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Friday, January 13th - Disease Models

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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
Summary:
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
Summary:
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. We 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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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. We generated near-isogenic lines 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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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 >
Summary:
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. Our 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
Summary:
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
Summary:
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, I determined the conservation of BBS sequences 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. I present evidence 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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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
Summary:
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, we assessed the efficiency of six minimal promoters and comprehensively interrogated the compatibility of the promoters with developmental and regeneration enhancers. 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
Summary:
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
Summary:
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.

Friday, December 30th - Gonads

Siddall, N. A., Casagranda, F., Johanson, T. M., Dominado, N., Heaney, J., Sutherland, J. M., McLaughlin, E. A. and Hime, G. R. (2022). MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function. Cell Death Discov 8(1): 455. PubMed ID: 36371343
Summary:
The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. This study has identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). FSC maintenance and escort cell function were shown to specifically require the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. It was further demonstrated that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, this study shows that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.
Garlovsky, M. D., Sandler, J. A. and Karr, T. L. (2022). Functional Diversity and Evolution of the Drosophila Sperm Proteome. Mol Cell Proteomics 21(10): 100281. PubMed ID: 35985624
Summary:
Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. This study provides an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and this study provides the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. Nine of the 16 Y-linked proteins were identified, including known testis-specific male fertility factors. Almost one-half of known Drosophila ribosomal proteins were identified in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, the expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. A significant fraction of 'sperm-associated Sfps' are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, these results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.
O'Brien, C. E., Younger, S. H., Jan, L. Y. and Jan, Y. N. (2023). The GARP complex prevents sterol accumulation at the trans-Golgi network during dendrite remodeling. J Cell Biol 222(1). PubMed ID: 36239632
Summary:
Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, both complexes were found to be required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, genes that regulate sterols and related lipids at the TGN were investigated. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.
Finger, D. S., Williams, A. E., Holt, V. V. and Ables, E. T. (2022). Novel roles for RNA binding proteins squid, hephaesteus, and Hrb27C in Drosophila oogenesis. Dev Dyn. PubMed ID: 36308715
Summary:
Reproductive capacity in many organisms is maintained by germline stem cells (GSCs). A complex regulatory network influences stem cell fate, including intrinsic factors, local signals, and hormonal and nutritional cues. Posttranscriptional regulatory mechanisms ensure proper cell fate transitions, promoting germ cell differentiation to oocytes. As essential RNA binding proteins with constitutive functions in RNA metabolism, heterogeneous nuclear ribonucleoproteins (hnRNPs) have been implicated in GSC function and axis specification during oocyte development. HnRNPs support biogenesis, localization, maturation, and translation of nascent transcripts. Whether and individual hnRNPs specifically regulate GSC function has yet to be explored. This study demonstratea that hnRNPs are expressed in distinct patterns in the Drosophila germarium. Three hnRNPs, squid, hephaestus, and Hrb27C are cell-autonomously required in GSCs for their maintenance. Although these hnRNPs do not impact adhesion of GSCs to adjacent cap cells, squid and hephaestus (but not Hrb27C) are necessary for proper bone morphogenetic protein signaling in GSCs. Moreover, Hrb27C promotes proper GSC proliferation, whereas hephaestus promotes cyst division. It is concluded find that hnRNPs are independently and intrinsically required in GSCs for their maintenance in adults. These results support the model that hnRNPs play unique roles in stem cells essential for their self-renewal and proliferation.
Sotillos, S., von der Decken, I., Domenech Mercade, I., Srinivasan, S., Sirokha, D., Livshits, L., Vanni, S., Nef, S., Biason-Lauber, A., Rodríguez Gutierrez, D. and Castelli-Gair Hombria, J. (2022). A conserved function of Human DLC3 and Drosophila Cv-c in testis development. Elife 11. PubMed ID: 36326091
Summary:
The identification of genes affecting gonad development is essential to understand the mechanisms causing Variations/Differences in Sex Development (DSD). Recently, a DLC3 mutation was associated with male gonadal dysgenesis in 46,XY DSD patients. This study has investigated the requirement of Cv-c, the Drosophila ortholog of DLC3, in Drosophila gonad development, as well as the functional capacity of DLC3 human variants to rescue cv-c gonad defects. Cv-c was shown to be required to maintain testis integrity during fly development. Cv-c and human DLC3 can perform the same function in fly embryos, as flies carrying wild type but not patient DLC3 variations can rescue gonadal dysgenesis, suggesting functional conservation. It was also demonstrated that the StART domain mediates Cv-c's function in the male gonad independently from the GAP domain's activity. This work demonstrates a role for DLC3/Cv-c in male gonadogenesis and highlights a novel StART domain mediated function required to organize the gonadal mesoderm and maintain its interaction with the germ cells during testis development.
Milas, A., de-Carvalho, J. and Telley, I. A. (2023). Follicle cell contact maintains main body axis polarity in the Drosophila melanogaster oocyte. J Cell Biol 222(2). PubMed ID: 36409222
Summary:
In Drosophila melanogaster, the anterior-posterior body axis is maternally established and governed by differential localization of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the oocyte posterior end, while Par-3/Bazooka is excluded there but maintains its localization along the remaining oocyte cortex. Past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, neither the molecular identity nor the nature of the signal is known. This study provides evidence that mechanical contact of posterior follicle cells (PFCs) with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. Furthermore, evidence is provided that PFC contact maintains Par-1 and oskar mRNA localization and microtubule cytoskeleton polarity in the oocyte. These observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex.

Thursday, December 29th - Larval and Adult Neural Development

Lobb-Rabe, M., DeLong, K., Salazar, R. J., Zhang, R., Wang, Y. and Carrillo, R. A. (2022). Dpr10 and Nocte are required for Drosophila motor axon pathfinding. Neural Dev 17(1): 10. PubMed ID: 36271407
Summary:
The paths axons travel to reach their targets and the subsequent synaptic connections they form are highly stereotyped. How cell surface proteins (CSPs) mediate these processes is not completely understood. The Drosophila neuromuscular junction (NMJ) is an ideal system to study how pathfinding and target specificity are accomplished, as the axon trajectories and innervation patterns are known and easily visualized. Dpr10 is a CSP required for synaptic partner choice in the neuromuscular and visual circuits and for axon pathfinding in olfactory neuron organization. This study shows that Dpr10 is also required for motor axon pathfinding. To uncover how Dpr10 mediates this process, immunoprecipitation followed by mass spectrometry were used to identify Dpr10 associated proteins. One of these, Nocte, is an unstructured, intracellular protein implicated in circadian rhythm entrainment. nocte expression in larvae was mapped; it was found to be widely expressed in neurons, muscles, and glia. Cell-specific knockdown suggests nocte is required presynaptically to mediate motor axon pathfinding. Additionally, nocte and dpr10 genetically interact to control NMJ assembly, suggesting that they function in the same molecular pathway. Overall, these data reveal novel roles for Dpr10 and its newly identified interactor, Nocte, in motor axon pathfinding and provide insight into how CSPs regulate circuit assembly.
Manoim, J. E., Davidson, A. M., Weiss, S., Hige, T. and Parnas, M. (2022). Lateral axonal modulation is required for stimulus-specific olfactory conditioning in Drosophila. Curr Biol 32(20): 4438-4450. PubMed ID: 36130601
Summary:
Effective and stimulus-specific learning is essential for animals' survival. Two major mechanisms are known to aid stimulus specificity of associative learning. One is accurate stimulus-specific representations in neurons. The second is a limited effective temporal window for the reinforcing signals to induce neuromodulation after sensory stimuli. However, these mechanisms are often imperfect in preventing unspecific associations; different sensory stimuli can be represented by overlapping populations of neurons, and more importantly, the reinforcing signals alone can induce neuromodulation even without coincident sensory-evoked neuronal activity. This paper reports a crucial neuromodulatory mechanism that counteracts both limitations and is thereby essential for stimulus specificity of learning. In Drosophila, olfactory signals are sparsely represented by cholinergic Kenyon cells (KCs), which receive dopaminergic reinforcing input. KCs were found to have numerous axo-axonic connections mediated by the muscarinic type-B receptor (mAChR-B). By using functional imaging and optogenetic approaches, it was shown that these axo-axonic connections suppress both odor-evoked calcium responses and dopamine-evoked cAMP signals in neighboring KCs. Strikingly, behavior experiments demonstrate that mAChR-B knockdown in KCs impairs olfactory learning by inducing undesired changes to the valence of an odor that was not associated with the reinforcer. Thus, this local neuromodulation acts in concert with sparse sensory representations and global dopaminergic modulation to achieve effective and accurate memory formation.
Hou, Y. N., Zhang, Y. Y., Wang, Y. R., Wu, Z. M., Luan, Y. X. and Wei, Q. (2022). IFT52 plays an essential role in sensory cilia formation and neuronal sensory function in Drosophila. Insect Sci. PubMed ID: 36326027
Summary:
Cilia are microtubule-based, hair-like organelles involved in sensory function or motility, playing critical roles in many physiological processes such as reproduction, organ development, and sensory perception. In insects, cilia are restricted to certain sensory neurons and sperms, being important for chemical and mechanical sensing, and fertility. Although great progress has been made regarding the mechanism of cilia assembly, the formation of insect cilia remains poorly understand, even in the insect model organism Drosophila. Intraflagellar transport (IFT) is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia. This study investigated the role of IFT52, a core component of IFT-B, in cilia/flagellar formation of Drosophila. Drosophila IFT52 is distributed along the sensory neuronal cilia, and is essential for sensory cilia formation. Deletion of Ift52 results in severe defects in cilia-related sensory behaviors. It should be noted that IFT52 is not detected in spermatocyte cilia or sperm flagella of Drosophila. Accordingly, ift52 mutants can produce sperms with normal motility, supporting a dispensable role of IFT in Drosophila sperm flagella formation. Altogether, IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects.
Jang, W., Lim, J. Y., Kang, S., Kim, M., Hwang, S. W. and Kim, C. (2022). Drosophila ppk19 encodes a proton-gated and mechanosensitive ion channel. Sci Rep 12(1): 18346. PubMed ID: 36319833
Summary:
In Drosophila larvae, nociceptive mdIV sensory neurons detect diverse noxious stimuli and prompt a nociceptive rolling response. Intriguingly, the same neurons also regulate stereotyped larval movement. The channels responsible for transducing these stimuli into electric signals are not yet fully identified. This study undertook genetic and electrophysiological analysis of Ppk19, a member of the Deg/ENaC family of cationic channels. ppk19 mutants exhibited an impaired nociceptive rolling response upon mechanical force and acid, but no impairment in response to noxious temperature and gentle touch. Mutants also exhibited defective larval movement. RNAi against ppk19 in mdIV neurons likewise produced larvae with defects in mechanical and acid nociception and larval movement, but no impairment in detection of heat and gentle touch. Cultured cells transfected with ppk19 produced currents in acid and hypotonic solution, suggesting that ppk19 encodes an ion channel that responds to acid and cell swelling. Taken together, these findings suggest that Ppk19 acts in mdIV neurons as a proton- and mechano-gated ion channel to mediate acid- and mechano-responsive nociception and larval movement.
Ohhara, Y. and Yamanaka, N. (2022). Internal sensory neurons regulate stage-specific growth in Drosophila. Development 149(21). PubMed ID: 36227580
Summary:
Animals control their developmental schedule in accordance with internal states and external environments. In Drosophila larvae, it is well established that nutrient status is sensed by different internal organs, which in turn regulate production of insulin-like peptides and thereby control growth. In contrast, the impact of the chemosensory system on larval development remains largely unclear. A genetic screen was performed to identify gustatory receptor (Gr) neurons regulating growth and development; Gr28a-expressing neurons were found to be required for proper progression of larval growth. Gr28a is expressed in a subset of peripheral internal sensory neurons, which directly extend their axons to insulin-producing cells (IPCs) in the central nervous system. Silencing of Gr28a-expressing neurons blocked insulin-like peptide release from IPCs and suppressed larval growth during the mid-larval period. These results indicate that Gr28a-expressing neurons promote larval development by directly regulating growth-promoting endocrine signaling in a stage-specific manner.
Rylee, J., Mahato, S., Aldrich, J., Bergh, E., Sizemore, B., Feder, L. E., Grega, S., Helms, K., Maar, M., Britt, S. G. and Zelhof, A. C. (2022). A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation. G3 (Bethesda) 12(11). PubMed ID: 36218412
Summary:
Drosophila rhabdomeric terminal photoreceptor differentiation is an extended process taking several days to complete. Following ommatidial patterning by the morphogenetic furrow, photoreceptors are sequentially recruited and specified, and terminal differentiation begins. Key events of terminal differentiation include the establishment of apical and basolateral domains, rhabdomere and stalk formation, inter-rhabdomeral space formation, and expression of phototransduction machinery. This paper reports an RNAi screen to identify additional molecules and cellular pathways required for photoreceptor terminal differentiation. First, several eye-specific GAL4 drivers for correct spatial and temporal specificity and identified Pph13-GAL4 were tested to find the most appropriate GAL4 line for the screen. Lines available through the Transgenic RNAi Project were screened, and lines were isolated that when combined with Pph13-GAL4 resulted in the loss of the deep pseudopupil, as a readout for abnormal differentiation. In the end, 6,189 lines were screened, representing 3,971 genes, and 64 genes were identified, illuminating potential new regulatory molecules and cellular pathways for the differentiation and organization of Drosophila rhabdomeric photoreceptors.

Wednesday, December 28th - Larval and Adult Development

Shimell, M. and O'Connor, M. B. (2022). The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster. MicroPubl Biol 2022. PubMed ID: 35991292
Summary:
The steroid hormone 20-hydroxyecdysone (20E) is essential for proper development and the timing of intermediary stage transitions in insects. As a result, there is intense interest in identifying and defining the roles of the ws and signaling pathways that regulate 20E production in the prothoracic gland (PG), the major endocrine organ of juvenile insect phases. Transcriptomics is one powerful tool that has been used to identify novel genes that are up- or down-regulated in the PG which may contribute to 20E regulation. Additional functional characterization of putative regulatory candidate genes typically involves qRT-PCR and/or RNAi mediated knockdown of the candidate mRNA in the PG to assess whether the gene's expression shows temporal regulation in the PG and whether its expression is essential for proper 20E production and the correct timing of developmental transitions. While these methods have proved fruitful for identifying novel regulators of 20E production, characterizing the null phenotype of putative regulatory genes is the gold standard for assigning gene function since RNAi is known to generate various types of "off target" effects. This study describes the genetic null mutant phenotype of the Drosophila melanogaster Cyp6t3 gene. Cyp6t3 was originally identified as a differentially regulated gene in a PG microarray screen and assigned a place in the "Black Box" step of the E biosynthetic pathway based on RNAi mediated knockdown phenotypes and rescue experiments involving feeding of various intermediate compounds of the E biosynthetic pathway. In contrast, it was found that Crispr generated null mutations in Cyp6t3 are viable and have normal developmental timing. Therefore, it is concluded that Cyp6t3 is not required for E production under typical lab growth conditions and therefore is not an obligate enzymatic component of the Black Box.
Wu, J. J., Chen, F., Yang, R., Shen, C. H., Ze, L. J., Jin, L. and Li, G. Q. (2022). Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle. Biology (Basel) 11(11). PubMed ID: 36358341
Summary:
Ecdysone-induced protein 93F (E93) plays triple roles during post-embryonic development in insects whose juvenile instars are more than four. However, it only acts as a specifier of adult structures in Drosophila flies whose larval instars are fixed at three. In this study, the functions were determined of E93 in the eggplant lady beetle (Henosepilachna vigintioctopunctata), which has four larval instars. E93 was shown to be abundantly expressed at the prepupal and pupal stages. A precocious inhibition of the juvenile hormone signal by RNA interference (RNAi) of HvKr-h1 or HvHairy, two vital downstream developmental effectors, at the penultimate instar larval stage increased the expression of E93, Conversely, ingestion of JH by the third-instar larvae stimulated the expression of HvKr-h1 but repressed the transcription of either HvE93X1 or HvE93X2. However, disturbance of the JH signal neither drove premature metamorphosis nor caused supernumerary instars. In contrast, depletion of E93 at the third- and fourth-instar larval and prepupal stages severely impaired pupation and caused a larval-pupal mixed phenotype: pupal spines and larval scoli were simultaneously presented on the cuticle. RNAi of E93 at the pupal stage affected adult eclosion. When the beetles had suffered from a dsE93 injection at the fourth-instar larval and pupal stages, a few resultant adults emerged, with separated elytra, abnormally folded hindwings, a small body size and short appendages. Taken together, these results suggest the larval instars are fixed in H. vigintioctopunctata; E93 serves as a repressor of larval characters and a specifier of adult structures during the larval-pupal-adult transition.
Katti, P., Ajayi, P. T., Aponte, A., Bleck, C. K. E. and Glancy, B. (2022). Identification of evolutionarily conserved regulators of muscle mitochondrial network organization. Nat Commun 13(1): 6622. PubMed ID: 36333356
Summary:
Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they might affect contractile fiber-type. This study shows that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm, identified transcription factors H15 and cut as potential mitochondrial network regulators. This study demonstrated H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, it was found that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors cut, salm, and H15.
Frendo-Cumbo, S., Li, T., Ammendolia, D. A., Coyaud, E., Laurent, E. M. N., Liu, Y., Bilan, P. J., Polevoy, G., Raught, B., Brill, J. A., Klip, A. and Brumell, J. H. (2022). DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling. iScience 25(10): 105188. PubMed ID: 36248734
Summary:
Cell proliferation is dependent on growth factors insulin and IGF1. This study sought to identify interactors of IRS1, the most proximal mediator of insulin/IGF1 signaling, that regulate cell proliferation. Using proximity-dependent biotin identification (BioID), 40 proteins were detecteddisplaying proximal interactions with IRS1, including DCAF7 and its interacting partners DYRK1A and DYRK1B. In HepG2 cells, DCAF7 knockdown attenuated cell proliferation by inducing cell cycle arrest at G2. DCAF7 expression was required for insulin-stimulated AKT phosphorylation, and its absence promoted nuclear localization of the transcription factor FOXO1. DCAF7 knockdown induced expression of FOXO1-target genes implicated in G2 cell cycle inhibition, correlating with G2 cell cycle arrest. In Drosophila melanogaster, wing-specific knockdown of DCAF7/wap caused smaller wing size and lower wing cell number; the latter recovered upon double knockdown of wap and dfoxo. It is proposed that DCAF7 regulates cell proliferation and cell cycle via IRS1-FOXO1 signaling, of relevance to whole organism growth.
Liu, P., Guo, Y., Xu, W., Song, S., Li, X., Wang, X., Lu, J., Guo, X., Richardson, H. E. and Ma, X. (2022). Ptp61F integrates Hippo, TOR, and actomyosin pathways to control three-dimensional organ size. Cell Rep 41(7): 111640. PubMed ID: 36384105
Summary:
Precise organ size control is fundamental for all metazoans, but how organ size is controlled in a three-dimensional (3D) way remains largely unexplored at the molecular level. This study screened and identified Drosophila Ptp61F as a pivotal regulator of organ size that integrates the Hippo pathway, TOR pathway, and actomyosin machinery. Pathologically, Ptp61F loss synergizes with Ras(V12) to induce tumorigenesis. Physiologically, Ptp61F depletion increases body size and drives neoplastic intestinal tumor formation and stem cell proliferation. Ptp61F also regulates cell contractility and myosin activation and controls 3D cell shape by reducing cell height and horizontal cell size. Mechanistically, Ptp61F forms a complex with Expanded (Ex) and increases endosomal localization of Ex and Yki. Furthermore, it was demonstrated that PTPN2, the conserved human ortholog of Ptp61F, can functionally substitute for Ptp61F in Drosophila. This work defines Ptp61F as an essential determinant that controls 3D organ size under both physiological and pathological conditions.
Summary:
Guo, X., Zhang, Y., Huang, H. and Xi, R. (2022). A hierarchical transcription factor cascade regulates enteroendocrine cell diversity and plasticity in Drosophila. Nat Commun 13(1): 6525. PubMed ID: 36316343
Summary:
Enteroendocrine cells (EEs) represent a heterogeneous cell population in intestine and exert endocrine functions by secreting a diverse array of neuropeptides. Although many transcription factors (TFs) required for specification of EEs have been identified in both mammals and Drosophila, it is not understood how these TFs work together to generate this considerable subtype diversity. This study showed that EE diversity in adult Drosophila is generated via an "additive hierarchical TF cascade". Specifically, a combination of a master TF, a secondary-level TF and a tertiary-level TF constitute a "TF code" for generating EE diversity. A high degree of post-specification plasticity of EEs was found, as changes in the code-including as few as one distinct TF-allow efficient switching of subtype identities. This study thus reveals a hierarchically-organized TF code that underlies EE diversity and plasticity in Drosophila, which can guide investigations of EEs in mammals and inform their application in medicine.

Tuesday, December 27th - RNA and Transposons

Jouravleva, K., Golovenko, D., Demo, G., Dutcher, R. C., Hall, T. M. T., Zamore, P. D. and Korostelev, A. A. (2022). Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB. Mol Cell 82(21): 4049-4063.e4046. PubMed ID: 36182693
Summary:
In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. This study reports six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs‑PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. These reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer‑1/Loqs‑PB heterodimer. The Dicer-1 dsRBD and three Loqs‑PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. These data suggest a model for how the Dicer‑1⋅Loqs‑PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
Liu, W., Liang, W., Xiong, X. P., Li, J. L. and Zhou, R. (2022). A circular RNA Edis-Relish-castor axis regulates neuronal development in Drosophila. PLoS Genet 18(10): e1010433. PubMed ID: 36301831
Summary:
Circular RNAs (circRNAs) are a new group of noncoding/regulatory RNAs that are particularly abundant in the nervous system, however, their physiological functions are underexplored. This study reports that the brain-enriched circular RNA Edis (Ect4-derived immune suppressor) plays an essential role in neuronal development in Drosophila. Depletion of Edis in vivo causes defects in axonal projection patterns of mushroom body (MB) neurons in the brain, as well as impaired locomotor activity and shortened lifespan of adult flies. In addition, it was found that the castor gene, which encodes a transcription factor involved in neurodevelopment, is upregulated in Edis knockdown neurons. Notably, castor overexpression phenocopies Edis knockdown, and reducing castor levels suppresses the neurodevelopmental phenotypes in Edis-depleted neurons. Furthermore, chromatin immunoprecipitation analysis reveals that the transcription factor Relish, which plays a key role in regulating innate immunity signaling, occupies a pair of sites at the castor promoter, and that both sites are required for optimal castor gene activation by either immune challenge or Edis depletion. Lastly, Relish mutation and/or depletion can rescue both the castor gene hyperactivation phenotype and neuronal defects in Edis knockdown animals. It is concluded that the circular RNA Edis acts through Relish and castor to regulate neuronal development.
Destefanis, F., Manara, V., Santarelli, S., Zola, S., Brambilla, M., Viola, G., Maragno, P., Signoria, I., Viero, G., Pasini, M. E., Penzo, M. and Bellosta, P. (2022). Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition. J Cell Sci. PubMed ID: 36314272
Summary:
NOC1 is a nucleolar protein necessary in yeast for both transport and maturation of ribosomal subunits. This study showed that Drosophila NOC1 is necessary for rRNAs maturation and for a correct animal development. Its ubiquitous downregulation results in a dramatic decrease in polysome level and of protein synthesis. NOC1 expression in multiple organs, such as the prothoracic gland and the fat body, is necessary for their proper functioning. Reduction of NOC1 in epithelial cells from the imaginal discs results in clones that die by apoptosis, an event that is partially rescued in a M/+ background, suggesting that reduction of NOC1 induces the cells to become less fit and to acquire a loser state. NOC1 downregulation activates the pro-apoptotic eiger-JNK pathway and leads to an increase of Xrp1 that results in Dilp8 upregulation. These data underline NOC1 as an essential gene in ribosome biogenesis and highlight its novel functions in the control of growth and cell competition.
Cassella, L. and Ephrussi, A. (2022). Subcellular spatial transcriptomics identifies three mechanistically different classes of localizing RNAs. Nat Commun 13(1): 6355. PubMed ID: 36289223
Summary:
Intracellular RNA localization is a widespread and dynamic phenomenon that compartmentalizes gene expression and contributes to the functional polarization of cells. Thus far, mechanisms of RNA localization identified in Drosophila have been based on a few RNAs in different tissues, and a comprehensive mechanistic analysis of RNA localization in a single tissue is lacking. By subcellular spatial transcriptomics this study has identified RNAs localized in the apical and basal domains of the columnar follicular epithelium (FE) and the mechanisms mediating their localization were analyzed. Whereas the dynein/BicD/Egl machinery controls apical RNA localization, basally-targeted RNAs require kinesin-1 to overcome a default dynein-mediated transport. Moreover, a non-canonical, translation- and dynein-dependent mechanism mediates apical localization of a subgroup of dynein-activating adaptor-encoding RNAs (BicD, Bsg25D, hook). Altogether, this study identifies at least three mechanisms underlying RNA localization in the FE, and suggests a possible link between RNA localization and dynein/dynactin/adaptor complex formation in vivo.
Khyzha, N., Henikoff, S. and Ahmad, K. (2022). Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation. Nat Methods 19(11): 1383-1392. PubMed ID: 36192462
Summary:
Whereas techniques to map chromatin-bound proteins are well developed, mapping chromatin-associated RNAs remains a challenge. This study describes Reverse Transcribe and Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tagmented by Tn5 transposases for downstream sequencing. The utility of RT&Tag in Drosophila cells for capturing the noncoding RNA roX2 with the dosage compensation complex and maturing transcripts associated with silencing histone modifications was demonstrated. It was also shown that RT&Tag can detect N6-methyladenosine-modified mRNAs and that genes producing methylated transcripts are characterized by extensive promoter pausing of RNA polymerase II. The high efficiency of in situ antibody tethering and tagmentation makes RT&Tag especially suitable for rapid low-cost profiling of chromatin-associated RNAs.
Li, Y., Yang, X., Zhao, Z. and Du, J. (2022). SRP54 mediates circadian rhythm-related, temperature-dependent gene expression in Drosophila. Genomics 114(6): 110512. PubMed ID: 36273743
Summary:
Recent studies have shown that alternative splicing (AS) plays an important role in regulating circadian rhythm. However, it is not clear whether clock neuron-specific AS is circadian rhythm dependent and what genetic and environmental factors mediate the circadian control of AS. By genome-wide RNA sequencing, SRP54 was identified is one of the Clock (Clk) dependent alternative splicing factors. Genetic interaction between Clock and SRP54 alleles showed that the enhancement of the circadian phenotype increased with temperature, being strongest at 29 °C and weakest at 18 °C. The alternative splicing and differential gene expression profile of Clock and SRP54 overlapped with the circadian-related gene profiles identified in various genome-wide studies, indicating that SRP54 is involved in circadian rhythm. By analyzing of the RNA-seq results at different temperatures, it was found that the roles of Clock and SRP54 are temperature dependent. Multiple novel temperature-dependent transcripts not documented in current databases were also found.

Monday, December 26th - Disease Models

Dewey, E. B., Holsclaw, J. K., Saghaey, K., Wittmer, M. E. and Sekelsky, J. (2022). The Effect of Repeat Length on Marcal1-Dependent Single-Strand Annealing in Drosophila. Genetics. PubMed ID: 36303322
Summary:
Proper repair of DNA double strand breaks (DSBs) is essential to maintenance of genomic stability and avoidance of genetic disease. Organisms have many ways of repairing DSBs, including use of homologous sequences through homology-directed repair (HDR). While HDR repair is often error-free, in single-strand annealing (SSA) homologous repeats flanking a DSB are annealed to one another, leading to deletion of one repeat and the intervening sequences. Studies in yeast have shown a relationship between the length of the repeat and SSA efficacy. This study sought to determine the effects of homology length on SSA in Drosophila, as Drosophila uses a different annealing enzyme (Marcal1) than yeast. Using an in vivo SSA assay, it was shown that 50 base pairs (bp) is insufficient to promote SSA and that 500-2000 bp is required for maximum efficiency. Loss of Marcal1 generally followed the same homology length trend as wild-type flies, with SSA frequencies reduced to about a third of wild-type frequencies regardless of homology length. Interestingly, a difference was found in SSA rates between 500 bp homologies that align to the annealing target either nearer or further from the DSB, a phenomenon that may be explained by Marcal1 dynamics. This study gives insights into Marcal1 function and provides important information to guide design of genome engineering strategies that use SSA to integrate linear DNA constructs into a chromosomal DSB.
Kapur, I., Boulier, E. L. and Francis, N. J. (2022). Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding. Epigenomes 6(4). PubMed ID: 36412795
Summary:
The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. Three distinct low-complexity regions were identified in Ph based on sequence composition. The role of each of these sequences was tested in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. These data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. These results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. These observations highlight the complexity of regulation of biological condensates housed in single proteins.
Richards, L., Lord, C. L., Benton, M. L., Capra, J. A. and Nordman, J. T. (2022). Nucleoporins facilitate ORC loading onto chromatin. Cell Rep 41(6): 111590. PubMed ID: 36351393
Summary:
The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. This study perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, it was found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. This work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation.
Reddy, H. M., Randall, T. A., Cipressa, F., Porrazzo, A., Cenci, G., Frydrychova, R. C. and Mason, J. M. (2022). Identification of the Telomere elongation Mutation in Drosophila. Cells 11(21). PubMed ID: 36359878
Summary:
Length maintenance of Telomeres in Drosophila relies on the transposition of the specialized retrotransposons Het-A, TART, and TAHRE, rather than on the activity of the enzyme telomerase as it occurs in most other eukaryotic organisms. The length of the telomeres in Drosophila thus depends on the number of copies of these transposable elements. Previous work has led to the isolation of a dominant mutation, Tel(1), that caused a several-fold elongation of telomeres. In this study, the Tel(1) mutation was molecularly identified by a combination of transposon-induced, site-specific recombination and next-generation sequencing. Recombination located Tel(1) to a 15 kb region in 92A. Comparison of the DNA sequence in this region with the Drosophila Genetic Reference Panel of wild-type genomic sequences delimited Tel(1) to a 3 bp deletion inside intron 8 of Ino80. Furthermore, CRISPR/Cas9-induced deletions surrounding the same region exhibited the Tel(1) telomere phenotype, confirming a strict requirement of this intron 8 gene sequence for a proper regulation of Drosophila telomere length.
Klucnika, A., Mu, P., Jezek, J., McCormack, M., Di, Y., Bradshaw, C. R. and Ma, H. (2023). REC drives recombination to repair double-strand breaks in animal mtDNA. J Cell Biol 222(1). PubMed ID: 36355348
Summary:
Mechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, this study identified that REC-an MCM helicase that drives meiotic recombination in the nucleus-also localizes to mitochondria in Drosophila. REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. It was further shown that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.
Takeuchi, C., Yokoshi, M., Kondo, S., Shibuya, A., Saito, K., Fukaya, T., Siomi, H. and Iwasaki, Y. W. (2022). Mod(mdg4) variants repress telomeric retrotransposon HeT-A by blocking subtelomeric enhancers. Nucleic Acids Res. PubMed ID: 36373634
Summary:
Telomeres in Drosophila are composed of sequential non-LTR retrotransposons HeT-A, TART and TAHRE. Although they are repressed by the PIWI-piRNA pathway or heterochromatin in the germline, the regulation of these retrotransposons in somatic cells is poorly understood. This study demonstrated that specific splice variants of Mod(mdg4) repress HeT-A by blocking subtelomeric enhancers in ovarian somatic cells. Among the variants, it was found that the Mod(mdg4)-N variant represses HeT-A expression the most efficiently. Subtelomeric sequences bound by Mod(mdg4)-N block enhancer activity within subtelomeric TAS-R repeats. This enhancer-blocking activity is increased by the tandem association of Mod(mdg4)-N to repetitive subtelomeric sequences. In addition, the association of Mod(mdg4)-N couples with the recruitment of RNA polymerase II to the subtelomeres, which reinforces its enhancer-blocking function. These findings provide novel insights into how telomeric retrotransposons are regulated by the specific variants of insulator proteins associated with subtelomeric sequences.

Thursday, December 23rd - Signaling

Towarnicki, S. G., Youngson, N. A., Corley, S. M., St John, J. C., Melvin, R. G., Turner, N., Morris, M. J. and Ballard, J. W. O. (2022). Ancestral dietary change alters the development of Drosophila larvae through MAPK signalling. Fly (Austin) 16(1): 299-311. PubMed ID: 35765944
Summary:
Studies in a broad range of animal species have revealed phenotypes that are caused by ancestral life experiences, including stress and diet. Ancestral dietary macronutrient composition and quantity (over- and under-nutrition) have been shown to alter descendent growth, metabolism and behaviour. Molecules have been identified in gametes that are changed by ancestral diet and are required for transgenerational effects. However, there is less understanding of the developmental pathways altered by inherited molecules during the period between fertilization and adulthood. To investigate this non-genetic inheritance, this study exposed great grand-parental and grand-parental generations to defined protein to carbohydrate (P:C) dietary ratios. Descendent developmental timing was consistently faster in the period between the embryonic and pupal stages when ancestors had a higher P:C ratio diet. Transcriptional analysis revealed extensive and long-lasting changes to the MAPK signalling pathway, which controls growth rate through the regulation of ribosomal RNA transcription. Pharmacological inhibition of both MAPK and rRNA pathways recapitulated the ancestral diet-induced developmental changes. This work provides insight into non-genetic inheritance between fertilization and adulthood.
Hu, L., Brichalli, W., Li, N., Chen, S., Cheng, Y., Liu, Q., Xiong, Y. and Yu, J. (2022). Myotubularin functions through actomyosin to interact with the Hippo pathway. EMBO Rep: e55851. PubMed ID: 36285521
Summary:
The Hippo pathway is an evolutionarily conserved developmental pathway that controls organ size by integrating diverse regulatory inputs, including actomyosin-mediated cytoskeletal tension. Despite established connections between the actomyosin cytoskeleton and the Hippo pathway, the upstream regulation of actomyosin in the Hippo pathway is less defined. This study identified the phosphoinositide-3-phosphatase Myotubularin (Mtm) as a novel upstream regulator of actomyosin that functions synergistically with the Hippo pathway during growth control. Mechanistically, Mtm regulates membrane phospholipid PI(3)P dynamics, which, in turn, modulates actomyosin activity through Rab11-mediated vesicular trafficking. PI(3)P dynamics were revealed to be a novel mode of upstream regulation of actomyosin and established Rab11-mediated vesicular trafficking as a functional link between membrane lipid dynamics and actomyosin activation in the context of growth control. This study also shows that MTMR2, the human counterpart of Drosophila Mtm, has conserved functions in regulating actomyosin activity and tissue growth, providing new insights into the molecular basis of MTMR2-related peripheral nerve myelination and human disorders.
Miguez, D. G., Iannini, A., Garcia-Morales, D. and Casares, F. (2022). Patterning on the move: the effects of Hh morphogen source movement on signaling dynamics. Development. PubMed ID: 36355083
Summary:
Morphogens of the Hh-family trigger gene expression changes of receiving cells in a concentration-dependent manner to regulate their identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its logic includes a negative feedback loop involving the Hh receptor Ptc. Using experiments and computational models the different spatial signaling profiles downstream of Hh was studied and compared in several developing Drosophila organs. The spatial distribution of Ptc and the activator transcription factor CiA in wing, antenna and ocellus show similar features, but markedly different from that in the compound eye (CE). It is proposed that these two profile types represent two time points along the signaling dynamics, and that the interplay between the spatial displacement of the Hh source in the CE and the negative feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results show how the interaction between spatial and temporal dynamics of signaling and differentiation processes may contribute to the informational versatility of the conserved Hh signaling pathway.
Fangninou, F. F., Yu, Z., Li, Z., Guadie, A., Li, W., Xue, L. and Yin, D. (2023). Metastatic effects of environmental carcinogens mediated by MAPK and UPR pathways with an in vivo Drosophila Model. J Hazard Mater 441: 129826. PubMed ID: 36084456
Summary:
Metastasis includes tumor invasion and migration and underlies over 90% of cancer mortality. The metastatic effects of environmental carcinogens raised serious health concerns. However, the underlying mechanisms remained poorly studied. In the present study, an in vivo Ras(V12)/lgl(-/-) model of the fruitfly, Drosophila melanogaster, with an 8-day exposure was employed to explore the metastatic effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126), perfluorooctanoic acid (PFOA) and cadmium chloride (CdCl(2)). At 1.0 mg/L, PCB126, PFOA, and CdCl(2) significantly increased tumor invasion rates by 1.32-, 1.33-, and 1.29-fold of the control, respectively. They also decreased the larval body weight and locomotion behavior. Moreover, they commonly disturbed the expression levels of target genes in MAPK and UPR pathways, and their metastatic effects were significantly abolished by the addition of p38 inhibitor (SB203580), JNK inhibitor (SP600125) and IRE1 inhibitor (KIRA6). Notably, the addition of the IRE inhibitor significantly influenced sna/E-cad pathway which is essential in both p38 and JNK regulations. The results demonstrated an essential role of sna/E-cad in connecting the effects of carcinogens on UPR and MAPK regulations and the resultant metastasis.
Gerlach, S. U., de Vreede, G. and Bilder, D. (2022). PTP10D-mediated cell competition is not obligately required for elimination of polarity-deficient clones. Biol Open 11(11). PubMed ID: 36355597
Summary:
Animal organs maintain tissue integrity and ensure removal of aberrant cells through several types of surveillance mechanisms. One prominent example is the elimination of polarity-deficient mutant cells within developing Drosophila imaginal discs. This has been proposed to require heterotypic cell competition dependent on the receptor tyrosine phosphatase PTP10D within the mutant cells. This study reports experiments to test this requirement in various contexts and found that PTP10D is not obligately required for the removal of scribble (scrib) mutant and similar polarity-deficient cells. These experiments used identical stocks with which another group can detect the PTP10D requirement, and the results do not vary under several husbandry conditions including high and low protein food diets. Although it was not possible to identify the source of the discrepant results, it is suggested that the role of PTP10D in polarity-deficient cell elimination may not be absolute.
Mukherjee, S., Calvi, B. R., Hundley, H. A. and Sokol, N. S. (2022). MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine. Cell Rep 41(3): 111495. PubMed ID: 36261011
Summary:
Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood. This study reports that commitment to the terminally differentiated enterocyte (EC) cell fate is under microRNA control. An intestinally enriched microRNA, miR-956, fine-tunes Notch signaling activity specifically in intermediate, enteroblast (EB) progenitor cells to control EC differentiation. This study further identified insensitive mRNA as a target of miR-956 that regulates EB/EC ratios by repressing Notch activity in EBs. In summary, this study highlights a post-transcriptional gene-regulatory mechanism for controlling differentiation in an adult intestinal stem cell lineage.

Wednesday, December 21st - Neural Development and Function

Turrel, O., Ramesh, N., Escher, M. J. F., Pooryasin, A. and Sigrist, S. J. (2022). Transient active zone remodeling in the Drosophila mushroom body supports memory. Curr Biol 32(22): 4900-4913. PubMed ID: 36327980
Summary:
Elucidating how the distinct components of synaptic plasticity dynamically orchestrate the distinct stages of memory acquisition and maintenance within neuronal networks remains a major challenge. Specifically, plasticity processes tuning the functional and also structural state of presynaptic active zone (AZ) release sites are widely observed in vertebrates and invertebrates, but their behavioral relevance remains mostly unclear. This study provides evidence that a transient upregulation of presynaptic AZ release site proteins supports aversive olfactory mid-term memory in the Drosophila mushroom body (MB). Upon paired aversive olfactory conditioning, AZ protein levels (ELKS-family BRP/(m)unc13-family release factor Unc13A) increased for a few hours with MB-lobe-specific dynamics. Kenyon cell (KC, intrinsic MB neurons)-specific knockdown (KD) of BRP did not affect aversive olfactory short-term memory (STM) but strongly suppressed aversive mid-term memory (MTM). Different proteins crucial for the transport of AZ biosynthetic precursors (transport adaptor Aplip1/Jip-1; kinesin motor IMAC/Unc104; small GTPase Arl8) were also specifically required for the formation of aversive olfactory MTM. Consistent with the merely transitory increase of AZ proteins, BRP KD did not interfere with the formation of aversive olfactory long-term memory (LTM; i.e., 1 day). These data suggest that the remodeling of presynaptic AZ refines the MB circuitry after paired aversive conditioning, over a time window of a few hours, to display aversive olfactory memories.
Lorber, C., Leleux, S., Stanewsky, R. and Lamaze, A. (2022). Light triggers a network switch between circadian morning and evening oscillators controlling behaviour during daily temperature cycles. PLoS Genet 18(11): e1010487. PubMed ID: 36367867
Summary:
Proper timing of rhythmic locomotor behavior is the consequence of integrating environmental conditions and internal time dictated by the circadian clock. Rhythmic environmental input like daily light and temperature changes (called Zeitgeber) reset the molecular clock and entrain it to the environmental time zone the organism lives in. This study attempted to entrain the clock with a Zeitgeber different from the environmental input used for phasing the behavior. 150 clock neurons in the Drosophila melanogaster brain control different aspects of the daily activity rhythms and are organized in various clusters, During regular 12 hr light: 12 hr dark cycles at constant mild temperature (LD 25°C, LD being the Zeitgeber), so called morning oscillator (MO) neurons control the increase of locomotor activity just before lights-on, while evening oscillator (EO) neurons regulate the activity increase at the end of the day, a few hours before lights-off. This study, using 12 h: 12 h 25°C:16°C temperature cycles as Zeitgeber, attempted to look at the impact of light on phasing locomotor behavior. While in constant light and 25°C:16°C temperature cycles (LLTC), flies show an unimodal locomotor activity peak in the evening, during the same temperature cycle, but in the absence of light (DDTC), the phase of the activity peak is shifted to the morning. This study shows that the EO is necessary for synchronized behavior in LLTC but not for entraining the molecular clock of the other clock neuronal groups, while the MO controls synchronized morning activity in DDTC. Interestingly, the data suggest that the influence of the EO on the synchronization increases depending on the length of the photoperiod (constant light vs 12h of light). Hence, these results show that effects of different environmental cues on clock entrainment and activity phase can be separated, allowing to decipher their integration by the circadian clock.
Giri, R., Brady, S., Papadopoulos, D. K. and Carthew, R. W. (2022). Single-cell Senseless protein analysis reveals metastable states during the transition to a sensory organ fate. iScience 25(10): 105097. PubMed ID: 36157584
Summary:
Cell fate decisions can be envisioned as bifurcating dynamical systems, and the decision that Drosophila cells make during sensory organ differentiation has been described as such. This study extended these studies by focusing on the Senseless protein which orchestrates sensory cell fate transitions. Wing cells contain intermediate Senseless numbers before their fate transition, after which they express much greater numbers of Senseless molecules as they differentiate. However, the dynamics are inconsistent with it being a simple bistable system. Cells with intermediate Senseless are best modeled as residing in four discrete states, each with a distinct protein number and occupying a specific region of the tissue. Although the states are stable over time, the number of molecules in each state vary with time. The fold change in molecule number between adjacent states is invariant and robust to absolute protein number variation. Thus, cells transitioning to sensory fates exhibit metastability with relativistic properties.
Aymanns, F., Chen, C. L. and Ramdya, P. (2022). Descending neuron population dynamics during odor-evoked and spontaneous limb-dependent behaviors. Elife 11. PubMed ID: 36286408
Summary:
Deciphering how the brain regulates motor circuits to control complex behaviors is an important, long-standing challenge in neuroscience. In the fly, Drosophila melanogaster, this is coordinated by a population of ~ 1100 descending neurons (DNs). Activating only a few DNs is known to be sufficient to drive complex behaviors like walking and grooming. However, what additional role the larger population of DNs plays during natural behaviors remains largely unknown. For example, they may modulate core behavioral commands or comprise parallel pathways that are engaged depending on sensory context. This study evaluated these possibilities by recording populations of nearly 100 DNs in individual tethered flies while they generated limb-dependent behaviors, including walking and grooming. The largest fraction of recorded DNs encode walking while fewer are active during head grooming and resting. A large fraction of walk-encoding DNs encode turning and far fewer weakly encode speed. Although odor context does not determine which behavior-encoding DNs are recruited, a few DNs encode odors rather than behaviors. Lastly, this study illustrated how one can identify individual neurons from DN population recordings by using their spatial, functional, and morphological properties. These results set the stage for a comprehensive, population-level understanding of how the brain's descending signals regulate complex motor actions.
Zhao, Y., Ke, S., Cheng, G., Lv, X., Chang, J. and Zhou, W. (2022). Direction Selectivity of TmY Neurites in Drosophila. Neurosci Bull. PubMed ID: 36399278
Summary:
The perception of motion is an important function of vision. Neural wiring diagrams for extracting directional information have been obtained by connectome reconstruction. Direction selectivity in Drosophila is thought to originate in T4/T5 neurons through integrating inputs with different temporal filtering properties. Through genetic screening based on synaptic distribution, a new type of TmY neuron, termed TmY-ds, was isolated that forms reciprocal synaptic connections with T4/T5 neurons. Its neurites responded to grating motion along the four cardinal directions and showed a variety of direction selectivity. Intriguingly, its direction selectivity originated from temporal filtering neurons rather than T4/T5. Genetic silencing and activation experiments showed that TmY-ds neurons are functionally upstream of T4/T5. These results suggest that direction selectivity is generated in a tripartite circuit formed among these three neurons-temporal filtering, TmY-ds, and T4/T5 neurons, in which TmY-ds plays a role in the enhancement of direction selectivity in T4/T5 neurons.
Thiagarajan, D., Eberl, F., Veit, D., Hansson, B. S., Knaden, M. and Sachse, S. (2022). Aversive Bimodal Associations Differently Impact Visual and Olfactory Memory Performance in Drosophila. iScience 25(12): 105485. PubMed ID: 36404920
Summary:
Animals form sensory associations and store them as memories to guide behavioral decisions. Although unimodal learning has been studied extensively in insects, it is important to explore sensory cues in combination because most behaviors require multimodal inputs. This study optimized the T-maze to employ both visual and olfactory cues in a classical aversive learning paradigm in Drosophila melanogaster. In contrast to unimodal training, bimodal training evoked a significant short-term visual memory after a single training trial. Interestingly, the same protocol did not enhance short-term olfactory memory and even had a negative impact. However, compromised long-lasting olfactory memory significantly improved after bimodal training. This study demonstrates that the effect of bimodal integration on learning is not always beneficial and is conditional upon the formed memory strengths.It is postulated that flies utilize information on a need-to basis: bimodal training augments weakly formed memories while stronger associations are impacted differently.

Tuesday, December 20th - Behavior

Yu, Z., Shi, J., Jiang, X., Song, Y., Du, J. and Zhao, Z. (2022). Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae. Pest Manag Sci. PubMed ID: 36396604
Summary:
The feeding of pests is one of the important reasons for losses of agricultural crop yield. This study aimed to reveal how juvenile hormone participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. Larvae of O. furnacalis exhibit a daily circadian rhythm on feeding, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a juvenile hormone (JH) active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by the brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide to O. furnacalis that controls feeding and metabolism. The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control.
Cellini, B. and Mongeau, J. M. (2022). Nested mechanosensory feedback actively damps visually guided head movements in Drosophila. Elife 11. PubMed ID: 36259536
Summary:
Executing agile locomotion requires animals to integrate sensory feedback, often from multiple sources. For example, human gaze is mediated by multiple feedback loops that integrate visual and vestibular information. A central challenge in studying biological feedback loops is that they are nested and dynamically coupled. This study developed a framework based on control theory for unraveling nested feedback systems and apply it to study gaze stabilization in the fruit fly (Drosophila). By combining experimental and mathematical methods to manipulate control topologies, this study uncovered the role of body-generated mechanosensory feedback nested within visual feedback in the control of head movements. It was discovered that visual feedback changed the tuning of head movements across visual motion frequencies whereas mechanosensory feedback damped head movements. Head saccades had slower dynamics when the body was free to move, further pointing to the role of damping via mechanosensory feedback. By comparing head responses between self-generated and externally generated body motion, a nonlinear gating of mechanosensory feedback was revealed that is motor-context dependent. Altogether, these findings reveal the role of nested feedback loops in flies and uncover mechanisms that reconcile differences in head kinematics between body-free and body-fixed flies. This framework is generalizable to biological and robotic systems relying on nested feedback control for guiding locomotion.
Wang, W., Dweck, H. K. M., Talross, G. J. S., Zaidi, A., Gendron, J. M. and Carlson, J. R. (2022). Sugar sensation and mechanosensation in the egg-laying preference shift of Drosophila suzukii. Elife 11. PubMed ID: 36398882
Summary:
The agricultural pest Drosophila suzukii differs from most other Drosophila species in that it lays eggs in ripe, rather than overripe, fruit. Previously, it was shown that changes in bitter taste sensation accompanied this adaptation. This study shows that D. suzukii has also undergone a variety of changes in sweet taste sensation. D. suzukii has a weaker preference than Drosophila melanogaster for laying eggs on substrates containing all three primary fruit sugars: sucrose, fructose, and glucose. Major subsets of D. suzukii taste sensilla have lost electrophysiological responses to sugars. Expression of several key sugar receptor genes is reduced in the taste organs of D. suzukii. By contrast, certain mechanosensory channel genes, including no mechanoreceptor potential C, are expressed at higher levels in the taste organs of D. suzukii, which has a higher preference for stiff substrates. Finally, it was found that D. suzukii responds differently from D. melanogaster to combinations of sweet and mechanosensory cues. Thus, the two species differ in sweet sensation, mechanosensation, and their integration, which are all likely to contribute to the differences in their egg-laying preferences in nature.
Fenk, L. M., Avritzer, S. C., Weisman, J. L., Nair, A., Randt, L. D., Mohren, T. L., Siwanowicz, I. and Maimon, G. (2022). Muscles that move the retina augment compound eye vision in Drosophila. Nature. PubMed ID: 36289333
Summary:
Most animals have compound eyes, with tens to thousands of lenses attached rigidly to the exoskeleton. A natural assumption is that all of these species must resort to moving either their head or their body to actively change their visual input. However, classic anatomy has revealed that flies have muscles poised to move their retinas under the stable lenses of each compound eye. This study shows that Drosophila use their retinal muscles to smoothly track visual motion, which helps to stabilize the retinal image, and also to perform small saccades when viewing a stationary scene. When the retina moves, visual receptive fields shift accordingly, and even the smallest retinal saccades activate visual neurons. Using a head-fixed behavioural paradigm, it was found that Drosophila perform binocular, vergence movements of their retinas-which could enhance depth perception-when crossing gaps, and impairing the physiology of retinal motor neurons alters gap-crossing trajectories during free behaviour. That flies evolved an ability to actuate their retinas suggests that moving the eye independently of the head is broadly paramount for animals. The similarities of smooth and saccadic movements of the Drosophila retina and the vertebrate eye highlight a notable example of convergent evolution.
Deere, J. U. and Devineni, A. V. (2022). Taste cues elicit prolonged modulation of feeding behavior in Drosophila. iScience 25(10): 105159. PubMed ID: 36204264
Summary:
Taste cues regulate immediate feeding behavior, but their ability to modulate future behavior has been less well studied. Pairing one taste with another can modulate subsequent feeding responses through associative learning, but this requires simultaneous exposure to both stimuli. This study investigated whether exposure to one taste modulates future responses to other tastes even when they do not overlap in time. Using Drosophila, it was found that brief exposure to sugar enhanced future feeding responses, whereas bitter exposure suppressed them. This modulation relies on neural pathways distinct from those that acutely regulate feeding or mediate learning-dependent changes. Sensory neuron activity was required not only during initial taste exposure but also afterward, suggesting that ongoing sensory activity may maintain experience-dependent changes in downstream circuits. Thus, the brain stores a memory of each taste stimulus after it disappears, enabling animals to integrate information as they sequentially sample different taste cues that signal local food quality.
Fischer, P. J. and Schnell, B. (2022). Multiple mechanisms mediate the suppression of motion vision during escape maneuvers in flying Drosophila. iScience 25(10): 105143. PubMed ID: 36185378
Summary:
During voluntary behaviors, animals need to disable any reflexes that could interfere with the intended movements. With the optomotor response, flies stabilize a straight flight path by correcting for unintended deviations sensed as the panoramic motion of the surround. HS cells of the fly are thought to mediate optomotor responses to horizontal motion. During spontaneous flight turns, an efference copy acts on HS cells with the right sign to counteract the visual input elicited by the fly's own behavior. This study investigated whether looming-elicited turns in flying Drosophila have a similar effect on HS cells. Looming stimuli themselves can influence the processing of panoramic motion stimuli in HS cells and that an inhibitory efference copy suppresses excitatory motion responses during turns in both directions, but only in a subset of HS cells. These findings support the notion that the processing of sensory information is finely tuned to behavioral context.

Monday, December 19th - - Neural Development and Function

Seroka, A., Lai, S. L. and Doe, C. Q. (2022). Transcriptional profiling from whole embryos to single neuroblast lineages in Drosophila. Dev Biol 489: 21-33. PubMed ID: 35660371
Summary:
Embryonic development results in the production of distinct tissue types, and different cell types within each tissue. A major goal of developmental biology is to uncover the "parts list" of cell types that comprise each organ. Single cell RNA sequencing (scRNA-seq) of the Drosophila embryo was performed to identify the genes that characterize different cell and tissue types during development. Three different timepoints were assayed, revealing a coordinated change in gene expression within each tissue. Interestingly, the elav and Mhc genes, whose protein products are widely used as markers for neurons and muscles, respectively, were found to exhibit broad pan-embryonic expression, indicating the importance of post-transcriptional regulation. Next focus was placed on the central nervous system (CNS), where genes were identified whose expression is enriched at each stage of neuronal differentiation: from neural progenitors, called neuroblasts, to their immediate progeny ganglion mother cells (GMCs), followed by new-born neurons, young neurons, and the most mature neurons. Finally, it was asked whether the clonal progeny of a single neuroblast (NB7-1) share a similar transcriptional identity. Surprisingly, it was found that clonal identity does not lead to transcriptional clustering, showing that neurons within a lineage are diverse, and that neurons with a similar transcriptional profile (e.g. motor neurons, glia) are distributed among multiple neuroblast lineages. Although each lineage consists of diverse progeny, it was possible to identify a previously uncharacterized gene, Fer3, as an excellent marker for the NB7-1 lineage. Within the NB7-1 lineage, neurons which share a temporal identity (e.g. Hunchback, Kruppel, Pdm, and Castor temporal transcription factors in the NB7-1 lineage) have shared transcriptional features, allowing for the identification of candidate novel temporal factors or targets of the temporal transcription factors. In conclusion, this study has characterized the embryonic transcriptome for all major tissue types and for three stages of development, as well as the first transcriptomic analysis of a single, identified neuroblast lineage, finding a lineage-enriched transcription factor.
Theodorou, V., Stefanaki, A., Drakos, M., Triantafyllou, D. and Delidakis, C. (2022). ASC proneural factors are necessary for chromatin remodeling during neuroectodermal to neuroblast fate transition to ensure the timely initiation of the neural stem cell program. BMC Biol 20(1): 107. PubMed ID: 35549704
Summary:
In both Drosophila and mammals, the achaete-scute (ASC/ASCL) proneural bHLH transcription factors are expressed in the developing central and peripheral nervous systems, where they function during specification and maintenance of the neural stem cells in opposition to Notch signaling. However, the impact of ASC on chromatin dynamics during neural stem cell generation remains elusive. This study investigated the chromatin changes accompanying neural commitment using an integrative genetics and genomics methodology. ASC factors were found to bind equally strongly to two distinct classes of cis-regulatory elements: open regions remodeled earlier during maternal to zygotic transition by Zelda and less accessible, Zelda-independent regions. Both classes of cis-elements exhibit enhanced chromatin accessibility during neural specification and correlate with transcriptional regulation of genes involved in a variety of biological processes necessary for neuroblast function/homeostasis. This study identified an ASC-Notch regulated TF network that includes likely prime regulators of neuroblast function. Using a cohort of ASC target genes, it is reported that ASC null neuroblasts are defectively specified, remaining initially stalled, unable to divide, and lacking expression of many proneural targets. When mutant neuroblasts eventually start proliferating, they produce compromised progeny. Reporter lines driven by proneural-bound enhancers display ASC dependency, suggesting that the partial neuroblast identity seen in the absence of ASC genes is likely driven by other, proneural-independent, cis-elements. Neuroblast impairment and the late differentiation defects of ASC mutants are corrected by ectodermal induction of individual ASC genes but not by individual members of the TF network downstream of ASC. However, in wild-type embryos, the induction of individual members of this network induces CNS hyperplasia, suggesting that they synergize with the activating function of ASC to consolidate the chromatin dynamics that promote neural specification. This study has demonstrate that ASC proneural transcription factors are indispensable for the timely initiation of the neural stem cell program at the chromatin level by regulating a large number of enhancers in the vicinity of neural genes. This early chromatin remodeling is crucial for both neuroblast homeostasis as well as future progeny fidelity.
Manoim, J. E., Davidson, A. M., Weiss, S., Hige, T. and Parnas, M. (2022). Lateral axonal modulation is required for stimulus-specific olfactory conditioning in Drosophila. Curr Biol. PubMed ID: 36130601
Summary:
Effective and stimulus-specific learning is essential for animals' survival. Two major mechanisms are known to aid stimulus specificity of associative learning. One is accurate stimulus-specific representations in neurons. The second is a limited effective temporal window for the reinforcing signals to induce neuromodulation after sensory stimuli. However, these mechanisms are often imperfect in preventing unspecific associations; different sensory stimuli can be represented by overlapping populations of neurons, and more importantly, the reinforcing signals alone can induce neuromodulation even without coincident sensory-evoked neuronal activity. This study reports a crucial neuromodulatory mechanism that counteracts both limitations and is thereby essential for stimulus specificity of learning. In Drosophila, olfactory signals are sparsely represented by cholinergic Kenyon cells (KCs), which receive dopaminergic reinforcing input. This study found that KCs have numerous axo-axonic connections mediated by the muscarinic type-B receptor (mAChR-B). By using functional imaging and optogenetic approaches, this study showed that these axo-axonic connections suppress both odor-evoked calcium responses and dopamine-evoked cAMP signals in neighboring KCs. Strikingly, behavior experiments demonstrate that mAChR-B knockdown in KCs impairs olfactory learning by inducing undesired changes to the valence of an odor that was not associated with the reinforcer. Thus, this local neuromodulation acts in concert with sparse sensory representations and global dopaminergic modulation to achieve effective and accurate memory formation.
Deng, Q., Wang, C., Koe, C. T., Heinen, J. P., Tan, Y. S., Li, S., Gonzalez, C., Sung, W. K. and Wang, H. (2022). Parafibromin governs cell polarity and centrosome assembly in Drosophila neural stem cells. PLoS Biol 20(10): e3001834. PubMed ID: 36223339
Summary:
Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. This study demonstrated that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, human Parafibromin was shown to rescue the ectopic NSC phenotype in Drosophila hyx mutant brains. This study also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. These data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers.
Carrasco, J., Mateos, F. and Hilgers, V. (2022). A critical developmental window for ELAV/Hu-dependent mRNA signatures at the onset of neuronal differentiation. Cell Rep 41(4): 111542. PubMed ID: 36288718 Cell-type-specific gene regulatory programs are essential for cell differentiation and function. In animal neurons, the highly conserved ELAV/Hu family of proteins promotes alternative splicing and polyadenylation of mRNA precursors to create unique neuronal transcript isoforms. This study assessed transcriptome profiles and neurogenesis success in Drosophila models engineered to express differing levels of ELAV activity in the course of development. The ELAV-mediated establishment of a subset of neuronal mRNA isoforms at the onset of neuron differentiation constitutes a developmental bottleneck that cannot be overcome later by the nuclear activation of the paralog found in neurons (FNE). Loss of ELAV function outside of that critical time window results in neurological defects. FNE, when activated early enough, can restore ELAV-dependent neuronal mRNA isoforms and fully rescue development. These findings demonstrate the essential role of robust cellular strategies to maintain ELAV activity and intact neuronal signatures in neurogenesis and neuronal function.
Summary:
Velten, J., Gao, X., Van Nierop, Y. S. P., Domsch, K., Agarwal, R., Bognar, L., Paulsen, M., Velten, L. and Lohmann, I. (2022). Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos. Mol Syst Biol 18(3): e10255. PubMed ID: 35225419
Summary:
The correct wiring of neuronal circuits is one of the most complex processes in development, since axons form highly specific connections out of a vast number of possibilities. This study investigated Drosophila embryonic motoneurons using single-cell genomics, imaging, and genetics. A cell-specific combination of homeodomain transcription factors and downstream immunoglobulin domain proteins is expressed in individual cells and plays an important role in determining cell-specific connections between differentiated motoneurons and target muscles. Genetic evidence is provided for a functional role of five homeodomain transcription factors and four immunoglobulins in the neuromuscular wiring. Knockdown and ectopic expression of these homeodomain transcription factors induces cell-specific synaptic wiring defects that are partly phenocopied by genetic modulations of their immunoglobulin targets. Taken together, these data suggest that homeodomain transcription factor and immunoglobulin molecule expression could be directly linked and function as a crucial determinant of neuronal circuit structure.

Friday, December 16th - Adult Physiology

Saha, S., Spinelli, L., Castro Mondragon, J. A., Kervadec, A., Lynott, M., Kremmer, L., Roder, L., Krifa, S., Torres, M., Brun, C., Vogler, G., Bodmer, R., Colas, A. R., Ocorr, K. and Perrin, L. (2022). Genetic architecture of natural variation of cardiac performance from flies to humans. Elife 11. PubMed ID: 36383075
Summary:
Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. This study investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome-wide associations studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that this study identified were used to map potential regulatory non-coding regions, which in turn were employed to predict transcription factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart-specific knockdown. Additionally, this study showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, it was shown that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, correlations were documented between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 (Drosophila Poxm) and EGR2 (Drosophila Stripe) in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.
Guo, X., Yu, Z. and Yin, D. (2023). Sex-dependent obesogenic effect of tetracycline on Drosophila melanogaster deteriorated by dysrhythmia. J Environ Sci (China) 124: 472-480. PubMed ID: 36182155
Summary:
Antibiotics have been identified as obesogens contributing to the prevalence of obesity. Moreover, their environmental toxicity shows sex dependence, which might also explain the sex-dependent obesity observed. Yet, the direct evidence for such a connection and the underlying mechanisms remain to be explored. In this study, the effects of tetracycline, which is a representative antibiotic found in both environmental and food samples, on Drosophila melanogaster were studied with consideration of both sex and circadian rhythms (represented by the eclosion rhythm). Results showed that in morning-eclosed adults, tetracycline significantly stimulated the body weight of females (AM females) at 0.1, 1.0, 10.0 and 100.0 μg/L, while tetracycline only stimulated the body weight of males (AM males) at 1.0 μg/L. In the afternoon-eclosed adults, tetracycline significantly stimulated the body weight of females (PM females) at 0.1, 1.0 and 100.0 μg/L, while it showed more significant stimulation in males (PM males) at all concentrations. Notably, the stimulation levels were the greatest in PM males among all the adults. The results showed the clear sex dependence of the obesogenic effects, which was diminished by dysrhythmia. Further biochemical assays and clustering analysis suggested that the sex- and rhythm-dependent obesogenic effects resulted from the bias toward lipogenesis against lipolysis. Moreover, they were closely related to the preference for the energy storage forms of lactate and glucose and also to the presence of excessive insulin, with the involvement of glucolipid metabolism. Such relationships indicated potential bridges between the obesogenic effects of pollutants and other diseases, e.g., cancer and diabetes.
Malita, A., Kubrak, O., Koyama, T., Ahrentløv, N., Texada, M. J., Nagy, S., Halberg, K. V. and Rewitz, K. (2022). A gut-derived hormone suppresses sugar appetite and regulates food choice in Drosophila. Nat Metab. PubMed ID: 36344765
Summary:
Animals must adapt their dietary choices to meet their nutritional needs. How these needs are detected and translated into nutrient-specific appetites that drive food-choice behaviours is poorly understood. This study shows that enteroendocrine cells of the adult female Drosophila midgut sense nutrients and in response release neuropeptide F (NPF), which is an ortholog of mammalian neuropeptide Y-family gut-brain hormones. Gut-derived NPF acts on glucagon-like adipokinetic hormone (AKH) signalling to induce sugar satiety and increase consumption of protein-rich food, and on adipose tissue to promote storage of ingested nutrients. Suppression of NPF-mediated gut signalling leads to overconsumption of dietary sugar while simultaneously decreasing intake of protein-rich yeast. Furthermore, gut-derived NPF has a female-specific function in promoting consumption of protein-containing food in mated females. Together, these findings suggest that gut NPF-to-AKH signalling modulates specific appetites and regulates food choice to ensure homeostatic consumption of nutrients, providing insight into the hormonal mechanisms that underlie nutrient-specific hungers.
Semaniuk, U. V., Gospodaryov, D. V., Strilbytska, O. M., Kucharska, A. Z., Sokol-Lętowska, A., Burdyliuk, N. I., Storey, K. B., Bayliak, M. M. and Lushchak, O. (2022). Chili-supplemented food decreases glutathione-S-transferase activity in Drosophila melanogaster females without a change in other parameters of antioxidant system. Redox Rep 27(1): 221-229. PubMed ID: 36200601
Summary:
Many plant-derived anti-aging preparations influence antioxidant defense system. Consumption of food supplemented with chili pepper powder was found to extend lifespan in the fruit fly, Drosophila melanogaster. The present study aimed to test a connection between life-extending effect of chili powder and antioxidant defense system of D. melanogaster. Flies were reared for 15 days in the mortality cages on food with 0% (control), 0.04%, 0.12%, 0.4%, or 3% chili powder. Antioxidant and related enzymes, as well as oxidative stress indices were measured. Female flies that consumed chili-supplemented food had a 40-60% lower glutathione-S-transferase (GST) activity as compared with the control cohort. Activity of superoxide dismutase (SOD) was about 37% higher in males that consumed food with 3% chili powder in comparison with the control cohort. Many of the parameters studied were sex-dependent. It is concluded that consumption of chili-supplemented food extends lifespan in fruit fly cohorts in a concentration- and gender-dependent manner. However, this extension is not mediated by a strengthening of antioxidant defenses. Consumption of chili-supplemented food does not change the specific relationship between antioxidant and related enzymes in D. melanogaster, and does not change the linkage of the activities of these enzymes to fly gender.
Ritchie, I. T., Needles, K. T., Leigh, B. A., Kaur, R. and Bordenstein, S. R. (2022). Transgenic cytoplasmic incompatibility persists across age and temperature variation in Drosophila melanogaster. iScience 25(11): 105327. PubMed ID: 36304111
Summary:
Environmental stressors can impact the basic biology and applications of host-microbe symbioses. For example, Wolbachia symbiont densities and cytoplasmic incompatibility (CI) levels can decline in response to extreme temperatures and host aging. To investigate whether transgenic expression of CI-causing cif genes overcomes the environmental sensitivity of CI, transgenic male flies were exposed to low and high temperatures as well as aging treatments. The results indicate that transgenic cif expression induces nearly complete CI regardless of temperature and aging, despite severe weakening of Wolbachia-based wild-type CI. Strong CI levels correlate with higher levels of cif transgene expression in young males. Altogether, these results highlight that transgenic CI persists against common environmental pressures and may be relevant for future control applications involving the cifA and cifB transgenes.
Bohere, J., Eldridge-Thomas, B. L. and Kolahgar, G. (2022). Vinculin recruitment to α-catenin halts the differentiation and maturation of enterocyte progenitors to maintain homeostasis of the Drosophila intestine. Elife 11. PubMed ID: 36269226
Summary:
Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the Drosophila intestinal epithelium, this study investigated the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. Vinculin was found to regulated by &alpha-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, it was shown that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number.

Thursday, December 15th - Synapse and Vesicles

Muttathukunnel, P., Frei, P., Perry, S., Dickman, D. and Muller, M. (2022). Rapid homeostatic modulation of transsynaptic nanocolumn rings. Proc Natl Acad Sci U S A 119(45): e2119044119. PubMed ID: 36322725
Summary:
Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, it was found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. Distinct phases of structural transsynaptic homeostatic plasticity were revealed, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy.
Swope, R. D., Hertzler, J. I., Stone, M. C., Kothe, G. O. and Rolls, M. M. (2022). The exocyst complex is required for developmental and regenerative neurite growth in vivo. Dev Biol 492: 1-13. PubMed ID: 36162553
Summary:
The exocyst complex is an important regulator of intracellular trafficking and tethers secretory vesicles to the plasma membrane. Understanding of its role in neuron outgrowth remains incomplete, and previous studies have come to different conclusions about its importance for axon and dendrite growth, particularly in vivo. To investigate exocyst function in vivo Drosophila sensory neurons were used as a model system. To bypass early developmental requirements in other cell types, neuron-specific RNAi was used to target seven exocyst subunits. Initial neuronal development proceeded normally in these backgrounds, however, this was considered to be due to residual exocyst function. To probe neuronal growth capacity at later times after RNAi initiation, laser microsurgery was used to remove axons or dendrites and prompt regrowth. Exocyst subunit RNAi reduced axon regeneration, although new axons could be specified. In control neurons, a vesicle trafficking marker often concentrated in the new axon, but this pattern was disrupted in Sec6 RNAi neurons. Dendrite regeneration was also severely reduced by exocyst RNAi, even though the trafficking marker did not accumulate in a strongly polarized manner during normal dendrite regeneration. The requirement for the exocyst was not limited to injury contexts as exocyst subunit RNAi eliminated dendrite regrowth after developmental pruning. It is concluded that the exocyst is required for injury-induced and developmental neurite outgrowth, but that residual protein function can easily mask this requirement.
Csizmadia, T., Dosa, A., Farkas, E., Csikos, B. V., Kriska, E. A., Juhász, G. and Low, P. (2022). Developmental program-independent secretory granule degradation in larval salivary gland cells of Drosophila. Traffic. PubMed ID: 36353974
Summary:
Both constitutive and regulated secretion require cell organelles that are able to store and release the secretory cargo. During development, the larval salivary gland of Drosophila initially produces high amount of glue-containing small immature secretory granules, which then fuse with each other and reach their normal 3-3.5 μm in size. Following the burst of secretion, obsolete glue granules directly fuse with late endosomes or lysosomes by a process called crinophagy, which leads to fast degradation and recycling of the secretory cargo. However, hindering of endosome-to-TGN retrograde transport in these cells causes abnormally small glue granules which are not able to fuse with each other. This study shows that loss of function of the SNARE genes Syntaxin 16 (Syx16) and Synaptobrevin (Syb), the small GTPase Rab6 and the GARP tethering complex members Vps53 and Scattered (Vps54) all involved in retrograde transport causes intense early degradation of immature glue granules via crinophagy independently of the developmental program. Moreover, silencing of these genes also provokes secretory failure and accelerated crinophagy during larval development. These results provide a better understanding of the relations among secretion, secretory granule maturation and degradation and paves the way for further investigation of these connections in other metazoans.
Crosby, D. and Lee, T. H. (2022). Membrane fusion by Drosophila atlastin does not require GTP hydrolysis. Mol Biol Cell 33(14): br23. PubMed ID: 36129776
Summary:
Atlastin (ATL) GTPases undergo trans dimerization and a power strokelike crossover conformational rearrangement to drive endoplasmic reticulum membrane fusion. Fusion depends on GTP, but the role of nucleotide hydrolysis has remained controversial. For instance, nonhydrolyzable GTP analogs block fusion altogether, suggesting a requirement for GTP hydrolysis in ATL dimerization and crossover, but this leaves unanswered the question of how the ATL dimer is disassembled after fusion. The truncated cytoplasmic domain of wild-type Drosophila ATL (DATL) and a novel hydrolysis-deficient D127N variant were recently used in single turnover assays to reveal that dimerization and crossover consistently precede GTP hydrolysis, with hydrolysis coinciding more closely with dimer disassembly. Moreover, while nonhydrolyzable analogs can bind the DATL G domain, they fail to fully recapitulate the GTP-bound state. This predicted that nucleotide hydrolysis would be dispensable for fusion. This study reports that the D127N variant of full-length DATL drives both outer and inner leaflet membrane fusion with little to no detectable hydrolysis of GTP. However, the trans dimer fails to disassemble and subsequent rounds of fusion fail to occur. These findings confirm that ATL mediated fusion is driven in the GTP-bound state, with nucleotide hydrolysis serving to reset the fusion machinery for recycling.
Choudhury, S. D., Dwivedi, M. K., Pippadpally, S., Patnaik, A., Mishra, S., Padinjat, R. and Kumar, V. (2022). AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila. eNeuro 9(5). PubMed ID: 36180220
Summary:
Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. This study shows that α2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila. Ultrastructural analysis of α2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. Mutations in α2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the α2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of α2-adaptin mutations. A model is proposed in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.
Araujo, M., Tavares, A., Vieira, D. V., Telley, I. A. and Oliveira, R. A. (2023). Endoplasmic reticulum membranes are continuously required to maintain mitotic spindle size and forces. Life Sci Alliance 6(1). PubMed ID: 36379670
Summary:
Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. This study shows that acute disruption of the ER around spindle poles affects mitotic spindle size and function in Drosophila syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. When centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, this study found that nuclear separation and distribution are affected by ER disruption. These results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions.

Wednesday, December 14th - Disease Models

Zhou, Z. D., Saw, W. T., Ho, P. G. H., Zhang, Z. W., Zeng, L., Chang, Y. Y., Sun, A. X. Y., Ma, D. R., Wang, H. Y., Zhou, L., Lim, K. L. and Tan, E. K. (2022). The role of tyrosine hydroxylase-dopamine pathway in Parkinson's disease pathogenesis. Cell Mol Life Sci 79(12): 599. PubMed ID: 36409355
Summary:
Parkinson's disease (PD) is characterized by selective and progressive dopamine (DA) neuron loss in the substantia nigra and other brain regions, with the presence of Lewy body formation. Most PD cases are sporadic, whereas monogenic forms of PD have been linked to multiple genes, including Leucine kinase repeat 2 (LRRK2) and PTEN-induced kinase 1 (PINK1), two protein kinase genes involved in multiple signaling pathways. There is increasing evidence to suggest that endogenous DA and DA-dependent neurodegeneration have a pathophysiologic role in sporadic and familial PD. This study generated patient-derived dopaminergic neurons and human midbrain-like organoids (hMLOs), transgenic (TG) mouse and Drosophila models, expressing both mutant and wild-type (WT) LRRK2 and PINK1. Using these models,the effect of LRRK2 and PINK1 on tyrosine hydroxylase (TH)-DA pathway was studied. PD-linked LRRK2 mutations were able to modulate TH-DA pathway, resulting in up-regulation of DA early in the disease which subsequently led to neurodegeneration. The LRRK2-induced DA toxicity and degeneration were abrogated by wild-type (WT) PINK1 (but not PINK1 mutations), and early treatment with a clinical-grade drug, α-methyl-L-tyrosine (α-MT), a TH inhibitor, was able to reverse the pathologies in human neurons and TG Drosophila models. Opposing effects between LRRK2 and PINK1 on TH expression were also identified, suggesting that functional balance between these two genes may regulate the TH-DA pathway. These findings highlight the vital role of the TH-DA pathway in PD pathogenesis. LRRK2 and PINK1 have opposing effects on the TH-DA pathway, and its balance affects DA neuron survival. LRRK2 or PINK1 mutations can disrupt this balance, promoting DA neuron demise. These findings provide support for potential clinical trials using TH-DA pathway inhibitors in early or prodromic PD.
Tibashailwa, N., Stephano, F., Shadrack, D. M., Munissi, J. J. E. and Nyandoro, S. S. (2022). Neuroprotective potential of cinnamoyl derivatives against Parkinson's disease indicators in Drosophila melanogaster and in silico models. Neurotoxicology. PubMed ID: 36410467
Summary:
Parkinson's disease (PD) is a movement disorder resulting from the loss of dopaminergic neurons over time. While there is no cure for PD, available conventional therapies aid to manage the motor symptoms. Natural products (NPs) derived from plants are among the most potent alternative therapies for PD. This study explored the neuroprotective potential of selected cinnamoyl derivatives namely toussaintine A (1), E-toussaintine E (2), asperphenamate (3) and julocrotine (4) against PD indicators using rotenone-challenged Drosophila melanogaster and in silico models. The compounds were first assessed for their toxicity preceding treatment experiments. Adult flies (aged 1-4 days) were exposed to varying concentrations of the compounds for 7 days. During the experiment, the mortality of flies was observed, and the lethal concentration (LC(50)) of each tested compound was determined. The LC(50) values were found to be 50.1, 55.6, 513.5, and 101.0μM for compounds 1, 2, 3, and 4, respectively. For seven days, flies were exposed to 500μM of rotenone and co-fed with a chosen dose of 40μM of each test compound in the diet. Using a negative geotaxis test, rotenone-challenged flies exhibited compromised climbing ability in comparison to control flies, the condition that was reversed by the action of studied compounds. Rotenone exposure also elevated malondialdehyde levels in the brain tissues, as measured by lipid peroxidation, when compared to control flies. In flies exposed to rotenone and co-fed with the compounds, this effect was lessened. In flies exposed to rotenone, mRNA levels of antioxidant enzymes such as superoxide dismutase and catalase were raised but were normalized in flies treated with the investigated compounds. Moreover, in-silico studies examined the inhibitory ability of compounds 1 - 4 against selected PD molecular targets, revealing the strong power of toussaintine A (1) against Adenosine receptor 2 (A2AR) and monoamine oxidase B. Thus, theser findings suggest that cinnamoyl derivatives have neuroprotective potential via reducing the oxidative burden and improving locomotor ability after toxin invectives. In particular, compound 1 at lower doses can simultaneously be a potential inhibitor of A2AR and an anti-oxidative mediator in the development of anti-PD agents.
Shit, B., Prakash, A., Sarkar, S., Vale, P. F. and Khan, I. (2022). Ageing leads to reduced specificity of antimicrobial peptide responses in Drosophila melanogaster. Proc Biol Sci 289(1987): 20221642. PubMed ID: 36382522
Summary:
Evolutionary theory predicts a late-life decline in the force of natural selection, possibly leading to late-life deregulations of the immune system. A potential outcome of such deregulations is the inability to produce specific immunity against target pathogens. This possibility was tested by infecting multiple Drosophila melanogaster lines (with bacterial pathogens) across age groups, where either individual or different combinations of Imd- and Toll-inducible antimicrobial peptides (AMPs) were deleted using CRISPR gene editing. A high degree of non-redundancy and pathogen-specificity of AMPs was demonstrated in young flies: in some cases, even a single AMP could confer complete resistance. However, ageing led to drastic reductions in such specificity to target pathogens, warranting the action of multiple AMPs across Imd and Toll pathways. Moreover, use of diverse AMPs either lacked survival benefits or even accompanied survival costs post-infection. These features were also sexually dimorphic: females required a larger repertoire of AMPs than males but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-repertoire was accompanied with ageing-induced downregulation of negative-regulators of the Imd pathway and damage to renal function post-infection, as features of poorly regulated immunity. Overall, this study highlights the potentially non-adaptive role of ageing in producing less-specific AMP responses, across sexes and pathogens.
Singh, A. and Agrawal, N. (2022). Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease. Metab Brain Dis 37(8): 2783-2792. PubMed ID: 36121619
Summary:
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder marked by progressive neuronal atrophy, particularly in striatum and cerebral cortex. Although predominant manifestations of the disease include loss in the triad of motor, cognitive and behavioral capabilities, metabolic dysfunction in patients and HD models are being increasingly recognized. Patients display progressive body weight loss, which aggravates the disease and leads to cachexia in the terminal stages. Using the Drosophila model of HD, it was reported earlier that diseased flies exhibit an atypical pattern of lipid gain and loss with progression along with exhibiting extensive mitochondrial dysfunction, impaired calcium homeostasis and heightened apoptosis in the fatbody. This study first monitored the structural changes that abdominal fatbody undergoes with disease progression. Further, the transcriptional changes of key metabolic genes in whole fly were checked as well as genes regulating mitochondrial function, apoptosis, autophagy and calcium homeostasis in the abdominal fatbody. Extensive alterations were found in whole-body and fatbody-specific transcriptional profile of the diseased flies, which was in consort with their stage-specific physiological state. Additionally, lysosome-mediated autophagy in the fatbody of diseased flies was also checked in order to ascertain the mechanisms contributing to fatbody atrophy at the terminal stage. Interestingly, elevated autophagy was found in fatbody of flies throughout disease progression. This study provides new insights into the effect on peripheral metabolism due to degeneration of neurons in the neurodegenerative disease, thereby discerns novel mechanisms leading to cachexia in diseased flies and advocates for the need of managing metabolic dysfunctions in HD.
Martinez, P., Patel, H., You, Y., Jury, N., Perkins, A., Lee-Gosselin, A., Taylor, X., You, Y., Viana Di Prisco, G., Huang, X., Dutta, S., Wijeratne, A. B., Redding-Ochoa, J., Shahid, S. S., Codocedo, J. F., Min, S., Landreth, G. E., Mosley, A. L., Wu, Y. C., McKinzie, D. L., Rochet, J. C., Zhang, J., Atwood, B. K., Troncoso, J. and Lasagna-Reeves, C. A. (2022). Bassoon contributes to tau-seed propagation and neurotoxicity. Nat Neurosci. PubMed ID: 36344699
Summary:
Tau aggregation is a defining histopathological feature of Alzheimer's disease and other tauopathies. However, the cellular mechanisms involved in tau propagation remain unclear. This study performed an unbiased quantitative proteomic analysis to identify proteins that specifically interact with this tau seed. Bassoon (BSN), a presynaptic scaffolding protein, was identified as an interactor of the tau seed isolated from a mouse model of tauopathy, and from Alzheimer's disease and progressive supranuclear palsy postmortem samples. BSN was shown to exacerbate tau seeding and toxicity in both mouse and Drosophila models for tauopathy, and that BSN downregulation decreases tau spreading and overall disease pathology, rescuing synaptic and behavioral impairments and reducing brain atrophy. These findings improve the understanding of how tau seeds can be stabilized by interactors such as BSN. Inhibiting tau-seed interactions is a potential new therapeutic approach for neurodegenerative tauopathies.
Meng, Q., Xu, Y., Li, Y. and Wang, Y. (2022). Novel studies on Drosophila melanogaster model reveal the roles of JNK-Jak/STAT axis and intestinal microbiota in insulin resistance. J Drug Target: 1-8. PubMed ID: 36343203
Summary:
The JNK pathway play a critical role in insulin resistance induced by a long-term high-sugar diet. However, the roles of up- and downstream molecules of the JNK pathway in insulin resistance are less known in vertebrates and invertebrates. As a classical organism in biological research, Drosophila melanogaster has been widely applied to the studies of mechanism of insulin resistance. Based on previous studies, this study found a novel predictive mechanism of the formation of insulin resistance in D. melanogaster. JNK activated by high-sugar diet and dysregulated intestinal microbiota could mediate inflammation, and then the activated JNK released Upd3, which in turn stimulated Jak/STAT pathway to release ImpL2. ImpL2 can compete with Drosophila insulin-like peptides (Dilps) for binding with the insulin receptor and inhibit the activation of insulin pathway. This study reviewed novel studies on the insulin signalling pathway based on the D. melanogaster model. The findings support the hypothesis. Therefore how a long-term high-sugar diet disrupts intestinal microbiota to induce inflammation and the disruption of JNK-Jak/STAT axis. This description may offer some new clues to the formation of insulin resistance.

Tuesday, December 13th - Gene, Protein, and Enzyme Structure and Function

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
Summary:
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. This study screened all 43 Drosophila eIFs and 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 is 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.
Yushkova, E. A. (2022). The effects of transpositions of functional I retrotransposons depend on the conditions and dose of parental exposure. Int J Radiat Biol: 1-13. PubMed ID: 36318749
Summary:
Transposable elements (TEs) cause destabilization of animal genomes. I retrotransposons of Drosophila melanogaster, as well as human LINE1 retrotransposons, are sources of intra- and interindividual diversity and responses to the action of internal and external factors. The aim of this study was to investigate the response to irradiation for the offspring of Drosophila melanogaster with the increased activity of inherited functional I elements. The material used was dysgenic Drosophila females with active I retrotransposons obtained as a result of crossing irradiated/non-irradiated parents of a certain genotype. Non-dysgenic females (without functional I elements) were used as controls. The effects of different conditions (irradiation of both parents simultaneously or separately) and doses (1-100 Gy) of parental irradiation have been assessed by analyzing SF-sterility, DNA damage and lifespan. The presence of full-size I retrotransposons was determined by PCR analysis. The maternal exposure and exposure of both parents are efficient in contrast with paternal exposure. Irradiation of mothers reduces the reproductive potential and viability of their female offspring which undergo high activity of functional I retrotransposons. Though I retrotranspositions negatively affect the female gonads, irradiation of the paternal line can increase the lifespan of SF-sterile females. Radiation stress in the range of 1-100 Gy increases DNA fragmentation in both somatic and germ cells of the ovaries with high I-retrotransposition. These results allow for the specificity of the radiation-induced behavior of I retrotransposons and their role in survival under conditions of strong radiation stress.
Nadimpalli, H. P., Guitart, T., Coll, O. and Gebauer, F. (2022). Ataxin-2, Twenty-four, and Dicer-2 are components of a noncanonical cytoplasmic polyadenylation complex. Life Sci Alliance 5(12). PubMed ID: 36114004
Summary:
Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. Evidence has been reported for an alternative noncanonical, CPEB-independent complex in Drosophila, of which the RNA-interference factor Dicer-2 is a component. This study investigate Dicer-2 mRNA targets and protein cofactors in cytoplasmic polyadenylation. Using RIP-Seq analysis, hundreds of potential Dicer-2 target transcripts were identified, ∼60% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Complex analyses indicated that both factors form an RNA-independent complex with Dicer-2 and mediate interactions of Dicer-2 with Wispy. Functional poly(A)-test analyses showed that Twenty-four and Ataxin-2 are required for cytoplasmic polyadenylation of a subset of Dicer-2 targets. These results reveal components of a novel cytoplasmic polyadenylation complex that operates during Drosophila early embryogenesis.
Lee, S., Chen, Y. C., Gillen, A. E., Taliaferro, J. M., Deplancke, B., Li, H. and Lai, E. C. (2022). Diverse cell-specific patterns of alternative polyadenylation in Drosophila. Nat Commun 13(1): 5372. PubMed ID: 36100597
Summary:
Most genes in higher eukaryotes express isoforms with distinct 3' untranslated regions (3' UTRs), generated by alternative polyadenylation (APA). Since 3' UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. Previous work used 3'-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. This study exploited comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3' UTRs across >250 adult Drosophila cell types. The cellular bases of multiple tissue-specific APA/ALE programs were revealed, such as 3' UTR lengthening in differentiated neurons and 3' UTR shortening in spermatocytes and spermatids. Dynamic 3' UTR patterns were traced across cell lineages, including in the male germline, and new APA patterns were discovered in the intestinal stem cell lineage. Finally, expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors were correllated in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3' isoforms across the major cell types of this widely-studied model organism.
Wang, L., Tracy, L., Su, W., Yang, F., Feng, Y., Silverman, N. and Zhang, Z. Z. Z. (2022). Retrotransposon activation during Drosophila metamorphosis conditions adult antiviral responses. Nat Genet. PubMed ID: 36396707
Summary:
Retrotransposons are one type of mobile genetic element that abundantly reside in the genomes of nearly all animals. Their uncontrolled activation is linked to sterility, cancer and other pathologies, thereby being largely considered detrimental. This study reports that, within a specific time window of development, retrotransposon activation can license the host's immune system for future antiviral responses. mdg4 (also known as Gypsy) retrotransposon selectively becomes active during metamorphosis at the Drosophila pupal stage. At this stage, mdg4 activation educates the host's innate immune system by inducing the systemic antiviral function of the nuclear factor-κB protein Relish in a dSTING-dependent manner. Consequently, adult flies with mdg4, Relish or dSTING silenced at the pupal stage are unable to clear exogenous viruses and succumb to viral infection. Altogether, these data reveal that hosts can establish a protective antiviral response that endows a long-term benefit in pathogen warfare due to the developmental activation of mobile genetic elements. [email protected]
Li, H. and Gavis, E. R. (2022). Drosophila FMRP controls miR-276-mediated regulation of nejire mRNA for space filling dendrite development. G3 (Bethesda). PubMed ID: 36102801
Summary:
MicroRNAs (miRNAs) are enriched in neurons and play important roles in dendritic spine development and synaptic plasticity. miRNA activity is controlled by a wide range of RNA-binding proteins (RBPs). Fragile X mental retardation protein (FMRP), a highly conserved RBP, has been linked to miRNA-mediated gene regulation in axonal development and dendritic spine formation. Using Drosophila larval sensory neurons, this study shows that a FMRP-associated miRNA, miR-276, functions in FMRP-mediated space filling dendrite morphogenesis. Using EGFP miRNA sensors, it was demonstrated that FMRP likely acts by regulating miR-276a RNA targeting rather than by modulating miRNA levels. Supporting this conclusion, miR-276a co-immunoprecipitated with FMRP and this association was dependent on the FMRP KH domains. By testing putative targets of the FMRP-miR-276a regulatory axis, nejire (nej) was identified as a FMRP-associated mRNA and, using EGFP reporters, showed that the nej 3' UTR is a target of miR-276a in vivo. Genetic analysis places nej downstream of the FMRP-miR-276a pathway in regulating dendrite patterning. Together, these findings support a model in which FMRP facilitates miR-276a-mediated control of nej for proper dendrite space filling morphology, and shed light on miRNA-dependent dendrite developmental pathology of Fragile X syndrome.

Monday, December 12th - Gene, Protein, and Enzyme Structure and Function

Bonche, R., Smolen, P., Chessel, A., Boisivon, S., Pisano, S., Voigt, A., Schaub, S., Theroned, P. and Pizette, S. (2022). Regulation of the collagen IV network by the basement membrane protein perlecan is crucial for squamous epithelial cell morphogenesis and organ architecture. Matrix Biol. PubMed ID: 36343860
Summary:
All epithelia have their basal side in contact with a specialized extracellular matrix, the basement membrane (BM). During development, the BM contributes to the shaping of epithelial organs via its mechanical properties. These properties rely on two core components of the BM, collagen type IV and perlecan/HSPG2, which both interact with another core component, laminin, the initiator of BM assembly. While collagen type IV supplies the BM with rigidity to constrain the tissue, perlecan antagonizes this effect. With the use of a hypomorphic allele, this study showed that the depletion of Trol (Drosophila perlecan) affects the morphogenesis of the three epithelia, but particularly that of the squamous one. The planar surface of the squamous epithelium (SE) becomes extremely narrow, due to a function for Trol in the control of the squamous shape of its cells. Furthermore, it was found that the lack of Trol impairs the biogenesis of the BM of the SE by modifying the structure of the collagen type IV lattice. Through atomic force microscopy and laser surgery, it was demonstrated that Trol provides elasticity to the SE's BM, thereby regulating the mechanical properties of the SE. Moreover, it was shown that Trol acts via collagen type IV, since the global reduction in the trol mutant context of collagen type IV or the enzyme that cross-links its 7S -but not the enzyme that cross-links its NC1- domain substantially restores the morphogenesis of the SE. In addition, a stronger decrease in collagen type IV achieved by the overexpression of the matrix metalloprotease 2 exclusively in the BM of the SE, significantly rescuing the organization of the two other epithelia. These data thus sustain a model in which Trol counters the rigidity conveyed by collagen type IV to the BM of the SE, via the regulation of the NC1-dependent assembly of its scaffold, allowing the spreading of the squamous cells, spreading which is compulsory for the architecture of the whole organ.
Amstrup Bay, A., Bæk, I., Loeschcke, V. and Givskov Sorensena, J. (2022). A functional study of the role of Turandot genes in Drosophila melanogaster: An emerging candidate mechanism for inducible heat tolerance. J Insect Physiol: 104456. PubMed ID: 36396076
Summary:
Plastic responses to heat stress have been shown to temporarily increase heat stress tolerance in many small ectotherms. Heat shock proteins (Hsps) have previously been shown to play a role in this induced heat stress tolerance. The heat shock response is fast but short lived, with the cellular Hsp concentration peaking within a few hours after induction. Induced heat stress tolerance, on the other hand, peaks 16-32 hours after induction. Therefore, the inducible heat stress response must depend on additional mechanisms. The Turandot gene family has been suggested as a candidate. It contains eight genes that are all upregulated to some degree following heat stress in Drosophila melanogaster. Previously, Turandot A (totA) and Turandot X (totX) have been linked to induced heat stress tolerance. This study aimed to investigate the temporal dynamics of Turandot expression and the functional role of totA and totC for heat stress tolerance. This was done by assaying the temporal heat tolerance and Turandot gene expression after a heat insult, and by exposing Turandot gene knock down flies to a range of heat hardening treatments, and evaluating the effects on heat tolerance. Successful gene knock down was verified by gene expression assays. In addition, expression of hsp70A was included. Both totA, totC, and hsp70A expression increased following a heat hardening treatment, while the results for totX were less clear. The expression of totC temporally co-occurred with and was functionally linked to increased heat tolerance. Expression of totA did not have a significant effect on heat stress tolerance. The complexity of inducible heat tolerance was underlined by the result that knock down of Turandot genes led to increased expression of hsp70. The results suggest that heat tolerance is determined by the interaction between several mechanisms, of which Turandot genes constitute one such mechanism.
Hoedjes, K. M., Kostic, H., Keller, L. and Flatt, T. (2022). Natural alleles at the Doa locus underpin evolutionary changes in Drosophila lifespan and fecundity. Proc Biol Sci 289(1986): 20221989. PubMed ID: 36350205
Summary:
'Evolve and resequence' (E&R) studies in Drosophila melanogaster have identified many candidate loci underlying the evolution of ageing and life history, but experiments that validate the effects of such candidates remain rare. In a recent E&R study several alleles were identified of the LAMMER kinase Darkener of apricot (Doa) as candidates for evolutionary changes in lifespan and fecundity. This study used two complementary approaches to confirm a functional role of Doa in life-history evolution. First, transgenic RNAi was used to study the effects of Doa at the whole-gene level. Ubiquitous silencing of expression in adult flies reduced both lifespan and fecundity, indicating pleiotropic effects. Second, to characterize segregating variation at Doa, four candidate single nucleotide polymorphisms (SNPs; Doa-1, -2, -3, -4) were examined using a genetic association approach. Three candidate SNPs had effects that were qualitatively consistent with expectations based on the E&R study: Doa-2 pleiotropically affected both lifespan and late-life fecundity; Doa-1 affected lifespan (but not fecundity); and Doa-4 affected late-life fecundity (but not lifespan). Finally, the last candidate allele (Doa-3) also affected lifespan, but in the opposite direction from predicted.
Hildebrandt, K., Kloppel, C., Gogel, J., Hartenstein, V. and Walldorf, U. (2022). Orthopedia expression during Drosophila melanogaster nervous system development and its regulation by microRNA-252. Dev Biol 492: 87-100. PubMed ID: 36179878
Summary:
During brain development of Drosophila melanogaster many transcription factors are involved in regulating neural fate and morphogenesis. In this study it was shown that the transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, plays an important role in this process. Otp is expressed in a stable pattern in defined lineages from mid-embryonic stages into the adult brain and therefore a very stable marker for these lineages. The abundance of the two different otp transcripts in the brain and hindgut during development was determined using qPCR. CRISPR/Cas9 generated otp mutants of the longer protein form significantly affect the expression of Otp in specific areas. An otp enhancer trap strain was generated by gene targeting and reintegration of Gal4 that mimics the complete expression of otp during development except the embryonic hindgut expression. Since in the embryo, the expression of Otp is posttranscriptionally regulated, putative miRNAs interacting with the otp 3'UTR were sought; microRNA-252 was identified as a candidate. Further analyses with mutated and deleted forms of the microRNA-252 interacting sequence in the otp 3'UTR demonstrate an in vivo interaction of microRNA-252 with the otp 3'UTR. An effect of this interaction is seen in the adult brain, where Otp expression is partially abolished in a knockout strain of microRNA-252. These results show that Otp is another important factor for brain development in Drosophila melanogaster.
Goupil, A., Heinen, J. P., Salame, R., Rossi, F., Reina, J., Pennetier, C., Simon, A., Skorski, P., Louzao, A., Bardin, A. J., Basto, R. and Gonzalez, C. (2022). Illuminati: a form of gene expression plasticity in Drosophila neural stem cells. Development 149(22). PubMed ID: 36399062
Summary:
While testing for genome instability in Drosophila as reported by unscheduled upregulation of UAS-GFP in cells that co-express GAL80 and GAL4, it was noticed that, as expected, background levels were low in most developing tissues. However, GFP-positive clones were frequent in the larval brain. Most of these clones originated from central brain neural stem cells. Using imaging-based approaches and genome sequencing, it was shown that these unscheduled clones do not result from chromosome loss or mutations in GAL80. This phenomenon was named 'Illuminati'. Illuminati is strongly enhanced in brat tumors and is also sensitive to environmental conditions such as food content and temperature. Illuminati is suppressed by Su(var)2-10, but it is not significantly affected by several modifiers of position effect variegation or Gal4::UAS variegation. It is conclude that Illuminati identifies a previously unknown type of functional instability that may have important implications in development and disease.
Wu, X., Gupta, K. and Swartz, K. J. (2022). Mutations within the selectivity filter reveal that Kv1 channels have distinct propensities to slow inactivate. J Gen Physiol 154(11). PubMed ID: 36197416
Summary:
Voltage-activated potassium (Kv) channels open in response to membrane depolarization and subsequently inactivate through distinct mechanisms. For the model Shaker Kv channel from Drosophila, fast N-type inactivation is thought to occur by a mechanism involving blockade of the internal pore by the N-terminus, whereas slow C-type inactivation results from conformational changes in the ion selectivity filter in the external pore. Although structures of the closely related Shaker and Kv1.2 channels containing mutations that promote slow inactivation both support a mechanism involving dilation of the outer selectivity filter, mutations in the outer pores of these two Kv channels have been reported to have markedly distinct effects on slow inactivation, raising questions about the extent to which slow inactivation is related in both channels. This study characterized the influence of a series of mutations within the external pore of Shaker and Kv1.2 channels and observed many distinct mutant phenotypes. Mutations at four positions near the selectivity filter promote inactivation less dramatically in Kv1.2 when compared to Shaker, and they identify one key variable position (T449 in Shaker and V381 in Kv1.2) underlying the different phenotypes in the two channels. Collectively, these results suggest that Kv1.2 is less prone to inactivate compared to Shaker, yet support a common mechanism of inactivation in the two channels.

Friday, December 9th - Evolution

Yassin, A., Gidaszewski, N., Debat, V. and David, J. R. (2022). Long-term evolution of quantitative traits in the Drosophila melanogaster species subgroup. Genetica 150(6): 343-353. PubMed ID: 36242716
Summary:
Quantitative genetics aims at untangling the genetic and environmental effects on phenotypic variation. Trait heritability, which summarizes the relative importance of genetic effects, is estimated at the intraspecific level, but theory predicts that heritability could influence long-term evolution of quantitative traits. The phylogenetic signal concept bears resemblance to heritability and it has often been called species-level heritability. Under certain conditions, such as trait neutrality or contribution to phylogenesis, within-species heritability and between-species phylogenetic signal should be correlated. This study investigated the potential relationship between these two concepts by examining the evolution of multiple morphological traits for which heritability has been estimated in Drosophila melanogaster. Specifically, 42 morphological traits in both sexes on a phylogeny were analysed, inferred from 22 nuclear genes for nine species of the melanogaster subgroup. Pagel's λ was used as a measurement of phylogenetic signal because it is the least influenced by the number of analysed taxa. Pigmentation traits showed the strongest concordance with the phylogeny, but no correlation was found between phylogenetic signal and heritability estimates mined from the literature. Data was obtained for multiple climatic variables inferred from the geographical distribution of each species. Phylogenetic regression of quantitative traits on climatic variables showed a significantly positive correlation with heritability. Convergent selection, the response to which depends on the trait heritability, may have led to the null association between phylogenetic signal and heritability for morphological traits in Drosophila. The possible causes of discrepancy between both statistics was discussed and caution against their confusion in evolutionary biology.
Martinez-Gomez, L., Cerdan-Velez, D., Abascal, F. and Tress, M. L. (2022). Origins and evolution of human tandem duplicated exon substitution events. Genome Biol Evol. PubMed ID: 36346145
Summary:
The mutually exclusive splicing of tandem duplicated exons produces protein isoforms that are identical save for a homologous region that allows for the fine tuning of protein function. Tandem duplicated exon substitution events are rare, yet highly important alternative splicing events. Most events are ancient, their isoforms are highly expressed, and they have significantly more pathogenic mutations than other splice event. This study analysed the physicochemical properties and functional roles of the homologous polypeptide regions produced by the 236 tandem duplicated exon substitutions annotated in the human gene set. Most important structural and functional residues in these homologous regions are maintained, and most changes are conservative rather than drastic. Three quarters of the isoforms produced from tandem duplicated exon substitution events are tissue specific, particularly in nervous and cardiac tissues, and tandem duplicated exon substitution events are enriched in functional terms related to structures in the brain and skeletal muscle. Considerable evidence was found for the convergent evolution of tandem duplicated exon substitution events in vertebrates, arthropods and nematodes. Twelve human gene families have orthologues with tandem duplicated exon substitution events in both Drosophila melanogaster and Caenorhabditis elegans. Six of these gene families are ion transporters, suggesting that tandem exon duplication in genes that control the flow of ions into the cell has an adaptive benefit. The ancient origins, the strong indications of tissue-specific functions, and the evidence of convergent evolution suggest that these events may have played important roles in the evolution of animal tissues and organs.
Cao, T., Akhter, S. and Jin, J. P. (2022). Early Divergence of the C-Terminal Variable Region of Troponin T Via a Pair of Mutually Exclusive Alternatively Spliced Exons Followed by a Selective Fixation in Vertebrate Heart. J Mol Evol 90(6): 452-467. PubMed ID: 36171395
Summary:
Troponin T (TnT) is the thin filament anchoring subunit of troponin complex and plays an organizer role in the Ca(2+)-regulation of striated muscle contraction. From an ancestral gene emerged ~ 700 million years ago in Bilateria, three homologous genes have evolved in vertebrates to encode muscle type-specific isoforms of TnT. Alternative splicing variants of TnT are present in vertebrate and invertebrate muscles to add functional diversity. While the C-terminal region of TnT is largely conserved, it contains an alternatively spliced segment emerged early in C. elegans, which has evolved into a pair of mutually exclusive exons in arthropods (10A and 10B of Drosophila TpnT gene) and vertebrates (16 and 17 of fast skeletal muscle Tnnt3 gene). The C-terminal alternatively spliced segment of TnT interfaces with the other two subunits of troponin with functional significance. The vertebrate cardiac TnT gene that emerged from duplication of the fast TnT gene has eliminated this alternative splicing by the fixation of an exon 17-like constitutive exon, indicating a functional value in slower and rhythmic contractions. The vertebrate slow skeletal muscle TnT gene that emerged from duplication of the cardiac TnT gene has the exon 17-like structure conserved, indicating its further function in sustained and fatigue resistant contractions. This functionality-based evolution is consistent with the finding that exon 10B-encoded segment of Drosophila TnT homologous to the exon 17-encoded segment of vertebrate fast TnT is selectively expressed in insect heart and leg muscles. The evolution of the C-terminal variable region of TnT demonstrates a submolecular mechanism in modifying striated muscle contractility and for the treatment of muscle and heart diseases.
Singh, K., Arun Samant, M. and Prasad, N. G. (2022). Evolution of cross-tolerance in Drosophila melanogaster as a result of increased resistance to cold stress. Sci Rep 12(1): 19536. PubMed ID: 36376445
Summary:
Cold stress is a critical environmental challenge that affects an organism's fitness-related traits. In Drosophila, increased resistance to specific environmental stress may lead to increased resistance to other kinds of stress. This study aimed to understand whether increased cold stress resistance in Drosophila melanogaster can facilitate their ability to tolerate other environmental stresses. This study used successfully selected replicate populations of D. melanogaster against cold shock and their control population. The present work investigated egg viability and mating frequency with and without heat and cold shock conditions in the selected and their control populations. Resistance to cold shock, heat shock, desiccation, starvation, and survival post-challenge with Staphylococcus succinus subsp. succinus PK-1 were also examined in the selected and their control populations. After cold-shock treatment, it was found a 1.25 times increase in egg viability and a 1.57 times increase in mating frequency in the selected populations compared to control populations. Moreover, more males (0.87 times) and females (1.66 times) of the selected populations survived under cold shock conditions relative to their controls. After being subjected to heat shock, the selected population's egg viability and mating frequency increased by 0.30 times and 0.57 times, respectively, compared to control populations. Additionally, more selected males (0.31 times) and females (0.98 times) survived under heat shock conditions compared to the control populations. Desiccation resistance slightly increased in the females of the selected populations relative to their control, but no change was observed in the case of males. Starvation resistance decreased in males and females of the selected populations compared to their controls. These findings suggest that the increased resistance to cold shock correlates with increased tolerance to heat stress, but this evolved resistance comes at a cost, with decreased tolerance to starvation.
Winbush, A. and Singh, N. D. (2022). Variation in fine scale recombination rate in temperature-evolved Drosophila melanogaster populations in response to selection. G3 (Bethesda). PubMed ID: 35961026
Summary:
Meiotic recombination plays a critical evolutionary role in maintaining fitness in response to selective pressures due to changing environments. Variation in recombination rate has been observed amongst and between species and populations and within genomes across numerous taxa. Studies have demonstrated a link between changes in recombination rate and selection, but the extent to which fine scale recombination rate varies between evolved populations during the evolutionary period in response to selection is under active research. This study utilize a set of three temperature-evolved Drosophila melanogaster populations that were shown to have diverged in several phenotypes, including recombination rate, based on the temperature regime in which they evolved. Using whole genome sequencing data from these populations, LD-based fine scale recombination maps wee generated for each population. With these maps, recombination rates and patterns were comparedamong the three populations; they have diverged at fine scales but are conserved at broader scales. A correlation was was further demonstrated between recombination rates and genomic variation in the three populations. Lastly, variation was shown in localized regions of enhanced recombination rates, termed warm-spots, between the populations with these warm-spots and associated genes overlapping areas previously shown to have diverged in the three populations due to selection. These data support the existence of recombination modifiers in these populations which are subject to selection during evolutionary change.
Tanaka, K., Barmina, O., Thompson, A., Massey, J. H., Kim, B. Y., Suvorov, A. and Kopp, A. (2022). Evolution and development of male-specific leg brushes in Drosophilidae. Dev Genes Evol. PubMed ID: 35939093
Summary:
The origin, diversification, and secondary loss of sexually dimorphic characters are common in animal evolution. In some cases, structurally and functionally similar traits have evolved independently in multiple lineages. Prominent examples of such traits include the male-specific grasping structures that develop on the front legs of many dipteran insects. This report describes the evolution and development of one of these structures, the male-specific "sex brush." The sex brush is composed of densely packed, irregularly arranged modified bristles and is found in several distantly related lineages in the family Drosophilidae. Phylogenetic analysis using 250 genes from over 200 species provides modest support for a single origin of the sex brush followed by many secondary losses; however, independent origins of the sex brush cannot be ruled out completely. Sex brushes were shown to develop in very similar ways in all brush-bearing lineages. The dense packing of brush hairs is explained by the specification of bristle precursor cells at a near-maximum density permitted by the lateral inhibition mechanism, as well as by the reduced size of the surrounding epithelial cells. In contrast to the female and the ancestral male condition, where bristles are arranged in stereotypical, precisely spaced rows, cell migration does not contribute appreciably to the formation of the sex brush. The complex phylogenetic history of the sex brush can make it a valuable model for investigating coevolution of sex-specific morphology and mating behavior.

Thursday, December 8th - Apoptosis

Zhang, J., Zhang, W., Wei, L., Zhang, L., Liu, J., Huang, S., Li, S., Yang, W. and Li, K. (2022). E93 promotes transcription of RHG genes to initiate apoptosis during Drosophila salivary gland metamorphosis. Insect Sci. PubMed ID: 36281570
Summary:
20-hydroxyecdysone (20E) induced transcription factor E93 is important for larval-adult transition, which functions in programmed cell death of larval obsolete tissues, and the formation of adult new tissues. However, the apoptosis-related genes directly regulated by E93 are still ambiguous. In this study, an E93 mutation fly strain was obtained by clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-mediated long exon deletion to investigate whether and how E93 induces apoptosis during larval tissues metamorphosis. The transcriptional profile of E93 was consistent with 3 RHG (rpr, hid, and grim) genes and the effector caspase gene drice, and all their expressions peaked at the initiation of apoptosis during the degradation of salivary glands. The transcription expression of 3 RHG genes decreased and apoptosis was blocked in E93 mutation salivary gland during metamorphosis. In contrast, E93 overexpression promoted the transcription of 3 RHG genes, and induced advanced apoptosis in the salivary gland. Moreover, E93 not only enhance the promoter activities of the 3 RHG genes in Drosophila Kc cells in vitro, but also in the salivary gland in vivo. These results demonstrated that 20E induced E93 promotes the transcription of RHG genes to trigger apoptosis during obsolete tissues degradation at metamorphosis in Drosophila.
Clarembaux-Badell, L., Baladron-de-Juan, P., Gabilondo, H., Rubio-Ferrera, I., Millan, I., Estella, C., Valverde-Ortega, F. S., Cobeta, I. M., Thor, S. and Benito-Sipos, J. (2022). Dachshund acts with Abdominal-B to trigger programmed cell death in the Drosophila central nervous system at the frontiers of Abd-B expression. Dev Neurobiol 82(6): 495-504. PubMed ID: 35796156
Summary:
A striking feature of the nervous system pertains to the appearance of different neural cell subtypes at different axial levels. Studies in the Drosophila central nervous system reveal that one mechanism underlying such segmental differences pertains to the segment-specific removal of cells by programmed cell death (PCD). One group of genes involved in segment-specific PCD is the Hox homeotic genes. However, while segment-specific PCD is highly precise, Hox gene expression is evident in gradients, raising the issue of how the Hox gene function is precisely gated to trigger PCD in specific segments at the outer limits of Hox expression. The Drosophila Va neurons are initially generated in all nerve cord segments but removed by PCD in posterior segments. Va PCD is triggered by the posteriorly expressed Hox gene Abdominal-B (Abd-B). However, Va PCD is highly reproducible despite exceedingly weak Abd-B expression in the anterior frontiers of its expression. This study found that the transcriptional cofactor Dachshund supports Abd-B-mediated PCD in its anterior domain. In vivo bimolecular fluorescence complementation analysis lends support to the idea that the Dachshund/Abd-B interplay may involve physical interactions. These findings provide an example of how combinatorial codes of transcription factors ensure precision in Hox-mediated PCD in specific segments at the outer limits of Hox expression.
Long, M. and McWilliams, T. G. (2022). Lipid droplets promote efficient mitophagy. Autophagy: 1-2. PubMed ID: 35939345
Summary:
Mitophagy neutralizes defective mitochondria via lysosomal elimination. Increased levels of mitophagy hallmark metabolic transitions and are induced by iron depletion, yet its metabolic basis has not been studied in-depth. How mitophagy integrates with different homeostatic mechanisms to support metabolic integrity is incompletely understood. This study examined metabolic adaptations in cells treated with deferiprone (DFP), a therapeutic iron chelator known to induce PINK1-PRKN-independent mitophagy. Iron depletion was found to profoundly rewired the cellular metabolome, remodeling lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurs upstream of mitochondrial turnover, with many LDs bordering mitochondria upon iron chelation. Surprisingly, DGAT1 inhibition restricts mitophagy in vitro by lysosomal dysfunction. Genetic depletion of mdy/DGAT1 in vivo impairs neuronal mitophagy and locomotor function in Drosophila, demonstrating the physiological relevance of these findings.
Jiao, D., Chen, Y., Wang, Y., Sun, H., Shi, Q., Zhang, L., Zhao, X., Liu, Y., He, H., Lv, Z., Liu, C., Zhang, P., Gao, K., Huang, Y., Li, Y., Li, L. and Wang, C. (2022). DCAF12 promotes apoptosis and inhibits NF-κB activation by acting as an endogenous antagonist of IAPs. Oncogene 41(21): 3000-3010. PubMed ID: 35459779
Summary:
Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival and appear to neutralize the cell death machinery by binding pro-apoptotic caspases. dcaf12 was recently identified as an apoptosis regulator in Drosophila. However, the underlying molecular mechanisms are unknown. HThis study revealed that human DCAF12 homolog binds multiple IAPs, including XIAP, cIAP1, cIAP2, and BRUCE, through recognition of BIR domains in IAPs. The pro-apoptotic function of DCAF12 is dependent on its capacity to bind IAPs. In response to apoptotic stimuli, DCAF12 translocates from the nucleus to the cytoplasm, where it blocks the interaction between XIAP and pro-apoptotic caspases to facilitate caspase activation and apoptosis execution. Similarly, DCAF12 suppresses NF-κB activation in an IAP binding-dependent manner. Moreover, DCAF12 acts as a tumor suppressor to restrict the malignant phenotypes of cancer cells. Together, these results suggest that DCAF12 is an evolutionarily conserved IAP antagonist.
Damulewicz, M., Szypulski, K. and Pyza, E. (2022). Glia-Neurons Cross-Talk Regulated Through Autophagy. Front Physiol 13: 886273. PubMed ID: 35574462
Summary:
Autophagy is a self-degradative process which plays a role in removing misfolded or aggregated proteins, clearing damaged organelles, but also in changes of cell membrane size and shape. The aim of this phenomenon is to deliver cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle (autophagosome), that fuses with a lysosome to form autolysosome, where cargo is degraded by proteases. Products of degradation are transported back to the cytoplasm, where they can be re-used. This study showed that autophagy is important for proper functioning of the glia and that it is involved in the regulation of circadian structural changes in processes of the pacemaker neurons. This effect is mainly observed in astrocyte-like glia, which play a role of peripheral circadian oscillators in the Drosophila brain.
Ciesielski, H. M., Nishida, H., Takano, T., Fukuhara, A., Otani, T., Ikegawa, Y., Okada, M., Nishimura, T., Furuse, M. and Yoo, S. K. (2022). Erebosis, a new cell death mechanism during homeostatic turnover of gut enterocytes. PLoS Biol 20(4): e3001586. PubMed ID: 35468130
Summary:
Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. This study discovered a previously uncharacterized, new phenomenon, which was name erebosis based on the ancient Greek word erebos ("complete darkness"), in the gut enterocytes of adult Drosophila. Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. It is hypothesized that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut.

Wednesday, December 7th -Evolution

Kinoshita, S., Takarada, K., Kinoshita, Y. and Inoue, Y. H. (2022). Drosophila hemocytes recognize lymph gland tumors of mxc mutants and activate the innate immune pathway in a reactive oxygen species-dependent manner. Biol Open 11(11). PubMed ID: 36226812
Summary:
Mechanisms of cancer cell recognition and elimination by the innate immune system remains unclear. The immune signaling pathways are activated in the fat body to suppress the tumor growth in mxcmbn1 hematopoietic tumor mutants in Drosophila by inducing antimicrobial peptides (AMP). This study investigated the regulatory mechanism underlying the activation in the mutant. Firstly, it was found that reactive oxygen species (ROS) accumulated in the hemocytes due to induction of dual oxidase and one of its activators. This was required for the AMP induction and the tumor growth suppression. Next, more hemocytes transplanted from normal larvae were associated with the mutant tumor than normal lymph glands (LGs). Matrix metalloproteinase 1 and 2 (MMP2) were highly expressed in the tumors. The basement membrane components in the tumors were reduced and ultimately lost inside. Depletion of the MMP2 rather than MMP1 resulted in a significantly reduced AMP expression in the mutant larvae. The hemocytes may recognize the disassembly of basement membrane in the tumors and activate the ROS production. These findings highlight the mechanism via which macrophage-like hemocytes recognize tumor cells and subsequently convey the information to induce AMPs in the fat body. They contribute to uncover the role of innate immune system against cancer.
Shaka, M., Arias-Rojas, A., Hrdina, A., Frahm, D. and Iatsenko, I. (2022). Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster. PLoS Pathog 18(9): e1010825. PubMed ID: 36084158
Summary:
Bacteria from the genus Providencia are ubiquitous Gram-negative opportunistic pathogens, causing "travelers' diarrhea", urinary tract, and other nosocomial infections in humans. Some Providencia strains have also been isolated as natural pathogens of Drosophila melanogaster. This study investigated the virulence factors of a representative Providencia species-P. alcalifaciens. A P. alcalifaciens transposon mutant library was generated, and an unbiased forward genetics screen was performed in vivo for attenuated mutants. The screen uncovered 23 mutants with reduced virulence. The vast majority of them had disrupted genes linked to lipopolysaccharide (LPS) synthesis or modifications. These LPS mutants were sensitive to cationic antimicrobial peptides (AMPs) in vitro and their virulence was restored in Drosophila mutants lacking most AMPs. Thus, LPS-mediated resistance to host AMPs is one of the virulence strategies of P. alcalifaciens. Another subset of P. alcalifaciens attenuated mutants exhibited increased susceptibility to reactive oxygen species (ROS) in vitro and their virulence was rescued by chemical scavenging of ROS in flies prior to infection. Using genetic analysis, it was found that the enzyme Duox specifically in hemocytes is the source of bactericidal ROS targeting P. alcalifaciens. Consistently, the virulence of ROS-sensitive P. alcalifaciens mutants was rescued in flies with Duox knockdown in hemocytes. Therefore, these genes function as virulence factors by helping bacteria to counteract the ROS immune response. This reciprocal analysis of host-pathogen interactions between D. melanogaster and P. alcalifaciens identified that AMPs and hemocyte-derived ROS are the major defense mechanisms against P. alcalifaciens, while the ability of the pathogen to resist these host immune responses is its major virulence mechanism.
Vincent, C. M., Beckwith, E. J., Simoes da Silva, C. J., Pearson, W. H., Kierdorf, K., Gilestro, G. F. and Dionne, M. S. (2022). Infection increases activity via Toll dependent and independent mechanisms in Drosophila melanogaster. PLoS Pathog 18(9): e1010826. PubMed ID: 36129961
Summary:
Host behavioural changes are among the most apparent effects of infection. 'Sickness behaviour' can involve a variety of symptoms, including anorexia, depression, and changed activity levels. Using a real-time tracking and behavioural profiling platform, this study shows that in Drosophila melanogaster, several systemic bacterial infections cause significant increases in physical activity and that the extent of this activity increase is a predictor of survival time in some lethal infections. Using multiple bacteria and D. melanogaster immune and activity mutants, this study showed that increased activity is driven by at least two different mechanisms. Increased activity after infection with Micrococcus luteus, a Gram-positive bacterium rapidly cleared by the immune response, strictly requires the Toll ligand spatzle. In contrast, increased activity after infection with Francisella novicida, a Gram-negative bacterium that cannot be cleared by the immune response, is entirely independent of both Toll and the parallel IMD pathway. The existence of multiple signalling mechanisms by which bacterial infections drive increases in physical activity implies that this effect may be an important aspect of the host response.
Tang, C., Kurata, S. and Fuse, N. (2022). Genetic dissection of innate immune memory in Drosophila melanogaster. Front Immunol 13: 857707. PubMed ID: 35990631
Summary:
Current studies have demonstrated that innate immunity possesses memory characteristics. Although the molecular mechanisms underlying innate immune memory have been addressed by numerous studies, genetic variations in innate immune memory and the associated genes remain unclear. This study explored innate immune memory in 163 lines of Drosophila melanogaster from the Drosophila Synthetic Population Resource. In this assay system, prior training with low pathogenic bacteria (Micrococcus luteus) increased the survival rate of flies after subsequent challenge with highly pathogenic bacteria (Staphylococcus aureus). This positive training effect was observed in most lines, but some lines exhibited negative training effects. Survival rates under training and control conditions were poorly correlated, suggesting that distinct genetic factors regulate training effects and normal immune responses. Subsequent quantitative trait loci analysis suggested that four loci containing 80 genes may be involved in regulating innate immune memory. Among them, Adgf-A, which encodes an extracellular adenosine deaminase-related growth factor, was shown to be associated with training effects. These findings help to elucidate the genetic architecture of innate immune memory in Drosophila and may provide insight for new therapeutic treatments aimed at boosting immunity.
Ding, S. D., Leitao, A. B., Day, J. P., Arunkumar, R., Phillips, M., Zhou, S. O. and Jiggins, F. M. (2022). Trans-regulatory changes underpin the evolution of the Drosophila immune response. PLoS Genet 18(11): e1010453. PubMed ID: 36342922
Summary:
When an animal is infected, the expression of a large suite of genes is changed, resulting in an immune response that can defend the host. Despite much evidence that the sequence of proteins in the immune system can evolve rapidly, the evolution of gene expression is comparatively poorly understood. This study therefore investigated the transcriptional response to parasitoid wasp infection in Drosophila simulans and D. sechellia. Although these species are closely related, there has been a large scale divergence in the expression of immune-responsive genes in their two main immune tissues, the fat body and hemocytes. Many genes, including those encoding molecules that directly kill pathogens, have cis regulatory changes, frequently resulting in large differences in their expression in the two species. However, these changes in cis regulation overwhelmingly affected gene expression in immune-challenged and uninfected animals alike. Divergence in the response to infection was controlled in trans. It is argued that altering trans-regulatory factors, such as signalling pathways or immune modulators, may allow natural selection to alter the expression of large numbers of immune-responsive genes in a coordinated fashion.
Yoo, T. J., Sup Shim, M., Bang, J., Kim, J. H. and Lee, B. Jae (2022). SPS1 deficiency-triggered PGRP-LC and Toll expression controls innate immunity in Drosophila S2 cells. Biol Open 11(8). PubMed ID: 35723425
Summary:
Selenophosphate synthetase 1 (SPS1) is an essential gene for the cell growth and embryogenesis in Drosophila melanogaster. It has been reported that SPS1 deficiency stimulates the expression of genes responsible for the innate immune system, including antimicrobial peptides (AMPs), in Drosophila S2 cells. However, the underlying mechanism has not been elucidated. This study investigated the immune pathways that control the SPS1-deficiency-induced expression of AMPs in S2 cells. It was found that the activation of AMP expression is regulated by both immune deficiency (IMD) and the Toll pathway. Double knockdown of the upstream genes of each pathway with SPS1 showed that the peptidoglycan recognition protein-LC (PGRP-LC) and Toll genes are targeted by SPS1 for regulating these pathways. It was also found that the IMD and Toll pathway regulate AMP expression by cross-talking. The levels of PGRP-LC and Toll mRNAs were upregulated upon Sps1 knockdown (6.±0.36 and 3.2±0.45-fold, respectively, n=3). Overexpression of each protein also upregulated AMPs. Interestingly, PGRP-LC overexpression upregulated AMP more than Toll overexpression. These data strongly suggest that SPS1 controls the innate immune system of D. melanogaster through regulating PGRP-LC and Toll expression.

Tuesday, December 6th - Chromatin, DNA and Chromosome

Messina, G., Prozzillo, Y., Monache, F. D., Santopietro, M. V. and Dimitri, P. (2022). Evolutionary conserved relocation of chromatin remodeling complexes to the mitotic apparatus. BMC Biol 20(1): 172. PubMed ID: 35922843
Summary:
ATP-dependent chromatin remodeling complexes are multi-protein machines highly conserved across eukaryotic genomes. They control sliding and displacing of the nucleosomes, modulating histone-DNA interactions and making nucleosomal DNA more accessible to specific binding proteins during replication, transcription, and DNA repair, which are processes involved in cell division. The SRCAP and p400/Tip60 chromatin remodeling complexes in humans and the related Drosophila Tip60 complex belong to the evolutionary conserved INO80 family, whose main function is promoting the exchange of canonical histone H2A with the histone variant H2A in different eukaryotic species. Some subunits of these complexes were additionally shown to relocate to the mitotic apparatus and proposed to play direct roles in cell division in human cells. However, whether this phenomenon reflects a more general function of remodeling complex components and its evolutionary conservation remains unexplored. This study has combined cell biology, reverse genetics, and biochemical approaches to study the subcellular distribution of a number of subunits belonging to the SRCAP and p400/Tip60 complexes and assess their involvement during cell division progression in HeLa cells. Interestingly, beyond their canonical chromatin localization, the subunits under investigation accumulate at different sites of the mitotic apparatus (centrosomes, spindle, and midbody), with their depletion yielding an array of aberrant outcomes of mitosis and cytokinesis, thus causing genomic instability. Importantly, this behavior was conserved by the Drosophila melanogaster orthologs tested, despite the evolutionary divergence between fly and humans has been estimated at approximately 780 million years ago. Overall, these results support the existence of evolutionarily conserved diverse roles of chromatin remodeling complexes, whereby subunits of the SRCAP and p400/Tip60 complexes relocate from the interphase chromatin to the mitotic apparatus, playing moonlighting functions required for proper execution of cell division.
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
Summary:
The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. 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 considered 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.
Bonnet, J., Boichenko, I., Kalb, R., Le Jeune, M., Maltseva, S., Pieropan, M., Finkl, K., Fierz, B. and Muller, J. (2022). PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction. Genes Dev. PubMed ID: 36357125
Summary:
The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. This study shows that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.
Kapur, I., Boulier, E. L. and Francis, N. J. (2022). Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding. Epigenomes 6(4). PubMed ID: 36412795
Summary:
The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. It is therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. In this study identified three distinct low-complexity regions in Ph based on sequence composition. The role of each of these sequences was systematically tested in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. These data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. The results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. These observations highlight the complexity of regulation of biological condensates housed in single proteins.
Voortman, L., Anderson, C., Urban, E., Yuan, L., Tran, S., Neuhaus-Follini, A., Derrick, J., Gregor, T. and Johnston, R. J., Jr. (2022). Temporally dynamic antagonism between transcription and chromatin compaction controls stochastic photoreceptor specification in flies. Dev Cell 57(15): 1817-1832 PubMed ID: 35835116
Summary:
Stochastic mechanisms diversify cell fates during development. How cells randomly choose between two or more fates remains poorly understood. In the Drosophila eye, the random mosaic of two R7 photoreceptor subtypes is determined by expression of the transcription factor Spineless (Ss). This study investigated how cis-regulatory elements and trans factors regulate nascent transcriptional activity and chromatin compaction at the ss gene locus during R7 development. The ss locus is in a compact state in undifferentiated cells. An early enhancer drives transcription in all R7 precursors, and the locus opens. In differentiating cells, transcription ceases and the ss locus stochastically remains open or compacts. In Ss(ON) R7s, ss is open and competent for activation by a late enhancer, whereas in Ss(OFF) R7s, ss is compact, and repression prevents expression. These results suggest that a temporally dynamic antagonism, in which transcription drives large-scale decompaction and then compaction represses transcription, controls stochastic fate specification.
George, S., Blum, H. R., Torres-Zelada, E. F., Estep, G. N., Hegazy, Y. A., Speer, G. M. and Weake, V. M. (2022). The interaction between the Dbf4 ortholog Chiffon and Gcn5 is conserved in Dipteran insect species. Insect Mol Biol 31(6): 734-746. PubMed ID: 35789507
Summary:
Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, yeast two-hybrid assays were performed using Chiffon-B and Gcn5 from a variety of insect species; Chiffon-B and Gcn5 were found to interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. It is proposed propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.

Monday, December 5th - Disease Models

Kim, Y. A., Siddiqui, T., Blaze, J., Cosacak, M. I., Winters, T., Kumar, A., Tein, E., Sproul, A. A., Teich, A. F., Bartolini, F., Akbarian, S., Kizil, C., Hargus, G. and Santa-Maria, I. (2022). RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation. Acta Neuropathol. PubMed ID: 36357715
Summary:
Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. This study shows NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, decreased NSun2 protein expression and an increased ratio of pTau/NSun2 were observed in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, this study confirmed NSun2 deficiency results in tau hyperphosphorylation. It was also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AβO). Notably, AβO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avet was observed that age affects loading into RISC for a small number of miRNAs.
G, N. L., Ko, S., Peng, O., Bognar, B., Khmelkov, M., H, S. B. and Tower, J. (2022). A screen of small molecule and genetic modulators of life span in female Drosophila identifies etomoxir, RH5849 and unanticipated temperature effects. Fly (Austin) 16(1): 397-413. PubMed ID: 36412257
Summary:
Mifepristone increases life span in female Drosophila melanogaster, and its molecular target(s) remain unclear. In this study small molecule and genetic interventions were tested for ability to mimic mifepristone, or to decrease life span in a way that can be rescued by mifepristone. Etomoxir inhibits lipid metabolism, and significantly increased life span in virgin and mated females, but not males, at 50 μM concentration. Pioglitazone is reported to activate both mammalian PPAR γ and its Drosophila homolog Eip75B. Pioglitazone produced minor and inconsistent benefits for female Drosophila life span, and only at the lowest concentrations tested. Ecdysone is a Drosophila steroid hormone reported to regulate responses to mating, and RH5849 is a potent mimic of ecdysone. RH5849 reduced virgin female life span, and this was partly rescued by mifepristone. Mifepristone did not compete with RH5849 for activation of an ecdysone receptor (EcR)-responsive transgenic reporter, indicating that the relevant target for mifepristone is not EcR. The conditional GAL4/GAL80ts system was used in attempt to test the effect of an Eip75B RNAi construct on female life span. However, the 29C temperature used for induction reduced or eliminated mating-induced midgut hypertrophy, the negative life span effects of mating, and the positive life span effects of mifepristone. Even when applied after mating was complete, a shift to 29°C temperature reduced mating-induced midgut hypertrophy by half, and the life span effects of mating by 4.8-fold. Taken together, these results identify promising small molecules for further analysis, and inform the design of experiments involving the GAL4/GAL80ts system.
Gartland, S., Zeng, B. and Marr, M. T (2022). The small RNA landscape is stable with age and resistant to loss of dFOXO signaling in Drosophila. PLoS One 17(11): e0273590. PubMed ID: 36383505
Summary:
Small RNA pathways regulate many important processes including development, cellular physiology, aging, and innate immunity. The pathways illicit a form of posttranscriptional gene regulation that relies on small RNAs bound by the protein components of the RNA-induced silencing complexes (RISCs), which inhibit the expression of complementary RNAs. In Drosophila melanogaster, Argonaute 1 (Ago1) is the core RISC component in microRNA (miRNA) silencing, while Argonaute 2 (Ago2) is the core RISC component in small interfering RNA (siRNA) silencing. The expression of Ago1 and Ago2 is regulated by stress response transcription factor Forkhead box O (dFOXO) increasing siRNA silencing efficiency. dFOXO plays a role in multiple stress responses and regulates pathways important for longevity. This study used a next-generation sequencing approach to determine the effects of aging on small RNA abundance and RISC loading in male and female Drosophila. The impact of the loss of dFOXO on these processes was studied.The relative abundance of the majority of small RNAs does not change with age. Additionally, under normal growth conditions, the loss of dFOXO has little effect on the small RNA landscape. However, it was observed that age affects loading into RISC for a small number of miRNAs.
Inoshita, T., Liu, J. Y., Taniguchi, D., Ishii, R., Shiba-Fukushima, K., Hattori, N. and Imai, Y. (2022). Parkinson disease-associated Leucine-rich repeat kinase regulates UNC-104-dependent axonal transport of Arl8-positive vesicles in Drosophila. iScience 25(12): 105476. PubMed ID: 36404922
Summary:
Some Parkinson's disease (PD)-causative/risk genes, including the PD-associated kinase leucine-rich repeat kinase 2 (LRRK2), are involved in membrane dynamics. Although LRRK2 and other PD-associated genes are believed to regulate synaptic functions, axonal transport, and endolysosomal activity, it remains unclear whether a common pathological pathway exists. This study reports that the loss of Lrrk, an ortholog of human LRRK2, leads to the accumulation of the lysosome-related organelle regulator, Arl8 along with dense core vesicles at the most distal boutons of the neuron terminals in Drosophila. Moreover, the inactivation of a small GTPase Rab3 and altered Auxilin activity phenocopied Arl8 accumulation. The accumulation of Arl8-positive vesicles is UNC-104-dependent and modulated by PD-associated genes, Auxilin, VPS35, RME-8, and INPP5F, indicating that VPS35, RME-8, and INPP5F are upstream regulators of Lrrk. These results indicate that certain PD-related genes, along with LRRK2, drive precise neuroaxonal transport of dense core vesicles.
Han, Y. J. and Kim, K. (2022). Reduced oxidative stress suppresses neurotoxicity in the Drosophila model of TAF15-associated proteinopathies. Mol Brain 15(1): 93. PubMed ID: 36411469
Summary:
TATA-binding protein associated factor 15 (TAF15) is involved in the pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TAF15 accumulates as cytoplasmic aggregates in neurons and the clearance of aggregates may be a therapeutic strategy for ALS, the underlying mechanisms of TAF15 in ALS remain poorly understood. Recently, it was found that glutathione transferase omega 2 (GstO2) expression level is significantly reduced in the brain tissue of TAF15-expressing flies. This study demonstrated that GstO2 overexpression in TAF15-induced flies rescues the locomotive activity and neuromuscular junctional defects. Furthermore, TAF15 levels in both cytoplasm and nuclear fractions significantly decreased in the heads of GstO2 co-expressing flies. GstO2-co-overexpression in neurons caused a marked decrease in intracellular reactive oxygen species generation in TAF15-induced flies. These findings demonstrated that GstO2 was a pathogenic regulator of TAF15-associated proteinopathies. They help expand understanding of TAF15-associated ALS pathogenesis.
Lushchak, O., Strilbytska, O. and Storey, K. B. (2022). Gender-Specific Effects of Pro-longevity Interventions in Drosophila. jMech Ageing Dev: 111754. PubMed ID: 36375654
Summary:
Sex differences in lifespan are well recognized in the majority of animal species. For example, in male versus female Drosophila melanogaster there are significant differences in behavior and physiology. However, little is known about the underlying mechanisms of gender differences in responses to pro-longevity interventions in this model organism. This study summarized the existing data on the effects of nutritional and pharmacological anti-aging interventions such as nutrition regimens, diet and dietary supplementation on the lifespan of male and female Drosophila. Males and females have different sensitivities to interventions, and the effects are highly dependent on genetic background, mating, dose and exposure duration. Thia work highlights the importance of understanding the mechanisms that underlie the gender-specific effect of anti-aging manipulations. This will provide insight into how these benefits may be valuable for elucidating the primary physiological and molecular targets involved in aging and lifespan determination.

Thursday, December 1st -

Kadakia, N., Demir, M., Michaelis, B. T., DeAngelis, B. D., Reidenbach, M. A., Clark, D. A. and Emonet, T. (2022). Odour motion sensing enhances navigation of complex plumes. Nature. PubMed ID: 36352224
Summary:
Odour plumes in the wild are spatially complex and rapidly fluctuating structures carried by turbulent airflows. To successfully navigate plumes in search of food and mates, insects must extract and integrate multiple features of the odour signal, including odour identity, intensity and timing. Effective navigation requires balancing these multiple streams of olfactory information and integrating them with other sensory inputs, including mechanosensory and visual cues. Studies dating back a century have indicated that, of these many sensory inputs, the wind provides the main directional cue in turbulent plumes, leading to the longstanding model of insect odour navigation as odour-elicited upwind motion. This study shows that Drosophila melanogaster shape their navigational decisions using an additional directional cue-the direction of motion of odours-which they detect using temporal correlations in the odour signal between their two antennae. Using a high-resolution virtual-reality paradigm to deliver spatiotemporally complex fictive odours to freely walking flies, this study demonstrates that such odour-direction sensing involves algorithms analogous to those in visual-direction sensing. Combining simulations, theory and experiments, odour motion was shown to contain valuable directional information that is absent from the airflow alone, and that both Drosophila and virtual agents are aided by that information in navigating naturalistic plumes. The generality of these findings suggests that odour-direction sensing may exist throughout the animal kingdom and could improve olfactory robot navigation in uncertain environments.
Li, H. and Gavis, E. R. (2022). Drosophila FMRP controls miR-276-mediated regulation of nejire mRNA for space-filling dendrite development. G3 (Bethesda) 12(11). PubMed ID: 36102801
Summary:
MicroRNAs are enriched in neurons and play important roles in dendritic spine development and synaptic plasticity. MicroRNA activity is controlled by a wide range of RNA-binding proteins. FMRP, a highly conserved RNA-binding protein, has been linked to microRNA-mediated gene regulation in axonal development and dendritic spine formation. FMRP also participates in dendritic arbor morphogenesis, but whether and how microRNAs contribute to its function in this process remains to be elucidated. Using Drosophila larval sensory neurons, this study shows that a FMRP-associated microRNA, miR-276, functions in FMRP-mediated space-filling dendrite morphogenesis. Using EGFP microRNA sensors, FMRP was demonstrated to likely act by regulating miR-276a RNA targeting rather than by modulating microRNA levels. Supporting this conclusion, miR-276a coimmunoprecipitated with FMRP and this association was dependent on the FMRP KH domains. By testing putative targets of the FMRP-miR-276a regulatory axis, nejire as a FMRP-associated mRNA and, using EGFP reporters, showed that the nejire 3' untranslated region is a target of miR-276a in vivo. Genetic analysis places nejire downstream of the FMRP-miR-276a pathway in regulating dendrite patterning. Together, these findings support a model in which FMRP facilitates miR-276a-mediated control of nejire for proper dendrite space-filling morphology and shed light on microRNA-dependent dendrite developmental pathology of fragile X syndrome.
Kato, Y. S., Tomita, J. and Kume, K. (2022). Interneurons of fan-shaped body promote arousal in Drosophila. PLoS One 17(11): e0277918. PubMed ID: 36409701
Summary:
Sleep is required to maintain physiological functions and is widely conserved across species. To understand the sleep-regulatory mechanisms, sleep-regulating genes and neuronal circuits are studied in various animal species. In the sleep-regulatory neuronal circuits in Drosophila melanogaster, the dorsal fan-shaped body (dFB) is a major sleep-promoting region. However, other sleep-regulating neuronal circuits were not well identified. It was recently found that arousal-promoting T1 dopamine neurons, interneurons of protocerebral bridge (PB) neurons, and PB neurons innervating the ventral part of the FB form a sleep-regulatory circuit, which was named "the PB-FB pathway". In the exploration of other sleep-regulatory circuits, it was found that activation of FB interneurons, also known as pontine neurons, promoted arousal. FB interneurons had possible connections with the PB-FB pathway and dFB neurons. Ca2+ imaging revealed that FB interneurons received excitatory signals from the PB-FB pathway. The possible role of FB interneurons to regulate dFB neurons was demonstrated. These results suggested the role of FB interneurons in sleep regulation.
Knapp, E. M., Kaiser, A., Arnold, R. C., Sampson, M. M., Ruppert, M., Xu, L., Anderson, M. I., Bonanno, S. L., Scholz, H., Donlea, J. M. and Krantz, D. E. (2022). Mutation of the Drosophila melanogaster serotonin transporter dSERT impacts sleep, courtship, and feeding behaviors. PLoS Genet 18(11): e1010289. PubMed ID: 36409783
Summary:
The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, new loss of function mutations were developed in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and the newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D. melanogaster. Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.
Ikeda, K., Kataoka, M. and Tanaka, N. K. (2022). Nonsynaptic Transmission Mediates Light Context-Dependent Odor Responses in Drosophila melanogaster. J Neurosci 42(46): 8621-8628. PubMed ID: 36180227
Summary:
Recent connectome analyses of the entire synaptic circuit in the nervous system have provided tremendous insights into how neural processing occurs through the synaptic relay of neural information. Conversely, the extent to which ephaptic transmission which does not depend on the synapses contributes to the relay of neural information, especially beyond a distance between adjacent neurons and to neural processing remains unclear. This study shows that ephaptic transmission mediated by extracellular potential changes in female Drosophila melanogaster can reach >200 μm, equivalent to the depth of its brain. Furthermore, ephaptic transmission driven by retinal photoreceptor cells mediates light-evoked firing rate increases in olfactory sensory neurons. These results indicate that ephaptic transmission contributes to sensory responses that can change momentarily in a context-dependent manner.
Lee, H., Kostal, L., Kanzaki, R. and Kobayashi, R. (2022). Spike frequency adaptation facilitates the encoding of input gradient in insect olfactory projection neurons. Biosystems: 104802. PubMed ID: 36375712
Summary:
The olfactory system in insects has evolved to process the dynamic changes in the concentration of food odors or sex pheromones to localize the nutrients or conspecific mating partners. Experimental studies have suggested that projection neurons (PNs) in insects encode not only the stimulus intensity but also its rate-of-change (input gradient). This study aimed to develop a simple computational model for a PN to understand the mechanism underlying the coding of the rate-of-change information. The spike frequency adaptation was shown to be a potential key mechanism for reproducing the phasic response pattern of the PN in Drosophila. It was also demonstrated that this adaptation mechanism enables the PN to encode the rate-of-change of the input firing rate. Finally, the model predicts that the PN exhibits the intensity-invariant response for the pulse and ramp odor stimulus. These results suggest that the developed model is useful for investigating the coding principle underlying olfactory information processing in insects.

Wednesday, December 1st - Adult and Larval Physiology and Metabolism

Lee, J., Yun, H. M., Han, G., Lee, G. J., Jeon, C. O. and Hyun, S. (2022). A bacteria-regulated gut peptide determines host dependence on specific bacteria to support host juvenile development and survival. BMC Biol 20(1): 258. PubMed ID: 36397042
Summary:
Commensal microorganisms have a significant impact on the physiology of host animals, including Drosophila. Lactobacillus and Acetobacter, the two most common commensal bacteria in Drosophila, stimulate fly development and growth, but the mechanisms underlying their functional interactions remain elusive. This study found that imaginal morphogenesis protein-Late 2 (Imp-L2), a Drosophila homolog of insulin-like growth factor binding protein 7, is expressed in gut enterocytes in a bacteria-dependent manner, determining host dependence on specific bacteria for host development. Imp-L2 mutation abolished the stimulatory effects of Lactobacillus, but not of Acetobacter, on fly larval development. The lethality of the Imp-L2 mutant markedly increased under axenic conditions, which was reversed by Acetobacter, but not Lactobacillus, re-association. The host dependence on specific bacteria was determined by Imp-L2 expressed in enterocytes, which was repressed by Acetobacter, but not Lactobacillus. Mechanistically, Lactobacillus and Acetobacter differentially affected steroid hormone-mediated Imp-L2 expression and Imp-L2-specific FOXO regulation. These findings may provide a way how host switches dependence between different bacterial species when benefiting from varying microbiota.
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
Summary:
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 was found to not be required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. It was also seen that 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.
de Oliveira Dos Santos, A. M., Duarte, A. E., Costa, A. R., da Silva, A. A., Rohde, C., Silva, D. G., de Amorim E, M., da Cruz Santos, M. H., Pereira, M. G., Depra, M., de Santana, S. L., da Silva Valente, V. L. and Teixeira, C. S. (2022). Canavalia ensiformis lectin induced oxidative stress mediate both toxicity and genotoxicity in Drosophila melanogaster. Int J Biol Macromol 222(Pt B): 2823-2832. PubMed ID: 36228819
Summary:
Mannose/glucose-binding lectin from Canavalia ensiformis seeds (Concanavalin A - ConA) has several biological applications, such as mitogenic and antitumor activity. However, most of the mechanisms involved in the in vivo toxicity of ConA are not well known. In this study, the Drosophila melanogaster model was used to assess the toxicity and genotoxicity of different concentrations of native ConA (4.4, 17.5 and 70 microg/mL) in inhibited and denatured forms of ConA. The data show that native ConA affected: the survival, in the order of 30.6 %, and the locomotor performance of the flies; reduced cell viability to levels below 50 % (4.4 and 17.5 microg/mL); reduced nitric oxide levels; caused lipid peroxidation and increased protein and non-protein thiol content. In the Comet assay, native ConA (17.5 and 70 microg/mL) caused DNA damage higher than 50 %. In contrast, treatments with inhibited and denatured ConA did not affect oxidative stress markers and did not cause DNA damage. It is believed that protein-carbohydrate interactions between ConA and carbohydrates of the plasma membrane are probably the major events involved in these activities, suggesting that native ConA activates mechanisms that induce oxidative stress and consequently DNA damage.
Duarte, T., Silva, M. M., Michelotti, P., Barbosa, N. B. V., Feltes, B. C., Dorn, M., Rocha, J. and Corte, C. L. D. (2022). The Drosophila melanogaster ACE2 ortholog genes are differently expressed in obesity/diabetes and aging models: Implications for COVID-19 pathology. Biochim Biophys Acta Mol Basis Dis 1868(12): 166551. PubMed ID: 36116726
Summary:
The Spike glycoprotein of SARS-CoV-2, the virus responsible for coronavirus disease 2019, binds to its ACE2 receptor for internalization in the host cells. Elderly individuals or those with subjacent disorders, such as obesity and diabetes, are more susceptible to COVID-19 severity. Additionally, several SARS-CoV-2 variants appear to enhance the Spike-ACE2 interaction, which increases transmissibility and death. Considering that the fruit fly is a robust animal model in metabolic research and has two ACE2 orthologs, Ance and Acer, this work studied the effects of two hypercaloric diets (HFD and HSD) and aging on ACE2 orthologs mRNA expression levels in Drosophila melanogaster. To complement this work, the predicted binding affinity between the Spike protein with Ance and Acer was analyzed. Ance and Acer genes were shown to be differentially regulated and dependent on diet and age in adult flies. At the molecular level, Ance and Acer proteins exhibit the potential to bind to the Spike protein in different regions, as shown by a molecular docking approach. Acer, in particular, interacts with the Spike protein in the same region as in humans. Overall, it is suggested that the D. melanogaster is a promising animal model for translational studies on COVID-19 associated risk factors and ACE2.
Bosch, J. A., Ugur, B., Pichardo-Casas, I., Rabasco, J., Escobedo, F., Zuo, Z., Brown, B., Celniker, S., Sinclair, D. A., Bellen, H. J. and Perrimon, N. (2022). Two neuronal peptides encoded from a single transcript regulate mitochondrial complex III in Drosophila. Elife 11. PubMed ID: 36346220
Summary:
Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open-reading frames (smORFs). This study describes two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. Evidence is provided that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.
Katti, P., Ajayi, P. T., Aponte, A., Bleck, C. K. E. and Glancy, B. (2022). Identification of evolutionarily conserved regulators of muscle mitochondrial network organization. Nat Commun 13(1): 6622. PubMed ID: 36333356
Summary:
Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they might affect contractile fiber-type. This study shows that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm, identified transcription factors H15 and cut as potential mitochondrial network regulators. H15 was demonstrated to operate downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, 15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, this study found that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors Cut, Salm, and H15.
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