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Thursday December 30th - Adult Neural Development and Function

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Coquerel, Q. R. R., Demares, F., Geldenhuys, W. J., Le Ray, A. M., Breard, D., Richomme, P., Legros, C., Norris, E. and Bloomquist, J. R. (2021). Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor. Toxicon 201:141-147. PubMed ID: 34474068
Summary:
Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20-30 μM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC(50) value of about 1 μM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline.
Devineni, A. V., Deere, J. U., Sun, B. and Axel, R. (2021). Individual bitter-sensing neurons in Drosophila exhibit both ON and OFF responses that influence synaptic plasticity. Curr Biol. PubMed ID: 34731675
Summary:
The brain generates internal representations that translate sensory stimuli into appropriate behavior. In the taste system, different tastes activate distinct populations of sensory neurons. This study investigated the temporal properties of taste responses in Drosophila and discovered that different types of taste sensory neurons show striking differences in their response dynamics. Strong responses to stimulus onset (ON responses) and offset (OFF responses) were observed in bitter-sensing neurons in the labellum, whereas bitter neurons in the leg and other classes of labellar taste neurons showed only an ON response. Individual labellar bitter neurons generate both ON and OFF responses through a cell-intrinsic mechanism that requires canonical bitter receptors. A single receptor complex likely generates both ON and OFF responses to a given bitter ligand. These ON and OFF responses in the periphery are propagated to dopaminergic neurons that mediate aversive learning, and the presence of the OFF response impacts synaptic plasticity when bitter is used as a reinforcement cue. These studies reveal previously unknown features of taste responses that impact neural circuit function and may be important for behavior. Moreover, these studies show that OFF responses can dramatically influence timing-based synaptic plasticity, which is thought to underlie associative learning.
Hulse, B. K., Haberkern, H., Franconville, R., Turner-Evans, D. B., Takemura, S. Y., Wolff, T., Noorman, M., Dreher, M., Dan, C., Parekh, R., Hermundstad, A. M., Rubin, G. M. and Jayaraman, V. (2021). A connectome of the Drosophila central complex reveals network motifs suitable for flexible navigation and context-dependent action selection. Elife 10. PubMed ID: 34696823
Summary:
Flexible behaviors over long timescales are thought to engage recurrent neural networks in deep brain regions, which are experimentally challenging to study. In insects, recurrent circuit dynamics in a brain region called the central complex (CX) enable directed locomotion, sleep, and context- and experience-dependent spatial navigation. We describe the first complete electron-microscopy-based connectome of the Drosophila CX, including all its neurons and circuits at synaptic resolution. This study identified new CX neuron types, novel sensory and motor pathways, and network motifs that likely enable the CX to extract the fly's head-direction, maintain it with attractor dynamics, and combine it with other sensorimotor information to perform vector-based navigational computations. Numerous pathways were identified that may facilitate the selection of CX-driven behavioral patterns by context and internal state. The CX connectome provides a comprehensive blueprint necessary for a detailed understanding of network dynamics underlying sleep, flexible navigation, and state-dependent action selection.
Eschbach, C., Fushiki, A., Winding, M., Afonso, B., Andrade, I. V., Cocanougher, B. T., Eichler, K., Gepner, R., Si, G., Valdes-Aleman, J., Fetter, R. D., Gershow, M., Jefferis, G. S., Samuel, A. D., Truman, J. W., Cardona, A. and Zlatic, M. (2021). Circuits for integrating learned and innate valences in the insect brain. Elife 10. PubMed ID: 34755599
Summary:
Animal behavior is shaped both by evolution and by individual experience. Parallel brain pathways encode innate and learned valences of cues, but the way in which they are integrated during action-selection is not well understood. This study used electron microscopy to comprehensively map with synaptic resolution all neurons downstream of all Mushroom Body output neurons (encoding learned valences) and characterized their patterns of interaction with Lateral Horn neurons (encoding innate valences) in Drosophila larva. The connectome revealed multiple convergence neuron types that receive convergent Mushroom Body and Lateral Horn inputs. A subset of these receives excitatory input from positive-valence MB and LH pathways and inhibitory input from negative-valence MB pathways. Functional connectivity from LH and MB pathways and behavioral roles of two of these neurons was confirmed. These neurons encode integrated odor value and bidirectionally regulate turning. Based on this it is speculated that learning could potentially skew the balance of excitation and inhibition onto these neurons and thereby modulate turning. Together, this study provides insights into the circuits that integrate learned and innate valences to modify behavior.
Dhakal, S., Sang, J., Aryal, B. and Lee, Y. (2021). Ionotropic receptors mediate nitrogenous waste avoidance in Drosophila melanogaster. Commun Biol 4(1): 1281. PubMed ID: 34773080
Summary:
Ammonia and its amine-containing derivatives are widely found in natural decomposition byproducts. A biased chemoreceptor screening was conducted to investigate the mechanisms by which different concentrations of ammonium salt, urea, and putrescine in rotten fruits affect feeding and oviposition behavior. Three ionotropic receptors, including the two broadly required IR25a and IR76b receptors, were detected and the narrowly tuned IR51b receptor. These three IRs were fundamental in eliciting avoidance against nitrogenous waste products, which is mediated by bitter-sensing gustatory receptor neurons (GRNs). The aversion of nitrogenous wastes was evaluated by the cellular requirement by expressing Kir2.1 and behavioral recoveries of the mutants in bitter-sensing GRNs. Furthermore, by conducting electrophysiology assays, it was confirmed that ammonia compounds are aversive in taste as they directly activated bitter-sensing GRNs. Therefore, our findings provide insights into the ecological roles of IRs as a means to detect and avoid toxic nitrogenous waste products in nature.
Gruntman, E., Reimers, P., Romani, S. and Reiser, M. B. (2021). Non-preferred contrast responses in the Drosophila motion pathways reveal a receptive field structure that explains a common visual illusion. Curr Biol. PubMed ID: 34672960
Summary:
Diverse sensory systems, from audition to thermosensation, feature a separation of inputs into ON (increments) and OFF (decrements) signals. In the Drosophila visual system, separate ON and OFF pathways compute the direction of motion, yet anatomical and functional studies have identified some crosstalk between these channels. This well-studied circuit was used to ask whether the motion computation depends on ON-OFF pathway crosstalk. Using whole-cell electrophysiology, visual responses of T4 (ON) and T5 (OFF) cells were recorded and their composite ON-OFF receptive fields were mapped: it was found that they share a similar spatiotemporal structure. A biophysical model was fit to these receptive fields that accurately predicts directionally selective T4 and T5 responses to both ON and OFF moving stimuli. This model also provides a detailed mechanistic explanation for the directional preference inversion in response to the prominent reverse-phi illusion. Finally, the steering responses of tethered flying flies was used to validate the model's predicted effects of varying stimulus parameters on the behavioral turning inversion.

Wednesday, December 29th - Synapse and Vesicles

Giachello, C. N. G., Fan, Y. N., Landgraf, M. and Baines, R. A. (2021). Nitric oxide mediates activity-dependent change to synaptic excitation during a critical period in Drosophila. Sci Rep 11(1): 20286. PubMed ID: 34645891
Summary:
The emergence of coordinated network function during nervous system development is often associated with critical periods. These phases are sensitive to activity perturbations during, but not outside, of the critical period, that can lead to permanently altered network function for reasons that are not well understood. In particular, the mechanisms that transduce neuronal activity to regulating changes in neuronal physiology or structure are not known. This study take advantage of a recently identified invertebrate model for studying critical periods, the Drosophila larval locomotor system. Manipulation of neuronal activity during this critical period is sufficient to increase synaptic excitation and to permanently leave the locomotor network prone to induced seizures. Using genetics and pharmacological manipulations, this study identifies nitric oxide (NO)-signaling as a key mediator of activity. Transiently increasing or decreasing NO-signaling during the critical period mimics the effects of activity manipulations, causing the same lasting changes in synaptic transmission and susceptibility to seizure induction. Moreover, the effects of increased activity on the developing network are suppressed by concomitant reduction in NO-signaling and enhanced by additional NO-signaling. These data identify NO signaling as a downstream effector, providing new mechanistic insight into how activity during a critical period tunes a developing network (Giachello, 2021).
Feghhi, T., Hernandez, R. X., Stawarski, M., Thomas, C. I., Kamasawa, N., Lau, A. W. C. and Macleod, G. T. (2021). Computational modeling predicts ephemeral acidic microdomains followed by prolonged alkalinization in the glutamatergic synaptic cleft. Biophys J. PubMed ID: 34774503
Summary:
At chemical synapses, synaptic vesicles release their acidic contents into the cleft leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization, rather than acidification. It is study, using a reaction-diffusion scheme,pH dynamics were modeled at the Drosophila meuromuscular junction as glutamate, adenosine triphosphate (ATP) and protons (H(+)) are released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase (PMCA) activity and predicts substantial cleft acidification but only for fractions of a millisecond following neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 milliseconds, as the PMCA removes H(+) from the cleft in exchange for calcium ions (Ca(2+)) from adjacent pre- and postsynaptic compartments; thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis has little influence on the magnitude of acidification. Phosphate, but not bicarbonate buffering is effective at suppressing the magnitude and time course of the acid spike, while both buffering systems are effective at suppressing cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH transients accompanying neurotransmission.
Hung, Y. C., Huang, K. L., Chen, P. L., Li, J. L., Lu, S. H., Chang, J. C., Lin, H. Y., Lo, W. C., Huang, S. Y., Lee, T. T., Lin, T. Y., Imai, Y., Hattori, N., Liu, C. S., Tsai, S. Y., Chen, C. H., Lin, C. H. and Chan, C. C. (2021). UQCRC1 engages cytochrome c for neuronal apoptotic cell death. Cell Rep 36(12): 109729. PubMed ID: 34551295
Summary:
Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. This study recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. This study investigates the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases Cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. The findings identified a role for UQCRC1 in regulating cyt-c-induced apoptosis (Hung, 2021).
Mrestani, A., Pauli, M., Kollmannsberger, P., Repp, F., Kittel, R. J., Eilers, J., Doose, S., Sauer, M., Siren, A. L., Heckmann, M. and Paul, M. M. (2021. Active zone compaction correlates with presynaptic homeostatic potentiation. Cell Rep 37(1): 109770. PubMed ID: 34610300
Summary:
Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. This study use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. This study found compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier (Mretiani, 2021).
Lakatos, Z., Benko, P., Juhasz, G. and Lorincz, P. (2021). Drosophila Rab39 Attenuates Lysosomal Degradation. Int J Mol Sci 22(19). PubMed ID: 34638976
Summary:
=">Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, no failure was found in the early steps of endocytosis and autophagy. On the contrary, Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. These data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy.
Dar, G. H., Mendes, C. C., Kuan, W. L., Speciale, A. A., Conceicao, M., Gorgens, A., Uliyakina, I., Lobo, M. J., Lim, W. F., El Andaloussi, S., Mager, I., Roberts, T. C., Barker, R. A., Goberdhan, D. C. I., Wilson, C. and Wood, M. J. A. (2021). GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain. Nat Commun 12(1): 6666. PubMed ID: 34795295
Summary:
Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. This study demonstrates a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. This study observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain.

Tuesday, December 28th - Disease Models

Choi, H. J., Lee, J. Y., Cha, S. J., Han, Y. J., Yoon, J. H., Kim, H. J. and Kim, K. (2021). FUS-induced neurotoxicity is prevented by inhibiting GSK-3beta in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet. PubMed ID: 34605896.
Summary:
Amyotrophic lateral sclerosis (ALS)-linked mutations in fused in sarcoma (FUS; see Drosophila Cabeza) lead to the formation of cytoplasmic aggregates in neurons. They are believed play a critical role in the pathogenesis of FUS-associated ALS. Therefore, the clearance and degradation of cytoplasmic FUS aggregates in neurons may be considered a therapeutic strategy for ALS. However, the molecular pathogenic mechanisms behind FUS-associated ALS remain poorly understood. This paper reports GSK-3β as a potential modulator of FUS-induced toxicity. RNAi-mediated knockdown of Drosophila ortholog Shaggy in FUS-expressing flies suppresses defective phenotypes, including retinal degeneration, motor defects, motor neuron degeneration, and mitochondrial dysfunction. Furthermore, cytoplasmic FUS aggregates were significantly reduced by Shaggy knockdown. In addition, it was found that the levels of FUS proteins were significantly reduced by co-overexpression of Slimb, a F-box protein, in FUS-expressing flies, indicating that Slimb is critical for the suppressive effect of Shaggy/GSK-3β inhibition on FUS-induced toxicity in Drosophila. These findings revealed a novel mechanism of neuronal protective effect through SCFSlimb-mediated FUS degradation via GSK-3β inhibition, and provided in vivo evidence of the potential for modulating FUS-induced ALS progression using GSK-3β inhibitors.
Girard, V., Jollivet, F., Knittelfelder, O., Celle, M., Arsac, J. N., Chatelain, G., Van den Brink, D. M., Baron, T., Shevchenko, A., Kuhnlein, R. P., Davoust, N. and Mollereau, B. (2021). Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease. PLoS Genet 17(11): e1009921. PubMed ID: 34788284
Summary:
Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, a transgenic Drosophila model of PD was used, in which human αSyn is specifically expressed in photoreceptor neurons. It was first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic, and catabolic enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. It is proposed that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.
Aditi, K., Singh, A., Shakarad, M. N. and Agrawal, N. (2021). Management of altered metabolic activity in Drosophila model of Huntington's disease by curcumin. Exp Biol Med (Maywood): 15353702211046927. PubMed ID: 34743577
Summary:
Huntington's disease (HD) is a devastating polyglutamine disorder characterized by extensive neurodegeneration and metabolic abnormalities at systemic, cellular and intracellular levels. Metabolic alterations in HD manifest as abnormal body weight, dysregulated biomolecule levels, impaired adipocyte functions, and defective energy state which exacerbate disease progression and pose acute threat to the health of challenged individuals in form of insulin resistance, cardiovascular disease, and energy crisis. To mitigate disease symptoms, this study tested the efficacy of curcumin in Drosophila model of HD. Curcumin is the bioactive component of turmeric (Curcuma longa Linn), well-known for its ability to modulate metabolic activities. Curcumin was found to effectively managed abnormal body weight, dysregulated lipid content, and carbohydrate level in HD flies. In addition, curcumin administration lowered elevated reactive-oxygen-species levels in adult adipose tissue of diseased flies, and improved survival and locomotor function in HD flies at advanced disease stage. Altogether, these findings clearly suggest that curcumin efficiently attenuates metabolic derangements in HD flies and can prove beneficial in alleviating the complexities associated with HD.
Deng, X., Sun, X., Yue, W., Duan, Y., Hu, R., Zhang, K., Ni, J., Cui, J., Wang, Q., Chen, Y., Li, A. and Fang, Y. (2022). CHMP2B regulates TDP-43 phosphorylation and cytotoxicity independent of autophagy via CK1. J Cell Biol 221(1). PubMed ID: 34726688.
Summary:
The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy-endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2BIntron5 mutation. This study identified CHMP2B as a modifier of TDP-43-mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2BIntron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, this study found that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, this study uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, these findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.
Coni, S., Falconio, F. A., Marzullo, M., Munafo, M., Zuliani, B., Mosti, F., Fatica, A., Ianniello, Z., Bordone, R., Macone, A., Agostinelli, E., Perna, A., Matkovic, T., Sigrist, S., Silvestri, G., Canettieri, G. and Ciapponi, L. (2021). Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function. Elife 10. PubMed ID: 34517941.
Summary:
Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. This study shows that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. This study demonstrated that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, this study showed a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, evidence is provided that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. It is suggested that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions.
Habernig, L., Broeskamp, F., Aufschnaiter, A., Diessl, J., Peselj, C., Urbauer, E., Eisenberg, T., de Ory, A. and Buttner, S. (2021). Ca2+ administration prevents α-synuclein proteotoxicity by stimulating calcineurin-dependent lysosomal proteolysis. PLoS Genet 17(11): e1009911. PubMed ID: 34780474.
Summary:
The capacity of a cell to maintain proteostasis progressively declines during aging. Virtually all age-associated neurodegenerative disorders associated with aggregation of neurotoxic proteins are linked to defects in the cellular proteostasis network, including insufficient lysosomal hydrolysis. This study reports that proteotoxicity in yeast and Drosophila models for Parkinson's disease can be prevented by increasing the bioavailability of Ca2+, which adjusts intracellular Ca2+ handling and boosts lysosomal proteolysis. Heterologous expression of human α-synuclein (αSyn), a protein critically linked to Parkinson's disease, selectively increases total cellular Ca2+ content, while the levels of manganese and iron remain unchanged. Disrupted Ca2+ homeostasis results in inhibition of the lysosomal protease cathepsin D and triggers premature cellular and organismal death. External administration of Ca2+ reduces αSyn oligomerization, stimulates cathepsin D activity and in consequence restores survival, which critically depends on the Ca2+/calmodulin-dependent phosphatase calcineurin. In flies, increasing the availability of Ca2+ discloses a neuroprotective role of αSyn upon manganese overload. In sum, this study established a molecular interplay between cathepsin D and calcineurin that can be activated by Ca2+ administration to counteract αSyn proteotoxicity.

Monday, December 28th - Adult Physiology and Metabolism

Barrios, N., Farias, M. and Moita, M. A. (2021). Threat induces cardiac and metabolic changes that negatively impact survival in flies. Curr Biol. PubMed ID: 34710349
Summary:
Adjusting to a dynamic environment involves fast changes in the body's internal state, characterized by coordinated alterations in brain activity and physiological and motor responses. Threat-induced defensive states are a classic case of coordinated adjustment of bodily responses, cardiac regulation being one of the best characterized examples in vertebrates. A great deal is known regarding the neural basis of invertebrate defensive behaviors, mainly in Drosophila melanogaster. However, whether physiological changes accompany these remains unknown. This study sets out to describe the internal bodily state of fruit flies upon an inescapable threat and found cardiac acceleration during running and deceleration during freezing. In addition, this study found that freezing leads to increased cardiac pumping from the abdomen toward the head-thorax, suggesting mobilization of energy resources. Concordantly, threat-triggered freezing reduces sugar levels in the hemolymph and renders flies less resistant to starvation. The cardiac responses observed during freezing were absent during spontaneous immobility, underscoring the active nature of freezing response. Finally, this study shows that baseline cardiac activity predicts the amount of freezing upon threat. This work reveals a remarkable similarity with the cardiac responses of vertebrates, suggesting an evolutionarily convergent defensive state in flies. These findings are at odds with the widespread view that cardiac deceleration while freezing has first evolved in vertebrates and that it is energy sparing. Investigating the physiological changes coupled to defensive behaviors in the fruit fly has revealed that freezing is costly yet accompanied by cardiac deceleration and points to heart activity as a key modulator of defensive behaviors (Barrios, 2021).
Chokshi, R., Bennett, O., Zhelay, T. and Kozak, J. A. (2021). NSAIDs Naproxen, Ibuprofen, Salicylate, and Aspirin Inhibit TRPM7 Channels by Cytosolic Acidification. Front Physiol 12: 727549. PubMed ID: 34733174.
Summary:
Non-steroidal anti-inflammatory drugs (NSAIDs) are used for relieving pain and inflammation accompanying numerous disease states. The primary therapeutic mechanism of these widely used drugs is the inhibition of cyclooxygenase 1 and 2 (COX1, 2) enzymes that catalyze the conversion of arachidonic acid into prostaglandins. Transient receptor potential melastatin 7 (TRPM7) cation channels are highly expressed in T lymphocytes and are inhibited by Mg(2+), acidic pH, and polyamines. This study reports a novel effect of naproxen, ibuprofen, salicylate, and acetylsalicylate on TRPM7. At concentrations of 3-30mM, they reversibly inhibited TRPM7 channel currents. By measuring intracellular pH with the ratiometric indicator BCECF, it was found that at 300μM to 30mM, these NSAIDs reversibly acidified the cytoplasm in a concentration-dependent manner, and it is proposed that TRPM7 channel inhibition is a consequence of cytosolic acidification, rather than direct. NSAID inhibition of TRPM7 channels was slow, voltage-independent, and displayed use-dependence, increasing in potency upon repeated drug applications. The extent of channel inhibition by salicylate strongly depended on cellular PI(4,5)P(2) levels, as revealed when this phospholipid was depleted with voltage-sensitive lipid phosphatase (VSP). Salicylate inhibited heterologously expressed wildtype TRPM7 channels but not the S1107R variant, which is insensitive to cytosolic pH, Mg(2+), and PI(4,5)P(2) depletion. NSAID-induced acidification was also observed in Schneider 2 cells from Drosophila, an organism that lacks orthologous COX genes, suggesting that this effect is unrelated to COX enzyme activity. A 24-h exposure to 300μM-10mM naproxen resulted in a concentration-dependent reduction in cell viability. In addition to TRPM7, the described NSAID effect would be expected to apply to other ion channels and transporters sensitive to intracellular pH.
Ebanks, B., Wang, Y., Katyal, G., Sargent, C., Ingram, T. L., Bowman, A., Moisoi, N. and Chakrabarti, L. (2021). Exercising D. melanogaster Modulates the Mitochondrial Proteome and Physiology. The Effect on Lifespan Depends upon Age and Sex. Int J Mol Sci 22(21). PubMed ID: 34769041 .
Summary:
Ageing is a major risk factor for many of the most prevalent diseases, including neurodegenerative diseases, cancer, and heart disease. As the global population continues to age, behavioural interventions that can promote healthy ageing will improve quality of life and relieve the socioeconomic burden that comes with an aged society. Exercise is recognised as an effective intervention against many diseases of ageing, but we do not know the stage in an individual's lifetime at which exercise is most effective at promoting healthy ageing, and whether or not it has a direct effect on lifespan. This study exercised w(1118) Drosophila melanogaster, investigating the effects of sex and group size at different stages of their lifetime, and recorded their lifespan. Climbing scores at 30 days were measured to record differences in fitness in response to exercise. The mitochondrial proteome was assessed of w(1118) Drosophila that had been exercised for one week, alongside mitochondrial respiration measured using high-resolution respirometry, to determine changes in mitochondrial physiology in response to exercise. This study found that age-targeted exercise interventions improved the lifespan of both male and female Drosophila, and grouped males exercised in late life had improved climbing scores when compared with those exercised throughout their entire lifespan. The proteins of the electron transport chain were significantly upregulated in expression after one week of exercise, and complex-II-linked respiration was significantly increased in exercised Drosophila. Taken together, our findings provide a basis to test specific proteins, and complex II of the respiratory chain, as important effectors of exercise-induced healthy ageing
Dwivedi, S., D'Souza, L. C., Shetty, N. G., Raghu, S. V. and Sharma, A. (2021). Hsp27, a potential EcR target, protects nonylphenol-induced cellular and organismal toxicity in Drosophila melanogaster. Environ Pollut: 118484. PubMed ID: 34774861 .
Summary:
Deciphering the potential mechanism of chemical-induced toxicity enables us to alleviate the cellular and organismal dysfunction. The environmental presence of nonylphenol (endocrine disruptor) has a major health concern due to its widespread usage in our day-to-day life. The current study establishes a novel functional link among nonylphenol-induced oxidative stress, Heat shock protein 27 (Hsp27, member of stress protein family), and Ecdysone receptor (EcR, a nuclear receptor), which eventually coordinates the nonylphenol-induced sub-cellular and organismal level toxicity in a genetically tractable model Drosophila melanogaster. Drosophila larvae (wild-type) exposed to nonylphenol (0.05, 0.5 and 5.0 &mi;g/mL) showed a significant decrease in Hsp27 and EcR mRNA levels. In concurrence, reactive oxygen species (ROS) levels were increased with a corresponding decline in glutathione (GSH) level and Thioredoxin reductase (TrxR) activity. Increased lipid peroxidation (LPO), protein carbonyl (PC) contents, and cell death were also observed in a correlation with the nonylphenol concentration. Sub-cellular toxicity poses a negative organismal response, which was evident by delayed larval development and reduced Drosophila emergence. Subsequently, a positive genetic correlation (p < 0.001) between EcR and Hsp27 revealed that nonylphenol-dependent EcR reduction is a possible link for the downregulation of Hsp27. Further, Hsp27 overexpression in midgut cells showed a reduction in nonylphenol-induced intracellular ROS, LPO, PC content, and cell death through the TrxR mediated regenerative pathway and reduced GSH level improving the organismal response to the nonylphenol exposure. Altogether, the study elucidates the potential EcR-Hsp27 molecular interactions in mitigating the nonylphenol-induced cellular and organismal toxicity.
El Kholy, S., Wang, K., El-Seedi, H. R. and Al Naggar, Y. (2021). Dopamine Modulates Drosophila Gut Physiology, Providing New Insights for Future Gastrointestinal Pharmacotherapy. Biology (Basel) 10(10). PubMed ID: 34681083.
Summary:
Dopamine has a variety of physiological roles in the gastrointestinal tract (GI) through binding to Drosophila dopamine D1-like receptors (DARs) and/or adrenergic receptors and has been confirmed as one of the enteric neurotransmitters. To gain new insights into what could be a potential future promise for GI pharmacology, Drosophila was used as a model organism to investigate the effects of dopamine on intestinal physiology and gut motility. GAL4/UAS system was utilized to knock down specific dopamine receptors using specialized GAL4 driver lines targeting neurons or enterocytes cells to identify which dopamine receptor controls stomach contractions. DARs (Dop1R1 and Dop1R2) were shown by immunohistochemistry to be strongly expressed in all smooth muscles in both larval and adult flies, which could explain the inhibitory effect of dopamine on GI motility. Adult males' gut peristalsis was significantly inhibited by knocking down dopamine receptors Dop1R1, Dop1R2, and Dop2R, but female flies' gut peristalsis was significantly repressed by knocking down only Dop1R1 and Dop1R2. These findings also showed that dopamine drives PLC-β translocation from the cytoplasm to the plasma membrane in enterocytes for the first time. Overall, these data revealed the role of dopamine in modulating Drosophila gut physiology, offering us new insights for the future gastrointestinal pharmacotherapy of neurodegenerative diseases associated with dopamine deficiency.
Cong, B., Nakamura, M., Sando, Y., Kondo, T., Ohsawa, S. and Igaki, T. (2021). JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila. PLoS Genet 17(11): e1009893. PubMed ID: 34780467.
Summary:
Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. This study performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), was identified is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. These observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.

Friday, December 24th - Stem Cells

Nguyen, T. T. N., Shim, J. and Song, Y. H. (2021). Chk2-p53 and JNK in irradiation-induced cell death of hematopoietic progenitors and differentiated cells in Drosophila larval lymph gland. Biol Open 10(8). PubMed ID: 34328173.
Summary:
Ionizing radiation (IR) induces DNA double-strand breaks that activate the DNA damage response (DDR), which leads to cell cycle arrest, senescence, or apoptotic cell death. Understanding the DDR of stem cells is critical to tissue homeostasis and the survival of the organism. Drosophila hematopoiesis serves as a model system for sensing stress and environmental changes; however, their response to DNA damage remains largely unexplored. The Drosophila lymph gland is the larval hematopoietic organ, where stem-like progenitors proliferate and differentiate into mature blood cells called hemocytes. It was found that apoptotic cell death was induced in progenitors and hemocytes after 40 Gy irradiation, with progenitors showing more resistance to IR-induced cell death compared to hemocytes at a lower dose. Furthermore, it was found that Drosophila ATM (tefu), Chk2 (lok), p53, and reaper were necessary for IR-induced cell death in the progenitors. Notably, IR-induced cell death in mature hemocytes required tefu, Drosophila JNK (bsk), and reaper, but not lok or p53. In summary, this study found that DNA damage induces apoptotic cell death in the late third instar larval lymph gland and identified lok/p53-dependent and -independent cell death pathways in progenitors and mature hemocytes, respectively.
Roubinet, C., White, I. J. and Baum, B. (2021). Asymmetric nuclear division in neural stem cells generates sibling nuclei that differ in size, envelope composition, and chromatin organization. Curr Biol. PubMed ID: 34297912.
Summary:
Although nuclei are the defining features of eukaryotes, how the nuclear compartment is duplicated and partitioned during division is still do not fully understand. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, it was found that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a >30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fate determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate.
Liu, F., Zhao, H., Kong, R., Shi, L., Li, Z., Ma, R., Zhao, H. and Li, Z. (2021). Derlin-1 and TER94/VCP/p97 are required for intestinal homeostasis. J Genet Genomics. PubMed ID: 34547438.
Summary:
Adult stem cells are critical for the maintenance of residential tissue homeostasis and functions. However, the roles of cellular protein homeostasis maintenance in stem cell proliferation and tissue homeostasis are not fully understood. This study found that Derlin-1 and TER94/VCP/p97, components of the ER-associated degradation (ERAD) pathway, restrain intestinal stem cell proliferation to maintain intestinal homeostasis in adult Drosophila. Depleting any of them results in increased stem cell proliferation and midgut homeostasis disruption. Derlin-1 is specifically expressed in the ER of progenitors and its C-terminus is required for its function. Interestingly, it was found that increased stem cell proliferation is resulted from elevated ROS levels and activated JNK signaling in Derlin-1- or TER94-deficient progenitors. Further removal of ROS or inhibition of JNK signaling almost completely suppressed increased stem cell proliferation. Together, these data demonstrate that the ERAD pathway is critical for stem cell proliferation and tissue homeostasis. Thus this study provides insights into understanding of the mechanisms underlying cellular protein homeostasis maintenance (ER protein quality control) in tissue homeostasis and tumor development.
Herrera, S. C., Sainz de la Maza, D., Grmai, L., Margolis, S., Plessel, R., Burel, M., O'Connor, M., Amoyel, M. and Bach, E. A. (2021). Proliferative stem cells maintain quiescence of their niche by secreting the Activin inhibitor Follistatin. Dev Cell 56(16): 2284-2294.e2286. PubMed ID: 34363758.
Summary:
Aging causes stem cell dysfunction as a result of extrinsic and intrinsic changes. Decreased function of the stem cell niche is an important contributor to this dysfunction. The Drosophila testis was used to investigate what factors maintain niche cells. The testis niche comprises quiescent "hub" cells and supports two mitotic stem cell pools: germline stem cells and somatic cyst stem cells (CySCs). The cell-cycle-responsive Dp/E2f1 transcription factor was identified as a crucial non-autonomous regulator required in CySCs to maintain hub cell quiescence. Dp/E2f1 inhibits local Activin ligands through production of the Activin antagonist Follistatin (Fs). Inactivation of Dp/E2f1 or Fs in CySCs or promoting Activin receptor signaling in hub cells causes transdifferentiation of hub cells into fully functional CySCs. This Activin-dependent communication between CySCs and hub regulates the physiological decay of the niche with age and demonstrates that hub cell quiescence results from signals from surrounding stem cells.
Zhou, Y., Liu, J., Zhang, Y. and Liu, J. L. (2021). Drosophila intestinal homeostasis requires CTP synthase. Exp Cell Res 408(1): 112838. PubMed ID: 34560103.
Summary:
CTP synthase (CTPS) senses all four nucleotides and forms filamentous structures termed cytoophidia in all three domains of life. How CTPS and cytoophidia function in a developmental context, however, remains underexplored. This study reports that CTPS forms cytoophidia in a subset of cells in the Drosophila midgut. It was found that cytoophidia exist in intestinal stem cells (ISC) and enteroblasts in similar proportions. Both refeeding after starvation and feeding with dextran sulfate sodium (DSS) induce ISC proliferation and elongate cytoophidia. Knockdown of CTPS inhibits ISC proliferation. Remarkably, disruption of CTPS cytoophidia inhibits DSS-induced ISC proliferation. Taken together, these data suggest that both the expression level and the filament-form property of CTPS are crucial for intestinal homeostasis in Drosophila.
Reilein, A., Kogan, H. V., Misner, R., Park, K. S. and Kalderon, D. (2021). Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development. Elife 10. PubMed ID: 34590579 .
Summary:
Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. This study shows that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events.

Thursday December 23rd - Adult Neural Development and Function

Li, T., Fu, T. M., Wong, K. K. L., Li, H., Xie, Q., Luginbuhl, D. J., Wagner, M. J., Betzig, E. and Luo, L. (2021). Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging. Cell 184(20): 5107-5121. PubMed ID: 34551316.
Summary:
Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. This study used the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.
Guo, L., Wu, Y., Chang, H., Zhang, Z., Tang, H., Yu, Y., Xin, L., Liu, Y. and He, Y. (2021). Structure of cell-cell adhesion mediated by the Down syndrome cell adhesion molecule. Proc Natl Acad Sci U S A 118(39). PubMed ID: 34531300.
Summary:
The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. This study combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.
Nettnin, E. A., Sallese, T. R., Nasseri, A., Saurabh, S. and Cavanaugh, D. J. (2021). Dorsal clock neurons in Drosophila sculpt locomotor outputs but are dispensable for circadian activity rhythms. iScience 24(9): 103001. PubMed ID: 34505011
Summary:
The circadian system is comprised three components: a network of core clock cells in the brain that keeps time, input pathways that entrain clock cells to the environment, and output pathways that use this information to ensure appropriate timing of physiological and behavioral processes throughout the day. Core clock cells can be divided into molecularly distinct populations that likely make unique functional contributions. This study clarifies the role of the dorsal neuron 1 (DN1) population of clock neurons in the transmission of circadian information by the Drosophila core clock network. Using an intersectional genetic approach that allowed selective and comprehensive targeting of DN1 cells, this study shows the suppressing DN1 neuronal activity alters the magnitude of daily activity and sleep without affecting overt rhythmicity. This suggests that DN1 cells are dispensable for both the generation of circadian information and the propagation of this information across output circuits (Nettnin, 2021).
Romano, G., Holodkov, N., Klima, R. and Feiguin, F. (2021). TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila CNS. Sci Rep 11(1): 18761. PubMed ID: 34548578
Summary:
Alterations in the function of the RNA-binding protein TDP-43 are largely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease of the human motor system that leads to motoneurons degeneration and reduced life expectancy by molecular mechanisms not well known. In previous work, it was found found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible for converting glutamate to γ-aminobutyric acid (GABA), were downregulated in TBPH-null flies and motoneurons derived from ALS patients carrying mutations in TDP-43, suggesting that defects in the regulation of GAD1 may lead to neurodegeneration by affecting neurotransmitter balance. This study observed that TBPH was required for the regulation of GAD1 pre-mRNA splicing and the levels of GABA in the Drosophila central nervous system (CNS). Interestingly, this study discovered that pharmacological treatments aimed to potentiate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies, revealing novel mechanisms and therapeutic strategies in ALS.
Chen, C., Agrawal, S., Mark, B., Mamiya, A., Sustar, A., Phelps, J. S., Lee, W. A., Dickson, B. J., Card, G. M. and Tuthill, J. C. (2021). Functional architecture of neural circuits for leg proprioception in Drosophila. Curr Biol. PubMed ID: 34637749
Summary:
To effectively control their bodies, animals rely on feedback from proprioceptive mechanosensory neurons. In the Drosophila leg, different proprioceptor subtypes monitor joint position, movement direction, and vibration. This study investigate how these diverse sensory signals are integrated by central proprioceptive circuits. Signals for leg joint position and directional movement converge in second-order neurons, revealing pathways for local feedback control of leg posture. Distinct populations of second-order neurons integrate tibia vibration signals across pairs of legs, suggesting a role in detecting external substrate vibration. In each pathway, the flow of sensory information is dynamically gated and sculpted by inhibition. Overall, these results reveal parallel pathways for processing of internal and external mechanosensory signals, which are proposed to mediate feedback control of leg movement and vibration sensing, respectively. The existence of a functional connectivity map also provides a resource for interpreting connectomic reconstruction of neural circuits for leg proprioception.
Ramakrishnan, A. and Sheeba, V. (2021). Gap junction protein Innexin2 modulates the period of free-running rhythms in Drosophila melanogaster. iScience 24(9): 103011. PubMed ID: 34522854
Summary:
A neuronal circuit of ~150 neurons modulates rhythmic activity-rest behavior of Drosophila melanogaster. While it is known that coherent 24-hr rhythms in locomotion are brought about when 7 distinct neuronal clusters function as a network due to chemical communication amongst them, there are no reports of communication via electrical synapses made up of gap junctions. Innexins play crucial roles in determining the intrinsic period of activity-rest rhythms in flies. This study shows the presence of Innexin2 in the ventral lateral neurons, wherein RNAi-based knockdown of its expression slows down the speed of activity-rest rhythm along with alterations in the oscillation of a core-clock protein PERIOD and the output molecule Pigment dispersing factor. Specifically disrupting the channel-forming ability of Innexin2 causes period lengthening, suggesting that Innexin2 may function as hemichannels or gap junctions in the clock circuit (Ramakrishnan, 2021).

Wednesday, December 22nd - Wednesday Cytoskeleton and Junctions

Hu, C., Feng, P., Chen, M., Tang, Y. and Soba, P. (2022). Spatiotemporal changes in microtubule dynamics during dendritic morphogenesis. Fly (Austin) 16(1): 13-23. PubMed ID: 34609266
Summary:
Dendritic morphogenesis requires dynamic microtubules (MTs) to form a coordinated cytoskeletal network during development. Dynamic MTs are characterized by their number, polarity and speed of polymerization. Previous studies described a correlation between anterograde MT growth and terminal branch extension in Drosophila dendritic arborization (da) neurons, suggesting a model that anterograde MT polymerization provides a driving force for dendritic branching. This study recently found that the Ste20-like kinase Tao specifically regulates dendritic branching by controlling the number of dynamic MTs in a kinase activity-dependent fashion, without affecting MT polarity or speed. This finding raises the interesting question of how MT dynamics affects dendritic morphogenesis, and if Tao kinase activity is developmentally regulated to coordinate MT dynamics and dendritic morphogenesis. This study explored the possible correlation between MT dynamics and dendritic morphogenesis together with the activity changes of Tao kinase in C1da and C4da neurons during larval development. The data show that spatiotemporal changes in the number of dynamic MTs, but not polarity or polymerization speed, correlate with dendritic branching and Tao kinase activity. These findings suggest that Tao kinase limits dendritic branching by controlling the abundance of dynamic MTs and a novel model is proposed on how regulation of MT dynamics might influence dendritic morphogenesis (Hu, 2012).
Alzyoud, E., Vedelek, V., Rethi-Nagy, Z., Lipinszki, Z. and Sinka, R. (2021). Microtubule Organizing Centers Contain Testis-Specific γ-TuRC Proteins in Spermatids of Drosophila. Front Cell Dev Biol 9: 727264. PubMed ID: 34660584.
Summary:
Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary conserved protein complex, γ-Tubulin Ring Complex (γ-TuRC). γ-TuRC is part of the centrosome and basal body, which are the best-known microtubule-organizing centers. Centrosomes undergo intensive and dynamic changes during spermatogenesis, as they turn into basal bodies, a prerequisite for axoneme formation during spermatogenesis. This study describes the existence of a novel, tissue-specific γ-TuRC in Drosophila. Three genes (CG7716 (t-Grip84), CG18109 (t-Grip91), and CG32232 (t-Grip128) were characterized encoding testis-specific components of γ-TuRC (t-γ-TuRC) testis-specific Grip paralogs. Presence of t-γ-TuRC is essential to male fertility. The diverse subcellular distribution was shown of the t-γ-TuRC proteins during post-meiotic development, at first at the centriole adjunct and then also on the anterior tip of the nucleus, and finally, they appear in the tail region, close to the mitochondria. The physical interactions between the t-γ-TuRC members, γ-tubulin and Mozart1 were also proven. These results further indicate heterogeneity in γ-TuRC composition during spermatogenesis and suggest that the different post-meiotic microtubule organizing centers are orchestrated by testis-specific gene products, including t-γ-TuRC.
Lage, P. Z., Xi, Z., Lennon, J., Hunter, I., Chan, W. K., Carrancio, A. B., von Kriegsheim, A. and Jarman, A. P. (2021). The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly. Biol Open. PubMed ID: 34553759
Summary:
Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. This study reports the characterisation of the Drosophila orthologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, the only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly (Lage, 2021).
Deshpande, O., de-Carvalho, J., Vieira, D. V. and Telley, I. A. (2022).. Astral microtubule cross-linking safeguards uniform nuclear distribution in the Drosophila syncytium. J Cell Biol 221(1). PubMed ID: 34766978.
Summary:
The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin-driven cytoplasmic movement transporting nuclei or repulsion of neighbor nuclei driven by microtubule motors. This study shows that microtubule cross-linking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less-dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-inhibited embryos, following micromanipulation-assisted repositioning. A dimerization-deficient Feo abolishes nuclear separation in embryo explants, while the full-length protein rescues the genetic knockdown. It is concluded that Feo and Klp3A cross-linking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, this study illuminates an essential process of embryonic multicellularity.
Grintsevich, E. E., Ahmed, G., Ginosyan, A. A., Wu, H., Rich, S. K., Reisler, E. and Terman, J. R. (2021). Profilin and Mical combine to impair F-actin assembly and promote disassembly and remodeling. Nat Commun 12(1): 5542. PubMed ID: 34545088
Summary:
Cellular events require the spatiotemporal interplay between actin assembly and actin disassembly. Yet, how different factors promote the integration of these two opposing processes is unclear. In particular, cellular monomeric (G)-actin is complexed with profilin, which inhibits spontaneous actin nucleation but fuels actin filament (F-actin) assembly by elongation-promoting factors (formins, Ena/VASP). In contrast, site-specific F-actin oxidation by Mical promotes F-actin disassembly and release of polymerization-impaired Mical-oxidized (Mox)-G-actin. This study found that these two opposing processes connect with one another to orchestrate actin/cellular remodeling. Specifically, This study found that profilin binds Mox-G-actin, yet these complexes do not fuel elongation factors'-mediated F-actin assembly, but instead inhibit polymerization and promote further Mox-F-actin disassembly. Using Drosophila as a model system, studies show that similar profilin-Mical connections occur in vivo - where they underlie F-actin/cellular remodeling that accompanies Semaphorin-Plexin cellular/axon repulsion. Thus, profilin and Mical combine to impair F-actin assembly and promote F-actin disassembly, while concomitantly facilitating cellular remodeling and plasticity (Grintsevich, 2021).
Collins, M. A., Coon, L. A., Thomas, R., Mandigo, T. R., Wynn, E. and Folker, E. S. (2021). Ensconsin-dependent changes in microtubule organization and LINC complex-dependent changes in nucleus-nucleus interactions result in quantitatively distinct myonuclear positioning defects. Mol Biol Cell: mbcE21060324. PubMed ID: 34524872.
Summary:
Nuclear movement is a fundamental process of eukaryotic cell biology. Skeletal muscle presents an intriguing model to study nuclear movement because its development requires the precise positioning of multiple nuclei within a single cytoplasm. Furthermore, there is a high correlation between aberrant nuclear positioning and poor muscle function. Although many genes that regulate nuclear movement have been identified, the mechanisms by which these genes act is not known. Using Drosophila melanogaster muscle development as a model system, and a combination of live-embryo microscopy and laser ablation of nuclei, studies have found that clustered nuclei encompass at least two phenotypes that are caused by distinct mechanisms. Specifically, Ensconsin is necessary for productive force production to drive any movement of nuclei whereas Bocksbeutel and Klarsicht are necessary to form distinct populations of nuclei that move to different cellular locations. Mechanisitcally, Ensconsin regulates the number of growing microtubules that are used to move nuclei whereas Bocksbeutel and Klarsicht regulate interactions between nuclei (Collins, 2021).

Tuesday, December 21st - Enhancers and Transcriptional Regulation

Payankaulam, S., Hickey, S. L. and Arnosti, D. N. (2021). Cell cycle expression of polarity genes features Rb targeting of Vang. Cells Dev 169: 203747. PubMed ID: 34583062
Summary:
Specification of cellular polarity is vital to normal tissue development and function. Pioneering studies in Drosophila and C. elegans have elucidated the composition and dynamics of protein complexes critical for establishment of cell polarity, which is manifest in processes such as cell migration and asymmetric cell division. Conserved throughout metazoans, planar cell polarity (PCP) genes are implicated in disease, including neural tube closure defects associated with mutations in VANGL1/2. PCP protein regulation is well studied; however, relatively little is known about transcriptional regulation of these genes. Earlier study revealed an unexpected role for the fly Rbf1 retinoblastoma corepressor protein, a regulator of cell cycle genes, in transcriptional regulation of polarity genes. This study analyzes the physiological relevance of the role of E2F/Rbf proteins in the transcription of the key core polarity gene Vang. Targeted mutations to the E2F site within the Vang promoter disrupts binding of E2F/Rbf proteins in vivo, leading to polarity defects in wing hairs. E2F regulation of Vang is supported by the requirement for this motif in a reporter gene. Interestingly, the promoter is repressed by overexpression of E2F1, a transcription factor generally identified as an activator. Consistent with the regulation of this polarity gene by E2F and Rbf factors, expression of Vang and other polarity genes is found to peak in G2/M phase in cells of the embryo and wing imaginal disc, suggesting that cell cycle signals may play a role in regulation of these genes. These findings suggest that the E2F/Rbf complex mechanistically links cell proliferation and polarity (Payankaulam, 2021).
Bai, Y., Caussinus, E., Leo, S., Bosshardt, F., Myachina, F., Rot, G., Robinson, M. D. and Lehner, C. F. (2021). A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells. BMC Genomics 22(1): 771. PubMed ID: 34711176
Summary:
The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25°C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14-29°C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. This study analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. The identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range (Bai, 2021).
Chen, D., McManus, C. E., Radmanesh, B., Matzat, L. H. and Lei, E. P. (2021). Temporal inhibition of chromatin looping and enhancer accessibility during neuronal remodeling. Nat Commun 12(1): 6366. PubMed ID: 34737269
Summary:
During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, this study hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, this study identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. These results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation (Chen, 2021).
Petrenko, N. and Struhl, K. (2021). Comparison of transcriptional initiation by RNA polymerase II across eukaryotic species. Elife 10. PubMed ID: 34515029
Summary:
The preinitiation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TATA-binding protein (TBP) and TBP-associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity (Petrenko, 2021).
Lucas, T., Hafer, T. L., Zhang, H. G., Molotkova, N. and Kohwi, M. (2021). Discrete cis-acting element regulates developmentally timed gene-lamina relocation and neural progenitor competence in vivo. Dev Cell 56(18): 2649-2663.e2646. PubMed ID: 34529940
Summary:
hunchback gene movement to the lamina in Drosophila neuroblasts, this study identified a 250a bp intronic element (IE) both necessary and sufficient for relocation. The IE can target a reporter transgene to the lamina and silence it. Endogenously, however, hunchback is already repressed prior to relocation. Instead, IE-mediated relocation confers a heritably silenced gene state refractory to activation in descendent neurons, which terminates neuroblast competence to specify early-born identity. Surprisingly, this study found that the Polycomb group chromatin factors bind the IE and are required for lamina relocation, revealing a nuclear architectural role distinct from their well-known function in transcriptional repression. Together, these results uncover in vivo mechanisms underlying neuroblast competence and lamina association in heritable gene silencing (Lucas, 2021).
Kuang, Y., Pyo, A., Eafergan, N., Cain, B., Gutzwiller, L. M., Axelrod, O., Gagliani, E. K., Weirauch, M. T., Kopan, R., Kovall, R. A., Sprinzak, D. and Gebelein, B. (2021). Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor. PLoS Genet 17(9): e1009039. PubMed ID: 34559800.
Summary:
Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). This study tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. These findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

Monday, December 20th - Methods

Xu, C. S., Pang, S., Shtengel, G., Muller, A., Ritter, A. T., Hoffman, H. K., Takemura, S. Y., Lu, Z., Pasolli, H. A., Iyer, N., Chung, J., Bennett, D., Weigel, A. V., Freeman, M., van Engelenburg, S. B., Walther, T. C., Farese, R. V., Jr., Lippincott-Schwartz, J., Mellman, I., Solimena, M. and Hess, H. F. (2021). An open-access volume electron microscopy atlas of whole cells and tissues. Nature. PubMed ID: 34616045.
Summary:
Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, the volume that can be imaged with 4-nm voxels was increased by two orders of magnitude. This study presents a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and researchers are invited to explore this atlas and pose questions.
Guangming, G., Mei, C., Chenchen, Z., Wei, X. and Junhua, G. (2021). Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy. Anat Sci Int. PubMed ID: 34661863
Summary:
The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electron microscopy that only has a small field of view. This study reports an efficient method for acquiring the ultrastructural picture of neuromuscular junctions in Drosophila larva under electron microscopy. The procedure was as follows: first, the larval sample of body wall muscle was placed between the metal mesh and was dehydrated with alcohol and infiltrated with epoxy resin to prevent the sample from curling or bending, after it was dissected and fixed into thin slices. Second, the sample was embedded in resin into a flat sheet to facilitate the positioning of the muscles. Third, carefully and gradually remove the excess resin and the cuticle of the larvae, cut off both ends of the special body segment, and trim the excess specific muscles according to the recommended ratio of trimming muscles, which would reduce the workload exponentially. At last, the trimmed sample were prepared into serial about 1000 ultrathin sections that were about total 80 microns thickness, and 30-40 sections were gathered into a grid to stain with lead citrate and uranyl acetate. This method could also be applied to the other small and thin samples such as the Drosophila embryo, ventral nerve cord and brain.
Esquivel-Lopez, A., Arzate-Mejia, R., Perez-Molina, R. and Furlan-Magaril, M. (2021). In-Nucleus Hi-C in Drosophila Cells. J Vis Exp(175). PubMed ID: 34605807
Summary:
The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described in this study helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. In this study, the in-nucleus Hi-C method has been described in detail, providing important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements (Esquival-Lopez, 2021).
Meyer-Nava, S., Zurita, M. and Valadez-Graham, V. (2021). Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands. J Vis Exp(174). PubMed ID: 34487109
Summary:
Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. This study describes immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail (Meyer-Nava, 2021).
Cheng, J., Hsu, L. F., Juan, Y. H., Liu, H. P. and Lin, W. Y. (2021). Pathway-targeting gene matrix for Drosophila gene set enrichment analysis. PLoS One 16(10): e0259201. PubMed ID: 34710184
Summary:
Gene Set Enrichment Analysis (GSEA) is a powerful algorithm to determine biased pathways between groups based on expression profiling. However, for fruit fly, a popular animal model, gene matrixes for GSEA are unavailable. This study provides the pathway-targeting gene matrixes based on Reactome and KEGG database for fruit fly. An expression profiling containing neurons or glia of fruit fly was used to validate the feasibility of the generated gene matrixes. The gene matrixes were validated and characteristic neuronal and glial pathways were identified, including mRNA splicing and endocytosis. In conclusion, this study generated and validated the feasibility of Reactome and KEGG gene matrix files, which may benefit future profiling studies using Drosophila.
Abu, F. and Ohlstein, B. (2021). Monitoring Gut Acidification in the Adult Drosophila Intestine. J Vis Exp(176). PubMed ID: 34694296
Summary:
The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This study describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification (Abu, 2021).

Friday December 17th - Behavior

Chakraborty, A., Sgro, C. M. and Mirth, C. K. (2021). The proximate sources of genetic variation in body size plasticity: the relative contributions of feeding behaviour and development in Drosophila melanogaster. J Insect Physiol: 104321. PubMed ID: 34653505
Summary:
Body size is a key life-history trait that influences many aspects of an animal's biology and is shaped by a variety of factors, both genetic and environmental. While locally-adapted populations differ in the extent to which body size responds plastically to environmental conditions like diet, there is a limited understanding of what causes these differences. It was hypothesized that populations could differ in the way body size responds to nutrition either by modulating growth rate, development time, feeding rate, or a combination of the above. Using three locally-adapted populations of Drosophila melanogaster from along the east coast of Australia, body size plasticity was investigated across five different diets. How these populations differed in feeding behaviour and developmental timing on each of the diets was assessed. Population-specific plastic responses to nutrition was observed for body size and feeding rate, but not development time. However, differences in feeding rate did not fully explain the differences in the way body size responded to diet. Thus, it is concluded that body size variation in locally-adapted populations is shaped by a combination of growth rate and feeding behaviour. This paves the way for further studies that explore how differences in the regulation of the genetic pathways that control feeding behaviour and growth rate contribute to population-specific responses of body size to diet (Chakraborty, 2021).
Buchert, S. N., Murakami, P., Kalavadia, A. H., Reyes, M. T. and Sitaraman, D. (2021). Sleep correlates with behavioral decision making critical for reproductive output in Drosophila melanogaster. Comp Biochem Physiol A Mol Integr Physiol: 111114. PubMed ID: 34785379
Summary:
Balance between sleep, wakefulness and arousal is important for survival of organisms and species as a whole. While, the benefits of sleep both in terms of quantity and quality is widely recognized across species, sleep has a cost for organismal survival and reproduction. here the study focuses on how sleep duration, sleep depth and sleep pressure affect the ability of animals to engage in courtship and egg-laying behaviors critical for reproductive success. Using isogenic lines from the Drosophila Genetic Reference Panel with variable sleep phenotypes this study investigated the relationship between sleep and reproductive behaviors, courtship and oviposition. This study found that three out of five lines with decreased sleep and increased arousal phenotypes, showed increased courtship and decreased latency to court as compared to normal and long sleeping lines. However, the male courtship phenotype is dependent on context and genotype as some but not all long sleeping-low courting lines elevate their courtship in the presence of short sleeping-high courting flies. Sleep phenotypes were less variable and minimally susceptible to social experience. In addition to male courtship, Oviposition was found to be less sensitive to sleep length. Taken together this extensive behavioral analysis shows complex bidirectional interactions between genotype and environment and add to the growing evidence linking sleep duration and sleep-wake switch parameters to behavioral decision making critical to reproductive output (Buchert, 2021).
Williamson, M., Mitchell, A. and Condron, B. (2021). Birth temperature followed by a visual critical period determines cooperative group membership. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 34611741.
Summary:
Cooperative behavior often arises when a common exploitable resource is generated. Cooperation can provide equitable distribution and protection from raiding of a common resource such as processed food. Under crowded conditions in liquid food, Drosophila larvae adopt synchronized feeding behavior which provides a fitness benefit. A key for this synchronized feeding behavior is the visually guided alignment of a 1-2 s locomotion stride between adjacent larvae in a feeding cluster. The locomotion stride is thought to be set by embryonic incubation temperature. This raises a question as to whether sib larvae will only cluster efficiently if they hatch at the same temperature. To test this, larvae were first collected and incubated in outdoor conditions. Morning hatched lower temperature larvae move slower than their afternoon higher temperature sibs. Both temperature types synchronize but tend to exclude the other type of larvae from their clusters. In addition, fitness, as measured by adult wing size, is highest when larvae cluster with their own temperature type. Thus, the temperature at which an egg is laid sets a type of behavioral stamp or password which locks in membership for later cooperative feeding.
Ueno, T. and Takahashi, Y. (2021). Mitochondrial polymorphism shapes intrapopulation behavioural variation in wild Drosophila. Biol Lett 17(7): 20210194. PubMed ID: 34314641.
Summary:
Intrapopulation variation in behaviour, including activity, boldness and aggressiveness, is becoming more widely recognized and is hypothesized to substantially affect ecological and evolutionary dynamics. Although previous studies used candidate-gene approaches and genome-wide association analyses to identify genes correlated with variations in activity and aggressiveness, behavioural variation may not be fully captured in the nuclear genome, as it does not account for mitochondrial genomes. Mitochondrial genes encode products that are key regulators of the cellular energy-producing pathways in metabolic processes and are thought to play a significant role in life-history and reproductive traits. This study considered many isofemale lines of Drosophila immigrans established from two wild populations to investigate whether intrapopulation variation in the mitochondrial genome affected activity level within this species. Two major haplogroups in these populations, and activity levels in both larvae and adults differed significantly between the two haplogroups. This result indicated that intrapopulation variation in activity level may be partially controlled by mitochondrial genes, along with the interaction between nuclear and mitochondrial genes and the age of individual organisms.
Wu, Q., Park, S. J., Yang, M. and Ja, W. W. (2021). Vitamin preference in Drosophila. Curr Biol 31(15): R946-r947. PubMed ID: 34375595.
Summary:
Many animals rely on taste to identify nutritious foods and to avoid the consumption of harmful substances. The tastes of macronutrients, as well as of non-caloric micronutrients such as sodium and calcium, contribute to the regulation of ingestive behavior. Whether vitamins also affect feeding behavior through taste is less clear. This study shows that the fly Drosophila melanogaster has a strong preference for consuming a vitamin-containing diet: both sexes show a preference for folic acid, whereas only females show a preference for riboflavin. Females show a preference with vitamin concentrations as low as ∼10 nM - at least 50,000-fold lower than the concentration needed for sucrose preference. This female vitamin preference requires inputs from external and internal taste organs, suggesting that post-ingestive signals, in the absence of gustatory input, are insufficient to actuate preferential consumption of vitamin-containing diets. These studies demonstrate that vitamin perception is an important determinant of feeding behavior.
Werkhoven, Z., Bravin, A., Skutt-Kakaria, K., Reimers, P., Pallares, L. F., Ayroles, J. and de Bivort, B. L. (2021). The structure of behavioral variation within a genotype. Elife 10. PubMed ID: 34664550.
Summary:
Individual animals vary in their behaviors. This is true even when they share the same genotype and were reared in the same environment. Clusters of covarying behaviors constitute behavioral syndromes, and an individual's position along such axes of covariation is a representation of their personality. Despite these conceptual frameworks, the structure of behavioral covariation within a genotype is essentially uncharacterized and its mechanistic origins unknown. Passing hundreds of inbred Drosophila individuals through an experimental pipeline that captured hundreds of behavioral measures, sparse but significant correlations were found among small sets of behaviors. Thus, the space of behavioral variation has many independent dimensions. Manipulating the physiology of the brain, and specific neural populations, altered specific correlations. It was also observed that variation in gene expression can predict an individual's position on some behavioral axes. This work represents the first steps in understanding the biological mechanisms determining the structure of behavioral variation within a genotype.

Thursday, December 16th - Disease Models

Blommer, J., Fischer, M. C., Olszewski, A. R., Katzenberger, R. J., Ganetzky, B. and Wassarman, D. A. (2021). Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARgamma ortholog Eip75B. PLoS One 16(10): e0258873. PubMed ID: 34699541
Summary:
Traumatic brain injury (TBI) is a common neurological disorder whose outcomes vary widely depending on a variety of environmental factors, including diet. Using a Drosophila melanogaster TBI model that reproduces key aspects of TBI in humans, this study previously found that the diet consumed immediately following a primary brain injury has a substantial effect on the incidence of mortality within 24 h (early mortality). Flies that receive equivalent primary injuries have a higher incidence of early mortality when fed high-carbohydrate diets versus water. This study reports that flies fed high-fat ketogenic diet (KD) following TBI exhibited early mortality that was equivalent to that of flies fed water and that flies protected from early mortality by KD continued to show survival benefits weeks later. KD also has beneficial effects in mammalian TBI models, indicating that the mechanism of action of KD is evolutionarily conserved. To probe the mechanism, this study examined the effect of KD in flies mutant for Eip75B, an ortholog of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) that contributes to the mechanism of action of KD and has neuroprotective effects in mammalian TBI models. The incidence of early mortality of Eip75B mutant flies was found to be higher when they were fed KD than when they were fed water following TBI. These data indicate that Eip75B/PPARγ is necessary for the beneficial effects of KD following TBI. In summary, this work provides the first evidence that KD activates PPARγ to reduce deleterious outcomes of TBI and it demonstrates the utility of the fly TBI model for dissecting molecular pathways that contribute to heterogeneity in TBI outcomes (Blommer, 2021).
Azpurua, J., El-Karim, E. G., Tranquille, M. and Dubnau, J. (2021). A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia. PLoS Genet 17(11): e1009882. PubMed ID: 34723963
Summary:
Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer's disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, This study expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, this study conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, this screen also yielded genes involved in chromatin regulation and nuclear/import export - pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. The identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator (Azpurua, 2021).
Bouska, M. J. and Bai, H. (2021). Loxl2 is a mediator of cardiac aging in Drosophila melanogaster; genetically examining the role of aging clock genes. G3 (Bethesda). PubMed ID: 34734976
Summary:
Transcriptomic, proteomic, and methylation aging clocks demonstrate that aging has a predictable preset program, while Transcriptome Trajectory Turning Points indicate that the 20 to 40 age range in humans is the likely stage at which the progressive loss of homeostatic control, and in turn aging, begins to have detrimental effects. Turning points in this age range overlapping with human aging clock genes revealed five candidates that were hypothesized could play a role in aging or age-related physiological decline. To examine these gene's effects on lifespan and health-span, this study utilized whole body and heart specific gene knockdown of human orthologs in Drosophila melanogaster. Whole body Loxl2, fz3, and Glo1 RNAi positively affected lifespan as did heart-specific Loxl2 knockdown. Loxl2 inhibition concurrently reduced age-related cardiac arrythmia and collagen (Pericardin) fiber width. Loxl2 binds several transcription factors in humans and RT-qPCR confirmed that a conserved transcriptional target CDH1 (Drosophila CadN2), has expression levels which correlate with Loxl2 reduction in Drosophila. These results point to conserved pathways and multiple mechanisms by which inhibition of Loxl2 can be beneficial to heart health and organismal aging (Bouska, 2021).
Aditi, K., Singh, A., Shakarad, M. N. and Agrawal, N. (2021). Management of altered metabolic activity in Drosophila model of Huntington's disease by curcumin. Exp Biol Med (Maywood): 15353702211046927. PubMed ID: 34743577
Summary:
Huntington's disease (HD) is a devastating polyglutamine disorder characterized by extensive neurodegeneration and metabolic abnormalities at systemic, cellular and intracellular levels. Metabolic alterations in HD manifest as abnormal body weight, dysregulated biomolecule levels, impaired adipocyte functions, and defective energy state which exacerbate disease progression and pose acute threat to the health of challenged individuals in form of insulin resistance, cardiovascular disease, and energy crisis. To colossally mitigate disease symptoms, this study tested the efficacy of curcumin in Drosophila model of HD. Curcumin is the bioactive component of turmeric (Curcuma longa Linn), well-known for its ability to modulate metabolic activities. This study found that curcumin effectively managed abnormal body weight, dysregulated lipid content, and carbohydrate level in HD flies. In addition, curcumin administration lowered elevated reactive-oxygen-species levels in adult adipose tissue of diseased flies, and improved survival and locomotor function in HD flies at advanced disease stage. Altogether, these findings clearly suggest that curcumin efficiently attenuates metabolic derangements in HD flies and can prove beneficial in alleviating the complexities associated with HD (Aditi, 2021).
Agrawal, N., Lawler, K., Davidson, C. M., Keogh, J. M., Legg, R., Barroso, I., Farooqi, I. S. and Brand, A. H. (2021). Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila. PLoS Biol 19(11): e3001255. PubMed ID: 34748544
Summary:
The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. This study uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, this study identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. This study found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength in this approach in predicting novel human obesity genes and signalling pathways and their site of action (Agreawal, 2021).
Arsac, J. N., Sedru, M., Dartiguelongue, M., Vulin, J., Davoust, N., Baron, T. and Mollereau, B. (2021). Chronic Exposure to Paraquat Induces Alpha-Synuclein Pathogenic Modifications in Drosophila. Int J Mol Sci 22(21). PubMed ID: 34769043
Summary:
Parkinson's disease (PD) is characterized by the progressive accumulation of neuronal intracellular aggregates largely composed of α-Synuclein (αSyn) protein. The process of αSyn aggregation is induced during aging and enhanced by environmental stresses, such as the exposure to pesticides. Paraquat (PQ) is an herbicide which has been widely used in agriculture and associated with PD. PQ is known to cause an increased oxidative stress in exposed individuals but the consequences of such stress on αSyn conformation remains poorly understood. To study Syn pathogenic modifications in response to PQ, Drosophila expressing human αSyn were exposedto a chronic PQ protocol. It was first shown that PQ exposure and αSyn expression synergistically induced fly mortality. The exposure to PQ was also associated with increased levels of total and phosphorylated forms of αSyn in the Drosophila brain. Interestingly, PQ increased the detection of soluble αSyn in highly denaturating buffer but did not increase αSyn resistance to proteinase K digestion. These results suggest that PQ induces the accumulation of toxic soluble and misfolded forms of αSyn but that these toxic forms do not form fibrils or aggregates that are detected by the proteinase K assay. Collectively, these results demonstrate that Drosophila can be used to study the effect of PQ or other environmental neurotoxins on 34769043Syn driven pathology (Arsac, 2021).

Wednesday, December 15th - Enhancers and transcriptional regulation

Payankaulam, S., Hickey, S. L. and Arnosti, D. N. (2021). Cell cycle expression of polarity genes features Rb targeting of Vang. Cells Dev 169: 203747. PubMed ID: 34583062
Summary:
Specification of cellular polarity is vital to normal tissue development and function. Pioneering studies in Drosophila and C. elegans have elucidated the composition and dynamics of protein complexes critical for establishment of cell polarity, which is manifest in processes such as cell migration and asymmetric cell division. Conserved throughout metazoans, planar cell polarity (PCP) genes are implicated in disease, including neural tube closure defects associated with mutations in VANGL1/2. PCP protein regulation is well studied; however, relatively little is known about transcriptional regulation of these genes. Earlier study revealed an unexpected role for the fly Rbf1 retinoblastoma corepressor protein, a regulator of cell cycle genes, in transcriptional regulation of polarity genes. This study analyzes the physiological relevance of the role of E2F/Rbf proteins in the transcription of the key core polarity gene Vang. Targeted mutations to the E2F site within the Vang promoter disrupts binding of E2F/Rbf proteins in vivo, leading to polarity defects in wing hairs. E2F regulation of Vang is supported by the requirement for this motif in a reporter gene. Interestingly, the promoter is repressed by overexpression of E2F1, a transcription factor generally identified as an activator. Consistent with the regulation of this polarity gene by E2F and Rbf factors, expression of Vang and other polarity genes is found to peak in G2/M phase in cells of the embryo and wing imaginal disc, suggesting that cell cycle signals may play a role in regulation of these genes. These findings suggest that the E2F/Rbf complex mechanistically links cell proliferation and polarity (Payankaulam, 2021).
Bai, Y., Caussinus, E., Leo, S., Bosshardt, F., Myachina, F., Rot, G., Robinson, M. D. and Lehner, C. F. (2021). A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells. BMC Genomics 22(1): 771. PubMed ID: 34711176
Summary:
The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25°C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14-29°C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, this study analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. The identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range (Bai, 2021).
Chen, D., McManus, C. E., Radmanesh, B., Matzat, L. H. and Lei, E. P. (2021). Temporal inhibition of chromatin looping and enhancer accessibility during neuronal remodeling. Nat Commun 12(1): 6366. PubMed ID: 34737269
Summary:
During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, this study hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, this study identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. These results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation (Chen, 2021).
Petrenko, N. and Struhl, K. (2021). Comparison of transcriptional initiation by RNA polymerase II across eukaryotic species. Elife 10. PubMed ID: 34515029
Summary:
The preinitiation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TATA-binding protein (TBP) and TBP-associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity (Petrenko, 2021).
Lucas, T., Hafer, T. L., Zhang, H. G., Molotkova, N. and Kohwi, M. (2021). Discrete cis-acting element regulates developmentally timed gene-lamina relocation and neural progenitor competence in vivo. Dev Cell 56(18): 2649-2663.e2646. PubMed ID: 34529940
Summary:

The nuclear lamina is typically associated with transcriptional silencing, and peripheral relocation of genes highly correlates with repression. However, the DNA sequences and proteins regulating gene-lamina interactions are largely unknown. Exploiting the developmentally timed hunchback gene movement to the lamina in Drosophila neuroblasts, this study identified a 250a bp intronic element (IE) both necessary and sufficient for relocation. The IE can target a reporter transgene to the lamina and silence it. Endogenously, however, hunchback is already repressed prior to relocation. Instead, IE-mediated relocation confers a heritably silenced gene state refractory to activation in descendent neurons, which terminates neuroblast competence to specify early-born identity. Surprisingly, this study found that the Polycomb group chromatin factors bind the IE and are required for lamina relocation, revealing a nuclear architectural role distinct from their well-known function in transcriptional repression. Together, these results uncover in vivo mechanisms underlying neuroblast competence and lamina association in heritable gene silencing (Lucas, 2021).

Kuang, Y., Pyo, A., Eafergan, N., Cain, B., Gutzwiller, L. M., Axelrod, O., Gagliani, E. K., Weirauch, M. T., Kopan, R., Kovall, R. A., Sprinzak, D. and Gebelein, B. (2021). Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor. PLoS Genet 17(9): e1009039. PubMed ID: 34559800.
Summary:
Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. This study tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. These findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

Tuesday, December 13th - Signal Transduction

Boonen, B., Startek, J. B., Milici, A., López-Requena, A., Beelen, M., Callaerts, P. and Talavera, K. (2021). Activation of Drosophila melanogaster TRPA1 Isoforms by Citronellal and Menthol. Int J Mol Sci 22(20). PubMed ID: 34681657
Summary:
The transient receptor potential ankyrin 1 (TRPA1) cation channels function as broadly-tuned sensors of noxious chemicals in many species. Recent studies identified four functional TRPA1 isoforms in Drosophila melanogaster (dTRPA1(A) to (D)), but their responses to non-electrophilic chemicals are yet to be fully characterized. This study determined the behavioral responses of adult flies to the mammalian TRPA1 non-electrophilic activators citronellal and menthol, and characterized the effects of these compounds on all four dTRPA1 channel isoforms using intracellular Ca(2+) imaging and whole-cell patch-clamp recordings. Wild type flies avoided citronellal and menthol in an olfactory test and this behavior was reduced in dTrpA1 mutant flies. Both compounds activate all dTRPA1 isoforms in the heterologous expression system HEK293T, with the following sensitivity series: dTRPA1(C) = dTRPA1(D) > dTRPA1(A) >> dTRPA1(B) for citronellal and dTRPA1(A) > dTRPA1(D) > dTRPA1(C) > dTRPA1(B) for menthol. It is concluded that dTrpA1 is required for the normal avoidance of Drosophila melanogaster towards citronellal and menthol. All dTRPA1 isoforms are activated by both compounds, but the dTRPA1(B) is consistently the least sensitive. These findings may guide further studies on the physiological roles and the structural bases of chemical sensitivity of TRPA1 channels (Boonen, 2021).
Ma, J., Hou, C., Li, Y., Chen, S. and Wu, C. (2021). OGT Protein Interaction Network (OGT-PIN): A Curated Database of Experimentally Identified Interaction Proteins of OGT. Int J Mol Sci 22(17). PubMed ID: 34502531
Summary:
Interactions between proteins are essential to any cellular process and constitute the basis for molecular networks that determine the functional state of a cell. With the technical advances in recent years, an astonishingly high number of protein-protein interactions has been revealed. However, the interactome of O-linked N-acetylglucosamine transferase (OGT), the sole enzyme adding the O-linked β-N-acetylglucosamine (O-GlcNAc) onto its target proteins, has been largely undefined. To that end, this study collated OGT interaction proteins experimentally identified in the past several decades. Rigorous curation of datasets from public repositories and O-GlcNAc-focused publications led to the identification of up to 929 high-stringency OGT interactors from multiple species studied (including Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Arabidopsis thaliana, and others). Among them, 784 human proteins were found to be interactors of human OGT. Moreover, these proteins spanned a very diverse range of functional classes (e.g., DNA repair, RNA metabolism, translational regulation, and cell cycle), with significant enrichment in regulating transcription and (co)translation. This dataset demonstrates that OGT is likely a hub protein in cells. A webserver OGT-Protein Interaction Network (OGT-PIN) has also been created, which is freely accessible (Ma, 2021).
Audett, M. R., Johnson, E. L., McGory, J. M., Barcelos, D. M., Szalai, E. O., Przewloka, M. R. and Maresca, T. J. (2021). The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction. Mol Biol Cell: mbcE21060307T. PubMed ID: 34705493
Summary:
KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of D. melanogaster KNL1 (Spc105) has never been shown to bind MTs nor to recruit PP1. Furthermore, the phospho-regulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. This study resolved these apparent discrepancies are resolved using in vitro and cell based-assays. A phospho-regulatory circuit, which utilizes Aurora B kinase (ABK), promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag, and deletion/chimera mutants are used to define the interplay of MT- and PP1-binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction (Audett, 2021).
Jang, S., Chen, J., Choi, J., Lim, S. Y., Song, H., Choi, H., Kwon, H. W., Choi, M. S. and Kwon, J. Y. (2021). Spatiotemporal organization of enteroendocrine peptide expression in Drosophila. J Neurogenet: 1-12. PubMed ID: 34670462
Summary:
The digestion of food and absorption of nutrients occurs in the gut. The nutritional value of food and its nutrients is detected by enteroendocrine cells, and peptide hormones produced by the enteroendocrine cells are thought to be involved in metabolic homeostasis, but the specific mechanisms are still elusive. The enteroendocrine cells are scattered over the entire gastrointestinal tract and can be classified according to the hormones they produce. This study followed the changes in combinatorial expression of regulatory peptides in the enteroendocrine cells during metamorphosis from the larva to the adult fruit fly, and re-confirmed the diverse composition of enteroendocrine cell populations. Drosophila enteroendocrine cells appear to differentially regulate peptide expression spatially and temporally depending on midgut region and developmental stage. In the late pupa, Notch activity is known to determine which peptides are expressed in mature enteroendocrine cells of the posterior midgut. This study also found that the loss of Notch activity in the anterior midgut results in classes of enteroendocrine cells distinct from the posterior midgut. These results suggest that enteroendocrine cells that populate the fly midgut can differentiate into distinct subtypes that express different combinations of peptides, which likely leads to functional variety depending on specific needs (Jang, 2021).
Liu, T., Zhang, T., Nicolas, M., Boussicault, L., Rice, H., Soldano, A., Claeys, A., Petrova, I., Fradkin, L., De Strooper, B., Potier, M. C. and Hassan, B. A. (2021). The amyloid precursor protein is a conserved Wnt receptor. Elife 10. PubMed ID: 34515635
Summary:
The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. This study shows that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner (Liu, 2021).
Magny, E. G., Platero, A. I., Bishop, S. A., Pueyo, J. I., Aguilar-Hidalgo, D. and Couso, J. P. (2021). Pegasus, a small extracellular peptide enhancing short-range diffusion of Wingless. Nat Commun 12(1): 5660. PubMed ID: 34580289
Summary:
Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. This study reveals a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. These results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions (Magny, 2021).

Monday, December 13th - Adult and Larval Physiology and Metabolism

Kokki, K., Lamichane, N., Nieminen, A. I., Ruhanen, H., Morikka, J., Robciuc, M., Rovenko, B. M., Havula, E., Kakela, R. and Hietakangas, V. (2021). Metabolic gene regulation by Drosophila GATA transcription factor Grain. PLoS Genet 17(10): e1009855. PubMed ID: 34634038
Summary:
Nutrient-dependent gene regulation critically contributes to homeostatic control of animal physiology in changing nutrient landscape. In Drosophila, dietary sugars activate transcription factors (TFs), such as Mondo-Mlx, Sugarbabe and Cabut, which control metabolic gene expression to mediate physiological adaptation to high sugar diet. TFs that correspondingly control sugar responsive metabolic genes under conditions of low dietary sugar remain, however, poorly understood. This study identified a role for Drosophila GATA TF Grain in metabolic gene regulation under both low and high sugar conditions. De novo motif prediction uncovered a significant over-representation of GATA-like motifs on the promoters of sugar-activated genes in Drosophila larvae, which are regulated by Grain, the fly ortholog of GATA1/2/3 subfamily. grain expression is activated by sugar in Mondo-Mlx-dependent manner and it contributes to sugar-responsive gene expression in the fat body. On the other hand, grain displays strong constitutive expression in the anterior midgut, where it drives lipogenic gene expression also under low sugar conditions. Consistently with these differential tissue-specific roles, Grain deficient larvae display delayed development on high sugar diet, while showing deregulated central carbon and lipid metabolism primarily on low sugar diet. Collectively, this study provides evidence for the role of a metazoan GATA transcription factor in nutrient-responsive metabolic gene regulation in vivo.
Gunderson, J. T., Peppriell, A. E., Krout, I. N., Vorojeikina, D. and Rand, M. D. (2021). Neuroligin-1 is a mediator of methylmercury neuromuscular toxicity. Toxicol Sci. PubMed ID: 34546366
Summary:
Methylmercury (MeHg) is a developmental toxicant capable of eliciting neurocognitive and neuromuscular deficits in children with in utero exposure. Previous research in Drosophila melanogaster uncovered that developmental MeHg exposure simultaneously targets the developing musculature and innervating motor neuron in the embryo, along with identifying Drosophila neuroligin 1 (nlg1) as a gene associated with developmental MeHg sensitivity. Nlg1 and its transsynaptic partner neurexin 1 (Nrx1) are critical for axonal arborization and NMJ maturation. This study investigated the effects of MeHg exposure on indirect flight muscle (IFM) morphogenesis, innervation, and function via flight assays and monitored the expression of NMJ-associated genes to characterize the role of Nlg1 mediating the neuromuscular toxicity of MeHg. Developmental MeHg exposure reduced the innervation of the IFMs, which corresponded with reduced flight ability. In addition, nlg1 expression was selectively reduced during early metamorphosis, while a subsequent increase was observed in other NMJ-associated genes, including nrx1, in late metamorphosis. Developmental MeHg exposure also resulted in persistent reduced expression of most nlg and nrx genes during the first 11 days of adulthood. Transgenic modulation of nlg and nrx revealed that developing muscle is particularly sensitive to nlg1 levels, especially during the 20 - 36-hour window of metamorphosis with reduced nlg1 expression resulting in adult flight deficits. Muscle-specific overexpression of nlg1 partially rescued MeHg-induced deficits in eclosion and flight. This study identified Nlg1 as a muscle-specific, NMJ structural component, that can mediate MeHg neuromuscular toxicity resulting from early life exposure (Gunderson, 2021).
Atienza-Manuel, A., Castillo-Mancho, V., De Renzis, S., Culi, J. and Ruiz-Gomez, M. (2021). Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes. Development. PubMed ID: 34738617
Summary:
The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. This study shows that Drosophila CUBAM is a tripartite complex composed of dAmnionless and two cubilin paralogues Cubilin and Cubilin-2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Noteworthy, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD-positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes argue for a possible unexpected conserved role of this endocytic receptor in vertebrate SD remodelling (Atienza-Manuel, 2021).
Nazario-Yepiz, N. O., Fernandez Sobaberas, J., Lyman, R., Campbell, M. R., Shankar, V., Anholt, R. R. H. and Mackay, T. F. C. (2021). Physiological and metabolomic consequences of reduced expression of the Drosophila brummer triglyceride Lipase. PLoS One 16(9): e0255198. PubMed ID: 34547020
Summary:
Disruption of lipolysis has widespread effects on intermediary metabolism and organismal phenotypes. Defects in lipolysis can be modeled in Drosophila melanogaster through genetic manipulations of brummer (bmm), which encodes a triglyceride lipase orthologous to mammalian Adipose Triglyceride Lipase. RNAi-mediated knock-down of bmm in all tissues or metabolic specific tissues results in reduced locomotor activity, altered sleep patterns and reduced lifespan. Metabolomic analysis on flies in which bmm is downregulated reveals a marked reduction in medium chain fatty acids, long chain saturated fatty acids and long chain monounsaturated and polyunsaturated fatty acids, and an increase in diacylglycerol levels. Elevated carbohydrate metabolites and tricarboxylic acid intermediates indicate that impairment of fatty acid mobilization as an energy source may result in upregulation of compensatory carbohydrate catabolism. bmm downregulation also results in elevated levels of serotonin and dopamine neurotransmitters, possibly accounting for the impairment of locomotor activity and sleep patterns. Physiological phenotypes and metabolomic changes upon reduction of bmm expression show extensive sexual dimorphism. Altered metabolic states in the Drosophila model are relevant for understanding human metabolic disorders, since pathways of intermediary metabolism are conserved across phyla (Nazario-Yepiz, 2021).
Kay, A. R., Eberl, D. F. and Wang, J. W. (2021). Myogenic contraction of a somatic muscle powers rhythmic flow of hemolymph through Drosophila antennae and generates brain pulsations. J Exp Biol 224(20). PubMed ID: 34585241
Summary:
Hemolymph is driven through the antennae of Drosophila melanogaster by the rhythmic contraction of muscle 16 (m16), which runs through the brain. Contraction of m16 results in the expansion of an elastic ampulla, opening ostia and filling the ampulla. Relaxation of the ampullary membrane forces hemolymph through vessels into the antennae. This study shows that m16 is an auto-active rhythmic somatic muscle. The activity of m16 leads to the rapid perfusion of the antenna by hemolymph. In addition, it leads to the rhythmic agitation of the brain, which could be important for clearing the interstitial space (Kay, 2021).
Hocaoglu, H., Wang, L., Yang, M., Yue, S. and Sieber, M. (2021). Heritable shifts in redox metabolites during mitochondrial quiescence reprogramme progeny metabolism. Nat Metab 3(9): 1259-1274. PubMed ID: 34545253
Summary:
This study shows that the precocious onset of mitochondrial respiratory quiescence causes a reprogramming of progeny metabolic state. The premature onset of mitochondrial respiratory quiescence drives the lowering of Drosophila oocyte NAD+ levels. NAD+ depletion in the oocyte leads to reduced methionine cycle production of the methyl donor S-adenosylmethionine in embryos and lower levels of histone H3 lysine 27 trimethylation, resulting in enhanced intestinal lipid metabolism in progeny. In addition, this study shows that triggering cellular quiescence in mammalian cells and chemotherapy-resistant human cancer cell models induces cellular reprogramming events identical to those seen in Drosophila, suggesting a conserved metabolic mechanism in systems reliant on quiescent cells (Hocaoglu, 2021).

Friday, December 10th - Disease Models

Medvedeva, A. V., Tokmatcheva, E. V., Kaminskaya, A. N., Vasileva, S. A., Nikitina, E. A., Zhuravlev, S. A., Zakharov, G. A., Zatsepina, O. G. and Savvateeva-Popova, E. V. (2021). Parent-of-origin effects on nuclear chromatin organization and behavior in a Drosophila model for Williams-Beuren Syndrome. Vavilovskii Zhurnal Genet Selektsii 25(5): 472-485. PubMed ID: 34595370
Summary:
Prognosis of neuropsychiatric disorders in progeny requires consideration of individual parent-of-origin effects (POEs) relying on the nerve cell nuclear 3D chromatin architecture and impact of parent-specific miRNAs. Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting, or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMP dependent, the second relies on actin remodeling by small GTPase Rac1 - LIMK1 (LIM-kinase 1). Simple experimental model systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies. This study has developed a Drosophila model for Williams-Beuren Syndrome (WBS): a mutant agn(ts3) of the agnostic locus (X:11AB) harboring the dlimk1 gene. The agn(ts3) mutation drastically increases the frequency of ectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affects locomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between agn(ts3) and the wild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal gene origin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with the occurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat. Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-term memory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Several parameters of locomotion demonstrate heterosis. These data indicate that the agn(ts3) locus is capable of trans-regulating gene activity via POEs on the chromatin nuclear organization, thereby affecting behavior (Medvedeva, 2021).
Rimal, S., Li, Y., Vartak, R., Geng, J., Tantray, I., Li, S., Huh, S., Vogel, H., Glabe, C., Grinberg, L. T., Spina, S., Seeley, W. W., Guo, S. and Lu, B. (2021). Inefficient quality control of ribosome stalling during APP synthesis generates CAT-tailed species that precipitate hallmarks of Alzheimer's disease. Acta Neuropathol Commun 9(1): 169. PubMed ID: 34663454
Summary:
Amyloid precursor protein (APP) metabolism is central to Alzheimer's disease (AD) pathogenesis, but the key etiological driver remains elusive. Recent failures of clinical trials targeting amyloid-β (Aβ) peptides, the proteolytic fragments of amyloid precursor protein (APP) that are the main component of amyloid plaques, suggest that the proteostasis-disrupting, key pathogenic species remain to be identified. Previous studies suggest that APP C-terminal fragment (APP.C99) can cause disease in an Aβ-independent manner. The mechanism of APP.C99 pathogenesis is incompletely understood. Drosophila models were used to expressing APP.C99 with the native ER-targeting signal of human APP, expressing full-length human APP only, or co-expressing full-length human APP and β-secretase (BACE), to investigate mechanisms of APP.C99 pathogenesis. Key findings are validated in mammalian cell culture models, mouse 5xFAD model, and postmortem AD patient brain materials. This study found that ribosomes stall at the ER membrane during co-translational translocation of APP.C99, activating ribosome-associated quality control (RQC) to resolve ribosome collision and stalled translation. Stalled APP.C99 species with C-terminal extensions (CAT-tails) resulting from inadequate RQC are prone to aggregation, causing endolysosomal and autophagy defects and seeding the aggregation of amyloid β peptides, the main component of amyloid plaques. Genetically removing stalled and CAT-tailed APP.C99 rescued proteostasis failure, endolysosomal/autophagy dysfunction, neuromuscular degeneration, and cognitive deficits in AD models. The finding of RQC factor deposition at the core of amyloid plaques from AD brains further supports the central role of defective RQC of ribosome collision and stalled translation in AD pathogenesis. These findings demonstrate that amyloid plaque formation is the consequence and manifestation of a deeper level proteostasis failure caused by inadequate RQC of translational stalling and the resultant aberrantly modified APP.C99 species, previously unrecognized etiological drivers of AD and newly discovered therapeutic targets (Rimal, 2021).
Roemmich, A. J., Vu, T., Lukacsovich, T., Hawkins, C., Schutte, S. S. and O'Dowd, D. K. (2021). Seizure Phenotype and Underlying Cellular Defects in Drosophila Knock-In Models of DS (R1648C) and GEFS+ (R1648H) SCN1A Epilepsy. eNeuro 8(5). PubMed ID: 34475263
Summary:
Mutations in the voltage-gated sodium channel gene SCN1A are associated with human epilepsy disorders, but how most of these mutations alter channel properties and result in seizures is unknown. This study focuses on two different mutations occurring at one position within SCN1A R1648C (R-C) is associated with the severe disorder Dravet syndrome, and R1648H (R-H), with the milder disorder GEFS+. To explore how these different mutations contribute to distinct seizure disorders, Drosophila lines with the R-C or R-H mutation, or R1648R (R-R) control substitution in the fly sodium channel gene para were generated by CRISPR-Cas9 gene editing. The R-C and R-H mutations are homozygous lethal. Animals heterozygous for R-C or R-H mutations displayed reduced life spans and spontaneous and temperature-induced seizures not observed in R-R controls. Electrophysiological recordings from adult GABAergic neurons in R-C and R-H mutants revealed the appearance of sustained neuronal depolarizations and altered firing frequency that were exacerbated at elevated temperature. The only significant change observed in underlying sodium currents in both R-C and R-H mutants was a hyperpolarized deactivation threshold at room and elevated temperature compared with R-R controls. Since this change is constitutive, it is likely to interact with heat-induced changes in other cellular properties to result in the heat-induced increase in sustained depolarizations and seizure activity. Further, the similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines, suggests that disease symptoms of different severity associated with these mutations in humans could be due in large part to differences in genetic background (Roemmich, 2021).
Parkhitko, A. A., Wang, L., Filine, E., Jouandin, P., Leshchiner, D., Binari, R., Asara, J. M., Rabinowitz, J. D. and Perrimon, N. (2021). A genetic model of methionine restriction extends Drosophila health- and lifespan. Proc Natl Acad Sci U S A 118(40). PubMed ID: 34588310
Summary:
Loss of metabolic homeostasis is a hallmark of aging and is characterized by dramatic metabolic reprogramming. To analyze how the fate of labeled methionine is altered during aging, This study applied C5-Methionine labeling to Drosophila and demonstrated significant changes in the activity of different branches of the methionine metabolism as flies age. Whether targeted degradation of methionine metabolism components would "reset" methionine metabolism flux and extend the fly lifespan was tested. Specifically, this study created transgenic flies with inducible expression of Methioninase, a bacterial enzyme capable of degrading methionine and revealed methionine requirements for normal maintenance of lifespan. Also demonstrated was the microbiota-derived methionine is an alternative and important source in addition to food-derived methionine. In this genetic model of methionine restriction (MetR), this study also demonstrate that either whole-body or tissue-specific Methioninase expression can dramatically extend Drosophila health- and lifespan and exerts physiological effects associated with MetR. Interestingly, while previous dietary MetR extended lifespan in flies only in low amino acid conditions, MetR from Methioninase expression extends lifespan independently of amino acid levels in the food. Finally, because impairment of the methionine metabolism has been previously associated with the development of Alzheimer's disease, this study compared methionine metabolism reprogramming between aging flies and a Drosophila model relevant to Alzheimer's disease, and found that overexpression of human Tau caused methionine metabolism flux reprogramming similar to the changes found in aged flies. Altogether, this study highlights Methioninase as a potential agent for health- and lifespan extension (Parkhitko, 2021).
Rapisarda, A., Bargiela, A., Llamusi, B., Pont, I., Estrada-Tejedor, R., Garcia-Espana, E., Artero, R. and Perez-Alonso, M. (2021). Defined D-hexapeptides bind CUG repeats and rescue phenotypes of myotonic dystrophy myotubes in a Drosophila model of the disease. Sci Rep 11(1): 19417. PubMed ID: 34593893
Summary:
In Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic double-stranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2; see Drosophila Muscleblind) and trigger other DM1-related pathogenesis pathway defects. This paper characterizes four D-amino acid hexapeptides identified together with abp1, a peptide previously shown to stabilize CUG RNA in its single-stranded conformation. With the generalized sequence cpy(a/t)(q/w)e, these related peptides improved three MBNL-regulated exon inclusions in DM1-derived cells. Subsequent experiments showed that these compounds generally increased the relative expression of MBNL1 and its nuclear-cytoplasmic distribution, reduced hyperactivated autophagy, and increased the percentage of differentiated (Desmin-positive) cells in vitro. All peptides rescued atrophy of indirect flight muscles in a Drosophila model of the disease, and partially rescued muscle function according to climbing and flight tests. Investigation of their mechanism of action supports that all four compounds can bind to CUG repeats with slightly different association constant, but binding did not strongly influence the secondary structure of the toxic RNA in contrast to abp1. Finally, molecular modeling suggests a detailed view of the interactions of peptide-CUG RNA complexes useful in the chemical optimization of compounds (Rapisarda, 2021).
Johnson, S. L., Libohova, K., Blount, J. R., Sujkowski, A. L., Prifti, M. V., Tsou, W. L. and Todi, S. V. (2021). Targeting the VCP-binding motif of ataxin-3 improves phenotypes in Drosophila models of Spinocerebellar Ataxia Type 3. Neurobiol Dis 160: 105516. PubMed ID: 34563642
Summary:
Of the family of polyglutamine (polyQ) neurodegenerative diseases, Spinocerebellar Ataxia Type 3 (SCA3) is the most common. Like other polyQ diseases, SCA3 stems from abnormal expansions in the CAG triplet repeat of its disease gene resulting in elongated polyQ repeats within its protein, ataxin-3. Various ataxin-3 protein domains contribute to its toxicity, including the valosin-containing protein (VCP)-binding motif (VBM). A previous study reported that VCP, a homo-hexameric protein, enhances pathogenic ataxin-3 aggregation and exacerbates its toxicity. These findings led to an exploration of the impact of targeting the SCA3 protein by utilizing a decoy protein comprising the N-terminus of VCP (N-VCP) that binds ataxin-3's VBM. The notion was that N-VCP would reduce binding of ataxin-3 to VCP, decreasing its aggregation and toxicity. This study found that expression of N-VCP in Drosophila melanogaster models of SCA3 ameliorated various phenotypes, coincident with reduced ataxin-3 (see Drosophila Ataxin-2)aggregation. This protective effect was specific to pathogenic ataxin-3 and depended on its VBM. Increasing the amount of N-VCP resulted in further phenotype improvement. This work highlights the protective potential of targeting the VCP-ataxin-3 interaction in SCA3, a key finding in the search for therapeutic opportunities for this incurable disorder (Johnson, 2021).

Thursday, December 9th - Gonads

Soffers, J. H. M., Alcantara, S. G., Li, X., Shao, W., Seidel, C. W., Li, H., Zeitlinger, J., Abmayr, S. M. and Workman, J. L. (2021). The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters. PLoS Genet 17(11): e1009668. PubMed ID: 34807910
Summary:
The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. This study analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. Depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. These data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.
Landis, G. N., Hilsabeck, T. A. U., Bell, H. S., Ronnen-Oron, T., Wang, L., Doherty, D. V., Tejawinata, F. I., Erickson, K., Vu, W., Promislow, D. E. L., Kapahi, P. and Tower, J. (2021). Mifepristone Increases Life Span of Virgin Female Drosophila on Regular and High-fat Diet Without Reducing Food Intake. Front Genet 12: 751647. PubMed ID: 34659367.
Summary:
The synthetic steroid mifepristone is reported to have anti-obesity and anti-diabetic effects in mammals on normal and high-fat diets (HFD). Previous work showed that mifepristone blocks the negative effect on life span caused by mating in female Drosophila melanogaster. This study asked if mifepristone could protect virgin females from the life span-shortening effect of HFD. Mifepristone increased life span of virgin females on regular media, as well as on media supplemented with either 2.5 or 5% coconut oil. Food intake was not reduced in any assay, and was significantly increased by mifepristone in half of the assays. To ask if mifepristone might rescue virgin females from all life span-shortening stresses, the oxidative stressor paraquat was tested, and mifepristone produced little to no rescue. Analysis of extant metabolomics and transcriptomics data suggested similarities between effects of mifepristone in virgin and mated females, including reduced tryptophan breakdown and similarities to dietary restriction. Bioinformatics analysis identified candidate mifepristone targets, including transcription factors Paired and Extra-extra. In addition to shortening life span, mating also causes midgut hypertrophy and activation of the lipid metabolism regulatory factor SREBP. Mifepristone blocked the increase in midgut size caused by mating, but did not detectably affect midgut size in virgins. Finally, mating increased activity of a SREBP reporter in abdominal tissues, as expected, but reporter activity was not detectably reduced by mifepristone in either mated or virgin females. Mifepristone increases life span of virgin females on regular and HFD without reducing food intake. Metabolomics and transcriptomics analyses suggest some similar effects of mifepristone between virgin and mated females, however reduced midgut size was observed only in mated females.
Ishibashi, J. R., Keshri, R., Taslim, T. H., Brewer, D. K., Chan, T. C., Lyons, S., McManamen, A. M., Chen, A., Del Castillo, D. and Ruohola-Baker, H. (2021). Chemical Genetic Screen in Drosophila Germline Uncovers Small Molecule Drugs That Sensitize Stem Cells to Insult-Induced Apoptosis. Cells 10(10). PubMed ID: 34685753
Summary:
Cancer stem cells, in contrast to their more differentiated daughter cells, can endure genotoxic insults, escape apoptosis, and cause tumor recurrence. Understanding how normal adult stem cells survive and go to quiescence may help identify druggable pathways that cancer stem cells have co-opted. This study utilized a genetically tractable model for stem cell survival in the Drosophila gonad to screen drug candidates and probe chemical-genetic interactions. This study employs three levels of small molecule screening: a medium-throughput primary screen in male germline stem cells (GSCs), a secondary screen with irradiation and protein-constrained food in female GSCs, and a tertiary screen in breast cancer organoids in vitro. This study uncover a series of small molecule drug candidates that may sensitize cancer stem cells to apoptosis. This study assessed these small molecules for chemical-genetic interactions in the germline and identified the NF-kappaB pathway as an essential and druggable pathway in GSC quiescence and viability. This study demonstrates the power of the Drosophila stem cell niche as a model system for targeted drug discovery (Ishibashi, 2021).
Lawlor, M. A., Cao, W. and Ellison, C. E. (2021). A transposon expression burst accompanies the activation of Y-chromosome fertility genes during Drosophila spermatogenesis. Nat Commun 12(1): 6854. PubMed ID: 34824217
Summary:
Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of Drosophila testis are not well understood. Here, we reanalyze publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We develop a method for identification of TE and host gene expression modules and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are specifically enriched on the Y chromosome and depleted on the X chromosome, relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y chromosome-specific transcriptional programs.
Reilein, A., Kogan, H. V., Misner, R., Park, K. S. and Kalderon, D. (2021). Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development. Elife 10. PubMed ID: 34590579
Summary:
Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. This study shows that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events (Reilein, 2021).
ElMaghraby, M. F., Tirian, L., Senti, K. A., Meixner, K. and Brennecke, J. (2021). A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary. Genetics. PubMed ID: 34718559
Summary:
Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. This study established a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. Constitutive, pan-germline RNAi was compared with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows investigation of the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. This work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center (ElMaghraby, 2021).

Wednesday, December 8th - Evolution

Harrison, B. R., Hoffman, J. M., Samuelson, A., Raftery, D. and Promislow, D. E. L. (2021). Modular Evolution of the Drosophila Metabolome. Mol Biol Evol. PubMed ID: 34662414
Summary:
Comparative phylogenetic studies offer a powerful approach to study the evolution of complex traits. While much effort has been devoted to the evolution of the genome and to organismal phenotypes, until now relatively little work has been done on the evolution of the metabolome, despite the fact that it is composed of the basic structural and functional building blocks of all organisms. This study explored variation in metabolite levels across 50 million years of evolution in the genus Drosophila, employing a common garden design to measure the metabolome within and among 11 species of Drosophila. This study found that both sex and age have dramatic and evolutionarily conserved effects on the metabolome. This study also found substantial evidence that many metabolite pairs covary after phylogenetic correction, and that such metabolome coevolution is modular. Some of these modules are enriched for specific biochemical pathways and show different evolutionary trajectories, with some showing signs of stabilizing selection. Both observations suggest that functional relationships may ultimately cause such modularity. These coevolutionary patterns also differ between sexes and are affected by age. This study also explored the relevance of modular evolution to fitness by associating modules with lifespan variation measured in the same common garden. Several modules associated with lifespan were found, particularly in the metabolome of older flies. Oxaloacetate levels in older females appear to coevolve with lifespan, and a lifespan-associated module in older females suggests that metabolic associations could underlie 50 million years of lifespan evolution (Harrison, 2021).
Marin, P., Jaquet, A., Picarle, J., Fablet, M., Merel, V., Delignette-Muller, M. L., Ferrarini, M. G., Gibert, P. and Vieira, C. (2021). Phenotypic and Transcriptomic Responses to Stress Differ According to Population Geography in an Invasive Species. Genome Biol Evol 13(9). PubMed ID: 34505904
Summary:
Adaptation to rapid environmental changes must occur within a short-time scale. In this context, studies of invasive species may provide insights into the underlying mechanisms of rapid adaptation as these species have repeatedly encountered and adapted to novel environmental conditions. This study investigated how invasive and noninvasive genotypes of Drosophila suzukii deal with oxidative stress at the phenotypic and molecular levels. Also studied was the impact of transposable element (TE) insertions on the gene expression in response to stress. Results show that flies from invasive areas (France and the United States) live longer in natural conditions than the ones from native Japanese areas. As expected, lifespan for all genotypes was significantly reduced following exposure to paraquat, but this reduction varied among genotypes (genotype-by-environment interaction) with invasive genotypes appearing more affected by exposure than noninvasive ones. A transcriptomic analysis of genotypes upon paraquat treatment detected many genes differentially expressed (DE). Although a small core set of genes were DE in all genotypes following paraquat exposure, much of the response of each genotype was unique. Moreover, it was shown that TEs were not activated after oxidative stress and DE genes were significantly depleted of TEs. In conclusion, it is likely that transcriptomic changes are involved in the rapid adaptation to local environments. This study provides new evidence that in the decade since the invasion from Asia, the sampled genotypes in Europe and the United States of D. suzukii diverged from the ones from the native area regarding their phenotypic and genomic response to oxidative stress (Marin, 2021).
Reddiex, A. J. and Chenoweth, S. F. (2021). Integrating genomics and multivariate evolutionary quantitative genetics: a case study of constraints on sexual selection in Drosophila serrata. Proc Biol Sci 288(1960): 20211785. PubMed ID: 34641732
Summary:
In evolutionary quantitative genetics, the genetic variance-covariance matrix, G, and the vector of directional selection gradients, β, are key parameters for predicting multivariate selection responses and genetic constraints. Historically, investigations of G and β have not overlapped with those dissecting the genetic basis of quantitative traits. Thus, it remains unknown whether these parameters reflect pleiotropic effects at individual loci. This study integrates multivariate genome-wide association study (GWAS) with G and β estimation in a well-studied system of multivariate constraint: sexual selection on male cuticular hydrocarbons (CHCs) in Drosophila serrata. In a panel of wild-derived re-sequenced lines, this study augments genome-based restricted maximum likelihood to estimate G alongside multivariate single nucleotide polymorphism (SNP) effects, detecting 532 significant associations from 1 652 276 SNPs. Constraint was evident, with β lying in a direction of G with low evolvability. Interestingly, minor frequency alleles typically increased male CHC-attractiveness suggesting opposing natural selection on β. SNP effects were significantly misaligned with the major eigenvector of G, gmax, but well aligned to the second and third eigenvectors g2 and g3. Potential factors leading to these varied results including multivariate stabilizing selection and mutational bias. This framework may be useful as researchers increasingly access genomic methods to study multivariate selection responses in wild populations (Reddiex, 2021).
Gomez-Llano, M., Scott, E. and Svensson, E. I. (2021). The importance of pre- and postcopulatory sexual selection promoting adaptation to increasing temperatures. Curr Zool 67(3): 321-327. PubMed ID: 34616924
Summary:
Global temperatures are increasing rapidly affecting species globally. Understanding if and how different species can adapt fast enough to keep up with increasing temperatures is of vital importance. One mechanism that can accelerate adaptation and promote evolutionary rescue is sexual selection. Two different mechanisms by which sexual selection can facilitate adaptation are pre- and postcopulatory sexual selection. However, the relative effects of these different forms of sexual selection in promoting adaptation are unknown. This study presents the results from an experimental study in which fruit flies Drosophila melanogaster were exposed to either no mate choice or 1 of 2 different sexual selection regimes (pre- and postcopulatory sexual selection) for 6 generations, under different thermal regimes. Populations showed evidence of thermal adaptation under precopulatory sexual selection, but this effect was not detected in the postcopulatory sexual selection and the no choice mating regime. This study further demonstrates that sexual dimorphism decreased when flies evolved under increasing temperatures, consistent with recent theory predicting more sexually concordant selection under environmental stress. These results suggest an important role for precopulatory sexual selection in promoting thermal adaptation and evolutionary rescue (Gomez-Llano, 2021).
Keaney, T. A., Jones, T. M. and Holman, L. (2021). Sexual selection can partly explain low frequencies of Segregation Distorter alleles. Proc Biol Sci 288(1959): 20211190. PubMed ID: 34583584
Summary:
The Segregation Distorter (SD) allele found in Drosophila melanogaster distorts Mendelian inheritance in heterozygous males by causing developmental failure of non-SD spermatids, such that greater than 90% of the surviving sperm carry SD. This within-individual advantage should cause SD to fix, and yet SD is typically rare in wild populations. this study explores whether this paradox can be resolved by sexual selection, by testing if males carrying three different variants of SD suffer reduced pre- or post-copulatory reproductive success. This study finds that males carrying the SD allele are just as successful at securing matings as control males, but that one SD variant (SD-5) reduces sperm competitive ability and increases the likelihood of female remating. The results inform a theoretical model; It was discovered that sexual selection could limit SD to natural frequencies when sperm competitive ability and female remating rate equalled the values observed for SD-5. However, sexual selection was unable to explain natural frequencies of the SD allele when the model was parameterized with the values found for two other SD variants, indicating that sexual selection alone is unlikely to explain the rarity of SD (Keaney, 2021).
Jackson, B. and Charlesworth, B. (2021). Evidence for a force favoring GC over AT at short intronic sites in Drosophila simulans and Drosophila melanogaster. G3 (Bethesda) 11(9). PubMed ID: 34544137
Summary:
Population genetics studies often make use of a class of nucleotide site free from selective pressures, in order to make inferences about population size changes or natural selection at other sites. If such neutral sites can be identified, they offer the opportunity to avoid any confounding effects of selection. This study investigates evolution at putatively neutrally evolving short intronic sites in natural populations of Drosophila melanogaster and Drosophila simulans, in order to understand the properties of spontaneous mutations and the extent of GC-biased gene conversion in these species. Use of data on the genetics of natural populations is advantageous because it integrates information from large numbers of individuals over long timescales. In agreement with direct evidence from observations of spontaneous mutations in Drosophila was found, this study found a bias in the spectrum of mutations toward AT basepairs. In addition, a find that this bias is stronger in the D. melanogaster lineage than in the D. simulans lineage. The evidence for GC-biased gene conversion in Drosophila has been equivocal. This study provides evidence for a weak force favoring GC in both species, which is correlated with the GC content of introns and is stronger in D. simulans than in D. melanogaster (Jackson, 2021).

Tuesday, December 7th - Adult Development

Lam, G., Nam, H. J., Velentzas, P. D., Baehrecke, E. H. and Thummel, C. S. (2021). Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis. Dev Biol. PubMed ID: 34648816.
Summary:
Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 h after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. This study shows that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. It was also shown that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation.
Rand, D. A., Raju, A., Sa¡ez, M., Corson, F. and Siggia, E. D. (2021). Geometry of gene regulatory dynamics. Proc Natl Acad Sci U S A 118(38). PubMed ID: 34518231
Summary:
Embryonic development leads to the reproducible and ordered appearance of complexity from egg to adult. The successive differentiation of different cell types that elaborate this complexity results from the activity of gene networks and was likened by Waddington to a flow through a landscape in which valleys represent alternative fates. Geometric methods allow the formal representation of such landscapes and codify the types of behaviors that result from systems of differential equations. Results from Smale and coworkers imply that systems encompassing gene network models can be represented as potential gradients with a Riemann metric, justifying the Waddington metaphor. This study extends the representation to include parameter dependence and enumerate all three-way cellular decisions realizable by tuning at most two parameters, which can be generalized to include spatial coordinates in a tissue. All diagrams of cell states vs. model parameters are thereby enumerated. The study unifies a number of standard models for spatial pattern formation by expressing them in potential form (i.e., as topographic elevation). Turing systems appear nonpotential, yet in suitable variables the dynamics are low dimensional and potential. A time-independent embedding recovers the original variables. Lateral inhibition is described by a saddle point with many unstable directions. A model for the patterning of the Drosophila eye appears as relaxation in a bistable potential. Geometric reasoning provides intuitive dynamic models for development that are well adapted to fit time-lapse data (Rand, 2021).
Juarez-Carreno, S., Vallejo, D. M., Carranza-Valencia, J., Palomino-Schatzlein, M., Ramon-Canellas, P., Santoro, R., de Hartog, E., Ferres-Marco, D., Romero, A., Peterson, H. P., Ballesta-Illan, E., Pineda-Lucena, A., Dominguez, M. and Morante, J. (2021). Body-fat sensor triggers ribosome maturation in the steroidogenic gland to initiate sexual maturation in Drosophila. Cell Rep 37(2): 109830. PubMed ID: 34644570
Summary:
Fat stores are critical for reproductive success and may govern maturation initiation. This study reports signaling and sensing fat sufficiency for sexual maturation commitment requires the lipid carrier apolipophorin in fat cells and Sema1a in the neuroendocrine prothoracic gland (PG). Larvae lacking apolpp or Sema1a fail to initiate maturation despite accruing sufficient fat stores, and they continue gaining weight until death. Mechanistically, sensing peripheral body-fat levels via the apolipophorin/Sema1a axis regulates endocytosis, endoplasmic reticulum remodeling, and ribosomal maturation for the acquisition of the PG cells' high biosynthetic and secretory capacity. Downstream of apolipophorin/Sema1a, leptin-like upd2 triggers the cessation of feeding and initiates sexual maturation. Human Leptin in the insect PG substitutes for upd2, preventing obesity and triggering maturation downstream of Sema1a. Data shows how peripheral fat levels regulate the control of the maturation decision-making process via remodeling of endomembranes and ribosomal biogenesis in gland cells (Juarez, 2021).
Ostala, C. M., Esteban, N., Lapez-Varea, A. and de Celis, J. F. (2021). Functional requirements of protein kinases and phosphatases in the development of the Drosophila melanogaster wing. G3 (Bethesda). PubMed ID: 34599799
Summary:
Protein kinases and phosphatases constitute a large family of conserved enzymes that control a variety of biological processes by regulating the phosphorylation state of target proteins. They play fundamental regulatory roles during cell cycle progression and signaling, among other key aspects of multicellular development. The complement of protein kinases and phosphatases includes approximately 326 members in Drosophila, and they have been the subject of several functional screens searching for novel components of signaling pathways and regulators of cell division and survival. These approaches have been carried out mostly in cell cultures using RNA interference to evaluate the contribution of each protein in different functional assays, and have contributed significantly to assign specific roles to the corresponding genes. The results are described of an evaluation of the Drosophila complement of kinases and phosphatases using the wing as a system to identify their functional requirements in vivo. This study also describes the results of several modifying screens aiming to identify among the set of protein kinases and phosphatases additional components or regulators of the activities of the Epidermal Growth Factor and Insulin receptors signaling pathways (Ostala, 2021).
Ozsoy, E. D., Yilmaz, M., Patlar, B., Emecen, G., Durmaz, E., Magwire, M. M., Zhou, S., Huang, W., Anholt, R. R. H. and Mackay, T. F. C. (2021). Epistasis for head morphology in Drosophila melanogaster. G3 (Bethesda) 11(10). PubMed ID: 34568933
Summary:
Epistasis-gene-gene interaction-is common for mutations with large phenotypic effects in humans and model organisms. Epistasis impacts quantitative genetic models of speciation, response to natural and artificial selection, genetic mapping, and personalized medicine. However, the existence and magnitude of epistasis between alleles with small quantitative phenotypic effects are controversial and difficult to assess. This study used the Drosophila melanogaster Genetic Reference Panel of sequenced inbred lines to evaluate the magnitude of naturally occurring epistasis modifying the effects of mutations in jing and inv, two transcription factors that have subtle quantitative effects on head morphology as homozygotes. This study found significant epistasis for both mutations and performed single marker genome-wide association analyses to map candidate modifier variants and loci affecting head morphology. A subset of these loci was significantly enriched for a known genetic interaction network, and mutations of the candidate epistatic modifier loci also affect head morphology (Ozsoy, 2021).
Lapez-Varea, A., Ostale, C. M., Vega-Cuesta, P., Ruiz-Gomez, A., Organista, M. F., Martan, M., Hevia, C. F., Molnar, C., de Celis, J., Culi, J., Esteban, N. and de Celis, J. F. (2021). Genome-wide Phenotypic RNAi Screen in the Drosophila Wing: Global Parameters. G3 (Bethesda). PubMed ID: 34599819
Summary:
This study has screened a collection of UAS-RNAi lines targeting 10920 Drosophila protein-coding genes for phenotypes in the adult wing. 3653 genes (33%) were identified whose knock-down causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist in changes in wing size, vein differentiation and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. This study also defined 16 functional categories encompassing the most relevant aspect of each protein function, and assigned each Drosophila gene to one of these functional groups. This allowed identification of which mutant phenotypes are enriched within each functional group. Finally, this study used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them (Lapez-Varea, 2021).

Monday, December 6th - RNA and Transposons

Horiuchi, K., Kawamura, T. and Hamakubo, T. (2021). Wilms' Tumor 1-Associating Protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences. J Biol Chem: 101248. PubMed ID: 34582888
Summary:
Wilms' tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various process of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. This study demonstrates using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. this study also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3'-end processing complex. Taken together, these results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences (Horiuchi, 2021).
Prudencio, P., Savisaar, R., Rebelo, K., Goncalo Martinho, R. and Carmo-Fonseca, M. (2021). Transcription and splicing dynamics during early Drosophila development. RNA. PubMed ID: 34667107
Summary:
Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. This study adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. These results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. This study found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. This study included the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, this study found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. This study further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, these data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo (Prudencio, 2021).
Nakamura, K., Nakao, T., Mori, T., Ohno, S., Fujita, Y., Masaoka, K., Sakabayashi, K., Mori, K., Tobimatsu, T. and Sera, T. (2021). Necessity of Flanking Repeats R1' and R8' of Human Pumilio1 Protein for RNA Binding. Biochemistry 60(40): 3007-3015. PubMed ID: 34541851
Summary:
Human Pumilio (hPUM see Drosophila Pumilio) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1' and R8', however, remain unclear. This study reports how the repeats contribute to overall RNA binding. The first step was to prepare three mutants in which R1' and/or R8' were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1' contributed more than R8', unlike Drosophila PUM. It was further investigated which amino acid residues of R1' or R8' were responsible for RNA binding. With detailed analysis of the protein tertiary structure, this study found a hydrophobic core in each of the repeats. All hydrophobic amino residues in each core to alanine were mutated during this process. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1' had a significant influence. In the present study, it was demonstrated that the flanking R1' and R8' repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1'-R1 interactions may stabilize the whole hPUM structure (Nakamura, 2021).
Nirala, N. K., Li, Q., Ghule, P. N., Chen, H. J., Li, R., Zhu, L. J., Wang, R., Rice, N. P., Mao, J., Stein, J. L., Stein, G. S., van Wijnen, A. J. and Ip, Y. T. (2021). Hinfp is a guardian of the somatic genome by repressing transposable elements. Proc Natl Acad Sci U S A 118(41). PubMed ID: 34620709
Summary:
Germ cells possess the Piwi-interacting RNA pathway to repress transposable elements and maintain genome stability across generations. Transposable element mobilization in somatic cells does not affect future generations, but nonetheless can lead to pathological outcomes in host tissues. This study shows the loss of function of the conserved zinc-finger transcription factor Hinfp causes dysregulation of many host genes and derepression of most transposable elements. There is also substantial DNA damage in somatic tissues of Drosophila after loss of Hinfp. Interference of transposable element mobilization by reverse-transcriptase inhibitors can suppress some of the DNA damage phenotypes. The key cell-autonomous target of Hinfp in this process is Histone1, which encodes linker histones essential for higher-order chromatin assembly. Transgenic expression of Hinfp or Histone1, but not Histone4 of core nucleosome, is sufficient to rescue the defects in repressing transposable elements and host genes. Loss of Hinfp enhances Ras-induced tissue growth and aging-related phenotypes. Therefore, Hinfp is a physiological regulator of Histone1-dependent silencing of most transposable elements, as well as many host genes, and serves as a venue for studying genome instability, cancer progression, neurodegeneration, and aging (Nirala, 2021).
Roy, M., Viginier, B., Mayeux, C. A., Ratinier, M. and Fablet, M. (2021). Infections by Transovarially Transmitted DMelSV in Drosophila Have No Impact on Ovarian Transposable Element Transcripts but Increase Their Amounts in the Soma. Genome Biol Evol 13(9). PubMed ID: 34498066
Summary:
Transposable elements (TEs) are genomic parasites, which activity is tightly controlled in germline cells. Using Sindbis virus, it was recently demonstrated that viral infections affect TE transcript amounts in somatic tissues. However, the strongest evolutionary impacts are expected in gonads, because that is where the genomes of the next generations lie. This aspect was investigated using the Drosophila melanogaster Sigma virus. It is particularly relevant in the genome/TE interaction given its tropism to ovaries, which is the organ displaying the more sophisticated TE control pathways. Results in Drosophila simulans flies allowed confirmation of the existence of a strong homeostasis of the TE transcriptome in ovaries upon infection, which, however, rely on TE-derived small RNA modulations. In addition, this study performed a meta-analysis of RNA-seq data and propose that the immune pathway that is triggered upon viral infection determines the direction of TE transcript modulation in somatic tissues (Roy, 2021).
Kunar, R. and Roy, J. K. (2021). The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms. Cell Tissue Res. PubMed ID: 34536141.
Summary:
The Drosophila genome codes for two decapping proteins, DCP1 and DCP2, out of which DCP2 is the active decapping enzyme. The present endeavour explores the endogenous promoter firing, transcript and protein expression of DCP2 in Drosophila wherein, besides a ubiquitous expression across development, an active expression paradigm during dorsal closure and a plausible moonlighting expression in the Corazonin neurons of the larval brain were identified. It was also demonstrated that the ablation of DCP2 leads to embryonic lethality and defects in vital morphogenetic processes whereas a knockdown of DCP2 in the Corazonin neurons reduces the sensitivity to ethanol in adults, thereby ascribing novel regulatory roles to DCP2. These findings unravel novel putative roles for DCP2 and identify it as a candidate for studies on the regulated interplay of essential molecules during early development in Drosophila, nay the living world.

Monday December 5th - Gonads

McDonough-Goldstein, C. E., Whittington, E., McCullough, E. L., Buel, S. M., Erdman, S., Pitnick, S. and Dorus, S. (2021). Pronounced postmating response in the Drosophila female reproductive tract fluid proteome. Mol Cell Proteomics: 100156. PubMed ID: 34597791
Summary:
Fertility depends on the progression of complex and coordinated postmating processes within the extracellular environment of the female reproductive tract (FRT). Molecular interactions between ejaculate and FRT proteins regulate many of these processes, including sperm motility, migration, storage, and modification, along with concurrent changes in the female. Although extensive progress has been made in the proteomic characterization of male-derived components of sperm and seminal fluid, investigations into the FRT have remained more limited. To achieve a comparable level of knowledge regarding female-derived proteins that comprise the reproductive environment, this study utilized semiquantitative mass spectrometry-based proteomics to study the composition of the FRT tissue and, separately, the luminal fluid, before and after mating in Drosophila melanogaster. This approach leveraged whole-fly isotopic labelling to delineate female proteins from those transferred male ejaculate proteins. the results revealed several characteristics that distinguish the FRT fluid proteome from the FRT tissue proteome: the fluid proteome is encoded by genes with higher overall levels of FRT gene expression and tissue specificity, including many genes with enriched expression in the fat body, fluid-biased proteins are enriched for metabolic functions and the fluid exhibits pronounced postmating compositional changes. The dynamic mating-induced proteomic changes in the FRT fluid informs understanding of secretory mechanisms of the FRT, serves as a foundation for establishing female contributions to the ejaculate-female interactions that regulate fertility and highlights the importance of applying proteomic approaches to characterize the composition and dynamics of the FRT environment (McDonough-Goldstein, 2021).
Ohsako, T., Shirakami, M., Oiwa, K., Ibaraki, K., Karr, T. L., Tomaru, M., Sanuki, R. and Takano-Shimizu-Kouno, T. (2021). The Drosophila Neprilysin 4 gene is essential for sperm function following sperm transfer to females. Genes Genet Syst. PubMed ID: 34556622
Summary:
Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. This story focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. This study examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, this study induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, this study concluded that the Nep4 gene is essential for sperm function following sperm transfer to females (Ohsako, 2021).
Macartney, E. L., Zeender, V., Meena, A., De Nardo, A. N., Bonduriansky, R. and Lupold, S. (2021). Sperm depletion in relation to developmental nutrition and genotype in Drosophila melanogaster. Evolution. PubMed ID: 34617270
Summary:
Nutrient limitation during development can restrict the ability of adults to invest in costly fitness traits, and genotypes can vary in their sensitivity to developmental nutrition. However, little is known about how genotype and nutrition affect male ability to maintain ejaculate allocation and achieve fertilization across successive matings. Using 17 isogenic lines of Drosophila melanogaster, This study investigated how variation in developmental nutrition affects males' abilities to mate, transfer sperm, and sire offspring when presented with successive virgin females. This study found that, with each successive mating, males required longer to initiate copulation, transferred fewer sperm, and sired fewer offspring. Males reared on a low-nutrient diet transferred fewer sperm than those reared on nutritionally superior diets, but the rate at which males depleted their sperm, as well as their reproductive performance, was largely independent of diet. Genotype and the genotype x diet interaction explained little of the variation in these male reproductive traits. Results show that sperm depletion can occur rapidly and impose substantial fitness costs for D. melanogaster males across multiple genotypes and developmental environments (Macartney, 2021).
Nallasivan, M. P., Haussmann, I. U., Civetta, A. and Soller, M. (2021). Channel nuclear pore protein 54 directs sexual differentiation and neuronal wiring of female reproductive behaviors in Drosophila. BMC Biol 19(1): 226. PubMed ID: 34666772
Summary:
Female reproductive behaviors and physiology change profoundly after mating. In Drosophila, the post-mating response induced by male-derived sex-peptide in females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. This study use a genetic approach to further characterize the molecular and cellular architecture of the sex-peptide response in Drosophila females. Screening for mutations that affect the sensitivity to sex-peptide, this study identified the channel nuclear pore protein Nup54 gene as an essential component for mediating the sex-peptide response, with viable mutant alleles leading to the inability of laying eggs and reducing receptivity upon sex-peptide exposure. Nup54 directs correct wiring of eight adult brain neurons that express pickpocket and are required for egg-laying, while additional channel Nups also mediate sexual differentiation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and promoter variants alter nucleo-cytoplasmic shuttling. These results implicate nuclear pore functionality to neuronal wiring underlying the sex-peptide response and sexual differentiation as a response to sexual conflict arising from male-derived sex-peptide to direct the female post-mating response (Nallasivan, 2021).
Sankaranarayanan, M., Emenecker, R. J., Wilby, E. L., Jahnel, M., Trussina, I., Wayland, M., Alberti, S., Holehouse, A. S. and Weil, T. T. (2021). Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development. Dev Cell. PubMed ID: 34655524
Summary:
Ribonucleoprotein condensates can exhibit diverse physical states in vitro and in vivo. Despite considerable progress, the relevance of condensate physical states for in vivo biological function remains limited. This study investigated the physical properties of processing bodies (P bodies) and their impact on mRNA storage in mature Drosophila oocytes. This study shows that the conserved DEAD-box RNA helicase Me31B forms viscous P body condensates, which adopt an arrested physical state. This study demonstrates that structurally distinct proteins and protein-protein interactions, together with RNA, regulate the physical properties of P bodies. Using live imaging and in situ hybridization, this study shows that the arrested state and integrity of P bodies support the storage of bicoid (bcd) mRNA and that egg activation modulates P body properties, leading to the release of bcd for translation in the early embryo. Together, this work provides an example of how physical states of condensates regulate cellular function in development (Sankaranarayanan, 2021).
Fajner, V., Giavazzi, F., Sala, S., Oldani, A., Martini, E., Napoletano, F., Parazzoli, D., Cesare, G., Cerbino, R., Maspero, E., Vaccari, T. and Polo, S. (2021). Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles. Nat Commun 12(1): 5488. PubMed ID: 34531401
Summary:
Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. This study demonstrates that Hecw (CG42797), a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons (Fajner, 2021).

Thursday, December 2nd - Chromatin

Shaheen, N., Akhtar, J., Umer, Z., Khan, M. H. F., Bakhtiari, M. H., Saleem, M., Faisal, A. and Tariq, M. (2021). Polycomb Requires Chaperonin Containing TCP-1 Subunit 7 for Maintaining Gene Silencing in Drosophila. Front Cell Dev Biol 9: 727972. PubMed ID: 34660585
Summary:
In metazoans, heritable states of cell type-specific gene expression patterns linked with specialization of various cell types constitute transcriptional cellular memory. Evolutionarily conserved Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the transcriptional cellular memory by maintaining heritable patterns of repressed and active expression states, respectively. Although chromatin structure and modifications appear to play a fundamental role in maintenance of repression by PcG, the precise targeting mechanism and the specificity factors that bind PcG complexes to defined regions in chromosomes remain elusive. This study reports a serendipitous discovery that uncovers an interplay between Polycomb (Pc) and chaperonin containing T-complex protein 1 (TCP-1) subunit 7 (CCT7) of TCP-1 ring complex (TRiC) chaperonin in Drosophila. CCT7 interacts with Pc at chromatin to maintain repressed states of homeotic and non-homeotic targets of PcG, which supports a strong genetic interaction observed between Pc and CCT7 mutants. Depletion of CCT7 results in dissociation of Pc from chromatin and redistribution of an abundant amount of Pc in cytoplasm. It is proposed that CCT7 is an important modulator of Pc, which helps Pc recruitment at chromatin, and compromising CCT7 can directly influence an evolutionary conserved epigenetic network that supervises the appropriate cellular identities during development and homeostasis of an organism.
Laghmach, R., Di Pierro, M. and Potoyan, D. A. (2021). The interplay of chromatin phase separation and lamina interactions in nuclear organization. Biophys J. PubMed ID: 34653387
Summary:
The genetic material of eukaryotes is segregated into transcriptionally active euchromatin and silent heterochromatin compartments. The spatial arrangement of chromatin compartments evolves over the course of cellular life in a process that remains poorly understood. The latest nuclear imaging experiments reveal a number of dynamical signatures of chromatin that are reminiscent of active multi-phase liquids. This includes the observations of viscoelastic response, coherent motions, Ostwald ripening, and coalescence of chromatin compartments. There is also growing evidence that liquid-liquid phase separation of protein and nucleic acid components is the underlying mechanism for the dynamical behavior of chromatin. In order to dissect the organizational and dynamical implications of chromatin's liquid behavior, this study has devised a phenomenological field-theoretic model of the nucleus as a multi-phase condensate of liquid chromatin types. Employing the liquid chromatin model of the Drosophila nucleus, an extensive set of simulations with an objective to shed light on the dynamics and chromatin patterning observed in the latest nuclear imaging experiments. Simulations reveal the emergence of experimentally detected mesoscale chromatin channels and spheroidal droplets which arise from the dynamic interplay of chromatin type to type interactions and intermingling of chromosomal territories. In addition, coherent motions of chromatin domains observed in displacement correlation spectroscopy measurements which are explained within the framework of this model was quantitatively reproduced by phase separation of chromatin types operating within constrained intra-chromosomal and inter-chromosomal boundaries. Finally, this study illuminates the role of heterochromatin-lamina interactions in the nuclear organization by showing that these interactions enhance the mobility of euchromatin and indirectly introduce correlated motions of heterochromatin droplets (Laghmach, 2021).
Shaukat, A., Khan, M. H. F., Ahmad, H., Umer, Z. and Tariq, M. (2021). Interplay Between BALL and CREB Binding Protein Maintains H3K27 Acetylation on Active Genes in Drosophila. Front Cell Dev Biol 9: 740866. PubMed ID: 34650987
Summary:
CREB binding protein (CBP) is a multifunctional transcriptional co-activator that interacts with a variety of transcription factors and acts as a histone acetyltransferase. In Drosophila, CBP mediated acetylation of histone H3 lysine 27 (H3K27ac) is a known hallmark of gene activation regulated by trithorax group proteins (trxG). Recently, it has been shown that a histone kinase Ballchen (BALL) substantially co-localizes with H3K27ac at trxG target loci and is required to maintain gene activation in Drosophila. This study reports a previously unknown interaction between BALL and CBP, which positively regulates H3K27ac. Analysis of genome-wide binding profile of BALL and CBP reveals major overlap and their co-localization at actively transcribed genes. This study shows that BALL biochemically interacts with CBP and depletion of BALL results in drastic reduction in H3K27ac. Together, these results demonstrate a previously unknown synergy between BALL and CBP and reveals a potentially new pathway required to maintain gene activation during development.
Napoletano, F., Ferrari Bravo, G., Voto, I. A. P., Santin, A., Celora, L., Campaner, E., Dezi, C., Bertossi, A., Valentino, E., Santorsola, M., Rustighi, A., Fajner, V., Maspero, E., Ansaloni, F., Cancila, V., Valenti, C. F., Santo, M., Artimagnella, O. B., Finaurini, S., Gioia, U., Polo, S., Sanges, R., Tripodo, C., Mallamaci, A., Gustincich, S., d'Adda di Fagagna, F., Mantovani, F., Specchia, V. and Del Sal, G. (2021). The prolyl-isomerase PIN1 is essential for nuclear Lamin-B structure and function and protects heterochromatin under mechanical stress. Cell Rep 36(11): 109694. PubMed ID: 34525372
Summary:
Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. This study shows that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. This study demonstrated that the PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1α (HP1α). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies (Napoletano, 2021).
Sharp, K. A., Khoury, M. J., Wirtz-Peitz, F. and Bilder, D. (2021). Evidence for a nuclear role for Drosophila Dlg as a regulator of the NURF complex. Mol Biol Cell 32(21): ar23. PubMed ID: 34495684.
Summary:
Scribble (Scrib), Discs-large (Dlg), and Lethal giant larvae (Lgl) are basolateral regulators of epithelial polarity and tumor suppressors whose molecular mechanisms of action remain unclear. This study used proximity biotinylation to identify proteins localized near Dlg in the Drosophila wing imaginal disc epithelium. In addition to expected membrane- and cytoskeleton-associated protein classes, nuclear proteins were prevalent in the resulting mass spectrometry dataset, including all four members of the nucleosome remodeling factor (NURF) chromatin remodeling complex. Subcellular fractionation demonstrated a nuclear pool of Dlg and proximity ligation confirmed its position near the NURF complex. Genetic analysis showed that NURF activity is also required for the overgrowth of dlg tumors, and this growth suppression correlated with a reduction in Hippo pathway gene expression. Together, these data suggest a nuclear role for Dlg in regulating chromatin and transcription through a more direct mechanism than previously thought.
Ibarra-Morales, D., Rauer, M. Quarato, P. Rabbani, L. Zenk, F. Schulte-Sasse, Cardamon, F. Gomez-Auli, Cecere, G. and Iovino, N. (2021). Histone variant H2A.Z regulates zygotic genome activation. Nat Commun 12(1):7002. PubMed ID: 34853314 During embryogenesis, the genome shifts from transcriptionally quiescent to extensively active in a process known as Zygotic Genome Activation (ZGA). In Drosophila, the pioneer factor Zelda is known to be essential for the progression of development; still, it regulates the activation of only a small subset of genes at ZGA. However, thousands of genes do not require Zelda, suggesting that other mechanisms exist. By conducting GRO-seq, HiC and ChIP-seq in Drosophila embryos, this study demonstrated that up to 65% of zygotically activated genes are enriched for the histone variant H2A.Z. H2A.Z enrichment precedes ZGA and RNA Polymerase II loading onto chromatin. In vivo knockdown of maternally contributed Domino, a histone chaperone and ATPase, reduces H2A.Z deposition at transcription start sites, causes global downregulation of housekeeping genes at ZGA, and compromises the establishment of the 3D chromatin structure. It is inferred that H2A.Z is essential for the de novo establishment of transcriptional programs during ZGA via chromatin reorganization.

Wednesday, December 1st - Adult Neural Development and Function

Huang, S. K., Whitney, P. H., Dutta, S., Shvartsman, S. Y. and Rushlow, C. A. (2021. Spatial organization of transcribing loci during early genome activation in Drosophila. Curr Biol. PubMed ID: 34614388
Summary:
The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the entire genome. Using 1.5-h-old Drosophila embryos undergoing the first wave of genome activation, This study detected ~110 discrete "speckles" of RNA polymerase II (RNA Pol II) per nucleus, two of which were larger and localized to the histone locus bodies (HLBs). In the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda (Zld) 70% fewer speckles were present; however, the HLBs tended to be larger than wild-type (WT) HLBs, indicating that RNA Pol II accumulates at the HLBs in the absence of robust early-gene transcription. This study observed a uniform distribution of distances between active genes in the nuclei of both WT and zld mutant embryos, indicating that early co-regulated genes do not cluster into nuclear sub-domains. However, in instances whereby transcribing genes did come into close 3D proximity (within 400 nm), they were found to have distinct RNA Pol II speckles. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, the data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model, where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems (Huang, 2021).
Flores, C. C., Loschky, S. S., Marshall, W., Spano, G. M., Cenere, M. M., Tononi, G. and Cirelli, C. (2021). Identification of Ultrastructural Signatures of Sleep and Wake in the Fly Brain. Sleep. PubMed ID: 34536282
Summary:
The cellular consequences of sleep loss are poorly characterized. In the pyramidal neurons of mouse frontal cortex this study found that mitochondria and secondary lysosomes occupy a larger proportion of the cytoplasm after chronic sleep restriction compared to sleep, consistent with increased cellular burden due to extended wake. For each morphological parameter the within-animal variance was high, suggesting that the effects of sleep and sleep loss vary greatly among neurons. However, the analysis was based on 4-5 mice/group and a single section/cell. this study applied serial block-face scanning electron microscopy to identify signatures of sleep and sleep loss in the Drosophila brain. Stacks of images were acquired and used to obtain full 3D reconstructions of the cytoplasm and nucleus of 263 Kenyon cells from adult flies collected after a night of sleep (S) or after 11 hours (SD11) or 35 hours (SD35) of sleep deprivation (9 flies/group). Relative to S flies, SD35 flies showed increased density of dark clusters of chromatin and of Golgi apparata and a trend increase in the percent of cell volume occupied by mitochondria, consistent with increased need for energy and protein supply during extended wake. Logistic regression models could assign each neuron to the correct experimental group with good accuracy, but in each cell nuclear and cytoplasmic changes were poorly correlated, and within-fly variance was substantial in all experimental groups. Together, these results support the presence of ultrastructural signatures of sleep and sleep loss but underscore the complexity of their effects at the single-cell level (Flores, 2021).
Lone, S. R., Potdar, S., Venkataraman, A., Sharma, N., Kulkarni, R., Rao, S., Mishra, S., Sheeba, V. and Sharma, V. K. (2021). Mechanosensory Stimulation via Nanchung Expressing Neurons Can Induce Daytime Sleep in Drosophila. J Neurosci. PubMed ID: 34635540
Summary:
The neuronal and genetic bases of sleep, a phenomenon considered crucial for well-being of organisms, has been under investigation using the model organism Drosophila melanogaster. Although sleep is a state where sensory threshold for arousal is greater, it is known that certain kinds of repetitive sensory stimuli, such as rocking, can indeed promote sleep in humans. This study reports that orbital motion-aided mechanosensory stimulation promotes sleep of male and female Drosophila, independent of the circadian clock, but controlled by the homeostatic system. Mechanosensory receptor nanchung (Nan)-expressing neurons in the chordotonal organs mediate this sleep induction: flies in which these neurons are either silenced or ablated display significantly reduced sleep induction on mechanosensory stimulation. Transient activation of the Nan-expressing neurons also enhances sleep levels, confirming the role of these neurons in sleep induction. This study also reveals that certain regions of the antennal mechanosensory and motor center in the brain are involved in conveying information from the mechanosensory structures to the sleep centers. Thus, for the first time, this study shows that a circadian clock-independent pathway originating from peripherally distributed mechanosensors can promote daytime sleep of flies Drosophila melanogaster (Lone, 2021).
Krzeptowski, W., Walkowicz, L., Krzeptowska, E., Motta, E., Witek, K., Szramel, J., Al Abaquita, T., Baster, Z., Rajfur, Z., Rosato, E., Stratoulias, V., Heino, T. I. and Pyza, E. M. (2021). Mesencephalic Astrocyte-Derived Neurotrophic Factor Regulates Morphology of Pigment-Dispersing Factor-Positive Clock Neurons and Circadian Neuronal Plasticity in Drosophila melanogaster. Front Physiol 12: 705183. PubMed ID: 34646147
Summary:
Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is one of a few neurotrophic factors described in Drosophila melanogaster (DmMANF) but its function is still poorly characterized. The present study found that DmMANF is expressed in different clusters of clock neurons. In particular, the PDF-positive large (l-LNv) and small (s-LNv) ventral lateral neurons, the CRYPTOCHROME-positive dorsal lateral neurons (LNd), the group 1 dorsal neurons posterior (DN1p) and different tim-positive cells in the fly's visual system. Importantly, DmMANF expression in the ventral lateral neurons is not controlled by the clock nor it affects its molecular mechanism. However, silencing DmMANF expression in clock neurons affects the rhythm of locomotor activity in light:dark and constant darkness conditions. Such phenotypes correlate with abnormal morphology of the dorsal projections of the s-LNv and with reduced arborizations of the l-LNv in the medulla of the optic lobe. Additionally, it was shown that DmMANF is important for normal morphology of the L2 interneurons in the visual system and for the circadian rhythm in the topology of their dendritic tree. These results indicate that DmMANF is important not only for the development of neurites but also for maintaining circadian plasticity of neurons.
Lin, H. W., Chen, C. C., de Belle, J. S., Tully, T. and Chiang, A. S. (2021). CREBA and CREBB in two identified neurons gate long-term memory formation in Drosophila. Proc Natl Acad Sci U S A 118(37). PubMed ID: 34507985
Summary:
Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. This study demonstrates enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. These experiments show that this process is regulated by protein-gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins.
Almeida, M. P., Lago Solis, B., Stickley, L., Feidler, A. and Nagoshi, E. (2021). Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP-PKA signaling. Nat Commun 12(1): 5758. PubMed ID: 34599173
Summary:
Various behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila melanogaster, in which only ~0.1% of the brain's neurons contain circadian clocks. Clock neurons transmit the timing information to a plethora of non-clock neurons via poorly understood mechanisms. This study addresses the molecular underpinning of this phenomenon by profiling circadian gene expression in non-clock neurons that constitute the mushroom body, the center of associative learning and sleep regulation. This study shows that circadian clocks drive rhythmic expression of hundreds of genes in mushroom body neurons, including the Neurofibromin 1 (Nf1) tumor suppressor gene and Pka-C1. Circadian clocks also drive calcium rhythms in mushroom body neurons via NF1-cAMP/PKA-C1 signaling, eliciting higher mushroom body activity during the day than at night, thereby promoting daytime wakefulness. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep (Almeida, 2021). Home page: The Interactive Fly© 2020 Thomas B. Brody, Ph.D.

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