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Current papers in developmental biology and gene function





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Thursday, August 17th

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Shilts, J. and Broadie, K. (2017). Secreted tissue inhibitor of matrix metalloproteinase restricts trans-synaptic signaling to coordinate synaptogenesis. J Cell Sci [Epub ahead of print]. PubMed ID: 28576972
Summary:
Synaptogenesis is coordinated by trans-synaptic signals that traverse the specialized synaptomatrix between pre- and postsynaptic cells. Matrix metalloproteinase (Mmp) activity sculpts this environment, balanced by secreted Tissue inhibitors of Mmp (Timp). This study used the reductionist Drosophila matrix metalloproteome to test consequences of eliminating all Timp regulatory control of Mmp activity at the neuromuscular junction (NMJ). Using in situ zymography, Timp was found to limit Mmp activity at the NMJ terminal and shape extracellular proteolytic dynamics surrounding individual synaptic boutons. In newly-generated timp null mutants, NMJs exhibit architectural overelaboration with supernumerary synaptic boutons. With cell-targeted RNAi and rescue studies, postsynaptic Timp was found to limit presynaptic architecture. Functionally, timp nulls exhibit compromised synaptic vesicle cycling, with reduced, lower fidelity activity. NMJ defects manifest in impaired locomotor function. Mechanistically, Timp was found to limit BMP trans-synaptic signaling and the downstream synapse-to-nucleus signal transduction. Pharmacologically restoring Mmp inhibition in timp nulls corrects BMP signaling and synaptic properties. Genetically restoring BMP signaling in timp nulls corrects NMJ structure and motor function. Thus, Timp inhibition of Mmp proteolytic activity restricts BMP trans-synaptic signaling to coordinate synaptogenesis.
Wagner, N. (2017). Ultrastructural comparison of the Drosophila larval and adult ventral abdominal neuromuscular junction. J Morphol [Epub ahead of print]. PubMed ID: 28444917
Summary:
Drosophila melanogaster has recently emerged as model system for studying synaptic transmission and plasticity during adulthood, aging and neurodegeneration. However, still little is known about the basic neuronal mechanisms of synaptic function in the adult fly. Per se, adult Drosophila neuromuscular junctions should be highly suited for studying these aspects as they allow for genetic manipulations in combination with ultrastructural and electrophysiological analyses. Although different neuromuscular junctions of the adult fly have been described during the last years, a direct ultrastructural comparison with their larval counterpart is lacking. The present study was designed to close this gap by providing a detailed ultrastructural comparison of the larval and the adult neuromuscular junction of the ventrolongitudinal muscle. Assessment of several parameters revealed similarities but also major differences in the ultrastructural organisation of the two model neuromuscular junctions. While basic morphological parameters are retained from the larval into the adult stage, the analysis discovered major differences of potential functional relevance in the adult: The electron-dense membrane apposition of the presynaptic and postsynaptic membrane is shorter, the subsynaptic reticulum is less elaborated and the number of synaptic vesicles at a certain distance of the presynaptic membrane is higher.
Kiragasi, B., Wondolowski, J., Li, Y. and Dickman, D. K. (2017). A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation. Cell Rep 19(13): 2694-2706. PubMed ID: 28658618
Summary:
Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, this study has identified the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D to be required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, this study demonstrated that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. It is proposed that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity.
Mosca, T. J., Luginbuhl, D. J., Wang, I. E. and Luo, L. (2017). Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons. Elife 6. PubMed ID: 28606304
Summary:
Precise coordination of synaptic connections ensures proper information flow within circuits. The activity of presynaptic organizing molecules signaling to downstream pathways is essential for such coordination, though such entities remain incompletely known. This study shows that LRP4 (CG8909), a conserved transmembrane protein known for its postsynaptic roles, functions presynaptically as an organizing molecule. In the Drosophila brain, LRP4 localizes to the nerve terminals at or near active zones. Loss of presynaptic LRP4 reduces excitatory (not inhibitory) synapse number, impairs active zone architecture, and abolishes olfactory attraction - the latter of which can be suppressed by reducing presynaptic GABAB receptors. LRP4 overexpression increases synapse number in excitatory and inhibitory neurons, suggesting an instructive role and a common downstream synapse addition pathway. Mechanistically, LRP4 functions via the conserved kinase SRPK79D to ensure normal synapse number and behavior. This highlights a presynaptic function for LRP4, enabling deeper understanding of how synapse organization is coordinated.

Wednesday, August 16th

Moose, D. L., Haase, S. J., Aldrich, B. T. and Lear, B. C. (2017). The narrow abdomen ion channel complex is highly stable and persists from development into adult stages to promote behavioral rhythmicity. Front Cell Neurosci 11: 159. PubMed ID: 28634443
Summary:
The sodium leak channel Narrow abdomen (NA)/ NALCN is an important component of circadian pacemaker neuronal output. In Drosophila, rhythmic expression of the NA channel regulator Nlf-1 in a subset of adult pacemaker neurons has been proposed to contribute to circadian regulation of channel localization or activity. This study restricted expression of Drosophila NA channel subunits or the Nlf-1 regulator to either development or adulthood using the temperature-inducible tubulin-GAL80ts system. Surprisingly, it was found that developmental expression of endogenous channel subunits and Nlf-1 is sufficient to promote robust rhythmic behavior in adults. Moreover, channel complex proteins produced during development persist in the Drosophila head with little decay for at least 5-7 days in adults. In contrast, restricting either endogenous or transgenic gene expression to adult stages produces only limited amounts of the functional channel complex. These data indicate that much of the NA channel complex that functions in adult circadian neurons is normally produced during development, and that the channel complex is very stable in most neurons in the Drosophila brain. Based on these findings, it is proposed that circadian regulation of NA channel function in adult pacemaker neurons is mediated primarily by post-translational mechanisms that are independent of Nlf-1.
Seki, Y., Dweck, H. K. M., Rybak, J., Wicher, D., Sachse, S. and Hansson, B. S. (2017). Olfactory coding from the periphery to higher brain centers in the Drosophila brain. BMC Biol 15(1): 56. PubMed ID: 28666437
Summary:
Odor information is processed through multiple receptor-glomerular channels in the first order olfactory center, the antennal lobe (AL), then reformatted into higher brain centers and eventually perceived by the fly. To reveal the logic of olfaction, it is fundamental to map odor representations from the glomerular channels into higher brain centers. This study characterized odor response profiles of AL projection neurons (PNs) originating from 31 glomeruli using whole cell patch-clamp recordings in Drosophila melanogaster. Odor representation from olfactory sensory neurons to PNs is generally conserved, while transformation of odor tuning curves is glomerulus-dependent. Reconstructions of PNs reveal that attractive and aversive odors are represented in different clusters of glomeruli in the AL. These separate representations are preserved into higher brain centers, where attractive and aversive odors are segregated into two regions in the lateral horn and partly separated in the mushroom body calyx. This study reveals spatial representation of odor valence coding from the AL to higher brain centers. These results provide a global picture of the olfactory circuit design underlying innate odor-guided behavior.
Placais, P. Y., de Tredern, E., Scheunemann, L., Trannoy, S., Goguel, V., Han, K. A., Isabel, G. and Preat, T. (2017). Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory. Nat Commun 8: 15510. PubMed ID: 28580949
Summary:
Efficient energy use has constrained the evolution of nervous systems. However, it is unresolved whether energy metabolism may resultantly regulate major brain functions. The observation that Drosophila flies double their sucrose intake at an early stage of long-term memory formation initiated the investigation of how energy metabolism intervenes in this process. Cellular-resolution imaging of energy metabolism reveals a concurrent elevation of energy consumption in neurons of the mushroom body, the fly's major memory centre. Strikingly, upregulation of mushroom body energy flux is both necessary and sufficient to drive long-term memory formation. This effect is triggered by a specific pair of dopaminergic neurons afferent to the mushroom bodies, via the D5-like DAMB dopamine receptor. Hence, dopamine signalling mediates an energy switch in the mushroom body that controls long-term memory encoding. These data thus point to an instructional role for energy flux in the execution of demanding higher brain functions.
Selcho, M., Millan, C., Palacios-Munoz, A., Ruf, F., Ubillo, L., Chen, J., Bergmann, G., Ito, C., Silva, V., Wegener, C. and Ewer, J. (2017). Central and peripheral clocks are coupled by a neuropeptide pathway in Drosophila. Nat Commun 8: 15563. PubMed ID: 28555616
Summary:
circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. This study dissected the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. The central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals.

Tuesday, August 15th

Parsons, L. M., Grzeschik, N. A., Amaratunga, K., Burke, P., Quinn, L. M. and Richardson, H. E. (2017). A kinome RNAi screen in Drosophila identifies novel genes interacting with Lgl, aPKC and Crb cell polarity genes in epithelial tissues. G3 (Bethesda) 7(8):2497-2509. PubMed ID: 28611255
Summary:
In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, a boutique collection of RNAi stocks targeting the kinome was screened for their capacity to modify Drosophila 'cell polarity' eye and wing phenotypes. Initially kinase or phosphatase genes were identified whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). Next a secondary screen was conducted to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, JNK, and Notch signalling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways, not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras and lipid/Phospho-inositol-3-kinase (PI3K) signalling pathways. Additionally, it was demonstrated that the 'nutrient sensing' kinases, Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, this screen has revealed novel cell-polarity interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.
Sap, K. A., Bezstarosti, K., Dekkers, D. H., Voets, O. and Demmers, J. A. (2017). Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA mediated subunit knockdown in Drosophila. J Proteome Res. PubMed ID: 28665616
Summary:
The ubiquitin-proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin in order to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. This study investigated the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in Drosophila S2 cells. dsRNA mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, the overall impact on the ubiquitinome being most dramatic. The abundances of approx. 10% of all proteins are increased, while the abundances of the far majority of over 14 thousand detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Since this is the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the Drosophila community.
Ma, X., Lu, J. Y., Dong, Y., Li, D., Malagon, J. N. and Xu, T. (2017). PP6 disruption synergizes with oncogenic Ras to promote JNK-dependent tumor growth and invasion. Cell Rep 19(13): 2657-2664. PubMed ID: 28658615
Summary:
RAS genes are frequently mutated in cancers, yet an effective treatment has not been developed, partly because of an incomplete understanding of signaling within Ras-related tumors. To address this, a genetic screen was performed in Drosophila, aiming to find mutations that cooperate with oncogenic Ras (RasV12) to induce tumor overgrowth and invasion. fiery mountain (fmt; CG10289), a regulatory subunit of the protein phosphatase 6 (PP6) complex, was identified as a tumor suppressor that synergizes with RasV12 to drive c-Jun N-terminal kinase (JNK)-dependent tumor growth and invasiveness. Fmt was shown to negatively regulate JNK upstream of dTAK1. It was further demonstrated that disruption of PpV, the catalytic subunit of PP6, mimics fmt loss-of-function-induced tumorigenesis. Finally, Fmt synergizes with PpV to inhibit JNK-dependent tumor progression. These data here further highlight the power of Drosophila as a model system to unravel molecular mechanisms that may be relevant to human cancer biology.
Manavalan, M. A., Jayasinghe, V. R., Grewal, R. and Bhat, K. M. (2017). The glycosylation pathway is required for the secretion of Slit and for the maintenance of the Slit receptor Robo on axons. Sci Signal 10(484). PubMed ID: 28634210
Summary:
Slit proteins act as repulsive axon guidance cues by activating receptors of the Roundabout (Robo) family. During early neurogenesis in Drosophila melanogaster, Slit prevents the growth cones of longitudinal tract neurons from inappropriately crossing the midline, thus restricting these cells to trajectories parallel to the midline. Slit is expressed in midline glial cells, and Robo is present in longitudinal axon tracts and growth cones. This study shows that the enzyme Mummy (Mmy) controls Slit-Robo signaling through mechanisms that affected both the ligand and the receptor. Mmy was required for the glycosylation of Slit, which was essential for Slit secretion. Mmy was also required for maintaining the abundance and spatial distribution of Robo through an indirect mechanism that was independent of Slit secretion. Moreover, secretion of Slit was required to maintain the fasciculation and position of longitudinal axon tracts, thus maintaining the hardwiring of the nervous system. Thus, Mmy is required for Slit secretion and for maintaining Robo abundance and distribution in the developing nervous system in Drosophila.
Li, C., Li, B., Ma, S., Lu, P. and Chen, K. (2017). Dusky works upstream of Four-jointed and Forked in wing morphogenesis in Tribolium castaneum. Insect Mol Biol. PubMed ID: 28677915
Summary:
Dusky (dy) is required for cytoskeletal reorganization during wing morphogenesis in Drosophila melanogaster, but which genes participate together with dy for wing morphogenesis has remained unclear. In Tribolium castaneum, dy is highly expressed at the late embryonic stage. Tissue-specific expression analysis indicated high expression levels of dy in the epidermis, head and fat body of late-stage larvae. RNA interference (RNAi) targeting dy significantly decreased adult wing size and caused improper folding of the elytra. Meanwhile, dy knockdown reduced the transcription of four-jointed (fj) and forked (f). These results show that fj RNAi reduces adult wing size and that silencing f results in abnormal wing folding in T. castaneum. Interestingly, knocking down fj and f simultaneously phenocopies dy RNAi, suggesting that dy probably acts upstream of fj and f to regulate wing morphogenesis in T. castaneum.
Rives-Quinto, N., Franco, M., de Torres-Jurado, A. and Carmena, A. (2017). canoe and scribble loss synergizes causing tumor-like overgrowth via Ras activation in neural stem cells and epithelia. Development [Epub ahead of print]. PubMed ID: 28619817
Summary:
Over the past decade an intriguing connection between asymmetric cell division, stem cells and tumorigenesis has emerged. Neuroblasts, the neural stem cells of the Drosophila central nervous system, divide asymmetrically and constitute an excellent paradigm for further investigating that connection. This study shows that the simultaneous loss of the asymmetric cell division regulators Canoe (Afadin in mammals) and Scribble in neuroblast clones leads to tumor-like overgrowth through both a severe disruption of the asymmetric cell division process and a canoe loss-mediated Ras-PI3K-Akt activation. Moreover, canoe loss also interacts synergistically with scribble to promote overgrowth in epithelial tissues, here just by activating the Ras-Raf-MAPK pathway. Finally scribble functionally related genes discs large and lethal (2) giant larvae were shown to contribute to repress the Ras-MAPK signaling cascade in epithelia. Hence, this work uncovers novel cooperative interactions between all these well-conserved tumor suppressors to ensure a tight regulation of the Ras signaling pathway.

Monday, August 14th

Murata, S., Brockmann, A. and Tanimura, T. (2017). Pharyngeal stimulation with sugar triggers local searching behavior in Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 28684466
Summary:
Foraging behavior is essential for all organisms to find food containing nutritional chemicals. A hungry fly of Drosophila melanogaster performs local searching behavior after drinking a small amount of sugar solution. Using video tracking this study examined how the searching behavior is regulated in D. melanogaster. A small amount of highly concentrated sugar solution was found to induce a long-lasting searching behavior. After the intake of sugar solution, a fly moved around in circles and repeatedly returned to the position where the sugar droplet had been placed. The non-nutritious sugar, D-arabinose, but not the non-sweet nutritious sugar, D-sorbitol, was effective in inducing the behavior, indicating that sweet sensation is essential. Furthermore, pox-neuro mutant flies with no external taste bristles showed local searching behavior, suggesting the involvement of the pharyngeal taste organ. Experimental activation of pharyngeal sugar-sensitive gustatory receptor neurons by capsaicin using the Gal4/UAS system induced local searching behavior. In contrast, inhibition of pharyngeal sugar-responsive gustatory receptor neurons abolished the searching behavior. Together these results indicate that in Drosophila the pharyngeal taste-receptor neurons trigger searching behavior immediately after ingestion.
McConnell, M. W. and Fitzpatrick, M. J. (2017). 'Foraging' for a place to lay eggs: A genetic link between foraging behaviour and oviposition preferences. PLoS One 12(6): e0179362. PubMed ID: 28622389
Summary:
Gravid female arthropods in search of egg-laying substrates embark on foraging-like forays: they survey the environment assessing multiple patches, tasting each with their tarsi and proboscis, and then, if interested, they deposit an egg (or eggs). In fruit flies, Drosophila melanogaster, allelic variation in the foraging gene (for) underlies the rover/sitter foraging behaviour polymorphism. Rover flies (forR) are more active foragers (both within and between food patches) compared to sitters (fors). In nematodes, Caenorhabditis elegans, a mutation in egl-4, the ortholog of for, leads to aberrations in egg laying. Given this and the notion that females may 'forage' for a place to oviposit, it was hypothesized that for may underlie egg-laying decisions in the fruit fly. Indeed, when given a choice between patches of low- and high-nutrient availability, rovers lay significantly more eggs on the low-nutrient patches than sitters and also a sitter mutant (fors2). This study confirmed the role of for by inducing rover-like oviposition preferences in a sitter fly using the transgenic overexpression of for-mRNA in the nervous system.
Liang, X., Holy, T. E. and Taghert, P. H. (2017). A series of suppressive signals within the Drosophila circadian neural circuit generates sequential daily outputs. Neuron 94(6): 1173-1189.e1174. PubMed ID: 28552314
Summary:
The Drosophila circadian neural circuit was studied using whole-brain imaging in vivo. Five major groups of pacemaker neurons display synchronized molecular clocks, yet each exhibits a distinct phase of daily Ca2+ activation. Light and neuropeptide pigment dispersing factor (PDF) from morning cells (s-LNv) together delay the phase of the evening (LNd) group by approximately 12 hr; PDF alone delays the phase of the DN3 group by approximately 17 hr. Neuropeptide sNPF, released from s-LNv and LNd pacemakers, produces Ca2+ activation in the DN1 group late in the night. The circuit also features negative feedback by PDF to truncate the s-LNv Ca2+ wave and terminate PDF release. Both PDF and sNPF suppress basal Ca2+ levels in target pacemakers with long durations by cell-autonomous actions. Thus, light and neuropeptides act dynamically at distinct hubs of the circuit to produce multiple suppressive events that create the proper tempo and sequence of circadian pacemaker neuronal activities.
Chouhan, N. S., Wolf, R. and Heisenberg, M. (2017). Starvation promotes odor/feeding-time associations in flies. Learn Mem 24(7): 318-321. PubMed ID: 28620079
Summary:
Starvation causes a motivational state that facilitates diverse behaviors such as feeding, walking, and search. Starved Drosophila can form odor/feeding-time associations but the role of starvation in encoding of "time" is poorly understood. This study shows that the extent of starvation is correlated with the fly's ability to establish odor/feeding-time memories. Prolonged starvation promotes odor/feeding-time associations after just a single cycle of reciprocal training. Starvation is also show to be required for acquisition but is dispensable for retrieval of odor/feeding-time memory. Finally, even with extended starvation, a functional circadian oscillator is indispensable for establishing odor/feeding-time memories.

Sunday, August 13th

Yenigun, V. B., Sirito, M., Amcheslavky, A., Czernuszewicz, T., Colonques-Bellmunt, J., Garcia-Alcover, I., Wojciechowska, M., Bolduc, C., Chen, Z., Lopez Castel, A., Krahe, R. and Bergmann, A. (2017). (CCUG)n RNA toxicity in a Drosophila model for myotonic dystrophy type 2 (DM2) activates apoptosis. Dis Model Mech. PubMed ID: 28623239
Summary:
The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions -- (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP. Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, a transgenic DM2 model was developed in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCTG)16, in muscle and retinal cells led to formation of (CCUG) ribonuclear inclusions and mis-splicing of genes implicated in the DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, while CUGBP1/CELF1 complementation did not. Flies with (CCUG)106 displayed strong disruption of the external eye morphology and the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, two chemical compounds were tested that have shown therapeutic potential in DM1 models. While treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. These data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. The Drosophila DM2 model may provide a convenient tool for in vivo drug screening.
M'Angale, P. G. and Staveley, B. E. (2017). A loss of Pdxk model of Parkinson disease in Drosophila can be suppressed by Buffy. BMC Res Notes 10(1): 205. PubMed ID: 28606139
Summary:
The identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene has led to a study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of Drosophila. The multitude of biological functions attributable to the vitamers of vitamin B6 catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk, and this study used RNAi to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. Any association was further investigated between this enhanced disease risk gene with the established PD model induced by expression of alpha-synuclein in the same neurons. The pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, were relied on to rescue the Pdxk-induced phenotypes. Ddc-Gal4, which drives expression in both dopaminergic and serotonergic neurons, was used to drive the expression of Pdxk RNA interference in DA neurons of Drosophila. The inhibition of Pdxk in the alpha-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when Pdxk was inhibited in the developing eye using GMR-Gal4, it was found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the alpha-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array. It is concluded that though Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD.
Meng, H., Yamashita, C., Shiba-Fukushima, K., Inoshita, T., Funayama, M., Sato, S., Hatta, T., Natsume, T., Umitsu, M., Takagi, J., Imai, Y. and Hattori, N. (2017). Loss of Parkinson's disease-associated protein CHCHD2 affects mitochondrial crista structure and destabilizes Cytochrome c. Nat Commun 8: 15500. PubMed ID: 28589937
Summary:
Mutations in CHCHD2 have been identified in some Parkinson's disease (PD) cases. To understand the physiological and pathological roles of CHCHD2, this study manipulated the expression of CHCHD2 in Drosophila and mammalian cells. The loss of CHCHD2 in Drosophila causes abnormal matrix structures and impaired oxygen respiration in mitochondria, leading to oxidative stress, dopaminergic neuron loss and motor dysfunction with age. These PD-associated phenotypes are rescued by the overexpression of the translation inhibitor 4E-BP and by the introduction of human CHCHD2 but not its PD-associated mutants. CHCHD2 is upregulated by various mitochondrial stresses, including the destabilization of mitochondrial genomes and unfolded protein stress, in Drosophila. CHCHD2 binds to cytochrome c along with a member of the Bax inhibitor-1 superfamily, MICS1, and modulated cell death signalling, suggesting that CHCHD2 dynamically regulates the functions of cytochrome c in both oxidative phosphorylation and cell death in response to mitochondrial stress.
Hosaka, Y., Inoshita, T., Shiba-Fukushima, K., Cui, C., Arano, T., Imai, Y. and Hattori, N. (2017). Reduced TDP-43 expression improves neuronal activities in a Drosophila model of Perry syndrome. EBioMedicine [Epub ahead of print]. PubMed ID: 28625517
Summary:
Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. This study reports that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43 TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities.

Saturday, August 12th

Bataille, L., Boukhatmi, H., Frendo, J. L. and Vincent, A. (2017). Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles. BMC Biol 15(1): 48. PubMed ID: 28599653
Summary:
A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs), such as Apterous, Even-Skipped and Slouch/S59, expressed by its FC. This study addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. It is concluded that transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells.
Sallee, M. D., Littleford, H. E. and Greenwald, I. (2017). A bHLH code for sexually dimorphic form and function of the C. elegans somatic gonad. Curr Biol 27(12): 1853-1860 e1855. PubMed ID: 28602651
Evolutionary Homolog Study
How sexually dimorphic gonads are generated is a fundamental question at the interface of developmental and evolutionary biology. In C. elegans, sexual dimorphism in gonad form and function largely originates in different apportionment of roles to three regulatory cells of the somatic gonad primordium in young larvae. Their essential roles include leading gonad arm outgrowth, serving as the germline niche, connecting to epithelial openings, and organizing reproductive organ development. The development and function of the regulatory cells in both sexes requires the basic-helix-loop-helix (bHLH) transcription factor HLH-2, the sole ortholog of the E proteins mammalian E2A and Drosophila Daughterless, yet how they adopt different fates to execute their different roles has been unknown. This study shows that each regulatory cell expresses a distinct complement of bHLH-encoding genes-and therefore distinct HLH-2:bHLH dimers-and formulate a "bHLH code" hypothesis for regulatory cell identity. This hypothesis is supported by showing that the bHLH gene complement is both necessary and sufficient to confer particular regulatory cell fates. Strikingly, prospective regulatory cells can be directly reprogrammed into other regulatory cell types simply by loss or ectopic expression of bHLH genes, and male-to-female and female-to-male transformations indicate that the code is instructive for sexual dimorphism. The bHLH code appears to be embedded in a bow-tie regulatory architecture, wherein sexual, positional, temporal, and lineage inputs connect through bHLH genes to diverse outputs for terminal features and provides a plausible mechanism for the evolutionary plasticity of gonad form seen in nematodes.
Kushnir, T., Mezuman, S., Bar-Cohen, S., Lange, R., Paroush, Z. and Helman, A. (2017). Novel interplay between JNK and Egfr signaling in Drosophila dorsal closure. PLoS Genet 13(6): e1006860. PubMed ID: 28628612
Summary:
Dorsal closure (DC) is a developmental process in which two contralateral epithelial sheets migrate to seal a large hole in the dorsal ectoderm of the Drosophila embryo. Two signaling pathways act sequentially to orchestrate this dynamic morphogenetic process. First, c-Jun N-terminal kinase (JNK) signaling activity in the dorsal-most leading edge (LE) cells of the epidermis induces expression of decapentaplegic (dpp). Second, Dpp, a secreted TGF-beta homolog, triggers cell shape changes in the adjacent, ventrally located lateral epidermis, that guide the morphogenetic movements and cell migration mandatory for DC. This study uncovered a cell non-autonomous requirement for the Epidermal growth factor receptor (Egfr) pathway in the lateral epidermis for sustained dpp expression in the LE. Specifically, it was demonstrated that Egfr pathway activity in the lateral epidermis prevents expression of the gene scarface (scaf), encoding a secreted antagonist of JNK signaling. In embryos with compromised Egfr signaling, upregulated Scaf causes reduction of JNK activity in LE cells, thereby impeding completion of DC. These results identify a new developmental role for Egfr signaling in regulating epithelial plasticity via crosstalk with the JNK pathway.
Olivares-Castineira, I. and Llimargas, M. (2017). EGFR controls Drosophila tracheal tube elongation by intracellular trafficking regulation. PLoS Genet 13(7): e1006882. PubMed ID: 28678789
Summary:
Development is governed by a few conserved signalling pathways. Amongst them, the EGFR pathway is used reiteratively for organ and tissue formation, and when dysregulated can lead to cancer and metastasis. Given its relevance, identifying its downstream molecular machinery and understanding how it instructs cellular changes is crucial. This study approached this issue in the respiratory system of Drosophila. A new role was identified for EGFR restricting the elongation of the tracheal Dorsal Trunk. EGFR was found to regulate the apical determinant Crumbs and the extracellular matrix regulator Serpentine, two factors previously known to control tube length. EGFR regulates the organisation of endosomes in which Crb and Serp proteins are loaded. These results are consistent with a role of EGFR in regulating Retromer/WASH recycling routes. Furthermore, this study provides new insights into Crb trafficking and recycling during organ formation. This work connects cell signalling, trafficking mechanisms and morphogenesis and suggests that the regulation of cargo trafficking can be a general outcome of EGFR activation.

Friday, August 11th

Obadia, B., Guvener, Z. T., Zhang, V., Ceja-Navarro, J. A., Brodie, E. L., Ja, W. W. and Ludington, W. B. (2017). Probabilistic invasion underlies natural gut microbiome stability. Curr Biol [Epub ahead of print]. PubMed ID: 28625783
Summary:
Species compositions of gut microbiomes impact host health, but the processes determining these compositions are largely unknown. An unexplained observation is that gut species composition varies widely between individuals but is largely stable over time within individuals. Stochastic factors during establishment may drive these alternative stable states (colonized versus non-colonized), which can influence susceptibility to pathogens, such as Clostridium difficile. A precise, high-throughput technique revealed stable between-host variation in colonization when individual germ-free flies were fed their own natural commensals (including the probiotic Lactobacillus plantarum). Some flies were colonized while others remained germ-free even at extremely high bacterial doses. Thus, alternative stable states of colonization exist even in this low-complexity model of host-microbe interactions. These alternative states are driven by a fundamental asymmetry between the inoculum population and the stably colonized population that is mediated by spatial localization and a population bottleneck, which makes stochastic effects important by lowering the effective population size. Prior colonization with other bacteria reduced the chances of subsequent colonization, thus increasing the stability of higher-diversity guts. Therefore, stable gut diversity may be driven by inherently stochastic processes, which has important implications for combatting infectious diseases and for stably establishing probiotics in the gut.
Garcia, J. F., Carbone, M. A., Mackay, T. F. C. and Anholt, R. R. H. (2017). Regulation of Drosophila lifespan by bellwether promoter alleles. Sci Rep 7(1): 4109. PubMed ID: 28646164
Summary:
Longevity varies among individuals, but how natural genetic variation contributes to variation in lifespan is poorly understood. Drosophila melanogaster presents an advantageous model system to explore the genetic underpinnings of longevity, since its generation time is brief and both the genetic background and rearing environment can be precisely controlled. The bellwether (blw) gene encodes the alpha subunit of mitochondrial ATP synthase. Since metabolic rate may influence lifespan, this study investigated whether alternative haplotypes in the blw promoter affect lifespan when expressed in a co-isogenic background. 521 bp upstream promoter sequences containing the alternative SNP haplotypes (G/T and A/G) were amplified, and promoter activity was assessed both in vitro and in vivo using a luciferase reporter system. The AG haplotype showed significantly greater expression of luciferase than the GT haplotype. A blw cDNA construct driven by either the AG or GT haplotype promoter was driven in transgenic flies, and the AG haplotype was shown to results in greater blw cDNA expression and a significant decrease in lifespan relative to the GT promoter haplotype, in male flies only. Thus, the results show that naturally occurring regulatory variants of blw affect lifespan in a sex-specific manner.
Hazegh, K. E., Nemkov, T., D'Alessandro, A., Diller, J. D., Monks, J., McManaman, J. L., Jones, K. L., Hansen, K. C. and Reis, T. (2017). An autonomous metabolic role for Spen. PLoS Genet 13(6): e1006859. PubMed ID: 28640815
Summary:
Preventing obesity requires a precise balance between deposition into and mobilization from fat stores, but regulatory mechanisms are incompletely understood. Drosophila Split ends (Spen) is the founding member of a conserved family of RNA-binding proteins involved in transcriptional regulation and frequently mutated in human cancers. This study found that manipulating Spen expression alters larval fat levels in a cell-autonomous manner. Spen-depleted larvae had defects in energy liberation from stores, including starvation sensitivity and major changes in the levels of metabolic enzymes and metabolites, particularly those involved in beta-oxidation. Spenito, a small Spen family member, counteracted Spen function in fat regulation. Finally, mouse Spen and Spenito transcript levels scaled directly with body fat in vivo, suggesting a conserved role in fat liberation and catabolism. This study demonstrates that Spen is a key regulator of energy balance and provides a molecular context to understand the metabolic defects that arise from Spen dysfunction.
Garcia, C. J., Khajeh, J., Coulanges, E., Chen, E. I. and Owusu-Ansah, E. (2017). Regulation of mitochondrial complex I biogenesis in Drosophila flight muscles. Cell Rep 20(1): 264-278. PubMed ID: 28683319
Summary:
The flight muscles of Drosophila are highly enriched with mitochondria, but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. This study reports the mechanism of CI biogenesis in Drosophila flight muscles and shows that it proceeds via the formation of approximately 315, approximately 550, and approximately 815 kDa CI assembly intermediates. Additionally, specific roles were defined for several CI subunits in the assembly process. In particular, dNDUFS5 was shown to be required for converting an approximately 700 kDa transient CI assembly intermediate into the approximately 815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. These findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans.

Thursday, August 10th

Moshe, A. and Kaplan, T. (2017). Genome-wide search for Zelda-like chromatin signatures identifies GAF as a pioneer factor in early fly development. Epigenetics Chromatin 10(1): 33. PubMed ID: 28676122
Summary:
The protein Zelda was shown to play a key role in early Drosophila development, binding thousands of promoters and enhancers prior to maternal-to-zygotic transition (MZT), and marking them for transcriptional activation. Zelda has been shown to act through specific chromatin patterns of histone modifications to mark developmental enhancers and active promoters. Intriguingly, some Zelda sites still maintain these chromatin patterns in Drosophila embryos lacking maternal Zelda protein. This suggests that additional Zelda-like pioneer factors may act in early fly embryos. A computational method was developed to analyze and refine the chromatin landscape surrounding early Zelda peaks, using a multichannel spectral clustering. This allowed characterization their chromatin patterns through MZT (mitotic cycles 8-14). Specifically, focus was placed on H3K4me1, H3K4me3, H3K18ac, H3K27ac, and H3K27me3 and three different classes of chromatin signatures were identified, matching "promoters," "enhancers" and "transiently bound" Zelda peaks. Then the genome was further scanned using these chromatin patterns and additional loci - with no Zelda binding- were identified that show similar chromatin patterns, resulting with hundreds of Zelda-independent putative enhancers. These regions were found to be enriched with GAGA factor (GAF, Trl) and are typically located near early developmental zygotic genes. Overall this analysis suggests that GAF, together with Zelda, plays an important role in activating the zygotic genome. The computational approach offers an efficient algorithm for characterizing chromatin signatures around some loci of interest and allows a genome-wide identification of additional loci with similar chromatin patterns.
Martin, R. L., Maiorano, J., Beitel, G. J., Marko, J. F., McVicker, G. and Fondufe-Mittendorf, Y. N. (2017). A comparison of nucleosome organization in Drosophila cell lines. PLoS One 12(6): e0178590. PubMed ID: 28570602
Summary:
Changes in the distribution of nucleosomes along the genome influence chromatin structure and impact gene expression by modulating the accessibility of DNA to transcriptional machinery. This study compared genome-wide nucleosome positioning and occupancy in five different Drosophila tissue-specific cell lines, and in reconstituted chromatin, and then tests were performed for correlations between nucleosome positioning, transcription factor binding motifs, and gene expression. Nucleosomes in all cell lines were positioned in accordance with previously known DNA-nucleosome interactions, with helically repeating A/T di-nucleotide pairs arranged within nucleosomal DNAs and AT-rich pentamers generally excluded from nucleosomal DNA. Nucleosome organization in all cell lines differed markedly from in vitro reconstituted chromatin, with highly expressed genes showing strong nucleosome organization around transcriptional start sites. Importantly, comparative analysis identified genomic regions that exhibited cell line-specific nucleosome enrichment or depletion. Further analysis of these regions identified 91 out of 16,384 possible heptamer sequences that showed differential nucleosomal occupation between cell lines, and 49 of the heptamers matched one or more known transcription factor binding sites. These results demonstrate that there is differential nucleosome positioning between these Drosophila cell lines and therefore identify a system that could be used to investigate the functional significance of differential nucleosomal positioning in cell type specification.
Jox, T., Buxa, M. K., Bohla, D., Ullah, I., Macinkovic, I., Brehm, A., Bartkuhn, M. and Renkawitz, R. (2017). Drosophila CP190- and dCTCF-mediated enhancer blocking is augmented by SUMOylation. Epigenetics Chromatin. 10: 32. PubMed ID: 28680483d
Summary:

Chromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. This study addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation.

Dutta, P. and Li, W. X. (2017). The SERTAD protein Taranis plays a role in Polycomb-mediated gene repression. PLoS One 12(6): e0180026. PubMed ID: 28665982
Summary:

The Polycomb group (PcG) proteins have been implicated in epigenetic transcriptional repression in development, stem cell maintenance and in cancer. The chromodomain protein Polycomb (Pc) is a key member of the PcG. Pc binds to the histone mark, trimethylated histone 3 lysine 27 (H3K27me3), to initiate transcriptional repression. How PcG proteins are recruited to target loci is not fully understood. This study shows that the Drosophila SERTA domain protein Taranis (Tara) is involved in transcriptional regulation of Pc target genes. Embryos lacking Tara exhibit a partial homeotic transformation of cuticular the segments, a phenotype associated with the loss of Pc function. Moreover, Drosophila embryos homozygous for a tara hypomorphic allele also misexpress engrailed, a Pc-regulated gene, and this phenotype is associated with the loss of Pc binding to the cis response element in the engrailed enhancer. In relation to that, Pc recruitment is reduced on the salivary gland polytene chromosomes and specifically at the engrailed locus. These results suggest that Tara might be required for positioning Pc to a subset of its target genes.

Wednesday, August 9th

LaFerriere, H. and Zars, T. (2017). The Drosophila melanogaster tribbles pseudokinase is necessary for proper memory formation. Neurobiol Learn Mem 144: 68-76. PubMed ID: 28669782
Summary:
The tribbles (trbl) pseudokinases play important roles in signaling and physiology in multiple contexts, ranging from innate immunity to cancer, suggesting fundamental cellular functions for the trbl gene products. Despite expression of the trbl pseudokinases in the nervous systems of invertebrate and vertebrate animals, and evidence that they have a function within mouse and human dopamine neurons, there is no clear case for a function of a Trbl protein that influences behavior. Indeed, the first and only evidence for this type of function comes from Drosophila melanogaster, where a mutation of the single trbl gene was identified in a genetic screen for short-term memory mutant flies. The current study tested flies containing multiple trbl mutant alleles and potential transgenic rescue in both operant place memory and classical olfactory memory paradigms. Genetic complementation tests and transgenic rescue of memory phenotypes in both paradigms show that the D. melanogaster trbl pseudokinase is essential for proper memory formation. Expression analysis with a polyclonal antiserum against Trbl shows that the protein is expressed widely in the fly brain, with higher expression in the cellular rind than the neuropil. Rescue of the behavioral phenotype with transgenic expression indicates the trbl function can be localized to a subset of the nervous system. Thus, this study provides the first compelling case for the function of a trbl pseudokinase in the regulation of behavior.
Lark, A., Kitamoto, T. and Martin, J. R. (2017). Modulation of neuronal activity in the Drosophila mushroom body by DopEcR, a unique dual receptor for ecdysone and dopamine. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28554773
Summary:
G-protein-coupled receptors (GPCRs) for steroid hormones mediate unconventional steroid signaling. Drosophila DopEcR is a GPCR that responds to both ecdysone (the major steroid hormone in insects) and dopamine, regulating multiple second messenger systems. Recent studies have revealed that DopEcR is preferentially expressed in the nervous system and involved in behavioral regulation. This study utilized the bioluminescent Ca2+-indicator GFP-aequorin to monitor the nicotine-induced Ca2+-response within the mushroom bodies (MB), a higher-order brain center in flies, and examined how DopEcR modulates these Ca2+-dynamics. The results show that in DopEcR knockdown flies, the nicotine-induced Ca2+-response in the MB was significantly enhanced selectively in the medial lobes. Application of DopEcR's ligands, ecdysone and dopamine, had different effects on nicotine-induced Ca2+-responses in the MB: ecdysone enhanced activity in the calyx and cell body region in a DopEcR-dependent manner, whereas dopamine reduced activity in the medial lobes independently of DopEcR. Finally, flies with reduced DopEcR function in the MB were shown to display decreased locomotor activity. This behavioral phenotype of DopEcR-deficient flies may be partly due to their enhanced MB activity, since the MB have been implicated in the suppression of locomotor activity. Overall, these data suggest that DopEcR is involved in region-specific modulation of Ca2+ dynamics within the MB, which may play a role in behavioral modulation.
Lovick, J. K., Omoto, J. J., Ngo, K. T. and Hartenstein, V. (2017). Development of the anterior visual input pathway to the Drosophila central complex. J Comp Neurol. PubMed ID: 28675433
Summary:
The anterior visual pathway (AVP) conducts visual information from the medulla of the optic lobe via the anterior optic tubercle (AOTU) and bulb (BU) to the ellipsoid body (EB) of the central complex. This paper analyzes the formation of the AVP from early larval to adult stages. The immature fiber tracts of the AVP, formed by secondary neurons of lineages DALcl1/2 and DALv2, assemble into structurally distinct primordia of the AOTU, BU, and EB within the late larval brain. During the early pupal period (P6-P48) these primordia grow in size and differentiate into the definitive subcompartments of the AOTU, BU, and EB. The primordium of the EB has a complex composition. DALv2 neurons form the anterior EB primordium, which starts out as a bilateral structure, then crosses the midline between P6 and P12, and subsequently bends to adopt the ring shape of the mature EB. Columnar neurons of the central complex, generated by the type II lineages DM1-4, form the posterior EB primordium. Starting out as an integral part of the fan-shaped body (FB) primordium, the posterior EB primordium moves forward and merges with the anterior EB primordium. This paper documents the extension of neuropil glia around the nascent EB and BU and analyzes the relationship of primary and secondary neurons of the AVP lineages.
Knecht, Z. A., Silbering, A. F., Cruz, J., Yang, L., Croset, V., Benton, R. and Garrity, P. A. (2017). Ionotropic Receptor-dependent moist and dry cells control hygrosensation in Drosophila. Elife 6. PubMed ID: 28621663
Summary:
Insects use hygrosensation (humidity sensing) to avoid desiccation and, in vectors such as mosquitoes, to locate vertebrate hosts. Sensory neurons activated by either dry or moist air ('dry cells' and 'moist cells') have been described in many insects, but their behavioral roles and the molecular basis of their hygrosensitivity remain unclear. It has been reported that Drosophila hygrosensation relies on three Ionotropic Receptors (IRs) required for dry cell function: IR25a, IR93a and IR40a. This paper reports the discovery of Drosophila moist cells and shows that they require IR25a and IR93a together with IR68a, a conserved, but orphan IR. Both IR68a- and IR40a-dependent pathways drive hygrosensory behavior: each is important for dry-seeking by hydrated flies and together they underlie moist-seeking by dehydrated flies. These studies reveal that humidity sensing in Drosophila, and likely other insects, involves the combined activity of two molecularly related but neuronally distinct hygrosensing systems.

Tuesday, August 8th

Gonzalez, D. A., et al. (2017). The Arf6 activator Efa6/PSD3 confers regional specificity and modulates ethanol consumption in Drosophila and humans. Mol Psychiatry [Epub ahead of print]. PubMed ID: 28607459
Summary:
The ubiquitously expressed small GTPase Arf6 is required for normal ethanol-induced sedation in adult Drosophila. This behavioral response also requires Efa6, one of three Drosophila Arf6 guanine exchange factors. Ethanol-naive Arf6 and Efa6 mutants were sensitive to ethanol-induced sedation and lacked rapid tolerance upon re-exposure to ethanol, when compared with wild-type flies. In contrast to wild-type flies, both Arf6 and Efa6 mutants preferred alcohol-containing food without prior ethanol experience. An analysis of the human ortholog of Arf6 and orthologs of Efa6 (PSD1-4) revealed that the minor G allele of single nucleotide polymorphism (SNP) rs13265422 in PSD3, as well as a haplotype containing rs13265422, was associated with an increased frequency of drinking and binge drinking episodes in adolescents. The same haplotype was also associated with increased alcohol dependence in an independent European cohort. Unlike the ubiquitously expressed human Arf6 GTPase, PSD3 localization is restricted to the brain, particularly the prefrontal cortex (PFC). Functional magnetic resonance imaging revealed that the same PSD3 haplotype was also associated with a differential functional magnetic resonance imaging signal in the PFC during a Go/No-Go task, which engages PFC-mediated executive control. This analysis therefore suggests that PSD3 confers regional specificity to ubiquitous Arf6 in the PFC to modulate human alcohol-drinking behaviors.
Hautbergue, G. M., et al. (2017). SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits. Nat Commun 8: 16063. PubMed ID: 28677678
Summary:
Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. This study shows that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. it was further demonstrated that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, this study shows that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.
Sekiya, M., Maruko-Otake, A., Hearn, S., Sakakibara, Y., Fujisaki, N., Suzuki, E., Ando, K. and Iijima, K. M. (2017). EDEM function in ERAD protects against chronic ER Proteinopathy and age-related physiological decline in Drosophila. Dev Cell 41(6): 652-664.e655. PubMed ID: 28633019
Summary:
The unfolded protein response (UPR), which protects cells against accumulation of misfolded proteins in the ER, is induced in several age-associated degenerative diseases. However, sustained UPR activation has negative effects on cellular functions and may worsen disease symptoms. It remains unknown whether and how UPR components can be utilized to counteract chronic ER proteinopathies. This study found that promotion of ER-associated degradation (ERAD) through upregulation of ERAD-enhancing alpha-mannosidase-like proteins [EDEMs; EDEM1 (CG3810) and EDEM2 (CG5682)] protected against chronic ER proteinopathy without inducing toxicity in a Drosophila model. ERAD activity in the brain decreased with aging, and upregulation of EDEMs suppressed age-dependent behavioral decline and extended the lifespan without affecting the UPR gene expression network. Intriguingly, EDEM mannosidase activity was dispensable for these protective effects. Therefore, upregulation of EDEM function in the ERAD protects against ER proteinopathy in vivo and thus represents a potential therapeutic target for chronic diseases.
Lim, N. R., Shohayeb, B., Zaytseva, O., Mitchell, N., Millard, S. S., Ng, D. C. H. and Quinn, L. M. (2017). Glial-specific functions of microcephaly protein WDR62 and interaction with the mitotic kinase AURKA are essential for Drosophila brain growth. Stem Cell Reports [Epub ahead of print]. PubMed ID: 28625535
Summary:
The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. This study used Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. It was further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.

Monday, August 7th

Garcia Garcia, E., Little, J. C. and Kalderon, D. (2017). The exon junction complex and Srp54 contribute to Drosophila Hedgehog signaling via ci RNA splicing. Genetics [Epub ahead of print]. PubMed ID: 28637711
Summary:
Hedgehog (Hh) regulates the Cubitus interruptus (Ci) transcription factor in Drosophila melanogaster by activating full-length Ci-155 and blocking processing to Ci-75 repressor. However, the interplay between regulation of Ci-155 levels and activity, as well as processing-independent mechanisms that affect Ci-155 levels have not been explored extensively. This study has identified Mago Nashi (Mago) and Y14 core Exon Junction Complex (EJC) proteins, as well as the Srp54 splicing factor as modifiers of Hh pathway activity under sensitized conditions. Mago inhibition reduced Hh pathway activity by altering the splicing pattern of ci to reduce Ci-155 levels. Srp54 inhibition also affected pathway activity by reducing ci RNA levels but additionally altered Ci-155 levels and activity independently of ci splicing. Further tests using ci transgenes and ci mutations confirmed evidence from studying the effects of Mago and Srp54 that relatively small changes in the level of Ci-155 primary translation product alter Hh pathway activity under a variety of sensitized conditions. ci transgenes lacking intron sequences or the presumed translation initiation codon were used for an alternatively spliced ci RNA to provide further evidence that Mago acts principally by modulating the levels of the major ci RNA encoding Ci-155, and to show that ci introns are necessary to support production of sufficient Ci-155 for robust Hh signaling and may also be important mediators of regulatory inputs.
Li, Q., Kellner, D. A., Hatch, H. A. M., Yumita, T., Sanchez, S., Machold, R. P., Frank, C. A. and Stavropoulos, N. (2017). Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function. PLoS Genet 13(5): e1006815. PubMed ID: 28558011
Summary:
Sleep is an ancient animal behavior that is regulated similarly in species ranging from flies to humans. Various genes that regulate sleep have been identified in invertebrates, but whether the functions of these genes are conserved in mammals remains poorly explored. Drosophila insomniac (inc) mutants exhibit severely shortened and fragmented sleep. Inc protein physically associates with the Cullin-3 (Cul3) ubiquitin ligase, and neuronal depletion of Inc or Cul3 strongly curtails sleep, suggesting that Inc is a Cul3 adaptor that directs the ubiquitination of neuronal substrates that impact sleep. Three proteins similar to Inc exist in vertebrates-KCTD2, KCTD5, and KCTD17-but are uncharacterized within the nervous system and their functional conservation with Inc has not been addressed. This study shows that Inc and its mouse orthologs exhibit striking biochemical and functional interchangeability within Cul3 complexes. Remarkably, KCTD2 and KCTD5 restore sleep to inc mutants, indicating that they can substitute for Inc in vivo and engage its neuronal targets relevant to sleep. Inc and its orthologs localize similarly within fly and mammalian neurons and can traffic to synapses, suggesting that their substrates may include synaptic proteins. Consistent with such a mechanism, inc mutants exhibit defects in synaptic structure and physiology, indicating that Inc is essential for both sleep and synaptic function. These findings reveal that molecular functions of Inc are conserved through ~600 million years of evolution and support the hypothesis that Inc and its orthologs participate in an evolutionarily conserved ubiquitination pathway that links synaptic function and sleep regulation.
Hall, E. T., Pradhan-Sundd, T., Samnani, F. and Verheyen, E. M. (2017). The Protein Phosphatase 4 complex promotes the Notch pathway and wingless transcription. Biol Open [Epub ahead of print]. PubMed ID: 28652317
Summary:
The Wnt/Wingless (Wg) pathway controls cell fate specification, tissue differentiation and organ development across organisms. Using an in vivo RNAi screen to identify novel kinase and phosphatase regulators of the Wg pathway, subunits of the serine threonine phosphatase Protein phosphatase 4 (PP4) were identifed. Knockdown of the catalytic and the regulatory subunits of PP4 cause reductions in the Wg pathway targets Senseless and Distal-less. PP4 regulates the Wg pathway by controlling Notch-driven wg transcription. Genetic interaction experiments identified that PP4 likely promotes Notch signaling within the nucleus of the Notch-receiving cell. Although the PP4 complex is implicated in various cellular processes, its role in the regulation of Wg and Notch pathways was previously uncharacterized. This study identifies a novel role of PP4 in regulating Notch pathway, resulting in aberrations in Notch-mediated transcriptional regulation of the Wingless ligand. Furthermore, it was shown that PP4 regulates proliferation independent of its interaction with Notch.
Jossin, Y., Lee, M., Klezovitch, O., Kon, E., Cossard, A., Lien, W. H., Fernandez, T. E., Cooper, J. A. and Vasioukhin, V. (2017). Llgl1 connects cell polarity with cell-cell adhesion in embryonic neural stem cells. Dev Cell 41(5): 481-495.e485. PubMed ID: 28552558
Evolutionary Homolog Study
Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. Mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1fl/fl;), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. This study shows that LLGL1 directly binds to and promotes internalization of N-cadherin (see Drosophila N-cadherin), and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells.

Sunday, August 6th

Ballinger, M. J. and Perlman, S. J. (2017). Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila. PLoS Pathog 13(7): e1006431. PubMed ID: 28683136
Summary:
While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. This study tested the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. Strong evidence was found for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the alpha-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, was found to escape protection by Spiroplasma, and its ribosomes do not show high levels of depurination. It was also shown that fly ribosomes show little evidence of targeting by RIPs. Finally, the genome of D. neotestacea's defensive Spiroplasma was found to encode a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.
Osada, N., Miyagi, R. and Takahashi, A. (2017). Cis- and trans-regulatory effects on gene expression in a natural population of Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28615283
Summary:
Cis- and trans-regulatory mutations are important contributors to transcriptome evolution. Quantifying their relative contributions to intraspecific variation in gene expression is essential for understanding the population genetic processes that underlie evolutionary changes in gene expression. This study has examined this issue by quantifying genome-wide allele specific expression (ASE) variation using a crossing scheme that produces F1 hybrids between 18 different Drosophila melanogaster strains sampled from the Drosophila Genetic Reference Panel (DGRP) and a reference strain from another population. Head and body samples from F1 adult females were subjected to RNA-seq and the subsequent ASE quantification. Cis- and trans-regulatory effects on expression variation were estimated from these data. A higher proportion of genes showed significant cis-regulatory variation (~28%) than those showed significant trans-regulatory variation (~9%). The sizes of cis-regulatory effects on expression variation were 1.98 and 1.88 times larger than trans-regulatory effects in heads and bodies, respectively. A generalized linear model analysis revealed that both cis- and trans-regulated expression variation was strongly associated with nonsynonymous nucleotide diversity and tissue specificity. Interestingly, trans-regulated variation showed a negative correlation with local recombination rate. Also, analysis on proximal transposon element (TE) insertions suggested that they affect transcription levels of ovary-expressed genes more pronouncedly than genes not expressed in the ovary, possibly due to defense mechanisms against TE mobility in the germline. Collectively, this detailed quantification of ASE variations from a natural population has revealed a number of new relationships between genomic factors and the effects of cis- and trans-regulatory factors on expression variation.
Gibert, J. M., Blanco, J., Dolezal, M., Nolte, V., Peronnet, F. and Schlotterer, C. (2017). Strong epistatic and additive effects of linked candidate SNPs for Drosophila pigmentation have implications for analysis of genome-wide association studies results. Genome Biol 18(1): 126. PubMed ID: 28673357
Summary:
The mapping resolution of genome-wide association studies (GWAS) is limited by historic recombination events and effects are often assigned to haplotype blocks rather than individual SNPs. It is not clear how many of the SNPs in the block, and which ones, are causative. Drosophila pigmentation is a powerful model to dissect the genetic basis of intra-specific and inter-specific phenotypic variation. Three tightly linked SNPs in the t-MSE enhancer have been identified in three D. melanogaster populations as major contributors to female abdominal pigmentation. This enhancer controls the expression of the pigmentation gene tan (t) in the abdominal epidermis. Two of the three SNPs were confirmed in an independent study using the D. melanogaster Genetic Reference Panel established from a North American population. This study determined the functional impact of SNP1, SNP2, and SNP3 using transgenic lines to test all possible haplotypes in vivo. All three candidate SNPs contribute to female Drosophila abdominal pigmentation. Interestingly, only two SNPs agree with the effect predicted by GWAS; the third one goes in the opposite direction because of linkage disequilibrium between multiple functional SNPs. The experimental design uncovered strong additive effects for the three SNPs, but significant epistatic effects explaining up to 11% of the total variation. These results suggest that linked causal variants are important for the interpretation of GWAS and functional validation is needed to understand the genetic architecture of traits.
Leung, W., et al. (2017). Retrotransposons are the major contributors to the expansion of the Drosophila ananassae Muller F element. G3 (Bethesda). PubMed ID: 28667019
Summary:
The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (~5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in Drosophila ananassae. To identify the major contributors to the expansion of the F element and to assess their impact, the genome sequence was improved and the genes in a 1.4 Mb region of the D. ananassae F element, and a 1.7 Mb region from the D element were annotated for comparison. Transposons (particularly LTR and LINE retrotransposons) were found to be major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F element genes exhibit distinct characteristics compared to D element genes (e.g., larger coding spans, larger introns, more coding exons, lower codon bias), but these differences are exaggerated in D. ananassae. Compared to D. melanogaster, the codon bias observed in D. ananassae F element genes can primarily be attributed to mutational biases instead of selection. The 5' ends of F element genes in both species are enriched in H3K4me2 while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves understanding of how transposons can affect genome size and how genes can function within highly repetitive domains.
Morimoto, J., Ponton, F., Tychsen, I., Cassar, J. and Wigby, S. (2017). Interactions between the developmental and adult social environments mediate group dynamics and offspring traits in Drosophila melanogaster. Sci Rep 7(1): 3574. PubMed ID: 28620201
Summary:
Developmental conditions can strongly influence adult phenotypes and social interactions, which in turn affect key evolutionary processes such as sexual selection and sexual conflict. While the implications of social interactions in phenotypically mixed populations at the individual level are increasingly well known, how these effects influence the fate of groups remains poorly understood, which limits understanding of the broader ecological implications. To address this problem this study manipulated adult phenotypes and social composition in Drosophila melanogaster - by experimentally manipulating the larval density of the group-members - and measured a range of group-level outcomes across the lifespan of groups. Adult groups composed of exclusively low larval-density individuals showed high courtship levels, and low early reproductive rates, group growth rates, offspring mass and offspring eclosion success, relative to high larval-density or mixed larval-density groups. Furthermore, high larval-density groups had lower survival. Offspring mass increased with time, but at a reduced rate in groups when male group members (but not females) were from a mixture of larval-densities; peak reproductive rates were also earlier in these groups. These results suggest that that variation in developmental conditions experienced by adult group members can modify the reproductive output of groups.
de Lima, L. G., Svartman, M. and Kuhn, G. C. S. (2017). Dissecting the satellite DNA landscape in three cactophilic Drosophila sequenced genomes. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28659292
Summary:
Eukayote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet poorly studied) genomic components. This study used high throughput sequencing data from three cactophilic Drosophila species, D. buzzatii, D. seriema and D. mojavensis, to access and study their whole satDNA landscape. Five satDNAs were identified, three previously described (pBuM, DBC-150 and CDSTR198) and two novel ones (CDSTR138 and CDSTR130). Only pBuM is shared among all three species. The satDNA repeat length falls within only two classes, between 130-200bp or between 340-390bp. FISH on metaphase and polytene chromosomes revealed the presence of satDNA arrays in at least one of the following genomic compartments: centromeric, telomeric, subtelomeric or dispersed along euchromatin. The chromosomal distribution ranges from a single chromosome to almost all chromosomes of the complement. Interspersion were revealed between pBuM and CDSTR130 in the microchromosomes of D. mojavensis. Phylogenetic analyses showed that the pBuM satDNA underwent concerted evolution at both interspecific and intraspecific levels. Based on RNAseq data, transcription activity was found for pBuM (in D. mojavensis) and CDSTR198 (in D. buzzatii) in all five analyzed developmental stages, most notably in pupae and adult males. These data revealed that cactophilic Drosophila present the lowest amount of satDNAs (1.9% to 2.9%) within the Drosophila genus reported so far.

Saturday, August 5th

Merk, K., Breinig, M., Bottcher, R., Krebs, S., Blum, H., Boutros, M. and Forstemann, K. (2017). Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks. PLoS Genet 13(6): e1006861. PubMed ID: 28628606
Summary:
DNA double-strand breaks trigger the production of locus-derived siRNAs. In flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. A genome-wide RNAi-screen was performed in cultured Drosophila cells, that revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. This observation was validated through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene's first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. It is concluded that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster. Since the splicing factors identified in the screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks.
Kunzelmann, S. and Forstemann, K. (2017). Reversible perturbations of gene regulation after genome editing in Drosophila cells. PLoS One 12(6): e0180135. PubMed ID: 28658280
Summary:
The prokaryotic phage defense CRISPR/cas-system has developed into a versatile toolbox for genome engineering and genetic studies in many organisms. While many efforts were spent on analyzing the consequences of off-target effects, only few studies addressed side-effects that occur due to the targeted manipulation of the genome. This study shows that the CRISPR/cas9-mediated integration of an epitope tag in combination with a selection cassette can trigger an siRNA-mediated, epigenetic genome surveillance pathway in Drosophila melanogaster cells. After homology-directed insertion of the sequence coding for the epitope tag and the selection marker, a moderate level of siRNAs covering the inserted sequence and extending into the targeted locus was detected. This response affected protein levels less than two-fold and it persisted even after single cell cloning. However, removal of the selection cassette abolished the siRNA generation, demonstrating that this response is reversible. Consistently, marker-free genome engineering did not trigger the same surveillance mechanism. These two observations indicate that the selection cassette that was employed induces an aberrant transcriptional arrangement and ultimately sets off the siRNA production. There have been prior concerns about undesirable effects induced by selection markers, but fortunately it was possible to show that at least one of the epigenetic changes reverts as the marker gene is excised. Although the effects observed were rather weak (less than twofold de-repression upon ago2 or dcr-2 knock-down), it is recommended that when selection markers are used during genome editing, a strategy for their subsequent removal should always be included.
Fast, I., Hewel, C., Wester, L., Schumacher, J., Gebert, D., Zischler, H., Berger, C. and Rosenkranz, D. (2017). Temperature-responsive miRNAs in Drosophila orchestrate adaptation to different ambient temperatures. RNA [Epub ahead of print]. PubMed ID: 28630141
Summary:
The majority of Drosophila genes are expressed in a temperature-dependent manner, but the way in which small RNAs may contribute to this effect is completely unknown, since ideas of how small RNA transcriptomes change as a function of temperature are lacking. Applying high throughput sequencing techniques complemented by quantitative real-time PCR experiments, this study demonstrates that altered ambient temperature induces drastic, but reversible changes in sequence composition and total abundance of both, miRNA- and piRNA populations. Further, mRNA sequencing reveals that the expression of miRNAs and their predicted target transcripts correlates inversely, suggesting that temperature-responsive miRNAs drive adaptation to different ambient temperatures on the transcriptome level. Finally, shifts in temperature were shown to affect both, primary and secondary piRNA pools, and the observed aberrations are consistent with altered expression levels of the involved Piwi-pathway factors. It was further reasoned that enhanced ping-pong processing at 29 ° C is driven by dissolved RNA secondary structures at higher temperatures, uncovering target sites that are not accessible at low temperatures. Together, these results show that small RNAs are an important part of epigenetic regulatory mechanisms that ensure homeostasis and adaptation under fluctuating environmental conditions.
Kristo, I., Bajusz, C., Borsos, B. N., Pankotai, T., Dopie, J., Jankovics, F., Vartiainen, M. K., Erdelyi, M. and Vilmos, P. (2017). The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28554770
Summary:
Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. This study shows that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on these results it is proposed that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.
Hara, M., Petrova, B. and Orr-Weaver, T. L. (2017). Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation. Elife 6. PubMed ID: 28555567
Summary:
The oocyte-to-embryo transition involves extensive changes in mRNA translation, regulated in Drosophila by the PNG kinase complex whose activity is shown in this study to be under precise developmental control. Despite presence of the catalytic PNG subunit and the PLU and GNU activating subunits in the mature oocyte, GNU is phosphorylated at Cyclin B/CDK1 sites and unable to bind PNG and PLU. In vitro phosphorylation of GNU by CyclinB/CDK1 blocks activation of PNG. Meiotic completion promotes GNU dephosphorylation and PNG kinase activation to regulate translation. The critical regulatory effect of phosphorylation is shown by replacement in the oocyte with a phosphorylation-resistant form of GNU, which promotes PNG-GNU complex formation, elevation of Cyclin B, and meiotic defects consistent with premature PNG activation. After PNG activation GNU is destabilized, thus inactivating PNG. This short-lived burst in kinase activity links development with maternal mRNA translation and ensures irreversibility of the oocyte-to-embryo transition.
Kan, L., Grozhik, A. V., Vedanayagam, J., Patil, D. P., Pang, N., Lim, K. S., Huang, Y. C., Joseph, B., Lin, C. J., Despic, V., Guo, J., Yan, D., Kondo, S., Deng, W. M., Dedon, P. C., Jaffrey, S. R. and Lai, E. C. (2017). The m6A pathway facilitates sex determination in Drosophila. Nat Commun 8: 15737. PubMed ID: 28675155
Summary:
The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. This study establish the Drosophila system to study the m6A pathway. miCLIP was applied to map m6A across embryogenesis, characterize its m6A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, the transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.

Friday, August 4th

Hidalgo, S., Molina-Mateo, D., Escobedo, P., Zarate, R. V., Fritz, E., Fierro, A., Perez, E. G., Iturriaga-Vasquez, P., Reyes-Parada, M., Varas, R., Fuenzalida-Uribe, N. and Campusano, J. M. (2017). Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors. ACS Chem Neurosci [Epub ahead of print]. PubMed ID: 28665105
Summary:
A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. This study has attempted advance understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. A mutant for Drosophila SERT (dSERT) was characterized, and additionally a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), was used whose mechanism of action involves the SERT protein. The results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior and decreased levels in an anxiety-related parameter, centrophobism. It was also shown that 4-MTA increases the negative chemotaxis towards a strong aversive odorant, Benzaldehyde. The neurochemical data suggest that this effect is mediated by dSERT and depends on 4-MTA-increased release of serotonin in the fly brain. The in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum neurochemical, in-silico and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila.
Koemans, T. S., Oppitz, C., Donders, R. A. T., van Bokhoven, H., Schenck, A., Keleman, K. and Kramer, J. M. (2017). Drosophila courtship conditioning as a measure of learning and memory. J Vis Exp(124) [Epub ahead of print]. PubMed ID: 28605393
Summary:
Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naive flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized in this study. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.
Hu, C., Petersen, M., Hoyer, N., Spitzweck, B., Tenedini, F., Wang, D., Gruschka, A., Burchardt, L. S., Szpotowicz, E., Schweizer, M., Guntur, A. R., Yang, C. H. and Soba, P. (2017). Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior. Nat Neurosci [Epub ahead of print]. PubMed ID: 28604684
Summary:
Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, this study uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. It was further shown that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. These findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.
Kozlov, A., Jaumouille, E., Machado Almeida, P., Koch, R., Rodriguez, J., Abruzzi, K. C. and Nagoshi, E. (2017). A screening of UNF targets identifies Rnb, a novel regulator of Drosophila circadian rhythms. J Neurosci [Epub ahead of print]. PubMed ID: 28592698
Summary:
Behavioral circadian rhythms are controlled by multi-oscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly Drosophila melanogaster are regulated differently in clock neuron subclasses to support their specific functions. The nuclear receptor unfulfilled (unf) represents a regulatory node that provides the small ventral Lateral Neurons (s-LNvs) unique characteristics as the master pacemaker. Previous work has shown that UNF interacts with the s-LNv molecular clocks by regulating transcription of the core clock gene period (per). To gain more insight into the mechanisms by which UNF contributes to the functioning of the circadian master pacemaker, this study identified UNF target genes using chromatin immunoprecipitation. The data demonstrate that a previously uncharacterized gene CG7837, which this study has termed R and B (Rnb), acts downstream of UNF to regulate the function of s-LNvs as the master circadian pacemaker. Mutations and LNv-targeted adult-restricted knockdown of Rnb impair locomotor rhythms. RNB localizes to the nucleus and its loss-of-function blunts the molecular rhythms and output rhythms of the s-LNvs, particularly the circadian rhythms in PDF accumulation and axonal arbor remodeling. These results establish a second pathway by which UNF interacts with the molecular clocks in the s-LNvs and highlight the mechanistic differences in the molecular clockwork within the pacemaker circuit.

Thursday, August 3rd

Jin, P., Bulkley, D., Guo, Y., Zhang, W., Guo, Z., Huynh, W., Wu, S., Meltzer, S., Cheng, T., Jan, L. Y., Jan, Y. N. and Cheng, Y. (2017). Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547(7661): 118-122. PubMed ID: 28658211
Summary:
Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels. The other is the tether model: force is transmitted via a tether to gate the channel. The transient receptor potential (TRP) channel NOMPC is important for mechanosensation-related behaviours such as locomotion, touch and sound sensation across different species including Caenorhabditis elegans, Drosophila and zebrafish. NOMPC is the founding member of the TRPN subfamily, and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton. Thus, a goal of studying NOMPC is to reveal the underlying mechanism of force-induced gating, which could serve as a paradigm of the tether model. NOMPC fulfils all the criteria that apply to mechanotransduction channels and has 29 ankyrin repeats, the largest number among TRP channels. A key question is how the long ankyrin repeat domain is organized as a tether that can trigger channel gating. This study presents a de novo atomic structure of Drosophila NOMPC determined by single-particle electron cryo-microscopy. Structural analysis suggests that the ankyrin repeat domain of NOMPC resembles a helical spring, suggesting its role of linking mechanical displacement of the cytoskeleton to the opening of the channel. The NOMPC architecture underscores the basis of translating mechanical force into an electrical signal within a cell.
Greenblatt Ben-El, R. T., Hassan, A. and Salzberg, A. (2017). Loss of thrombospondin reveals a possible role for the extracellular matrix in chordotonal cap cell elongation. Int J Dev Biol 61(3-4-5): 311-318. PubMed ID: 28621428
Summary:
In the Drosophila larva, major proprioceptive input is provided to the brain by sub-epidermal stretch receptors called chordotonal organs (ChO). Similarly to the body wall muscle that needs to be attached on both of its sides to the larval exoskeleton in order to generate movement, the sensory unit of a ChO must be stably anchored to the cuticle on both of its sides in order to sense the relative displacement of body parts. Through an RNAi screen this study has identified thrombospondin (Tsp), a secreted calcium binding glycoprotein, as a critical component in the anchoring of ChOs to the cuticle. The Tsp protein starts to accumulate in the extracellular matrix (ECM) surrounding the ChO attachment cells towards the end of embryogenesis and that it becomes highly concentrated at the attachment junction during larval stages. In the absence of Tsp, the ChO's accessory cells fail to form a stable junction with their epidermal attachment cells and organ integrity is not maintained. Tsp is a known player in the establishment of the myotendinous junctions in both invertebrates and vertebrates. Thus, these findings extend the known similarities between muscle-attachment and ChO-attachment cells. In addition to its role in establishing the ChO attachment junctions, Tsp was found to affect ligament cell migration and cap cell elongation. Most interestingly, the Tsp protein was found to decorate the ChO cap cells along their entire length, suggesting that the elongated cap cells are supported by the ECM to which they attach via integrin-based, Tsp-dependent, adhesion plaques. The ECM enwrapping the cap cells is probably important for keeping the cap cells fasciculate and may also provide mechanical support that allows the extremely elongated cells to maintain tension.
Gorur-Shandilya, S., Demir, M., Long, J., Clark, D. A. and Emonet, T. (2017). Olfactory receptor neurons use gain control and complementary kinetics to encode intermittent odorant stimuli. Elife 6. PubMed ID: 28653907
Summary:
Insects find food and mates by navigating odorant plumes that can be highly intermittent, with intensities and durations that vary rapidly over orders of magnitude. Much is known about olfactory responses to pulses and steps, but it remains unclear how olfactory receptor neurons (ORNs) detect the intensity and timing of natural stimuli, where the absence of scale in the signal makes detection a formidable olfactory task. By stimulating Drosophila ORNs in vivo with naturalistic and Gaussian stimuli, this study shows that ORNs adapt to stimulus mean and variance, and that adaptation and saturation contribute to naturalistic sensing. Mean-dependent gain control followed the Weber-Fechner relation and occurred primarily at odor transduction, while variance-dependent gain control occurred at both transduction and spiking. Transduction and spike generation possessed complementary kinetic properties, that together preserved the timing of odorant encounters in ORN spiking, regardless of intensity. Such scale-invariance could be critical during odor plume navigation.
Herzmann, S., Krumkamp, R., Rode, S., Kintrup, C. and Rumpf, S. (2017). PAR-1 promotes microtubule breakdown during dendrite pruning in Drosophila. Embo J 36(13): 1981-1991. PubMed ID: 28554895
Summary:
Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. This study shows that the kinase PAR-1 is required for pruning and dendritic microtubule breakdown. The data show that neurons lacking PAR-1 fail to break down dendritic microtubules, and PAR-1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR-1 is a known Tau kinase, and genetic interactions suggest that PAR-1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila. Finally, PAR-1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. These results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning.

Wednesday, August 2nd

Huang, J., Reilein, A. and Kalderon, D. (2017). Yorkie and Hedgehog independently restrict BMP production in Escort cells to permit germline differentiation in the Drosophila ovary. Development. PubMed ID: 28619819
Summary:
Multiple signaling pathways guide the behavior and differentiation of both germline stem cells (GSCs) and somatic stem cells (FSCs) in the Drosophila germarium, necessitating careful control of signal generation, range and responses. Signal integration involves Escort Cells (ECs), which promote differentiation of the GSC derivatives they envelop, provide niche signals for FSCs and derive directly from FSCs in adults. Hedgehog (Hh) signaling induces the Hippo pathway effector Yorkie (Yki) to promote proliferation and maintenance of FSCs but Hh also signals to ECs, which are quiescent. This study shows that in ECs both Hh and Yki limit production of BMP ligands to allow germline differentiation. Loss of Yki produced a more severe germarial phenotype than loss of Hh signaling and principally induced a different BMP ligand. Moreover, Yki activity reporters and epistasis tests showed that Yki does not mediate the key actions of Hh signaling in ECs. Thus, both the coupling and output of Hh and Yki signaling pathways differ between FSCs and ECs despite their proximity and the fact that FSCs give rise directly to ECs.
Hsu, T. H., Yang, C. Y., Yeh, T. H., Huang, Y. C., Wang, T. W. and Yu, J. Y. (2017). The Hippo pathway acts downstream of the Hedgehog signaling to regulate follicle stem cell maintenance in the Drosophila ovary. Sci Rep 7(1): 4480. PubMed ID: 28667262
Summary:
The Hippo pathway is conserved and plays important roles in organ size control. The core components of the Hippo pathway are two kinases Hippo (Hpo), Warts (Wts), and a transcription-co-activator Yorkie (Yki). Yki activity is regulated by phosphorylation, which affects its nuclear localization and stability. To determine the role of the Hippo pathway in stem cells, this study examined follicle stem cells (FSCs) in the Drosophila ovary. Yki is detected in the nucleus of FSCs. Knockdown of yki in the follicle cell lineage leads to a disruption of the follicular epithelium. Mitotic clones of FSCs mutant for hpo or wts are maintained in the niche and tend to replace the other FSCs, and FSCs mutant for yki are rapidly lost, demonstrating that the Hippo pathway is both required and sufficient for FSC maintenance. Using genetic interaction analyses, the Hedgehog pathway was demonstrated to act upstream of the Hippo pathway in regulating FSC maintenance. The nuclear localization of Yki is enhanced when the Hedgehog signaling is activated. Furthermore, a constitutively active but not a wild-type Yki promotes FSC maintenance as activation of the Hedgehog signaling does, suggesting that the Hedgehog pathway regulates Yki through a post-translational mechanism in maintaining FSCs.
Inaba, M., Sorenson, D. R., Kortus, M., Salzmann, V. and Yamashita, Y. M. (2017). Merlin is required for coordinating proliferation of two stem cell lineages in the Drosophila testis. Sci Rep 7(1): 2502. PubMed ID: 28566755
Summary:
Although the mechanisms that balance self-renewal and differentiation of a stem cell lineage have been extensively studied, it remains poorly understood how tissues that contain multiple stem cell lineages maintain balanced proliferation among distinct lineages: when stem cells of a particular lineage proliferate, how do the other lineages respond to maintain the correct ratio of cells among linages? This study shows that Merlin (Mer), a homolog of the human tumor suppressor neurofibromatosis 2, is required to coordinate proliferation of germline stem cells (GSCs) and somatic cyst stem cells (CySCs) in the Drosophila testis. Mer mutant CySCs fail to coordinate their proliferation with that of GSCs in multiple settings, and can be triggered to undergo tumorous overproliferation. Mer executes its function by stabilizing adherens junctions. Given the known role of Mer in contact-dependent inhibition of proliferation, it is proposed that the proliferation of CySCs are regulated by crowdedness, or confluency, of cells in their lineage with respect to that of germline, thereby coordinating the proliferation of two lineages.
Hamada-Kawaguchi, N. and Yamamoto, D. (2017). Ovarian polarity and cell shape determination by Btk29A in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 28639397
Summary:
Drosophila Btk29A is a Tec family nonreceptor tyrosine kinase, the ortholog of which causes X-linked agammagluburinemia in humans when mutant. In Btk29AficP mutant ovaries, multiple defects are observed: extra polar cells form ectopically; osk mRNA fails to accumulate posteriorly in mature oocytes; the shape and alignment of follicle cells are grossly distorted. All these phenotypes are rescued by selectively overexpressing the type 2 isoform of wild-type Btk29A in follicle cells. Expression of certain proteins enriched in adherens junctions is markedly affected in Btk29AficP mutants; the anterior-posterior gradient normally observed in the expression of DE-Cadherin and Armadillo are lost and Canoe is sequestered from adherens junctions. Intriguingly, tyrosine phosphorylation of Canoe is reduced in Btk29AficP mutants. It is proposed that Btk29A is required for the establishment of egg chamber polarity presumably through the regulation of subcellular localization of its downstream proteins, including Cno.

Tuesday, August 1st

Davis, T. L. and Rebay, I. (2017). Antagonistic regulation of the second mitotic wave by Eyes absent-Sine oculis and Combgap coordinates proliferation and specification in the Drosophila retina. Development. PubMed ID: 28619818
Summary:
The transition from proliferation to specification is fundamental to the development of appropriately patterned tissues. In the developing Drosophila eye, Eyes absent (Eya) and Sine oculis (So) orchestrate the progression of progenitor cells from asynchronous cell division to G1 arrest and neuronal specification at the morphogenetic furrow. This study uncovered a novel role for Eya and So in promoting cell cycle exit in the Second Mitotic Wave (SMW), a synchronized, terminal cell division that occurs several hours after passage of the furrow. Combgap (Cg), a zinc-finger transcription factor, antagonizes Eya-So function in the SMW. Based on Cg's ability to attenuate Eya-So transcriptional output in vivo and in cultured cells and on meta-analysis of their chromatin occupancy profiles, it is speculated that Cg limits Eya-So activation of select target genes posterior to the furrow to ensure properly timed mitotic exit. This work supports a model in which context-specific modulation of transcriptional activity enables Eya and So to promote both entry into and exit from the cell cycle in a distinct spatiotemporal sequence.
Dahal, G. R., Pradhan, S. J. and Bates, E. A. (2017). Inwardly rectifying potassium channels regulate Dpp release in the Drosophila wing disc. Development [Epub ahead of print]. PubMed ID: 28684627
Summary:
Loss of embryonic ion channel function leads to morphological defects, but the underlying reason for these defects remains elusive. This study shows that inwardly-rectifying potassium (Irk) channels regulate release of a Drosophila bone morphogenetic protein (BMP/Dpp) in the developing fly wing and this is necessary for developmental signaling. Inhibition of Irk channels decreases the incidence of distinct Dpp-GFP release events above baseline fluorescence while leading to broader distribution of Dpp-GFP. Work by others in different cell types show Irk channels regulate peptide release by modulating membrane potential and calcium levels. This study found calcium transients in the developing wing and inhibition of Irk channels reduces the duration and amplitude of calcium transients. Depolarization with high extracellular potassium evokes Dpp release. Taken together, these data implicate Irk channels as a requirement for regulated release of Dpp, highlighting the importance of the temporal pattern of Dpp presentation for morphogenesis of the wing.
Barrio, L. and Milan, M. (2017). Boundary Dpp promotes growth of medial and lateral regions of the Drosophila wing. Elife 6. PubMed ID: 28675372
Summary:
The gradient of Decapentaplegic (Dpp) in the Drosophila wing has served as a paradigm to characterize the role of morphogens in regulating patterning. However, the role of this gradient in regulating tissue size is a topic of intense debate as proliferative growth is homogenous. This study combined the Gal4/UAS system and a temperature-sensitive Gal80 molecule to induce RNAi-mediated depletion of dpp and characterise the spatial and temporal requirement of Dpp in promoting growth. Dpp emanating from the AP compartment boundary was shown to be required throughout development to promote growth by regulating cell proliferation and tissue size. Dpp regulates growth and proliferation rates equally in central and lateral regions of the developing wing appendage and reduced levels of Dpp affects similarly the width and length of the resulting wing. Evidence is presented supporting the proposal that graded activity of Dpp is not an absolute requirement for wing growth.
Heinze, S. D., Kohlbrenner, T., Ippolito, D., Meccariello, A., Burger, A., Mosimann, C., Saccone, G. and Bopp, D. (2017). CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus. Sci Rep 7(1): 4582. PubMed ID: 28676649
Summary:
The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). This study demonstrates that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, two mutant MdY alleles were identified that contain lesions predicted to result in premature truncation of the MdY open reading frame. Wildtype MdY was targeted by CRISPR-Cas9 RNPs and new mutant alleles were generated alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. Further evidence was found for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. This work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model
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