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Current papers in developmental biology and gene function


Tuesday, January 31st, 2023 - Larval and Adult Physiology and Metabolism

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Ding, K., Barretto, E. C., Johnston, M., Lee, B., Gallo, M. and Grewal, S. S. (2022). Transcriptome analysis of FOXO-dependent hypoxia gene expression identifies Hipk as a regulator of low oxygen tolerance in Drosophila. G3 (Bethesda). PubMed ID: 36200850
When exposed to low oxygen or hypoxia, animals must alter their metabolism and physiology to ensure proper cell-, tissue- and whole-body level adaptations to their hypoxic environment. These alterations often involve changes in gene expression. While extensive work has emphasized the importance of the HIF-1 alpha transcription factor on controlling hypoxia gene expression, less is known about other transcriptional mechanisms. Previous work has identified the transcription factor FOXO as a regulator of hypoxia tolerance in Drosophila larvae and adults. This study used an RNA-sequencing approach to identify FOXO-dependent changes in gene expression that are associated with these tolerance effects. Hypoxia was found to alter the expression of over 2000 genes, and approximately 40% of these gene expression changes required FOXO. Hypoxia exposure led to a FOXO-dependent increase in genes involved in cell signaling, such as kinases, GTPase regulators, and regulators of the Hippo/Yorkie pathway. Among these, homeodomain-interacting protein kinase (Hipk) was identified as being required for hypoxia survival. It was also found that hypoxia suppresses the expression of genes involved in ribosome synthesis and egg production, and hypoxia suppresses tRNA synthesis and mRNA translation and reduces female fecundity. Among the downregulated genes, FOXO was found to be required for suppression of many ribosomal protein genes and genes involved in oxidative phosphorylation, pointing to a role for FOXO in limiting energetically costly processes such as protein synthesis and mitochondrial activity upon hypoxic stress. This work uncovers a widespread role for FOXO in mediating hypoxia changes in gene expression.
Savola, E., Vale, P. F. and Walling, C. A. (2022). Larval diet affects adult reproduction, but not survival, independent of the effect of injury and infection in Drosophila melanogaster. J Insect Physiol 142: 104428. PubMed ID: 35932926
Early-life conditions have profound effects on many life-history traits, where early-life diet affects both juvenile development, and adult survival and reproduction. Early-life diet also has consequences for the ability of adults to withstand environmental challenges such as starvation, temperature and desiccation. However, it is less well known how early-life diet influences the consequences of infection in adults. This study tested whether varying the larval diet of female Drosophila melanogaster (through altering protein to carbohydrate ratio, P:C) influences the long-term consequences of injury and infection with the bacterial pathogen Pseudomonasentomophila. Given previous work manipulating adult dietary P:C, it was predicted that adults from larvae raised on higher P:C diets would have increased reproduction, but shorter lifespans and an increased rate of ageing, and that the lowest larval P:C diets would be particularly detrimental for adult survival in infected individuals. For larval development, it was predicted that low P:C would lead to a longer development time and lower viability. It was found that early-life and lifetime egg production were highest at intermediate to high larval P:C diets, but this was independent of injury and infection. There was no effect of larval P:C on adult survival. Larval development was quickest on intermediate P:C and egg-to-pupae and egg-to-adult viability were slightly higher on higher P:C. Overall, despite larval P:C affecting several measured traits, no evidence was seen that larval P:C altered the consequence of infection or injury for adult survival or early-life and lifetime reproduction. Taken together, these data suggest that larval diets appear to have a limited impact on the adult life history consequences of infection.
Galikova, M. and Klepsatel, P. (2022). Ion transport peptide regulates energy intake, expenditure, and metabolic homeostasis in Drosophila. Genetics. PubMed ID: 36190340
In mammals, energy homeostasis is regulated by the antagonistic action of hormones insulin and glucagon. However, in contrast to the highly conserved insulin, glucagon is absent in most invertebrates. Although there are several endocrine regulators of energy expenditure and catabolism (such as the Adipokinetic hormone), no single invertebrate hormone with all of the functions of glucagon has been described so far. This study used genetic gain- and loss-of-function experiments to show that the Drosophila gene Ion transport peptide (ITP) codes for a novel catabolic regulator that increases energy expenditure, lowers fat and glycogen reserves, and increases glucose and trehalose. Intriguingly, ITP has additional functions reminiscent of glucagon, such as inhibition of feeding and transit of the meal throughout the digestive tract. Furthermore, ITP interacts with the well-known signaling via the Adipokinetic hormone (AKH); ITP promotes the pathway by stimulating AKH secretion and transcription of the receptor AkhR. The genetic manipulations of ITP on standard and AKH deficient backgrounds showed that the AKH peptide mediates the hyperglycemic and hypertrehalosemic effects of ITP, while the other metabolic functions of ITP seem to be AKH-independent. In addition, ITP is necessary for critical processes such as development, starvation-induced foraging, reproduction, and average lifespan. Altogether, this work describes a novel master regulator of fly physiology with functions closely resembling mammalian glucagon.
Johnstone, P. S., Ogueta, M., Akay, O., Top, I., Syed, S., Stanewsky, R. and Top, D. (2022). Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster. Elife 11. PubMed ID: 36190119
Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. This study developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila; the method was termed Locally Activatable BioLuminescence, or LABL. Using this method, it was observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. While peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. It was also demonstrated that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. These results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo.
Liu, J., Zhang, Y., Zhou, Y., Wang, Q. Q., Ding, K., Zhao, S., Lu, P. and Liu, J. L. (2022). Cytoophidia coupling adipose architecture and metabolism. Cell Mol Life Sci 79(10): 534. PubMed ID: 36180607
Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. This study shows that the metabolic enzyme CTP synthase (CTPS) forms filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, causes impaired adipocyte adhesion and defective adipose tissue architecture. Moreover, CTPS influences integrin distribution and dot-like deposition of type IV collagen (Col IV). Col IV-integrin signaling reciprocally regulates the assembly of cytoophidia in adipocytes. These results demonstrate that a positive feedback signaling loop containing both cytoophidia and integrin adhesion complex couple tissue architecture and metabolism in Drosophila adipose tissue.
Kelly, K. P., Alassaf, M., Sullivan, C. E., Brent, A. E., Goldberg, Z. H., Poling, M. E., Dubrulle, J. and Rajan, A. (2022). Fat body phospholipid state dictates hunger-driven feeding behavior. Elife 11. PubMed ID: 36201241
Diet-induced obesity leads to dysfunctional feeding behavior. However, the precise molecular nodes underlying diet-induced feeding motivation dysregulation are poorly understood. Using a longitudinal high-sugar regime in Drosophila, this study sought to address how diet-induced changes in adipocyte lipid composition regulate feeding behavior. It was observed that subjecting adult Drosophila to a prolonged high-sugar diet degrades the hunger-driven feeding response. Lipidomics analysis reveals that longitudinal exposure to high-sugar diets significantly alters whole-body phospholipid profiles. By performing a systematic genetic screen for phospholipid enzymes in adult fly adipocytes, Pect was identified as a critical regulator of hunger-driven feeding. Pect is a rate-limiting enzyme in the phosphatidylethanolamine (PE) biosynthesis pathway and the fly ortholog of human PCYT2. Disrupting Pect activity only in the Drosophila fat cells causes insulin resistance, dysregulated lipoprotein delivery to the brain, and a loss of hunger-driven feeding. Previously human studies have noted a correlation between PCYT2/Pect levels and clinical obesity. Now, these unbiased studies in Drosophila provide causative evidence for adipocyte Pect function in metabolic homeostasis. Altogether, this study has uncovered that PE phospholipid homeostasis regulates hunger response.

Monday, January 30th - Signaling

Mo, L., Li, R., He, C., Chen, Q., Xu, C., Shen, L., Chen, K. and Wu, Y. (2022). Hedgehog pathway is negatively regulated during the development of Drosophila melanogaster PheRS-m (Drosophila homologs gene of human FARS2) mutants. Hum Cell. PubMed ID: 36205831
Hereditary spastic paraplegia (HSP) is a neurodegeneration disease, one of the reasons is caused by autosomal recessive missense mutation of the karyogene that encodes phenylalanyl-tRNA synthetase 2, mitochondrial (FARS2). However, the molecular mechanism underlying FARS2-mediated HSP progression is unknown. Mitochondrial phenylalanyl-tRNA synthetase gene (PheRS-m) is the Drosophila melanogaster homolog gene of human FARS2. This study constructed a Drosophila HSP missense mutation model and a PheRS-m knockout model. Some of the mutant fly phenotypes included developmental delay, shortened lifespan, wing-structure abnormalities and decreased mobility. RNA-sequencing results revealed a relationship between abnormal phenotypes and the hedgehog (Hh) pathway. A qRT-PCR assay was used to determine the key genes (ptc, hib, and slmb) of the Hh pathway that exhibited increased expression during different developmental stages. Hh signaling transduction was shown to be negatively regulated during the developmental stages of PheRS-m mutants but positively regulated during adulthood. By inducing the agonist and inhibitor of Hh pathway in PheRS-m larvae, the developmental delay in mutants can be partly salvaged or postponed. Collectively, these findings indicate that Hh signaling negatively regulates the development of PheRS-m mutants, subsequently leading to developmental delay.
Fechner, J., Ketelhut, M., Maier, D., Preiss, A. and Nagel, A. C. (2022). The Binding of CSL Proteins to Either Co-Activators or Co-Repressors Protects from Proteasomal Degradation Induced by MAPK-Dependent Phosphorylation. Int J Mol Sci 23(20). PubMed ID: 36293193
The primary role of Notch is to specify cellular identities, whereby the cells respond to amazingly small changes in Notch signalling activity. Hence, dosage of Notch components is crucial to regulation. Central to Notch signal transduction are CSL proteins: together with respective cofactors, they mediate the activation or the silencing of Notch target genes. CSL proteins are extremely similar amongst species regarding sequence and structure. It was noticed that the fly homologue suppressor of hairless (Su(H)) is stabilised in transcription complexes. Using specific transgenic fly lines and HeLa RBPJ(KO) cells evidence is provided that Su(H) is subjected to proteasomal degradation with a half-life of about two hours if not protected by binding to co-repressor hairless or co-activator Notch. Moreover, Su(H) stability is controlled by MAPK-dependent phosphorylation, matching earlier data for RBPJ in human cells. The homologous murine and human RBPJ proteins, however, are largely resistant to degradation in this system. Mutating presumptive protein contact sites, however, sensitised RBPJ for proteolysis. Overall, these data highlight the similarities in the regulation of CSL protein stability across species and imply that turnover of CSL proteins may be a conserved means of regulating Notch signalling output directly at the level of transcription.
Zhou, M., Han, Y., Wang, B., Cho, Y. S. and Jiang, J. (2022). Dose-dependent phosphorylation and activation of Hh pathway transcription factors. Life Sci Alliance 5(11). PubMed ID: 36271509
Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. This study shows that graded Hh in Drosophila induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. This study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition.
Neal, S. J., Zhou, Q. and Pignoni, F. (2022). Protein Phosphatase 2A with B' specificity subunits regulates the Hippo-Yorkie signaling axis in the Drosophila eye disc. J Cell Sci 135(20). PubMed ID: 36205125
Hippo-Yorkie (Hpo-Yki) signaling is central to diverse developmental processes. Although its redeployment has been amply demonstrated, its context-specific regulation remains poorly understood. The Drosophila eye disc is a continuous epithelium folded into two layers, the peripodial epithelium (PE) and the retinal progenitor epithelium. Here, Yki acts in the PE, first to promote PE identity by suppressing retina fate, and subsequently to maintain proper disc morphology. In the latter process, loss of Yki results in the displacement of a portion of the differentiating retinal epithelium onto the PE side. Protein Phosphatase 2A (PP2A) complexes comprising different substrate-specificity B-type subunits govern the Hpo-Yki axis in this context. These include holoenzymes containing the B'' subunit Cka and those containing the B' subunits Wdb or Wrd. Whereas PP2A(Cka), as part of the STRIPAK complex, is known to regulate Hpo directly, PP2A(Wdb) acts genetically upstream of the antagonistic activities of the Hpo regulators Sav and Rassf. These in vivo data provide the first evidence of PP2A(B') heterotrimer function in Hpo pathway regulation and reveal pathway diversification at distinct developmental times in the same tissue.
Happ, J. T., Arveseth, C. D., Bruystens, J., Bertinetti, D., Nelson, I. B., Olivieri, C., Zhang, J., Hedeen, D. S., Zhu, J. F., Capener, J. L., Brockel, J. W., Vu, L., King, C. C., Ruiz-Perez, V. L., Ge, X., Veglia, G., Herberg, F. W., Taylor, S. S. and Myers, B. R. (2022). A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction. Nat Struct Mol Biol 29(10): 990-999. PubMed ID: 36202993
The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. This study showed that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, it was demonstrated that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, these findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.
Stevens, A. C., O'Connor, J. T., Pumford, A. D., Page-McCaw, A. and Hutson, M. S. (2022). A mathematical model of calcium signals around laser-induced epithelial wounds. Mol Biol Cell: mbcE22080361. PubMed ID: 36322412
Cells around epithelial wounds must first become aware of the wound's presence in order to initiate the wound healing process. An initial response to an epithelial wound is an increase in cytosolic calcium followed by complex calcium signaling events. While these calcium signals are driven by both physical and chemical wound responses, cells around the wound will all be equipped with the same cellular components to produce and interact with the calcium signals. This study developed a mathematical model in the context of laser-ablation of the Drosophila pupal notum that integrates tissue-level damage models with a cellular calcium signaling toolkit. The model replicates experiments in the contexts of control wounds as well as knockdowns of specific cellular components, but it also provides new insights that are not easily accessible experimentally. The model suggests that cell-cell variability is necessary to produce calcium signaling events observed in experiments, it quantifies calcium concentrations during wound-induced signaling events, and it shows that intercellular transfer of the molecule IP(3) is required to coordinate calcium signals across distal cells around the wound. The mathematical model developed in this study serves as a framework for quantitative studies in both wound signaling and calcium signaling in the Drosophila system.

Friday, January 13th - Disease Models

Lu, S., Ma, M., Mao, X., Bacino, C. A., Jankovic, J., Sutton, V. R., Bartley, J. A., Wang, X., Rosenfeld, J. A., Beleza-Meireles, A., Chauhan, J., Pan, X., Li, M., Liu, P., Prescott, K., Amin, S., Davies, G., Wangler, M. F., Dai, Y. and Bellen, H. J. (2022). De novo variants in FRMD5 are associated with developmental delay, intellectual disability, ataxia, and abnormalities of eye movement. Am J Hum Genet 109(10): 1932-1943. PubMed ID: 36206744
Proteins containing the FERM (four-point-one, ezrin, radixin, and moesin) domain link the plasma membrane with cytoskeletal structures at specific cellular locations and have been implicated in the localization of cell-membrane-associated proteins and/or phosphoinositides. FERM domain-containing protein 5 (FRMD5) localizes at cell adherens junctions and stabilizes cell-cell contacts. To date, variants in FRMD5 have not been associated with a Mendelian disease in OMIM. This study describes eight probands with rare heterozygous missense variants in FRMD5 who present with developmental delay, intellectual disability, ataxia, seizures, and abnormalities of eye movement. The variants are de novo in all for whom parental testing was available (six out of eight probands), and human genetic datasets suggest that FRMD5 is intolerant to loss of function (LoF). It was found that the fly ortholog of FRMD5, CG5022 (dFrmd), is expressed in the larval and adult central nervous systems where it is present in neurons but not in glia. dFrmd LoF mutant flies are viable but are extremely sensitive to heat shock, which induces severe seizures. The mutants also exhibit defective responses to light. The human FRMD5 reference (Ref) cDNA rescues the fly dFrmd LoF phenotypes. In contrast, all the FRMD5 variants tested in this study (c.340T>C, c.1051A>G, c.1053C>G, c.1054T>C, c.1045A>C, and c.1637A>G) behave as partial LoF variants. In addition, the results indicate that two variants that were tested have dominant-negative effects. In summary, the evidence supports that the observed variants in FRMD5 cause neurological symptoms in humans.
Liu, W., Lim, K. L. and Tan, E. K. (2022). Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila. Transl Neurodegener 11(1): 44. PubMed ID: 36253844
Aberrant aggregation of α-synuclein (α-syn) is a key pathological feature of Parkinson's disease (PD), but the precise role of intestinal α-syn in the progression of PD is unclear. In a number of genetic Drosophila models of PD, α-syn was frequently ectopically expressed in the neural system to investigate the pathobiology. This study investigated the potential role of intestinal α-syn in PD pathogenesis using a Drosophila model. Human α-syn was overexpressed in Drosophila guts, and life span, survival, immunofluorescence and climbing were evaluated. Immunofluorescence, Western blotting and reactive oxygen species (ROS) staining were performed to assess the effects of intestinal α-syn on intestinal dysplasia. High-throughput RNA and 16S rRNA gene sequencing, quantitative RT-PCR, immunofluorescence, and ROS staining were performed to determine the underlying molecular mechanism. It was found that the midgut α-syn alone recapitulated many phenotypic and pathological features of PD, including impaired life span, loss of dopaminergic neurons, and progressive motor defects. The intestine-derived α-syn disrupted intestinal homeostasis and accelerated the onset of intestinal ageing. Moreover, intestinal expression of α-syn induced dysbiosis, while microbiome depletion was efficient to restore intestinal homeostasis and ameliorate the progression of PD. Intestinal α-syn triggered ROS, and eventually led to the activation of the dual oxidase (DUOX)-ROS-Jun N-terminal Kinase (JNK) pathway. In addition, α-syn from both the gut and the brain synergized to accelerate the progression of PD. The intestinal expression of α-syn recapitulates many phenotypic and pathologic features of PD, and induces dysbiosis that aggravates the pathology through the DUOX-ROS-JNK pathway in Drosophila. These findings provide new insights into the role of intestinal α-syn in PD pathophysiology.
Lee, B., Choi, B., Park, Y., Jang, S., Yuan, C., Lim, C., Lee, J. H., Song, G. J. and Cho, K. S. (2022). Roles of ZnT86D in Neurodevelopment and Pathogenesis of Alzheimer Disease in a Drosophila melanogaster Model. Int J Mol Sci 23(19). PubMed ID: 36233134
Zinc is a fundamental trace element essential for numerous biological processes, and zinc homeostasis is regulated by the Zrt-/Irt-like protein (ZIP) and zinc transporter (ZnT) families. ZnT7 is mainly localized in the Golgi apparatus and endoplasmic reticulum (ER) and transports zinc into these organelles. Although previous studies have reported the role of zinc in animal physiology, little is known about the importance of zinc in the Golgi apparatus and ER in animal development and neurodegenerative diseases. This study demonstrate that ZnT86D, a Drosophila ortholog of ZnT7, plays a pivotal role in the neurodevelopment and pathogenesis of Alzheimer disease (AD). When ZnT86D was silenced in neurons, the embryo-to-adult survival rate, locomotor activity, and lifespan were dramatically reduced. The toxic phenotypes were accompanied by abnormal neurogenesis and neuronal cell death. Furthermore, knockdown of ZnT86D in the neurons of a Drosophila AD model increased apoptosis and exacerbated neurodegeneration without significant changes in the deposition of amyloid beta plaques and susceptibility to oxidative stress. Taken together, these results suggest that an appropriate distribution of zinc in the Golgi apparatus and ER is important for neuronal development and neuroprotection and that ZnT7 is a potential protective factor against AD.
Ma, M., Zhang, X., Zheng, Y., Lu, S., Pan, X., Mao, X., Pan, H., Chung, H. L., Wang, H., Guo, H. and Bellen, H. J. (2022). The fly homolog of SUPT16H, a gene associated with neurodevelopmental disorders, is required in a cell-autonomous fashion for cell survival. Hum Mol Genet. PubMed ID: 36255738
SUPT16H encodes the large subunit of the FACT complex, which functions as a nucleosome organizer during transcription. This study identified two individuals from unrelated families carrying de novo missense variants in SUPT16H. The probands exhibit global developmental delay, intellectual disability, epilepsy, facial dysmorphism, and brain structural abnormalities. Drosophila was used to characterize these two variants, p.T171I and p.G808R. Loss of the fly ortholog, dre4, causes lethality at an early developmental stage. RNAi-mediated knockdown of dre4 in either glia or neurons causes severely reduced eclosion and longevity. Tissue-specific knockdown of dre4 in the eye or wing leads to the loss of these tissues whereas overexpression of SUPT16H has no dominant effect. Moreover, expression of the reference SUPT16H significantly rescues the loss-of-function phenotypes in the nervous system as well as wing and eye. In contrast, expression of SUPT16H p.T171I or p.G808R rescues the phenotypes poorly, indicating that the variants are partial loss-of-function alleles. While previous studies argued that the developmental arrest caused by loss of dre4 is due to impaired ecdysone production in the prothoracic gland, the data show that dre4 is required for proper cell growth and survival in multiple tissues in a cell-autonomous manner. Altogether, these data indicate that the de novo loss-of-function variants in SUPT16H are indeed associated with developmental and neurological defects observed in the probands.
Larasati, Y. A., Savitsky, M., Koval, A., Solis, G. P., Valnohova, J. and Katanaev, V. L. (2022). Restoration of the GTPase activity and cellular interactions of Gα(o) mutants by Zn(2+) in GNAO1 encephalopathy models. Sci Adv 8(40): eabn9350. PubMed ID: 36206333
De novo point mutations in GNAO1, gene encoding the major neuronal G protein Gα(o), have recently emerged in patients with pediatric encephalopathy having motor, developmental, and epileptic dysfunctions. Half of clinical cases affect codons Gly(203), Arg(209), or Glu(246); these mutations accelerate GTP uptake and inactivate GTP hydrolysis through displacement Gln(205) critical for GTP hydrolysis, resulting in constitutive GTP binding by Gα(o). However, the mutants fail to adopt the activated conformation and display aberrant interactions with signaling partners. Through high-throughput screening of approved drugs, rhis study identifie zinc pyrithione and Zn(2+) as agents restoring active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with negligible effects on wild-type Gα(o). A Drosophila model is described of GNAO1 encephalopathy where dietary zinc restores the motor function and longevity of the mutant flies. Zinc supplements are approved for diverse human neurological conditions. This work provides insights into the molecular etiology of GNAO1 encephalopathy and defines a potential therapy for the patients.
Karagas, N. E., Gupta, R., Rastegari, E., Tan, K. L., Leung, H. H., Bellen, H. J., Venkatachalam, K. and Wong, C. O. (2022). Loss of Activity-Induced Mitochondrial ATP Production Underlies the Synaptic Defects in a Drosophila Model of ALS. J Neurosci 42(42): 8019-8037. PubMed ID: 36261266
Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapbP58S) ) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, this study demonstrate the suppression of vapbP58S) -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca(2+) release channels or the inhibition Ca(2+)/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapbP58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca(2+)]. The Ca(2+) dyshomeostasis is attributed to delayed extrusion of cytosolic Ca(2+) Suggesting that this defect in Ca(2+) extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapbP58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapbP58S -expressing motor neurons.

Thursday, January 12 - Immune response

Wu, P. J. and Yan, S. J. (2022). HP1a-mediated heterochromatin formation promotes antimicrobial responses against Pseudomonas aeruginosa infection. BMC Biol 20(1): 234. PubMed ID: 36266682
Pseudomonas aeruginosa is a Gram-negative bacterium that causes severe infectious disease in diverse host organisms, including humans. Effective therapeutic options for P. aeruginosa infection are limited due to increasing multidrug resistance and it is therefore critical to understand the regulation of host innate immune responses to guide development of effective therapeutic options. The epigenetic mechanisms by which hosts regulate their antimicrobial responses against P. aeruginosa infection remain unclear. This study used Drosophila melanogaster to investigate the role of heterochromatin protein 1a (HP1a), a key epigenetic regulator, and its mediation of heterochromatin formation in antimicrobial responses against PA14, a highly virulent P. aeruginosa strain. Animals with decreased heterochromatin levels showed less resistance to P. aeruginosa infection. In contrast, flies with increased heterochromatin formation, either in the whole organism or specifically in the fat body-an organ important in humoral immune response-showed greater resistance to P. aeruginosa infection, as demonstrated by increased host survival and reduced bacterial load. Increased heterochromatin formation in the fat body promoted the antimicrobial responses via upregulation of fat body immune deficiency (imd) pathway-mediated antimicrobial peptides (AMPs) before and in the middle stage of P. aeruginosa infection. The fat body AMPs were required to elicit HP1a-mediated antimicrobial responses against P. aeruginosa infection. Moreover, the levels of heterochromatin in the fat body were downregulated in the early stage, but upregulated in the middle stage, of P. aeruginosa infection. These data indicate that HP1a-mediated heterochromatin formation in the fat body promotes antimicrobial responses by epigenetically upregulating AMPs of the imd pathway. This study provides novel molecular, cellular, and organismal insights into new epigenetic strategies targeting heterochromatin that have the potential to combat P. aeruginosa infection.
Fuse, N., Okamori, C., Okaji, R., Tang, C., Hirai, K. and Kurata, S. (2022). Transcriptome features of innate immune memory in Drosophila. PLoS Genet 18(10): e1010005. PubMed ID: 36252180
Immune memory is the ability of organisms to elicit potentiated immune responses at secondary infection. Current studies have revealed that similar to adaptive immunity, innate immunity exhibits memory characteristics (called "innate immune memory"). Although epigenetic reprogramming plays an important role in innate immune memory, the underlying mechanisms have not been elucidated, especially at the individual level. This study established experimental systems for detecting innate immune memory in Drosophila melanogaster. Training infection with low-pathogenic bacteria enhanced the survival rate of the flies at subsequent challenge infection with high-pathogenic bacteria. Among low-pathogenic bacteria, Micrococcus luteus (Ml) and Salmonella typhimurium (St) exerted apparent training effects in the fly but exhibited different mechanisms of action. Ml exerted training effects even after its clearance from flies, while live St persisted in the flies for a prolonged duration. RNA sequencing (RNA-Seq) analysis revealed that Ml training enhanced the expression of the immune-related genes under the challenge condition but not under the non-challenge condition. In contrast, St training upregulated the expression of the immune-related genes independent of challenge. These results suggest that training effects with Ml and St are due to memory and persistence of immune responses, respectively. Furthermore, the gene involved in immune memory was souvhg, and a candidate gene, Ada2b, was identified which encodes a component of the histone modification complex. The Ada2b mutant suppressed Ml training effects on survival and disrupted the expression of some genes under the training + challenge condition. These results suggest that the gene expression regulated by Ada2b may contribute to innate immune memory in Drosophila.
Xu, R., Lou, Y., Tidu, A., Bulet, P., Heinekamp, T., Martin, F., Brakhage, A., Li, Z., Liegeois, S. and Ferrandon, D. (2022). The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins. EMBO Rep: e56036. PubMed ID: 36322050
Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. Mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.
Deshpande, R., Lee, B. and Grewal, S. S. (2022). Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the immune deficiency signaling pathway. G3 (Bethesda) 12(11). PubMed ID: 35781508
When infected by intestinal pathogenic bacteria, animals initiate both local and systemic defence responses. These responses are required to reduce pathogen burden and also to alter host physiology and behavior to promote infection tolerance, and they are often mediated through alterations in host gene expression. This study used transcriptome profiling to examine gene expression changes induced by enteric infection with the Gram-negative bacteria Pseudomonas entomophila in adult female Drosophila. Infection was found to induce a strong upregulation of metabolic gene expression, including gut and fat body-enriched genes involved in lipid transport, lipolysis, and beta-oxidation, as well as glucose and amino acid metabolism genes. Furthermore, the classic innate immune deficiency (Imd)/Relish/NF-KappaB pathway were not required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. Enteric infection with Pseudomonas entomophila downregulates the expression of many transcription factors and cell-cell signaling molecules, particularly those previously shown to be involved in gut-to-brain and neuronal signaling. Moreover, as with the metabolic genes, these changes occurred largely independent of the Imd pathway. Together, this study identifies many metabolic, signaling, and transcription factor gene expression changes that may contribute to organismal physiological and behavioral responses to enteric pathogen infection.
Pavlidaki, A., Panic, R., Monticelli, S., Riet, C., Yuasa, Y., Cattenoz, P. B., Nait-Oumesmar, B. and Giangrande, A. (2022). An anti-inflammatory transcriptional cascade conserved from flies to humans. Cell Rep 41(3): 111506. PubMed ID: 36261018
Innate immunity is an ancestral process that can induce pro- and anti-inflammatory states. A major challenge is to characterize transcriptional cascades that modulate the response to inflammation. Since the Drosophila glial cells missing (Gcm) transcription factor has an anti-inflammatory role, this study explored its regulation and evolutionary conservation. The murine Gcm2 (mGcm2) gene was shown to be expressed in a subpopulation of aged microglia (chronic inflammation) and upon lysophosphatidylcholine (LPC)-induced central nervous system (CNS) demyelination (acute inflammation). Moreover, mGcm2 conditional knockout mice show an increased inflammatory phenotype upon aging or LPC injection, and hGCM2 is expressed in active demyelinating lesions of patients with multiple sclerosis. Finally, Drosophila Gcm expression is induced upon aging and acute challenge, and its overexpression decreases the inflammatory phenotype. Altogether, these data indicate that the inducible Gcm cascade is conserved from flies to humans and represents a potential therapeutic target in the control of the inflammatory response.
Masuzzo, A., Maniere, G., Grosjean, Y., Kurz, L. and Royet, J. (2022). Bacteria-Derived Peptidoglycan Triggers a Noncanonical Nuclear Factor-kappaB-Dependent Response in Drosophila Gustatory Neurons. J Neurosci 42(41): 7809-7823. PubMed ID: 36414007
Probing the external world is essential for eukaryotes to distinguish beneficial from pathogenic micro-organisms. If it is clear that the main part of this task falls to the immune cells, recent work shows that neurons can also detect microbes, although the molecules and mechanisms involved are less characterized. In Drosophila, detection of bacteria-derived peptidoglycan by pattern recognition receptors of the peptidoglycan recognition protein (PGRP) family expressed in immune cells triggers nuclear factor-κB (NF-κB)/immune deficiency (IMD)-dependent signaling. This study shows that one PGRP protein, called PGRP-LB, is expressed in bitter gustatory neurons of proboscises. In vivo calcium imaging in female flies reveals that the PGRP/IMD pathway is cell-autonomously required in these neurons to transduce the peptidoglycan signal. It was finally shown that NF-κB/IMD pathway activation in bitter-sensing gustatory neurons influences fly behavior. This demonstrates that a major immune response elicitor and signaling module are required in the peripheral nervous system to sense the presence of bacteria in the environment.

Wednesday, January 11th - RNA and Transposons

Castillo, D. M. and Moyle, L. C. (2022). Hybrid incompatibility between Drosophila virilis and D. lummei is stronger in the presence of transposable elements. J Evol Biol 35(10): 1319-1334. PubMed ID: 35988129
Mismatches between parental genomes in selfish elements are frequently hypothesized to underlie hybrid dysfunction and drive speciation. However, because the genetic basis of most hybrid incompatibilities is unknown, testing the contribution of selfish elements to reproductive isolation is difficult. This study evaluated the role of transposable elements (TEs) in hybrid incompatibilities between Drosophila virilis and D. lummei by experimentally comparing hybrid incompatibility in a cross where active TEs are present in D. virilis (TE+) and absent in D. lummei, to a cross where these TEs are absent from both D. virilis (TE-) and D. lummei genotypes. Using genomic data, copy number differences in TEs were confirmed between the D. virilis (TE+) strain and both the D. virilis (TE-) strain and D. lummei. F1 postzygotic reproductive isolation was observed exclusively in the interspecific cross involving TE+ D. virilis but not in crosses involving TE- D. virilis. This mirrors intraspecies dysgenesis where atrophied testes only occur when TE+ D. virilis is the paternal parent. A series of backcross experiments, that accounted for alternative models of hybrid incompatibility, showed that both F1 hybrid incompatibility and intrastrain dysgenesis are consistent with the action of TEs rather than genic interactions. Thus, these data suggest that this TE mechanism manifests as two different incompatibility phenotypes. A further Y-autosome interaction contributes to additional, sex-specific, inviability in one direction of this cross-combination. These experiments demonstrate that TEs that cause intraspecies dysgenesis can increase reproductive isolation between closely related lineages, thereby adding to the processes that consolidate speciation.
Dorador, A. P., Dalikova, M., Cerbin, S., Stillman, C. M., Zych, M. G., Hawley, R. S., Miller, D. E., Ray, D. A., Funikov, S. Y., Evgen'ev, M. B. and Blumenstiel, J. P. (2022). Paramutation-like Epigenetic Conversion by piRNA at the Telomere of Drosophila virilis. Biology (Basel) 11(10). PubMed ID: 36290385
First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. This paper characterizes a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. This paper shows that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation.
Baumgartner, L., Handler, D., Platzer, S. W., Yu, C., Duchek, P. and Brennecke, J. (2022). The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters. Elife 11. PubMed ID: 36193674
RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. This study identifed a member of the diverse family of zinc finger associated domain (ZAD)-C(2)H(2) zinc finger proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric Satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. These findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.
Song, Z., Lin, J., Su, R., Ji, Y., Jia, R., Li, S., Shan, G. and Huang, C. (2022). eIF3j inhibits translation of a subset of circular RNAs in eukaryotic cells. Nucleic Acids Res. PubMed ID: 36330957
Increasing studies have revealed that a subset of circular RNAs (circRNAs) harbor an open reading frame and can act as protein-coding templates to generate functional proteins that are closely associated with multiple physiological and disease-relevant processes, and thus proper regulation of synthesis of these circRNA-derived proteins is a fundamental cellular process required for homeostasis maintenance. However, how circRNA translation initiation is coordinated by different trans-acting factors remains poorly understood. In particular, the impact of different eukaryotic translation initiation factors (eIFs) on circRNA translation and the physiological relevance of this distinct regulation have not yet been characterized. In this study, all 43 Drosophila eIFs were screened and it was revealed the conflicting functions of eIF3 subunits in the translational control of the translatable circRNA circSfl: eIF3 is indispensable for circSfl translation, while the eIF3-associated factor eIF3j is the most potent inhibitor. Mechanistically, the binding of eIF3j to circSfl promotes the disassociation of eIF3. The C-terminus of eIF3j and an RNA regulon within the circSfl untranslated region (UTR) are essential for the inhibitory effect of eIF3j. Moreover, the physiological relevance of eIF3j-mediated circSfl translation repression in response to heat shock was revealed. Finally, additional translatable circRNAs were identified to be similarly regulated in an eIF3j-dependent manner. Altogether, this study provides a significant insight into the field of cap-independent translational regulation and undiscovered functions of eIF3.
Xiong, X. P., Liang, W., Liu, W., Xu, S., Li, J. L., Tito, A., Situ, J., Martinez, D., Wu, C., Perera, R. J., Zhang, S. and Zhou, R. (2022). The circular RNA Edis regulates neurodevelopment and innate immunity. PLoS Genet 18(10): e1010429. PubMed ID: 36301822
Circular RNAs (circRNAs) are widely expressed in eukaryotes. However, only a subset has been functionally characterized. This study identified and validated a collection of circRNAs in Drosophila, and showed that depletion of the brain-enriched circRNA Edis (circ_Ect4) causes hyperactivation of antibacterial innate immunity both in cultured cells and in vivo. Notably, Edis depleted flies display heightened resistance to bacterial infection and enhanced pathogen clearance. Conversely, ectopic Edis expression blocks innate immunity signaling. In addition, inactivation of Edis in vivo leads to impaired locomotor activity and shortened lifespan. Remarkably, these phenotypes can be recapitulated with neuron-specific depletion of Edis, accompanied by defective neurodevelopment. Furthermore, inactivation of Relish suppresses the innate immunity hyperactivation phenotype in the fly brain. Moreover, evidence is provided that Edis encodes a functional protein that associates with and compromises the processing and activation of the immune transcription factor Relish. Importantly, restoring Edis expression or ectopic expression of Edis-encoded protein suppresses both innate immunity and neurodevelopment phenotypes elicited by Edis depletion. Thus, this study establishes Edis as a key regulator of neurodevelopment and innate immunity.
Liu, J., Jin, T., Ran, L., Zhao, Z., Zhu, R., Xie, G. and Bi, X. (2022). Profiling ATM regulated genes in Drosophila at physiological condition and after ionizing radiation. Hereditas 159(1): 41. PubMed ID: 36271387
ATM (ataxia-telangiectasia mutated) protein kinase is highly conserved in metazoan, and plays a critical role at DNA damage response, oxidative stress, metabolic stress, immunity, RNA biogenesis etc. Systemic profiling of ATM regulated genes, including protein-coding genes, miRNAs, and long non-coding RNAs, will greatly improve understanding of ATM functions and its regulation.  This study shows: 1) differentially expressed protein-coding genes, miRNAs, and long non-coding RNAs in atm mutated flies were identified at physiological condition and after X-ray irradiation. 2) functions of differentially expressed genes in atm mutated flies, regardless of protein-coding genes or non-coding RNAs, are closely related with metabolic process, immune response, DNA damage response or oxidative stress. 3) these phenomena are persistent after irradiation. 4) there is a cross-talk regulation towards miRNAs by ATM, E2f1, and p53 during development and after irradiation. 5) knock-out flies or knock-down flies of most irradiation-induced miRNAs were sensitive to ionizing radiation. This study provides a valuable resource of protein-coding genes, miRNAs, and long non-coding RNAs, for understanding ATM functions and regulations. This work provides the new evidence of inter-dependence among ATM-E2F1-p53 for the regulation of miRNAs.

Tuesday, January 10th - Larval and Adult Physiology and Metabolism

D'Souza, L. C., Kuriakose, N., Raghu, S. V., Kabekkodu, S. P. and Sharma, A. (2022). ROS-directed activation of Toll/NF-κB in the hematopoietic niche triggers benzene-induced emergency hematopoiesis. Free Radic Biol Med 193(Pt 1): 190-201. PubMed ID: 36216301
Hematopoietic stem cells/progenitor cells (HSC/HPCs) orchestrate the hematopoietic process, effectively regulated by the hematopoietic niche under normal and stressed conditions. The hematopoietic niche provides various soluble factors which influence the differentiation and self-renewal of HSC/HSPs. Unceasing differentiation/proliferation/high metabolic activity of HSC/HPCs makes them susceptible to damage by environmental toxicants like benzene. Oxidative stress, epigenetic modifications, and DNA damage in the HSC/HPCs are the key factors of benzene-induced hematopoietic injury. However, the role of the hematopoietic niche in benzene-induced hematopoietic injury/response is still void. Therefore, the current study aims to unravel the role of the hematopoietic niche in benzene-induced hematotoxicity using a genetically tractable model, Drosophila melanogaster. The lymph gland is a dedicated hematopoietic organ in Drosophila larvae. A group of 30-45 cells called the posterior signaling center (PSC) in the lymph gland acts as a niche that regulates Drosophila HSC/HPCs maintenance. Benzene exposure to Drosophila larvae (48 h) resulted in aberrant hemocyte production, especially hyper-differentiation of lamellocytes followed by premature lymph gland dispersal and reduced adult emergence upon developmental exposure. Subsequent genetic experiments revealed that benzene-induced lamellocyte production and premature lymph gland dispersal were PSC mediated. The genetic experiments further showed that benzene generates Dual oxidase (Duox)-dependent Reactive Oxygen Species (ROS) in the PSC, activating Toll/NF-κB signaling, which is essential for the aberrant hemocyte production, lymph gland dispersal, and larval survival. Together, this study establishes a functional perspective of the hematopoietic niche in a benzene-induced hematopoietic emergency in a genetic model, Drosophila, which might be relevant to higher organisms
Sivakumar, S., Miellet, S., Clarke, C. and Hartley, P. S. (2022). Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover. J Insect Physiol 143: 104453. PubMed ID: 36341969
Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte's ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes. This work aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm. These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans.
Chakraborty, A., Walter, G. M., Monro, K., Alves, A. N., Mirth, C. K. and Sgro, C. M. (2022). Within-population variation in body size plasticity in response to combined nutritional and thermal stress is partially independent from variation in development time. J Evol Biol. PubMed ID: 36208146
Ongoing climate change has forced animals to face changing thermal and nutritional environments. Animals can adjust to such combinations of stressors via plasticity. Body size is a key trait influencing organismal fitness, and plasticity in this trait in response to nutritional and thermal conditions varies among genetically diverse, locally adapted populations. The standing genetic variation within a population can also influence the extent of body size plasticity. Near-isogenic lines were generated from a newly collected population of Drosophila melanogaster at the mid-point of east coast Australia and assayed body size for all lines in combinations of thermal and nutritional stress. Isogenic lines showed distinct underlying patterns of body size plasticity in response to temperature and nutrition that were often different from the overall population response. Then, whether plasticity in development time could explain, and therefore regulate, variation in body size to these combinations of environmental conditions was tested. Five genotypes were selected that showed the greatest variation in response to combined thermal and nutritional stress, and the correlation between response of developmental time and body size was assessed. While significant genetic variation was found in development time plasticity, it was a poor predictor of body size among genotypes. These results therefore suggest that multiple developmental pathways could generate genetic variation in body size plasticity. This study emphasizes the need to better understand genetic variation in plasticity within a population, which will help determine the potential for populations to adapt to ongoing environmental change.
Fernandez-Acosta, M., Romero, J. I., Bernabo, G., Velazquez-Campos, G. M., Gonzalez, N., Mares, M. L., Werbajh, S., Avendano-Vazquez, L. A., Rechberger, G. N., Kühnlein, R. P., Marino-Buslje, C., Cantera, R., Rezaval, C. and Ceriani, M. F. (2022). orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control. BMC Biol 20(1): 233. PubMed ID: 36266680
Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. This study identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, it was demonstrated that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, this study shows that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of β-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.
Kellermann, V., Overgaard, J., Sgrò, C. M. and Hoffmann, A. A. (2022). Phylogenetic and environmental patterns of sex differentiation in physiological traits across Drosophila species. J Evol Biol 35(11): 1548-1557. PubMed ID: 36196885
Sex-based differences in physiological traits may be influenced by both evolutionary and environmental factors. This study used male and female flies from >80 Drosophila species reared under common conditions to examine variance in a number of physiological traits including size, starvation, desiccation and thermal tolerance. Sex-based differences for desiccation and starvation resistance were comparable in magnitude to those for size, with females tending to be relatively more resistant than males. In contrast thermal resistance showed low divergence between the sexes. Phylogenetic signal was detected for measures of divergence between the sexes, such that species from the Sophophora clade showed larger differences between the sexes than species from the Drosophila clade. This study also found that sex-based differences in desiccation resistance, body size and starvation resistance were weakly associated with climate (annual mean temperature/precipitation seasonality) but the direction and association with environment depended on phylogenetic position. The results suggest that divergence between the sexes can be linked to environmental factors, while an association with phylogeny suggests sex-based differences persist over long evolutionary time-frames.
Campos-Blazquez, J. P., Schuth, N., Garay, E., Clark, A. H., Vogelsang, U., Nachtegaal, M., Contreras, R. G., Quintanar, L. and Missirlis, F. (2022). Chloroquine disrupts zinc storage granules in primary Malpighian tubule cells of Drosophila melanogaster.. Metallomics 14(10). PubMed ID: 36151967
Contrasting reports exist in the literature regarding the effect of chloroquine treatment on cellular zinc uptake or secretion. This study tested the effect of chloroquine administration in the Drosophila model organism. Larvae grown on a diet supplemented with 2.5 mg/ml chloroquine were shown to lose up to 50% of their stored zinc and around 10% of their total potassium content. This defect in chloroquine-treated animals correlates with the appearance of abnormal autophagolysosomes in the principal cells of the Malpighian tubules, where zinc storage granules reside. It was further shown that the reported increase of Fluozin-3 fluorescence following treatment of cells with 300 μM chloroquine for 1 h may not reflect increased zinc accumulation, since a similar treatment in Madin-Darby canine kidney cells results in a 36% decrease in their total zinc content. Thus, chloroquine should not be considered a zinc ionophore. Zinc supplementation plus chloroquine treatment restored zinc content both in vivo and in vitro, without correcting autophagic or other ionic alterations, notably in potassium, associated with the chloroquine treatment. It is suggest that chloroquine or hydroxychloroquine administration to patients could reduce intracellular zinc storage pools and be part of the drug's mechanism of action.

Monday, January 8th - Chromatin, DNA Replication, and Chromosome Dynamics

Starczak, M., Gawronski, M., Wasilow, A., Mijewski, P., Olinski, R. and Gackowski, D. (2022). Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions. Sci Rep 12(1): 17552. PubMed ID: 36266436
In this study, the level of DNA modifications was investigated in three developmental stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). Analysis was carried out using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. This method made it possible, for the first time, to analyze a broad spectrum of DNA modifications in the three stages of Drosophila. Each stage was characterized by a specific modification pattern, and the levels of these compounds fluctuated throughout the D. melanogaster life cycle. The level of DNA modification was also compared between insects bred at 25 °C (optimal temperature) and at 18 °C, and the groups differed significantly. The profound changes in N6-methyladenine and 5-hydroxymethyluracil levels during the Drosophila life cycle and as a result of breeding temperature changes indicate that these DNA modifications can play important regulatory roles in response to environmental changes and/or biological conditions. Moreover, the supplementation of Schneider 2 cells with 1 mM L-ascorbic acid caused a time-dependent increase in the level of 5-(hydroxymethyl)-2'-deoxyuridine. These data suggest that a certain pool of this compound may arise from the enzymatic activity of the dTET protein.
McKowen, J. K., Avva, S., Maharjan, M., Duarte, F. M., Tome, J. M., Judd, J., Wood, J. L., Negedu, S., Dong, Y., Lis, J. T. and Hart, C. M. (2022). The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex. G3 (Bethesda) 12(11). PubMed ID: 36029240
The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. This study shows that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. The effect of this interaction on gene expression and chromatin structure was examined using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. The results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. The possibility is discussed of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) were identified as candidate redundant factors.
Zhang, X., Wu, X., Peng, J., Sun, A., Guo, Y., Fu, P. and Gao, G. (2022). Cis- and trans-regulation by histone H4 basic patch R17/R19 in metazoan development. Open Biol 12(11): 220066. PubMed ID: 36382370
The histone H4 basic patch is critical for chromatin structure and regulation of the chromatin machinery. However, the biological roles of these positively charged residues and the mechanisms by which they regulate gene expression remain unclear. This study used histone mutagenesis to investigate the physiological function and downstream regulatory genes of H4 residues R17 and R19 in Drosophila. All histone mutations, including R17A/E/H and R19A/E/H (R17 and R19 of H4 are substituted by A, E and H respectively), result in a range of growth defects and abnormalities in chromosomal high-order structures, whereas R17E mutation is embryonic lethal. RNA-seq demonstrates that downregulated genes in both R17A and R19A show significant overlap and are enriched in development-related pathways. In addition, Western and cytological analyses showed that the R17A mutation resulted in a significant reduction in H4K16 acetylation and male offspring, implying that the R17 may be involved in male dosage compensation mechanisms. R19 mutation on the other hand strongly affect Gpp (Dot1 homologue in flies)-mediated H3K79 methylation, possibly through histone crosstalk. Together these results provide insights into the differential impacts of positive charges of H4 basic patch R17/R19 on regulation of gene transcription during developmental processes.
Sureka, R., Avvaru, A. K., Sowpati, D. T., Pathak, R. U. and Mishra, R. K. (2022). Structural and developmental dynamics of Matrix associated regions in Drosophila melanogaster genome. BMC Genomics 23(1): 725. PubMed ID: 36284304
Eukaryotic genome is compartmentalized into structural and functional domains. One of the concepts of higher order organization of chromatin posits that the DNA is organized in constrained loops that behave as independent functional domains. Nuclear Matrix (NuMat), a ribo-proteinaceous nucleoskeleton, provides the structural basis for this organization. DNA sequences located at base of the loops are known as the Matrix Attachment Regions (MARs). NuMat relates to multiple nuclear processes and is partly cell type specific in composition. It is a biochemically defined structure and several protocols have been used to isolate the NuMat where some of the steps have been critically evaluated. These sequences play an important role in genomic organization it is imperative to know their dynamics during development and differentiation. This study looked into the dynamics of MARs when the preparation process is varied and during embryonic development of D. melanogaster. A subset of MARs termed as "Core-MARs" present abundantly in pericentromeric heterochromatin, are constant unalterable anchor points as they associate with NuMat through embryonic development and are independent of the isolation procedure. Euchromatic MARs are dynamic and reflect the transcriptomic profile of the cell. New MARs are generated by nuclear stabilization, and during development, mostly at paused RNA polymerase II promoters. Paused Pol II MARs depend on RNA transcripts for NuMat association. These data reveals the role of MARs in functionally dynamic nucleus and contributes to the current understanding of nuclear architecture in genomic context.
Cho, C. Y., Kemp, J. P., Jr., Duronio, R. J. and O'Farrell, P. H. (2022). Coordinating transcription and replication to mitigate their conflicts in early Drosophila embryos. Cell Rep 41(3): 111507. PubMed ID: 36261005
Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. It hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2 min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. It is proposed that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes.
Chetverina, D. A., Gorbenko, F. V., Lomaev, D. V., Georgiev, P. G. and Erokhin, M. M. (2022). Recruitment to Chromatin of (GA)n-Associated Factors GAF and Psq in the Transgenic Model System Depends on the Presence of Architectural Protein Binding Sites. Dokl Biochem Biophys 506(1): 210-214. PubMed ID: 36303054
Polycomb group (PcG) repressors and Trithorax group (TrxG) activators of transcription are essential for the proper development and maintenance of gene expression profiles in multicellular organisms. In Drosophila, PcG/TrxG proteins interact with DNA elements called PRE (Polycomb response elements). Previous work has shown that the repressive activity of inactive PRE in transgenes can be induced by architectural protein-binding sites. It was shown that the induction of repression is associated with the recruitment of PcG/TrxG proteins, including the DNA-binding factors Pho and Combgap. The present study the association of the two other PRE DNA-binding factors, GAF and Psq, with bxdPRE in the presence and absence of sites for architectural proteins. As a result, it was shown that both factors can be efficiently recruited to the bxdPRE only in the presence of adjacent binding sites for architectural proteins Su(Hw), CTCF, or Pita.

Friday, January 6th - Disease Models

Cao, H., Tang, J., Liu, Q., Huang, J. and Xu, R. (2022). Autism-like behaviors regulated by the serotonin receptor 5-HT2B in the dorsal fan-shaped body neurons of Drosophila melanogaster. Eur J Med Res 27(1): 203. PubMed ID: 36253869
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and repetitive stereotyped behaviors. Previous studies have reported an association of serotonin or 5-hydroxytryptamine (5-HT) with ASD, but the specific receptors and neurons by which serotonin modulates autistic behaviors have not been fully elucidated. RNAi-mediated knockdown was done to destroy the function of tryptophan hydroxylase (Trh) and all the five serotonin receptors. Given that ubiquitous knockdown of 5-HT2B showed significant defects in social behaviors, the CRISPR/Cas9 system was used to knock out the 5-HT2B receptor gene. Social space assays and grooming assays were the major methods used to understand the role of serotonin and related specific receptors in autism-like behaviors of Drosophila melanogaster. A close relationship was identified between serotonin and autism-like behaviors reflected by increased social space distance and high-frequency repetitive behavior in Drosophila. The binary expression system was further utilized to knock down all the five 5-HT receptors; the 5-HT2B receptor was observed to act as the main receptor responsible for the normal social space and repetitive behavior in Drosophila for the specific serotonin receptors underlying the regulation of these two behaviors. These data also showed that neurons in the dorsal fan-shaped body (dFB), which expressed 5-HT2B, were functionally essential for the social behaviors of Drosophila. Collectively, these data suggest that serotonin levels and the 5-HT2B receptor are closely related to the social interaction and repetitive behavior of Drosophila. Of all the 5 serotonin receptors, 5-HT2B receptor in dFB neurons is mainly responsible for serotonin-mediated regulation of autism-like behaviors.
Sen, A. and Cox, R. T. (2022). Loss of Drosophila Clueless differentially affects the mitochondrial proteome compared to loss of Sod2 and Pink1. Front Physiol 13: 1004099. PubMed ID: 36388112
Mitochondria contain their own DNA, mitochondrial DNA, which encodes thirteen proteins. However, mitochondria require thousands of proteins encoded in the nucleus to carry out their many functions. Identifying the definitive mitochondrial proteome has been challenging as methods isolating mitochondrial proteins differ and different tissues and organisms may have specialized proteomes. Mitochondrial diseases arising from single gene mutations in nucleus encoded genes could affect the mitochondrial proteome, but deciphering which effects are due to loss of specific pathways or to accumulated general mitochondrial damage is difficult. To identify specific versus general effects, this study has taken advantage of mutations in three Drosophila genes, clueless, Sod2, and Pink1, which are required for mitochondrial function through different pathways. Changes were measired in each mutant's mitochondrial proteome using quantitative tandem mass tag mass spectrometry. This analysis identified protein classes that are unique to each mutant and those shared between them, suggesting that some changes in the mitochondrial proteome are due to general mitochondrial damage whereas others are gene specific. For example, clueless mutants had the greatest number of less and more abundant mitochondrial proteins whereas loss of all three genes increased stress and metabolism proteins. This study is the first to directly compare in vivo steady state levels of mitochondrial proteins by examining loss of three pathways critical for mitochondrial function. These data could be useful to understand disease etiology, and how mutations in genes critical for mitochondrial function cause specific mitochondrial proteomic changes as opposed to changes due to generalized mitochondrial damage.
Keshav, N., Ammankallu, R., Shashidhar, Paithankar, J. G., Baliga, M. S., Patil, R. K., Kudva, A. K. and Raghu, S. V. (2022). Dextran sodium sulfate alters antioxidant status in the gut affecting the survival of Drosophila melanogaster. 3 Biotech 12(10): 280. PubMed ID: 36275361
Inflammatory bowel disease (IBD) is a group of disorders characterized by chronic inflammation in the intestine. Several studies confirmed that oxidative stress induced by an enormous amount of reactive free radicals triggers the onset of IBD. Currently, there is an increasing trend in the global incidence of IBD and it is coupled with a lack of adequate long-term therapeutic options. At the same time, progress in research to understand the pathogenesis of IBD has been hampered due to the absence of adequate animal models. Currently, the toxic chemical Dextran Sulfate Sodium (DSS) induced gut inflammation in rodents is widely perceived as a good model of experimental colitis or IBD. Drosophila melanogaster, a genetic animal model, shares ~ 75% sequence similarity to genes causing different diseases in humans and also has conserved digestion and absorption features. Therefore, the current study used Drosophila as a model system to induce and investigate DSS-induced colitis. Anatomical, biochemical, and molecular analyses were performed to measure the levels of inflammation and cellular disturbances in the gastrointestinal (GI) tract of Drosophila. This study shows that DSS-induced inflammation lowers the levels of antioxidant molecules, affects the life span, reduces physiological activity and induces cellular damage in the GI tract mimicking pathophysiological features of IBD in Drosophila. Such a DSS-induced Drosophila colitis model can be further used for understanding the molecular pathology of IBD and screening novel drugs.
Suzuta, S., Nishida, H., Ozaki, M., Kohno, N., Le, T. D. and Inoue, Y. H. (2022). smin suppresses progression of muscle aging via activation of the AMP kinase-mediated pathways in Drosophila adults. Eur Rev Med Pharmacol Sci 26(21): 8039-8056. PubMed ID: 36394755
Metformin, a medicine used for the treatment of type 2 diabetes, was previously reported to suppress age-dependent hyperproliferation of intestinal stem cells in Drosophila. This study aimed to investigate its anti-aging effects on other tissues, such as adult muscle and elucidate the mechanisms underlying the anti-ageing effect. To evaluate the anti-muscle ageing effect of Metformin, ubiquitinated protein aggregates accumulated in adult muscle as the flies age was visualized by immunostaining, and the total pixel size of the aggregates was measured. Continuous metformin feeding significantly extended the lifespan of Drosophila adults. Furthermore, the feeding suppressed the aging-dependent accumulation of ubiquitinated aggregates in adult muscle. To delineate the mechanism through which metformin influences the muscle aging phenotype, the constitutively active AMPK was induced specifically in the muscles; the activation of the AMPK-mediated pathway was sufficient for the anti-aging effect of Metformin. Furthermore, the AMPK-mediated downregulation of Tor-mediated pathways, subsequent induction of an eIF-4E inhibitor were involved in the effect. These genetic data suggested that the metformin effect is related to the partial suppression of protein synthesis in ribosomes. Furthermore, metformin stimulated autophagy induction in adult muscles. These results suggest that metformin can be regarded as an anti-aging compound in Drosophila muscle. The stimulation of autophagy was also involved in the anti-aging effect, which delayed the progression of muscle aging in Drosophila adults.
Cheng, A., Liu, C., Ye, W., Huang, D., She, W., Liu, X., Fung, C. P., Xu, N., Suen, M. C., Ye, W., Sung, H. H. Y., Williams, I. D., Zhu, G. and Qian, P. Y. (2022). Selective C9orf72 G-Quadruplex-Binding Small Molecules Ameliorate Pathological Signatures of ALS/FTD Models. J Med Chem 65(19): 12825-12837. PubMed ID: 36226410
The G-quadruplex (G4) forming C9orf72 GGGGCC (G4C2) expanded hexanucleotide repeat (EHR) is the predominant genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Developing selective G4-binding ligands is challenging due to the conformational polymorphism and similarity of G4 structures. Three first-in-class marine natural products were identified, chrexanthomycin A (cA), chrexanthomycin B (cB), and chrexanthomycin C (cC), with remarkable bioactivities. Thereinto, cA shows the highest permeability and lowest cytotoxicity to live cells. NMR titration experiments and in silico analysis demonstrate that cA, cB, and cC selectively bind to DNA and RNA G4C2 G4s. Notably, cA and cC dramatically reduce G4C2 EHR-caused cell death, diminish G4C2 RNA foci in (G4C2)(29)-expressing Neuro2a cells, and significantly eliminate ROS in HT22 cells. In (G4C2)(29)-expressing Drosophila, cA and cC significantly rescue eye degeneration and improve locomotor deficits. Overall, these findings reveal that cA and cC are potential therapeutic agents deserving further clinical study.
Vourkou, E., Paspaliaris, V., Bourouliti, A., Zerva, M. C., Prifti, E., Papanikolopoulou, K. and Skoulakis, E. M. C. (2022). Differential Effects of Human Tau Isoforms to Neuronal Dysfunction and Toxicity in the Drosophila CNS. Int J Mol Sci 23(21). PubMed ID: 36361774
Accumulation of highly post-translationally modified tau proteins is a hallmark of neurodegenerative disorders known as tauopathies, the most common of which is Alzheimer's disease. Although six tau isoforms are found in the human brain, the majority of animal and cellular tauopathy models utilize a representative single isoform. However, the six human tau isoforms present overlapping but distinct distributions in the brain and are differentially involved in particular tauopathies. These observations support the notion that tau isoforms possess distinct functional properties important for both physiology and pathology. To address this hypothesis, the six human brain tau isoforms were expressed singly in the Drosophila brain and their effects in an established battery of assays measuring neuronal dysfunction, vulnerability to oxidative stress and life span were systematically assessed comparatively. The results reveal isoform-specific effects clearly not attributed to differences in expression levels but correlated with the number of microtubule-binding repeats and the accumulation of a particular isoform in support of the functional differentiation of these tau isoforms. Delineation of isoform-specific effects is essential to understand the apparent differential involvement of each tau isoform in tauopathies and their contribution to neuronal dysfunction and toxicity.

Thursday, January 5th - Enzymes and Protein Expression, Evolution, Structure and Function

Koyanagi, M., Honda, H., Yokono, H., Sato, R., Nagata, T. and Terakita, A. (2022). Expression of a homologue of a vertebrate non-visual opsin Opn3 in the insect photoreceptors. Philos Trans R Soc Lond B Biol Sci 377(1862): 20210274. PubMed ID: 36058246
Insect vision starts with light absorption by visual pigments based on opsins that drive Gq-type G protein-mediated phototransduction. Since Drosophila, the most studied insect in vision research, has only Gq-coupled opsins, the Gq-mediated phototransduction has been solely focused on insect vision for decades. However, genome projects on mosquitos uncovered non-canonical insect opsin genes, members of the Opn3 or c-opsin group composed of vertebrate and invertebrate non-visual opsins. This study reports that a homologue of Opn3, MosOpn3 (Asop12) is expressed in eyes of a mosquito Anopheles stephensi. In situ hybridization analysis revealed that MosOpn3 is expressed in dorsal and ventral ommatidia, in which only R7 photoreceptor cells express MosOpn3. It was also found that Asop9, a Gq-coupled visual opsin, exhibited co-localization with MosOpn3. Spectroscopic analysis revealed that Asop9 forms a blue-sensitive opsin-based pigment. Thus, the Gi/Go-coupled opsin MosOpn3, which forms a green-sensitive pigment, is co-localized with Asop9, a Gq-coupled opsin that forms a blue-sensitive visual pigment. Since these two opsin-based pigments trigger different phototransduction cascades, the R7 photoreceptors could generate complex photoresponses to blue to green light. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods.
Liu, H., Wu, Y., Li, C., Tang, Q. and Zhang, Y. W. (2022). Molecular docking and biochemical validation of (-)-syringaresinol-4-O-β-D-apiofuranosyl-(1->2)-β-D-glucopyranoside binding to an allosteric site in monoamine transporters. Front Pharmacol 13: 1018473. PubMed ID: 36386236 >
Albizia julibrissin Durazz is one of the most common herbs used for depression and anxiety treatment, but its mechanism of action as an antidepressant or anxiolytic drug have not been fully understood. Previous work has isolated and identified one lignan glycoside compound from Albizia Julibrissin Durazz, (-)-syringaresinol-4-O-β-D-apiofuranosyl-(1>2)-β-D-glucopyranoside (SAG), that inhibited all three monoamine transporters with a mechanism of action different from that of the conventional antidepressants. This study, generated homology models for human dopamine transporter and human norepinephrine transporter, based on the X-ray structure of Drosophila dopamine transporter, and conducted the molecular docking of SAG to all three human monoamine transporters. The computational results indicated that SAG binds to an allosteric site (S2) that has been demonstrated to be formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule connected to the central site in these monoamine transporters. In addition, it wae demonstrated that SAG stabilizes a conformation of serotonin transporter with both the extracellular and cytoplasmic pathways closed. Furthermore, mutagenesis of the residues in both the allosteric and orthosteric sites was performed to biochemically validate SAG binding in all three monoamine transporters. These results are consistent with the molecular docking calculation and support the association of SAG with the allosteric site. It is expect that this herbal molecule could become a lead compound for the development of new therapeutic agents with a novel mechanism of action.
Dix, T. C., Haussmann, I. U., Brivio, S., Nallasivan, M. P., HadzHiev, Y., Muller, F., Müller, B., Pettitt, J. and Soller, M. (2022). CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals. Rna 28(10): 1377-1390. PubMed ID: 35970556
Cap methyltransferases (CMTrs) O methylate the 2' position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. This study showed that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation.
Taghert, P. H. (2022). The incidence of candidate binding sites for β-arrestin in Drosophila neuropeptide GPCRs. PLoS One 17(11): e0275410. PubMed ID: 36318573
To support studies of neuropeptide neuromodulation, beta-arrestin binding sites (BBS's) by evaluating the incidence of BBS sequences among the C terminal tails (CTs) of each of the 49 Drosophila melanogaster neuropeptide GPCRs. BBS were identified by matches with a prediction derived from structural analysis of rhodopsin:arrestin and vasopressin receptor: arrestin complexes. To increase the rigor of the identification, the conservation of BBS sequences was determined between two long-diverged species D. melanogaster and D. virilis. There is great diversity in the profile of BBS's in this group of GPCRs. Evidence is presented for conserved BBS's in a majority of the Drosophila neuropeptide GPCRs; notably some have no conserved BBS sequences. In addition, certain GPCRs display numerous conserved compound BBS's, and many GPCRs display BBS-like sequences in their intracellular loop (ICL) domains as well. Finally, 20 of the neuropeptide GPCRs are expressed as protein isoforms that vary in their CT domains. BBS profiles are typically different across related isoforms suggesting a need to diversify and regulate the extent and nature of GPCR:arrestin interactions. This work provides the initial basis to initiate future in vivo, genetic analyses in Drosophila to evaluate the roles of arrestins in neuropeptide GPCR desensitization, trafficking and signaling.
Dai, S., Qu, L., Li, J., Zhang, Y., Jiang, L., Wei, H., Guo, M., Chen, X. and Chen, Y. (2022). Structural insight into the ligand binding mechanism of aryl hydrocarbon receptor. Nat Commun 13(1): 6234. PubMed ID: 36266304
The aryl hydrocarbon receptor (AHR), a member of the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors, plays important roles in regulating xenobiotic metabolism, cellular differentiation, stem cell maintenance, as well as immunity. More recently, AHR has gained significant interest as a drug target for the development of novel cancer immunotherapy drugs. Detailed understanding of AHR-ligand binding has been hampered for decades by the lack of a three-dimensional structure of the AHR PAS-B domain. A present multiple crystal structures of the Drosophila AHR PAS-B domain, including its apo, ligand-bound, and AHR nuclear translocator (ARNT) PAS-B-bound forms. Together with biochemical and cellular assays, these data reveal structural features of the AHR PAS-B domain, provide insights into the mechanism of AHR ligand binding, and provide the structural basis for the future development of AHR-targeted therapeutics.
Kovacs, L., Fatalska, A. and Glover, D. M. (2022). Targeting Drosophila Sas6 to mitochondria reveals its high affinity for Gorab. Biol Open 11(11). PubMed ID: 36331102
The ability to relocalize proteins to defined subcellular locations presents a powerful tool to examine protein-protein interactions that can overcome a tendency of non-targeted exogenous proteins to form inaccessible aggregates. This study shows that a 24-amino-acid sequence from the Drosophila proapoptotic protein Hid's tail anchor (HTA) domain can target exogenous proteins to the mitochondria in Drosophila cells. This HTA tag was used to target the Drosophila centriole cartwheel protein Sas6 to the mitochondria, and show that both exogenous and endogenous Gorab can be co-recruited from the Golgi to the new mitochondrial site. This accords with a previous observation that monomeric Drosophila Gorab binds Sas6 to become centriole associated with a 50-fold greater affinity than dimeric Gorab binds Rab6 to become localized at the Golgi. Strikingly, Drosophila Sas6 can bind both Drosophila Gorab and its human GORAB ortholog, whereas human SAS6 is unable to bind either GORAB or Gorab. These findings in relation to the evolutionary conservation of Gorab and the divergence of Sas6, possibly reflecting known differences in persistence of the cartwheel in the centriole duplication cycle of fly and human cells.

Thursday, January 4th - Adult Neural Development and Function

Yamagata, N., Imanishi, Y., Wu, H., Kondo, S., Sano, H. and Tanimoto, H. (2022). Nutrient responding peptide hormone CCHamide-2 consolidates appetitive memory. Front Behav Neurosci 16: 986064. PubMed ID: 36338876
CCHamide-2 (CCHa2) is a protostome excitatory peptide ortholog known for various arthropod species. In fruit flies, CCHa2 plays a crucial role in the endocrine system, allowing peripheral tissue to communicate with the central nervous system to ensure proper development and the maintenance of energy homeostasis. Since the formation of odor-sugar associative long-term memory (LTM) depends on the nutrient status in an animal, CCHa2 may play an essential role in linking memory and metabolic systems. This study shows that CCHa2 signals are important for consolidating appetitive memory by acting on the rewarding dopamine neurons. Genetic disruption of CCHa2 using mutant strains abolished appetitive LTM but not short-term memory (STM). A post-learning thermal suppression of CCHa2 expressing cells impaired LTM. In contrast, a post-learning thermal activation of CCHa2 cells stabilized STM induced by non-nutritious sugar into LTM. The receptor of CCHa2, CCHa2-R, was expressed in a subset of dopamine neurons that mediate reward for LTM. In accordance, the receptor expression in these dopamine neurons was required for LTM specifically. It is thus concluded that CCHa2 conveys a sugar nutrient signal to the dopamine neurons for memory consolidation. This finding establishes a direct interplay between brain reward and the putative endocrine system for long-term energy homeostasis.
Xie, N. and Gross, A. D. (2022). Muscarinic acetylcholine receptor activation synergizes the knockdown and toxicity of GABA-gated chloride channel insecticides. Pest Manag Sci 78(11): 4599-4607. PubMed ID: 35841135
Pest management requires continual identification of new physiological targets and strategies to control pests affecting agriculture and public/animal health. It is proposed that the muscarinic system is a target for agrochemicals because of its physiological importance. Unlike the muscarinic system, gamma-amino butyric acid (GABA) receptors are an established insecticide target. This study investigated target-site synergism using small molecule probes (agonist and antagonist) against the muscarinic system and their ability to enhance the toxicity of GABAergic insecticides in Drosophila melanogaster. Oral delivery of pilocarpine (muscarinic agonist) enhanced the toxicity of dieldrin, fipronil, and lindane, resulting in synergist ratios (SRs) between 4-32-fold (orally delivered) or between 2-67-fold when insecticides were topically applied. The synergism between pilocarpine and the GABA-insecticides was greater than the synergism observed with atropine (muscarinic antagonist), and was greater, or comparable, to the synergism observed with the metabolic inhibitor piperonyl butoxide. In addition to lethality, pilocarpine increased the knockdown of lindane. The mechanism of synergism was also investigated in the central nervous system using extracellular electrophysiology, where pilocarpine (3 μmo/L) lowered the half-maximal inhibitory concentration (IC(50)) of lindane and fipronil's IC(50). It is concluded that convergence of the cellular function between the muscarinic and GABAergic systems enhanced the insecticidal activity of GABA receptor blocking insecticides through the modulation of the central nervous system (CNS).
Shin, M. and Venton, B. J. (2022). Fast-Scan Cyclic Voltammetry (FSCV) Reveals Behaviorally Evoked Dopamine Release by Sugar Feeding in the Adult Drosophila Mushroom Body. Angew Chem Int Ed Engl 61(44): e202207399. PubMed ID: 35989453
Drosophila melanogaster, the fruit fly, is an excellent model organism for studying dopaminergic mechanisms and simple behaviors, but methods to measure dopamine during behavior are needed. This study developed fast-scan cyclic voltammetry (FSCV) to track in vivo dopamine during sugar feeding. First, acetylcholine stimulation was used to evaluate the feasibility of in vivo measurements in an awake fly. Next, sugar feeding was tested by placing sucrose solution near the fly proboscis. In the mushroom body medial tip, 1 pmol acetylcholine and sugar feeding released 0.49±0.04 μM and 0.31#177;0.06 μM dopamine, respectively but sugar-evoked release lasted longer than with acetylcholine. Administering the dopamine transporter inhibitor nisoxetine or D2 receptor antagonist flupentixol significantly increased sugar-evoked dopamine. This study develops FSCV to measure behaviorally evoked release in fly, enabling Drosophila studies of neurochemical control of reward, learning, and memory behaviors.
Xie, J., Han, Y., Liang, Y., Peng, L. and Wang, T. (2022). Drosophila HisT is a specific histamine transporter that contributes to histamine recycling in glia. Sci Adv 8(43): eabq1780. PubMed ID: 36288320
Histamine is an important monoamine neurotransmitter that regulates multiple physiological activities in both vertebrates and invertebrates. Clearance and recycling of histamine are critical for sustaining histaminergic transmission. However, unlike other monoamine neurotransmitters, a histamine-specific transporter capable of clearing histamine from the synaptic cleft has not been identified. Through an in vitro histamine uptake screening, this study identified an epithelial glia-expressing transporter, HisT (Histamine Transporter), that specifically transports histamine into cells. HisT misexpression in both pre- and postsynaptic neurons revealed a critical in vivo role for HisT in histamine transport and synaptic transmission. Last, null hist alleles were generated, and key physiological roles were demonstrated of HisT in maintaining histamine pools and sustaining visual transmission when the de novo synthesis of histamine synthesis was reduced. This work identifies the first transporter that specifically recycles histamine and further indicates that the histamine clearance pathway may involve both the uptake-1 and uptake-2 transport systems.
Zhao, Y., Duan, J., Han, Z., Engstrom, Y. and Hartenstein, V. (2022). Identification of a GABAergic neuroblast lineage modulating sweet and bitter taste sensitivity. Curr Biol. PubMed ID: 36347251
In Drosophila melanogaster, processing of gustatory information and controlling feeding behavior are executed by neural circuits located in the subesophageal zone (SEZ) of the brain. Gustatory receptor neurons (GRNs) project their axons in the primary gustatory center (PGC), which is located in the SEZ. To address the function of the PGC, detailed information is needed about the different classes of gustatory interneurons that frame the PGC. This work screened large collections of driver lines for SEZ interneuron-specific labeling and subsequently used candidate lines to access the SEZ neuroblast lineages. 130 Gal4 lines were converted to LexA drivers, and functional screening was carried out using calcium imaging. One neuroblast lineage, TRdm, was found whose neurons responded to both sweet and bitter tastants and formed green fluorescent protein (GFP) reconstitution across synaptic partners (GRASP)-positive synapses with sweet sensory neurons. TRdm neurons express the inhibitory transmitter GABA, and silencing these neurons increases appetitive feeding behavior. These results demonstrate that TRdm generates a class of inhibitory local neurons that control taste sensitivity in Drosophila.
Turner, M. H., Krieger, A., Pang, M. M. and Clandinin, T. R. (2022). Visual and motor signatures of locomotion dynamically shape a population code for feature detection in Drosophila. Elife 11. PubMed ID: 36300621
Natural vision is dynamic: as an animal moves, its visual input changes dramatically. How can the visual system reliably extract local features from an input dominated by self-generated signals? In Drosophila, diverse local visual features are represented by a group of projection neurons with distinct tuning properties. This study describes a connectome-based volumetric imaging strategy to measure visually evoked neural activity across this population. Local visual features are shown to be jointly represented across the population, and a shared gain factor improves trial-to-trial coding fidelity. A subset of these neurons, tuned to small objects, is modulated by two independent signals associated with self-movement, a motor-related signal, and a visual motion signal associated with rotation of the animal. These two inputs adjust the sensitivity of these feature detectors across the locomotor cycle, selectively reducing their gain during saccades and restoring it during intersaccadic intervals. This work reveals a strategy for reliable feature detection during locomotion.

Wednesday, January 3rd - Enhancers and Transcriptional Regulation

Yoshioka, Y., Anzai, K., Kowada, R., Hiratsuka, K., Hirayabu, T., Yasuda, M., Ohkawa, Y., Sato, T., Suyama, M., Yoshida, H. and Yamaguchi, M. (2022). Drosophila transcription factor NF-Y suppresses transcription of the lipase 4 gene, a key gene for lipid storage. Exp Cell Res 420(1): 113307. PubMed ID: 36028059
The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB, and NF-YC. Although it is suggested that NF-Y activity is essential for normal tissue homeostasis, survival, and metabolic function, its precise role in lipid metabolism is not clarified yet. In Drosophila, eye disc specific knockdown of Drosophila NF-YA (dNF-YA) induced aberrant morphology of the compound eye, the rough eye phenotype in adults and mutation of the lipase 4 (lip4) gene suppressed the rough eye phenotype. RNA-seq analyses with dNF-YA knockdown third instar larvae identified the lip4 gene as one of the genes that are up-regulated by the dNF-YA knockdown. Three dNF-Y-binding consensuses were found in the 5'flanking region of the lip4 gene, and a chromatin immunoprecipitation assay with the specific anti-dNF-YA IgG demonstrated dNF-Y binding to this genomic region. The luciferase transient expression assay with cultured Drosophila S2 cells and the lip4 promoter-luciferase fusion genes with and without mutations in the dNF-Y-binding consensuses showed that each of the three dNF-Y consensus sequences negatively regulated lip4 gene promoter activity. Consistent with these results, qRT-PCR analysis with the dNF-YA knockdown third instar larvae revealed that endogenous lip4 mRNA levels were increased by the knockdown of dNF-YA in vivo. The specific knockdown of dNF-YA in the fat body with the collagen-GAL4 driver resulted in smaller oil droplets in the fat body cells. Collectively, these results suggest that dNF-Y is involved in lipid storage through its negative regulation of lip4 gene transcription.
Uyehara, C. M., Leatham-Jensen, M. and McKay, D. J. (2022). Opportunistic binding of EcR to open chromatin drives tissue-specific developmental responses. Proc Natl Acad Sci U S A 119(40): e2208935119. PubMed ID: 36161884
Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and ultraspiracle proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. This study investigates the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates morphogenesis. EcR was found to function bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, it was found that EcR does not control chromatin accessibility at genomic targets. It is concluded that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.
Hauptman, G., Reichert, M. C., Abdal Rhida, M. A. and Evans, T. A. (2022). Characterization of enhancer fragments in Drosophila robo2. Fly (Austin) 16(1): 312-346. PubMed ID: 36217698
Receptor proteins of the Roundabout (Robo) family regulate axon guidance decisions during nervous system development. Among the three Drosophila robo family genes (robo1, robo2 and robo3), robo2 displays a dynamic expression pattern and regulates multiple axon guidance outcomes, including preventing midline crossing in some axons, promoting midline crossing in others, forming lateral longitudinal axon pathways, and regulating motor axon guidance. The identity and location of enhancer elements regulating robo2's complex and dynamic expression pattern in different neural cell types are unknown. This study characterize a set of 17 transgenic lines expressing GAL4 under the control of DNA sequences derived from noncoding regions in and around robo2, to identify enhancers controlling specific aspects of robo2 expression in the embryonic ventral nerve cord. Individual fragments were idetified that confer expression in specific cell types where robo2 is known to function, including early pioneer neurons, midline glia and lateral longitudinal neurons. These results indicate that robo2's dynamic expression pattern is specified by a combination of enhancer elements that are active in different subsets of cells. robo2 expression in lateral longitudinal axons represents two genetically separable subsets of neurons, and compare their axon projections with each other and with Fasciclin II (FasII), a commonly used marker of longitudinal axon pathways. In addition, a general description is provided of each fragment's expression in embryonic tissues outside of the nervous system, to serve as a resource for other researchers interested in robo2 expression and its functional roles outside the central nervous system.
Begeman, I. J., Emery, B., Kurth, A. and Kang, J. (2022). Regeneration and developmental enhancers are differentially compatible with minimal promoters. Dev Biol 492: 47-58. PubMed ID: 36167150
Enhancers and promoters are cis-regulatory elements that control gene expression. Enhancers are activated in a cell type-, tissue-, and condition-specific manner to stimulate promoter function and transcription. Zebrafish have emerged as a powerful animal model for examining the activities of enhancers derived from various species through transgenic enhancer assays, in which an enhancer is coupled with a minimal promoter. However, the efficiency of minimal promoters and their compatibility with multiple developmental and regeneration enhancers have not been systematically tested in zebrafish. Thus, the efficiency of six minimal promoters were assessed and the compatibility of the promoters with developmental and regeneration enhancers was comprehensively interrogated. Thus study found that the fos minimal promoter and Drosophila synthetic core promoter (DSCP) yielded high rates of leaky expression that may complicate the interpretation of enhancer assays. Notably, the adenovirus E1b promoter, the zebrafish lepb 0.8-kb (P0.8) and lepb 2-kb (P2) promoters, and a new zebrafish synthetic promoter (ZSP) that combines elements of the E1b and P0.8 promoters drove little or no ectopic expression, making them suitable for transgenic assays. This study also found significant differences in compatibility among specific combinations of promoters and enhancers, indicating the importance of promoters as key regulatory elements determining the specificity of gene expression. This study provides guidelines for transgenic enhancer assays in zebrafish to aid in the discovery of functional enhancers regulating development and regeneration.
Stevens, C. A., Stott, H. L., Desai, S. V. and Yakoby, N. (2022). Shared cis-regulatory modules control expression of the tandem paralogs midline and H15 in the follicular epithelium. Development 149(22). PubMed ID: 36278857
The posterior end of the follicular epithelium is patterned by midline (MID) and its paralog H15, the Drosophila homologs of the mammalian Tbx20 transcription factor. Two perviously identified cis-regulatory modules (CRMs) recapitulate the endogenous pattern of mid in the follicular epithelium. Using CRISPR/Cas9 genome editing, this study demonstrated redundant activity of these mid CRMs. Although the deletion of either CRM alone generated marginal change in mid expression, the deletion of both CRMs reduced expression by 60%. Unexpectedly, the deletion of the 5' proximal CRM of mid eliminated H15 expression. Interestingly, expression of these paralogs in other tissues remained unaffected in the CRM deletion backgrounds. These results suggest that the paralogs are regulated by a shared CRM that coordinates gene expression during posterior fate determination. The consistent overlapping expression of mid and H15 in various tissues may indicate that the paralogs could also be under shared regulation by other CRMs in these tissues.
Whitney, P. H., Shrestha, B., Xiong, J., Zhang, T. and Rushlow, C. A. (2022). Shadow enhancers modulate distinct transcriptional parameters that differentially effect downstream patterning events. Development 149(21). PubMed ID: 36264246
Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called 'shadow enhancers', which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, but the biological significance of this remains unclear. This study characterize the shadow enhancer pair controlling the gene short gastrulation (sog). Either the intronic proximal enhancer or the upstream distal enhancer were removed, and sog transcriptional kinetics were monitored. Notably, each enhancer differs in sog spatial expression, timing of activation and RNA Polymerase II loading rates. In addition, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. Whereas activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, the downstream signaling consequences resulting from the loss of either enhancer were monitored, changes in tissue patterning were found that can be explained by the differences in transcriptional kinetics measured.
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