiae. Insect Mol Biol. PubMed ID: 29520896
Haemocytes respond to infection by phagocytosing pathogens, producing the enzymes that drive the phenoloxidase-based melanization cascade, secreting lytic factors, and producing other humoral proteins. A subset of haemocytes, called periostial haemocytes, aggregate on the surface of the heart of mosquitoes and kill pathogens in areas of high haemolymph flow. Periostial haemocytes are always present, but an infection induces the recruitment of additional haemocytes to these regions. This study tested whether members of the Nimrod gene family are involved in the periostial immune response of the African malaria mosquito, Anopheles gambiae. Using organismal manipulations, RNA interference (RNAi) and microscopy, it was shown that, following an infection with Escherichia coli, nimrod - the orthologue of Drosophila NimB2 - is not involved in periostial responses. At 4 h postinfection, however, RNAi-based knockdown of draper results in a marginal increase in the number of periostial haemocytes and a doubling of E. coli accumulation at the periostial regions. Finally, at 24 h postinfection, knockdown of eater decreases the number of periostial haemocytes and decreases the phagocytosis of E. coli on the surface of the heart. Phagocytosis of bacteria is more prevalent in the periostial regions of the mid abdominal segments, and knockdown of draper, nimrod or eater does not alter this distribution. Finally, knockdown of Nimrod family genes did not have a meaningful effect on the accumulation of melanin at the periostial regions. This study identifies roles for eater and draper in the functional integration of the mosquito immune and circulatory systems.
Varjak, M., Dietrich, I., Sreenu, V. B., Till, B. E., Merits, A., Kohl, A. and Schnettler, E. (2018). Spindle-E acts antivirally against Alphaviruses in mosquito Cells. Viruses 10(2). PubMed ID: 29463033
Mosquitoes transmit several human- and animal-pathogenic alphaviruses (Togaviridae family). In alphavirus-infected mosquito cells two different types of virus-specific small RNAs are produced as part of the RNA interference response: short-interfering (si)RNAs and PIWI-interacting (pi)RNAs. The siRNA pathway is generally thought to be the main antiviral pathway. Although an antiviral activity has been suggested for the piRNA pathway its role in host defences is not clear. Knock down of key proteins of the piRNA pathway (Ago3 and Piwi5) in Aedes aegypti-derived cells reduced the production of alphavirus chikungunya virus (CHIKV)-specific piRNAs but had no effect on virus replication. In contrast, knock down of the siRNA pathway key protein Ago2 resulted in an increase in virus replication. Similar results were obtained when expression of Piwi4 was silenced. Knock down of the helicase Spindle-E (SpnE), an essential co-factor of the piRNA pathway in Drosophila melanogaster, resulted in increased virus replication indicating that SpnE acts as an antiviral against alphaviruses such as CHIKV and the related Semliki Forest virus (SFV). Surprisingly, this effect was found to be independent of the siRNA and piRNA pathways in Ae. aegypti cells and specific for alphaviruses. This suggests a small RNA-independent antiviral function for this protein in mosquitoes.
Poirier, E. Z., Goic, B., Tome-Poderti, L., Frangeul, L., Boussier, J., Gausson, V., Blanc, H., Vallet, T., Loyd, H., Levi, L. I., Lanciano, S., Baron, C., Merkling, S. H., Lambrechts, L., Mirouze, M., Carpenter, S., Vignuzzi, M. and Saleh, M. C. (2018). Dicer-2-dependent generation of viral DNA from defective genomes of RNA viruses modulates antiviral immunity in insects. Cell Host Microbe 23(3): 353-365.e358. PubMed ID: 29503180
The RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. This study found that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. It is suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.
Harumoto, T., Fukatsu, T. and Lemaitre, B. (2018). Common and unique strategies of male killing evolved in two distinct Drosophila symbionts. Proc Biol Sci 285(1875). PubMed ID: 29563258
Male killing is a selfish reproductive manipulation caused by symbiotic bacteria, where male offspring of infected hosts are selectively killed. The underlying mechanisms and the process of their evolution are of great interest not only in terms of fundamental biology, but also their potential applications. The two bacterial Drosophila symbionts, Wolbachia and Spiroplasma, have independently evolved male-killing ability. This raises the question whether the underlying mechanisms share some similarities or are specific to each bacterial species. This study analysed pathogenic phenotypes of D. bifasciata infected with its natural male-killing Wolbachia strain and compare them with those of D. melanogaster infected with male-killing Spiroplasma. Male progeny infected with the Wolbachia strain died during embryogenesis with abnormal apoptosis. Interestingly, male-killing Wolbachia infection induces DNA damage and segregation defects in the dosage-compensated chromosome in male embryos, which are reminiscent of the phenotypes caused by male-killing Spiroplasma in D. melanogaster. By contrast, host neural development seems to proceed normally unlike male-killing Spiroplasma infection. These results demonstrate that the dosage-compensated chromosome is a common target of two distinct male killers, yet Spiroplasma uniquely evolved the ability to damage neural tissue of male embryos.
Harumoto, T. and Lemaitre, B. (2018). Male-killing toxin in a bacterial symbiont of Drosophila. Nature. PubMed ID: 29720654
Several lineages of symbiotic bacteria in insects selfishly manipulate host reproduction to spread in a population, often by distorting host sex ratios. Spiroplasma poulsonii is a helical and motile, Gram-positive symbiotic bacterium that resides in a wide range of Drosophila species. A notable feature of S. poulsonii is male killing, whereby the sons of infected female hosts are selectively killed during development. Although male killing caused by S. poulsonii has been studied since the 1950s, its underlying mechanism is unknown. This study identified an S. poulsonii protein, designated Spaid, whose expression induces male killing. Overexpression of Spaid in D. melanogaster kills males but not females, and induces massive apoptosis and neural defects, recapitulating the pathology observed in S. poulsonii-infected male embryos. The data suggest that Spaid targets the dosage compensation machinery on the male X chromosome to mediate its effects. Spaid contains ankyrin repeats and a deubiquitinase domain, which are required for its subcellular localization and activity. Moreover, a laboratory mutant strain of S. poulsonii was found with reduced male-killing ability and a large deletion in the spaid locus. This study has uncovered a bacterial protein that affects host cellular machinery in a sex-specific way, which is likely to be the long-searched-for factor responsible for S. poulsonii-induced male killing.

Monday, May 14th - RNA biology

Zhang, Y Liu, W., Li, R., Gu, J., Wu, P., Peng, C., Ma, J., Wu, L., Yu, Y. and Huang, Y. (2018). Structural insights into the sequence-specific recognition of Piwi by Drosophila Papi. Proc Natl Acad Sci U S A. PubMed ID: 29531043
The Tudor domain-containing (Tdrd) family proteins play a critical role in transposon silencing in animal gonads by recognizing the symmetrically dimethylated arginine (sDMA) on the (G/A)R motif of the N-terminal of PIWI family proteins via the eTud domains. Papi, also known as "Tdrd2," is involved in Zucchini-mediated PIWI-interacting RNA (piRNA) 3'-end maturation. Intriguingly, a recent study showed that, in papi mutant flies, only Piwi-bound piRNAs increased in length, and not Ago3-bound or Aub-bound piRNAs. However, the molecular and structural basis of the Papi-Piwi complex is still not fully understood, which limits mechanistic understanding of the function of Papi in piRNA biogenesis. This study determined the crystal structures of Papi-eTud in the apo form and in complex with a peptide containing unmethylated or dimethylated R10 residues. Structural and biochemical analysis showed that the Papi interaction region on the Drosophila Piwi contains an RGRRR motif (R7-R11) distinct from the consensus (G/A)R motif recognized by canonical eTud. Mass spectrometry results indicated that Piwi is the major binding partner of Papi in vivo. The papi mutant flies suffered from both fertility and transposon-silencing defects, supporting the important role conferred to Papi in piRNA 3' processing through direct interaction with Piwi proteins.
Wakisaka, K. T., Ichiyanagi, K., Ohno, S. and Itoh, M. (2018). Association of zygotic piRNAs derived from paternal P elements with hybrid dysgenesis in Drosophila melanogaster. Mob DNA 9: 7. PubMed ID: 29441132
P-element transposition in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster. Maternally deposited piRNAs suppress P-element transposition in the progeny, linking them to P-M phenotypes; however, the role of zygotic piRNAs derived from paternal P elements is poorly understood. This study investigated the genomic constitution and P-element piRNA production derived from fathers. As a result, males were characterized of naturally derived Q, M' and P strains, which show different capacities for the P-element mobilizations introduced after hybridizations with M-strain females. The amounts of piRNAs produced in ovaries of F1 hybrids varied among the strains and were influenced by the characteristics of the piRNA clusters that harbored the P elements. Importantly, while both the Q- and M'-strain fathers restrict the P-element mobilization in ovaries of their daughters, the Q-strain fathers supported the production of the highest piRNA expression in the ovaries of their daughters, and the M' strain carries KP elements in transcriptionally active regions directing the highest expression of KP elements in their daughters. Interestingly, the zygotic P-element piRNAs, but not the KP element mRNA, contributed to the variations in P transposition immunity in the granddaughters. It is concluded that the piRNA-cluster-embedded P elements and the transcriptionally active KP elements from the paternal genome are both important suppressors of P element activities that are co-inherited by the progeny.
Sriskanthadevan-Pirahas, S., Deshpande, R., Lee, B. and Grewal, S. S. (2018). Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila. PLoS Genet 14(2): e1007202. PubMed ID: 29401457
The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. This study reports on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. Activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. Pol III function was also shown to be required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. The transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead, Ras signalling was shown to promote Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. It is proposed that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.
You, S., Fulga, T. A., Van Vactor, D. and Jackson, F. R. (2018). Regulation of circadian behavior by astroglial microRNAs in Drosophila. Genetics 208(3): 1195-1207. PubMed ID: 29487148
This study describes a genome-wide microRNA (miRNA)-based screen to identify brain glial cell functions required for circadian behavior. To identify glial miRNAs that regulate circadian rhythmicity, a collection of 'miR-sponges' was employed to inhibit miRNA function in a glia-specific manner. The initial screen identified 20 glial miRNAs that regulate circadian behavior. Two miRNAs, miR-263b and miR-274, were studied in detail; both function in adult astrocytes to regulate behavior. Astrocyte-specific inhibition of miR-263b or miR-274 in adults acutely impairs circadian locomotor activity rhythms with no effect on glial or clock neuronal cell viability. To identify potential RNA targets of miR-263b and miR-274, 35 predicted miRNA targets were screenexd, employing RNA interference-based approaches. Glial knockdown of two putative miR-274 targets, CG4328 and MESK2, resulted in significantly decreased rhythmicity. Homology of the miR-274 targets to mammalian counterparts suggests mechanisms that might be relevant for the glial regulation of rhythmicity.
Boukhatmi, H. and Bray, S. (2018). A population of adult satellite-like cells in Drosophila is maintained through a switch in RNA-isoforms. Elife 7. PubMed ID: 29629869
Adult stem cells are important for tissue maintenance and repair. One key question is how such cells are specified and then protected from differentiation for a prolonged period. Investigating the maintenance of Drosophila muscle progenitors (MPs) this study demonstrated that it involves a switch in zfh1/ZEB1 RNA-isoforms. Differentiation into functional muscles is accompanied by expression of miR-8/miR-200, which targets the major zfh1-long RNA isoform and decreases Zfh1 protein. Through activity of the Notch pathway, a subset of MPs produce an alternate zfh1-short isoform, which lacks the miR-8 seed site. Zfh1 protein is thus maintained in these cells, enabling them to escape differentiation and persist as MPs in the adult. There, like mammalian satellite cells, they contribute to muscle homeostasis. Such preferential regulation of a specific RNA isoform, with differential sensitivity to miRs, is a powerful mechanism for maintaining a population of poised progenitors and may be of widespread significance.
Guo, J., Tang, H. W., Li, J., Perrimon, N. and Yan, D. (2018). Xio is a component of the Drosophila sex determination pathway and RNA N(6)-methyladenosine methyltransferase complex. Proc Natl Acad Sci U S A 115(14): 3674-3679. PubMed ID: 29555755
N(6)-methyladenosine (m(6)A), the most abundant chemical modification in eukaryotic mRNA, has been implicated in Drosophila sex determination by modifying Sex-lethal (Sxl) pre-mRNA and facilitating its alternative splicing. This study identified a sex determination gene, CG7358, and rename it xio according to its loss-of-function female-to-male transformation phenotype. xio encodes a conserved ubiquitous nuclear protein of unknown function. Xio colocalizes and interacts with all previously known m(6)A writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15), and loss of xio is associated with phenotypes that resemble other m(6)A factors, such as sexual transformations, Sxl splicing defect, held-out wings, flightless flies, and reduction of m(6)A levels. Thus, Xio encodes a member of the m(6)A methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several m(6)A writer factors, the function of Xio in the m(6)A pathway is likely evolutionarily conserved.

Friday, May 11th - Larval and adult development

Shimell, M., Pan, X., Martin, F. A., Ghosh, A. C., Leopold, P., O'Connor, M. B. and Romero, N. M. (2018). Prothoracicotropic hormone modulates environmental adaptive plasticity through the control of developmental timing. Development 145(6). PubMed ID: 29467242
Adult size and fitness are controlled by a combination of genetics and environmental cues. In Drosophila, growth is confined to the larval phase and final body size is impacted by the duration of this phase, which is under neuroendocrine control. The neuropeptide prothoracicotropic hormone (PTTH) has been proposed to play a central role in controlling the length of the larval phase through regulation of ecdysone production, a steroid hormone that initiates larval molting and metamorphosis. This was tested by examining the consequences of null mutations in the Ptth gene for Drosophila development. Loss of Ptth causes several developmental defects, including a delay in developmental timing, increase in critical weight, loss of coordination between body and imaginal disc growth, and reduced adult survival in suboptimal environmental conditions such as nutritional deprivation or high population density. These defects are caused by a decrease in ecdysone production associated with altered transcription of ecdysone biosynthetic genes. Therefore, the PTTH signal contributes to coordination between environmental cues and the developmental program to ensure individual fitness and survival.
Camuglia, J. M., Mandigo, T. R., Moschella, R., Mark, J., Hudson, C. H., Sheen, D. and Folker, E. S. (2018). An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and myotendinous junction structure. Skelet Muscle 8(1): 12. PubMed ID: 29625624
This study used larval locomotion as an assay to identify novel regulators of skeletal muscle function. This assay was combined with muscle-specific depletion of 82 genes to identify genes that impact muscle function by their expression in muscle cells. It was shown that 12/82 tested genes regulate muscle function. Intriguingly, the disruption of five genes caused an increase in muscle function. The data from this screen was extended, and the mechanism was tested by which the strongest hit, fascin (singed), impacted muscle function. Compared to controls, animals in which singed expression was disrupted with either a mutant allele or muscle-specific expression of RNAi had fewer muscles, smaller muscles, muscles with fewer nuclei, and muscles with disrupted myotendinous junctions. However, expression of RNAi against singed only after the muscle had finished embryonic development did not recapitulate any of these phenotypes. These data suggest that muscle function is reduced due to impaired myoblast fusion, muscle growth, and muscle attachment. Together, these data demonstrate the utility of Drosophila larval locomotion as an assay for the identification of novel regulators of muscle development and implicate fascin as necessary for embryonic muscle development.
Zhu, J., Ordway, A., Weber, L., Buddika, K. and Kumar, J. P. (2018). Polycomb group (Pc-G) proteins and Pax6 cooperate to inhibit in vivo reprogramming of the developing Drosophila eye. Development [Epub ahead of print]. PubMed ID: 29530880
How different cells and tissues commit and determine their fates has been a central question in developmental biology since the seminal embryological experiments conducted by Wilhelm Roux and Hans Driesch in sea urchins and frogs. This study demonstrates that Polycomb group (PcG) proteins maintain Drosophila eye specification by suppressing the activation of alternative fate choices. The loss of PcG in the developing eye results in a cellular reprogramming event in which the eye is redirected to a wing fate. This fate transformation occurs with either the individual loss of Pc or the simultaneous reduction of Pho-repressive complex and Pax6. Interestingly, the requirement for retinal selector genes is limited to Pax6, as the removal of more downstream members does not lead to the eye-wing transformation. Distinct PcG complexes are required during different developmental windows during eye formation. These findings build on earlier observations that the eye can be reprogrammed to initiate head epidermis, antennal, and leg development.
Wang, Y., Berger, J. and Moussian, B. (2018). Trynity models a tube valve in the Drosophila larval airway system. Dev Biol [Epub ahead of print]. PubMed ID: 29518377
Terminal differentiation of an organ is the last step in development that enables the organism to survive in the outside world after birth. Terminal differentiation of the insect tracheae that ends with filling the tubular network with gas is not fully understood at the tissue level. This study demonstrates that yet unidentified valves at the end of the tracheal system of the fruit fly Drosophila melanogaster embryo are important elements allowing terminal differentiation of this organ. Formation of these valves depends on the function of the zona pellucida protein Trynity (Tyn). The tracheae of tyn mutant embryos that lack these structures do not fill with gas. Additionally, external material penetrates into the tracheal tubes indicating that the tyn spiracles are permanently open. It is concluded that the tracheal endings have to be closed to ensure gas-filling. It is speculated that according to physical models closing of the tubular tracheal network provokes initial increase of the internal hydrostatic pressure necessary for gas generation through cavitation when the pressure is subsequently decreased.
Gibert, J. M., Mouchel-Vielh, E. and Peronnet, F. (2018). Pigmentation pattern and developmental constraints: flight muscle attachment sites delimit the thoracic trident of Drosophila melanogaster. Sci Rep 8(1): 5328. PubMed ID: 29593305
In their seminal paper published in 1979, Gould and Lewontin argued that some traits arise as by-products of the development of other structures and not for direct utility in themselves. This study shows that this applies to the trident, a pigmentation pattern observed on the thorax of Drosophila melanogaster. Using reporter constructs, it was shown that the expression domain of several genes encoding pigmentation enzymes follows the trident shape. This domain is complementary to the expression pattern of stripe (sr), which encodes an essential transcription factor specifying flight muscle attachment sites. sr limits the expression of these pigmentation enzyme genes to the trident by repressing them in its own expression domain, i.e. at the flight muscle attachment sites. Evidence is given that repression of not only yellow but also other pigmentation genes, notably tan, is involved in the trident shape. The flight muscle attachment sites and sr expression patterns are remarkably conserved in dipterans reflecting the essential role of sr. The data suggest that the trident is a by-product of flight muscle attachment site patterning that arose when sr was co-opted for the regulation of pigmentation enzyme coding genes.
Schurmann, S., Steffes, G., Manikowski, D., Kastl, P., Malkus, U., Bandari, S., Ohlig, S., Ortmann, C., Rebollido-Rios, R., Otto, M., Nusse, H., Hoffmann, D., Klambt, C., Galic, M., Klingauf, J. and Grobe, K. (2018). Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing. Elife 7. PubMed ID: 29522397
Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, this study shows that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions.

Thursday, May 10th - Drosophila Disease Models

Donnelly, K. M. and Pearce, M. M. P. (2018). Monitoring cell-to-cell transmission of prion-like protein aggregates in Drosophila melanogaster. J Vis Exp(133). PubMed ID: 29578503
Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. A Drosophila melanogaster model was established that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach described in this study can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.
Edenharter, O., Schneuwly, S. and Navarro, J. A. (2018). Mitofusin-dependent ER stress triggers glial dysfunction and nervous system degeneration in a Drosophila model of Friedreich's ataxia. Front Mol Neurosci 11: 38. PubMed ID: 29563863
Friedreich's ataxia (FRDA) is the most important recessive ataxia in the Caucasian population. It is caused by a deficit of the mitochondrial<.a> protein frataxin. A forward genetic screen was carried out to analyze genetic interactions between genes controlling mitochondrial homeostasis and Drosophila frataxin. The screen has identified silencing of Drosophila mitofusin (Marf) as a suppressor of FRDA phenotypes in glia. Drosophila Marf is known to play crucial roles in mitochondrial fusion, mitochondrial degradation and in the interface between mitochondria and endoplasmic reticulum (ER). The results indicated that frataxin-deficiency had a small impact on mitochondrial morphology but enhanced mitochondrial clearance and altered the ER stress response in Drosophila. Remarkably, it was demonstrated that downregulation of Marf suppresses ER stress in frataxin-deficient cells and this is sufficient to improve locomotor dysfunction, brain degeneration and lipid dyshomeostasis in the FRDA model. In agreement, chemical reduction of ER stress by means of two different compounds was sufficient to ameliorate the effects of frataxin deficiency in three different fly FRDA models. Altogether, these results strongly suggest that the protection mediated by Marf knockdown in glia is mainly linked to its role in the mitochondrial-ER tethering and not to mitochondrial dynamics or mitochondrial degradation and that ER stress is a novel and pivotal player in the progression and etiology of FRDA. This work might define a new pathological mechanism in FRDA, linking mitochondrial dysfunction due to frataxin deficiency and mitofusin-mediated ER stress, which might be responsible for characteristic cellular features of the disease and also suggests ER stress as a therapeutic target.
Wen, D. T., Zheng, L., Yang, F., Li, H. Z. and Hou, W. Q. (2018). Endurance exercise prevents high-fat-diet induced heart and mobility premature aging and dsir2 expression decline in aging Drosophila. Oncotarget 9(7): 7298-7311. PubMed ID: 29484111
High-Fat-Diet (HFD)-induced obesity is a major contributor to heart and mobility premature aging and mortality in both Drosophila and humans. The dSir2 genes are closely related to aging, but there are few directed reports showing that whether HFD could inhibit the expression dSir2 genes. Endurance exercise can prevent fat accumulation and reverse HFD-induced cardiac dysfunction. Endurance also delays age-relate functional decline. It is unclear whether lifetime endurance exercise can combat lifetime HFD-induced heart and mobility premature aging, and relieve the harmful HFD-induced influence on the dSir2 gene and lifespan yet. In this study, flies are fed a HFD and trained from when they are 1 week old until they are 5 weeks old. Then, triacylglycerol levels, climbing index, cardiac function, lifespan, and dSir2 mRNA expressions are measured. Endurance exercise was shown to improve climbing capacity, cardiac contraction, and dSir2 expression, and it reduces body and heart triacylglycerol levels, heart fibrillation, and mortality in both HFD and aging flies. So, lifelong endurance exercise delays HFD-induced accelerated age-related locomotor impairment, cardiac dysfunction, death, and dSir2 expression decline, and prevents HFD-induced premature aging in Drosophila.
Zamurrad, S., Hatch, H. A. M., Drelon, C., Belalcazar, H. M. and Secombe, J. (2018). A Drosophila model of intellectual disability caused by mutations in the histone demethylase KDM5. Cell Rep 22(9): 2359-2369. PubMed ID: 29490272
Mutations in KDM5 family histone demethylases cause intellectual disability in humans. However, the molecular mechanisms linking KDM5-regulated transcription and cognition remain unknown. This study establish Drosophila as a model to understand this connection by generating a fly strain harboring an allele in the fly KDM5C homolog Little imaginal discs, analogous to a disease-causing missense mutation in human KDM5C (kdm5A512P). Transcriptome analysis of kdm5A512P flies revealed a striking downregulation of genes required for ribosomal assembly and function and a concomitant reduction in translation. kdm5A512P flies also showed impaired learning and/or memory. Significantly, the behavioral and transcriptional changes in kdm5(A512P) flies were similar to those specifically lacking demethylase activity. These data suggest that the primary defect of the kdm5A512P mutation is a loss of histone demethylase activity and reveal an unexpected role for this enzymatic function in gene activation. Because translation is critical for neuronal function, it is proposed that this defect contributes to the cognitive defects of kdm5A512P flies.
Foriel, S., Beyrath, J., Eidhof, I., Rodenburg, R. J., Schenck, A. and Smeitink, J. A. M. (2018). Feeding difficulties, a key feature of the Drosophila NDUFS4 mitochondrial disease model. Dis Model Mech 11(3). PubMed ID: 29590638
Mitochondrial diseases are associated with a wide variety of clinical symptoms and variable degrees of severity. Patients with such diseases generally have a poor prognosis and often an early fatal disease outcome. With an incidence of 1 in 5000 live births and no curative treatments available, relevant animal models to evaluate new therapeutic regimes for mitochondrial diseases are urgently needed. By knocking down ND-18, the unique Drosophila ortholog of NDUFS4, an accessory subunit of the NADH:ubiquinone oxidoreductase (Complex I), this study developed and characterized several dNDUFS4 models that recapitulate key features of mitochondrial disease. Like in humans, the dNDUFS4 KD flies display severe feeding difficulties, an aspect of mitochondrial disorders that has so far been largely ignored in animal models. The impact of this finding, and an approach to overcome it, are discussed in the context of interpreting disease model characterization and intervention studies.
Bhide, S., Trujillo, A. S., O'Connor, M. T., Young, G. H., Cryderman, D. E., Chandran, S., Nikravesh, M., Wallrath, L. L. and Melkani, G. C. (2018). Increasing autophagy and blocking Nrf2 suppress laminopathy-induced age-dependent cardiac dysfunction and shortened lifespan. Aging Cell: e12747. PubMed ID: 29575479
Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age-dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A-type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age-dependent heart defects are not well understood. This study modeled human disease-causing mutations in the Drosophila Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Simultaneous over-expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies.

Wednesday, May 9th - Adult Nutrition and Physiology

Olufs, Z. P. G., Loewen, C. A., Ganetzky, B., Wassarman, D. A. and Perouansky, M. (2018). Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster. Sci Rep 8(1): 2348. PubMed ID: 29402974
Genetic variability affects the response to numerous xenobiotics but its role in the clinically-observed irregular responses to general anesthetics remains uncertain. To investigate the pharmacogenetics of volatile general anesthetics (VGAs), a Serial Anesthesia Array apparatus was developed to expose multiple Drosophila melanogaster samples to VGAs, and behavioral assays were carried out to determine pharmacokinetic and pharmacodynamic properties of VGAs. The VGAs isoflurane and sevoflurane were studied in four wild type strains from the Drosophila Genetic Reference Panel, two commonly used laboratory strains (Canton S and w1118), and a mutant in Complex I of the mitochondrial electron transport chain (ND2360114). In all seven strains, isoflurane was more potent than sevoflurane, as predicted by their relative lipid solubilities, and emergence from isoflurane was slower than from sevoflurane, reproducing cardinal pharmacokinetic and pharmacodynamic properties in mammals. In addition, ND2360114 flies were more sensitive to both agents, as observed in worms, mice, and humans carrying Complex I mutations. Moreover, substantial variability was found among the fly strains both in absolute and in relative pharmacokinetic and pharmacodynamic profiles of isoflurane and sevoflurane. These data indicate that naturally occurring genetic variations measurably influence cardinal pharmacologic properties of VGAs and that flies can be used to identify relevant genetic variations.
Sannino, D. R., Dobson, A. J., Edwards, K., Angert, E. R. and Buchon, N. (2018). The Drosophila melanogaster gut microbiota provisions thiamine to its host. MBio 9(2). PubMed ID: 29511074
The microbiota of Drosophila melanogaster has a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. This study explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to support D. melanogaster at different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, it was found that the interaction of thiamine concentration and microbiota did not affect the longevity of adult D. melanogaster Likewise, this interplay did not have an impact on egg production. However, it was determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, it was determined that Acetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relative Acetobacter pasteurianus also rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that the D. melanogaster microbiota functions to provision thiamine to its host in a low-thiamine environment.
Obata, F., Fons, C. O. and Gould, A. P. (2018). Early-life exposure to low-dose oxidants can increase longevity via microbiome remodelling in Drosophila. Nat Commun 9(1): 975. PubMed ID: 29515102
Environmental stresses experienced during development exert many long-term effects upon health and disease. For example, chemical oxidants or genetic perturbations that induce low levels of reactive oxygen species can extend lifespan in several species. In some cases, the beneficial effects of low-dose oxidants are attributed to adaptive protective mechanisms such as mitohormesis, which involve long-term increases in the expression of stress response genes. This study shows in Drosophila that transient exposure to low concentrations of oxidants during development leads to an extension of adult lifespan. Surprisingly, this depends upon oxidants acting in an antibiotic-like manner to selectively deplete the microbiome of Acetobacter proteobacteria. The presence of Acetobacter species, such as A. aceti, in the indigenous microbiota increases age-related gut dysfunction and shortens lifespan. This study demonstrates that low-dose oxidant exposure during early life can extend lifespan via microbiome remodelling rather than mitohormesis.
Yamada, T., Habara, O., Kubo, H. and Nishimura, T. (2018). Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila. Development 145(6). PubMed ID: 29467247
Adapting to changes in food availability is a central challenge for survival. Glucose is an important resource for energy production, and therefore many organisms synthesize and retain sugar storage molecules. In insects, glucose is stored in two different forms: the disaccharide trehalose and the branched polymer glycogen. Glycogen is synthesized and stored in several tissues, including in muscle and the fat body. Despite the major role of the fat body as a center for energy metabolism, the importance of its glycogen content remains unclear. This study showed that glycogen metabolism is regulated in a tissue-specific manner under starvation conditions in the fruit fly Drosophila. The mobilization of fat body glycogen in larvae is independent of Adipokinetic hormone (Akh, the glucagon homolog) but is regulated by sugar availability in a tissue-autonomous manner. Fat body glycogen plays a crucial role in the maintenance of circulating sugars, including trehalose, under fasting conditions. These results demonstrate the importance of fat body glycogen as a metabolic safeguard in Drosophila.
Davies, L. R., Schou, M. F., Kristensen, T. N. and Loeschcke, V. (2018). Linking developmental diet to adult foraging choice in Drosophila melanogaster. J Exp Biol [Epub ahead of print]. PubMed ID: 29666197
Rather than maximizing intake of available macronutrients, insects increase intake of some nutrients and restrict intake of others. This selective consumption influences, and potentially optimizes developmental time, reproduction and lifespan of the organism. Studies so far have focused on discriminating between protein and carbohydrate and the consequences on fitness components at different life stages. However, it is largely unknown if and how the developmental diets, which may entail habitat specific nutrient restrictions, affect the selective consumption of adults. Adult female D. melanogaster were shown to opt for the same protein to carbohydrate (P:C) ratio regardless of their developmental diet (P:C ratio of 1:1, 1:4 or 1:8). Males choose a diet that makes up for deficiencies; when protein is low during development, males increase protein consumption despite this being detrimental to starvation resistance. The sexual dimorphism in foraging choice could be due to the different energetic requirements of males and females. To investigate the effect of developmental diet on lifespan once an adult nutritional environment had been established, a no choice experiment was conducted. Here adult lifespan increased as P:C ratio decreased irrespective of developmental diet, thus demonstrating a 'cancelling out' effect of nutritional environment experienced during early life stages. This study provides novel insights into how developmental diet is linked to adult diet by presenting evidence for sexual dimorphism in foraging choice as well as life stage dependency of diet on lifespan.
Dobson, A. J., He, X., Blanc, E., Bolukbasi, E., Feseha, Y., Yang, M. and Piper, M. D. W. (2018). Tissue-specific transcriptome profiling of Drosophila reveals roles for GATA transcription factors in longevity by dietary restriction. NPJ Aging Mech Dis 4: 5. PubMed ID: 29675265
Dietary restriction (DR) extends animal lifespan, but imposes fitness costs. This phenomenon depends on dietary essential amino acids (EAAs) and TOR signalling, which exert systemic effects. However, the roles of specific tissues and cell-autonomous transcriptional regulators in diverse aspects of the DR phenotype are unknown. Manipulating relevant transcription factors (TFs) specifically in lifespan-limiting tissues may separate the lifespan benefits of DR from the early-life fitness costs. This study systematically analysed transcription across organs of Drosophila subjected to DR or low TOR and predict regulatory TFs. Roles were predicted and validated for the evolutionarily conserved GATA family of TFs, and conservation of this signal was identified in mice. Importantly, restricting knockdown of the GATA TF serpent to specific fly tissues recapitulated the benefits but not the costs of DR. Together, these data indicate that the GATA TFs mediate effects of dietary amino acids on lifespan, and that by manipulating them in specific tissues it is possible to reap the fitness benefits of EAAs, decoupled from a cost to longevity.

Tuesday, May 8th - Larval and Adult Neural Development

Gorska-Andrzejak, J., Chwastek, E. M., Walkowicz, L. and Witek, K. (2018). On variations in the level of PER in glial clocks of Drosophila optic lobe and its negative regulation by PDF signaling. Front Physiol 9: 230. PubMed ID: 29615925
The level of the core protein of the circadian clock Period (PER) expressed by glial peripheral oscillators depends on their location in the Drosophila optic lobe. It appears to be controlled by the ventral lateral neurons (LNvs) that release the circadian neurotransmitter Pigment Dispersing Factor (PDF). Glial cells of the distal medulla neuropil (dMnGl) that lie in the vicinity of the PDF-releasing terminals of the LNvs possess receptors for PDF (PDFRs) and express PER at significantly higher level than other types of glia. Surprisingly, the amplitude of PER molecular oscillations in dMnGl is increased twofold in PDF-free environment, that is in Pdf0 mutants. The Pdf0 mutants also reveal an increased level of glia-specific protein REPO in dMnGl. The photoreceptors of the compound eye (R-cells) of the PDF-null flies, on the other hand, exhibit de-synchrony of PER molecular oscillations, which manifests itself as increased variability of PER-specific immunofluorescence among the R-cells. Moreover, the daily pattern of expression of the presynaptic protein Bruchpilot (BRP) in the lamina terminals of the R-cells is changed in Pdf0 mutant. Considering that PDFRs are also expressed by the marginal glia of the lamina that surround the R-cell terminals, the LNv pacemakers appear to be the likely modulators of molecular cycling in the peripheral clocks of both the glial cells and the photoreceptors of the compound eye. Consequently, some form of PDF-based coupling of the glial clocks and the photoreceptors of the eye with the central LNv pacemakers must be operational.
Contreras, E. G., Sierralta, J. and Glavic, A. (2018). p53 is required for brain growth but is dispensable for resistance to nutrient restriction during Drosophila larval development. PLoS One 13(4): e0194344. PubMed ID: 29621246
Animal growth is influenced by the genetic background and the environmental circumstances. How genes promote growth and coordinate adaptation to nutrient availability is still an open question. p53 is a transcription factor that commands the cellular response to different types of stresses. In adult Drosophila melanogaster, p53 regulates the metabolic adaptation to nutrient restriction that supports fly viability. Furthermore, the larval brain is protected from nutrient restriction in a phenomenon called 'brain sparing'. Therefore, it is hypothesised that p53 may regulate brain growth and show a protective role over brain development under nutrient restriction. The function of p53 was studied during brain growth in normal conditions and in animals subjected to developmental nutrient restriction. p53 loss of function was shown to reduce animal growth and larval brain size. Endogenous p53 was expressed in larval neural stem cells, but its levels and activity were not affected by nutritional stress. Interestingly, p53 knockdown only in neural stem cells was sufficient to decrease larval brain growth. Finally, it was shown that in p53 mutant larvae under nutrient restriction, the energy storage levels were not altered, and these larvae generated adults with brains of similar size than wild-type animals. This study has demonstrated that p53 is required for proper growth of the larval brain. This developmental role of p53 does not have an impact on animal resistance to nutritional stress since brain growth in p53 mutants under nutrient restriction is similar to control animals.
Gabilondo, H., Rubio-Ferrera, I., Losada-Perez, M., Del Saz, D., Leon, Y., Molina, I., Torroja, L., D, W. A. and Benito-Sipos, J. (2018). Segmentally homologous neurons acquire two different terminal neuropeptidergic fates in the Drosophila nervous system. PLoS One 13(4): e0194281. PubMed ID: 29634720
This study identified the means by which segmentally homologous neurons acquire different neuropeptide fates in Drosophila. Ventral abdominal (Va)-neurons in the A1 segment of the ventral nerve cord express DH31 and AstA neuropeptides (neuropeptidergic fate I) by virtue of Ubx activity, whereas the A2-A4 Va-neurons express the Capa neuropeptide (neuropeptidergic fate II) under the influence of abdA. These different fates are attained through segment-specific programs of neural subtype specification undergone by segmentally homologous neurons. This is an attractive alternative by which Hox genes can shape Drosophila segmental neural architecture (more sophisticated than the previously identified binary "to live" or "not to live" mechanism). These data refine knowledge of the mechanisms involved in diversifying neuronal identity within the central nervous system.
Bruggemeier, B., Porter, M. A., Vigoreaux, J. O. and Goodwin, S. F. (2018). Female Drosophila melanogaster respond to song-amplitude modulations. Biol Open. PubMed ID: 29666051
Males in numerous animal species use mating songs to attract females and intimidate competitors. This study demonstrates that modulations in song amplitude are behaviourally relevant in the fruit fly Drosophila. D. melanogaster females prefer amplitude modulations typical of melanogaster song over other modulations, which suggests that amplitude modulations are processed auditorily by D. melanogaster. This work demonstrates that receivers can decode messages in amplitude modulations, complementing the recent finding that male flies actively control song amplitude. To describe amplitude modulations, the concept of song amplitude structure (SAS) is proposed and similarities and differences to amplitude modulation with distance (AMD) are discussed.
Alavizargar, A., Berti, C., Ejtehadi, M. R. and Furini, S. (2018). Molecular dynamics simulations of Orai reveal how the third transmembrane segment contributes to hydration and Ca(2+) selectivity in Calcium Release-Activated Calcium Channels. J Phys Chem B 122(16): 4407-4417. PubMed ID: 29600712
Calcium release-activated calcium (CRAC) channels open upon depletion of Ca(2+) from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Molecular dynamics and Brownian dynamics simulations were employed to analyze how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and mutated channels revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270, and Brownian dynamics simulations proved that the altered configuration of residues K270 in the mutated channel impairs selectivity to Ca(2+) over Na(+). The crevices of water molecules, revealed by molecular dynamics simulations, are perfectly located to contribute to the dynamics of the hydrophobic gate and the basic gate, suggesting a possible role in channel opening and in selectivity function.
DeVault, L., Li, T., Izabel, S., Thompson-Peer, K. L., Jan, L. Y. and Jan, Y. N. (2018). Dendrite regeneration of adult Drosophila sensory neurons diminishes with aging and is inhibited by epidermal-derived matrix metalloproteinase 2. Genes Dev 32(5-6): 402-414. PubMed ID: 29563183
Dendrites possess distinct structural and functional properties that enable neurons to receive information from the environment as well as other neurons. Despite their key role in neuronal function, current understanding of the ability of neurons to regenerate dendrites is lacking. This study characterizes the structural and functional capacity for dendrite regeneration in vivo in adult animals and examines the effect of neuronal maturation on dendrite regeneration. Focus was placed on the class IV dendritic arborization (c4da) neuron of the Drosophila sensory system, which has a dendritic arbor that undergoes dramatic remodeling during the first 3 d of adult life and then maintains a relatively stable morphology thereafter. Using a laser severing paradigm, regeneration was monitored after acute and spatially restricted injury. The capacity for regeneration was found to be present in adult neurons but diminished as the animal aged. Regenerated dendrites recovered receptive function. Furthermore, it was found that the regenerated dendrites show preferential alignment with the extracellular matrix (ECM). Finally, inhibition of ECM degradation by inhibition of matrix metalloproteinase 2 (Mmp2) to preserve the extracellular environment characteristics of young adults led to increased dendrite regeneration. These results demonstrate that dendrites retain regenerative potential throughout adulthood and that regenerative capacity decreases with aging.

Monday, May 7th - Embryonic CNS Development

Bonneaud, N., Layalle, S., Colomb, S., Jourdan, C., Ghysen, A., Severac, D., Dantec, C., Negre, N. and Maschat, F. (2017). Control of nerve cord formation by Engrailed and Gooseberry-neuro: A multi-step, coordinated process. Dev Biol. PubMed ID: 29097190
One way to better understand the molecular mechanisms involved in the construction of a nervous system is to identify the downstream effectors of major regulatory proteins. It has been shown that Engrailed (EN) and Gooseberry-Neuro (GsbN) transcription factors act in partnership to drive the formation of posterior commissures in the central nervous system of Drosophila. This report identifies genes regulated by both EN and GsbN through chromatin immunoprecipitation ("ChIP on chip") and transcriptome experiments, combined to a genetic screen relied to the gene dose titration method. The genomic-scale approaches allowed definition of 175 potential targets of EN-GsbN regulation. A subset of these genes was chosen to examine ventral nerve cord (VNC) defects; half of the mutated targets show clear VNC phenotypes when doubly heterozygous with en or gsbn mutations, or when homozygous. This strategy revealed new groups of genes never described for their implication in the construction of the nerve cord. Their identification suggests that, to construct the nerve cord, EN-GsbN may act at three levels, in: (1) sequential control of the attractive-repulsive signaling that ensures contralateral projection of the commissural axons, (2) temporal control of the translation of some mRNAs, (3) regulation of the capability of glial cells to act as commissural guideposts for developing axons. These results illustrate how an early, coordinated transcriptional control may orchestrate the various mechanisms involved in the formation of stereotyped neuronal networks. They also validate the overall strategy to identify genes that play crucial role in axonal pathfinding.
Rickert, C., Luer, K., Vef, O. and Technau, G. M. (2018). Progressive derivation of serially homologous neuroblast lineages in the gnathal CNS of Drosophila. PLoS One 13(2): e0191453. PubMed ID: 29415052
Along the anterior-posterior axis the central nervous system is subdivided into segmental units (neuromeres) the composition of which is adapted to their region-specific functional requirements. In Drosophila melanogaster each neuromere is formed by a specific set of identified neural stem cells (neuroblasts, NBs). In the thoracic and anterior abdominal region of the embryonic ventral nerve cord segmental sets of NBs resemble the ground state (2nd thoracic segment, which does not require input of homeotic genes), and serial (segmental) homologs generate similar types of lineages. The three gnathal head segments form a transitional zone between the brain and the ventral nerve cord. It has been shown recently that although all NBs of this zone are serial homologs of NBs in more posterior segments, they progressively differ from the ground state in anterior direction (labial > maxillary > mandibular segment) with regard to numbers and expression profiles. To study the consequences of their derived characters the embryonic lineages of gnathal NBs were traced using the Flybow and DiI-labelling techniques. For a number of clonal types serial homology is rather clearly reflected by their morphology (location and projection patterns) and cell specific markers, despite of reproducible segment-specific differences. However, many lineages, particularly in the mandibular segment, show a degree of derivation that impedes their assignment to ground state serial homologs. These findings demonstrate that differences in gene expression profiles of gnathal NBs go along with anteriorly directed progressive derivation in the composition of their lineages. Furthermore, lineage sizes decrease from labial to mandibular segments, which in concert with decreasing NB-numbers lead to reduced volumes of gnathal neuromeres, most significantly in the mandibular segment.
Arbeille, E. and Bashaw, G. J. (2018). Brain Tumor promotes axon growth across the midline through interactions with the microtubule stabilizing protein Apc2. PLoS Genet 14(4): e1007314. PubMed ID: 29617376
Commissural axons must cross the midline to establish reciprocal connections between the two sides of the body. This process is highly conserved between invertebrates and vertebrates and depends on guidance cues and their receptors to instruct axon trajectories. The DCC family receptor Frazzled (Fra) signals chemoattraction and promotes midline crossing in response to its ligand Netrin. However, in Netrin or fra mutants, the loss of crossing is incomplete, suggesting the existence of additional pathways. This study has identified Brain Tumor (Brat), a tripartite motif protein, as a new regulator of midline crossing in the Drosophila CNS. Genetic analysis indicates that Brat acts independently of the Netrin/Fra pathway. In addition, through its B-Box domains, Brat acts cell autonomously to regulate the expression and localization of Adenomatous polyposis coli-2 (Apc2), a key component of the Wnt canonical signaling pathway, to promote axon growth across the midline. Genetic evidence indicates that the role of Brat and Apc2 to promote axon growth across the midline is independent of Wnt and Beta-catenin-mediated transcriptional regulation. Instead, it is proposed that Brat promotes midline crossing through directing the localization or stability of Apc2 at the plus ends of microtubules in navigating commissural axons. These findings define a new mechanism in the coordination of axon growth and guidance at the midline.
Yaghmaeian Salmani, B., Monedero Cobeta, I., Rakar, J., Bauer, S., Curt, J. R., Starkenberg, A. and Thor, S. (2018). Evolutionarily conserved anterior expansion of the central nervous system promoted by a common PcG-Hox program. Development. PubMed ID: 29530878
A conserved feature of the central nervous system (CNS) is the prominent expansion of anterior regions (brain) when compared to posterior (nerve cord). The cellular and regulatory processes driving anterior CNS expansion are not well understood in any bilaterian species. This expansion was addressed in Drosophila and mouse. Compared to the nerve cord, the brain, in both Drosophila and mouse, displays extended progenitor proliferation, more elaborate daughter cell proliferation and more rapid cell cycle speed. These features contribute to anterior CNS expansion in both species. With respect to genetic control, enhanced brain proliferation is severely reduced by ectopic Hox gene expression, by either Hox misexpression or by loss of Polycomb Group (PcG) function. Strikingly, in PcG mutants, early CNS proliferation appears unaffected, whereas subsequently, brain proliferation is severely reduced. Hence, a conserved PcG-Hox program promotes the anterior expansion of the CNS. The profound differences in proliferation and in the underlying genetic mechanisms between brain and nerve cord lend support to the emerging concept of separate evolutionary origins of these two CNS regions.
Alvarez, J. A. and Diaz-Benjumea, F. J. (2018). Origin and specification of type II neuroblasts in the Drosophila embryo. Development 145(7). PubMed ID: 29567672
In Drosophila, neural stem cells or neuroblasts (NBs) acquire different identities according to their site of origin in the embryonic neuroectoderm. Their identity determines the number of times they will divide and the types of daughter cells they will generate. All NBs divide asymmetrically, with type I NBs undergoing self-renewal and generating another cell that will divide only once more. By contrast, a small set of NBs in the larval brain, type II NBs, divides differently, undergoing self-renewal and generating an intermediate neural progenitor (INP) that continues to divide asymmetrically several more times, generating larger lineages. This study analysed the origin of type II NBs and how they are specified. The results indicate that these cells originate in three distinct clusters in the dorsal protocerebrum during stage 12 of embryonic development. Moreover, it appears that their specification requires the combined action of EGFR signalling and the activity of the related genes buttonhead and Drosophila Sp1. In addition, it was also shown that the INPs generated in the embryo enter quiescence at the end of embryogenesis, resuming proliferation during the larval stage.
Dinges, N., Morin, V., Kreim, N., Southall, T. D. and Roignant, J. Y. (2017). Comprehensive characterization of the complex lola locus reveals a novel role in the octopaminergic pathway via tyramine beta-Hydroxylase regulation. Cell Rep 21(10): 2911-2925. PubMed ID: 29212035
Longitudinals lacking (lola) is one of the most complex genes in Drosophila melanogaster, encoding up to 20 protein isoforms that include key transcription factors involved in axonal pathfinding and neural reprogramming. Most previous studies have employed loss-of-function alleles that disrupt lola common exons, making it difficult to delineate isoform-specific functions. To overcome this issue, this study generated isoform-specific mutants for all isoforms using CRISPR/Cas9. This enabled study of specific isoforms with respect to previously characterized roles for Lola and to demonstrate a specific function for one variant in axon guidance via activation of the microtubule-associated factor Futsch. Importantly, a role was revealed for a second variant in preventing neurodegeneration via the positive regulation of a key enzyme of the octopaminergic pathway. Thus, this comprehensive study expands the functional repertoire of Lola functions, and it adds insights into the regulatory control of neurotransmitter expression in vivo.

Friday, May 4th - Adult Neural Function

Yu, D., Tan, Y., Chakraborty, M., Tomchik, S. and Davis, R. L. (2018). Elongator complex is required for long-term olfactory memory formation in Drosophila. Learn Mem 25(4): 183-196. PubMed ID: 29545390
The evolutionarily conserved Elongator Complex associates with RNA polymerase II for transcriptional elongation. Elp3 is the catalytic subunit, contains histone acetyltransferase activity, and is associated with neurodegeneration in humans. Elp1 is a scaffolding subunit and when mutated causes familial dysautonomia. This study shows that elp3 and elp1 are required for aversive long-term olfactory memory in Drosophila. RNAi knockdown of elp3 in adult mushroom bodies impairs long-term memory (LTM) without affecting earlier forms of memory. RNAi knockdown with coexpression of elp3 cDNA reverses the impairment. Similarly, RNAi knockdown of elp1 impairs LTM and coexpression of elp1 cDNA reverses this phenotype. The LTM deficit in elp3 and elp1 knockdown flies is accompanied by the abolishment of a LTM trace, which is registered as increased calcium influx in response to the CS+ odor in the alpha-branch of mushroom body neurons. Coexpression of elp1 or elp3 cDNA rescues the memory trace in parallel with LTM. These data show that the Elongator complex is required in adult mushroom body neurons for long-term behavioral memory and the associated long-term memory trace.
Zhang, X., Li, Q., Wang, L., Liu, Z. J. and Zhong, Y. (2018). Active Protection: Learning-Activated Raf/MAPK Activity Protects Labile Memory from Rac1-Independent Forgetting. Neuron [Epub ahead of print]. PubMed ID: 29551489
Active forgetting explains the intrinsic instability of a labile memory lasting for hours. However, how such memory maintains stability against unwanted disruption is not completely understood. This study reports a learning-activated active protection mechanism that enables labile memory to resist disruptive sensory experiences in Drosophila. Aversive olfactory conditioning activates mitogen-activated protein kinase (MAPK) transiently in the mushroom-body gamma lobe, where labile-aversive memory is stored. This increased MAPK activity significantly prolongs labile memory retention and enhances its resistance to disruption induced by heat shock, electric shock, or odor reactivation. Such experience-induced forgetting cannot be prevented by inhibition of Rac1 activity. Instead, protection of Rac1-independent forgetting correlates with non-muscle myosin II activity and persistence of learning-induced presynaptic structural changes. Increased Raf/MAPK activity, together with suppressed Rac1 activity, completely blocks labile memory decay. Thus, learning not only leads to memory formation, but also activates active protection and active forgetting to regulate the formed memory.
Zhang, B., Sato, K. and Yamamoto, D. (2018). Ecdysone signaling regulates specification of neurons with a male-specific neurite in Drosophila. Biol Open 7(2). PubMed ID: 29463514
Some mAL neurons in the male brain form the ipsilateral neurite (ILN[+]) in a manner dependent on FruBM, a male-specific transcription factor. FruBM represses robo1 transcription, allowing the ILN to form. The proportion of ILN[+]-mALs in all observed single cell clones dropped from approximately 90% to approximately 30% by changing the heat-shock timing for clone induction from 4-5 days after egg laying (AEL) to 6-7 days AEL, suggesting that the ILN[+]-mALs are produced predominantly by young neuroblasts. Upon EcR-A knockdown, ILN[+]-mALs were produced at a high rate (approximately 60%), even when heat shocked at 6-7 days AEL, yet EcR-B1 knockdown reduced the proportion of ILN[+]-mALs to approximately 30%. Immunoprecipitation assays in S2 cells demonstrated that EcR-A and EcR-B1 form a complex with FruBM. robo1 reporter transcription was repressed by FruBM and ecdysone counteracted FruBM. It is suggested that ecdysone signaling modulates the FruBM action to produce an appropriate number of male-type neurons.
Wu, C. L., Chang, C. C., Wu, J. K., Chiang, M. H., Yang, C. H. and Chiang, H. C. (2018). Mushroom body glycolysis is required for olfactory memory in Drosophila. Neurobiol Learn Mem 150: 13-19. PubMed ID: 29477608
Glucose catabolism, also known as glycolysis, is important for energy generation and involves a sequence of enzymatic reactions that convert a glucose molecule into two pyruvate molecules. The glycolysis process generates adenosine triphosphate as a byproduct. This study investigated whether glycolysis plays a role in maintaining neuronal functions in the Drosophila mushroom bodies (MBs), which are generally accepted to be an olfactory learning and memory center. The data showed that individual knockdown of glycolytic enzymes in the MBs, including hexokinase (HexA), phosphofructokinase (Pfk), or pyruvate kinase (PyK), disrupts olfactory memory. Whole-mount brain immunostaining indicated that pyruvate kinase is strongly expressed in the MB alphabeta, α'β', and γ neuron subsets. It is concluded that HexA, Pfk, and PyK are required in each MB neuron subset for olfactory memory formation. The data therefore indicates that glucose catabolism in the MBs is important for olfactory memory formation in Drosophila.
Yamazaki, D., Hiroi, M., Abe, T., Shimizu, K., Minami-Ohtsubo, M., Maeyama, Y., Horiuchi, J. and Tabata, T. (2018). Two parallel pathways assign opposing odor valences during Drosophila memory formation. Cell Rep 22(9): 2346-2358. PubMed ID: 29490271
During olfactory associative learning in Drosophila, odors activate specific subsets of intrinsic mushroom body (MB) neurons. Coincident exposure to either rewards or punishments is thought to activate extrinsic dopaminergic neurons, which modulate synaptic connections between odor-encoding MB neurons and MB output neurons to alter behaviors. However, this study identifies two classes of intrinsic MB γ neurons based on cAMP response element (CRE)-dependent expression, γCRE-p and γCRE-n, which encode aversive and appetitive valences. γCRE-p and γCRE-n neurons act antagonistically to maintain neutral valences for neutral odors. Activation or inhibition of either cell type upsets this balance, toggling odor preferences to either positive or negative values. The mushroom body output neurons, MBON-;gamma'5β'2a/&beta'2mp and MBON-γ2α'1, mediate the actions of γCRE-p and γCRE-n neurons. The data indicate that MB neurons encode valence information, as well as odor information, and this information is integrated through a process involving MBONs to regulate learning and memory.
Zanini, D., Giraldo, D., Warren, B., Katana, R., Andres, M., Reddy, S., Pauls, S., Schwedhelm-Domeyer, N., Geurten, B. R. H. and Gopfert, M. C. (2018). Proprioceptive Opsin Functions in Drosophila Larval Locomotion. Neuron. PubMed ID: 29551493
Animals rely on mechanosensory feedback from proprioceptors to control locomotory body movements. Unexpectedly, this study found that this movement control requires visual opsins. Disrupting the Drosophila opsins NINAE or Rh6 impaired larval locomotion and body contractions, independently of light and vision. Opsins were detected in chordotonal proprioceptors along the larval body, localizing to their ciliated dendrites. Loss of opsins impaired mechanically evoked proprioceptor spiking and cilium ultrastructure. Without NINAE or Rh6, NOMPC mechanotransduction channels leaked from proprioceptor cilia and ciliary Inactive (Iav) channels partly disappeared. Locomotion is shown to require opsins in proprioceptors, and the receptors are found to express the opsin gene Rh7, in addition to ninaE and Rh6. Besides implicating opsins in movement control, this documents roles of non-ciliary, rhabdomeric opsins in cilium organization, providing a model for a key transition in opsin evolution and suggesting that structural roles of rhabdomeric opsins preceded their use for light detection.

Thursday, May 3rd - Cytoskeleton and Junctions

Tingler, M., Kurz, S., Maerker, M., Ott, T., Fuhl, F., Schweickert, A., LeBlanc-Straceski, J. M., Noselli, S. and Blum, M. (2018). A conserved role of the Unconventional Myosin 1d in laterality determination. Curr Biol 28(5): 810-816.e813. PubMed ID: 29478852
Evolutionary Homolog Study
Anatomical and functional asymmetries are widespread in the animal kingdom. In vertebrates, many visceral organs are asymmetrically placed. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question. Nodal signaling is widely used, and many, but not all, vertebrates use cilia for symmetry breaking. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextral rotation of chiral organs. The role of myo1d in left-right (LR) axis formation was studied in Xenopus. Morpholino oligomer-mediated myo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila, acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited in myo1d morphants, and the ATF2 reporter gene for non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, these data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry.
Tubman, E., He, Y., Hays, T. S. and Odde, D. J. (2018). Kinesin-5 mediated chromosome congression in insect spindles. Cell Mol Bioeng 11(1): 25-36. PubMed ID: 29552234
The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment "congression" at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, this study sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles. RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Drosophila melanogaster S2 cells to test for inhibited chromosome congression. Klp61F RNAi, Klp67A RNAi, and control metaphase mitotic spindles expressing fluorescent tubulin and fluorescent Cid were imaged, and their fluorescence distributions were compared. RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression. This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis.
Walther, R. F., Burki, M., Pinal, N., Rogerson, C. and Pichaud, F. (2018). Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor. J Cell Sci. PubMed ID: 29507112
In Drosophila epithelial cells, apical exclusion of Bazooka/Par3 defines the position of the Zonula Adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. This study shows that the small GTPase Rap1, its effector AF6/Canoe (Cno) and the Cdc42-effector Pak4/Mushroom bodies tiny (Mbt), converge in regulating epithelial E-Cadherin, and Bazooka retention at the ZA. Furthermore, the results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating their functions during ZA morphogenesis are interlinked. In this context, the Rap1-GEF Dizzy was found to be enriched at the ZA and the results suggest it promotes Rap1 activity during ZA morphogenesis. Altogether, it is proposed the Dizzy, Rap1/Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis.
Verboon, J. M., Decker, J. R., Nakamura, M. and Parkhurst, S. M. (2018). Wash exhibits context dependent phenotypes and, along with the WASH Regulatory Complex, regulates Drosophila oogenesis. J Cell Sci. PubMed ID: 29549166
WASH, a Wiskott Aldrich Syndrome (WAS) family protein, has many cell and developmental roles related to its function as a branched actin nucleation factor. Similar to mammalian WASH, which is embryonic lethal, Drosophila Wash was found to be essential for oogenesis and larval development. Recently, however, Drosophila wash was reported to be homozygous viable. This study verified that the original wash null allele harbors an unrelated lethal background mutation, however, this unrelated lethal mutation does not contribute to any Wash oogenesis phenotypes. Significantly, it was found that the homozygous wash null allele retains partial lethality leading to non-Mendelian inheritance, that this allele's functions are subject to its specific genetic background, and that the homozygous stock rapidly accumulates modifications that allow it to become robust. Accordingly, Wash was found to play an important role in oogenesis and also that this role involves the WASH Regulatory Complex. Finally, this study showed that another WAS protein, SCAR/WAVE, plays a similar role in oogenesis and that it is upregulated as one of the modifications that allows the wash allele to survive in the homozygous state.
Wang, H., Qiu, Z., Xu, Z., Chen, S. J., Luo, J., Wang, X. and Chen, J. (2018). aPKC is a key polarity molecule coordinating the function of three distinct cell polarities during collective migration. Development. PubMed ID: 29636381
Apical-basal polarity is a hallmark of epithelia and it needs to be remodeled when epithelial cells undergo morphogenetic cell movements. This study used border cells in Drosophila ovary to address how the apical-basal polarity is remodeled and turned into front-back, apical-basal and inside-outside polarities, during collective migration. Crumbs (Crb) complex is required for the generation of the three distinct but inter-connected cell polarities of border cells. Specifically, Crb complex, together with Par complex and the endocytic recycling machinery, ensures a strict distribution control of two distinct populations of aPKC at the inside apical junction and near the outside lateral membrane respectively. Interestingly, aPKC distributed near the outside lateral membrane interacts with Tiam1/Sif and promotes the Rac-induced protrusions, whereas alteration of the aPKC distribution pattern changed protrusion formation pattern, leading to disruption of all three polarities. Therefore, this study demonstrates that aPKC, spatially controlled by Crb complex, is a key polarity molecule coordinating the generation of three distinct but inter-connected cell polarities during collective migration.
Yadav, R., Nisha and Sarkar, S. (2018). Drosophila globin1 is required for maintenance of the integrity of F-actin based cytoskeleton during development. Exp Cell Res. PubMed ID: 29524391
Hemoglobins (Hbs) are evolutionarily conserved small globular proteins with characteristic 3-over-3 alpha-helical sandwich structure that is typically known as "globin fold". Hbs have been found to be involved in diverse biological functions and the characteristic property of oxygen transportation is relatively a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2, and glob3) and it was previously reported that adequate expression of glob1 is required for various aspects of development, and also to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of glob1 gene in Drosophila development. A dynamic expression pattern of glob1 in larval tissues is largely concentrated around F-actin rich structures and also co-precipitates. Reduced expression of glob1 leads to developmental abnormalities which appeared to be largely mediated by inappropriately formed F-actin based cytoskeletal structures. Subsequent analysis in FLP/FRT mediated somatic clones establishes specific role of Drosophila glob1 in maintenance of the integrity of F-actin based cytoskeleton during development. For the first time, interaction is reported between Glob1 and actin, and a novel role of glob1 is proposed in maintenance of F-actin based cytoskeleton in Drosophila.

Wednesday, May 2nd - Adult Behavior

Tapanainen, R., Parker, D. J. and Kankare, M. (2018). Photosensitive alternative splicing of the circadian clock gene timeless is population specific in a cold-adapted Fly, Drosophila montana. G3 (Bethesda). PubMed ID: 29472309
To function properly, organisms must adjust their physiology, behavior and metabolism in response to a suite of varying environmental conditions. One of the central regulators of these changes is organisms' internal circadian clock, and recent evidence has suggested that the clock genes are also important in the regulation of seasonal adjustments. In particular, thermosensitive splicing of the core clock gene timeless in a cosmopolitan fly, Drosophila melanogaster, has implicated this gene to be involved in thermal adaptation. To further investigate this link, the splicing of timeless was examined in a northern malt fly species, Drosophila montana, which can withstand much colder climatic conditions than its southern relative. Northern and southern populations from two different continents (North America and Europe) were examined to find out whether and how the splicing of this gene varies in response to different temperatures and day lengths. Interestingly, it was found that the expression of timeless splice variants was sensitive to differences in light conditions, and while the flies of all study populations showed a change in the usage of splice variants in constant light compared to LD 22:2, the direction of the shift varied between populations. Overall, these findings suggest that the splicing of timeless in northern Drosophila montana flies is photosensitive, rather than thermosensitive and highlights the value of studying multiple species and populations in order to gain perspective on the generality of gene function changes in different kinds of environmental conditions.
Yadlapalli, S., Jiang, C., Bahle, A., Reddy, P., Meyhofer, E. and Shafer, O. T. (2018). Circadian clock neurons constantly monitor environmental temperature to set sleep timing. Nature 555(7694): 98-102. PubMed ID: 29466329
Circadian clocks coordinate behaviour, physiology and metabolism with Earth's diurnal cycle. These clocks entrain to both light and temperature cycles, and daily environmental temperature oscillations probably contribute to human sleep patterns. However, the neural mechanisms through which circadian clocks monitor environmental temperature and modulate behaviour remain poorly understood. This study has elucidate how the circadian clock neuron network of Drosophila melanogaster processes changes in environmental temperature. In vivo calcium-imaging techniques demonstrate that the posterior dorsal neurons 1 (DN1ps), which are a discrete subset of sleep-promoting clock neurons, constantly monitor modest changes in environmental temperature. These neurons are acutely inhibited by heating and excited by cooling; this is an unexpected result when considering the strong correlation between temperature and light, and the fact that light excites clock neurons. The DN1ps rely on peripheral thermoreceptors located in the chordotonal organs and the aristae. The DN1ps and their thermosensory inputs are required for the normal timing of sleep in the presence of naturalistic temperature cycles. These results identify the DN1ps as a major gateway for temperature sensation into the circadian neural network, which continuously integrates temperature changes to coordinate the timing of sleep and activity.
Li, X., Ishimoto, H. and Kamikouchi, A. (2018). Auditory experience controls the maturation of song discrimination and sexual response in Drosophila. Elife 7. PubMed ID: 29555017
In birds and higher mammals, auditory experience during development is critical to discriminate sound patterns in adulthood. However, the neural and molecular nature of this acquired ability remains elusive. In fruit flies, acoustic perception has been thought to be innate. This study reports, surprisingly, that auditory experience of a species-specific courtship song in developing Drosophila shapes adult song perception and resultant sexual behavior. Preferences in the song-response behaviors of both males and females were tuned by social acoustic exposure during development. This study examined the molecular and cellular determinants of this social acoustic learning and found that GABA signaling acting on the GABAA receptor Rdl in the pC1 neurons, the integration node for courtship stimuli, regulated auditory tuning and sexual behavior. These findings demonstrate that maturation of auditory perception in flies is unexpectedly plastic and is acquired socially, providing a model to investigate how song learning regulates mating preference in insects.
Qiao, B., Li, C., Allen, V. W., Shirasu-Hiza, M. and Syed, S. (2018). Automated analysis of long-term grooming behavior in Drosophila using a k-nearest neighbors classifier. Elife 7. PubMed ID: 29485401
Despite being pervasive, the control of programmed grooming is poorly understood. This study addressed this gap by developing a high-throughput platform that allows long-term detection of grooming in Drosophila melanogaster. In this method, a k-nearest neighbors algorithm automatically classifies fly behavior and finds grooming events with over 90% accuracy in diverse genotypes. The data show that flies spend ~13% of their waking time grooming, driven largely by two major internal programs. One of these programs regulates the timing of grooming and involves the core circadian clock components cycle, clock, and period. The second program regulates the duration of grooming and, while dependent on cycle and clock, appears to be independent of period. This emerging dual control model in which one program controls timing and another controls duration, resembles the two-process regulatory model of sleep. Together, this quantitative approach presents the opportunity for further dissection of mechanisms controlling long-term grooming in Drosophila.
Szuperak, M., Churgin, M. A., Borja, A. J., Raizen, D. M., Fang-Yen, C. and Kayser, M. S. (2018). A sleep state in Drosophila larvae required for neural stem cell proliferation. Elife 7. PubMed ID: 29424688
Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. This study has identified a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, sleep loss in larvae was found to impair cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation.
Toepfer, F., Wolf, R. and Heisenberg, M. (2018). Multi-stability with ambiguous visual stimuli in Drosophila orientation behavior. PLoS Biol 16(2): e2003113. PubMed ID: 29438378
It is widely accepted for humans and higher animals that vision is an active process in which the organism interprets the stimulus. To find out whether this also holds for lower animals, an ambiguous motion stimulus was designed that serves as something like a multi-stable perception paradigm in Drosophila behavior. Confronted with a uniform panoramic texture in a closed-loop situation in stationary flight, the flies adjust their yaw torque to stabilize their virtual self-rotation. To make the visual input ambiguous, a second texture was examined. Both textures got a rotatory bias to move into opposite directions at a constant relative angular velocity. The results indicate that the fly now had three possible frames of reference for self-rotation: either of the two motion components as well as the integrated motion vector of the two. In this ambiguous stimulus situation, the flies generated a continuous sequence of behaviors, each one adjusted to one or another of the three references.
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