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

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





ARCHIVE

Monday, February 28th, 2022 - Disease Models

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Deshpande, P., Chen, C. Y., Yeates, C., Chen, C. H., Kango-Singh, M. and Singh, A. (2021). miR-277 targets hid to ameliorate Aβ42-mediated neurodegeneration in Drosophila eye model of Alzheimer's Disease. Alzheimers Dement 17 Suppl 2: e058678. Article URL
Summary:
Alzheimer's disease (AD), an age-related progressive neurodegenerative disorder, exhibits reduced cognitive functions with no cure to date. One of the reasons for AD is the extracellular accumulation of Amyloid-beta 42 (Aβ42) plaques. Misexpression of human Aβ42 in the developing retina of Drosophila exhibits AD-like neuropathology. Accumulation of Aβ42 plaque(s) triggers aberrant signaling resulting in neuronal cell death by unknown mechanism(s). Alzheimer's disease (AD), an age-related progressive neurodegenerative disorder, exhibits reduced cognitive functions with no cure to date. One of the reasons for AD is the extracellular accumulation of Amyloid-beta 42 (Aβ42) plaques. Misexpression of human Aβ42 in the developing retina of Drosophila exhibits AD-like neuropathology. Accumulation of Aβ42 plaque(s) triggers aberrant signaling resulting in neuronal cell death by unknown mechanism(s). Gain-of-function of mir-277 rescues Aβ42 mediated neurodegeneration whereas loss-of-function of mir-277 enhances Aβ42 mediated neurodegeneration. Moreover, misexpression of higher levels of mir-277 in the GMR>Aβ42 background restores the retinal axonal targeting indicating functional rescue. Furthermore, this study has identified head involution defective (hid) as one of the targets of mir-277. The hid transcript levels are decreased by one third when mir-277 is misexpressed in the GMR>Aβ42 background in comparison to the GMR>Aβ42 fly model. This study provides a mechanism of how mir-277 modulates Aβ42 mediated neurodegeneration by regulating hid transcript levels and demonstrate its neuroprotective role in Aβ42-mediated neuropathology.
Atrian, F., Ramirez, P. and Frost, B. (2021). Investigating circular RNA-mediated neurotoxicity in tauopathies. Alzheimers Dement 17 Suppl 2: e058483. Article URL
Summary:
Circular RNAs (circRNA) are a subclass of non-coding RNAs with a covalently closed loop structure that are formed via non-canonical splicing. CircRNA biogenesis is regulated by N6-methyladenosine (m(6) A), a post transcriptional RNA modification. Elevated levels of circRNAs and abnormal m(6) A modification significantly correlate with development of Alzheimer's disease. Previous studies have shown that tau-induced dysfunction of lamin causes nuclear polymorphisms including invaginations and blebs. Polyadenylated RNA accumulates within nuclear invaginations in the context of tauopathy, and genetic and pharmacologic reduction of RNA export reduce RNA accumulation within invaginations and suppresses tau neurotoxicity. The role of m(6) A in circRNA accumulation, and their relationship with aging and tauopathy are currently unknown. Based on the association between nuclear polymorphism and RNA export, alongside the global enrichment of circRNAs and disrupted RNA methylation in Alzheimer's disease brains, it was hypothesized that m(6) A-dependent accumulation of circRNAs in tauopathies sequester complementary RNAs and RNA binding proteins into large inclusions that trigger RNA export via nuclear blebbing. It was found that circRNAs accumulate in brains of a Drosophila model of tauopathy and that RNAi-mediated reduction of mbl, which is particularly enriched in its circular form in the brain, significantly suppresses tau-induced neurotoxicity. RNAi-mediated reduction of an m(6) A writer and reader significantly reduces circMbl biogenesis and neuronal death in tau transgenic Drosophila. circMbl lines the nuclear blebs that contain large inclusions in brains of tau transgenic Drosophila. It is concluded that Tau-induced accumulation of circRNA is mediated by aberrant m(6) A modification. Presence of circMbl-lined nuclear buds suggests a potential role of nuclear blebs in circRNA nuclear export.
Cheng, K. C., Hwang, Y. L. and Chiang, H. C. (2022). The double-edged sword effect of HDAC6 in Abeta toxicities. Faseb j 36(1): e22072. PubMed ID: 34907598
Summary:
Alzheimer's disease (AD) is marked by cognitive impairment, massive cell death, and reduced life expectancy. Pathologically, accumulated beta-amyloid (Aβ) aggregates and hyperphosphorylated tau protein is the hallmark of the disease. Although changes in cellular function and protein accumulates have been demonstrated in many different AD animal models, the molecular mechanism involved in different cellular functions and the correlation and causative relation between different protein accumulations remain elusive. In vivo genetic studies revealed that the molecular mechanisms involved in memory loss and lifespan shortening are different and that tau plays an essential role in mediating Aβ-induced early death. When the first deacetylase (DAC) domain of histone deacetylase 6 (HDAC6) activity was increased, it regulated cortactin deacetylation to reverse Aβ-induced learning and memory deficit, but with no effect on the lifespan of the Aβ flies. On the other hand, an increased amount of the second DAC domain of HDAC6 promoted tau phosphorylation to facilitate Aβ-induced lifespan shortening without affecting learning performance in the Aβ flies.These data also confirmed decreased acetylation in two major HDAC6 downstream proteins, suggesting increased HDAC6 activity in Aβ flies. These data established the double-edged sword effect of HDAC6 activity in Aβ-induced pathologies. Not only did memory loss and lifespan shortening in Aβ flies segregated, but also evidence is provided to link the Aβ with tau signaling.
Cho, Y. H., Kim, G. H. and Park, J. J. (2021). Mitochondrial aconitase 1 regulates age-related memory impairment via autophagy/mitophagy-mediated neural plasticity in middle-aged flies. Aging Cell 20(12): e13520. PubMed ID: 34799973
Summary:
Age-related memory impairment (AMI) occurs in many species, including humans. The underlying mechanisms are not fully understood. In wild-type Drosophila (w1118), AMI appears in the form of a decrease in learning (3-min memory) from middle age (30 days after eclosion [DAE]). in vivo, DNA microarray, and behavioral screen studies were performed to identify genes controlling both lifespan and AMI and mitochondrial Acon1 (mAcon1) was selected. mAcon1 expression in the head of w(1118) decreased with age. Neuronal overexpression of mAcon1 extended its lifespan and improved AMI. Neuronal or mushroom body expression of mAcon1 regulated the learning of young (10 DAE) and middle-aged flies. Interestingly, acetyl-CoA and citrate levels increased in the heads of middle-aged and neuronal mAcon1 knockdown flies. Acetyl-CoA, as a cellular energy sensor, is related to autophagy. Autophagy activity and efficacy determined by the positive and negative changes in the expression levels of Atg8a-II and p62 were proportional to the expression level of mAcon1. Levels of the presynaptic active zone scaffold protein Bruchpilot were inversely proportional to neuronal mAcon1 levels in the whole brain. Furthermore, mAcon1 overexpression in Kenyon cells induced mitophagy labeled with mt-Keima and improved learning ability. Both processes were blocked by pink1 knockdown. Taken together, these results imply that the regulation of learning and AMI by mAcon1 occurs via autophagy/mitophagy-mediated neural plasticity.
Atas-Ozcan, H., Brault, V., Duchon, A. and Herault, Y. (2021). Dyrk1a from Gene Function in Development and Physiology to Dosage Correction across Life Span in Down Syndrome. Genes (Basel) 12(11). PubMed ID: 34828439
Summary:
Down syndrome is the main cause of intellectual disabilities with a large set of comorbidities from developmental origins but also that appeared across life span. Investigation of the genetic overdosage found in Down syndrome, due to the trisomy of human chromosome 21, has pointed to one main driver gene, the Dual-specificity tyrosine-regulated kinase 1A (Dyrk1a). Dyrk1a is a murine homolog of the Drosophila minibrain gene. It has been found to be involved in many biological processes during development and in adulthood. Further analysis showed its haploinsufficiency in mental retardation disease 7 and its involvement in Alzheimer's disease. DYRK1A plays a role in major developmental steps of brain development, controlling the proliferation of neural progenitors, the migration of neurons, their dendritogenesis and the function of the synapse. Several strategies targeting the overdosage of DYRK1A in DS with specific kinase inhibitors have showed promising evidence that DS cognitive conditions can be alleviated. Nevertheless, providing conditions for proper temporal treatment and to tackle the neurodevelopmental and the neurodegenerative aspects of DS across life span is still an open question.
Bordet, G., Lodhi, N., Kossenkov, A. and Tulin, A. (2021). Age-Related Changes of Gene Expression Profiles in Drosophila. Genes (Basel) 12(12). PubMed ID: 34946931
Summary:
An individual's gene expression profile changes throughout their life. This change in gene expression is shaped by differences in physiological needs and functions between the younger and older organism. Despite intensive studies, the aging process is not fully understood, and several genes involved in this process may remain to be identified. This paper reports a transcriptomic analysis of Drosophila melanogaster using microarrays. The expression profiles of two-day-old female adult flies were compared with those of 45-day-old flies. 1184 genes were identified with pronounced differences in expression level between young and old age groups. Most genes involved in muscle development/maintenance that display different levels of expression with age were downregulated in older flies. Many of these genes contributed to sarcomere formation and function. Several of these genes were functionally related to direct and indirect flight muscles; some of them were exclusively expressed in these muscles. Conversely, several genes involved in apoptosis processes were upregulated in aging flies. In addition, several genes involved in resistance to toxic chemicals were upregulated in aging flies, which is consistent with a global upregulation of the defense response system in aging flies. Finally, 12 genes were randomly selected among 232 genes with unknown function and transgenic flies expressing recombinant proteins fused with GFP protein were generated to determine their subcellular expression. It was also found that the knockdown of some of those 12 genes can affect the lifespan of flies.

Friday, February 25th - Chromatin and DNA Repair

Torres-Campana, D., Horard, B., Denaud, S., Benoit, G., Loppin, B. and Orsi, G. A. (2022). Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain. PLoS Genet 18(1): e1009615. PubMed ID: 34982772
Summary:
The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. This study identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, it was found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, it was shown that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, this study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.
Liaw, G. J. (2022). Polycomb repressive complex 1 initiates and maintains tailless repression in Drosophila embryo. Biochim Biophys Acta Gene Regul Mech 1865(2): 194786. PubMed ID: 35032681 l
Summary:
Maternally-deposited morphogens specify the fates of embryonic cells via hierarchically regulating the expression of zygotic genes that encode various classes of developmental regulators. Once the cell fates are determined, Polycomb-group proteins frequently maintain the repressed state of the genes. This study investigates how Polycomb-group proteins repress the expression of tailless, which encodes a developmental regulator in Drosophila embryo. Previous studies have shown that maternal Tramtrack69 facilitates maternal GAGA-binding factor and Heat shock factor binding to the torso response element (tor-RE) to initiate tailless repression in the stage-4 embryo. Chromatin-immunoprecipitation and genetic-interaction studies exhibit that maternally-deposited Polycomb repressive complex 1 (PRC1) recruited by the tor-RE-associated Tramtrack69 represses tailless expression in the stage-4 embryo. A noncanonical Polycomb-group response element (PRE) is mapped to the tailless proximal region. High levels of Bric-a-brac, Tramtrack, and Broad (BTB)-domain proteins are fundamental for maintaining tailless repression in the stage-8 to -10 embryos. Trmtrack69 sporadically distributes in the linear BTB-domain oligomer, which recruits and retains a high level of PRC1 near the GCCAT cluster for repressing tll expression in the stage-14 embryos. Disrupting the retention of PRC1 decreases the levels of PRC1 and Pleiohomeotic protein substantially on the PRE and causes tailless derepression in the stage-14 embryo. Furthermore, the retained PRC1 potentially serves as a second foundation for assembling the well-characterized polymer of the Sterile alpha motif domain in Polyhomeotic protein, which compacts chromatin to maintain the repressed state of tailless in the embryos after stage 14.
Bottcher, R., Schmidts, I., Nitschko, V., Duric, P. and Forstemann, K. (2022). RNA polymerase II is recruited to DNA double-strand breaks for dilncRNA transcription in Drosophila. RNA Biol 19(1): 68-77. PubMed ID: 34965182
Summary:
DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. This paper provides nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. This study thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells.
Chang, C. W., Shen, Y. C. and Yan, S. J. (2021). HP1a-mediated heterochromatin formation inhibits high dietary sugar-induced tumor progression. Cell Death Dis 12(12): 1130. PubMed ID: 34866135
Summary:
High dietary sugar (HDS) is a modern dietary concern that involves excessive consumption of carbohydrates and added sugars, and increases the risk of metabolic disorders and associated cancers. However, epigenetic mechanisms by which HDS induces tumor progression remain unclear. This study investigated the role of heterochromatin, an important yet poorly understood part of the epigenome, in HDS-induced tumor progression of Drosophila Ras/Src and Ras/scrib tumor systems. Increased heterochromatin formation was found with overexpression of heterochromatin protein 1a (HP1a), specifically in tumor cells, not only decreases HDS-induced tumor growth/burden but also drastically improves survival of Drosophila with HDS and Ras/Src or Ras/scrib tumors. Moreover, HDS reduces heterochromatin levels in tumor cells. Mechanistically, this study demonstrated that increased heterochromatin formation decreases wingless (wg) and Hippo (Hpo) signaling, thereby promoting apoptosis, via inhibition of Yorkie (Yki) nuclear accumulation and upregulation of apoptotic genes, and reduces DNA damage in tumor cells under HDS. Taken together, this work identified a novel epigenetic mechanism by which HP1a-mediated heterochromatin formation suppresses HDS-induced tumor progression likely by decreasing wingless and Hippo signaling, increasing apoptosis, and maintaining genome stability. This model explains that the molecular, cellular, and organismal aspects of HDS-aggravated tumor progression are dependent on heterochromatin formation, and highlights heterochromatin as a therapeutic target for cancers associated with HDS-induced metabolic disorders.
Azpiazu, N. and Morata, G. (2022). Chromatin remodelling and retrotransposons activities during regeneration in Drosophila. Dev Biol 482: 7-16. PubMed ID: 34822846
Summary:
Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling. This study analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila. In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. Euchromatin and heterochromatin histone marks were examined during the process, and it was found that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. The transcription of two retrotransposons, Roo and F-element in the regenerating cells is described. A temporal correlation was established between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process.
Cheng, J., Allgeyer, E. S., Richens, J. H., Dzafic, E., Palandri, A., Lewkow, B., Sirinakis, G. and St Johnston, D. (2021). A single-molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila. J Cell Sci 134(24). PubMed ID: 34806753
Summary:
Single-molecule localization microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues because of high background from out-of-focus emitters and optical aberrations. This study describes a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-point accumulation for imaging in nanoscale topography (PAINT) routinely gives 30 nm resolution or better at depths greater than 20 μm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to what is seen in cultured cells. Lamin Dm0 shows a complementary localization to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C causes the nuclear pores to distribute more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Given that nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organization and contribute to the disease phenotypes caused by human lamin A/C laminopathies.

Thursday, February 24th - Enhancers and Transcriptional regulation

Yang, T. H., Yang, Y. C. and Tu, K. C. (2022). regCNN: identifying Drosophila genome-wide cis-regulatory modules via integrating the local patterns in epigenetic marks and transcription factor binding motifs. Comput Struct Biotechnol J 20: 296-308. PubMed ID: 35035784
Summary:
Transcription regulation in metazoa is controlled by the binding events of transcription factors (TFs) or regulatory proteins on specific modular DNA regulatory sequences called cis-regulatory modules (CRMs). Understanding the distributions of CRMs on a genomic scale is essential for constructing the metazoan transcriptional regulatory networks that help diagnose genetic disorders. While traditional reporter-assay CRM identification approaches can provide an in-depth understanding of functions of some CRM, these methods are usually cost-inefficient and low-throughput. It is generally believed that by integrating diverse genomic data, reliable CRM predictions can be made. Hence, researchers often first resort to computational algorithms for genome-wide CRM screening before specific experiments. However, current existing in silico methods for searching potential CRMs were restricted by low sensitivity, poor prediction accuracy, or high computation time from TFBS composition combinatorial complexity. To overcome these obstacles, this study designed a novel CRM identification pipeline called regCNN by considering the base-by-base local patterns in TF binding motifs and epigenetic profiles. On the test set, regCNN shows an accuracy/auROC of 84.5%/92.5% in CRM identification. And by further considering local patterns in epigenetic profiles and TF binding motifs, it can accomplish 4.7% (92.5%-87.8%) improvement in the auROC value over the average value-based pure multi-layer perceptron model. It was also demonstrated that regCNN outperforms all currently available tools by at least 11.3% in auROC values. Finally, regCNN is verified to be robust against its resizing window hyperparameter in dealing with the variable lengths of CRMs.
Hildebrandt, K., Kolb, D., Kloppel, C., Kaspar, P., Wittling, F., Hartwig, O., Federspiel, J., Findji, I. and Walldorf, U. (2022). Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development. Hereditas 159(1): 2. PubMed ID: 34983686
Summary:
The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. This study conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. A new hbn(KOGal4) enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. Several enhancers were characterized that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, deletions of four of these enhancer regions were genereted through gene targeting and analysed their effects on the expression and function of hbn. The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.
Chen, J., Huang, Y. and Qi, G. (2021). LncRNA-IRAR-mediated regulation of insulin receptor transcripts in Drosophila melanogaster during nutritional stress. Insect Mol Biol. PubMed ID: 34923706
Summary:
The insulin signalling pathway plays a crucial role in regulating the metabolism of sugars, fats and proteins in cells, thereby affecting the growth, metabolism, reproduction and ageing of organisms. However, little is known about the functions of long non-coding RNAs (lncRNAs) in the regulation of insulin receptors under stress conditions in insects. This study showed that insulin receptor-associated lncRNA (IRAR) regulates insulin receptor transcripts in response to nutritional stress in Drosophila melanogaster. Genome editing by CRISPR-Cas9 showed reduced sensitivity of IRAR mutants to environmental nutritional changes. In contrast, the sensitivity of mutants overexpressing tubulin-gal4 > IRAR increased under low nutrition. The pupation and eclosion timings in IRAR mutants were significantly delayed with an increase in insulin concentration compared with that in the w1118 group. In addition, the expression pattern of IRAR was almost consistent with that of the four transcripts of the insulin receptor from the embryonic period to the adult period. RNA immunoprecipitation assay showed the direct regulation of insulin receptor transcripts by IRAR to the through FOXO binding under nutritional stress. This is the first study that describes a model of lncRNA-mediated development regulation through insulin receptor transcripts.
Janssens, J., Aibar, S., Taskiran, II, Ismail, J. N., Gomez, A. E., Aughey, G., Spanier, K. I., De Rop, F. V., González-Blas, C. B., Dionne, M., Grimes, K., Quan, X. J., Papasokrati, D., Hulselmans, G., Makhzami, S., De Waegeneer, M., Christiaens, V., Southall, T. and Aerts, S. (2022). Decoding gene regulation in the fly brain. Nature 601(7894): 630-636. PubMed ID: 34987221
Summary:
The Drosophila brain is a frequently used model in neuroscience. Single-cell transcriptome analysis, three-dimensional morphological classification and electron microscopy mapping of the connectomehave revealed an immense diversity of neuronal and glial cell types that underlie an array of functional and behavioural traits in the fly. The identities of these cell types are controlled by gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. this study characterize GRNs at the cell-type level in the fly brain, the chromatin accessibility of 240,919 single cells was characterized spanning 9 developmental timepoints and integrated these data with single-cell transcriptomes. More than 95,000 regulatory regions were identified that are used in different neuronal cell types, of which 70,000 are linked to developmental trajectories involving neurogenesis, reprogramming and maturation. For 40 cell types, uniquely accessible regions were associated with their expressed transcription factors and downstream target genes through a combination of motif discovery, network inference and deep learning, creating enhancer GRNs. The enhancer architectures revealed by DeepFlyBrain lead to a better understanding of neuronal regulatory diversity and can be used to design genetic driver lines for cell types at specific timepoints, facilitating their characterization and manipulation.
Dunipace, L., Newcomb, S. and Stathopoulos, A. (2022). brinker levels regulated by a promoter proximal element support germ cell homeostasis. Development PubMed ID: 35037688
Summary:
Limiting BMP signalling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signalling in both the Drosophila embryo and wing disc is the Brinker (Brk) transcription factor, yet the expression and potential role of brk in the germarium has not previously been described. This study found that brk expression requires a promoter-proximal element (PPE), to both support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to precisely regulate brk levels. Using PPE mutants as well as tissue specific RNAi and overexpression, this study shows that altering brk expression within either the soma or germline affects germ cell homeostasis. Remarkably, it was found that Decapentaplegic (Dpp), the main BMP ligand and Brk's canonical antagonist, is upregulated by Brk in the escort cells of the germarium demonstrating that Brk can positively regulate this pathway.
Peng, Q., Chen, J., Wang, R., Zhu, H., Han, C., Ji, X. and Pan, Y. (2022). The sex determination gene doublesex regulates expression and secretion of the basement membrane protein Collagen IV. J Genet Genomics. PubMed ID: 35017121
Summary:
The highly conserved doublesex (dsx) and doublesex/mab-3 related (Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, Dsx(M) in males and Dsx(F) in females, which function differentially and often oppositely to establish sexual dimorphism. This study report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane (BM) protein Collagen IV in the fat body. Dsx isoforms regulate the expression of Collagen IV in the fat body and its secretion into the BM of other tissues. The procollagen lysyl hydroxylase (dPlod) gene, which is involved in the biosynthesis of Collagen IV, was identified as a direct target of Dsx. It was further shown that Dsx regulates Collagen IV through dPlod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen IV and remotely establish sexual dimorphism.

Wednesday, February 23rd - RNA and Transposons -

Fefelova, E. A., Pleshakova, I. M., Mikhaleva, E. A., Pirogov, S. A., Poltorachenko, V. A., Abramov, Y. A., Romashin, D. D., Shatskikh, A. S., Blokh, R. S., Gvozdev, V. A. and Klenov, M. S. (2022). Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon. Nucleic Acids Res 50(2): 867-884. PubMed ID: 35037046
Summary:
Eukaryotic genomes harbor hundreds of rRNA genes, many of which are transcriptionally silent. In Drosophila, some rDNA repeats contain insertions of the R2 retrotransposon, which is capable to be transcribed only as part of pre-rRNA molecules. rDNA units with R2 insertions are usually inactivated, although R2 expression may be beneficial in cells with decreased rDNA copy number. This study found that R2-inserted rDNA units are enriched with HP1a and H3K9me3 repressive mark, whereas disruption of the heterochromatin components slightly affects their silencing in ovarian germ cells. Surprisingly, a dramatic upregulation of R2-inserted rRNA genes was observed in ovaries lacking Udd (Under-developed) or other subunits (TAF1b and TAF1c-like) of the SL1-like complex, which is homologues to mammalian Selective factor 1 (SL1) involved in rDNA transcription initiation. Derepression of rRNA genes with R2 insertions was accompanied by a reduction of H3K9me3 and HP1a enrichment. It is suggested that the impairment of the SL1-like complex affects a mechanism of selective activation of intact rDNA units which competes with heterochromatin formation. It is also proposed that R2 derepression may serve as an adaptive response to compromised rRNA synthesis.
Carnesecchi, J., Boumpas, P., van Nierop, Y. S. P., Domsch, K., Pinto, H. D., Borges Pinto, P. and Lohmann, I. (2022). The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II. Nucleic Acids Res 50(2): 763-783. PubMed ID: 34931250
Summary:
Transcription factors (TFs) play a pivotal role in cell fate decision by coordinating gene expression programs. Although most TFs act at the DNA layer, few TFs bind RNA and modulate splicing. Yet, the mechanistic cues underlying TFs activity in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), this work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct outcomes in defined cellular contexts. These results demonstrate a new RNA-binding ability of Ubx. The N51 amino acid of the DNA-binding Homeodomain is non-essential for RNA interaction in vitro, but is required for RNA interaction in vivo and Ubx splicing activity. Moreover, mutation of the N51 amino acid weakens the interaction between Ubx and active RNA Polymerase II (Pol II). These results reveal that Ubx regulates elongation-coupled splicing, which could be coordinated by a dynamic interplay with active Pol II on chromatin. Overall, this work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.
Dong, H., Xu, B., Guo, P., Zhang, J., Yang, X., Li, L., Fu, Y., Shi, J., Zhang, S., Zhu, Y., Shi, Y., Zhou, F., Bian, L., You, W., Shi, F., Yang, X., Huang, J., He, H. and Jin, Y. (2022). Hidden RNA pairings counteract the "first-come, first-served" splicing principle to drive stochastic choice in Dscam1 splice variants. Sci Adv 8(4): eabm1763. PubMed ID: 35080968
Summary:
Drosophila melanogaster Dscam1 encodes 38,016 isoforms via mutually exclusive splicing; however, the regulatory mechanism behind this is not fully understood. This study found a set of hidden RNA secondary structures that balance the stochastic choice of Dscam1 splice variants (designated balancer RNA secondary structures). In vivo mutational analyses revealed the dual function of these balancer interactions in driving the stochastic choice of splice variants, through enhancement of the inclusion of distal exon 6s by cooperating with docking site-selector pairing to form a stronger multidomain pre-mRNA structure and through simultaneous repression of the inclusion of proximal exon 6s by antagonizing their docking site-selector pairings. Thus, this study provides an elegant molecular model based on competition and cooperation between two sets of docking site-selector and balancer pairings, which counteracts the "first-come, first-served" principle. These findings provide conceptual and mechanistic insight into the dynamics and functions of long-range RNA secondary structures.
Berloco, M. F., Minervini, C. F., Moschetti, R., Palazzo, A., Viggiano, L. and Marsano, R. M. (2021). Evidence of the Physical Interaction between Rpl22 and the Transposable Element Doc5, a Heterochromatic Transposon of Drosophila melanogaster. Genes (Basel) 12(12). PubMed ID: 34946947
Summary:
Chromatin is a highly dynamic biological entity that allows for both the control of gene expression and the stabilization of chromosomal domains. Given the high degree of plasticity observed in model and non-model organisms, it is not surprising that new chromatin components are frequently described. This work tested the hypothesis that the remnants of the Doc5 transposable element, which retains a heterochromatin insertion pattern in the melanogaster species complex, can be bound by chromatin proteins, and thus be involved in the organization of heterochromatic domains. Using the Yeast One Hybrid approach, Rpl22 was found to be a potential interacting protein of Doc5. The observed interaction was further tested through an Electrophoretic Mobility Shift Assay, uncovering that the N-terminal portion of the protein is sufficient to interact with Doc5. However, in situ localization of the native protein failed to detect Rpl22 association with chromatin. The results obtained are discussed in the light of the current knowledge on the extra-ribosomal role of ribosomal protein in eukaryotes, which suggests a possible role of Rpl22 in the determination of the heterochromatin in Drosophila.
Giordani, G., Cavaliere, V., Gargiulo, G., Lattanzi, G. and Andrenacci, D. (2021).. Retrotransposons Down- and Up-Regulation in Aging Somatic Tissues. Cells 11(1). PubMed ID: 35011640
Summary:
The transposon theory of aging hypothesizes the activation of transposable elements (TEs) in somatic tissues with age, leading to a shortening of the lifespan. It is thought that TE activation in aging produces an increase in DNA double-strand breaks, contributing to genome instability and promoting the activation of inflammatory responses. To investigate how TE regulation changes in somatic tissues during aging, this study analyzed the expression of some TEs, as well as a source of small RNAs that specifically silence the analyzed TEs; the Drosophila cluster named flamenco. Significant variations was found in the expression levels of all the analyzed TEs during aging, with a trend toward reduction in middle-aged adults and reactivation in older individuals that suggests dynamic regulation during the lifespan.
Takase, N., Otsu, M., Hirakata, S., Ishizu, H., Siomi, M. C. and Kawai, G. (2022). T-hairpin structure found in the RNA element involved in piRNA biogenesis. RNA. PubMed ID: 34987083
Summary:
PIWI-interacting RNAs (piRNAs) repress transposons to protect the germline genome from DNA damage caused by transposon transposition. In Drosophila, the Traffic jam (Tj) mRNA is consumed to produce piRNA in its 3' UTR. A cis element located within the 3'-UTR, Tj-cis, is necessary for piRNA biogenesis. This study analyzed the structure of the Tj-cis RNA, a 100 nt RNA corresponding to the Tj-cis element, by the SHAPE and NMR analyses and found that a stable hairpin structure formed in the 5' half of the Tj-cis RNA. The tertiary structure of the 16 nt stable hairpin was analyzed by NMR, and a novel stem-loop structure, the T-hairpin, was found. In the T-hairpin, four uridine residues are exposed to the solvent, suggesting that this stem loop is the target of Yb protein, a Tudor domain-containing piRNA biogenesis factor. The piRNA biogenesis assay showed that both the T-hairpin and the 3' half are required for the function of the Tj-cis element, suggesting that both the T-hairpin and the 3' half are recognized by Yb protein.

Tuesday, February 22nd - Adult neural development and function

Avetisyan, A., Glatt, Y., Cohen, M., Timerman, Y., Aspis, N., Nachman, A., Halachmi, N., Preger-Ben Noon, E. and Salzberg, A. (2021). Delilah, prospero, and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors. Elife 10. PubMed ID: 34964712
Summary:
Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. This work describea a gene regulatory network consisting of three transcription factors-Prospero (Pros), D-Pax2, and Delilah (Dei)-that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila. D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei. Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. It was further shown that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ.
Zhou, B., Li, Z., Kim, S., Lafferty, J. and Clark, D. A. (2022). Shallow neural networks trained to detect collisions recover features of visual loom-selective neurons. Elife 11. PubMed ID: 35023828
Summary:
Animals have evolved sophisticated visual circuits to solve a vital inference problem: detecting whether or not a visual signal corresponds to an object on a collision course. Such events are detected by specific circuits sensitive to visual looming, or objects increasing in size. Various computational models have been developed for these circuits, but how the collision-detection inference problem itself shapes the computational structures of these circuits remains unknown. Inspired by the distinctive structures of LPLC2 neurons (ultra-selective looming detecting neuron, lobula plate/lobula columnar, type II (LPLC2) in Drosophila) in the visual system of Drosophila, this study built anatomically-constrained shallow neural network models and trained them to identify visual signals that correspond to impending collisions. Surprisingly, the optimization arrives at two distinct, opposing solutions, only one of which matches the actual dendritic weighting of LPLC2 neurons. Both solutions can solve the inference problem with high accuracy when the population size is large enough. The LPLC2-like solutions reproduces experimentally observed LPLC2 neuron responses for many stimuli, and reproduces canonical tuning of loom sensitive neurons, even though the models are never trained on neural data. Thus, LPLC2 neuron properties and tuning are predicted by optimizing an anatomically-constrained neural network to detect impending collisions. More generally, these results illustrate how optimizing inference tasks that are important for an animal's perceptual goals can reveal and explain computational properties of specific sensory neurons.
Yamaguchi, S. T., Tomita, J. and Kume, K. (2022). Insulin signaling in clock neurons regulates sleep in Drosophila. Biochem Biophys Res Commun 591: 44-49. PubMed ID: 34998032
Summary:
Sleeprelates to numerous biological functions, including metabolism. Both dietary conditions and genes related to metabolism are known to affect sleep behavior. Insulin signaling is well conserved across species including the fruit fly and relates to both metabolism and sleep. However, the neural mechanism of sleep regulation by insulin signaling is poorly understood. This paper reports that insulin signaling in specific neurons regulates sleep in Drosophila melanogaster. This study analyzed the sleep behavior of flies with the mutation in insulin-like ligands expressed in the brain and found that three insulin-like ligands participate in sleep regulation with some redundancy. Twenty-one Gal4 drivers were used to express a dominant-negative form of the insulin receptor (InR DN) in various neurons including circadian clock neurons, which express the clock gene, and the pars intercerebralis (PI). Inhibition of insulin signaling in the anterior dorsal neuron group 1 (DN1a) decreased sleep. Additionally, the same manipulation in PI also decreased sleep. Pan-neuronal induced expression of InR DN also decreased sleep. These results suggested that insulin signaling in DN1a and PI regulates sleep.
Zadeh-Haghighi, H. and Simon, C. (2022). Radical pairs can explain magnetic field and lithium effects on the circadian clock. Sci Rep 12(1): 269. PubMed ID: 34997158
Summary:
Drosophila's circadian clock can be perturbed by magnetic fields, as well as by lithium administration. Cryptochromes are critical for the circadian clock. Further, the radical pairs in cryptochrome also can explain magnetoreception in animals. Based on a simple radical pair mechanism model of the animal magnetic compass, this study shows that both magnetic fields and lithium can influence the spin dynamics of the naturally occurring radical pairs and hence modulate the circadian clock's rhythms. Using a simple chemical oscillator model for the circadian clock, this study showed that the spin dynamics influence a rate in the chemical oscillator model, which translates into a change in the circadian period. This model can reproduce the results of two independent experiments, magnetic field and lithium effects on the circadian clock. The model predicts that stronger magnetic fields would shorten the clock's period. It is also predicted that lithium influences the clock in an isotope-dependent manner. Furthermore, the model also predicts that magnetic fields and hyperfine interactions modulate oxidative stress. The findings of this work suggest that the quantum nature of radical pairs might play roles in the brain, as another piece of evidence in addition to recent results on xenon anesthesia and lithium effects on hyperactivity.
Yao, Z. and Scott, K. (2022). Serotonergic neurons translate taste detection into internal nutrient regulation. Neuron. PubMed ID: 35051377
Summary:
The nervous and endocrine systems coordinately monitor and regulate nutrient availability to maintain energy homeostasis. Sensory detection of food regulates internal nutrient availability in a manner that anticipates food intake, but sensory pathways that promote anticipatory physiological changes remain unclear. This study identified serotonergic (5-HT) neurons as critical mediators that transform gustatory detection by sensory neurons into the activation of insulin-producing cells and enteric neurons in Drosophila. One class of 5-HT neurons responds to gustatory detection of sugars, excites insulin-producing cells, and limits consumption, suggesting that they anticipate increased nutrient levels and prevent overconsumption. A second class of 5-HT neurons responds to gustatory detection of bitter compounds and activates enteric neurons to promote gastric motility, likely to stimulate digestion and increase circulating nutrients upon food rejection. These studies demonstrate that 5-HT neurons relay acute gustatory detection to divergent pathways for longer-term stabilization of circulating nutrients.
Abdelrahman, N. Y., Vasilaki, E. and Lin, A. C. (2021). Compensatory variability in network parameters enhances memory performance in the Drosophila mushroom body. Proc Natl Acad Sci U S A 118(49). PubMed ID: 34845010
Summary:
Neural circuits use homeostatic compensation to achieve consistent behavior despite variability in underlying intrinsic and network parameters. However, it remains unclear how compensation regulates variability across a population of the same type of neurons within an individual and what computational benefits might result from such compensation. These questions were addressed in the Drosophila mushroom body, the fly's olfactory memory center. In a computational model, this study showed that under sparse coding conditions, memory performance is degraded when the mushroom body's principal neurons, Kenyon cells (KCs), vary realistically in key parameters governing their excitability. However, memory performance is rescued while maintaining realistic variability if parameters compensate for each other to equalize KC average activity. Such compensation can be achieved through both activity-dependent and activity-independent mechanisms. Finally, it was shown that correlations predicted by the model's compensatory mechanisms appear in the Drosophila hemibrain connectome. These findings reveal compensatory variability in the mushroom body and describe its computational benefits for associative memory.

Monday, February 21st - Adult Physiology

Pavkovic-Lucic, S., Trajkovic, J., Milicic, D., Anđelkovic, B., Lucic, L., Savic, T. and Vujisic, L. (2022). "Scent of a fruit fly": Cuticular chemoprofiles after mating in differently fed Drosophila melanogaster (Diptera: Drosophilidae) strains. Arch Insect Biochem Physiol: e21866. PubMed ID: 35020218
Summary:
In the world of complex smells in natural environment, feeding and mating represent two important olfactory-guided behaviors in Drosophila melanogaster (Diptera: Drosophilidae). Diet affects the chemoprofile composition of the individuals, which, indirectly, may significantly affect their mating success. In this study, chemoprofiles of recently mated flies belonging to four D. melanogaster strains, which were fed for many generations on different substrates (standard cornmeal-S strain; banana-B strain; carrot-C strain; tomato-T strain) were identified and quantified. In total, 67 chemical compounds were identified: 48 compounds were extracted from males maintained on banana and carrot, and 47 compounds from males maintained on cornmeal and tomato substrates, while total of 60 compounds were identified in females from all strains. The strains and the sexes significantly differed in qualitative nature of their chemoprofiles after mating. Significant differences in the relative amount of three major male pheromones (cis-vaccenyl acetate-cVA, (Z)-7-pentacosene, and (Z)-7-tricosene) and in female pheromone (Z,Z)-7,11-nonacosadiene among strains were also recorded. Furthermore, multivariate analysis of variance (MANOVA) pointed to significant differences between virgin and mated individuals of all strains and within both sexes. Differences in some of the well known sex pheromones were also identified when comparing their relative amount before and after mating. The presence of typical male pheromones in females, and vice versa may indicate their bidirectional transfer during copulation. These results confirm significant effect of mating status on cuticular hydrocarbon (CHC) phenotypes in differently fed D. melanogaster flies.
Rodrigues, L. R., Zwoinska, M. K., Axel, W. W. R. and Snook, R. R. (2022). The genetic basis and adult reproductive consequences of developmental thermal plasticity. J Anim Ecol. PubMed ID: 35060127
Summary:
Increasing temperature and thermal variability generates profound selection on populations. Given the fast rate of environmental change, understanding the role of plasticity and genetic adaptation in response to increasing temperatures is critical. This may be especially true for thermal effects on reproductive traits in which thermal fertility limits at high temperatures may be lower than for survival traits. A panel of Drosophila (the Drosophila Genetic Reference Panel; DGRP) was used in which male fertility performance was previously defined as either showing relatively little (status = "high" performing lines) or substantial ("low" performing lines) decline when exposed to increasing developmental temperatures. Developmental thermal stress impacted the means and thermal reaction norms of all reproductive traits except offspring sex-ratio. Mating success declined as temperature increased with no difference between high and low lines whereas increasing temperature resulted in declines for both male and female fertility and productivity but depended on line status. Fertility and offspring number were positively correlated within and between the sexes, but males were more affected than females. 933 SNPs were identified with significant evolved genetic differentiation between high and low lines. 54 of these lie within genomic windows of overall high differentiation, have significant effects of genotype on the male thermal reaction norm for productivity and are associated with 16 genes enriched for phenotypes affecting reproduction, stress responses and autophagy in Drosophila and other organisms. These results illustrate considerable plasticity in male thermal limits on several reproductive traits following development at high temperature, and differentiated loci with relevant phenotypic effects were identified that may contribute to this population variation.
Singh, A., Kandi, A. R., Jayaprakashappa, D., Thuery, G., Purohit, D. J., Huelsmeier, J., Singh, R., Pothapragada, S. S., Ramaswami, M. and Bakthavachalu, B. (2022). The Transcriptional Response to Oxidative Stress is Independent of Stress-Granule Formation. Mol Biol Cell: mbcE21080418. PubMed ID: 34985933
Summary:
Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this, this study characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3-hours predominantly resulted in induction or upregulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for chaperones, including HSP70s, small heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. It is suggested that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.
Welch, C., Johnson, E., Tupikova, A., Anderson, J., Tinsley, B., Newman, J., Widman, E., Alfareh, A., Davis, A., Rodriguez, L., Visger, C., Miller-Schulze, J. P., Lee, W. and Mulligan, K. (2022). Bisphenol a affects neurodevelopmental gene expression, cognitive function, and neuromuscular synaptic morphology in Drosophila melanogaster. Neurotoxicology 89: 67-78. PubMed ID: 35041872
Summary:
Bisphenol A (BPA) is an environmentally prevalent endocrine disrupting chemical that can impact human health and may be an environmental risk factor for neurodevelopmental disorders. BPA has been associated with behavioral impairment in children and a variety of neurodevelopmental phenotypes in model organisms. This study used Drosophila melanogaster to explore the consequences of developmental BPA exposure on gene expression, cognitive function, and synapse development. Transcriptome analysis indicated neurodevelopmentally relevant genes were predominantly downregulated by BPA. Among the misregulated genes were those with roles in learning, memory, and synapse development, as well as orthologs of human genes associated with neurodevelopmental and neuropsychiatric disorders. To examine how gene expression data corresponded to behavioral and cellular phenotypes, a predator-response behavioral paradigm was used, and it was found that BPA disrupts visual perception. Further analysis using conditioned courtship suppression showed that BPA impairs associative learning. Finally, synapse morphology within the larval neuromuscular junction was examined; BPA significantly increased the number of axonal branches. Given that these findings align with studies of BPA in mammalian model organisms, this data indicates that BPA impairs neurodevelopmental pathways that are functionally conserved from invertebrates to mammals. Further, because Drosophila do not possess classic estrogen receptors or estrogen, this research suggests that BPA can impact neurodevelopment by molecular mechanisms distinct from its role as an estrogen mimic.
Sun, Y., Liu, R., Bi, Y., Feng, X., Wang, J., Li, T., Wu, H., Zhang, C. and Sun, Y. (2022). Characterization of sleep-related neurochemicals in the different developmental stages and insomnia models of Drosophila melanogaster. Biomed Chromatogr: e5341. PubMed ID: 35045589
Summary:
Neurotransmitters play an important role in regulating the physiological activity of the animal, especially in emotion and sleep. While nucleotides are involved in almost all cellular processes. However, the characteristics of sleep-related neurochemicals under different life cycles and environment remain poorly understood. A rapid and sensitive analytical method was established with LC-MS/MS to determine eight endogenous neurochemicals in Drosophila melanogaster and the levels of neurochemicals in the different developmental stages of Drosophila melanogaster were evaluated. The results indicated that there were significant discrepancies among different stages, especially from pupal stage to adult. The levels of these compounds in caffeine-induced insomnia model of Drosophila melanogaster were investigated. Compared with normal group the eight endogenous metabolites did not fluctuate significantly in insomnia Drosophila melanogaster, which may be due to the mechanism of caffeine-induced insomnia through other pathways, such as adenosine. The results provide a reference for decoding of neurochemicals involved in the development of the full cycle of mammalian life and exploration of insomnia even other mental diseases induced by exogenous substances in the future.
Lee, S. H. and Kim, E. Y. (2021). Short-term maintenance on a high-sucrose diet alleviates aging-induced sleep fragmentation in drosophila. Anim Cells Syst (Seoul) 25(6): 377-386. PubMed ID: 35059137
Summary:
Sleep is a fundamental behavior in an animal's life influenced by many internal and external factors, such as aging and diet. Critically, poor sleep quality places people at risk of serious medical conditions. Because aging impairs quality of sleep, measures to improve sleep quality for elderly people are needed. Given that diet can influence many aspects of sleep, this study investigated whether a high-sucrose diet (HSD) affected aging-induced sleep fragmentation using the fruit fly, Drosophila melanogaster. Drosophila is a valuable model for studying sleep due to its genetic tractability and many similarities with mammalian sleep. Total sleep duration, sleep bout numbers (SBN), and average sleep bout length (ABL) were compared between young and old flies on a normal sucrose diet (NSD) or HSD. On the NSD, old flies slept slightly more and showed increased SBN and reduced ABL, indicating increased sleep fragmentation. Short-term maintenance of flies in HSD (up to 8 days), but not long-term maintenance (up to 35 days), suppressed aging-induced sleep fragmentation. This study provides meaningful strategies for preventing the deterioration of sleep quality in the elderly.

Friday, February 18th - Methods

Pak, E. S. and Murashov, A. K. (2021). Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning. J Vis Exp(176). PubMed ID: 34723949
Summary:
This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.
Yang, T. H., Yang, Y. C. and Tu, K. C. (2022). regCNN: identifying Drosophila genome-wide cis-regulatory modules via integrating the local patterns in epigenetic marks and transcription factor binding motifs. Comput Struct Biotechnol J 20: 296-308. PubMed ID: 35035784
Summary:
Transcription regulation in metazoa is controlled by the binding events of transcription factors (TFs) or regulatory proteins on specific modular DNA regulatory sequences called cis-regulatory modules (CRMs). Understanding the distributions of CRMs on a genomic scale is essential for constructing the metazoan transcriptional regulatory networks that help diagnose genetic disorders. While traditional reporter-assay CRM identification approaches can provide an in-depth understanding of functions of some CRM, these methods are usually cost-inefficient and low-throughput. It is generally believed that by integrating diverse genomic data, reliable CRM predictions can be made. Hence, researchers often first resort to computational algorithms for genome-wide CRM screening before specific experiments. However, current existing in silico methods for searching potential CRMs were restricted by low sensitivity, poor prediction accuracy, or high computation time from TFBS composition combinatorial complexity. To overcome these obstacles, a novel CRM identification pipeline called regCNN was developed by considering the base-by-base local patterns in TF binding motifs and epigenetic profiles. On the test set, regCNN shows an accuracy/auROC of 84.5%/92.5% in CRM identification. And by further considering local patterns in epigenetic profiles and TF binding motifs, it can accomplish 4.7% (92.5%-87.8%) improvement in the auROC value over the average value-based pure multi-layer perceptron model. It was also demonstrated that regCNN outperforms all currently available tools by at least 11.3% in auROC values. Finally, regCNN is verified to be robust against its resizing window hyperparameter in dealing with the variable lengths of CRMs.
Liu, J., Liu, Y., Chen, W., Ding, Y., Lan, X. and Liu, W. (2021). Purification of Endogenous Drosophila Transient Receptor Potential Channels. J Vis Exp(178). PubMed ID: 35037653l
Summary:
Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca(2+))-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase Cβ/No receptor potential A (PLCβ/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.
Mok, J. W. and Choi, K. W. (2021). Ex-vivo Microtubule Stability Assay Using Drosophila Wing Disc. Bio Protoc 11(23): e4241. PubMed ID: 35005086
Summary:
Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various in vitro assays for microtubule stability have been developed to identify and characterize proteins involved in controlling microtubule stability. This study introduced a simple ex-vivo assay for identifying potential microtubule regulators in the wing imaginal disc of Drosophila melanogaster. This assay utilizes silicon rhodamine-tubulin (SiR-Tub) as a cell-permeable fluorogenic dye for labeling microtubules. In an attempt to increase the sensitivity of the screen, an assay was designed using a sensitized microtubule condition. Wing discs are treated with SiR-Tub followed by demecolcine, a microtubule inhibitor, to partially label impaired microtubules. Under this sensitized condition, it was possible to test whether overexpression or downregulation of a gene can enhance or suppress the weakened SiR-Tub labeling. This assay allows highly sensitive detection of microtubules in developing larval tissues. Hence, it provides a useful tool for identifying new microtubule regulators in both unfixed and fixed imaginal discs in Drosophila. This strategy may also be applied to characterize microtubule regulators in tissues from other model organisms.
Aragon, M. J., Mok, A. T., Shea, J., Wang, M., Kim, H., Barkdull, N., Xu, C. and Yapici, N. (2022). Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle. Elife 11. PubMed ID: 35073257
Summary:
A multiphoton imaging method was developed to capture neural structure and activity in behaving flies through the intact cuticles. Measurements show that the fly head cuticle has surprisingly high transmission at wavelengths > 900 nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing or removing the air sacs, multiphoton imaging of the fly brain was performed through the intact cuticle. Anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body γ-lobes was demonstrated in behaving flies short-term and long-term. The through-cuticle imaging method developed in this study extends the time limits of in vivo imaging in flies and opens new ways to capture neural structure and activity from the fly brain.
Steinfath, E., Palacios-Munoz, A., Rottschafer, J. R., Yuezak, D. and Clemens, J. (2021). Fast and accurate annotation of acoustic signals with deep neural networks. Elife 10. PubMed ID: 34723794
Summary:
Acoustic signals serve communication within and across species throughout the animal kingdom. Studying the genetics, evolution, and neurobiology of acoustic communication requires annotating acoustic signals: segmenting and identifying individual acoustic elements like syllables or sound pulses. To be useful, annotations need to be accurate, robust to noise, and fast. This paper introduces DeepAudioSegmenter (DAS), a method that annotates acoustic signals across species based on a deep-learning derived hierarchical presentation of sound. The accuracy, robustness, and speed of DAS was demonstrated using acoustic signals with diverse characteristics from insects, birds, and mammals. DAS comes with a graphical user interface for annotating song, training the network, and for generating and proofreading annotations. The method can be trained to annotate signals from new species with little manual annotation and can be combined with unsupervised methods to discover novel signal types. DAS annotates song with high throughput and low latency for experimental interventions in realtime. Overall, DAS is a universal, versatile, and accessible tool for annotating acoustic communication signals.

Thursday, February 17th - Behavior

Churchill, E. R., Dytham, C., Bridle, J. R. and Thom, M. D. F. (2021). Social and physical environment independently affect oviposition decisions in Drosophila. Behav Ecol 32(6): 1391-1399. PubMed ID: 34949961
Summary:
In response to environmental stimuli, including variation in the presence of conspecifics, genotypes show highly plastic responses in behavioral and physiological traits influencing reproduction. Although extensively documented in males, such female responses are rather less studied. It is expected that females would be highly responsive to environmental variation and to differentially allocate resources to increase offspring fitness, given the major contribution of mothers to offspring number, size, and developmental conditions. Using Drosophila melanogaster, this study (1) manipulated exposure to conspecific females, which mothers could use to anticipate the number of potential mates and larval density, and; (2) tested how this interacts with the spatial distribution of potential oviposition sites, with females from higher densities expected to prefer clustered resources that can support a larger number of larvae. It was found that high density females were slower to start copulating and reduced their copulation duration, the opposite effect to that observed in males. There was a parallel, perhaps related, effect on egg production: females previously housed in groups laid fewer eggs than those housed in solitude. Resource patchiness also influenced oviposition behavior: females preferred aggregated substrate, which attracted more females to lay eggs. However, no interaction was found between prior housing conditions and resource patchiness, indicating that females did not perceive the value of different resource distributions differently when exposed to environments that could signal expected levels of larval competition. This study shows that, although exposure to consexual competition changes copulatory behaviors of females, the distribution of oviposition resources has a greater effect on oviposition decisions.
Borrero-Echeverry, F., Solum, M., Trona, F., Becher, P. G., Wallin, E. A., Bengtsson, M., Witzgall, P. and Lebreton, S. (2022). The female sex pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster. J Insect Physiol 137: 104355. PubMed ID: 35007554
Summary:
Specific mate communication and recognition underlies reproduction and hence speciation. This study provides new insights in Drosophila melanogaster premating olfactory communication. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues. Female-produced, species-specific volatile pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone (Z)-11-octadecenyl acetate (cVA) interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males. Two isoforms of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. However, only Z4-11Al mediates courtship in experienced males, not (R)-linalool. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds. Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in contrast to Z4-11Al. A blend of the food odour vinegar and cVA attracted females, while a blend of vinegar and female pheromone Z4-11Al attracted males, instead. Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects.
Jayaram, V., Kadakia, N. and Emonet, T. (2022). Sensing complementary temporal features of odor signals enhances navigation of diverse turbulent plumes. Elife 11. PubMed ID: 35072625
Summary:
It has been shown that during odor plume navigation, walking Drosophila melanogaster bias their motion upwind in response to both the frequency of their encounters with the odor, and the intermittency of the odor signal, which this study defines to be the fraction of time the signal is above a detection threshold. This study combined and simplified previous mathematical models that recapitulated these data to investigate the benefits of sensing both of these temporal features, and how these benefits depend on the spatiotemporal statistics of the odor plume. Through agent-based simulations, this study found that navigators that only use frequency or intermittency perform well in some environments - achieving maximal performance when gains are near those inferred from experiment - but fail in others. Robust performance across diverse environments requires both temporal modalities. However, a steep tradeoff was found when using both sensors simultaneously, suggesting a strong benefit to modulating how much each sensor is weighted, rather than using both in a fixed combination across plumes. Finally, it was shown that the circuitry of the Drosophila olfactory periphery naturally enables simultaneous intermittency and frequency sensing, enhancing robust navigation through a diversity of odor environments. Together, these results suggest that the first stage of olfactory processing selects and encodes temporal features of odor signals critical to real-world navigation tasks.
Turnell, B. R. and Reinhardt, K. (2022). Sperm metabolic rate predicts female mating frequency across Drosophila species. Evolution. PubMed ID: 35064568
Summary:
Female mating rates vary widely, even among closely related species, but the reasons for this variation are not fully understood. Across Drosophila species, female mating frequencies are positively associated with sperm length. This association may be due in part to sperm limitation, with longer-spermed species transferring fewer sperm, or to cryptic female choice. However, a previously overlooked factor is sperm metabolic rate, which may correlate with sperm length. If faster-metabolizing sperm accumulate age-related cellular damage more quickly, then females should remate sooner to obtain fresh sperm. Alternatively, frequent female mating may select for increased sperm competitiveness via increased metabolism. This study measured sperm metabolism across 13 Drosophila species and compared these measures to published data on female mating rate and on sperm length. Using fluorescent lifetime imaging microscopy, this study quantified NAD(P)H metabolism ex vivo, in intact organs. Phylogenetically controlled regression reveals that sperm metabolic rate is positively associated with sperm length and with female mating frequency. Path analysis shows sperm length driving sperm metabolism and sperm metabolism either driving or being driven by female mating rate. While the causal directionality of these relationships remains to be fully resolved, and the effect of sperm metabolism on sperm aging and/or sperm competitiveness remains to be established, these results demonstrate the importance of sperm metabolism in sexual selection.
Flores-Valle, A., Goncalves, P. J. and Seelig, J. D. (2021). Integration of sleep homeostasis and navigation in Drosophila. PLoS Comput Biol 17(7): e1009088. PubMed ID: 34252086
Summary:
During sleep, the brain undergoes dynamic and structural changes. In Drosophila, such changes have been observed in the central complex, a brain area important for sleep control and navigation. The connectivity of the central complex raises the question about how navigation, and specifically the head direction system, can operate in the face of sleep related plasticity. To address this question, this study develop a model that integrates sleep homeostasis and head direction. By introducing plasticity, the head direction system was shown to function in a stable way by balancing plasticity in connected circuits that encode sleep pressure. With increasing sleep pressure, the head direction system nevertheless becomes unstable and a sleep phase with a different plasticity mechanism is introduced to reset network connectivity. The proposed integration of sleep homeostasis and head direction circuits captures features of their neural dynamics observed in flies and mice.
Alsehli, A. M., Liao, S., Al-Sabri, M. H., Vasionis, L., Purohit, A., Behare, N., Clemensson, L. E., Williams, M. J. and Schioth, H. B. (2022). The Statin Target HMG-Coenzyme a Reductase (Hmgcr) Regulates Sleep Homeostasis in Drosophila. Pharmaceuticals (Basel) 15(1). PubMed ID: 35056136
Summary:
Statins, HMG Coenzyme A Reductase (HMGCR) inhibitors, are a first-line therapy, used to reduce hypercholesterolemia and the risk for cardiovascular events. While sleep disturbances are recognized as a side-effect of statin treatment, the impact of statins on sleep is under debate. Using Drosophila, this study discovered a novel role for Hmgcr in sleep modulation. Loss of pan-neuronal Hmgcr expression affects fly sleep behavior, causing a decrease in sleep latency and an increase in sleep episode duration. The pars intercerebralis (PI), equivalent to the mammalian hypothalamus, was identified as the region within the fly brain requiring Hmgcr activity for proper sleep maintenance. Lack of Hmgcr expression in the PI insulin-producing cells recapitulates the sleep effects of pan-neuronal Hmgcr knockdown. Conversely, loss of Hmgcr in a different PI subpopulation, the corticotropin releasing factor (CRF) homologue-expressing neurons (DH44 neurons), increases sleep latency and decreases sleep duration. Interestingly, loss of Hmgcr in the PI does not affect circadian rhythm, suggesting that Hmgcr regulates sleep by pathways distinct from the circadian clock. Taken together, these findings suggest that Hmgcr activity in the PI is essential for proper sleep homeostasis in flies.

Wednesday, February 16th - Larval and Adult Neural Development and Function

Takahashi, N., Zittrell, F., Hensgen, R. and Homberg, U. (2022). Receptive field structures for two celestial compass cues at the input stage of the central complex in the locust brain. J Exp Biol. PubMed ID: 35048987
Summary:
Successful navigation depends on an animal's ability to perceive its spatial orientation relative to visual surroundings. Heading direction in insects is represented in the central complex (CX), a navigation center in the brain, to generate steering commands. In insects that navigate relative to sky compass signals, CX neurons are tuned to celestial cues indicating the location of the sun. The desert locust CX contains a compass-like representation of two related celestial cues: the direction of unpolarized direct sunlight and the pattern of polarized light, which depends on the sun position. Whether congruent tuning to these two compass cues emerges within the CX network or is inherited from CX input neurons is unclear. To address this question, this study intracellularly recorded from GABA-immunoreactive TL neurons, input elements to the locust CX (corresponding to R neurons in Drosophila), while applying visual stimuli simulating unpolarized sunlight and polarized light across the hemisphere above the animal. TL neurons were shown to have large receptive fields for both types of stimuli. However, faithful integration of polarization angles across the dorsal hemisphere, or matched-filter ability to encode particular sun positions, was found in only two out of 22 recordings. Those two neurons also showed a good match in sun position coding through polarized and unpolarized light signaling, whereas 20 neurons showed substantial mismatch in signaling of the two compass cues. The data, therefore, suggest that considerable refinement of azimuth coding based on sky compass signals occurs at the synapses from TL neurons to postsynaptic CX compass neurons.
Prelic, S., Pal Mahadevan, V., Venkateswaran, V., Lavista-Llanos, S., Hansson, B. S. and Wicher, D. (2021). Functional Interaction Between Drosophila Olfactory Sensory Neurons and Their Support Cells. Front Cell Neurosci 15: 789086. PubMed ID: 35069116
Summary:
Insects detect volatile chemicals using antennae, which house a vast variety of olfactory sensory neurons (OSNs) that innervate hair-like structures called sensilla where odor detection takes place. In addition to OSNs, the antenna also hosts various support cell types. These include the triad of trichogen, tormogen, and thecogen support cells that lie adjacent to their respective OSNs. Little is known about the functional significance of the cells that support these neurons. To investigate the functional interaction between OSNs and support cells, optical and electrophysiological approaches were used in Drosophila. First, the distribution of various supporting cells were characterized using genetic markers. By means of an ex vivo antennal preparation and genetically-encoded Ca(2+) and K(+) indicators, the activation of these auxiliary cells during odor presentation in adult flies was study. Acute responses and distinct differences in Ca(2+) and K(+) fluxes were observed between support cell types. Finally, alterations were observed in OSN responses upon thecogen cell ablation in mature adults. Upon inducible ablation of thecogen cells, a gain was observed in mechanical responsiveness to mechanical stimulations during single-sensillum recording, but there was a lack of change to the neuronal resting activity. Taken together, these results demonstrate that support cells play a more active and responsive role during odor processing than previously thought. These observations thus reveal that support cells functionally interact with OSNs and may be important for the extraordinary ability of insect olfactory systems to dynamically and sensitively discriminate between odors in the turbulent sensory landscape of insect flight.
Marshall, Z. D. and Heckscher, E. S. (2022). The role of Even-skipped in Drosophila larval somatosensory circuit assembly. eNeuro. PubMed ID: 35031555
Summary:
Proper somatosensory circuit assembly is critical for processing somatosensory stimuli and for responding accordingly. In comparison to other sensory circuits (e.g., olfactory and visual), somatosensory circuits have unique anatomy and function. However, understanding of somatosensory circuit development lags far behind that of other sensory systems. For example, there are few identified transcription factors required for integration of interneurons into functional somatosensory circuits. This study examined one type of somatosensory interneuron, Even-skipped expressing Laterally placed interneurons (ELs) of the Drosophila larval nerve cord. Even-skipped (Eve) is a highly conserved, homeodomain transcription factor known to play a role in cell fate specification and neuronal axon guidance. Because marker genes are often functionally important in the cell types they define, this study deleted eve specifically from EL interneurons. On the cell biological level, using single neuron labeling, this study found eve plays several previously undescribed roles in refinement of neuron morphogenesis. Eve suppresses aberrant neurite branching, promotes axon elongation, and regulates dorsal-ventral dendrite position. On the circuit level, using optogenetics, calcium imaging, and behavioral analysis, it was found that eve is required in EL interneurons for the normal encoding of somatosensory stimuli and for normal mapping of outputs to behavior. It is concluded that eve coordinately regulates multiple aspects of EL interneuron morphogenesis and is critically required to properly integrate EL interneurons into somatosensory circuits. These data shed light on the genetic regulation of somatosensory circuit assembly.
Lu, W., Liu, Z., Fan, X., Zhang, X., Qiao, X. and Huang, J. (2022). Nicotinic acetylcholine receptor modulator insecticides act on diverse receptor subtypes with distinct subunit compositions. PLoS Genet 18(1): e1009920. PubMed ID: 35045067
Summary:
Insect nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of many insecticides, including neonicotinoids, which are the most widely used insecticides in the world. However, the development of resistance in pests and the negative impacts on bee pollinators affect the application of insecticides and have created a demand for alternatives. Thus, it is very important to understand the mode of action of these insecticides, which is not fully understood at the molecular level. This study systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunit mutants to eleven insecticides acting on nAChRs. The results showed that there are several subtypes of nAChRs with distinct subunit compositions that are responsible for the toxicity of different insecticides. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo. Moreover, spinosyns may act exclusively on the α6 homomeric pentamers but not any other nAChRs. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for the management of resistance and the development of novel insecticides targeting these important ion channels.
van Alphen, B., Stewart, S., Iwanaszko, M., Xu, F., Li, K., Rozenfeld, S., Ramakrishnan, A., Itoh, T. Q., Sisobhan, S., Qin, Z., Lear, B. C. and Allada, R. (2022). Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila. PLoS Biol 20(1): e3001456. PubMed ID: 35081110
Summary:
In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, this study developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, TBI-dependent behavior and lethality were examined in mutants of the master immune regulator NF-κB, important for AMP induction: while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.
Wei, J. Y., Chu, S. Y., Huang, Y. C., Chung, P. C. and Yu, H. H. (2022). Drosophila septin interacting protein 1 regulates neurogenesis in the early developing larval brain. Sci Rep 12(1): 292. PubMed ID: 34997175
Summary:
Neurogenesis in the Drosophila central brain progresses dynamically in order to generate appropriate numbers of neurons during different stages of development. Thus, a central challenge in neurobiology is to reveal the molecular and genetic mechanisms of neurogenesis timing. This study found that neurogenesis is significantly impaired when a novel mutation, Nuwa, is induced at early but not late larval stages. Intriguingly, when the Nuwa mutation is induced in neuroblasts of olfactory projection neurons (PNs) at the embryonic stage, embryonic-born PNs are generated, but larval-born PNs of the same origin fail to be produced. Through molecular characterization and transgenic rescue experiments, it was determined that Nuwa is a loss-of-function mutation in Drosophila septin interacting protein 1 (sip1). Furthermore, it was found that SIP1 expression is enriched in neuroblasts, and RNAi knockdown of sip1 using a neuroblast driver results in formation of small and aberrant brains. Finally, full-length SIP1 protein and truncated SIP1 proteins lacking either the N- or C-terminus display different subcellular localization patterns, and only full-length SIP1 can rescue the Nuwa-associated neurogenesis defect. Taken together, these results suggest that SIP1 acts as a crucial factor for specific neurogenesis programs in the early developing larval brain.

Tuesday, February 15th - Apoptosis

Valko, A., Perez-Pandolfo, S., Sorianello, E., Brech, A., Wappner, P. and Melani, M. (2021). Adaptation to hypoxia in Drosophila melanogaster requires autophagy. Autophagy: 1-12. PubMed ID: 34793268
Summary:
Macroautophagy/autophagy, a mechanism of degradation of intracellular material required to sustain cellular homeostasis, is exacerbated under stress conditions like nutrient deprivation, protein aggregation, organelle senescence, pathogen invasion, and hypoxia, among others. Detailed in vivo description of autophagic responses triggered by hypoxia is limited. This study characterized the autophagic response induced by hypoxia in Drosophila melanogaster. This process was found to be essential for Drosophila adaptation and survival because larvae with impaired autophagy are hypersensitive to low oxygen levels. Hypoxia triggers a bona fide autophagic response, as evaluated by several autophagy markers including Atg8, LysoTracker, Lamp1, Pi3K59F/Vps34 activity, transcriptional induction of Atg genes, as well as by transmission electron microscopy. Autophagy occurs in waves of autophagosome formation and maturation as hypoxia exposure is prolonged. Hypoxia-triggered autophagy is induced cell autonomously, and different tissues are sensitive to hypoxic treatments. Hypoxia-induced autophagy was found to depend on the basic autophagy machinery but not on the hypoxia master regulator sima/HIF1A. Overall, these studies lay the foundation for using D. melanogaster as a model system for studying autophagy under hypoxic conditions, which, in combination with the potency of genetic manipulations available in this organism, provides a platform for studying the involvement of autophagy in hypoxia-associated pathologies and developmentally regulated processes.
Gestuveo, R. J., Parry, R., Dickson, L. B., Lequime, S., Sreenu, V. B., Arnold, M. J., Khromykh, A. A., Schnettler, E., Lambrechts, L., Varjak, M. and Kohl, A. (2022). Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs. PLoS Pathog 18(1): e1010202. PubMed ID: 34990484
Summary:
The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. This study analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.
Medina, I., Calleja, M. and Morata, G. (2021). Tumorigenesis and cell competition in Drosophila in the absence of polyhomeotic function. Proc Natl Acad Sci U S A 118(45). PubMed ID: 34702735
Summary:
Cell competition is a homeostatic process that eliminates by apoptosis unfit or undesirable cells from animal tissues, including tumor cells that appear during the life of the organism. In Drosophila there is evidence that many types of oncogenic cells are eliminated by cell competition. One exception is cells mutant for polyhomeotic (ph), a member of the Polycomb family of genes; most of the isolated mutant ph clones survive and develop tumorous overgrowths in imaginal discs. To characterize the tumorigenic effect of the lack of ph, the growth was studied of different regions of the wing disc deficient in ph activity; the effect was restricted to the proximal appendage. Moreover, it was found that ph-deficient tissue is partially refractory to apoptosis. Then, the behavior of clones lacking ph function was studied; many suffered cell competition but were not completely eliminated. Unexpectedly, it was found that nonmutant cells also undergo cell competition when surrounded by ph-deficient cells, indicating that within the same tissue cell competition may operate in opposite directions. Two reasons are suggested for the incompleteness of cell competition inph mutant cells: 1) These cells are partially refractory to apoptosis, and 2) the loss of ph function alters the identity of imaginal cells and subsequently their cell affinities. It compromises the winner/loser interaction, a prerequisite for cell competition.
Malkeyeva, D., Kiseleva, E. and Fedorova, S. A. (2021). Loss of Hsp67Bc leads to autolysosome enlargement in the Drosophila brain. Cell Biol Int. PubMed ID: 34719095
Summary:
Hsp67Bc is a small heat shock protein found in Drosophila melanogaster. Apart from performing a function (common for all small heat shock proteins) of preventing aggregation of misfolded proteins, it is involved in macroautophagy regulation alongside the Starvin protein. Overexpression of the D. melanogaster Hsp67Bc gene has been shown to stimulate macroautophagy in S2 cell culture. Nonetheless, it has been unknown how the absence of the Hsp67Bc gene may affect it. The effect of Hsp67Bc gene deletion was studied on the macroautophagy induced by the pathogenic Wolbachia wMelPop strain in D. melanogaster. Wolbachia was detected inside autophagic vacuoles in fly neurons, thereby proving that these endosymbionts were being eliminated via macroautophagy. Nevertheless, no difference was registered in brain bacterial load between Hsp67Bc-null and control flies at all tested stages of ontogenesis. Moreover, the abundance of autophagic vacuoles was similar between neurons of the mutant and control flies, yet the cross-sectional area of autolysosomes on ultrathin sections was more than 1.5-fold larger in Hsp67Bc-null fly brains than in the control line. These findings suggest that the product of the Hsp67Bc gene does not participate in the initiation of endosymbiont-induced macroautophagy but may mediate autophagosome maturation: the deletion of the Hsp67Bc gene leads to the increase in autolysosome size.
Tsapras, P., Petridi, S., Chan, S., Geborys, M., Jacomin, A. C., Sagona, A. P., Meier, P. and Nezis, I. P. (2022). Selective autophagy controls innate immune response through a TAK1/TAB2/SH3PX1 axis. Cell Rep 38(4): 110286. PubMed ID: 35081354
Summary:
Selective autophagy is a catabolic route that turns over specific cellular material for degradation by lysosomes, and whose role in the regulation of innate immunity is largely unexplored. This study shows that the apical kinase of the Drosophila immune deficiency (IMD) pathway Tak1, as well as its co-activator Tab2, are both selective autophagy substrates that interact with the autophagy protein Atg8a. A role is presented for the Atg8a-interacting protein Sh3px1 in the downregulation of the IMD pathway, by facilitating targeting of the Tak1/Tab2 complex to the autophagy platform through its interaction with Tab2. These findings show the Tak1/Tab2/Sh3px1 interactions with Atg8a mediate the removal of the Tak1/Tab2 signaling complex by selective autophagy. This in turn prevents constitutive activation of the IMD pathway in Drosophila. This study provides mechanistic insight on the regulation of innate immune responses by selective autophagy.
Nandi, N., Zaidi, Z., Tracy, C. and Kramer, H. (2022). A phospho-switch at Acinus-Serine(437) controls autophagic responses to Cadmium exposure and neurodegenerative stress. Elife 11. PubMed ID: 35037620
Summary:
In Drosophila starvation-independent quality control autophagy is regulated by Acinus and the Cdk5-dependent phosphorylation of its serine(437). This study identified the phosphatase that counterbalances this activity. A genetic screen identified six phosphatases that genetically interacted with an Acinus gain-of-function model. Among these, loss of function of only one, the PPM-type phosphatase Nil (CG6036), enhanced pS437-Acinus levels. Cdk5-dependent phosphorylation of Acinus serine(437) in nil1 animals elevates neuronal autophagy and reduces the accumulation of polyQ proteins in a Drosophila Huntington's disease model. Consistent with previous findings that Cd(2+) inhibits PPM-type phosphatases, Cd(2+)-exposure elevated Acinus-serine(437) phosphorylation which was necessary for increased neuronal autophagy and protection against Cd(2+)-induced cytotoxicity. Together, these data establish the Acinus-S437 phospho-switch as critical integrator of multiple stress signals regulating neuronal autophagy.

Monday, February 14th - Stem Cells

Ishibashi, J. R., Keshri, R., Taslim, T. H., Brewer, D. K., Chan, T. C., Lyons, S., McManamen, A. M., Chen, A., Del Castillo, D. and Ruohola-Baker, H. (2021). Chemical Genetic Screen in Drosophila Germline Uncovers Small Molecule Drugs That Sensitize Stem Cells to Insult-Induced Apoptosis. Cells 10(10). PubMed ID: 34685753
Summary:
Cancer stem cells, in contrast to their more differentiated daughter cells, can endure genotoxic insults, escape apoptosis, and cause tumor recurrence. Understanding how normal adult stem cells survive and go to quiescence may help identify druggable pathways that cancer stem cells have co-opted. This study utilize a genetically tractable model for stem cell survival in the Drosophila gonad to screen drug candidates and probe chemical-genetic interactions. This study employed three levels of small molecule screening: (1) a medium-throughput primary screen in male germline stem cells (GSCs), (2) a secondary screen with irradiation and protein-constrained food in female GSCs, and (3) a tertiary screen in breast cancer organoids in vitro. This study uncover a series of small molecule drug candidates that may sensitize cancer stem cells to apoptosis. Further, these small molecules were tested for chemical-genetic interactions in the germline, and the NF-κB pathway was identified as an essential and druggable pathway in GSC quiescence and viability. This study demonstrates the power of the Drosophila stem cell niche as a model system for targeted drug discovery.
Thomas, A., Gallaud, E., Pascal, A., Serre, L., Arnal, I., Richard-Parpaillon, L., Savoian, M. S. and Giet, R. (2021). Peripheral astral microtubules ensure asymmetric furrow positioning in neural stem cells. Cell Rep 37(4): 109895. PubMed ID: 34706235
Summary:
Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly are governed by centralspindlin (Pavarotti and Tumbleweed) that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral microtubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.
Gadre, P., Nitsure, N., Mazumdar, D., Gupta, S. and Ray, K. (2021). The rates of stem cell division determine the cell cycle lengths of its lineage. iScience 24(11): 103232. PubMed ID: 34746698
Summary:
Adult stem cells and their transit-amplifying progeny alter their proliferation rates to maintain tissue homeostasis. To test how the division rates of stem cells and transit-amplifying progeny affect tissue growth and differentiation, a computation strategy was developed that estimates the average cell-cycle lengths (lifespans) of germline stem cells and their progeny from fixed-tissue demography in the Drosophila testis. Analysis of the wild-type data using this method indicated that during the germline transit-amplification, the cellular lifespans extend by nearly 1.3-fold after the first division and shrink by about 2-folds after the second division. Cell-autonomous perturbations of the stem cell lifespan accordingly altered the lifespans of successive transit-amplifying stages. Remarkably, almost 2-fold alterations in the lifespans of stem cells and their immediate daughters did not affect the subsequent differentiation. The results indicate that the early germline division rates can adjust the following division rates and the onset of differentiation.
Shi, L., Kong, R., Li, Z., Zhao, H., Ma, R., Bai, G., Li, J. and Li, Z. (2021). Identification of a new allele of O-fucosyltransferase 1 involved in Drosophila intestinal stem cell regulation. Biol Open 10(11). PubMed ID: 34731235
Summary:
Adult stem cells are critical for the maintenance of tissue homeostasis. However, how the proliferation and differentiation of intestinal stem cells (ISCs) are regulated remains not fully understood. This study found a mutant, stum 9-3, affecting the proliferation and differentiation of Drosophila adult ISCs in a forward genetic screen for factors regulating the proliferation and differentiation ISCs. stum 9-3 acts through the conserved Notch signaling pathway, upstream of the S2 cleavage of the Notch receptor. Interestingly, the phenotype of stum 9-3 mutant is not caused by disruption of stumble (stum), where the p-element is inserted. Detailed mapping, rescue experiments and mutant characterization show that stum 9-3 is a new allele of O-fucosyltransferase 1 (O-fut1). These results indicate that unexpected mutants with interesting phenotype could be recovered in forward genetic screens using known p-element insertion stocks.
Tang, R., Qin, P., Liu, X., Wu, S., Yao, R., Cai, G., Gao, J., Wu, Y. and Guo, Z. (2021). Intravital imaging strategy FlyVAB reveals the dependence of Drosophila enteroblast differentiation on the local physiology. Commun Biol 4(1): 1223. PubMed ID: 34697396
Summary:
Aging or injury in Drosophila intestine promotes intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation. However, the manner the local physiology couples with dynamic EB differentiation assessed by traditional lineage tracing method is still vague. Therefore, this study developed a 3D-printed platform "FlyVAB" for intravital imaging strategy that enables the visualization of the Drosophila posterior midgut at a single cell level across the ventral abdomen cuticle. Using ISCs in young and healthy midgut and enteroendocrine cells in age-associated hyperplastic midgut as reference coordinates, ISC-EB-enterocyte lineages were traced with Notch signaling reporter for multiple days. The results reveal a "differentiation-poised" EB status correlated with slow ISC divisions and a "differentiation-activated" EB status correlated with ISC hyperplasia and rapid EB to enterocyte differentiation. This FlyVAB imaging strategy opens the door to long-time intravital imaging of intestinal epithelium.
Tatapudy, S., Peralta, J. and Nystul, T. (2021). Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation. Development 148(22). PubMed ID: 35020878
Summary:
A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. This study found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and it, as well as other JNK pathway genes, were found to be essential for differentiation of FSC daughter cells. The data suggest that JNK signaling promotes differentiation by suppressing the activation of the EGFR effector, ERK. Loss of ben, but not the JNK kinase hemipterous, resulted in an upregulation of hedgehog signaling, increased proliferation and increased niche competition. Lastly, it was demonstrate that the hypercompetition phenotype caused by loss of ben is suppressed by decreasing the rate of proliferation or knockdown of the hedgehog pathway effector, Smoothened (Smo). Taken together, these findings reveal a new layer of regulation in which a single gene influences cell signaling at multiple stages of differentiation in the early FSC lineage.

Friday February 9th - Adult Physiology

Rass, M., Gizler, L., Bayersdorfer, F., Irlbeck, C., Schramm, M. and Schneuwly, S. (2022). The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family. PLoS One 17(1): e0262360. PubMed ID: 35030229
Summary:
Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-β signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, this study analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer.
Sun, G., Zhang, M., Chen, H. and Hochstrasser, M. (2022). The CinB Nuclease from wNo Wolbachia Is Sufficient for Induction of Cytoplasmic Incompatibility in Drosophila. mBio: e0317721. PubMed ID: 35073749
Summary:
Wolbachia is an obligate intracellular bacterium that can alter reproduction of its arthropod hosts, often through a mechanism called cytoplasmic incompatibility (CI). In CI, uninfected females fertilized by infected males yield few offspring, but if both are similarly infected, normal embryo viability results (called "rescue"). CI factors (Cifs) responsible for CI are pairs of proteins encoded by linked genes. The downstream gene in each pair encodes either a deubiquitylase (CidB) or a nuclease (CinB). The upstream gene products, CidA and CinA, bind their cognate enzymes with high specificity. Expression of CidB or CinB in yeast inhibits growth, but growth is rescued by expression of the cognate CifA protein. By contrast, transgenic Drosophila male germ line expression of both cifA and cifB was reported to be necessary to induce CI-like embryonic arrest; cifA expression alone in females is sufficient for rescue. This pattern, seen with genes from several Wolbachia strains, has been called the "2-by-1" model. This study shows that male germ line expression of the cinB gene alone, from a distinct clade of cif genes from wNo Wolbachia, is sufficient to induce nearly complete loss of embryo viability. This male sterility is fully rescued by cognate cinA(w)(No) expression in the female germ line. The proteins behave similarly in yeast. CinB(w)(No) toxicity depends on its nuclease active site. These results demonstrate that highly divergent CinB nucleases can induce CI, that rescue by cognate CifA factors is a general feature of Wolbachia CI systems, and that CifA is not strictly required in males for CI induction.
Logeay, R., Geminard, C., Lassus, P., Rodriguez-Vazquez, M., Kantar, D., Heron-Milhavet, L., Fischer, B., Bray, S. J., Colinge, J. and Djiane, A. (2022). Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila. Development. PubMed ID: 35005772
Summary:
Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, this study monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). The scrib mutation was shown to impact the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch dedicated transcription factor. The Notch-dependent neoplasms require however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1, and bZIP factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally this work highlights some Notch/scrib specificities, in particular the role of the PAR domain containing bZIP transcription factor and Notch direct target Pdp1 for neoplastic growth.
Ojha, R., Tantray, I., Rimal, S., Mitra, S., Cheshier, S. and Lu, B. (2022). Regulation of reverse electron transfer at mitochondrial complex I by unconventional Notch action in cancer stem cells. Dev Cell 57(2): 260-276. PubMed ID: 35077680
Summary:
Metabolic flexibility is a hallmark of many cancers where mitochondrial respiration is critically involved, but the molecular underpinning of mitochondrial control of cancer metabolic reprogramming is poorly understood. This study shows that reverse electron transfer (RET) through respiratory chain complex I (RC-I) is particularly active in brain cancer stem cells (CSCs). Although RET generates ROS, NAD(+)/NADH ratio turns out to be key in mediating RET effect on CSC proliferation, in part through the NAD(+)-dependent Sirtuin. Mechanistically, Notch acts in an unconventional manner to regulate RET by interacting with specific RC-I proteins containing electron-transporting Fe-S clusters and NAD(H)-binding sites. Genetic and pharmacological interference of Notch-mediated RET inhibited CSC growth in Drosophila brain tumor and mouse glioblastoma multiforme (GBM) models. These results identify Notch as a regulator of RET and RET-induced NAD(+)/NADH balance, a critical mechanism of metabolic reprogramming and a metabolic vulnerability of cancer that may be exploited for therapeutic purposes.
Soria, M. A., Cervantes, S. A. and Siemer, A. B. (2022). Calmodulin binds the N-terminus of the functional amyloid Orb2A inhibiting fibril formation. PLoS One 17(1): e0259872. PubMed ID: 35025866
Summary:
The cytoplasmic polyadenylation element-binding protein Orb2 is a key regulator of long-term memory (LTM) in Drosophila. The N-terminus of the Orb2 isoform A is required for LTM and forms cross-β fibrils on its own. However, this N-terminus is not part of the core found in ex vivo fibrils. Previous work showed that besides forming cross-β fibrils, the N-terminus of Orb2A binds anionic lipid membranes as an amphipathic helix. This study shows that the Orb2A N-terminus can similarly interact with calcium activated calmodulin (CaM) and that this interaction prevents fibril formation. Because CaM is a known regulator of LTM, this interaction could potentially explain the regulatory role of Orb2A in LTM.
Li, H., Zhao, Y., Zhang, X., Zhao, H., Li, W. and Wang, Q. (2022). Transcriptome-wide analysis of cellular immune response stimulated by nuclear input of different down syndrome cell adhesion molecule intracellular domains. Dev Comp Immunol 130: 104350. PubMed ID: 35051526
Summary:
In arthropods, Dscam produces multiple pathogen specific receptors via immune responsive alternative splicing, generating molecular complexity analogous to vertebrate antibodies. Fewer isoforms are produced by the exons encoding Dscam's intracellular domain (ICD); therefore, the present study aimed to determine the transcriptional response of Eriocheir sinensis to Dscam ICDs. In the group overexpressing all cytoplasmic tail exons (ICD-FL), 1401 differentially expressed genes (DEGs) were identified; overexpression of ICD constructs lacking exon-35 (ICD-Δ35) identified 413 DEGs; and overexpression of ICD constructs lacking exon-35 and exon-36 (ICD-Δ35 + 36) identified 22 DEGs. The DEGs were enriched in immunity and metabolism-related pathways. The transcriptomes of Drosophila S2 cells overexpressing different ICDs were then determined. Key immune, metabolic, and cell proliferation-regulated genes and gene networks were identified, providing insights into the membrane-to-nuclear signaling pathway of Dscam.

Thursday, February 10th - Gonads

Cridland, J. M., Majane, A. C., Zhao, L. and Begun, D. J. (2021). Population biology of accessory gland-expressed de novo genes in Drosophila melanogaster. Genetics. PubMed ID: 34791207
Summary:
Early work on de novo gene discovery in Drosophila was consistent with the idea that many such genes have male-biased patterns of expression, including a large number expressed in the testis. However, there has been little formal analysis of variation in the abundance and properties of de novo genes expressed in different tissues. This study investigated the population biology of recently evolved de novo genes expressed in the D. melanogaster accessory gland, a somatic male tissue that plays an important role in male and female fertility and the post mating response of females, using the same collection of inbred lines used previously to identify testis-expressed de novo genes, thus allowing for direct cross tissue comparisons of these genes in two tissues of male reproduction. Using RNA-seq data this study identified candidate de novo genes located in annotated intergenic and intronic sequence and determine the properties of these genes including chromosomal location, expression, abundance, and coding capacity. Generally, major differences were found between the tissues in terms of gene abundance and expression, though other properties such as transcript length and chromosomal distribution are more similar. Differences between regulatory mechanisms of de novo genes in the two tissues were also explored and how such differences may interact with selection to produce differences in D. melanogaster de novo genes expressed in the two tissues.
Hurtado, J., Almeida, F. C., Belliard, S. A., Revale, S. and Hasson, E. (2021). Research gaps and new insights in the evolution of Drosophila seminal fluid proteins. Insect Mol Biol. PubMed ID: 34747062
Summary:
While the striking effects of seminal fluid proteins (SFPs) on females are fairly conserved among Diptera, most SFPs lack detectable homologues among the SFP repertoires of phylogenetically distant species. How such a rapidly changing proteome conserves functions across taxa is a fascinating question. However, this and other pivotal aspects of SFPs' evolution remain elusive because discoveries on these proteins have been mainly restricted to the model Drosophila melanogaster. This study provides an overview of the current knowledge on the inter-specific divergence of the SFP repertoire in Drosophila and compile the increasing amount of relevant genomic information from multiple species. Capitalizing on the accumulated knowledge in D. melanogaster, novel sets of high-confidence SFP candidates and transcription factors are presented, presumptively involved in regulating the expression of SFPs. This study also addresses open questions by performing comparative genomic analyses that failed to support the existence of many conserved SFPs shared by most dipterans and indicated that gene co-option is the most frequent mechanism accounting for the origin of Drosophila SFP-coding genes. It is hoped this update establishes a starting point to integrate further data and thus widen the understanding of the intricate evolution of these proteins.
Colonnetta, M. M., Goyal, Y., Johnson, H. E., Syal, S., Schedl, P. and Deshpande, G. (2022). Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo. PLoS Genet 18(1): e1010002. PubMed ID: 34986144
Summary:
A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, this study shows that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, it was found that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.
Masukawa, M., Ishizaki, Y., Miura, H., Hayashi, M., Ota, R. and Kobayashi, S. (2021). Male-biased protein expression in primordial germ cells, identified through a comparative study of UAS vectors in Drosophila. Sci Rep 11(1): 21482. PubMed ID: 34728669
Summary:
In Drosophila, three types of UAS vectors (UASt, UASp, and UASz) are currently available for use with the Gal4-UAS system. They have been used successfully in somatic cells and germline cells from ovaries. However, it remains unclear whether they are functional in the germline cells of embryos, larvae, and adult testes. This study found that all three types of UAS vectors were functional in the germline cells of embryos and larvae and that the UASt and UASz vectors were active in the germline of the distal tip region in adult testes. Moreover, it was observed that protein expression from the UAS vectors was male-biased in germline cells of late embryos, whereas their respective mRNA expression levels were not. Furthermore, O-propargyl-puromycin (OPP) staining revealed that protein synthesis was male-biased in these germline cells. In addition, GO terms related to translation and ribosomal maturation were significantly enriched in the male germline. These observations show that translational activity is higher in male than in female germline cells. Therefore, it is proposed that male-biased protein synthesis may be responsible for the sex differences observed in the early germline.
Rowe, L. and Rockwell, A. L. (2022). Ubiquitous Knockdown of Mettl3 using TRiP.GL01126 Results in Spermatid Mislocalization During Drosophila Spermatogenesis. MicroPubl Biol 2022. PubMed ID: 35071998
Summary:
METTL3, the enzyme that catalyzes the m(6)A RNA modification in Drosophila is highly conserved and essential in various eukaryotic organisms. Mettl3 and its homologs have been linked to biological processes such as gametogenesis. This study focused on characterizing the role of METTL3 in Drosophila spermatogenesis. This study used the Gal4-UAS system to ubiquitously knockdown Mettl3 in both somatic cyst cells and germline cells. Using immunostaining and confocal microscopy, spermatid bundles were found to mislocalize in testes that contain the morphologically abnormal swollen apical tip. These result suggests Mettl3 knockdown using TRiP.GL01126 results in spermatogenesis aberrations.
Chakravarti, A., Thirimanne, H. N., Brown, S. and Calvi, B. R. (2022). Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control. Elife 11. PubMed ID: 35023826
Summary:
p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, it was found that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. In oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. These findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control.

Tuesday, February 9th - Disease Models

Kokotovic, T., Lenartowicz, E. M., Langeslag, M., Ciotu, C. I., Fell, C. W., Scaramuzza, A., Fischer, M. J. M., Kress, M., Penninger, J. M. and Nagy, V. (2022). Transcription factor Mesenchyme Homeobox Protein 2 (MEOX2) modulates nociceptor function. FEBS J. PubMed ID: 35029322
Summary:
Mesenchyme homeobox protein 2 (MEOX2) is a transcription factor involved in mesoderm differentiation, including development of bones, muscles, vasculature and dermatomes. Dysregulation was identified of MEOX2 in fibroblasts from Congenital Insensitivity to Pain (CIP) patients, and it was confirmed that buttonless, the Drosophila homologue of MEOX2, plays a role in nocifensive responses to noxious heat stimuli. To determine the importance of MEOX2 in the mammalian peripheral nervous system, a Meox2 heterozygous (Meox2(+/-)) mouse model was used to characterize its function in the sensory nervous system, and more specifically, in nociception. MEOX2 is expressed in the mouse dorsal root ganglia (DRG) and spinal cord, and localizes in the nuclei of a subset of sensory neurons. Functional studies of the mouse model, including behavioral, cellular and electrophysiological analyses, showed altered nociception encompassing impaired action potential initiation upon depolarization. Mechanistically, decreased expression was noted of Scn9a and Scn11a genes encoding Na(v) 1.7 and Na(v) 1.9 voltage gated sodium channels, respectively, that are crucial in subthreshold amplification and action potential initiation in nociceptors. Further transcriptomic analyses of Meox2(+/-) DRG revealed downregulation of a specific subset of genes including those previously associated with pain perception, such as PENK and NPY. Based on these observations, a novel role of MEOX2 in primary afferent nociceptor neurons is proposed for the maintenance of a transcriptional program required for proper perception of acute and inflammatory noxious stimuli.
Ribot, C., Soler, C., Chartier, A., Al Hayek, S., Nait-Saidi, R., Barbezier, N., Coux, O. and Simonelig, M. (2022). Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy. PLoS Genet 18(1): e1010015. PubMed ID: 35025870
Summary:
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. This study used a Drosophila model of OPMD, expression of alanine-expanded mammalian PABPN1 in Drosophila muscles, to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens a large number of UPS components were identified that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.
Heremans, I. P., Caligiore, F., Gerin, I., Bury, M., Lutz, M., Graff, J., Stroobant, V., Vertommen, D., Teleman, A. A., Van Schaftingen, E. and Bommer, G. T. (2022). Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite. Proc Natl Acad Sci U S A 119(4). PubMed ID: 35046029
Summary:
Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. This study reports that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. The glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 [or its orthologs (DJ-1α in Drosophila)] in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. This work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.
Jeon, Y. M., Kwon, Y., Lee, S., Kim, S., Jo, M., Lee, S., Kim, S. R., Kim, K. and Kim, H. J. (2021). Vitamin B12 Reduces TDP-43 Toxicity by Alleviating Oxidative Stress and Mitochondrial Dysfunction. Antioxidants (Basel) 11(1). PubMed ID: 35052586
Summary:
TAR DNA-binding protein 43 (TDP-43) is a member of an evolutionarily conserved family of heterogeneous nuclear ribonucleoproteins that modulate multiple steps in RNA metabolic processes. Cytoplasmic aggregation of TDP-43 in affected neurons is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Mislocalized and accumulated TDP-43 in the cytoplasm induces mitochondrial dysfunction and reactive oxidative species (ROS) production. This study shows that TDP-43- and rotenone-induced neurotoxicity in the human neuronal cell line SH-SY5Y were attenuated by hydroxocobalamin (Hb, vitamin B(12) analog) treatment. Although Hb did not affect the cytoplasmic accumulation of TDP-43, Hb attenuated TDP-43-induced toxicity by reducing oxidative stress and mitochondrial dysfunction. Moreover, a shortened lifespan and motility defects in TDP-43-expressing Drosophila were significantly mitigated by dietary treatment with hydroxocobalamin. Taken together, these findings suggest that oral intake of hydroxocobalamin may be a potential therapeutic intervention for TDP-43-associated proteinopathies.
Ji, J., Damschroder, D., Bessert, D., Lazcano, P., Wessells, R., Reynolds, C. A. and Greenberg, M. L. (2022). NAD supplementation improves mitochondrial performance of cardiolipin mutants. Biochim Biophys Acta Mol Cell Biol Lipids: 159094. PubMed ID: 35051613
Summary:
Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-β-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. This study identified abnormal NAD(+) metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD(+) precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function. Improved mitochondrial function in the Drosophila model was associated with restored exercise endurance, which suggests a potential therapeutic benefit of NAD(+) precursor supplementation in the management of BTHS patients.
Kaur, P., Chua, E. H. Z., Lim, W. K., Liu, J., Harmston, N. and Tolwinski, N. S. (2022). Wnt Signaling Rescues Amyloid Beta-Induced Gut Stem Cell Loss. Cells 11(2). PubMed ID: 35053396
Summary:
Patients with Alzheimer's disease suffer from a decrease in brain mass and a prevalence of amyloid-β plaques. These plaques are thought to play a role in disease progression, but their exact role is not entirely established. This study developed an optogenetic model to induce amyloid-β intracellular oligomerization to model distinct disease etiologies. This study examined the effect of Wnt signaling on amyloid in an optogenetic, Drosophila gut stem cell model. It was observed that Wnt activation rescues the detrimental effects of amyloid expression and oligomerization. The gene expression changes downstream of Wnt that contribute to this rescue was analyzed, and changes were found in aging related genes, protein misfolding, metabolism, and inflammation. It is proposed that Wnt expression reduces inflammation through repression of Toll activating factors. It was confirmed that chronic Toll activation reduces lifespan, but a decrease in the upstream activator Persephone extends it. It is proposed that the protective effect observed for lithium treatment functions, at least in part, through Wnt activation and the inhibition of inflammation.

Tuesday, February 8th - Adult Development

Wat, L. W., Chowdhury, Z. S., Millington, J. W., Biswas, P. and Rideout, E. J. (2021). Sex determination gene transformer regulates the male-female difference in Drosophila fat storage via the adipokinetic hormone pathway. Elife 10. PubMed ID: 34672260
Summary:
Sex differences in whole-body fat storage exist in many species. For example, Drosophila females store more fat than males. Yet, the mechanisms underlying this sex difference in fat storage remain incompletely understood. This study identified a key role for sex determination gene transformer (tra) in regulating the male-female difference in fat storage. Normally, a functional Tra protein is present only in females, where it promotes female sexual development. This study shows that loss of Tra in females reduced whole-body fat storage, whereas gain of Tra in males augmented fat storage. Tra's role in promoting fat storage was largely due to its function in neurons, specifically the Adipokinetic hormone (Akh)-producing cells (APCs). Analysis of Akh pathway regulation revealed a male bias in APC activity and Akh pathway function, where this sex-biased regulation influenced the sex difference in fat storage by limiting triglyceride accumulation in males. Importantly, Tra loss in females increased Akh pathway activity, and genetically manipulating the Akh pathway rescued Tra-dependent effects on fat storage. This identifies sex-specific regulation of Akh as one mechanism underlying the male-female difference in whole-body triglyceride levels, and provides important insight into the conserved mechanisms underlying sexual dimorphism in whole-body fat storage.
Melentev, P. A., Sharapenkov, E. G., Surina, N. V., Ivanova, E. A., Ryabova, E. V. and Sarantseva, S. V. (2021). Drosophila Lysophospholipase Gene swiss cheese Is Required for Survival and Reproduction. Insects 13(1). PubMed ID: 35055857
Summary:
Drosophila is widely used to analyse functions of different genes. The phosphatidylcholine lysophospholipase gene swiss cheese was initially shown to be important in the fruit fly nervous system. However, the role of this gene in non-nervous cell types has not been elucidated yet, and the evolutional explanation for the conservation of its function remains elusive. This study analyses the expression pattern and some aspects of the role of the swiss cheese gene in the fitness of Drosophila melanogaster. The spatiotemporal expression of swiss cheese throughout the fly development is described and the survival and productivity of swiss cheese mutants were analyzed. swiss cheese was found to be expressed in salivary glands, midgut, Malpighian tubes, adipocytes, and male reproductive system. Dysfunction of swiss cheese results in severe pupae and imago lethality and decline of fertility, which is impressive in males. The latter is accompanied with abnormalities of male locomotor activity and courtship behavior, accumulation of lipid droplets in testis cyst cells and decrease in spermatozoa motility. These results suggest that normal swiss cheese is important for Drosophila melanogaster fitness due to its necessity for both specimen survival and their reproductive success.
Melentev, P. A., Sharapenkov, E. G., Surina, N. V., Ivanova, E. A., Ryabova, E. V. and Sarantseva, S. V. (2021). Drosophila Lysophospholipase Gene swiss cheese Is Required for Survival and Reproduction. Insects 13(1). PubMed ID: 35055857
Summary:
Drosophila is widely used to analyse functions of different genes. The phosphatidylcholine lysophospholipase gene swiss cheese was initially shown to be important in the fruit fly nervous system. However, the role of this gene in non-nervous cell types has not been elucidated yet, and the evolutional explanation for the conservation of its function remains elusive. This study analyses the expression pattern and some aspects of the role of the swiss cheese gene in the fitness of Drosophila melanogaster. The spatiotemporal expression of swiss cheese throughout the fly development is described and the survival and productivity of swiss cheese mutants were analyzed. swiss cheese was found to be expressed in salivary glands, midgut, Malpighian tubes, adipocytes, and male reproductive system. Dysfunction of swiss cheese results in severe pupae and imago lethality and decline of fertility, which is impressive in males. The latter is accompanied with abnormalities of male locomotor activity and courtship behavior, accumulation of lipid droplets in testis cyst cells and decrease in spermatozoa motility. These results suggest that normal swiss cheese is important for Drosophila melanogaster fitness due to its necessity for both specimen survival and their reproductive success.
Rose, M., Domsch, K., Bartle-Schultheis, J., Reim, I. and Schaub, C. (2022). Twist regulates Yorkie activity to guide lineage reprogramming of syncytial alary muscles. Cell Rep 38(4): 110295. PubMed ID: 35081347
Summary:
Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. This study provides evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM.
Strassburger, K., Lutz, M., Muller, S. and Teleman, A. A. (2021). Ecdysone regulates Drosophila wing disc size via a TORC1 dependent mechanism. Nat Commun 12(1): 6684. PubMed ID: 34795214
Summary:
Most cells in a developing organ stop proliferating when the organ reaches a correct, final size. The underlying molecular mechanisms are not understood. This study finds that in Drosophila the hormone ecdysone controls wing disc size. To study how ecdysone affects wing size, endogenous ecdysone synthesis was inhibited and larvae were fed exogenous ecdysone in a dose-controlled manner. For any given ecdysone dose, discs stop proliferating at a particular size, with higher doses enabling discs to reach larger sizes. Termination of proliferation coincides with a drop in TORC1, but not Dpp or Yki signaling. Reactivating TORC1 bypasses the termination of proliferation, indicating that TORC1 is a main downstream effector causing proliferation termination at the maximal ecdysone-dependent size. Experimental manipulation of Dpp or Yki signaling can bypass proliferation termination in hinge and notum regions, but not the pouch, suggesting that the mechanisms regulating proliferation termination may be distinct in different disc regions.
Gallagher, K. D., Mani, M. and Carthew, R. W. (2022). Emergence of a geometric pattern of cell fates from tissue-scale mechanics in the Drosophila eye. Elife 11. PubMed ID: 35037852
Summary:
Pattern formation of biological structures involves the arrangement of different types of cells in an ordered spatial configuration. This study investigated the mechanism of patterning the Drosophila eye epithelium into a precise triangular grid of photoreceptor clusters called ommatidia. Previous studies had led to a long-standing biochemical model whereby a reaction-diffusion process is templated by recently formed ommatidia to propagate a molecular prepattern across the eye. This study finds that the templating mechanism is instead, mechanochemical in origin; newly born columns of differentiating ommatidia serve as a template to spatially pattern flows that move epithelial cells into position to form each new column of ommatidia. Cell flow is generated by a source and sink, corresponding to narrow zones of cell dilation and contraction respectively, that straddle the growing wavefront of ommatidia. The newly formed lattice grid of ommatidia cells are immobile, deflecting and focusing the flow of other cells. Thus, the self-organization of a regular pattern of cell fates in an epithelium is mechanically driven.

Monday, February 7th - Synapse and Vesicles

Wilkinson, E. C., Starke, E. L. and Barbee, S. A. (2021). Vps54 Regulates Lifespan and Locomotor Behavior in Adult Drosophila melanogaster. Front Genet 12: 762012. PubMed ID: 34712272
Summary:
Vps54 is an integral subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). A destabilizing missense mutation in Vps54 causes the age-progressive motor neuron (MN) degeneration, muscle weakness, and muscle atrophy observed in the wobbler mouse, an established animal model for human MN disease. It is currently unclear how the disruption of Vps54, and thereby the GARP complex, leads to MN and muscle phenotypes. To develop a new tool to address this question, this study has created an analogous model in Drosophila by generating novel loss-of-function alleles of the fly Vps54 ortholog (scattered/scat). Null scat mutant adults are viable but have a significantly shortened lifespan. Like phenotypes observed in the wobbler mouse, this study shows that scat mutant adults are male sterile and have significantly reduced body size and muscle area. Moreover, this study demonstrates that scat mutant adults have significant age-progressive defects in locomotor function. Interestingly, sexually dimorphic effects are seen, with scat mutant adult females exhibiting significantly stronger phenotypes. Finally, it was shown that scat interacts genetically with rab11 in MNs to control age-progressive muscle atrophy in adults. Together, these data suggest that scat mutant flies share mutant phenotypes with the wobbler mouse and may serve as a new genetic model system to study the cellular and molecular mechanisms underlying MN disease.
Kandasamy, S., Couto, K. and Thackeray, J. (2021). A docked mutation phenocopies dumpy oblique alleles via altered vesicle trafficking. PeerJ 9: e12175. PubMed ID: 34721959
Summary:
The Drosophila extracellular matrix protein Dumpy (Dpy) is one of the largest proteins encoded by any animal. One class of dpy mutations produces a characteristic shortening of the wing blade known as oblique (dpyo), due to altered tension in the developing wing. This study describes the characterization of docked (doc), a gene originally named because of an allele producing a truncated wing. This study shows that doc corresponds to the gene model CG5484, which encodes a homolog of the yeast protein Yif1 and plays a key role in ER to Golgi vesicle transport. Genetic analysis is consistent with a similar role for Doc in vesicle trafficking: docked alleles interact not only with genes encoding the COPII core proteins Sec23 and Sec13, but also with the SNARE proteins Synaptobrevin and Syntaxin. Further, it was demonstrated that the strong similarity between the doc1 and dpy0 wing phenotypes reflects a functional connection between the two genes; various dpy alleles were found to be sensitive to changes in dosage of genes encoding other vesicle transport components such as Sec13 and Sar1. Doc's effects on trafficking are not limited to Dpy; for example, reduced doc dosage disturbed Notch pathway signaling during wing blade and vein development. These results suggest a model in which the oblique wing phenotype in doc1 results from reduced transport of wild-type Dumpy protein; by extension, an additional implication is that the dpy0 alleles can themselves be explained as hypomorphs.
Camelo, C., Korte, A., Jacobs, T. and Luschnig, S. (2022). Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies. J Cell Sci. PubMed ID: 35019140
Summary:
Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. This study reports that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. Two distinct EV subpopulations are defined, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. It is proposed that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4.
Newman, Z. L., Bakshinskaya, D., Schultz, R., Kenny, S. J., Moon, S., Aghi, K., Stanley, C., Marnani, N., Li, R., Bleier, J., Xu, K. and Isacoff, E. Y. (2022). Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging. Nat Commun 13(1): 229. PubMed ID: 35017509
Summary:
Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (P(r)). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets P(r) remain to be defined. To address this, this study developed QuaSOR, a super-resolution method for determining P(r) from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. P(r) was mapped onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). P(r) varies greatly between synapses made by a single axon, the contribution of key AZ proteins to P(r) diversity was quantified, and that one of these, Complexin, was found to suppress spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission.
Sauvola, C. W., Akbergenova, Y., Cunningham, K. L., Aponte-Santiago, N. A. and Littleton, J. T. (2021). The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity. Elife 10. PubMed ID: 34713802
Summary:
Synaptic vesicle release probability (P(r)) is a key presynaptic determinant of synaptic strength established by cell intrinsic properties and further refined by plasticity. To characterize mechanisms that generate P(r) heterogeneity between distinct neuronal populations, this study examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in P(r) and synaptic plasticity. The decoy SNARE Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing P(r) to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic P(r) at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation (PHP).
Wagner, K., Smylla, T. K., Lampe, M., Krieg, J. and Huber, A. (2021). Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum. Traffic. PubMed ID: 34719094
Summary:
Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. This work reports a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. The results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD(3.1) mutants results in enhanced degradation of TRPL. In the retromer mutant vps35MH20 , TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD3.1 vps35MH20, PLD activity precedes retromer function. A model is proposed in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.

Friday, February 4th - Adult physiology

Klepsatel, P. and Galikova, M. (2022). Developmental temperature affects thermal dependence of locomotor activity in Drosophila. J Therm Biol 103: 103153. PubMed ID: 35027204
Summary:
In their natural environments, animals have to cope with fluctuations in numerous abiotic and biotic factors, and phenotypic plasticity can facilitate survival under such variable conditions. However, organisms may differ substantially in the ability to adjust their phenotypes in response to external factors. This study investigated how developmental temperature affects the thermal performance curve for locomotor activity in adult fruit flies (Drosophila melanogaster). The thermal dependence was examined of spontaneous activity in individuals originating from two natural populations (from tropical (India) and temperate climate zone (Slovakia)) that developed at three different temperatures (19 °C, 25 °C, and 29 °C). Firstly, developmental temperature was found to have a significant impact on overall activity - flies that developed at high temperature (29 °C) were, on average, less active than individuals that developed at lower temperatures. Secondly, developmental acclimation had a population-specific effect on the thermal optimum for activity. Whereas the optimal temperature was not affected by thermal conditions experienced during development in flies from India, developmental temperature shifted thermal optimum in flies from Slovakia. Thirdly, high developmental temperature broadened performance breadth in flies from the Indian population but narrowed it in individuals from the Slovak population. Finally, no consistent effect of acclimation temperature was detected on circadian rhythms of spontaneous activity. Altogether, these results demonstrate that developmental temperature can alter different parameters (maximum performance, thermal optimum, performance breadth) of the thermal performance curve for spontaneous activity. Since adult fruit flies are highly vagile, this sensitivity of locomotion to developmental conditions may be an important factor affecting fitness in changing environments.
Rodrigues, L. R., McDermott, H. A., Villanueva, I., Djukaric, J., Ruf, L. C., Amcoff, M. and Snook, R. R. (2021). Fluctuating heat stress during development exposes reproductive costs and putative benefits. J Anim Ecol. PubMed ID: 34775602
Summary:
This study tested how exposure to different average temperatures that either fluctuated or remained constant impacts different male reproductive performance traits and to assess adaptive potential to future heat stress. Advantage was taken of a set of Drosophila melanogaster isogenic lines of different genotypes, exposing them to four different thermal conditions. Subsequent male reproductive performance was measured for mating success, fertility, number of offspring produced, and offspring sex ratio, and the influence of thermal stress on estimated heritability and evolvability of these reproductive traits was calculated. Both costs and benefits to different thermal conditions on reproductive performance were found, with some responses varying between genotypes. Mating success improved under fluctuating benign temperature conditions and declined as temperature stress increased regardless of genotype. Fertility and productivity were severely reduced at fluctuating mean high temperature for all genotypes, but some genotypes were unaffected at constant high mean temperature. These more thermally robust genotypes showed a slight increase in productivity under the fluctuating benign condition compared to constant high temperature, despite both thermal conditions sharing the same temperature six hours daily. Increasing thermal stress resulted in higher heritability and evolvability. Overall, the effects of temperature on reproductive performance depended on the trait and genotype; performance of some traits slightly increased when high temperatures were experienced for short periods but decreased substantially even when experiencing a benign temperature for a portion of each day. While thermal stress increased genetic variation that could provide adaptive potential against climate warming, this is unlikely to compensate for the overall severe negative effect on reproductive performance as mean temperature and variance increase.
Schonborn, J. W., Stewart, F. A., Enriquez, K. M., Akhtar, I., Droste, A., Waschina, S. and Beller, M. (2021). Modeling Drosophila gut microbe interactions reveals metabolic interconnectivity. iScience 24(11): 103216. PubMed ID: 34712918
Summary:
A lot is known about varying gut microbiome compositions. Yet, how the bacteria affect each other remains elusive. In mammals, this is largely based on the sheer complexity of the microbiome with at least hundreds of different species. Thus, model organisms such as Drosophila melanogaster are commonly used to investigate mechanistic questions as the microbiome consists of only about 10 leading bacterial species. This study isolated gut bacteria from laboratory-reared Drosophila, sequenced their respective genomes, and used this information to reconstruct genome-scale metabolic models. With these, growth was similated in mono- and co-culture conditions and different media including a synthetic diet designed to grow Drosophila melanogaster. These simulations reveal a synergistic growth of some but not all gut microbiome members, which stems on the exchange of distinct metabolites including tricarboxylic acid cycle intermediates. Culturing experiments confirmed these predictions. This study thus demonstrates the possibility to predict microbiome-derived growth-promoting cross-feeding.
Hara, Y. and Yamamoto, D. (2021). Effects of Food and Temperature on Drosophila melanogaster Reproductive Dormancy as Revealed by Quantification of a GFP-Tagged Yolk Protein in the Ovary. Front Physiol 12: 803144. PubMed ID: 35046840
Summary:
When exposed to harsh environmental conditions, such as food scarcity and/or low temperature, Drosophila melanogaster females enter reproductive dormancy, a metabolic state that enhances stress resistance for survival at the expense of reproduction. Although the absence of egg chambers carrying yolk from the ovary has been used to define reproductive dormancy in this species, this definition is susceptible to false judgements of dormancy events: e.g. a trace amount of yolk could escape visual detection; a fly is judged to be in the non-dormancy state if it has a single yolk-containing egg chamber even when other egg chambers are devoid of yolk. In this study, an alternative method is proposed for describing the maturation state of oocytes, in which the amount of yolk in the entire ovary is quantified by the fluorescence intensity derived from GFP, which is expressed as a fusion with the major yolk protein Yp1. Yolk deposition was shown to increase with temperature with a sigmoidal function, and the quality of food substantially alters the maximum accumulation of yolk attainable at a given temperature. The Yp1::GFP reporter will serve as a reliable tool for quantifying the amount of yolk and provides a new means for defining the dormancy state in D. melanogaster.
Zhao, X. and Karpac, J. (2021). Glutamate metabolism directs energetic trade-offs to shape host-pathogen susceptibility in Drosophila. Cell Metab. PubMed ID: 34710355
Summary:
Individual hosts within populations often show inter-individual variation in their susceptibility to bacterial pathogen-related diseases. Utilizing Drosophila, this study highlighted that phenotypic variation in host-pathogen susceptibility within populations is driven by energetic trade-offs, facilitated by infection-mediated changes in glutamate metabolism. Furthermore, host-pathogen susceptibility is conditioned by life history, which adjusts immunometabolic sensing in muscles to direct vitamin-dependent reallocation of host energy substrates from the adipose tissue (i.e., a muscle-adipose tissue axis). Life history conditions inter-individual variation in the activation strength of intra-muscular NF-κB signaling. Limited intra-muscular NF-κB signaling activity allows for enhanced infection-mediated mitochondrial biogenesis and function, which stimulates glutamate dehydrogenase-dependent synthesis of glutamate. Muscle-derived glutamate acts as a systemic metabolite to promote lipid mobilization through modulating vitamin B enzymatic cofactor transport and function in the adipose tissue. This energy substrate reallocation improves pathogen clearance and boosts host survival. Finally, life history events that adjust energetic trade-offs can shape inter-individual variation in host-pathogen susceptibility after infection.
Ledru, M., Clark, C. A., Brown, J., Verghese, S., Ferrara, S., Goodspeed, A. and Su, T. T. (2022). Differential gene expression analysis identified determinants of cell fate plasticity during radiation-induced regeneration in Drosophila. PLoS Genet 18(1): e1009989. PubMed ID: 34990447
Summary:
Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. Previous work discovered a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. This study used a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

Thursday, February 3rd - Chromatin

Lan, K. Y. and Liao, B. Y. (2022). Epigenomic signatures on paralogous genes reveal underappreciated universality of active histone codes adopted across animals. Comput Struct Biotechnol J 20: 353-367. PubMed ID: 35035788
Summary:
The results of conventional gene-based analyses which combine epigenome and transcriptome data, including those conducted by the ENCODE/modENCODE projects, suggest various histone modifications performing regulatory functions in controlling mRNA expression (referred to as a histone code) in several model animals. While some histone codes were found to be universally adopted across organisms, "species-specific" histone codes have also been defined. This study found that the characterization of these histone codes was confounded by factors (e.g. gene essentiality, expression breadth) that are independent of, but correlated with, gene expression levels. Hence, this study attempted to decode histone marks in mouse (Mus musculus), fly (Drosophila melanogaster), and worm (Caenorhabditis elegans) genomes by examining ratios of RNA sequencing (and chromatin immunoprecipitation sequencing) intensities between paralog genes to remove confounding effects that would otherwise be present in a gene-based approach. With this paralog-based approach, associations between four histone modifications (H3K4me3, H3K27ac, H3K9ac, and H3K36me3) and gene expression are substantially revised. For example, it was demonstrated that H3K27ac and H3K9ac represent universal active marks in promoters, rather than worm-specific marks as previously reported. Second, acting regions of the studied active marks that are common across species (and across a wide range of tissues at different developmental stages) were found to extend beyond the previously defined regions. Thus, it appears that the active histone codes analyzed have a universality that has previously been underappreciated. These results suggested that these universal codes, including those previously considered species-specific, could have an ancient origin, and are important in regulating animal gene expression abundance.
Charidemou, E., Tsiarli, M. A., Theophanous, A., Yilmaz, V., Pitsouli, C., Strati, K., Griffin, J. L. and Kirmizis, A. (2022). Histone acetyltransferase NAA40 modulates acetyl-CoA levels and lipid synthesis. BMC Biol 20(1): 22. PubMed ID: 35057804
Summary:
Epigenetic regulation relies on the activity of enzymes that use sentinel metabolites as cofactors to modify DNA or histone proteins. Thus, fluctuations in cellular metabolite levels have been reported to affect chromatin modifications. However, whether epigenetic modifiers also affect the levels of these metabolites and thereby impinge on downstream metabolic pathways remains largely unknown. This study tested this notion by investigating the function of N-alpha-acetyltransferase 40 (NAA40), the enzyme responsible for N-terminal acetylation of histones H2A and H4, which has been previously implicated with metabolic-associated conditions such as age-dependent hepatic steatosis and calorie-restriction-mediated longevity. Using metabolomic and lipidomic approaches, this study found that depletion of NAA40 in murine hepatocytes leads to significant increase in intracellular acetyl-CoA levels, which associates with enhanced lipid synthesis demonstrated by upregulation in de novo lipogenesis genes as well as increased levels of diglycerides and triglycerides. Consistently, the increase in these lipid species coincide with the accumulation of cytoplasmic lipid droplets and impaired insulin signalling indicated by decreased glucose uptake. However, the effect of NAA40 on lipid droplet formation is independent of insulin. In addition, the induction in lipid synthesis is replicated in vivo in the Drosophila melanogaster larval fat body. Finally, supporting these results, this study found a strong association of NAA40 expression with insulin sensitivity in obese patients. Overall, these findings demonstrate that NAA40 affects the levels of cellular acetyl-CoA, thereby impacting lipid synthesis and insulin signalling. This study reveals a novel path through which histone-modifying enzymes influence cellular metabolism with potential implications in metabolic disorders.
Morgunova, V., Kordyukova, M., Mikhaleva, E. A., Butenko, I., Pobeguts, O. V. and Kalmykova, A. (2021). Loss of telomere silencing is accompanied by dysfunction of Polo kinase and centrosomes during Drosophila oogenesis and early development. PLoS One 16(10): e0258156. PubMed ID: 34624021
Summary:
Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. The mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila was studied. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. This study found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction (Morgunona, 2021).
Wolfe, J. C., Mikheeva, L. A., Hagras, H. and Zabet, N. R. (2021). An explainable artificial intelligence approach for decoding the enhancer histone modifications code and identification of novel enhancers in Drosophila. Genome Biol 22(1): 308. PubMed ID: 34749786
Summary:
Enhancers are non-coding regions of the genome that control the activity of target genes. Recent efforts to identify active enhancers experimentally and in silico have proven effective. While these tools can predict the locations of enhancers with a high degree of accuracy, the mechanisms underpinning the activity of enhancers are often unclear. Using machine learning (ML) and a rule-based explainable artificial intelligence (XAI) model, this study demonstrates that the location of known enhancers can be predicted in Drosophila with a high degree of accuracy. Most importantly, the rules of the XAI model were used to provide insight into the underlying combinatorial histone modifications code of enhancers. In addition, a large set of putative enhancers were identified that display the same epigenetic signature as enhancers identified experimentally. These putative enhancers are enriched in nascent transcription, divergent transcription and have 3D contacts with promoters of transcribed genes. However, they display only intermediary enrichment of mediator and cohesin complexes compared to previously characterised active enhancers. It was also found that 10-15% of the predicted enhancers display similar characteristics to super enhancers observed in other species. This study applied an explainable AI model to predict enhancers with high accuracy. Most importantly, it was found that different combinations of epigenetic marks characterise different groups of enhancers. Finally, a large set of putative enhancers was discovered which display similar characteristics with previously characterised active enhancers.
Chaouch, A., Berlandi, J., Chen, C. C. L., Frey, F., Badini, S., Harutyunyan, A. S., Chen, X., Krug, B., Hebert, S., Jeibmann, A., Lu, C., Kleinman, C. L., Hasselblatt, M., Lasko, P., Shirinian, M. and Jabado, N. (2021). Histone H3.3 K27M and K36M mutations de-repress transposable elements through perturbation of antagonistic chromatin marks. Mol Cell. PubMed ID: 34739871
Summary:
Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. This study shows that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon derepression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. These results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks (Chaouch, 2021).
Ghosh, S. and Lehner, C. F. (2022). Incorporation of CENP-A/CID into centromeres during early Drosophila embryogenesis does not require RNA polymerase II-mediated transcription. Chromosoma. PubMed ID: 35015118
Summary:
In many species, centromere identity is specified epigenetically by special nucleosomes containing a centromere-specific histone H3 variant, designated as CENP-A in humans and CID in Drosophila melanogaster. After partitioning of centromere-specific nucleosomes onto newly replicated sister centromeres, loading of additional CENP-A/CID into centromeric chromatin is required for centromere maintenance in proliferating cells. Analyses with cultured cells have indicated that transcription of centromeric DNA by RNA polymerase II is required for deposition of new CID into centromere chromatin. However, a dependence of centromeric CID loading on transcription is difficult to reconcile with the notion that the initial embryonic stages appear to proceed in the absence of transcription in Drosophila, as also in many other animal species. To address the role of RNA polymerase II-mediated transcription for CID loading in early Drosophila embryos, the effects of alpha-amanitin and triptolide on centromeric CID-EGFP levels were quantified. These analyses demonstrate that microinjection of these two potent inhibitors of RNA polymerase II-mediated transcription has at most a marginal effect on centromeric CID deposition during progression through the early embryonic cleavage cycles. Thus, it is concluded that at least during early Drosophila embryogenesis, incorporation of CID into centromeres does not depend on RNA polymerase II-mediated transcription.

Wednesday, January 2nd - Adult neural development and function

Lama, C., Love, C. R., Le, H. N., Waqar, M., Reeve, J. L., Lama, J. and Dauwalder, B. (2022). The nuclear receptor Hr46/Hr3 is required in the blood brain barrier of mature males for courtship. PLoS Genet 18(1): e1009519. PubMed ID: 35077443
Summary:
The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex-dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. In this study microarray analysis was carried out of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor Hr46/Hr3 was identified as a male-enriched BBB transcript. Hr46/Hr3 is best known for its essential roles in the ecdysone response during development and metamorphosis. This study demonstrated that Hr46/Hr3 is specifically required in the BBB cells for courtship behavior in mature males. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor Hr46/Hr3 in the regulation of male courtship, a novel function that is independent of its developmental role.
Chen, H. L., Motevalli, D., Stern, U. and Yang, C. H. (2022). A functional division of Drosophila sweet taste neurons that is value-based and task-specific. Proc Natl Acad Sci U S A 119(3). PubMed ID: 35031566
Summary:
Sucrose is an attractive feeding substance and a positive reinforcer for Drosophila. But Drosophila females have been shown to robustly reject a sucrose-containing option for egg-laying when given a choice between a plain and a sucrose-containing option in specific contexts. How the sweet taste system of Drosophila promotes context-dependent devaluation of an egg-laying option that contains sucrose, an otherwise highly appetitive tastant, is unknown. This study reports that devaluation of sweetness/sucrose for egg-laying is executed by a sensory pathway recruited specifically by the sweet neurons on the legs of Drosophila First, silencing just the leg sweet neurons caused acceptance of the sucrose option in a sucrose versus plain decision, whereas expressing the channelrhodopsin CsChrimson in them caused rejection of a plain option that was "baited" with light over another that was not. Analogous bidirectional manipulations of other sweet neurons did not produce these effects. Second, circuit tracing revealed that the leg sweet neurons receive different presynaptic neuromodulations compared to some other sweet neurons and were the only ones with postsynaptic partners that projected prominently to the superior lateral protocerebrum (SLP) in the brain. Third, silencing one specific SLP-projecting postsynaptic partner of the leg sweet neurons reduced sucrose rejection, whereas expressing CsChrimson in it promoted rejection of a light-baited option during egg-laying. These results uncover that the Drosophila sweet taste system exhibits a functional division that is value-based and task-specific, challenging the conventional view that the system adheres to a simple labeled-line coding scheme.
Zolin, A., Cohn, R., Pang, R., Siliciano, A. F., Fairhall, A. L. and Ruta, V. (2021). Context-dependent representations of movement in Drosophila dopaminergic reinforcement pathways. Nat Neurosci 24(11): 1555-1566. PubMed ID: 34697455
Summary:
Dopamine plays a central role in motivating and modifying behavior, serving to invigorate current behavioral performance and guide future actions through learning. This study examined how this single neuromodulator can contribute to such diverse forms of behavioral modulation. By recording from the dopaminergic reinforcement pathways of the Drosophila mushroom body during active odor navigation, this study reveals how their ongoing motor-associated activity relates to goal-directed behavior. Dopaminergic neurons were found to correlate with different behavioral variables depending on the specific navigational strategy of an animal, such that the activity of these neurons preferentially reflects the actions most relevant to odor pursuit. Furthermore, this study shows that these motor correlates are translated to ongoing dopamine release, and acutely perturbing dopaminergic signaling alters the strength of odor tracking. Context-dependent representations of movement and reinforcement cues are thus multiplexed within the mushroom body dopaminergic pathways, enabling them to coordinately influence both ongoing and future behavior.
Zhang, M. Y., Lear, B. C. and Allada, R. (2021). The microtubule associated protein tau suppresses the axonal distribution of PDF neuropeptide and mitochondria in circadian clock neurons. Hum Mol Genet. PubMed ID: 34750631
Summary:
Disrupted circadian rhythms is a prominent feature of multiple neurodegenerative diseases. Yet mechanisms linking Tau (see Drosophila Tau) to rhythmic behavior remain unclear. This study found that expression of a phosphomimetic human Tau mutant (TauE14) in Drosophila circadian pacemaker neurons disrupts free-running rhythmicity. While cell number and oscillations of the core clock protein PERIOD are unaffected in the small LNv (sLNv) neurons important for free running rhythms, a near complete loss of the major LNv neuropeptide pigment dispersing factor (PDF) in the dorsal axonal projections of the sLNvs. This was accompanied by a ~ 50% reduction in the area of the dorsal terminals and a modest decrease in cell body PDF levels. Expression of wild-type Tau also reduced axonal PDF levels but to a lesser extent than TauE14. TauE14 also induces a complete loss of mitochondria from these sLNv projections. However, mitochondria were increased in sLNv cell bodies in TauE14 flies. These results suggest that TauE14 disrupts axonal transport of neuropeptides and mitochondria in circadian pacemaker neurons, providing a mechanism by which Tau can disrupt circadian behavior prior to cell loss.
Henning, M., Ramos-Traslosheros, G., Gur, B. and Silies, M. (2022). Populations of local direction-selective cells encode global motion patterns generated by self-motion. Sci Adv 8(3): eabi7112. PubMed ID: 35044821
Summary:
Self-motion generates visual patterns on the eye that are important for navigation. These optic flow patterns are encoded by the population of local direction-selective cells in the mouse retina, whereas in flies, local direction-selective T4/T5 cells are thought to be uniformly tuned. How complex global motion patterns can be computed downstream is unclear. This study shows that the population of T4/T5 cells in Drosophila encodes global motion patterns. Whereas the mouse retina encodes four types of optic flow, the fly visual system encodes six. This matches the larger number of degrees of freedom and the increased complexity of translational and rotational motion patterns during flight. The four uniformly tuned T4/T5 subtypes described previously represent a local subset of the population. Thus, a population code for global motion patterns appears to be a general coding principle of visual systems that matches local motion responses to modes of the animal's movement.
Kaduskar, B., Kushwah, R. B. S., Auradkar, A., Guichard, A., Li, M., Bennett, J. B., Julio, A. H. F., Marshall, J. M., Montell, C. and Bier, E. (2022). Reversing insecticide resistance with allelic-drive in Drosophila melanogaster. Nat Commun 13(1): 291. PubMed ID: 35022402
Summary:
A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, common kdr mutations were generated in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. Differential sensitivities were identified to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, a CRISPR-based allelic-drive was applied to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.

Tuesday, February 1st - Immune response

Nayak, P., Kejriwal, A. and Ratnaparkhi, G. S. (2021). SUMOylation of Arginyl tRNA Synthetase Modulates the Drosophila Innate Immune Response. Front Cell Dev Biol 9: 695630. PubMed ID: 34660574
Summary:
SUMO conjugation of a substrate protein can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by Proteomics, but biological roles for SUMO conjugation for most targets remains elusive. The multi-aminoacyl tRNA synthetase complex (MARS) is a sensor and regulator of immune signaling. The proteins of this 1.2 MDa complex are targets of SUMO conjugation, in response to infection. Arginyl tRNA Synthetase (RRS), a member of the sub-complex II of MARS, is one such SUMO conjugation target. The sites for SUMO conjugation are Lys 147 and 383. Replacement of these residues by Arg (RRS (K147R,K383R)), creates a SUMO conjugation resistant variant (RRS (SCR)). Transgenic Drosophila lines for RRS (WT) and RRS (SCR) were generated by expressing these variants in a RRS loss of function (lof) animal, using the UAS-Gal4 system. The RRS-lof line was itself generated using CRISPR/Cas9 genome editing. Expression of both RRS (WT) and RRS (SCR) rescue the RRS-lof lethality. Adult animals expressing RRS (WT) and RRS (SCR) are compared and contrasted for their response to bacterial infection by gram positive M. luteus and gram negative Ecc15. This study finds that RRS (SCR), when compared to RRS (WT), shows modulation of the transcriptional response, as measured by quantitative 3' mRNA sequencing. This study uncovers a possible non-canonical role for SUMOylation of RRS, a member of the MARS complex, in host-defense (Nayak, 2021).
Carboni, A. L., Hanson, M. A., Lindsay, S. A., Wasserman, S. A. and Lemaitre, B. (2021). Cecropins contribute to Drosophila host defense against a subset of fungal and Gram-negative bacterial infection. Genetics. PubMed ID: 34791204
Summary:
Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding cecropins are strongly induced upon infection, pointing to their role in host-defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB and CecC) are expressed upon infection downstream of the Toll and Imd pathways. This study generated a short deletion ΔCecA-C removing the whole cecropin locus. Using the ΔCecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, this study addressed the function of cecropins in the systemic immune response. ΔCecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing ΔCecA-C in flies already lacking ten other AMP genes revealed a role for cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, this work provides the first genetic demonstration of a role for cecropins in insect host defense, and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (ΔAMP14) that lacks fourteen immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond (Carboni, 2021).
Mortimer, N. T., Fischer, M. L., Waring, A. L., Kr, P., Kacsoh, B. Z., Brantley, S. E., Keebaugh, E. S., Hill, J., Lark, C., Martin, J., Bains, P., Lee, J., Vrailas-Mortimer, A. D. and Schlenke, T. A. (2021). Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster. Proc Natl Acad Sci U S A 118(39). PubMed ID: 34544850
Summary:
In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. This study characterizes the Drosophila tuSz mutant strain, which mounts an aberrant immune response against its own fat body. This study demonstrates that this autoimmunity is the result of two mutations: 1) a mutation in the GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals (Mortimer, 2021).
Li, Y., Cai, J., Du, C., Lin, Y., Li, S., Ma, A. and Qin, Y. (2021). Bioinformatic Analysis and Antiviral Effect of Periplaneta americana Defensins. Virus Res: 198627. PubMed ID: 34785275
Summary:
Due to the lack of an adaptive immune system, insects rely on innate immune mechanisms to fight against pathogenic infections. Two major innate immune pathways, Toll and IMD, orchestrate anti-pathogen responses by regulating the expression of antimicrobial peptide (AMP) genes. Although the antifungal or antibacterial function of AMPs has been well characterized, the antiviral role of AMPs in insects remains largely unclear. Periplaneta americana (P. americana), or the American cockroach, is used in traditional Chinese medicine as an antiviral agent; however, the underlying mechanism of action of P. americana extracts is unclear. A previous study showed that the P. americana genome encodes multiple antimicrobial peptide genes. Based on these data, five novel P. americana defensins (PaDefensins) are predicted, and their primary structure, secondary structure, and physicochemical properties were analyzed. The putative antiviral, antifungal, antibacterial, and anticancer activities suggested that PaDefensin5 is a desirable therapeutic candidate against viral diseases. As the first experimental evidence of the antiviral effects of insect defensins, this study also showed the antiviral effect of PaDefensin5 in Drosophila Kc cells and Drosophila embryos in vivo . In conclusion, results of both in silico predictions and subsequent antiviral experiments suggested PaDefensin5 a promising antiviral drug.
Bahuguna, S., Atilano, M., Glittenberg, M., Lee, D., Arora, S., Wang, L., Zhou, J., Redhai, S., Boutros, M. and Ligoxygakis, P. (2022). Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila. PLoS Genet 18(1): e1009992. PubMed ID: 35007276
Summary:
The gut sets the immune and metabolic parameters for the survival of commensal bacteria. This study reports that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP, a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. Thise results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut.
Kanoh, H., Iwashita, S., Kuraishi, T., Goto, A., Fuse, N., Ueno, H., Nimura, M., Oyama, T., Tang, C., Watanabe, R., Hori, A., Momiuchi, Y., Ishikawa, H., Suzuki, H., Nabe, K., Takagaki, T., Fukuzaki, M., Tong, L. L., Yamada, S., Oshima, Y., Aigaki, T., Dow, J. A. T., Davies, S. A. and Kurata, S. (2021). cGMP signaling pathway that modulates NF-κB activation in innate immune responses. iScience 24(12): 103473. PubMed ID: 34988396
Summary:
The nuclear factor-kappa B (NF-κB) pathway is an evolutionarily conserved signaling pathway that plays a central role in immune responses and inflammation. This study shows that Drosophila NF-κB signaling is activated via a pathway in parallel with the Toll receptor by receptor-type guanylate cyclase, Gyc76C. Gyc76C produces cyclic guanosine monophosphate (cGMP) and modulates NF-κB signaling through the downstream Toll receptor components dMyd88, Pelle, Tube, and Dif/Dorsal (NF-κB). The cGMP signaling pathway comprises a membrane-localized cGMP-dependent protein kinase (cGK) called DG2 and protein phosphatase 2A (PP2A) and is crucial for host survival against Gram-positive bacterial infections in Drosophila. A membrane-bound cGK, PRKG2, also modulates NF-κB activation via PP2A in human cells, indicating that modulation of NF-κB activation in innate immunity by the cGMP signaling pathway is evolutionarily conserved.

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