What's hot today:
Current papers in developmental biology and gene function


Friday, May 31st, 2019 - Behavior

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Juneau, B. A., Stonemetz, J. M., Toma, R. F., Possidente, D. R., Heins, R. C. and Vecsey, C. G. (2019). Optogenetic activation of short neuropeptide F (sNPF) neurons induces sleep in Drosophila melanogaster. Physiol Behav 206: 143-156. PubMed ID: 30935941
Sleep abnormalities have widespread and costly public health consequences, yet there is only a rudimentary understanding of the events occurring at the cellular level in the brain that regulate sleep. Several key signaling molecules that regulate sleep across taxa come from the family of neuropeptide transmitters. For example, in Drosophila melanogaster, the neuropeptide Y (NPY)-related transmitter short neuropeptide F (sNPF) appears to promote sleep. This study utilized optogenetic activation of neuronal populations expressing sNPF to determine the causal effects of precisely timed activity in these cells on sleep behavior. Combining sNPF-GAL4 and UAS-Chrimson transgenes allowed activation of sNPF neurons using red light. Activating sNPF neurons for as little as 3 s at a time of day when most flies were awake caused a rapid transition to sleep that persisted for another 2+ hours following the stimulation. Changing the timing of red light stimulation to times of day when flies were already asleep caused the control flies to wake up (due to the pulse of light), but the flies in which sNPF neurons were activated stayed asleep through the light pulse, and then showed further increases in sleep at later points when they would have normally been waking up. Video recording of individual fly responses to short-term (0.5-20 s) activation of sNPF neurons demonstrated a clear light duration-dependent decrease in movement during the subsequent 4-min period. These results provide supportive evidence that sNPF-producing neurons promote long-lasting increases in sleep, and show for the first time that even brief periods of activation of these neurons can cause changes in behavior that persist after cessation of activation. Evidence is presented that sNPF neuron activation produces a homeostatic sleep drive that can be dissipated at times long after the neurons were stimulated. Future studies will determine the specific roles of sub-populations of sNPF-producing neurons, and will also assess how sNPF neurons act in concert with other neuronal circuits to control sleep.
Srivastava, M., Varma, V., Abhilash, L., Sharma, V. K. and Sheeba, V. (2019). Circadian clock properties and their relationships as a function of free-running period in Drosophila melanogaster. J Biol Rhythms: 748730419837767. PubMed ID: 30939971

The stability of circadian clock mechanisms under cyclic environments contributes to increased Darwinian fitness by accurately timing daily behavior and physiology. Earlier studies on biological clocks speculated that the timing of behavior and its accuracy are determined by the intrinsic period (tau) of the circadian clock under constant conditions, its stability, the period of the external cycle (T), and resetting of the clock by environmental time cues. However, most of these previous studies suffered from certain limitations, the major ones being a narrow range of examined tau values and a non-uniformity in the genetic background across the individuals tested. The data in this study rigorously test the following hypotheses by employing Drosophila melanogaster fruit flies with tau ranging from 17 to 30 h in a uniform genetic background. Tests were performed to see whether 1) precision (day-to-day stability of tau) is greater for clocks with tau close to 24 h; 2) accuracy (i.e., day-to-day stability of the phase relationship (psi), where psi is the duration between a phase of the rhythm and a phase of the external cycle) is greater for clocks with tau close to 24 h; 3) Psi is delayed with an increase in tau; and 4) Psi becomes more advanced with an increase in length of zeitgeber cycle (T). This study shows that precision is not always maximum for ~24-h clocks, but that accuracy is greatest when tau approximates T. Further, flies exhibit a delayed phase relationship with increasing tau and an advanced phase relationship under long T-cycles as compared with shorter T-cycles. Relationships between activity and rest durations are described and how these observations fit predictions from models of circadian entrainment. Overall, it is confirmed that accuracy and phase of entrained rhythm are governed by both intrinsic clock period and the length of the external cycle; however, it was found that the relationship between intrinsic period and precision does not fit previous predictions.

Hollis, B., Koppik, M., Wensing, K. U., Ruhmann, H., Genzoni, E., Erkosar, B., Kawecki, T. J., Fricke, C. and Keller, L. (2019). Sexual conflict drives male manipulation of female postmating responses in Drosophila melanogaster. Proc Natl Acad Sci U S A 116(17): 8437-8444. PubMed ID: 30962372
In many animals, females respond to mating with changes in physiology and behavior that are triggered by molecules transferred by males during mating. In Drosophila melanogaster, proteins in the seminal fluid are responsible for important female postmating responses, including temporal changes in egg production, elevated feeding rates and activity levels, reduced sexual receptivity, and activation of the immune system. It is unclear to what extent these changes are mutually beneficial to females and males or instead represent male manipulation. This study used an experimental evolution approach in which females are randomly paired with a single male each generation, eliminating any opportunity for competition for mates or mate choice and thereby aligning the evolutionary interests of the sexes. After >150 generations of evolution, males from monogamous populations elicited a weaker postmating stimulation of egg production and activity than males from control populations that evolved with a polygamous mating system. Males from monogamous populations did not differ from males from polygamous populations in their ability to induce refractoriness to remating in females, but they were inferior to polygamous males in sperm competition. Mating-responsive genes in both the female abdomen and head showed a dampened response to mating with males from monogamous populations. Males from monogamous populations also exhibited lower expression of genes encoding seminal fluid proteins, which mediate the female response to mating. Together, these results demonstrate that the female postmating response, and the male molecules involved in eliciting this response, are shaped by ongoing sexual conflict.
Highfill, C. A., Baker, B. M., Stevens, S. D., Anholt, R. R. H. and Mackay, T. F. C. (2019). Genetics of cocaine and methamphetamine consumption and preference in Drosophila melanogaster. PLoS Genet 15(5): e1007834. PubMed ID: 31107875
Illicit use of psychostimulants, such as cocaine and methamphetamine, constitutes a significant public health problem. Whereas neural mechanisms that mediate the effects of these drugs are well-characterized, genetic factors that account for individual variation in susceptibility to substance abuse and addiction remain largely unknown. Drosophila melanogaster can serve as a translational model for studies on substance abuse, since flies have a dopamine transporter that can bind cocaine and methamphetamine, and exposure to these compounds elicits effects similar to those observed in people, suggesting conserved evolutionary mechanisms underlying drug responses. This study used the D. melanogaster Genetic Reference Panel to investigate the genetic basis for variation in psychostimulant drug consumption, to determine whether similar or distinct genetic networks underlie variation in consumption of cocaine and methamphetamine, and to assess the extent of sexual dimorphism and effect of genetic context on variation in voluntary drug consumption. Quantification of natural genetic variation in voluntary consumption, preference, and change in consumption and preference over time for cocaine and methamphetamine uncovered significant genetic variation for all traits, including sex-, exposure- and drug-specific genetic variation. Genome wide association analyses identified both shared and drug-specific candidate genes, which could be integrated in genetic interaction networks. The effects were assessed of ubiquitous RNA interference (RNAi) on consumption behaviors for 34 candidate genes: all affected at least one behavior. Finally, RNAi knockdown in the nervous system was used to implicate dopaminergic neurons and the mushroom bodies as part of the neural circuitry underlying experience-dependent development of drug preference.
Onodera, Y., Ichikawa, R., Terao, K., Tanimoto, H. and Yamagata, N. (2019). Courtship behavior induced by appetitive olfactory memory. J Neurogenet: 1-9. PubMed ID: 30955396
Reinforcement signals such as food reward and noxious punishment can change diverse behaviors. This holds true in fruit flies, Drosophila melanogaster, which can be conditioned by an odor and sugar reward or electric shock punishment. Despite a wide variety of behavior modulated by learning, conditioned responses have been traditionally measured by altered odor preference in a choice, and other memory-guided behaviors have been only scarcely investigated. This study analyzed detailed conditioned odor responses of flies after sugar associative learning by employing a video recording and semi-automated processing pipeline. Trajectory analyses revealed that multiple behavioral components were altered along with conditioned approach to the rewarded odor. Notably, it was found that lateral wing extension, a hallmark of courtship behavior of D. melanogaster, was robustly increased specifically in the presence of the rewarded odor. Strikingly, genetic disruption of the mushroom body output did not impair conditioned courtship increase, while markedly weakening conditioned odor approach. These results highlight the complexity of conditioned responses and their distinct regulatory mechanisms that may underlie coordinated yet complex memory-guided behaviors in flies.
Ishikawa, Y., Okamoto, N., Yoneyama, Y., Maeda, N. and Kamikouchi, A. (2019). A single male auditory response test to quantify auditory behavioral responses in Drosophila melanogaster. J Neurogenet: 1-11. PubMed ID: 31106644
During courtship, males of the fruit fly Drosophila melanogaster and related species produce a courtship song comprised of sine and pulse songs by vibrating their wings. The pulse song increases female receptivity and male courtship activity, indicating that it functions as a sexual signal. One song parameter, interpulse interval (IPI), varies among closely related species. In D. melanogaster, a song with a conspecific IPI induces a stronger behavioral response than heterospecific songs, indicating the ability of the flies to discriminate conspecific IPI. To quantify the individual ability to discriminate a conspecific song, this study systematically analyzed the auditory response of single male flies to sound with various parameters. By quantifying the locomotor activity of single D. melanogaster males during sound exposure, increased locomotor activity was detected in response to pulse songs, but not to white noise or pure tone. The conspecific song evoked stronger response than the heterospecific songs, and ablation of their antennal receivers severely suppressed the locomotor increase. A pulse song with a small IPI variation evoked a continuous response, while the response to songs with highly variable IPIs tends to be rapidly decayed. This provides the first evidence that fruit flies discriminate IPI variations, which possibly inform the age and social contexts of the singer. Sister species, D. sechellia, exhibited a locomotor response to pulse song, while D. simulans exhibited no behavioral response. This suggests that auditory and other stimuli that elicit this behavioral response are diversified among Drosophila species.

Thursday, May 30th - Stem Cells

Kim, W., Cho, Y. S., Wang, X., Park, O., Ma, X., Kim, H., Gan, W., Jho, E. H., Cha, B., Jeung, Y. J., Zhang, L., Gao, B., Wei, W., Jiang, J., Chung, K. S. and Yang, Y. (2019). Hippo signaling is intrinsically regulated during cell cycle progression by APC/C(Cdh1). Proc Natl Acad Sci U S A. PubMed ID: 31000600
The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, this study has found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. It was further identified that the anaphase-promoting complex/cyclosome (APC/C)(Cdh1) E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, it was shown in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/C(Cdh1) represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.
Francis, D., Chanana, B., Fernandez, B., Gordon, B., Mak, T. and Palacios, I. M. (2019). YAP/Yorkie in the germline modulates the age-related decline of germline stem cells and niche cells. PLoS One 14(4): e0213327. PubMed ID: 30943201
The properties and behaviour of stem cells rely heavily on signaling from the local microenvironment. At the apical end of Drosophila testis, self-renewal and differentiation of germline stem cells (GSCs) are tightly controlled by distinct somatic cells that comprise a specialised stem cell niche known as the hub. The hub maintains GSC homeostasis through adhesion and cell signaling. The Salvador/Warts/Hippo (SWH) pathway, which suppresses the transcriptional co-activator YAP/Yki via a kinase cascade, is a known regulator of stem cell proliferation and differentiation. This study shows that increasing YAP/Yki expression in the germline, as well as reducing Warts levels, blocks the decrease of GSC numbers observed in aging flies, with only a small increase on their proliferation. An increased expression of YAP/Yki in the germline or a reduction in Warts levels also stymies an age-related reduction in hub cell number, suggesting a bilateral relationship between GSCs and the hub. Conversely, RNAi-based knockdown of YAP/Yki in the germline leads to a significant drop in hub cell number, further suggesting the existence of such a SC-to-niche relationship. All together, these data implicate the SWH pathway in Drosophila GSC maintenance and raise questions about its role in stem cell homeostasis in aging organisms.
Rodriguez-Fernandez, I. A., Qi, Y. and Jasper, H. (2019). Loss of a proteostatic checkpoint in intestinal stem cells contributes to age-related epithelial dysfunction. Nat Commun 10(1): 1050. PubMed ID: 30837466
A decline in protein homeostasis (proteostasis) has been proposed as a hallmark of aging. Somatic stem cells (SCs) uniquely maintain their proteostatic capacity through mechanisms that remain incompletely understood. This study describes and characterizes a 'proteostatic checkpoint' in Drosophila intestinal SCs (ISCs). Following a breakdown of proteostasis, ISCs coordinate cell cycle arrest with protein aggregate clearance by Atg8-mediated activation of the Nrf2-like transcription factor cap-n-collar C (CncC). CncC induces the cell cycle inhibitor Dacapo and proteolytic genes. The capacity to engage this checkpoint is lost in ISCs from aging flies, and it can be restored by treating flies with an Nrf2 activator, or by over-expression of CncC or Atg8a. This limits age-related intestinal barrier dysfunction and can result in lifespan extension. These findings identify a new mechanism by which somatic SCs preserve proteostasis, and highlight potential intervention strategies to maintain regenerative homeostasis.
Gervais, L., van den Beek, M., Josserand, M., Salle, J., Stefanutti, M., Perdigoto, C. N., Skorski, P., Mazouni, K., Marshall, O. J., Brand, A. H., Schweisguth, F. and Bardin, A. J. (2019). Stem cell proliferation is kept in check by the chromatin regulators Kismet/CHD7/CHD8 and Trr/MLL3/4. Dev Cell 49(4): 556-573.e556. PubMed ID: 31112698
Chromatin remodeling accompanies differentiation, however, its role in self-renewal is less well understood. This paper reports that in Drosophila, the chromatin remodeler Kismet/CHD7/CHD8 limits intestinal stem cell (ISC) number and proliferation without affecting differentiation. Stem-cell-specific whole-genome profiling of Kismet revealed its enrichment at transcriptionally active regions bound by RNA polymerase II and Brahma, its recruitment to the transcription start site of activated genes and developmental enhancers and its depletion from regions bound by Polycomb, Histone H1, and heterochromatin Protein 1. The Trithorax-related/MLL3/4 chromatin modifier regulates ISC proliferation, colocalizes extensively with Kismet throughout the ISC genome, and co-regulates genes in ISCs, including Cbl, a negative regulator of Epidermal Growth Factor Receptor (EGFR). Loss of kismet or trr leads to elevated levels of EGFR protein and signaling, thereby promoting ISC self-renewal. It is proposed that Kismet with Trr establishes a chromatin state that limits EGFR proliferative signaling, preventing tumor-like stem cell overgrowths.
Ke, Y. T. and Hsu, H. J. (2019). Generation of inducible gene-switched GAL4 expressed in the Drosophila female germline stem cell niche. G3 (Bethesda). PubMed ID: 31018943
Drosophila exhibits a short lifespan with well-characterized ovarian germline stem cells (GSCs) and niche compartments. However, no inducible tools for temporal and spatial control of gene expression in the GSC-niche unit have been previously developed for aging studies. Since GeneSwitch GAL4 is conveniently activated by the steroid RU486 (mifepristone), an enhancer-trap screen was conducted to isolate GeneSwitch GAL4 lines with expression in the GSC-niche unit. Six lines were identified with expression in germarial somatic cells, and two lines (#2305 and #2261) with expression in niche cap cells, the major constituent of the GSC niche. The use of lines #2305 or #2261 to overexpress Drosophila insulin-like peptide 2, which maintains GSC lifespan, in aged niche cap cells significantly delayed age-dependent GSC loss. These results support the notion that insulin signaling is beneficial for maintaining aged stem cells and also validate the utility of the GeneSwitch GAL4 lines for studying stem cell aging.
Otsuki, L. and Brand, A. H. (2019). Dorsal-ventral differences in neural stem cell quiescence are induced by p57(KIP2)/Dacapo. Dev Cell 49(2): 293-300.e293. PubMed ID: 30905769
Quiescent neural stem cells (NSCs) in the adult brain are regenerative cells that could be activated therapeutically to repair damage. It is becoming apparent that quiescent NSCs exhibit heterogeneity in their propensity for activation and in the progeny that they generate. It was discovered recently that NSCs undergo quiescence in either G0 or G2 in the Drosophila brain, challenging the notion that all quiescent stem cells are G0 arrested. G2-quiescent NSCs become activated prior to G0 NSCs. This study shows that the cyclin-dependent kinase inhibitor Dacapo (Dap; ortholog of p57(KIP2)) determines whether NSCs enter G0 or G2 quiescence during embryogenesis. The dorsal patterning factor, Muscle segment homeobox (Msh; ortholog of MSX1/2/3) binds directly to the Dap locus and induces Dap expression in dorsal NSCs, resulting in G0 arrest, while more ventral NSCs undergo G2 quiescence. These results reveal region-specific regulation of stem cell quiescence.

Wednesday, May 29th - Gonads

Chen, D. S., Delbare, S. Y. N., White, S. L., Sitnik, J., Chatterjee, M., DoBell, E., Weiss, O., Clark, A. G. and Wolfner, M. F. (2019). Female genetic contributions to sperm competition in Drosophila melanogaster. Genetics. PubMed ID: 31101677
In many species, sperm can remain viable in the reproductive tract of a female well beyond the typical interval to remating. This creates an opportunity for sperm from different males to compete for oocyte fertilization inside the female's reproductive tract. In Drosophila melanogaster, sperm characteristics and seminal fluid content affect male success in sperm competition. This study functionally tested 26 candidate genes implicated via a GWAS for their contribution to the female's role in sperm competition, measured as changes in the relative success of the first male to mate (P1). Of these 26 candidates, eight genes were identified that affect P1 when knocked down in females, and five of them were shown to do so when knocked down in the female nervous system. In particular, Rim knockdown in sensory pickpocket (ppk)(+) neurons lowered P1, confirming previously published results, and a novel candidate, caup, lowered P1 when knocked down in octopaminergic Tdc2(+) neurons. These results demonstrate that specific neurons in the female's nervous system play a functional role in sperm competition and expand understanding of the genetic, neuronal and mechanistic basis of female responses to multiple matings. It is proposed that these neurons in females are used to sense and integrate signals from courtship or ejaculates, to modulate sperm competition outcome accordingly.
Yu, J., Yan, Y., Luan, X., Qiao, C., Liu, Y., Zhao, D., Xie, B., Zheng, Q., Wang, M., Chen, W., Shen, C., He, Z., Hu, X., Huang, X., Li, H., Shao, Q., Chen, X., Zheng, B. and Fang, J. (2019). Srlp is crucial for the self-renewal and differentiation of germline stem cells via RpL6 signals in Drosophila testes. Cell Death Dis 10(4): 294. PubMed ID: 30931935
Self-renewal and differentiation in germline stem cells (GSCs) are tightly regulated by the stem cell niche and via multiple approaches. In a previous study, the novel GSC regulatory gene Srlp (CG5844) was screened in Drosophila testes. However, the underlying mechanistic links between Srlp and the stem cell niche remain largely undetermined. Using genetic manipulation of the Drosophila model, this study systematically analyzed the function and mechanism of Srlp in vivo and in vitro. In Drosophila, Srlp is an essential gene that regulates the self-renewal and differentiation of GSCs in the testis. In the in vitro assay, Srlp is found to control the proliferation ability and cell death in S2 cells, which is consistent with the phenotype observed in Drosophila testis. Furthermore, results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) reveal that RpL6 binds to Srlp. Srlp also regulates the expression of spliceosome and ribosome subunits and controls spliceosome and ribosome function via RpL6 signals. Collectively, these findings uncover the genetic causes and molecular mechanisms underlying the stem cell niche. This study provides new insights for elucidating the pathogenic mechanism of male sterility and the formation of testicular germ cell tumor.
Chen, K., Chen, S., Xu, J., Yu, Y., Liu, Z., Tan, A. and Huang, Y. (2019). Maelstrom regulates spermatogenesis of the silkworm, Bombyx mori. Insect Biochem Mol Biol 109: 43-51. PubMed ID: 30970276
Spermatogenesis is essential for the reproduction and a very large number of genes participate in this procession. The Maelstrom (Mael) is identified as essential for spermatogenesis in both Drosophila and mouse, though the mechanisms appear to differ. It was initially found that Mael gene is necessary for axis specification of oocytes in Drosophila, and recent studies suggested that Mael participates in the piRNA pathway. In this study, Bombyx mori Mael mutants were obtained by using a binary transgenic CRISPR/Cas9 system, and the function of Mael was analyzed in B. mori, a model lepidopteran insect. The results showed that BmMael is not necessary for piRNA pathway in the ovary of silkworm, whereas it might be essential for transposon elements (TEs) repression in testis. The BmMael mutation resulted in male sterility, and further analysis established that BmMael was essential for spermatogenesis. The spermatogenesis defects occurred in the elongation stage and resulted in nuclei concentration arrest. RNA-seq and qRT-PCR analyses demonstrated that spermatogenesis defects were associated with tight junctions and apoptosis. BmMael was not involved in the silkworm sex determination pathway. These data provide insights into the biological function of BmMael in male spermatogenesis and might be useful for developing novel methods to control lepidopteron pests.
Brantley, S. E. and Fuller, M. T. (2019). Somatic support cells regulate germ cell survival through the Baz/aPKC/Par6 complex. Development 146(8). PubMed ID: 30918053
Local signals and structural support from the surrounding cellular microenvironment play key roles in directing development in both embryonic organs and adult tissues. In Drosophila, male germ cells are intimately associated and co-differentiate with supporting somatic cells. This study shows that the function of the Baz/aPKC/Par6 apical polarity complex in somatic cyst cells is required stage specifically for survival of the germ cells they enclose. Although spermatogonia enclosed by cyst cells in which the function of the Par complex had been knocked down survived and proliferated, newly formed spermatocytes enclosed by cyst cells lacking Par complex proteins died soon after onset of meiotic prophase. Loss of Par complex function resulted in stage-specific overactivation of the Jun-kinase (JNK) pathway in cyst cells. Knocking down expression of JNK pathway components or the GTPase Rab35 in cyst cells lacking Par complex function rescued the survival of neighboring spermatocytes, suggesting that action of the apical polarity complex ensures germ cell survival by preventing JNK pathway activation, and that the mechanism by which cyst cells lacking Par complex function kill neighboring spermatocytes requires intracellular trafficking in somatic cyst cells.
Lu, Y., Yao, Y. and Li, Z. (2019). Ectopic Dpp signaling promotes stem cell competition through EGFR signaling in the Drosophila testis. Sci Rep 9(1): 6118. PubMed ID: 30992503
Stem cell competition could select the fittest stem cells and potentially control tumorigenesis. However, little is known about the underlying molecular mechanisms. This study has found that ectopic Decapentaplegic (Dpp) signal activation by expressing a constitutively active form of Thickveins (Tkv(CA)) in cyst stem cells (CySCs) leads to competition between CySCs and germline stem cells (GSCs) for niche occupancy and GSC loss. GSCs are displaced from the niche and undergo differentiation. Interestingly, it was found that induction of Tkv(CA) results in elevated expression of vein, which further activates Epidermal Growth Factor Receptor (EGFR) signaling in CySCs to promote their proliferation and compete GSCs out of the niche. These findings elucidate the important role of Dpp signaling in regulating stem cell competition and tumorigenesis, which could be shed light on tumorigenesis and cancer treatment in mammals.
Papagiannouli, F., Berry, C. W. and Fuller, M. T. (2019). The Dlg module and Clathrin-mediated endocytosis regulate EGFR signaling and cyst cell-germline coordination in the Drosophila testis. Stem Cell Reports. PubMed ID: 31006632
Tissue homeostasis and repair relies on proper communication of stem cells and their differentiating daughters with the local tissue microenvironment. In the Drosophila male germline adult stem cell lineage, germ cells proliferate and progressively differentiate enclosed in supportive somatic cyst cells, forming a small organoid, the functional unit of differentiation. This study show that cell polarity and vesicle trafficking influence signal transduction in cyst cells, with profound effects on the germ cells they enclose. The data suggest that the cortical components Dlg, Scrib, Lgl and the clathrin-mediated endocytic (CME) machinery downregulate epidermal growth factor receptor (EGFR) signaling. Knockdown of dlg, scrib, lgl, or CME components in cyst cells resulted in germ cell death, similar to increased signal transduction via the EGFR, while lowering EGFR or downstream signaling components rescued the defects. This work provides insights into how cell polarity and endocytosis cooperate to regulate signal transduction and sculpt developing tissues.

Tuesday, May 28th - Adult neural development and function

Yang, W. K., Chueh, Y. R., Cheng, Y. J., Siegenthaler, D., Pielage, J. and Chien, C. T. (2019). Epidermis-derived L1CAM homolog Neuroglian mediates dendrite enclosure and blocks heteroneuronal dendrite bundling. Curr Biol. PubMed ID: 31006568
Building sensory dendritic arbors requires branching, growth, spacing, and substrate support. The conserved L1CAM family of cell-adhesion molecules generates neuronal isoforms to regulate neurite development in various aspects. However, whether non-neuronal isoforms participate in any of these aspects is unclear. In Drosophila, the L1CAM homolog Neuroglian (Nrg) is expressed as two isoforms: the neuronal isoform Nrg180 on dendritic surfaces of dendritic arborization (da) neurons and the non-neuronal isoform Nrg167 in epidermis innervated by dendrites. Epidermal Nrg167 encircles dendrites by interactions with dendritic Nrg180 to support dendrite growth, stabilization, and enclosure inside epidermis. Interestingly, whereas Nrg180 forms homophilic interactions to facilitate axonal bundling, heteroneuronal dendrites in the same innervating field avoid bundling through unknown mechanisms to maintain individual dendritic patterns. This study shows that both epidermal Nrg167 depletion and neuronal Nrg180 overexpression can cause dendrite bundling, with genetic analyses suggesting that Nrg167-Nrg180 interactions antagonize Nrg180-Nrg180 homophilic interaction to prevent dendrite bundling. Furthermore, internalization of Nrg180 also participates in resolving dendrite bundling, as overexpression of endocytosis-defective Nrg180 and compromising endocytosis in neurons both exacerbated dendrite-bundling defects. Together, this study highlights the functional significance of substrate-derived Nrg167 in conferring dendrite stability, positioning, and avoidance.
Batra, S., Corcoran, J., Zhang, D. D., Pal, P., K, P. U., Kulkarni, R., Lofstedt, C., Sowdhamini, R. and Olsson, S. B. (2019). A functional agonist of insect olfactory receptors: Behavior, physiology and structure. Front Cell Neurosci 13: 134. PubMed ID: 31110474
Chemical signaling is ubiquitous and employs a variety of receptor types to detect the cacophony of molecules relevant for each living organism. Insects, the most diverse taxon, have evolved unique olfactory receptors with as little as 10% sequence identity between receptor types. This study has identified a promiscuous volatile, 2-methyltetrahydro-3-furanone (coffee furanone), that elicits chemosensory and behavioral activity across multiple insect orders and receptors. In vivo and in vitro physiology showed that coffee furanone was detected by roughly 80% of the recorded neurons expressing the insect-specific olfactory receptor complex in the antenna of Drosophila melanogaster, at concentrations similar to other known, and less promiscuous, ligands. Neurons expressing specialized receptors, other chemoreceptor types, or mutants lacking the complex entirely did not respond to this compound. This indicates that coffee furanone is a promiscuous ligand for the insect olfactory receptor complex itself and did not induce non-specific cellular responses. In addition, homology modeling and docking studies are presented with selected olfactory receptors that suggest conserved interaction regions for both coffee furanone and known ligands. Apart from its physiological activity, this known food additive elicits a behavioral response for several insects, including mosquitoes, flies, and cockroaches. A broad-scale behaviorally active molecule non-toxic to humans thus has significant implications for health and agriculture. Coffee furanone serves as a unique tool to unlock molecular, physiological, and behavioral relationships across this diverse receptor family and animal taxa.
Weiss, S., Melom, J. E., Ormerod, K. G., Zhang, Y. V. and Littleton, J. T. (2019). Glial Ca(2+) signaling links endocytosis to K(+) buffering around neuronal somas to regulate excitability. Elife 8. PubMed ID: 31025939
Glial-neuronal signaling at synapses is widely studied, but how glia interact with neuronal somas to regulate neuronal function is unclear. Drosophila cortex glia are restricted to brain regions devoid of synapses, providing an opportunity to characterize interactions with neuronal somas. Mutations in the cortex glial NCKX(zydeco) elevate basal Ca(2+), predisposing animals to seizure-like behavior. To determine how cortex glial Ca(2+) signaling controls neuronal excitability, an in-vivo modifier screen of the NCKX(zydeco) seizure phenotype was performed. This study showe that elevation of glial Ca(2+) causes hyperactivation of calcineurin-dependent endocytosis and accumulation of early endosomes. Knockdown of sandman, a K2P channel, recapitulates NCKX(zydeco) seizures. Indeed, sandman expression on cortex glial membranes is substantially reduced in NCKX(zydeco) mutants, indicating enhanced internalization of Sandman predisposes animals to seizures. These data provide an unexpected link between glial Ca(2+) signaling and the well-known role of glia in K(+) buffering as a key mechanism for regulating neuronal excitability.
Zhang, Y., Koe, C. T., Tan, Y. S., Ho, J., Tan, P., Yu, F., Sung, W. K. and Wang, H. (2019). The integrator complex prevents dedifferentiation of intermediate neural progenitors back into neural stem cells. Cell Rep 27(4): 987-996.e983. PubMed ID: 31018143
Mutations of the Integrator subunits are associated with neurodevelopmental disorders and cancers. However, their role during neural development is poorly understood. This study demonstrates that the Drosophila Integrator complex prevents dedifferentiation of intermediate neural progenitors (INPs) during neural stem cell (neuroblast) lineage development. Loss of intS5, intS8, and intS1 generated ectopic type II neuroblasts. INP-specific knockdown of intS8, intS1, and intS2 resulted in the formation of excess type II neuroblasts, indicating that Integrator prevents INP dedifferentiation. Cell-type-specific DamID analysis identified 1413 IntS5-binding sites in INPs, including zinc-finger transcription factor earmuff (erm). Furthermore, erm expression is lost in intS5 and intS8 mutant neuroblast lineages, and intS8 genetically interacts with erm to suppress the formation of ectopic neuroblasts. Taken together, these data demonstrate that the Drosophila Integrator complex plays a critical role in preventing INP dedifferentiation primarily by regulating a key transcription factor Erm that also suppresses INP dedifferentiation.
Zhang, Y., Tsang, T. K., Bushong, E. A., Chu, L. A., Chiang, A. S., Ellisman, M. H., Reingruber, J. and Su, C. Y. (2019). Asymmetric ephaptic inhibition between compartmentalized olfactory receptor neurons. Nat Commun 10(1): 1560. PubMed ID: 30952860
In the Drosophila antennal lobe, different subtypes of olfactory receptor neurons (ORNs) housed in the same sensory hair (sensillum) can inhibit each other non-synaptically. However, the mechanisms underlying this underexplored form of lateral inhibition remain unclear. This study used recordings from pairs of sensilla impaled by the same tungsten electrode to demonstrate that direct electrical ('ephaptic') interactions mediate lateral inhibition between ORNs. Intriguingly, within individual sensilla, it was found that ephaptic lateral inhibition is asymmetric such that one ORN exerts greater influence onto its neighbor. Serial block-face scanning electron microscopy of genetically identified ORNs and circuit modeling indicate that asymmetric lateral inhibition reflects a surprisingly simple mechanism: the physically larger ORN in a pair corresponds to the dominant neuron in ephaptic interactions. Thus, morphometric differences between compartmentalized ORNs account for highly specialized inhibitory interactions that govern information processing at the earliest stages of olfactory coding.
Zhu, S., Chen, R., Soba, P. and Jan, Y. N. (2019). JNK signaling coordinates with ecdysone signaling to promote pruning of Drosophila sensory neuron dendrites. Development 146(8). PubMed ID: 30936183
Developmental pruning of axons and dendrites is crucial for the formation of precise neuronal connections, but the mechanisms underlying developmental pruning are not fully understood. This study has investigated the function of JNK signaling in dendrite pruning using Drosophila class IV dendritic arborization (c4da) neurons as a model. Loss of JNK or its canonical downstream effectors Jun or Fos led to dendrite-pruning defects in c4da neurons. Interestingly, the data show that JNK activity in c4da neurons remains constant from larval to pupal stages but the expression of Fos is specifically activated by ecdysone receptor B1 (EcRB1) at early pupal stages, suggesting that ecdysone signaling provides temporal control of the regulation of dendrite pruning by JNK signaling. Thus, this work not only identifies a novel pathway involved in dendrite pruning and a new downstream target of EcRB1 in c4da neurons, but also reveals that JNK and Ecdysone signaling coordinate to promote dendrite pruning.

Friday, May 24th - Signaling

Irani, S., Sipe, S. N., Yang, W., Burkholder, N. T., Lin, B., Sim, K., Matthews, W. L., Brodbelt, J. S. and Zhang, Y. (2019). Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate CTD phosphatase during eukaryotic transcription. J Biol Chem. PubMed ID: 30971428
The C-terminal domain of RNA polymerase II (CTD) contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser5 (pSer5) is gradually dephosphorylated by CTD phosphatases, while Ser2 phosphorylation is accumulating. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, this study investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer2 and pSer5 CTDs. Ssu72 was found to dephosphorylates pSer5 effectively, but only has low activities toward pSer7 and pSer2. The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight beta-turn for recognition by Ssu72. It was also noted that residues flanking the SP motif, such as the bulky Tyr1 next to Ser2, prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr-1 further prohibited Ssu72 binding to pSer2 and thereby prevented untimely Ser2 dephosphorylation. These results reveal critical roles for Tyr1 in differentiating the phosphorylation states of Ser2/Ser5 of CTD in RNA polymerase II, which occur at different stages of transcription.
Kaur, A., Gourav, Kumar, S., Jaiswal, N., Vashisht, A., Kumar, D., Gahlay, G. K. and Mithu, V. S. (2019). NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3). Proteins. PubMed ID: 30958586
Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the beta2 -loop-alpha region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of (15) N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx(729) DPEEIIVLSDSD(740)), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by beta2 -loop-alpha structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of beta2 -loop-alpha region in dSmt3 is suspected to assist its interaction with Daxx12. These results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.
Yang, S. A., Portilla, J. M., Mihailovic, S., Huang, Y. C. and Deng, W. M. (2019). Oncogenic Notch tiggers neoplastic tumorigenesis in a transition-zone-like tissue microenvironment. Dev Cell. PubMed ID: 30982664
During the initial stages of tumorigenesis, the tissue microenvironment where the pro-tumor cells reside plays a crucial role in determining the fate of these cells. Transition zones, where two types of epithelial cells meet, are high-risk sites for carcinogenesis, but the underlying mechanism remains largely unclear. This study shows that persistent upregulation of Notch signaling induces neoplastic tumorigenesis in a transition zone between the salivary gland imaginal ring cells and the giant cells in Drosophila larvae. In this region, local endogenous JAK-STAT and JNK signaling creates a tissue microenvironment that is susceptible to oncogenic-Notch-induced tumorigenesis, whereas the rest of the salivary gland imaginal ring is refractory to Notch-induced tumor transformation. JNK signaling activates a matrix metalloprotease (MMP1) to promote Notch-induced tumorigenesis at the transition zone. These findings illustrate the significance of local endogenous inflammatory signaling in primary tumor formation.
Steinmetz, E. L., Dewald, D. N., Luxem, N. and Walldorf, U. (2019). Drosophila Homeodomain-interacting protein kinase (Hipk) phosphorylates the homeodomain proteins Homeobrain, Empty spiracles, and Muscle segment homeobox. Int J Mol Sci 20(8). PubMed ID: 31010135
The Drosophila homeodomain-interacting protein kinase (Hipk) is the fly representative of the well-conserved group of HIPKs in vertebrates. It was initially found through its characteristic interactions with homeodomain proteins. Hipk is involved in a variety of important developmental processes, such as the development of the eye or the nervous system. The present study set Hipk and the Drosophila homeodomain proteins Homeobrain (Hbn), Empty spiracles (Ems), and Muscle segment homeobox (Msh) in an enzyme-substrate relationship. These homeoproteins are transcription factors that function during Drosophila neurogenesis and are, at least in part, conserved in vertebrates. A physical interaction is revealed between Hipk and the three homeodomain proteins in vivo using bimolecular fluorescence complementation (BiFC). In the course of in vitro phosphorylation analysis and subsequent mutational analysis several Hipk phosphorylation sites of Hbn, Ems, and Msh were mapped. The phosphorylation of Hbn, Ems, and Msh may provide further insight into the function of Hipk during development of the Drosophila nervous system.
Sun, X., Sun, B., Cui, M. and Zhou, Z. (2019). HERC4 exerts an anti-tumor role through destabilizing the oncoprotein Smo. Biochem Biophys Res Commun. PubMed ID: 31010679
The GPCR-like transmembrane protein Smoothened (Smo) is an indispensable transducer in Hedgehog (Hh) pathway, its hyperactivation leads to several human cancers, including non-small cell lung cancer (NSCLC). The mechanism governing Smo stability still remains elusive. This study performed a modifier screening in Drosophila and found that the E3 ligase dHerc4 degrades dSmo. Depletion of dherc4 increases dSmo protein and activates Hh pathway. In addition, HERC4 is downregulated in NSCLC samples, negative correlating with Smo. HERC4 interacts with Smo reciprocally in NSCLC cells. Finally, knockdown of herc4 was shown to activate Hh pathway and promote NSCLC cell proliferation. Taken together, these studies have demonstrated that HERC4 acts as a tumor suppressor via destabilizing the oncoprotein Smo, and provided HERC4 as a promising therapeutic target for NSCLC treatment.
Lee, B., Barretto, E. C. and Grewal, S. S. (2019). TORC1 modulation in adipose tissue is required for organismal adaptation to hypoxia in Drosophila. Nat Commun 10(1): 1878. PubMed ID: 31015407
Animals often develop in environments where conditions such as food, oxygen and temperature fluctuate. The ability to adapt their metabolism to these fluctuations is important for normal development and viability. In most animals, low oxygen (hypoxia) is deleterious. However some animals can alter their physiology to tolerate hypoxia. This study shows that TORC1 modulation in adipose tissue is required for organismal adaptation to hypoxia in Drosophila. Hypoxia rapidly suppresses TORC1 signaling in Drosophila larvae via TSC-mediated inhibition of Rheb. This hypoxia-mediated inhibition of TORC1 specifically in the larval fat body is essential for viability. Moreover, these effects of TORC1 inhibition on hypoxia tolerance are mediated through remodeling of fat body lipid storage. These studies identify the larval adipose tissue as a key hypoxia-sensing tissue that coordinates whole-body development and survival to changes in environmental oxygen by modulating TORC1 and lipid metabolism.

Thursday, May 23rd - RNA and Transposons

Kofler, R. (2019). Dynamics of transposable element invasions with piRNA clusters. Mol Biol Evol. PubMed ID: 30968135
In mammals and invertebrates the proliferation of an invading transposable element (TE) is thought to be stopped by an insertion into a piRNA cluster. This study explored the dynamics of TE invasions under this trap model using computer simulations. piRNA clusters were found to confer a substantial benefit, effectively preventing extinction of host populations from a proliferation of deleterious TEs. TE invasions consists of three distinct phases: first the TE amplifies within the population, next TE proliferation is stopped by segregating cluster insertions and finally the TE is inactivated by fixation of a cluster insertion. Suppression by segregating cluster insertions is unstable and bursts of TE activity may yet occur. The transposition rate and the population size mostly influence the length of the phases but not the amount of TEs accumulating during an invasion. Solely the size of piRNA clusters was identified as a major factor influencing TE abundance. A single non-recombining cluster is more efficient in stopping invasions than clusters distributed over several chromosomes. Recombination among cluster sites makes it necessary that each diploid carries, on the average, four cluster insertions to stop an invasion. Surprisingly, negative selection in a model with piRNA clusters can lead to a novel equilibrium state, where TE copy numbers remain stable despite only some individuals in a population carrying a cluster insertion. In Drosophila melanogaster the trap model accounts for the abundance of TEs produced in the germline but fails to predict the abundance of TEs produced in the soma.
Liao, S. E., Kandasamy, S. K., Zhu, L. and Fukunaga, R. (2019). DEAD-box RNA helicase Belle post-transcriptionally promotes gene expression in an ATPase activity-dependent manner. RNA. PubMed ID: 30979781
Drosophila Belle (human ortholog DDX3) is a conserved DEAD-box RNA helicase implicated in regulating gene expression. However, the molecular mechanisms by which Belle/DDX3 regulates gene expression are poorly understood. A systematic mutational analysis was performed of Belle to determine the contributions of conserved motifs within Belle to its in vivo function. This study found that Belle RNA-binding and RNA-unwinding activities and intrinsically disordered regions (IDRs) are required for Belle in vivo function. Expression of Belle ATPase mutants that cannot bind, hydrolyze, or release ATP resulted in dominant toxic phenotypes. Mechanistically, Belle was found to upregulate reporter protein level when tethered to reporter mRNA, without corresponding changes at the mRNA level, indicating that Belle promotes translation of mRNA that it binds. Belle ATPase activity and N-terminal IDR were required for this translational promotion activity. It was also found that ectopic ovary expression of dominant Belle ATPase mutants decreases levels of cyclin proteins, including Cyclin B, without corresponding changes in their mRNA levels. Finally, Belle was found to bind endogenous cyclin B mRNA. It is proposed that Belle promotes translation of specific target mRNAs, including cyclin B mRNA, in an ATPase activity-dependent manner.
Lv, M., Yao, Y., Li, F., Xu, L., Yang, L., Gong, Q., Xu, Y. Z., Shi, Y., Fan, Y. J. and Tang, Y. (2019). Structural insights reveal the specific recognition of roX RNA by the dsRNA-binding domains of the RNA helicase MLE and its indispensable role in dosage compensation in Drosophila. Nucleic Acids Res 47(6): 3142-3157. PubMed ID: 30649456
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). This paper reports a 2.90A crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, this research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
Sokolova, O. A., Ilyin, A. A., Poltavets, A. S., Nenasheva, V. V., Mikhaleva, E. A., Shevelyov, Y. Y. and Klenov, M. S. (2019). Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production. Mol Biol Cell: mbcE17100591. PubMed ID: 30943101
In Drosophila ovarian somatic cells, piRNAs against transposable elements are mainly produced from the approximately 180-kb flamenco (flam) locus. flam transcripts are gathered into foci, located close to the nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA FISH, this study found that in the follicle cells of ovaries the 5'-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, while their extended downstream regions mostly overlap with Yb bodies. In flam(KG) mutant ovaries, flam transcripts containing the 1st and, partially, 2nd exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNAs from the protein-coding gene transcripts accumulate at higher levels in flam(KG) ovaries indicating that piRNA biogenesis may occur without Yb bodies. It is proposed that normally in follicle cells, flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which competitively decreases piRNA production from the protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.
Liu, Y., Wang, M., Liu, X., Quan, J., Fang, Y., Liu, Y., Qiu, Y., Yu, Y. and Zhou, X. (2019). Drosophila Trf4-1 involves in mRNA and primary miRNA transcription. Biochem Biophys Res Commun 511(4): 806-812. PubMed ID: 30837153
Drosophila Trf4-1 (DmTrf4-1) is a polyadenylation polymerase or terminal nucleotidyl transferase (PAP/TENT) that has been reported to add poly(A) tails to snRNAs in nucleus or mRNAs in cytoplasm. This study found that the loss of Trf4-1 resulted in the reduction of mRNAs and primary miRNAs (pri-miRNAs) in both Drosophila S2 cells and adult flies. Interestingly, the role of Trf4-1 in transcription is independent of its PAP/TENT activity. Moreover, using the chromatin immunoprecipitation assay, it was found that the loss of Trf4-1 led to abnormal RNA polymerase II accumulation and reduced H3K4me3 binding in promoter regions. Thus, this study indicates a positive role of Trf4-1 in the transcription of mRNAs and pri-miRNAs.
Wessels, H. H., Lebedeva, S., Hirsekorn, A., Wurmus, R., Akalin, A., Mukherjee, N. and Ohler, U. (2019). Global identification of functional microRNA-mRNA interactions in Drosophila. Nat Commun 10(1): 1626. PubMed ID: 30967537
MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. This study generated a transcriptome-wide in vivo map of miRNA-mRNA interactions in Drosophila, making use of single nucleotide resolution in Argonaute1 (AGO1) crosslinking and immunoprecipitation (CLIP) data. Absolute quantification of cellular miRNA levels presents the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two CLIP approaches, a similar predictive potential was found to unambiguously assign thousands of miRNA-mRNA pairs from AGO1 interaction data at unprecedented depth, achieving higher signal-to-noise ratios than with computational methods alone. Quantitative RNA-seq and sub-codon resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. This study provides the first comprehensive resource of miRNA target sites and their functional impact in Drosophila.

Wednesday, May 22nd - Adult Development

George, L. F., Pradhan, S. J., Mitchell, D., Josey, M., Casey, J., Belus, M. T., Fedder, K. N., Dahal, G. R. and Bates, E. A. (2019). Ion Channel Contributions to Wing Development in Drosophila melanogaster. G3 (Bethesda) 9(4): 999-1008. PubMed ID: 30733380
During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. A screen was carried out of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. This study identified 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in the screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of the screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in this screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. These results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development.
Uyehara, C. M. and McKay, D. J. (2019). Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31019084
The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. This study used the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. EcR binding was found to be temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.
Schultheis, D., Schwirz, J. and Frasch, M. (2019). RNAi screen in Tribolium reveals involvement of F-BAR proteins in myoblast fusion and visceral muscle morphogenesis in insects. G3 (Bethesda) 9(4): 1141-1151. PubMed ID: 30733382
In a large-scale RNAi screen in Tribolium castaneum for genes with knock-down phenotypes in the larval somatic musculature, one recurring phenotype was the appearance of larval muscle fibers that were significantly thinner than those in control animals. Several of the genes producing this knock-down phenotype corresponded to orthologs of Drosophila genes that are known to participate in myoblast fusion, particularly via their effects on actin polymerization. A new gene previously not implicated in myoblast fusion but displaying a similar thin-muscle knock-down phenotype was the Tribolium ortholog of Nostrin, which encodes an F-BAR and SH3 domain protein. Genetic studies of Nostrin and Cip4, a gene encoding a structurally related protein, in Drosophila show that the encoded F-BAR proteins jointly contribute to efficient myoblast fusion during larval muscle development. Together with the F-Bar protein Syndapin they are also required for normal embryonic midgut morphogenesis. In addition, Cip4 is required together with Nostrin during the profound remodeling of the midgut visceral musculature during metamorphosis. It is proposed that these F-Bar proteins help govern proper morphogenesis particularly of the longitudinal midgut muscles during metamorphosis.
van der Burg, K. R. L., Lewis, J. J., Martin, A., Nijhout, H. F., Danko, C. G. and Reed, R. D. (2019). Contrasting roles of transcription factors Spineless and EcR in the highly dynamic chromatin landscape of butterfly wing metamorphosis. Cell Rep 27(4): 1027-1038.e1023. PubMed ID: 31018121
Development requires highly coordinated changes in chromatin accessibility in order for proper gene regulation to occur. This study identified factors associated with major, discrete changes in chromatin accessibility during butterfly wing metamorphosis. By combining mRNA sequencing (mRNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and machine learning analysis of motifs, this study shows that distinct sets of transcription factors are predictive of chromatin opening at different developmental stages. The data suggest an important role for nuclear hormone receptors early in metamorphosis, whereas PAS-domain transcription factors are strongly associated with later chromatin opening. Chromatin immunoprecipitation sequencing (ChIP-seq) validation of select candidate factors showed spineless binding to be a major predictor of opening chromatin. Surprisingly, binding of ecdysone receptor (EcR), a candidate accessibility factor in Drosophila, was not predictive of opening but instead marked persistent sites. This work characterizes the chromatin dynamics of insect wing metamorphosis, identifies candidate chromatin remodeling factors in insects, and presents a genome assembly of the model butterfly Junonia coenia.
Powers, N. and Srivastava, A. (2019). JAK/STAT signaling is involved in air sac primordium development of Drosophila melanogaster. FEBS Lett 593(7): 658-669. PubMed ID: 30854626
The dorsal thoracic air sacs in fruit flies (Drosophila melanogaster) are functionally and developmentally comparable to human lungs. The progenitors of these structures, air sac primordia (ASPs), invasively propagate into wing imaginal disks, employing mechanisms similar to those that promote metastasis in malignant tumors. This study investigated whether JAK/STAT signaling plays a role in the directed morphogenesis of ASPs. JAK/STAT signaling occurs in ASP tip cells and misexpression of core components in the JAK/STAT signaling cascade significantly impedes ASP development. Upd2 was further identified as an activating ligand for JAK/STAT activity in the ASP. Together, these data constitute a considerable step forward in understanding the role of JAK/STAT signaling in ASPs and similar structures in mammalian models.
Wang, W., Peng, J., Li, Z., Wang, P., Guo, M., Zhang, T., Qian, W., Xia, Q. and Cheng, D. (2019). Transcription factor E93 regulates wing development by directly promoting Dpp signaling in Drosophila. Biochem Biophys Res Commun 513(1): 280-286. PubMed ID: 30954218
Transcription factor E93 is a steroid hormone ecdysone early response gene and plays crucial roles in both the degradation of larval tissues and the formation of adult organs during insect metamorphosis with the prepupal-pupal-adult transition. Specific knockdown of the E93 gene in the Drosophila wing disrupted wing development. ChIP-PCR analysis and dual-luciferase reporter assay confirmed that E93 can bind to the decapentaplegic (Dpp) promoter and enhanced its activity. Furthermore, the expressions of Dpp and other components in the Dpp signaling pathway were upregulated following E93 overexpression in Drosophila S2 cells but were decreased after E93 knockdown in the wing. Moreover, the impairment of the Dpp signaling pathway phenocopied the defects of E93 knockdown on wing development. Taken together, these results suggest that E93 modulates the Dpp signaling pathway to regulate wing development during Drosophila metamorphosis.

Tuesday, May 21st - Disease Models

Li, J., Suda, K., Ueoka, I., Tanaka, R., Yoshida, H., Okada, Y., Okamoto, Y., Hiramatsu, Y., Takashima, H. and Yamaguchi, M. (2019). Neuron-specific knockdown of Drosophila HADHB induces a shortened lifespan, deficient locomotive ability, abnormal motor neuron terminal morphology and learning disability. Exp Cell Res 379(2): 150-158. PubMed ID: 30953623
Mutations in the HADHB gene induce dysfunctions in the beta-oxidation of fatty acids and result in a Mitochondrial trifunctional protein (MTP) deficiency, which is characterized by clinical heterogeneity, such as cardiomyopathy and recurrent Leigh-like encephalopathy. In contrast, milder forms of HADHB mutations cause the later onset of progressive axonal peripheral neuropathy (approximately 50-80%) and myopathy with or without episodic myoglobinuria. The mechanisms linking neuronal defects in these diseases to the loss of HADHB function currently remain unclear. Drosophila has the CG4581 (dHADHB) gene as a single human HADHB homologue. This study established pan-neuron-specific dHADHB knockdown flies and examined their phenotypes. The knockdown of dHADHB shortened the lifespan of flies, reduced locomotor ability and also limited learning abilities. These phenotypes were accompanied by an abnormal synapse morphology at neuromuscular junctions (NMJ) and reduction in both ATP and ROS levels in central nervous system (CNS). The Drosophila NMJ synapses are glutamatergic that is similar to those in the vertebrate CNS. The present results reveal a critical role for dHADHB in the morphogenesis and function of glutamatergic neurons including peripheral neurons. The dHADHB knockdown flies established herein provide a useful model for investigating the pathological mechanisms underlying neuropathies caused by a HADHB deficiency.
Nixon, K. C. J., Rousseau, J., Stone, M. H., Sarikahya, M., Ehresmann, S., Mizuno, S., Matsumoto, N., Miyake, N., Baralle, D., McKee, S., Izumi, K., Ritter, A. L., Heide, S., Heron, D., Depienne, C., Titheradge, H., Kramer, J. M. and Campeau, P. M. (2019). A Syndromic neurodevelopmental disorder caused by mutations in SMARCD1, a core SWI/SNF subunit needed for context-dependent neuronal gene regulation in flies. Am J Hum Genet 104(4): 596-610. PubMed ID: 30879640
Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause neurodevelopmental disorders (NDDs). This paper reports on five individuals with mutations in SMARCD1; the individuals present with developmental delay, intellectual disability, hypotonia, feeding difficulties, and small hands and feet. Trio exome sequencing proved the mutations to be de novo in four of the five individuals. Mutations in other SWI/SNF components cause Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, or other syndromic and non-syndromic NDDs. Although the individuals presented in this study have dysmorphisms and some clinical overlap with these syndromes, they lack their typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, the Drosophila ortholog Bap60 was investigated in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom-body-specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, this study identified an NDD caused by SMARCD1 mutations and establish a role for the SMARCD1 ortholog Bap60 in the regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development when neuronal circuits that are required for learning and memory are formed.
Goodman, L. D., Prudencio, M., Srinivasan, A. R., Rifai, O. M., Lee, V. M., Petrucelli, L. and Bonini, N. M. (2019). eIF4B and eIF4H mediate GR production from expanded G4C2 in a Drosophila model for C9orf72-associated ALS. Acta Neuropathol Commun 7(1): 62. PubMed ID: 31023341
The discovery of an expanded (GGGGCC)n repeat (termed G4C2) within the first intron of C9orf72 in familial ALS/FTD (Amyotrophic Lateral Sclerosis/Frontotemporal Degeneration) has led to a number of studies showing that the aberrant expression of G4C2 RNA can produce toxic dipeptides through repeat-associated non-AUG (RAN-) translation. To reveal canonical translation factors that impact this process, an unbiased loss-of-function screen was performed in a G4C2 fly model that maintained the upstream intronic sequence of the human gene and contained a GFP tag in the Glycine Arginine (GR) reading frame. 11 of 48 translation factors were identified that impact production of the GR-GFP protein. Further investigations into two of these, eIF4B and eIF4H, revealed that downregulation of these factors reduced toxicity caused by the expression of expanded G4C2 and reduced production of toxic GR dipeptides from G4C2 transcripts. In patient-derived cells and in post-mortem tissue from ALS/FTD patients, eIF4H was found to be downregulated in cases harboring the G4C2 mutation compared to patients lacking the mutation and healthy individuals. Overall, these data define eIF4B and eIF4H as disease modifiers whose activity is important for RAN-translation of the GR peptide from G4C2-transcripts.
Lopez-Gonzalez, R., Yang, D., Pribadi, M., Kim, T. S., Krishnan, G., Choi, S. Y., Lee, S., Coppola, G. and Gao, F. B. (2019). Partial inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS/FTD. Proc Natl Acad Sci U S A. PubMed ID: 31019093
GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, a number of genetic modifiers of poly(GR) toxicity were identified. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G4C2 repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD.
von Stockum, S., Sanchez-Martinez, A., Corra, S., Chakraborty, J., Marchesan, E., Locatello, L., Da Re, C., Cusumano, P., Caicci, F., Ferrari, V., Costa, R., Bubacco, L., Rasotto, M. B., Szabo, I., Whitworth, A. J., Scorrano, L. and Ziviani, E. (2019). Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction. Life Sci Alliance 2(2). PubMed ID: 30988163
Aberrant mitochondrial dynamics disrupts mitochondrial function and contributes to disease conditions. A targeted RNA interference screen for deubiquitinating enzymes (DUBs) affecting protein levels of multifunctional mitochondrial fusion protein Mitofusin (MFN) identified USP8 prominently influencing MFN levels. Genetic and pharmacological inhibition of USP8 normalized the elevated MFN protein levels observed in PINK1 and Parkin-deficient models. This correlated with improved mitochondrial function, locomotor performance and life span, and prevented dopaminergic neurons loss in Drosophila PINK1 KO flies. This study has identified a novel target antagonizing pathologically elevated MFN levels, mitochondrial dysfunction, and dopaminergic neuron loss of a Drosophila model of mitochondrial dysfunction.
Lee, J., Bai, Y., Chembazhi, U. V., Peng, S., Yum, K., Luu, L. M., Hagler, L. D., Serrano, J. F., Chan, H. Y. E., Kalsotra, A. and Zimmerman, S. C. (2019). Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1. Proc Natl Acad Sci U S A. PubMed ID: 30975744
Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. This study reports intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of this multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

Monday, May 20th - Larval and Adult Neural Development and Function

Sturner, T., Tatarnikova, A., Mueller, J., Schaffran, B., Cuntz, H., Zhang, Y., Nemethova, M., Bogdan, S., Small, V. and Tavosanis, G. (2019). Transient localization of the Arp2/3 complex initiates neuronal dendrite branching in vivo. Development 146(7). PubMed ID: 30910826
The formation of neuronal dendrite branches is fundamental for the wiring and function of the nervous system. Indeed, dendrite branching enhances the coverage of the neuron's receptive field and modulates the initial processing of incoming stimuli. Complex dendrite patterns are achieved in vivo through a dynamic process of de novo branch formation, branch extension and retraction. The first step towards branch formation is the generation of a dynamic filopodium-like branchlet. The mechanisms underlying the initiation of dendrite branchlets are therefore crucial to the shaping of dendrites. Through in vivo time-lapse imaging of the subcellular localization of actin during the process of branching of Drosophila larva sensory neurons, combined with genetic analysis and electron tomography, this study has identified the Actin-related protein (Arp) 2/3 complex as the major actin nucleator involved in the initiation of dendrite branchlet formation, under the control of the activator WAVE and of the small GTPase Rac1. Transient recruitment of an Arp2/3 component marks the site of branchlet initiation in vivo. These data position the activation of Arp2/3 as an early hub for the initiation of branchlet formation.
Sun, L., Gao, Y., He, J., Cui, L., Meissner, J., Verbavatz, J. M., Li, B., Feng, X. and Liang, X. (2019). Ultrastructural organization of NompC in the mechanoreceptive organelle of Drosophila campaniform mechanoreceptors. Proc Natl Acad Sci U S A 116(15): 7343-7352. PubMed ID: 30918125
Mechanoreceptive organelles (MOs) are specialized subcellular entities in mechanoreceptors that transform extracellular mechanical stimuli into intracellular signals. Campaniform sensilla detect cuticular strain caused by muscular activities or external stimuli in Drosophila. Each campaniform sensillum has an MO located at the distal tip of its dendrite. This study analyzed the molecular architecture of the MOs in fly campaniform mechanoreceptors using electron microscopic tomography. Focus was placed on the ultrastructural organization of NompC (a force-sensitive channel) that is linked to the array of microtubules in these MOs via membrane-microtubule connectors (MMCs). NompC channels are arranged in a regular pattern, with their number increasing from the distal to the proximal end of the MO. Double-length MMCs in nompC (29+29ARs) confirm the ankyrin-repeat domain of NompC (NompC-AR) as a structural component of MMCs. The unexpected finding of regularly spaced NompC-independent linkers in nompC suggests that MMCs may contain non-NompC components. Localized laser ablation experiments on mechanoreceptor arrays in halteres suggest that MMCs bear tension, providing a possible mechanism for why the MMCs are longer when NompC-AR is duplicated or absent in mutants. Finally, mechanical modeling shows that upon cuticular deformation, sensillar architecture imposes a rotational activating force, with the proximal end of the MO, where more NOMPC channels are located, being subject to larger forces than the distal end.
Song, Y., Li, D., Farrelly, O., Miles, L., Li, F., Kim, S. E., Lo, T. Y., Wang, F., Li, T., Thompson-Peer, K. L., Gong, J., Murthy, S. E., Coste, B., Yakubovich, N., Patapoutian, A., Xiang, Y., Rompolas, P., Jan, L. Y. and Jan, Y. N. (2019). The Mechanosensitive ion channel Piezo inhibits axon regeneration. Neuron 102(2): 373-389. PubMed ID: 30819546
Neurons exhibit a limited ability of repair. Given that mechanical forces affect neuronal outgrowth, it is important to investigate whether mechanosensitive ion channels may regulate axon regeneration. This study shows that DmPiezo, a Ca(2+)-permeable non-selective cation channel, functions as an intrinsic inhibitor for axon regeneration in Drosophila. DmPiezo activation during axon regeneration induces local Ca(2+) transients at the growth cone, leading to activation of nitric oxide synthase and the downstream cGMP kinase Foraging or PKG to restrict axon regrowth. Loss of DmPiezo enhances axon regeneration of sensory neurons in the peripheral and CNS. Conditional knockout of its mammalian homolog Piezo1 in vivo accelerates regeneration, while its pharmacological activation in vitro modestly reduces regeneration, suggesting the role of Piezo in inhibiting regeneration may be evolutionarily conserved. These findings provide a precedent for the involvement of mechanosensitive channels in axon regeneration and add a potential target for modulating nervous system repair.
Miyamoto, T. and Amrein, H. (2019). Neuronal gluconeogenesis regulates systemic glucose homeostasis in Drosophila melanogaster. Curr Biol 29(8): 1263-1272.e1265. PubMed ID: 30930040
Gluconeogenesis is a well-established metabolic process whereby glucose is generated from small carbon molecules in the liver and kidney to maintain blood glucose levels. Expression of gluconeogenic genes has been reported in other organs of mammals and insects, where their function is not yet known. In the fruit fly, one of the gluconeogenic genes, glucose-6-phosphatase (G6P) is exclusively expressed in the CNS. Using a fluorescence resonance energy transfer (FRET)-based glucose sensor, this study shows that a small subset of neurons in the fly brain is capable of carrying out gluconeogenesis. Moreover, G6P mutant flies exhibit low whole-body glucose levels within 24 h of food deprivation. This phenotype can be mimicked by silencing G6P neurons and rescued by experimentally controlled activation in the absence of G6P. These results indicate that neural activity of G6P neurons, but not glucose production per se, is critical for glucose homeostasis. Lastly, it was observed that neuronal gluconeogenesis promotes anterograde neuropeptide distribution from the soma to axon terminals, suggesting that the generation of glucose facilitates neuropeptide transport. Together, this analysis reveals a novel role for gluconeogenesis in neuronal signaling.
Liang, X., Ho, M. C. W., Zhang, Y., Li, Y., Wu, M. N., Holy, T. E. and Taghert, P. H. (2019). Morning and evening circadian pacemakers independently drive premotor centers via a specific dopamine relay. Neuron. PubMed ID: 30981533
Many animals exhibit morning and evening peaks of locomotor behavior. In Drosophila, two corresponding circadian neural oscillators-M (morning) cells and E (evening) cells-exhibit a corresponding morning or evening neural activity peak. Yet little is known of the neural circuitry by which distinct circadian oscillators produce specific outputs to precisely control behavioral episodes. This study shows that ring neurons of the ellipsoid body (EB-RNs) display spontaneous morning and evening neural activity peaks in vivo: these peaks coincide with the bouts of locomotor activity and result from independent activation by M and E pacemakers. Further, M and E cells regulate EB-RNs via identified PPM3 dopaminergic neurons, which project to the EB and are normally co-active with EB-RNs. These in vivo findings establish the fundamental elements of a circadian neuronal output pathway: distinct circadian oscillators independently drive a common pre-motor center through the agency of specific dopaminergic interneurons.
Sales, E. C., Heckman, E. L., Warren, T. L. and Doe, C. Q. (2019). Regulation of subcellular dendritic synapse specificity by axon guidance cues. Elife 8. PubMed ID: 31012844
Neural circuit assembly occurs with subcellular precision, yet the mechanisms underlying this precision remain largely unknown. Subcellular synaptic specificity could be achieved by molecularly distinct subcellular domains that locally regulate synapse formation, or by axon guidance cues restricting access to one of several acceptable targets. These models have been addressed using two Drosophila neurons: the dbd sensory neuron and the A08a interneuron. In wild-type larvae, dbd synapses with the A08a medial dendrite but not the A08a lateral dendrite. dbd-specific overexpression of the guidance receptors Unc-5 or Robo-2 results in lateralization of the dbd axon, which forms anatomical and functional monosynaptic connections with the A08a lateral dendrite. It is concluded that axon guidance cues, not molecularly distinct dendritic arbors, are a major determinant of dbd-A08a subcellular synapse specificity.

Friday, May 17th - Evolution

Whittle, C. A. and Extavour, C. G. (2019). Selection shapes turnover and magnitude of sex-biased expression in Drosophila gonads. BMC Evol Biol 19(1): 60. PubMed ID: 30786879
Sex-biased gene expression is thought to drive the phenotypic differences in males and females in metazoans. Research on sex organs in Drosophila, employing original approaches and multiple-species contrasts, provides a means to gain insights into factors shaping the turnover and magnitude (fold-bias) of sex-biased expression. Using recent RNA-seq data, sex-biased gonadal expression in 10,740 protein coding sequences were examined in four species of Drosophila, D. melanogaster, D. simulans, D. yakuba and D. ananassae (5 to 44 My divergence). Using an approach wherein genes were identified with lineage-specific transitions (LSTs) in sex-biased status (amongst testis-biased, ovary-biased and unbiased; thus, six transition types) standardized to the number of genes with the ancestral state (S-LSTs), and those with clade-wide expression bias status, several key findings were revealed. First, the six categorical types of S-LSTs in sex-bias showed disparate rates of turnover, consistent with differential selection pressures. Second, the turnover in sex-biased status was largely unrelated to cross-tissue expression breadth, suggesting pleiotropy does not restrict evolution of sex-biased expression. Third, the fold-sex-biased expression, for both testis-biased and ovary-biased genes, evolved directionally over time toward higher values, a crucial finding that could be interpreted as a selective advantage of greater sex-bias, and sexual antagonism. Fourth, in terms of protein divergence, genes with LSTs to testis-biased expression exhibited weak signals of elevated rates of evolution (than ovary-biased) in as little as 5 My, which strengthened over time. Moreover, genes with clade-wide testis-specific expression (44 My), a status not observed for any ovary-biased genes, exhibited striking acceleration of protein divergence, which was linked to low pleiotropy. By studying LSTs and clade-wide sex-biased gonadal expression in a multi-species clade of Drosophila, this study describes evidence that interspecies turnover and magnitude of sex-biased expression have been influenced by selection. Further, whilst pleiotropy was not connected to turnover in sex-biased gonadal expression, it likely explains protein sequence divergence.
Howie, J. M., Mazzucco, R., Taus, T., Nolte, V. and Schlotterer, C. (2019). DNA motifs are not general predictors of recombination in two Drosophila sister species. Genome Biol Evol. PubMed ID: 30980655
Meiotic recombination is crucial for chromosomal segregation, and facilitates the spread of beneficial and removal of deleterious mutations. Recombination rates frequently vary along chromosomes and Drosophila melanogaster exhibits a remarkable pattern. Recombination rates gradually decrease towards centromeres and telomeres, with a dramatic impact on levels of variation in natural populations. Two close sister species, D. simulans and D. mauritiana do not only have higher recombination rates, but also exhibit a much more homogeneous recombination rate that only drops sharply very close to centromeres and telomeres. Because certain sequence motifs are associated with recombination rate variation in D. melanogaster, tests were performed to see whether the difference in recombination landscape between D. melanogaster and D. simulans can be explained by the genomic distribution of recombination-rate associated sequence motifs. The first high-resolution recombination map was constructed for D. simulans based on 189 haplotypes from a natural D. simulans population, and short sequence motifs linked with higher than average recombination in both sister species were sought. Five consensus motifs were identified, significantly associated with higher than average chromosome-wide recombination rates in at least one species and present in both. Testing fine resolution associations between motif density and recombination, strong and positive associations were found genome-wide over a range of scales in D. melanogaster, while the results were equivocal in D. simulans. Despite the strong association in D. melanogaster, decreasing density of these short-repeat motifs towards centromeres and telomeres was not found. It is concluded that the density of recombination-associated repeat motifs cannot explain the large-scale recombination landscape in D. melanogaster, nor the differences to D. simulans. The strong association seen for the sequence motifs in D. melanogaster likely reflects their impact influencing local differences in recombination rates along the genome.
Ng'oma, E., Fidelis, W., Middleton, K. M. and King, E. G. (2019). The evolutionary potential of diet-dependent effects on lifespan and fecundity in a multi-parental population of Drosophila melanogaster. Heredity (Edinb) 122(5): 582-594. PubMed ID: 30356225
The nutritional conditions experienced by a population have a major role in shaping trait evolution in many taxa. Constraints exerted by nutrient limitation or nutrient imbalance can influence the maximal value that fitness components such as reproduction and lifespan attains, and organisms may shift how resources are allocated to different structures and functions in response to changes in nutrition. Whether the phenotypic changes associated with changes in nutrition represent an adaptive response is largely unknown. Further, it is unclear whether the response of fitness components to diet even has the potential to evolve in most systems. This study used an admixed multi-parental population of Drosophila melanogaster reared in three different diet conditions to estimate quantitative genetic parameters for lifespan and fecundity. Significant genetic variation for both traits was found in these populations, and lifespan was shown to have moderate to high heritabilities within diets. Genetic correlations for lifespan between diets were significantly less than one, demonstrating a strong genotype by diet interaction. These findings demonstrate substantial standing genetic variation in this population that is comparable to natural populations and highlights the potential for adaptation to changing nutritional environments.
Srivastava, S., Avvaru, A. K., Sowpati, D. T. and Mishra, R. K. (2019). Patterns of microsatellite distribution across eukaryotic genomes. BMC Genomics 20(1): 153. PubMed ID: 30795733
Microsatellites, or Simple Sequence Repeats (SSRs), are short tandem repeats of 1-6 nt motifs present in all genomes. This study has identified ~ 685 million eukaryotic microsatellites and analyzed their genomic trends across 15 taxonomic subgroups from protists to mammals. The distribution of SSRs reveals taxon-specific variations in their exonic, intronic and intergenic densities. The analysis reveals the differences among non-related species and novel patterns uniquely demarcating closely related species. Several repeats common across subgroups are documented as well as rare SSRs that are excluded almost throughout evolution. Species-specific signatures were identified in pathogens like Leishmania as well as in cereal crops, Drosophila, birds and primates. Distinct SSRs preferentially exist as long repeating units in different subgroups; most unicellular organisms show no length preference for any SSR class, while many SSR motifs accumulate as long repeats in complex organisms, especially in mammals. This study presents a comprehensive analysis of SSRs across taxa at an unprecedented scale. The analysis indicates that the SSR composition of organisms with heterogeneous cell types is highly constrained, while simpler organisms such as protists, green algae and fungi show greater diversity in motif abundance, density and GC content. The microsatellite dataset generated in this work provides a large number of candidates for functional analysis and for studying their roles across the evolutionary landscape.
Palavicino-Maggio, C. B., Trannoy, S., Holton, K. M., Song, X., Li, K. and Nevo, E. (2019). Aggression and courtship differences found in Drosophila melanogaster from two different microclimates at Evolution Canyon, Israel. Sci Rep 9(1): 4084. PubMed ID: 30858499
Aggression and courtship behavior were examined of wild Drosophila melanogaster flies isolated from two contrasting microclimates found at Evolution Canyon in Mt. Carmel, Israel: an African-like dry tropical Slope (AS) and a European-like humid temperate Slope (ES), separated by 250 meters. Intraslope aggression between same sex fly pairings collected from the same slope was measured and compared. Both male and female flies displayed similar fighting abilities from both slopes. ES males, however, from the humid biome, showed a tendency to lunge more per aggressive encounter, compared with AS males from the dry biome. Interslope aggression was tested by pairing flies from opposite slopes. ES males displayed higher numbers of lunges, and won more fights against their AS opponents. Enhanced courtship performances were observed in ES compared to AS males. The fighting and courtship superiority seen in ES males could reinforce fitness and pre-mating reproductive isolation mechanisms that underlie incipient sympatric speciation. This may support an evolutionary advantage of adaptively divergent fruit fly aggression phenotypes from different environments.
Guichard, A., Haque, T., Bobik, M., Xu, X. S., Klanseck, C., Kushwah, R. B. S., Berni, M., Kaduskar, B., Gantz, V. M. and Bier, E. (2019). Efficient allelic-drive in Drosophila. Nat Commun 10(1): 1640. PubMed ID: 30967548
Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. This study generalizes this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. Two configurations for allelic-drive were tested: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. A phenomenon referred to as lethal-mosaicism was investigated that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process is referred to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.

Thursday, May 16th - Adult Physiology

Goda, T. and Hamada, F. N. (2019). Drosophila temperature preference rhythms: An innovative model to understand body temperature rhythms. Int J Mol Sci 20(8). PubMed ID: 31018551
Human body temperature increases during wakefulness and decreases during sleep. The body temperature rhythm (BTR) is a robust output of the circadian clock and is fundamental for maintaining homeostasis, such as generating metabolic energy and sleep, as well as entraining peripheral clocks in mammals. However, the mechanisms that regulate BTR are largely unknown. Drosophila are ectotherms, and their body temperatures are close to ambient temperature; therefore, flies select a preferred environmental temperature to set their body temperature. This study identified a novel circadian output, the temperature preference rhythm (TPR), in which the preferred temperature in flies increases during the day and decreases at night. TPR, thereby, produces a daily BTR. Fly TPR shares many features with mammalian BTR. Diuretic hormone 31 receptor (DH31R) was found to mediates Drosophila TPR, and the closest mouse homolog of DH31R, calcitonin receptor (Calcr), is essential for mice BTR. Importantly, both TPR and BTR are regulated in a distinct manner from locomotor activity rhythms, and neither DH31R nor Calcr regulates locomotor activity rhythms. These findings suggest that DH31R/Calcr is an ancient and specific mediator of BTR. Thus, understanding fly TPR will provide fundamental insights into the molecular and neural mechanisms that control BTR in mammals.
Matoo, O. B., Julick, C. R. and Montooth, K. L. (2019). Genetic variation for ontogenetic shifts in metabolism underlies physiological homeostasis in Drosophila. Genetics. PubMed ID: 30975764
This study found that the fundamental scaling relationship between mass and metabolic rate, as well as the oxidative capacity per mitochondria, were found to change significantly across development in the fruit fly Drosophila. However, mitochondrial respiration rate was maintained at similar levels across development. Furthermore, larvae clustered into two types-those that switched to aerobic, mitochondrial ATP production before the second instar and those that relied on anaerobic, glycolytic production of ATP through the second instar. Despite genetic variation for the timing of this metabolic shift, metabolic rate in second-instar larvae was more robust to genetic variation than was the metabolic rate of other instars. Larvae were found with a mitochondrial-nuclear incompatibility that disrupts mitochondrial function had increased aerobic capacity and relied more on anaerobic ATP production throughout development relative to larvae from wild-type strains. By taking advantage of both ways of making ATP, larvae with this mitochondrial-nuclear incompatibility maintained mitochondrial respiratory capacity, but also had higher levels of whole-body reactive oxygen species and decreased mitochondrial membrane potential, potentially as a physiological defense mechanism. Thus, genetic defects in core physiology can be buffered at the organismal level via physiological plasticity and natural populations may harbor genetic variation for distinct metabolic strategies in development that generate similar organismal outcomes.
Morciano, P., Di Giorgio, M. L., Porrazzo, A., Licursi, V., Negri, R., Rong, Y. and Cenci, G. (2019). Depletion of ATP-Citrate Lyase (ATPCL) affects chromosome integrity without altering histone acetylation in Drosophila mitotic cells. Front Physiol 10: 383. PubMed ID: 31019471
The Citrate Lyase (ACL) is the main cytosolic enzyme that converts the citrate exported from mitochondria by the SLC25A1 carrier in Acetyl Coenzyme A (acetyl-CoA) and oxaloacetate. Acetyl-CoA is a high-energy intermediate common to a large number of metabolic processes including protein acetylation reactions. This renders ACL a key regulator of histone acetylation levels and gene expression in diverse organisms including humans. This study found that depletion of ATPCL, the Drosophila ortholog of human ACL, reduced levels of Acetyl CoA but, unlike its human counterpart, does not affect global histone acetylation and gene expression. Nevertheless, reduced ATPCL levels caused evident, although moderate, mitotic chromosome breakage suggesting that this enzyme plays a partial role in chromosome stability. These defects did not increase upon X-ray irradiation, indicating that they are not dependent on an impairment of DNA repair. Interestingly, depletion of ATPCL drastically increased the frequency of chromosome breaks (CBs) associated to mutations in scheggia, which encodes the ortholog of the mitochondrial citrate carrier SLC25A1 that is also required for chromosome integrity and histone acetylation. These results indicate that ATPCL has a dispensable role in histone acetylation and prevents massive chromosome fragmentation when citrate efflux is altered.
Rudisill, S. S., Martin, B. R., Mankowski, K. M. and Tessier, C. R. (2019). Iron deficiency reduces synapse formation in the Drosophila clock circuit. Biol Trace Elem Res 189(1): 241-250. PubMed ID: 30022428
Iron serves as a critical cofactor for proteins involved in a host of biological processes. In most animals, dietary iron is absorbed in enterocytes and then disseminated for use in other tissues in the body. The brain is particularly dependent on iron. Altered iron status correlates with disorders ranging from cognitive dysfunction to disruptions in circadian activity. The exact role iron plays in producing these neurological defects, however, remains unclear. Invertebrates provide an attractive model to study the effects of iron on neuronal development since many of the genes involved in iron metabolism are conserved, and the organisms are amenable to genetic and cytological techniques. This study examined synapse growth specifically under conditions of iron deficiency in the Drosophila circadian clock circuit. Projections of the small ventrolateral clock neurons to the protocerebrum of the adult Drosophila brain are significantly reduced upon chelation of iron from the diet. This growth defect persists even when iron is restored to the diet. Genetic neuronal knockdown of ferritin 1 or ferritin 2, critical components of iron storage and transport, does not affect synapse growth in these cells. Together, these data indicate that dietary iron is necessary for central brain synapse formation in the fly and further validate the use of this model to study the function of iron homeostasis on brain development.
Sharrock, J., Estacio-Gomez, A., Jacobson, J., Kierdorf, K., Southall, T. D. and Dionne, M. S. (2019). fs(1)h controls metabolic and immune function and enhances survival via AKT and FOXO in Drosophila. Dis Model Mech 12(4). PubMed ID: 30910908
The Drosophila fat body is the primary organ of energy storage as well as being responsible for the humoral response to infection. Its physiological function is of critical importance to the survival of the organism; however, many molecular regulators of its function remain ill-defined. This study shows that the Drosophila melanogaster bromodomain-containing protein FS(1)H is required in the fat body for normal lifespan as well as metabolic and immune homeostasis. Flies lacking fat body fs(1)h exhibit short lifespan, increased expression of immune target genes, an inability to metabolize triglyceride, and low basal AKT activity, mostly resulting from systemic defects in insulin signalling. Removal of a single copy of the AKT-responsive transcription factor foxo normalises lifespan, metabolic function, uninduced immune gene expression and AKT activity. It is suggested that the promotion of systemic insulin signalling activity is a key in vivo function of fat body fs(1)h.
Rommelaere, S., Boquete, J. P., Piton, J., Kondo, S. and Lemaitre, B. (2019). The exchangeable apolipoprotein Nplp2 sustains lipid flow and heat acclimation in Drosophila. Cell Rep 27(3): 886-899.e886. PubMed ID: 30995484
In ectotherms, increased ambient temperature requires the organism to consume substantial amounts of energy to sustain a higher metabolic rate, prevent cellular damage, and respond to heat stress. This study identifies a heat-inducible apolipoprotein required for thermal acclimation in Drosophila. Neuropeptide-like precursor 2 (Nplp2) is an abundant hemolymphatic protein thought to be a neuropeptide. In contrast, Nplp2 contributes to lipid transport, functioning as an exchangeable apolipoprotein. More precisely, Nplp2-deficient flies accumulate lipids in their gut, have reduced fat stores, and display a dyslipoproteinemia, showing that Nplp2 is required for dietary lipid assimilation. Importantly, Nplp2 is induced upon thermal stress and contributes to survival upon heat stress. It is proposed that Nplp2 associates with lipoprotein particles under homeostatic and high energy-demand conditions to optimize fat transport and storage. This study also shows that modulation of the lipid uptake and transport machinery is part of an integrated cytoprotective response.

Wednesday, May 15th - Cytoskeleton and Junctions

Metivier, M., Monroy, B. Y., Gallaud, E., Caous, R., Pascal, A., Richard-Parpaillon, L., Guichet, A., Ori-McKenney, K. M. and Giet, R. (2019). Dual control of Kinesin-1 recruitment to microtubules by Ensconsin in Drosophila neuroblasts and oocytes. Development 146(8). PubMed ID: 30936181
Drosophila Ensconsin (also known as MAP7) controls spindle length, centrosome separation in brain neuroblasts (NBs) and asymmetric transport in oocytes. The control of spindle length by Ensconsin is Kinesin-1 independent but centrosome separation and oocyte transport require targeting of Kinesin-1 to microtubules by Ensconsin. However, the molecular mechanism used for this targeting remains unclear. Ensconsin contains a microtubule (MT)-binding domain (MBD) and a Kinesin-binding domain (KBD). Rescue experiments show that only full-length Ensconsin restores the spindle length phenotype. KBD expression rescues ensc centrosome separation defects in NBs, but not the fast oocyte streaming and the localization of Staufen and Gurken. Interestingly, the KBD can stimulate Kinesin-1 targeting to MTs in vivo and in vitro. It is proposed that a KBD and Kinesin-1 complex is a minimal activation module that increases Kinesin-1 affinity for MTs. Addition of the MBD present in full-length Ensconsin allows this process to occur directly on the MT and triggers higher Kinesin-1 targeting. This dual regulation by Ensconsin is essential for optimal Kinesin-1 targeting to MTs in oocytes, but not in NBs, illustrating the importance of adapting Kinesin-1 recruitment to different biological contexts.
Bell, K. M., Kronert, W. A., Huang, A., Bernstein, S. I. and Swank, D. M. (2019). The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production. J Physiol. PubMed ID: 30950055
Hypertrophic cardiomyopathy (HCM) is an inherited disease that causes thickening of the heart's ventricular walls. A generally accepted hypothesis for this phenotype is that myosin heavy chain HCM mutations increase muscle contractility. To test this hypothesis, an HCM myosin mutation, R249Q, was expressed in Drosophila indirect flight muscle (IFM), and myofibril structure, skinned fibre mechanical properties, and flight ability were assessed. Homozygous and heterozygous R249Q fibres showed decreased maximum power generation by 67% and 44%, respectively. Decreases in force and work and slower overall muscle kinetics caused homozygous fibres to produce less power. While heterozygous fibres showed no overall slowing of muscle kinetics, active force and work production dropped by 68% and 47%, respectively, which hindered power production. R249Q myosin slows attachment while speeding up detachment from actin, resulting in less time bound. Decreased IFM power output caused 43% and 33% decreases in Drosophila flight ability and 19% and 6% drops in wing beat frequency for homozygous and heterozygous flies, respectively. Overall, these results do not support the increased contractility hypothesis. Instead, these results suggest the ventricular hypertrophy for human R249Q mutation is a compensatory response to decreases in heart muscle power output.
Rauskolb, C., Cervantes, E., Madere, F. and Irvine, K. D. (2019). Organization and function of tension-dependent complexes at adherens junctions. J Cell Sci. PubMed ID: 30837288
Adherens junctions provide attachments between neighboring epithelial cells, and a physical link to the cytoskeleton, which enables them to sense and transmit forces and to initiate biomechanical signaling. Examination of the Ajuba LIM protein Jub in Drosophila embryos revealed that it is recruited to adherens junctions in tissues experiencing high levels of myosin activity, and that the pattern of Jub recruitment varies depending upon how tension is organized. In cells with high junctional myosin, Jub is recruited to puncta near intercellular vertices, which are distinct from Ena-containing puncta, but can overlap Vinculin-containing puncta. Roles were identified for Jub in modulating tension and cellular organization, which are shared with the cytohesin Steppke, and the cytohesin adapter Stepping stone. Jub and Stepping Stone together recruit Steppke to adherens junctions under tension. These observations establish Jub as a reporter of tension experienced at adherens junctions, and identify distinct types of tension-dependent and tension-independent junctional complexes. They also identify a role for Jub in mediating a feedback loop that modulates the distribution of tension and cellular organization in epithelia.
Das Gupta, P. T. and Narasimha, M. (2019). Cytoskeletal tension and Bazooka tune interface geometry to ensure fusion fidelity and sheet integrity during dorsal closure. Elife 8. PubMed ID: 30995201
Epithelial fusion establishes continuity between the separated flanks of epithelial sheets. Despite its importance in creating resilient barriers, the mechanisms that ensure stable continuity and preserve morphological and molecular symmetry upon fusion remain unclear. Using the segmented embryonic epidermis whose flanks fuse during Drosophila dorsal closure, this study demonstrates that epidermal flanks modulate cell numbers and geometry of their fusing fronts to achieve fusion fidelity. While fusing flanks become more matched for both parameters before fusion, differences persisting at fusion are corrected by modulating fusing front width within each segment to ensure alignment of segment boundaries. This study demonstrated that cell interfaces are remodelled from en-face contacts at fusion to an interlocking arrangement after fusion, and demonstrated that changes in interface length and geometry are dependent on the spatiotemporal regulation of cytoskeletal tension and Bazooka/Par3. This work uncovers genetically constrained and mechanically triggered adaptive mechanisms contributing to fusion fidelity and epithelial continuity.
Putz, S. M. (2019). Mbt/PAK4 together with SRC modulates N-Cadherin adherens junctions in the developing Drosophila eye. Biol Open 8(3). PubMed ID: 30885947
Tissue morphogenesis is accompanied by changes of adherens junctions (AJ). During Drosophila eye development, AJ reorganization includes the formation of isolated N-Cadherin AJ between photoreceptors R3/R4. Little is known about how these N-Cadherin AJ are established and maintained. This study focuses on the kinases Mbt/PAK4 and SRC, both known to alter E-Cadherin AJ across phyla. Drosophila p21-activated kinase Mbt and the non-receptor tyrosine kinases Src64 and Src42 regulate proper N-Cadherin AJ. N-Cadherin AJ elongation depends on SRC kinase activity. Cell culture experiments demonstrate binding of both Drosophila SRC isoforms to N-Cadherin and its subsequent tyrosine phosphorylation. In contrast, Mbt stabilizes but does not bind N-Cadherin in vitro Mbt is required in R3/R4 for zipping the N-Cadherin AJ between these cells, independent of its kinase activity and Cdc42-binding. The mbt phenotype can be reverted by mutations in Src64 and Src42 Because Mbt neither directly binds to SRC proteins nor has a reproducible influence on their kinase activity, the conclusion is that Mbt and SRC signaling converge on N-Cadherin. N-Cadherin AJ formation during eye development requires a proper balance between the promoting effects of Mbt and the inhibiting influences of SRC kinases.
Deneke, V. E., Puliafito, A., Krueger, D., Narla, A. V., De Simone, A., Primo, L., Vergassola, M., De Renzis, S. and Di Talia, S. (2019). Self-organized nuclear positioning synchronizes the cell cycle in Drosophila embryos. Cell. PubMed ID: 30982601
The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. This study shows that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. This study demonstrates that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. It is concluded that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

Tuesday, May 14th - Enhancers and gene regulation

Asma, H. and Halfon, M. S. (2019). Computational enhancer prediction: evaluation and improvements. BMC Bioinformatics 20(1): 174. PubMed ID: 30953451
This paper introduces pCRMeval, a pipeline for in silico evaluation of any enhancer prediction tools that are flexible enough to be applied to the Drosophila melanogaster genome. pCRMeval compares the result of predictions with the extensive existing knowledge of experimentally-validated Drosophila CRMs in order to estimate the precision and relative sensitivity of the prediction method. In the case of supervised prediction methods-when training data composed of validated CRMs are used-pCRMeval can also assess the sensitivity of specific training sets. This analysis demonstrates the utility of pCRMeval through evaluation of the SCRMshaw CRM prediction method and training data. By measuring the impact of different parameters on SCRMshaw performance, as assessed by pCRMeval, a more robust version of SCRMshaw, SCRMshaw_HD, was developed that improves the number of predictions while maintaining sensitivity and specificity. This analysis also demonstrates that SCRMshaw_HD, when applied to increasingly less well-assembled genomes, maintains its strong predictive power with only a minor drop-off in performance. This pCRMeval pipeline provides a general framework for evaluation that can be applied to any CRM prediction method, particularly a supervised method. While this study made use of it primarily to test and improve a particular method for CRM prediction, SCRMshaw, pCRMeval should provide a valuable platform to the research community not only for evaluating individual methods, but also for comparing between competing methods.
Bozek, M., Cortini, R., Storti, A. E., Unnerstall, U., Gaul, U. and Gompel, N. (2019). ATAC-seq reveals regional differences in enhancer accessibility during the establishment of spatial coordinates in the Drosophila blastoderm. Genome Res. PubMed ID: 30962180
Establishment of spatial coordinates during Drosophila embryogenesis relies on differential regulatory activity of axis patterning enhancers. Concentration gradients of activator and repressor transcription factors (TFs) provide positional information to each enhancer, which in turn promotes transcription of a target gene in a specific spatial pattern. However, the interplay between an enhancer regulatory activity and its accessibility as determined by local chromatin organization is not well understood. Chromatin accessibility was profiled with ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) in narrow, genetically tagged domains along the antero-posterior axis in the Drosophila blastoderm. One-quarter of the accessible genome displays significant regional variation in its ATAC-seq signal immediately after zygotic genome activation. Axis patterning enhancers are enriched among the most variable intervals, and their accessibility changes correlate with their regulatory activity. In an embryonic domain where an enhancer receives a net activating TF input and promotes transcription, it displays elevated accessibility in comparison to a domain where it receives a net repressive input. It is proposed that differential accessibility is a signature of patterning cis-regulatory elements in the Drosophila blastoderm, and potential mechanisms are discussed by which accessibility of enhancers may be modulated by activator and repressor TFs.
Shah, K., Cao, W. and Ellison, C. E. (2019). Adenine methylation in Drosophila is associated with the tissue-specific expression of developmental and regulatory genes. G3 (Bethesda). PubMed ID: 30988038 N6-methyladenine (6mA or m6dA) is a DNA modification that has long been known to play an important role in a variety of biological functions in prokaryotes. This modification has only recently been described in eukaryotes, where it seems to have evolved species-specific functions ranging from nucleosome positioning to transposon repression. In Drosophila, 6mA has been shown to be important for enforcing the tissue specificity of neuronal genes in the brain and suppressing transposable element expression in the ovaries. This study analyzes the raw signal data from nanopore sequencing to identify 6mA positions in the D. melanogaster genome at single-base resolution. This modification is enriched upstream from transcription start sites, within the introns and 3' UTRs of genes, as well as in simple repeats. These 6mA positions are enriched for sequence motifs that are recognized by known transcriptional activators involved in development, such as Bicoid and Caudal, and the genes that carry this modification are enriched for functions involved in development, regulation of transcription, and neuronal activity. These genes show high expression specificity in a variety of tissues besides the brain, suggesting that this modification may play a more general role in enforcing the specificity of gene expression across many tissues, throughout development, and between the sexes. Neves, A. and Eisenman, R. N. (2019). Distinct gene-selective roles for a network of core promoter factors in Drosophila neural stem cell identity. Biol Open 8(4). PubMed ID: 30948355
The transcriptional mechanisms that allow neural stem cells (NSC) to balance self-renewal with differentiation are not well understood. Employing an in vivo RNAi screen this study identified NSC-TAFs, a subset of nine TATA-binding protein associated factors (TAFs), as NSC identity genes in Drosophila. Depletion of NSC-TAFs results in decreased NSC clone size, reduced proliferation, defective cell polarity and increased hypersensitivity to cell cycle perturbation, without affecting NSC survival. Integrated gene expression and genomic binding analyses revealed that NSC-TAFs function with both TBP and TRF2, and that NSC-TAF-TBP and NSC-TAF-TRF2 shared target genes encode different subsets of transcription factors and RNA-binding proteins with established or emerging roles in NSC identity and brain development. Taken together, these results demonstrate that core promoter factors are selectively required for NSC identity in vivo by promoting cell cycle progression and NSC cell polarity. Because pathogenic variants in a subset of TAFs have all been linked to human neurological disorders, this work may stimulate and inform future animal models of TAF-linked neurological disorders.

Shokri, L., Inukai, S., Hafner, A., Weinand, K., Hens, K., Vedenko, A., Gisselbrecht, S. S., Dainese, R., Bischof, J., Furger, E., Feuz, J. D., Basler, K., Deplancke, B. and Bulyk, M. L. (2019). A comprehensive Drosophila melanogaster transcription factor interactome. Cell Rep 27(3): 955-970. PubMed ID: 30995488
Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, 1,983 protein-protein interactions (PPIs) were identified, more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, a subset of these interactions were verified using MITOMI and bimolecular fluorescence complementation assays. This interactome was combined with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. This analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. The utility of this Drosophila interactome in shedding light on human TF-TF interactions was demonstrated. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.
Shao, W., Alcantara, S. G. and Zeitlinger, J. (2019). Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing. Elife 8. PubMed ID: 31021316
RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. This study overcame this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, the pausing stability is shown to be influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing.

Monday, May 13th - Chromatin

Moreno-Moreno, O., Torras-Llort, M. and Azorin, F. (2019). The E3-ligases SCFPpa and APC/CCdh1 co-operate to regulate CENP-ACID expression across the cell cycle. Nucleic Acids Res 47(7): 3395-3406. PubMed ID: 30753559
Centromere identity is determined by the specific deposition of CENP-A, a histone H3 variant localizing exclusively at centromeres. Increased CENP-A expression, which is a frequent event in cancer, causes mislocalization, ectopic kinetochore assembly and genomic instability. Proteolysis regulates CENP-A expression and prevents its misincorporation across chromatin. How proteolysis restricts CENP-A localization to centromeres is not well understood. This study reports that, in Drosophila, CENP-ACID expression levels are regulated throughout the cell cycle by the combined action of SCFPpa and APC/CCdh1. SCFPpa regulates CENP-ACID expression in G1 and, importantly, in S-phase preventing its promiscuous incorporation across chromatin during replication. In G1, CENP-ACID expression is also regulated by APC/CCdh1. Cal1, the specific chaperone that deposits CENP-ACID at centromeres, protects CENP-ACID from SCFPpa-mediated degradation but not from APC/CCdh1-mediated degradation. These results suggest that, whereas SCFPpa targets the fraction of CENP-ACID that is not in complex with Cal1, APC/CCdh1 mediates also degradation of the Cal1-CENP-ACID complex and, thus, likely contributes to the regulation of centromeric CENP-ACID deposition
Baral, S. S. and DiMario, P. J. (2019). The Nopp140 gene in Drosophila melanogaster displays length polymorphisms in its large repetitive second exon. Mol Genet Genomics. PubMed ID: 31006039
Nopp140, often called the nucleolar and Cajal body phosphoprotein (NOLC1), is an evolutionarily conserved chaperone for the transcription and processing of rRNA during ribosome subunit assembly. Metazoan Nopp140 contains an amino terminal LisH dimerization domain and a highly conserved carboxyl domain. A large central domain consists of alternating basic and acidic motifs of low sequence complexity. Orthologous versions of Nopp140 contain variable numbers of repeating basic-acidic units. While vertebrate Nopp140 genes use multiple exons to encode the central domain, the Nopp140 gene in Drosophila uses exclusively exon 2 to encode the central domain. This study defined three overlapping repeat sequence patterns (P, P', and P'') within the central domain of D. melanogaster Nopp140. These repeat patterns are poorly conserved in other Drosophila species. A length polymorphism in exon 2 is described that pertains specifically to the P' pattern in D. melanogaster. The pattern displays either two or three 96 base pair repeats, respectively, referred to as Nopp140-Short and Nopp140-Long. Fly lines homozygous for one or the other allele, or heterozygous for both alleles, show no discernible phenotypes. PCR characterization of the long and short alleles shows a poorly defined, artifactual bias toward amplifying the long allele over the short allele. The significance of this polymorphism will be in discerning the largely unknown properties of Nopp140's large central domain in rDNA transcription and ribosome biogenesis.
Nilangekar, K., Murmu, N., Sahu, G. and Shravage, B. V. (2019). Generation and characterization of germline-specific autophagy and mitochondrial reactive oxygen species reporters in Drosophila. Front Cell Dev Biol 7: 47. PubMed ID: 31001531
Oogenesis is a fundamental process that forms the egg and, is crucial for the transmission of genetic information to the next generation. Drosophila oogenesis has been used extensively as a genetically tractable model to study organogenesis, niche-germline stem cell communication, and more recently reproductive aging including germline stem cell (GSC) aging. Autophagy, a lysosome-mediated degradation process, is implicated in gametogenesis and aging. However, there is a lack of genetic tools to study autophagy in the context of gametogenesis and GSC aging. This study describes the generation of three transgenic lines mcherry-Atg8a, GFP-Ref(2)P and mito-roGFP2-Orp1 (an H2O2 sensor) that are specifically expressed in the germline compartment including GSCs during Drosophila oogenesis. These transgenes are expressed from the nanos promoter and present a better alternative to UASp mediated overexpression of transgenes. These fluorescent reporters can be used to monitor and quantify autophagy, and the production of reactive oxygen species during oogenesis. These reporters provide a valuable tool that can be utilized in designing genetic screens to identify novel regulators of autophagy and redox homeostasis during oogenesis.
Arya, R., Gyonjyan, S., Harding, K., Sarkissian, T., Li, Y., Zhou, L. and White, K. (2019). A Cut/cohesin axis alters the chromatin landscape to facilitate neuroblast death. Development. PubMed ID: 30952666
Precise control of cell death in the nervous system is essential for development. Spatial and temporal factors activate the death of Drosophila neural stem cells (neuroblasts) by controlling the transcription of multiple cell death genes through a shared enhancer. The activity of this enhancer is controlled by abdominalA and Notch, but additional inputs are needed for proper specificity. This study shows that the Cut DNA binding protein is required for neuroblast death, regulating reaper and grim downstream of the shared enhancer and of abdominalA expression. cut loss accelerates the temporal progression of neuroblasts from a state of low overall levels of H3K27me3 to a higher H3K27me3 state. This is reflected in an increase in H3K27me3 modifications in the cell death gene locus in the CNS on cut knockdown. This study also shows that cut regulates the expression of the cohesin subunit Stromalin. Stromalin and the cohesin regulatory subunit NippedB are required for neuroblast death, and knockdown of Stromalin increases H3K27me3 levels in neuroblasts. Thus, Cut and cohesin regulate apoptosis in the developing nervous system by altering the chromatin landscape.
Cardozo Gizzi, A. M., Cattoni, D. I., Fiche, J. B., Espinola, S. M., Gurgo, J., Messina, O., Houbron, C., Ogiyama, Y., Papadopoulos, G. L., Cavalli, G., Lagha, M. and Nollmann, M. (2019). Microscopy-based chromosome conformation capture enables simultaneous visualization of genome organization and transcription in intact organisms. Mol Cell 74(1): 212-222.e215. PubMed ID: 30795893
Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. This paper presents multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed unveiling of the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.
Chathoth, K. T. and Zabet, N. R. (2019). Chromatin architecture reorganization during neuronal cell differentiation in Drosophila genome. Genome Res 29(4): 613-625. PubMed ID: 30709849
The organization of the genome into topologically associating domains (TADs) was shown to have a regulatory role in development and cellular function, but the mechanism involved in TAD establishment is still unclear. This study presents the first high-resolution contact map of Drosophila neuronal cells (BG3) and identifies different classes of TADs by comparing this to genome organization in embryonic cells (Kc167). Only some TADs are conserved in both cell lines, whereas the rest are cell-type-specific. This is supported by a change in the enrichment of architectural proteins at TAD borders, with BEAF-32 present in embryonic cells and CTCF in neuronal cells. Furthermore, strong divergent transcription was observed, together with RNA Polymerase II occupancy and an increase in DNA accessibility at the TAD borders. TAD borders that are specific to neuronal cells are enriched in enhancers controlled by neuronal-specific transcription factors. These results suggest that TADs are dynamic across developmental stages and reflect the interplay between insulators, transcriptional states, and enhancer activities.

Friday, May 10th - Adult CNS development and function

Lee, J., Iyengar, A. and Wu, C. F. (2019). Distinctions among electroconvulsion- and proconvulsant-induced seizure discharges and native motor patterns during flight and grooming: quantitative spike pattern analysis in Drosophila flight muscles. J Neurogenet: 1-18. PubMed ID: 30982417
In Drosophila, high-frequency electrical stimulation across the brain triggers a highly stereotypic repertoire of spasms. These electroconvulsive seizures (ECS) manifest as distinctive spiking discharges across the nervous system and can be stably assessed throughout the seizure repertoire in the large indirect flight muscles dorsal longitudinal muscles (DLMs) to characterize modifications in seizure-prone mutants. However, the relationships between ECS-spike patterns and native motor programs, including flight and grooming, are not known and their similarities and distinctions remain to be characterized. This study employed quantitative spike pattern analyses for the three motor patterns including: (1) overall firing frequency, (2) spike timing between contralateral fibers, and (3) short-term variability in spike interval regularity (CV2) and instantaneous firing frequency (ISI(-1)). This base-line information from wild-type (WT) flies facilitated quantitative characterization of mutational effects of major neurotransmitter systems: excitatory cholinergic (ChAT), inhibitory GABAergic (Rdl) and electrical (ShakB) synaptic transmission. The results provide an initial glimpse on the vulnerability of individual motor patterns to different perturbations. Marked alterationsof ECS discharge spike patterns were found in terms of either seizure threshold, spike frequency or spiking regularity. In contrast, no gross alterations during grooming and a small but noticeable reduction of firing frequency during Rdl mutant flight were found, suggesting a role for GABAergic modulation of flight motor programs. Picrotoxin (PTX), a known pro-convulsant that inhibits GABAA receptors, induced DLM spike patterns that displayed some features, e.g. left-right coordination and ISI(-1) range, that could be found in flight or grooming, but distinct from ECS discharges. These quantitative techniques may be employed to reveal overlooked relationships among aberrant motor patterns as well as their links to native motor programs.
Copf, T., Kamara, M. and Venkatesh, T. (2019). Axon length maintenance and synapse integrity are regulated by c-AMP-dependent protein kinase A (PKA) during larval growth of the Drosophila sensory neurons. J Neurogenet: 1-7. PubMed ID: 30955404
Axonal extension and synaptic targeting are usually completed during early development, but the axonal length and synaptic integrity need to be actively maintained during later developmental stages and the adult life. Failure in the axonal length maintenance and the subsequent axonal degeneration have been associated with neurological disorders, but currently little is known about the genetic factors controlling this process. This study has shown that regulated intracellular levels of cAMP-dependent protein kinase A (PKA) are critical for the axon maintenance during the transition from the early to the later larval stages in the Drosophila class IV dendritic arborization (da) sensory neurons. The data indicate that when the intracellular levels of PKA are increased via genetic manipulations, these peripheral neurons initially form synapses with wild-type appearance, at their predicted ventral nerve cord (VNC) target sites (in the first and second instar larval stages), but that their synapses disintegrate, and the axons retract and become fragmented in the subsequent larval stages (third larval stage). The affected axonal endings at the disintegrated synaptic sites still express the characteristic presynaptic and cytoskeletal markers such as Bruchpilot and Fascin, indicating that the synapse had been initially properly formed, but that it later lost its integrity. Finally, the phenotype is significantly more prominent in the axons of the neurons whose cell bodies are located in the posterior body segments. It is proposed that the reason for this is the fact that during the larval development the posterior neurons face a much greater challenge while trying to keep up with the fast-paced growth of the larval body, and that PKA is critical for this process. These data reveal PKA as a novel factor in the axonal length and synapse integrity maintenance in sensory neurons. These results could be of help in understanding neurological disorders characterized by destabilized synapses.
Chen, R., Prael, F., Li, Z., Delpire, E., Weaver, C. D. and Swale, D. (2019). Functional Coupling of K+-Cl- Cotransporter (KCC) to GABA-Gated Cl- Channels in the Central Nervous System of Drosophila melanogaster leads to altered drug sensitivities. ACS Chem Neurosci. PubMed ID: 30942574
GABAergic signaling is the cornerstone for fast synaptic inhibition of neural signaling in arthropods and mammals and is the molecular target for insecticides and pharmaceuticals, respectively. The K+-Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular Cl- concentration, yet the fundamental physiology, comparative physiology, and toxicological relevance of insect KCC is understudied. Considering this, electrophysiological, genetic, and pharmacological methods were employed to characterize the physiological underpinnings of KCC function to the Drosophila CNS. The data show that genetic ablation or pharmacological inhibition of KCC results in an increased spike discharge frequency and significantly (P<0.05) reduces the CNS sensitivity to gamma-aminobutyric acid (GABA). Further, simultaneous inhibition of KCC and ligand-gated chloride channel (LGCC) complex results in a significant (P<0.001) increase in CNS spontaneous activity over baseline firing rates that, taken together, supports functional coupling of KCC to LGCC function. Interestingly, 75% reduction in KCC mRNA did not alter basal neurotransmission levels indicating that only a fraction of the KCC population is required to maintain the Cl- ionic gradient when at rest, but prolonged synaptic activity increases the threshold for GABA-mediated inhibition and reduces nerve sensitivity to GABA. These data expand current knowledge regarding the physiological role of KCC in a model insect and provides the necessary foundation to develop KCC as a novel biochemical target of insecticides as well as complements existing research to provide a holistic understanding of the plasticity in mammalian health and disease.
Bulthuis, N., Spontak, K. R., Kleeman, B. and Cavanaugh, D. J. (2019). Neuronal activity in non-LNv clock cells is required to produce free-running rest:activity rhythms in Drosophila. J Biol Rhythms: 748730419841468. PubMed ID: 30994046
Circadian rhythms in behavior and physiology are produced by central brain clock neurons that can be divided into subpopulations based on molecular and functional characteristics. It has become clear that coherent behavioral rhythms result from the coordinated action of these clock neuron populations, but many questions remain regarding the organizational logic of the clock network. This study used targeted genetic tools in Drosophila to eliminate either molecular clock function or neuronal activity in discrete clock neuron subsets. Neuronal firing was found to be necessary across multiple clock cell populations to produce free-running rhythms of rest and activity. In contrast, such rhythms are much more subtly affected by molecular clock suppression in the same cells. These findings demonstrate that network connectivity can compensate for a lack of molecular oscillations within subsets of clock cells. It was further shown that small ventrolateral (sLNv) clock neurons, which have been characterized as master pacemakers under free-running conditions, cannot drive rhythms independent of communication between other cells of the clock network. In particular, an essential contribution of the dorsolateral (LNd) clock neurons was pinpointed and manipulations that affect LNd function were shown to reduce circadian rhythm strength without affecting molecular cycling in sLNv cells. These results suggest a hierarchical organization in which circadian information is first consolidated among one or more clock cell populations before accessing output pathways that control locomotor activity.
Jantrapirom, S., Cao, D. S., Wang, J. W., Hing, H., Tabone, C. J., Lantz, K., de Belle, J. S., Qiu, Y. T., Smid, H. M., Yamaguchi, M., Fradkin, L. G., Noordermeer, J. N. and Potikanond, S. (2019). Dystrophin is required for normal synaptic gain in the Drosophila olfactory circuit. Brain Res 1712: 158-166. PubMed ID: 30711401
The Drosophila olfactory system provides an excellent model to elucidate the neural circuits that control behaviors elicited by environmental stimuli. Despite significant progress in defining olfactory circuit components and their connectivity, little is known about the mechanisms that transfer the information from the primary antennal olfactory receptor neurons to the higher order brain centers. This study shows that the Dystrophin Dp186 isoform is required in the olfactory system circuit for olfactory functions. Using two-photon calcium imaging, this study found the reduction of calcium influx in olfactory receptor neurons (ORNs) and also the defect of GABAA mediated inhibitory input in the projection neurons (PNs) in Dp186 mutation. Moreover, the Dp186 mutant flies which display a decreased odor avoidance behavior were rescued by Dp186 restoration in the Drosophila olfactory neurons in either the presynaptic ORNs or the postsynaptic PNs. Therefore, these results revealed a role for Dystrophin, Dp 186 isoform in gain control of the olfactory synapse via the modulation of excitatory and inhibitory synaptic inputs to olfactory projection neurons.
Augustin, H., Zylbertal, A. and Partridge, L. (2019). A computational model of the escape response latency in the giant fiber system of Drosophila melanogaster. eNeuro 6(2). PubMed ID: 31001574
The giant fiber system (GFS) is a multi-component neuronal pathway mediating rapid escape response in the adult fruit-fly Drosophila melanogaster, usually in the face of a threatening visual stimulus. Two branches of the circuit promote the response by stimulating an escape jump followed by flight initiation. A recent work demonstrated an age-associated decline in the speed of signal propagation through the circuit, measured as the stimulus-to-muscle depolarization response latency. The decline is likely due to the diminishing number of inter-neuronal gap junctions in the GFS of ageing flies. This work presents a realistic conductance-based, computational model of the GFS that recapitulates the experimental results and identifies some of the critical anatomical and physiological components governing the circuit's response latency. According to this model, anatomical properties of the GFS neurons have a stronger impact on the transmission than neuronal membrane conductance densities. The model provides testable predictions for the effect of experimental interventions on the circuit's performance in young and ageing flies.

Thursday, May 9th - Immune Response

Chowdhury, M., Zhang, J., Xu, X. X., He, Z., Lu, Y., Liu, X. S., Wang, Y. F. and Yu, X. Q. (2019). An in vitro study of NF-kappaB factors cooperatively in regulation of Drosophila melanogaster antimicrobial peptide genes. Dev Comp Immunol 95: 50-58. PubMed ID: 30735676
An important innate immune response in Drosophila melanogaster is the production of antimicrobial peptides (AMPs). Expression of AMP genes is mediated by the Toll and immune deficiency (IMD) pathways via NF-kappaB transcription factors Dorsal, DIF and Relish. Dorsal and DIF act downstream of the Toll pathway, whereas Relish acts in the IMD pathway. Dorsal and DIF are held inactive in the cytoplasm by the IkappaB protein Cactus, while Relish contains an IkappaB-like inhibitory domain at the C-terminus. NF-kappaB factors normally form homodimers and heterodimers to regulate gene expression, but formation of heterodimers between Relish and DIF or Dorsal and the specificity and activity of the three NF-kappaB homodimers and heterodimers are not well understood. This study compared the activity of Rel homology domains (RHDs) of Dorsal, DIF and Relish in activation of Drosophila AMP gene promoters, demonstrated that Relish-RHD (Rel-RHD) interacted with both Dorsal-RHD and DIF-RHD, Relish-N interacted with DIF and Dorsal, and overexpression of individual RHD and co-expression of any two RHDs activated the activity of AMP gene promoters to various levels, suggesting formation of homodimers and heterodimers among Dorsal, DIF and Relish. Rel-RHD homodimers were stronger activators than heterodimers of Rel-RHD with either DIF-RHD or Dorsal-RHD, while DIF-RHD-Dorsal-RHD heterodimers were stronger activators than either DIF-RHD or Dorsal-RHD homodimers in activation of AMP gene promoters. The nucleotides at the 6th and 8th positions of the 3' half-sites of the kappaB motifs were identified that are important for the specificity and activity of NF-kappaB transcription factors.
Aalto, A. L., Mohan, A. K., Schwintzer, L., Kupka, S., Kietz, C., Walczak, H., Broemer, M. and Meinander, A. (2019). M1-linked ubiquitination by LUBEL is required for inflammatory responses to oral infection in Drosophila. Cell Death Differ 26(5): 860-876. PubMed ID: 30026495
Post-translational modifications such as ubiquitination play a key role in regulation of inflammatory nuclear factor-kappaB (NF-kappaB) signalling. The Drosophila IkappaB kinase gamma (IKKgamma) Kenny is a central regulator of the Drosophila Imd pathway responsible for activation of the NF-kappaB Relish. This study found that Drosophila E3 ligase and HOIL-1L interacting protein (HOIP) orthologue linear ubiquitin E3 ligase (LUBEL) catalyses formation of M1-linked linear ubiquitin (M1-Ub) chains in flies in a signal-dependent manner upon bacterial infection. Upon activation of the Imd pathway, LUBEL modifies Kenny with M1-Ub chains. Interestingly, the LUBEL-mediated M1-Ub chains seem to be targeted both directly to Kenny and to K63-linked ubiquitin chains conjugated to Kenny by DIAP2. This suggests that DIAP2 and LUBEL work together to promote Kenny-mediated activation of Relish. LUBEL-mediated M1-Ub chain formation is required for flies to survive oral infection with Gram-negative bacteria, for activation of Relish-mediated expression of antimicrobial peptide genes and for pathogen clearance during oral infection. Interestingly, LUBEL is not required for mounting an immune response against systemic infection, as Relish-mediated antimicrobial peptide genes can be expressed in the absence of LUBEL during septic injury. Finally, transgenic induction of LUBEL-mediated M1-Ub drives expression of antimicrobial peptide genes and hyperplasia in the midgut in the absence of infection. This suggests that M1-Ub chains are important for Imd signalling and immune responses in the intestinal epithelia, and that enhanced M1-Ub chain formation is able to drive chronic intestinal inflammation in flies.
Wen, Y., He, Z., Xu, T., Jiao, Y., Liu, X., Wang, Y. F. and Yu, X. Q. (2019). Ingestion of killed bacteria activates antimicrobial peptide genes in Drosophila melanogaster and protects flies from septic infection. Dev Comp Immunol 95: 10-18. PubMed ID: 30731096
Drosophila melanogaster possesses a sophisticated and effective immune system composed of humoral and cellular immune responses, and production of antimicrobial peptides (AMPs) is an important defense mechanism. Expression of AMPs is regulated by the Toll and IMD (immune deficiency) pathways. Production of AMPs can be systemic in the fat body or a local event in the midgut and epithelium. So far, most studies focus on systemic septic infection in adult flies and little is known about AMP gene activation after ingestion of killed bacteria. This study investigated activation of AMP genes in the wild-type w(1118), MyD88 and Imd mutant flies after ingestion of heat-killed Escherichia coli and Staphylococcus aureus. Ingestion of E. coli activated most AMP genes, including drosomycin and diptericin, in the first to third instar larvae and pupae, while ingestion of S. aureus induced only some AMP genes in some larval stages or in pupae. In adult flies, ingestion of killed bacteria activated AMP genes differently in males and females. Interestingly, ingestion of killed E. coli and S. aureus in females conferred resistance to septic infection by both live pathogenic Enterococcus faecalis and Pseudomonas aeruginosa, and ingestion of E. coli in males conferred resistance to P. aeruginosa infection. These results indicated that E. coli and S. aureus can activate both the Toll and IMD pathways, and systemic and local immune responses work together to provide Drosophila more effective protection against infection.
Istas, O., Greenhalgh, A. and Cooper, R. (2019). The effects of a bacterial endotoxin on behavior and sensory-CNS-Motor circuits in Drosophila melanogaster. Insects 10(4). PubMed ID: 31013568
The effect of bacterial sepsis on animal behavior and physiology is complex due to direct and indirect actions. The most common form of bacterial sepsis in humans is from gram-negative bacterial strains. The endotoxin (lipopolysaccharide, LPS) and/or associated peptidoglycans from the bacteria are the key agents to induce an immune response, which then produces a cascade of immunological consequences. However, there are direct actions of LPS and associated peptidoglycans on cells which are commonly overlooked. This study showed behavioral and neural changes in larval Drosophila fed commercially obtained LPS from Serratia marcescens. Locomotor behavior was not altered, but feeding behavior increased and responses to sensory tactile stimuli were decreased. In driving a sensory-central nervous system (CNS)-motor neural circuit in in-situ preparations, direct application of commercially obtained LPS initially increased evoked activity and then decreased and even stopped evoked responses in a dose-dependent manner. With acute LPS and associated peptidoglycans exposure (10 min), the depressed neural responses recovered within a few minutes after removal of LPS. Commercially obtained LPS induces a transitory hyperpolarization of the body wall muscles within seconds of exposure and alters activity within the CNS circuit. Thus, LPS and/or associated peptidoglycans have direct effects on body wall muscle without a secondary immune response.
Chen, K., Luan, X., Liu, Q., Wang, J., Chang, X., Snijders, A. M., Mao, J. H., Secombe, J., Dan, Z., Chen, J. H., Wang, Z., Dong, X., Qiu, C., Chang, X., Zhang, D., Celniker, S. E. and Liu, X. (2019). Drosophila histone demethylase KDM5 regulates social behavior through immune control and gut microbiota maintenance. Cell Host Microbe 25(4): 537-552.e538. PubMed ID: 30902578
Loss-of-function mutations in the histone demethylases KDM5A, KDM5B, or KDM5C are found in intellectual disability (ID) and autism spectrum disorders (ASD) patients. This study used the model organism Drosophila melanogaster to delineate how KDM5 contributes to ID and ASD. Reducing KDM5 causes intestinal barrier dysfunction and changes in social behavior that correlates with compositional changes in the gut microbiota. Therapeutic alteration of the dysbiotic microbiota through antibiotic administration or feeding with a probiotic Lactobacillus strain partially rescues the behavioral, lifespan, and cellular phenotypes observed in kdm5-deficient flies. Mechanistically, KDM5 was found to transcriptionally regulate component genes of the immune deficiency (IMD) signaling pathway and subsequent maintenance of host-commensal bacteria homeostasis in a demethylase-dependent manner. Together, this study uses a genetic approach to dissect the role of KDM5 in the gut-microbiome-brain axis and suggests that modifying the gut microbiome may provide therapeutic benefits for ID and ASD patients.
Kumada, K., Fuse, N., Tamura, T., Okamori, C. and Kurata, S. (2019). HSP70/DNAJA3 chaperone/cochaperone regulates NF-kappaB activity in immune responses. Biochem Biophys Res Commun. PubMed ID: 31005254
Nuclear factor kappa B (NF-kappaB) controls the transcription of various genes in response to immune stimuli. A previous study revealed that the Droj2/DNAJA3 cochaperone contributes to the NF-kappaB pathway in Drosophila and humans. In general, the cochaperone is associated with the 70-kDa heat shock protein (HSP70) chaperone and the complex supports the folding of diverse target proteins. The cochaperone/chaperone functions in the NF-kappaB pathway, however, are not clearly understood. This study reports that HSP70 proteins are involved in activating canonical NF-kappaB signaling during immune responses. In human cultured cells, HSP70 inhibitor destabilized the IKKbeta/IkappaBalpha/NF-kappaB p65 complex and dampened the phosphorylation of NF-kappaB p65 in response to flagellin stimulation. HSPA1A and HSPA8 were identified as the HSP70 family proteins that physically interact with DNAJA3, and established their requirement for the phosphorylation of NF-kappaB p65. Furthermore, as in flies with knockdown of Droj2, flies with knockdown of Hsc70-4, a Drosophila homolog of HSPA8, were more susceptible to infection. These results suggest that the chaperone/cochaperone complex regulates NF-kappaB immune signaling in an evolutionarily conserved manner.

Wednesday, May 8th - Behavior

Kepchia, D., Xu, P., Terryn, R., Castro, A., Schurer, S. C., Leal, W. S. and Luetje, C. W. (2019). Use of machine learning to identify novel, behaviorally active antagonists of the insect odorant receptor co-receptor (Orco) subunit. Sci Rep 9(1): 4055. PubMed ID: 30858563
Olfaction is a key component of the multimodal approach used by mosquitoes to target and feed on humans, spreading various diseases. Current repellents have drawbacks, necessitating development of more effective agents. In addition to variable odorant specificity subunits, all insect odorant receptors (ORs) contain a conserved Odorant receptor co-receptor (Orco) subunit which is an attractive target for repellent development. Orco directed antagonists allosterically inhibit odorant activation of ORs and previous work has shown that an airborne Orco antagonist could inhibit insect olfactory behavior. This study identified novel, volatile Orco antagonists. 83 structurally diverse compounds were functionally screened against Orco from Anopheles gambiae. Results were used for training machine learning models to rank probable activity of a library of 1280 odorant molecules. Functional testing of a representative subset of predicted active compounds revealed enrichment for Orco antagonists, many structurally distinct from previously known Orco antagonists. Novel Orco antagonist 2-tert-butyl-6-methylphenol (BMP) inhibited odorant responses in electroantennogram and single sensillum recordings in adult Drosophila melanogaster and inhibited OR-mediated olfactory behavior in D. melanogaster larvae. Structure-activity analysis of BMP analogs identified compounds with improved potency. These results provide a new approach to the discovery of behaviorally active Orco antagonists for eventual use as insect repellents/confusants.
Agrawal, P., Chung, P., Heberlein, U. and Kent, C. (2019). Enabling cell-type-specific behavioral epigenetics in Drosophila: a modified high-yield INTACT method reveals the impact of social environment on the epigenetic landscape in dopaminergic neurons. BMC Biol 17(1): 30. PubMed ID: 30967153
Epigenetic mechanisms play fundamental roles in brain function and behavior and stressors such as social isolation can alter animal behavior via epigenetic mechanisms. However, due to cellular heterogeneity, identifying cell-type-specific epigenetic changes in the brain is challenging. This study reports the first use of a modified isolation of nuclei tagged in specific cell type (INTACT) method in behavioral epigenetics of Drosophila melanogaster, a method called mini-INTACT. Using ChIP-seq on mini-INTACT purified dopaminergic nuclei, epigenetic signatures were identified in socially isolated and socially enriched Drosophila males. Social experience altered the epigenetic landscape in clusters of genes involved in transcription and neural function. Some of these alterations could be predicted by expression changes of four transcription factors (Hr38, sr, CrebA, and Cbt) and the prevalence of their binding sites in several clusters. These transcription factors were previously identified as activity-regulated genes, and their knockdown in dopaminergic neurons reduced the effects of social experience on sleep. This work work enables the use of Drosophila as a model for cell-type-specific behavioral epigenetics and establishes that social environment shifts the epigenetic landscape in dopaminergic neurons. Four activity-related transcription factors are required in dopaminergic neurons for the effects of social environment on sleep.
Struk, A. A., Mugon, J., Huston, A., Scholer, A. A., Stadler, G., Higgins, E. T., Sokolowski, M. B. and Danckert, J. (2019). Self-regulation and the foraging gene (PRKG1) in humans. Proc Natl Acad Sci U S A. PubMed ID: 30782798
Foraging is a goal-directed behavior that balances the need to explore the environment for resources with the need to exploit those resources. In Drosophila melanogaster, distinct phenotypes have been observed in relation to the foraging (for) gene, labeled the rover and sitter. Adult rovers explore their environs more extensively than do adult sitters. This study explored whether this distinction would be conserved in humans. A distinction was used from regulatory mode theory between those who "get on with it," so-called locomotors, and those who prefer to ensure they "do the right thing," so-called assessors. In this logic, rovers and locomotors share similarities in goal pursuit, as do sitters and assessors. Genetic variation in PRKG1, the human ortholog of for, is associated with preferential adoption of a specific regulatory mode. Next, participants performed a foraging task to see whether genetic differences associated with distinct regulatory modes would be associated with distinct goal pursuit patterns. Assessors tended to hug the boundary of the foraging environment, much like behaviors seen in Drosophila adult sitters. In a patchy foraging environment, assessors adopted more cautious search strategies maximizing exploitation. These results show that distinct patterns of goal pursuit are associated with particular genotypes of PRKG1, the human ortholog of for.
Churgin, M. A., Szuperak, M., Davis, K. C., Raizen, D. M., Fang-Yen, C. and Kayser, M. S. (2019). Quantitative imaging of sleep behavior in Caenorhabditis elegans and larval Drosophila melanogaster. Nat Protoc. PubMed ID: 30953041
Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, this study developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. This paper describes the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. Approaches are described for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, it is possible to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 microm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals.
Gong, C., Ouyang, Z., Zhao, W., Wang, J., Li, K., Zhou, P., Zhao, T., Zheng, N. and Gong, Z. (2019). A neuronal pathway that commands deceleration in Drosophila larval light-avoidance. Neurosci Bull. PubMed ID: 30810958
When facing a sudden danger or aversive condition while engaged in on-going forward motion, animals transiently slow down and make a turn to escape. The neural mechanisms underlying stimulation-induced deceleration in avoidance behavior are largely unknown. This study reports that in Drosophila larvae, light-induced deceleration was commanded by a continuous neural pathway that included prothoracicotropic hormone neurons, eclosion hormone neurons, and tyrosine decarboxylase 2 motor neurons (the PET pathway). Inhibiting neurons in the PET pathway led to defects in light-avoidance due to insufficient deceleration and head casting. On the other hand, activation of PET pathway neurons specifically caused immediate deceleration in larval locomotion. These findings reveal a neural substrate for the emergent deceleration response and provide a new understanding of the relationship between behavioral modules in animal avoidance responses.
Kudow, N., Kamikouchi, A. and Tanimura, T. (2019). Softness sensing and learning in Drosophila larvae. J Exp Biol. PubMed ID: 30833462
Mechanosensation provides animals with important sensory information in addition to olfaction and gustation during feeding behavior. This study used Drosophila larvae to investigate the role of softness sensing in behavior and learning. In the natural environment, Drosophila larvae need to dig into soft foods for feeding. Finding foods that are soft enough to dig into is likely to be essential for their survival. This study reports that Drosophila larvae can discriminate between different agar concentrations and prefer softer agar. Interestingly, it was shown that larvae on a harder place search for a softer place using memory associated with an odor and that they evaluate foods by balancing softness and sweetness. These findings suggest that Drosophila larvae integrate mechanosensory information with chemosensory input while foraging. Moreover, it was found that the larval preference for softness is affected by genetic background.

Tuesday, May 7th - RNA and Transposons

Erwin, A. A. and Blumenstiel, J. P. (2019). Aging in the Drosophila ovary: contrasting changes in the expression of the piRNA machinery and mitochondria but no global release of transposable elements. BMC Genomics 20(1): 305. PubMed ID: 31014230
Evolutionary theory indicates that the dynamics of aging in the soma and reproductive tissues may be distinct. Using mRNA sequencing data from late-stage egg chambers in Drosophila melanogaster, this study characterized the landscape of altered gene and transposable element expression in aged reproductive tissues. This allowed a test of the hypothesis that reproductive tissues may differ from somatic tissues in their response to aging. This study shows that age-related expression changes in late-stage egg chambers tend to occur in genes residing in heterochromatin, particularly on the largely heterochromatic 4th chromosome. However, these expression differences are seen as both decreases and increases during aging, inconsistent with a general loss of heterochromatic silencing. This study also identified an increase in expression of the piRNA machinery, suggesting an age-related increased investment in the maintenance of genome stability. A strong age-related reduction in the expression of mitochondrial transcripts was identified. However, no evidence was foud for global TE derepression in reproductive tissues. Rather, the observed effects of aging on TEs are primarily strain and family specific. These results identify unique responses in somatic versus reproductive tissue with regards to aging. As in somatic tissues, female reproductive tissues show reduced expression of mitochondrial genes. In contrast, the piRNA machinery shows increased expression during aging. Overall, these results also indicate that global loss of TE control observed in other studies may be unique to the soma and sensitive to genetic background and TE family.
Radion, E., Sokolova, O., Ryazansky, S., Komarov, P. A., Abramov, Y. and Kalmykova, A. (2019). The integrity of piRNA clusters is abolished by insulators in the Drosophila germline. Genes (Basel) 10(3). PubMed ID: 30862119
Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, the effects were studied of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. Transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.
Hanyu-Nakamura, K., Matsuda, K., Cohen, S. M. and Nakamura, A. (2019). Pgc suppresses the zygotically acting RNA decay pathway to protect germ plasm RNAs in the Drosophila embryo. Development 146(7). PubMed ID: 30890569
Specification of germ cells is pivotal to ensure continuation of animal species. In many animal embryos, germ cell specification depends on maternally supplied determinants in the germ plasm. Drosophila polar granule component (pgc) mRNA is a component of the germ plasm. pgc encodes a small protein that is transiently expressed in newly formed pole cells, the germline progenitors, where it globally represses mRNA transcription. pgc is also required for pole cell survival, but the mechanism linking transcriptional repression to pole cell survival remains elusive. This study reports that pole cells lacking pgc show premature loss of germ plasm mRNAs, including the germ cell survival factor nanos, and undergo apoptosis. pgc- pole cells misexpress multiple miRNA genes. Reduction of miRNA pathway activity in pgc- embryos partially suppressed germ plasm mRNA degradation and pole cell death, suggesting that Pgc represses zygotic miRNA transcription in pole cells to protect germ plasm mRNAs. Interestingly, germ plasm mRNAs are protected from miRNA-mediated degradation in vertebrates, albeit by a different mechanism. Thus, independently evolved mechanisms are used to silence miRNAs during germ cell specification.
Zheng, Z. Z., Sun, X., Zhang, B., Pu, J., Jiang, Z. Y., Li, M., Fan, Y. J. and Xu, Y. Z. (2019). Alternative splicing regulation of doublesex gene by RNA-binding proteins in the silkworm Bombyx mori. RNA Biol: 1-12. PubMed ID: 30836863
Doublesex is highly conserved and sex-specifically spliced in insect sex-determination pathways, and its alternative splicing (AS) is regulated by Transformer, an exonic splicing activator, in the model system of Drosophila melanogaster. However, due to the lack of a transformer gene, AS regulation of doublesex remains unclear in Lepidoptera, which contain the economically important silkworm Bombyx mori and thousands of agricultural pests. This study used yeast three-hybrid system to screen for RNA-binding proteins that recognize sex-specific exons 3 and 4 of silkworm doublesex (Bm-dsx); this approach identified BxRBP1/Lark binding to the exon 3, and BxRBP2/TBPH and BxRBP3/Aret binding to the exon 4. Investigation of tissues shows that BxRBP1 and BxRBP2 have no sex specificity, but BxRBP3 has - three of its four isoforms are expressed with a sex-bias. Using novel sex-specific silkworm cell lines, this study found that BxRBP1 and BxRBP3 directly interact with each other, and cooperatively function as splicing repressors. Over-expression of BxRBP1 and BxRBP3 isoforms efficiently inhibits splicing of the exons 3 and 4 in the female-specific cells and generates the male-specific isoform of Bm-dsx. It was also demonstrated that the sex-determination upstream gene Masc regulates alternatively transcribed BxRBP3 isoforms. Thus, this study identified a new regulatory mechanism of doublesex AS in the silkworm, revealing an evolutionary divergence in insect sex-determination.
Liu, Y., Wang, M., Liu, X., Quan, J., Fang, Y., Liu, Y., Qiu, Y., Yu, Y. and Zhou, X. (2019). Drosophila Trf4-1 involves in mRNA and primary miRNA transcription. Biochem Biophys Res Commun. PubMed ID: 30837153
Drosophila Trf4-1 (DmTrf4-1) is a polyadenylation polymerase or terminal nucleotidyl transferase (PAP/TENT) that has been reported to add poly(A) tails to snRNAs in nucleus or mRNAs in cytoplasm. This study found that the loss of Trf4-1 resulted in the reduction of mRNAs and primary miRNAs (pri-miRNAs) in both Drosophila S2 cells and adult flies. Interestingly, the role of Trf4-1 in transcription is independent of its PAP/TENT activity. Moreover, using the chromatin immunoprecipitation assay, the loss of Trf4-1 was found to lead to abnormal RNA polymerase II accumulation and reduced H3K4me3 binding in promoter regions. Thus, this study indicates a positive role of Trf4-1 in the transcription of mRNAs and pri-miRNAs.
Heber, S., Gaspar, I., Tants, J. N., Gunther, J., Moya, S. M. F., Janowski, R., Ephrussi, A., Sattler, M. and Niessing, D. (2019). Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains. Nat Commun 10(1): 1659. PubMed ID: 30971701
Throughout metazoans, Staufen (Stau) proteins are core factors of mRNA localization particles. Staufen proteins consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2)-like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that only dsRBDs 3-4 are necessary and sufficient for mRNA binding. This study shows that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient, all four dsRBDs recognize their target RNAs with high stability. Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization. In contrast, a rescue with mStau2 bearing RNA-binding mutations in dsRBD1-2 fails, confirming the physiological relevance of these findings. In summary, these data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau-family proteins of different species.

Monday, May 6th - Disease Models

Das, S., Kumar, P., Verma, A., Maiti, T. K. and Mathew, S. J. (2019). Myosin heavy chain mutations that cause Freeman-Sheldon syndrome lead to muscle structural and functional defects in Drosophila. Dev Biol 449(2): 90-98. PubMed ID: 30826400
Missense mutations in the MYH3 gene encoding myosin heavy chain-embryonic (MyHC-embryonic) have been reported to cause two skeletal muscle contracture syndromes, Freeman Sheldon Syndrome (FSS) and Sheldon Hall Syndrome (SHS). Two residues in MyHC-embryonic that are most frequently mutated, leading to FSS, R672 and T178, are evolutionarily conserved across myosin heavy chains in vertebrates and Drosophila. This study generated transgenic Drosophila expressing myosin heavy chain (Mhc) transgenes with the FSS mutations and characterized the effect of their expression on Drosophila muscle structure and function. The results indicate that expressing these mutant Mhc transgenes lead to structural abnormalities in the muscle, which increase in severity with age and muscle use. Flies expressing the FSS mutant Mhc transgenes in the muscle exhibit shortening of the inter-Z disc distance of sarcomeres, reduction in the Z-disc width, aberrant deposition of Z-disc proteins, and muscle fiber splitting. The ATPase activity of the three FSS mutant MHC proteins are reduced compared to wild type MHC, with the most severe reduction observed in the T178I mutation. Structurally, the FSS mutations occur close to the ATP binding pocket, disrupting the ATPase activity of the protein. Functionally, expression of the FSS mutant Mhc transgenes in muscle lead to significantly reduced climbing capability in adult flies. Thus, these findings indicate that the FSS contracture syndrome mutations lead to muscle structural defects and functional deficits in Drosophila, possibly mediated by the reduced ATPase activity of the mutant MHC proteins.
Solovev, I., Shegoleva, E., Fedintsev, A., Shaposhnikov, M. and Moskalev, A. (2019). Circadian clock genes' overexpression in Drosophila alters diet impact on lifespan. Biogerontology 20(2): 159-170. PubMed ID: 30470951
Diet restriction is one of the most accurately confirmed interventions which extend lifespan. Genes coding circadian core clock elements are known to be the key controllers of cell metabolism especially in aging aspect. The molecular mechanisms standing behind the phenomenon of diet-restriction-mediated life extension are connected to circadian clock either. This study investigated the effects of protein-rich and low-protein diets on lifespan observed in fruit flies overexpressing core clock genes (cry, per, Clk, cyc and tim). The majority of core clock genes being upregulated in peripheral tissues (muscles and fat body) on protein-rich diet significantly decrease the lifespan of male fruit flies from 5 to 61%. Nevertheless, positive increments of median lifespan were observed in both sexes, males overexpressing cry in fat body lived 20% longer on poor diet. Overexpression of per also on poor medium resulted in life extension in female fruit flies. Diet restriction reduces mortality caused by overexpression of core clock genes. Cox-regression model revealed that diet restriction seriously decreases mortality risks of flies which overexpress core clock genes. The hazard ratios are lower for flies overexpressing clock genes in fat body relatively to muscle-specific overexpression. The present work suggests a phenomenological view of how two peripheral circadian oscillators modify effects of rich and poor diets on lifespan and hazard ratios.
Wang, L., Davis, S. S., Borch Jensen, M., Rodriguez-Fernandez, I. A., Apaydin, C., Juhasz, G., Gibson, B. W., Schilling, B., Ramanathan, A., Ghaemmaghami, S. and Jasper, H. (2019). JNK modifies neuronal metabolism to promote proteostasis and longevity. Aging Cell: e12849. PubMed ID: 30810280
Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. This question was addressed by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. These animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. This study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants.
Senturk, M., Lin, G., Zuo, Z., Mao, D., Watson, E., Mikos, A. G. and Bellen, H. J. (2019). Ubiquilins regulate autophagic flux through mTOR signalling and lysosomal acidification. Nat Cell Biol 21(3): 384-396. PubMed ID: 30804504
Although the aetiology of amyotrophic lateral sclerosis (ALS) remains poorly understood, impaired proteostasis is a common feature of different forms of ALS. Mutations in genes encoding ubiquilins, UBQLN2 and UBQLN4, cause familial ALS. The role of ubiquilins in proteasomal degradation is well established, but their role in autophagy-lysosomal clearance is poorly defined. This study describes a crosstalk between endoplasmic reticulum stress, mTOR signalling and autophagic flux in Drosophila and mammalian cells lacking ubiquilins. Loss of ubiquilins leads to endoplasmic reticulum stress, impairs mTORC1 activity, promotes autophagy and causes the demise of neurons. Ubiquilin mutants were shown to display defective autophagic flux due to reduced lysosome acidification. Ubiquilins are required to maintain proper levels of the V0a/V100 subunit of the vacuolar H(+)-ATPase and lysosomal pH. Feeding flies acidic nanoparticles alleviates defective autophagic flux in ubiquilin mutants. Hence, these studies reveal a conserved role for ubiquilins as regulators of autophagy by controlling vacuolar H(+)-ATPase activity and mTOR signalling.
Solovev, I., Dobrovolskaya, E., Shaposhnikov, M., Sheptyakov, M. and Moskalev, A. (2019). Neuron-specific overexpression of core clock genes improves stress-resistance and extends lifespan of Drosophila melanogaster. Exp Gerontol 117: 61-71. PubMed ID: 30415070
Gene expression is much altered in aging. This study observed age-dependent decline of core clock genes' expression in the whole body of the fruit fly. It was hypothesized that inducible overexpression of clock genes (cry, per, tim, cyc and Clk) in the nervous system can improve healthspan of D. melanogaster. The lifespan of transgenic Drosophila was studied; life extension was shown for cry, per, cyc and tim genes. There were significant positive changes in the stress-resistance of flies overexpressing core clock genes in conditions of hyperthermia, hyperoxia, starvation and persistent lighting. The overexpression of per and cry restore circadian rhythms of locomotor activity. The results presented support the hypotheses that the compensation of circadian oscillator genes expression can improve the healthspan in Drosophila melanogaster.
Bernardoni, R., et al. (2019). A new BCR-ABL1 Drosophila model as a powerful tool to elucidate the pathogenesis and progression of chronic myeloid leukemia. Haematologica 104(4): 717-728. PubMed ID: 30409797
Chronic myeloid leukemia relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. This paper describes and validates a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. This study shows that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. This Drosophila model promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors.

Friday, May 3rd - Adult Physiology

Everman, E. R., McNeil, C. L., Hackett, J. L., Bain, C. L. and Macdonald, S. J. (2019). Dissection of complex, fitness-related traits in multiple Drosophila mapping populations offers insight into the genetic control of stress resistance. Genetics 211(4): 1449-1467. PubMed ID: 30760490
This study leveraged two complementary Drosophila melanogaster mapping panels to genetically dissect starvation resistance-an important fitness trait. Using >1600 genotypes from the multiparental Drosophila Synthetic Population Resource (DSPR), numerous starvation stress QTL were mapped that collectively explain a substantial fraction of trait heritability. Mapped QTL effects allowed estimation of DSPR founder phenotypes, predictions that were correlated with the actual phenotypes of these lines. A modest phenotypic correlation was observed between starvation resistance and triglyceride level, traits that have been linked in previous studies. However, overlap among QTL identified for each trait is low. Since it was also shown that DSPR strains with extreme starvation phenotypes differ in desiccation resistance and activity level, the data imply multiple physiological mechanisms contribute to starvation variability. The Drosophila Genetic Reference Panel (DGRP) was additionally exploited to identify sequence variants associated with starvation resistance. Consistent with prior work these sites rarely fall within QTL intervals mapped in the DSPR. This study offered a unique opportunity to directly compare association mapping results across laboratories since two other groups previously measured starvation resistance in the DGRP. Strong phenotypic correlations were found among studies, but extremely low overlap was found in the sets of genomewide significant sites. Despite this, the analyses revealed that the most highly associated variants from each study typically showed the same additive effect sign in independent studies, in contrast to otherwise equivalent sets of random variants. This consistency provides evidence for reproducible trait-associated sites in a widely used mapping panel, and highlights the polygenic nature of starvation resistance.
Rivera, O., McHan, L., Konadu, B., Patel, S., Sint Jago, S. and Talbert, M. E. (2019). A high-fat diet impacts memory and gene expression of the head in mated female Drosophila melanogaster. J Comp Physiol B 189(2): 179-198. PubMed ID: 30810797
Obesity predisposes humans to a range of life-threatening comorbidities, including type 2 diabetes and cardiovascular disease. Obesity also aggravates neural pathologies, such as Alzheimer's disease, but this class of comorbidity is less understood. When Drosophila melanogaster (flies) are exposed to high-fat diet (HFD) by supplementing a standard medium with coconut oil, they adopt an obese phenotype of decreased lifespan, increased triglyceride storage, and hindered climbing ability. The latter development has been previously regarded as a potential indicator of neurological decline in fly models of neurodegenerative disease. The objective of this study was to establish the obesity phenotype in Drosophila and identify a potential correlation, if any, between obesity and neurological decline through behavioral assays and gene expression microarray. Mated female w(1118) flies exposed to HFD were found to maintain an obese phenotype throughout adult life starting at 7 days, evidenced by increased triglyceride stores, diminished life span, and impeded climbing ability. While climbing ability worsened cumulatively between 7 and 14 days of exposure to HFD, there was no corresponding alteration in triglyceride content. Microarray analysis of the mated female w(1118) fly head revealed HFD-induced changes in expression of genes with functions in memory, metabolism, olfaction, mitosis, cell signaling, and motor function. Meanwhile, an Aversive Phototaxis Suppression assay in mated female flies indicated reduced ability to recall an entrained memory 6 h after training. Overall, these results support the suitability of mated female flies for examining connections between diet-induced obesity and nervous or neurobehavioral pathology, and provide many directions for further investigation.
Stobdan, T., Sahoo, D., Azad, P., Hartley, I., Heinrichsen, E., Zhou, D. and Haddad, G. G. (2019). High fat diet induces sex-specific differential gene expression in Drosophila melanogaster. PLoS One 14(3): e0213474. PubMed ID: 30861021
Currently about 2 billion adults globally are estimated to be overweight and ~13% of them are obese. High fat diet (HFD) is one of the major contributing factor to obesity, heart disease, diabetes and cancer. Recent findings on the role of HFD in inducing abnormalities in neurocognition and susceptibility to Alzheimer's disease are highly intriguing. Since fundamental molecular pathways are often conserved across species, studies involving Drosophila melanogaster as a model organism can provide insight into the molecular mechanisms involving human disease. In order to study some of such mechanisms in the central nervous system as well in the rest of the body, this study investigated the effect of HFD on the transcriptome in the heads and bodies of male and female flies kept on either HFD or regular diet (RD). Using comprehensive genomic analyses which include high-throughput transcriptome sequencing, pathway enrichment and gene network analyses, this study found that HFD induces a number of responses that are sexually dimorphic in nature. There was a robust transcriptional response consisting of a downregulation of stress-related genes in the heads and glycoside hydrolase activity genes in the bodies of males. In the females, the HFD led to an increased transcriptional change in lipid metabolism. A strong correlation also existed between the takeout gene and hyperphagic behavior in both males and females. It is concluded that a) HFD induces a differential transcriptional response between males and females, in heads and bodies and b) the non-dimorphic transcriptional response that was identified was associated with hyperphagia. Therefore, these data on the transcriptional responses in flies to HFD provides potentially relevant information to human conditions including obesity.
Pichaud, N., Berube, R., Cote, G., Belzile, C., Dufresne, F., Morrow, G., Tanguay, R. M., Rand, D. M. and Blier, P. U. (2019). Age dependent dysfunction of mitochondrial and ROS metabolism induced by mitonuclear mismatch. Front Genet 10: 130. PubMed ID: 30842791
Mitochondrial and nuclear genomes have to coevolve to ensure the proper functioning of the different mitochondrial complexes that are assembled from peptides encoded by both genomes. Mismatch between these genomes is believed to be strongly selected against due to the consequent impairments of mitochondrial functions and induction of oxidative stress. This study used a Drosophila model harboring an incompatibility between a mitochondrial tRNA(tyr) and its nuclear-encoded mitochondrial tyrosine synthetase to assess the cellular mechanisms affected by this incompatibility and to test the relative contribution of mitonuclear interactions and aging on the expression of impaired phenotypes. The results show that the mitochondrial tRNA mutation caused a decrease in mitochondrial oxygen consumption in the incompatible nuclear background but no effect with the compatible nuclear background. Mitochondrial DNA copy number increased in the incompatible genotype but that increase failed to rescue mitochondrial functions. The flies harboring mismatch between nuclear and mitochondrial genomes had almost three times the relative mtDNA copy number and fifty percent higher rate of hydrogen peroxide production compared to other genome combinations at 25 days of age. It was also found that aging exacerbated the mitochondrial dysfunctions. These results reveal the tight interactions linking mitonuclear mismatch to mitochondrial dysfunction, mitochondrial DNA regulation, ROS production and aging.
Davoodi, S., Galenza, A., Panteluk, A., Deshpande, R., Ferguson, M., Grewal, S. and Foley, E. (2019). The immune deficiency pathway regulates metabolic homeostasis in Drosophila. J Immunol 202(9): 2747-2759. PubMed ID: 30902902
Immune and metabolic pathways collectively influence host responses to microbial invaders, and mutations in one pathway frequently disrupt activity in another. This study used the Drosophila melanogaster model to characterize metabolic homeostasis in flies with modified immune deficiency (IMD) pathway activity. The IMD pathway is very similar to the mammalian TNF-alpha pathway, a key regulator of vertebrate immunity and metabolism. Persistent activation of IMD resulted in hyperglycemia, depleted fat reserves, and developmental delays, implicating IMD in metabolic regulation. Consistent with this hypothesis, imd mutants weigh more, are hyperlipidemic, and have impaired glucose tolerance. To test the importance of metabolic regulation for host responses to bacterial infection, insulin pathway mutants were challenged with lethal doses of several Drosophila pathogens. Loss-of-function mutations in the insulin pathway impacted host responses to infection in a manner that depends on the route of infection and the identity of the infectious microbe. Combined, these results support a role for coordinated regulation of immune and metabolic pathways in host containment of microbial invaders.
Glover, Z., Hodges, M. D., Dravecz, N., Cameron, J., Askwith, H., Shirras, A. and Broughton, S. J. (2019). Loss of angiotensin-converting enzyme-related (ACER) peptidase disrupts behavioural and metabolic responses to diet in Drosophila melanogaster. J Exp Biol 222(Pt 8). PubMed ID: 30940674
Drosophila Acer (Angiotensin-converting enzyme-related) encodes a member of the angiotensin-converting enzyme (ACE) family of metallopeptidases that in mammals play roles in the endocrine regulation of blood homeostasis. ACE is also expressed in adipose tissue, where it is thought to play a role in metabolic regulation. Drosophila ACER is expressed in the adult fat body of the head and abdomen and is secreted into the haemolymph. Acer null mutants have previously been found to have reduced night-time sleep and greater sleep fragmentation. ACER may thus be part of a signalling system linking metabolism with sleep. To further understand the role of ACER in response to diet, sleep and other nutrient-responsive phenotypes were measured in Acer null flies under different dietary conditions. Loss of Acer disrupted the normal response of sleep to changes in nutrition. Other nutrient-sensitive phenotypes, including survival and glycogen storage, were also altered in the Acer mutant but lipid storage was not. Although the physiological substrate of the ACER peptidase has not been identified, an alteration of the normal nutrient-dependent control of Drosophila insulin-like peptide 5 protein in the Acer mutant suggests insulin/IGF-like signalling as a candidate pathway modulated by ACER in the nutrient-dependent control of sleep, survival and metabolism.

Thursday, May 2nd - Signaling

Casas-Tinto, S. and Ferrus, A. (2019). Troponin-I mediates the localization of selected apico-basal cell polarity signaling proteins. J Cell Sci 132(8). PubMed ID: 30872455
Beyond its role in muscle contraction, Drosophila Troponin I (TnI; also known as Wings up A) is expressed in epithelial cells where it controls proliferation. TnI traffics between nucleus and cytoplasm through a sumoylation-dependent mechanism. This study addressed the role of TnI in the cytoplasm. TnI accumulates apically in epidermal cells and neuroblasts. TnI co-immunoprecipitates with Bazooka (also known as Par3) and Discs large (Dlg1, hereafter Dlg), two apico-basal polarity components. TnI depletion causes Baz and Dlg mislocalization; by contrast, the basolateral localization of Scribbled is not altered. In neuroblasts, TnI contributes to the polar localization of Miranda, while non-polar Dlg localization is not affected. Vertebrate phosphoinositide 3-kinase (PI3K) contributes to the apico-basal polarity of epithelia, but this study found that Drosophila PI3K depletion alters neither the apical localization of TnI or Bazooka, nor the basal localization of Dlg. Nevertheless, overexpressing PI3K prevents the defects seen upon TnI depletion. TnI loss-of-function disrupts cytoskeletal beta-Catenin, E-Cadherin and gamma-Tubulin, and causes an increase in DNA damage, as revealed by analyzing gammaH2Av. Previous work has shown that TnI depletion leads to apoptosis that can be suppressed by upregulating Sparc or downregulating Dronc. However, TnI-depleted cells expressing Sparc or downregulating Dronc, as well as those expressing p35 (also known as Cdk5alpha), that do not undergo apoptosis, still show DNA damage. This indicates that DNA damage is mechanistically independent of apoptosis induction. Thus, TnI binds certain apico-basal polarity signaling proteins in a cell type-dependent context, and this unveils a previously unsuspected diversity of mechanisms to allocate cell polarity factors.
Del Castillo, U., Gnazzo, M. M., Turpin, C. G. S., Nguyen, K. C. Q., Semaya, E., Lam, Y., de Cruz, M. A., Bembenek, J. N., Hall, D. H., Riggs, B., Gelfand, V. I. and Skop, A. R. (2019). Conserved role for Ataxin-2 in mediating ER dynamics. Traffic. PubMed ID: 30989774
Ataxin-2, a conserved RNA-binding protein, is implicated in the late-onset neurodegenerative disease Spinocerebellar ataxia type-2 (SCA2). SCA2 is characterized by shrunken dendritic arbors and torpedo-like axons within the Purkinje neurons of the cerebellum. Torpedo-like axons have been described to contain displaced Endoplasmic Reticulum (ER) in the periphery of the cell; however, the role of Ataxin-2 in mediating ER function in SCA2 is unclear. This study utilized the C. elegans and Drosophila homologs of Ataxin-2 (ATX-2 and DAtx2, respectively) to determine the role of Ataxin-2 in ER function and dynamics in embryos and neurons. Loss of ATX-2 and DAtx2 resulted in collapse of the ER in dividing embryonic cells and germline, and ultrastructure analysis revealed unique spherical stacks of ER in mature oocytes and fragmented and truncated ER tubules in the embryo. ATX-2 and DAtx2 reside in puncta adjacent to the ER in both C. elegans and Drosophila embryos. Lastly, depletion of DAtx2 in cultured Drosophila neurons recapitulated the shrunken dendritic arbor phenotype of SCA2. ER morphology and dynamics were severely disrupted in these neurons. Taken together, this study provides evidence that Ataxin-2 plays an evolutionary conserved role in ER dynamics and morphology in C. elegans and Drosophila embryos during development and in fly neurons, suggesting a possible SCA2 disease mechanism.
Wang, L., Sloan, M. A. and Ligoxygakis, P. (2019). Intestinal NF-kappaB and STAT signalling is important for uptake and clearance in a Drosophila-Herpetomonas interaction model. PLoS Genet 15(3): e1007931. PubMed ID: 30822306
Dipteran insects transmit serious diseases to humans, often in the form of trypanosomatid parasites. To accelerate research in more difficult contexts of dipteran-parasite relationships, this study examined the interaction of the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum. Parasite infection reduced fecundity but not lifespan in NF-kappaB/Relish-deficient flies. Gene expression analysis implicated the two NF-kappaB pathways Toll and Imd as well as STAT signalling. Tissue specific knock-down of key components of these pathways in enterocytes (ECs) and intestinal stem cells (ISCs) influenced initial numbers, infection dynamics and time of clearance. Herpetomonas triggered STAT activation and proliferation of ISCs. Loss of Relish suppressed ISCs, resulting in increased parasite numbers and delayed clearance. Conversely, overexpression of Relish increased ISCs and reduced uptake. Finally, loss of Toll signalling decreased EC numbers and enabled parasite persistence. This network of signalling may represent a general mechanism with which dipteran respond to trypanosomatids.
Chen, Z. (2019). The formation of the Thickveins (Tkv) gradient in Drosophila wing discs: A theoretical study. J Theor Biol. PubMed ID: 30998935
The development of wing imaginal disc is commonly adopted for the studies of patterning and growth which are two fundamental problems in developmental biology. Decapentaplegic (Dpp) signaling regulates several aspects of wing development, such as the anterior (A)-posterior (P) patterning, cellular growth rate, and cell adhesion. The distribution and activity of Dpp signaling are controlled in part by the expression level of its major type I receptor, Thickveins (Tkv). This paper focuses on theoretically investigating mechanisms by which the highly asymmetric pattern of Tkv is established in Drosophila wing disc. To that end, a mathematical model of Hh signaling and Dpp signaling is proposed and validated by comparisons with experimental observations. This model provides a comprehensive view of the formation of Tkv gradients in wing discs. It was found that engrailed (En), Hedgehog (Hh) signaling, and Dpp signaling cooperate to establish the asymmetric gradients of Tkv and pMad in the wing disc. Moreover, the model suggests a Brinker-mediated mechanism of Dpp-dependent repression of Tkv. Based on this mechanism, a couple of predicted experimental observations have been provided for further lab confirmation.
Dudzic, J. P., Hanson, M. A., Iatsenko, I., Kondo, S. and Lemaitre, B. (2019). More than black or white: Melanization and toll share regulatory serine proteases in Drosophila. Cell Rep 27(4): 1050-1061. PubMed ID: 31018123
The melanization response is an important defense mechanism in arthropods. This reaction is mediated by phenoloxidases (POs), which are activated by complex extracellular serine protease (SP) cascades. This study investigated the role of SPs in the melanization response using compound mutants in D. melanogaster; phenotypes were discovered that were previously concealed in single-mutant analyses. Two SPs, Hayan and Sp7, were found to activate the melanization response in different manners: Hayan is required for blackening wound sites, whereas Sp7 regulates an alternate melanization reaction responsible for the clearance of Staphylococcus aureus. Evidence is presented that Sp7 is regulated by SPs activating the Toll NF-kappaB pathway, namely ModSP and Grass. Additionally, a role was revealed for the combined action of Hayan and Psh in propagating Toll signaling downstream of pattern recognition receptors activating either Toll signaling or the melanization response.
Garcia-Morales, D., Navarro, T., Iannini, A., Pereira, P. S., Miguez, D. G. and Casares, F. (2019). Dynamic Hh signalling can generate temporal information during tissue patterning. Development 146(8). PubMed ID: 30918051
The differentiation of tissues and organs requires that cells exchange information in space and time. Spatial information is often conveyed by morphogens: molecules that disperse across receiving cells to generate signalling gradients. Cells translate such concentration gradients into space-dependent patterns of gene expression and cellular behaviour. But could morphogen gradients also convey developmental time? By investigating the developmental role of Hh on a component of the Drosophila visual system, the ocellar retina, this study discovered that ocellar cells use the non-linear gradient of Hh as a temporal cue, collectively performing the biological equivalent of a mathematical logarithmic transformation. In this way, a morphogen diffusing from a non-moving source is decoded as a wave of differentiating photoreceptors that travels at constant speed throughout the retinal epithelium.

Wednesday, May 1st - Adult Development

Deveci, D., Martin, F. A., Leopold, P. and Romero, N. M. (2019). AstA signaling functions as an evolutionary conserved mechanism timing juvenile to adult transition. Curr Biol 29(5): 813-822. PubMed ID: 30799245
The onset of sexual maturation is the result of a hormonal cascade peaking with the production of steroid hormones. In animals undergoing a program of determinate growth, sexual maturation also coincides with the attainment of adult size. The exact signals that time the onset of maturation and the mechanisms coupling growth and maturation remain elusive. This study shows that the Drosophila neuropeptide AstA and its receptor AstAR1 act as a brain trigger for maturation and juvenile growth. AstAR1 was identified in an RNAi-based genetic screen as a key regulator of sexual maturation. Its specific knockdown in prothoracicotropic hormone (PTTH)-producing neurons delays the onset of maturation by impairing PTTH secretion. In addition to its role in PTTH neurons, AstAR1 is required in the brain insulin-producing cells (IPCs) to promote insulin secretion and systemic growth. AstAR1 function is mediated by the AstA neuropeptide that is expressed in two bilateral neurons contacting the PTTH neurons and the IPCs. Silencing brain AstA expression delays the onset of maturation, therefore extending the growth period. However, no pupal overgrowth is observed, indicating that, in these conditions, the growth-promoting function of AstAR1 is also impaired. These data suggest that AstA/AstAR1 acts to coordinate juvenile growth with maturation. Interesting, AstA/AstAR1 is homologous to KISS/GPR54, a ligand-receptor signal required for human puberty, suggesting that an evolutionary conserved neural circuitry controls the onset of maturation.
Zhou, Z., Alegot, H. and Irvine, K. D. (2019). Oriented cell divisions are not required for Drosophila wing shape. Curr Biol 29(5): 856-864. PubMed ID: 30799243
Formation of correctly shaped organs is vital for normal function. The Drosophila wing has an elongated shape, which has been attributed in part to a preferential orientation of mitotic spindles along the proximal-distal axis. Orientation of mitotic spindles is believed to be a fundamental morphogenetic mechanism in multicellular organisms. A contribution of spindle orientation to wing shape was inferred from observations that mutation of Dachsous-Fat pathway genes results in both rounder wings and loss of the normal proximal-distal bias in spindle orientation. To directly evaluate the potential contribution of spindle orientation to wing morphogenesis, the consequences of loss of the Drosophila NuMA homolog Mud, which interacts with the dynein complex and has a conserved role in spindle orientation. Loss of Mud randomizes spindle orientation but does not alter wing shape. Analysis of growth and cell dynamics in developing discs and in ex vivo culture suggests that the absence of oriented cell divisions is compensated for by an increased contribution of cell rearrangements to wing shape. These results indicate that oriented cell divisions are not required for wing morphogenesis, nor are they required for the morphogenesis of other Drosophila appendages. Moreover, the results suggest that normal organ shape is not achieved through locally specifying and then summing up individual cell behaviors, like oriented cell division. Instead, wing shape might be specified through tissue-wide stresses that dictate an overall arrangement of cells without specifying the individual cell behaviors needed to achieve it.
Pitchers, W., Nye, J., Marquez, E. J., Kowalski, A., Dworkin, I. and Houle, D. (2019). A multivariate genome-wide association study of wing shape in Drosophila melanogaster. Genetics. PubMed ID: 30792267
Due to the complexity of genotype-phenotype relationships, simultaneous analyses of genomic associations with multiple traits will be more powerful and informative than a series of univariate analyses. In most cases, however, studies of genotype-phenotype relationships have been analyzed only one trait at a time. This paper reports the results of a fully integrated multivariate genome-wide association analysis of the shape of the Drosophila melanogaster wing in the Drosophila Genetic Reference Panel. Genotypic effects on wing shape were highly correlated between two different labs. 2,396 significant SNPs were found using a 5% FDR cutoff in the multivariate analyses, but just 4 significant SNPs were found in univariate analyses of scores on the first 20 principal component axes. One quarter of these initially significant SNPs retain their effects in regularized models that take into account population structure and linkage disequilibrium. A key advantage of multivariate analysis is that the direction of the estimated phenotypic effect is much more informative than in a univariate one. This fact was exploited to show that the effects of knockdowns of genes implicated in the initial screen were on average more similar than expected under a null model. A subset of SNP effects were replicable in an unrelated panel of inbred lines. Association studies that take a phenomic approach in considering many traits simultaneously are an important complement to the power of genomics.
Varga, G. I. B., Csordas, G., Cinege, G., Jankovics, F., Sinka, R., Kurucz, E., Ando, I. and Honti, V. (2019). Headcase is a repressor of lamellocyte fate in Drosophila melanogaster. Genes (Basel) 10(3). PubMed ID: 30841641
Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, this study investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. Naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, these findings contribute to the better understanding of HSC maintenance.
Boulan, L., Andersen, D., Colombani, J., Boone, E. and Leopold, P. (2019). Inter-organ growth coordination is mediated by the Xrp1-Dilp8 axis in Drosophila. Dev Cell. PubMed ID: 31006647
How organs scale with other body parts is not mechanistically understood. This question was addressed using the Drosophila imaginal disc model. When the growth of one disc domain is perturbed, other parts of the disc and other discs slow down their growth, maintaining proper inter-disc and intra-disc proportions. The relaxin-like Dilp8 is required for this inter-organ coordination. This work also reveals that the stress-response transcription factor Xrp1 plays a key role upstream of dilp8 in linking organ growth status with the systemic growth response. In addition, the small ribosomal subunit protein RpS12 is required to trigger Xrp1-dependent non-autonomous response. This work demonstrates that RpS12, Xrp1, and Dilp8 form an independent regulatory module that ensures intra- and inter-organ growth coordination during development.
Laddada, L., Jagla, K. and Soler, C. (2019). Odd-skipped and Stripe act downstream of Notch to promote the morphogenesis of long appendicular tendons in Drosophila. Biol Open 8(3). PubMed ID: 30796048
Multiple tissue interactions take place during the development of the limb musculoskeletal system. While appendicular myogenesis has been extensively studied, development of connective tissue associated with muscles has received less attention. In the developing Drosophila leg, tendon-like connective tissue arises from clusters of epithelial cells that invaginate into the leg cavity and then elongate to form internal tube-shape structures along which muscle precursors are distributed. This study shows that stripe-positive appendicular precursors of tendon-like connective tissue are set up among intersegmental leg joint cells expressing odd-skipped genes, and that Notch signaling is necessary and locally sufficient to trigger stripe expression. This study also finds that odd-skipped genes and stripe are both required downstream of Notch to promote morphogenesis of tube-shaped internal tendons of the leg.
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