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Current papers in developmental biology and gene function


Monday, January 31st, 2022 - Signaling

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Bakopoulos, D., Whisstock, J. C., Warr, C. G. and Johnson, T. K. (2022). Macrophage self-renewal is regulated by transient expression of PDGF- and VEGF-related factor 2. FEBS J. PubMed ID: 35066977
Macrophages are an ancient blood cell lineage critical for homeostasis and defence against pathogens. Although their numbers were long thought to be sustained solely by hematopoietic organs, it has recently become clear that their proliferation, or self-renewal, also plays a major role. In the Drosophila larva, macrophages undergo a phase of rapid self-renewal, making this an attractive model for elucidating the signals and regulatory mechanisms involved. However, a central self-renewal pathway has not been identified in this system. This study shows that the PDGF- and VEGF-receptor related (Pvr) pathway fulfils this role. The data show that two of the three known Pvr ligands, PDGF- and VEGF-related factor 2 (Pvf2) and Pvf3 are major determinants of overall macrophage numbers, yet they each act in a temporally independent manner and via distinct mechanisms. While Pvf3 is needed prior to the self-renewal period, Pvf2 is critical specifically for expanding the larval macrophage population. It was further shown that Pvf2 is a potent macrophage mitogen that is kept at limiting quantities by its transient expression in a remarkably small number of blood cells. Together, these data support a novel mechanism for the regulation of macrophage self-renewal rates by the dynamic transcriptional control of Pvf2. Given the strong parallels that exist between Drosophila and vertebrate macrophage systems, it is likely that a similar self-renewal control mechanism is at play across animal phyla.
Belyaeva, V., Wachner, S., Gyoergy, A., Emtenani, S., Gridchyn, I., Akhmanova, M., Linder, M., Roblek, M., Sibilia, M. and Siekhaus, D. (2022). Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila. PLoS Biol 20(1): e3001494. PubMed ID: 34990456
The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. This study found that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. This study thus identifies strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.
Ding, X., Li, Z., Lin, G., Li, W. and Xue, L. (2022). Toll-7 promotes tumour growth and invasion in Drosophila. Cell Prolif: e13188. PubMed ID: 35050535
Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. By using well-established Drosophila tumour models, this study identified Toll-7 as a novel regulator of tumour growth and invasion. Transgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Immunostaining and RT-qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0. Loss of Toll-7 suppressed Ras(V12) /lgl(-/-) -induced tumour growth and invasion, as well as cell polarity disruption-induced invasive cell migration, whereas expression of a constitutively active allele of Toll-7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr-JNK signalling is necessary and sufficient for Toll-7-induced invasive cell migration. Mechanistically, Toll-7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll-7 activates the EGFR-Ras signalling, which cooperates with the Egr-JNK signalling to promote Yki-mediated cell proliferation and tissue overgrowth. Finally, Toll-7 is necessary and sufficient for the proper maintenance of EGFR protein level. These findings characterized Toll-7 as a proto-oncogene that promotes tumour growth and invasion in Drosophila, shedding light on the pro-tumour function of mammalian Toll-like receptors (TLRs).
Ji, H., Sapar, M. L., Sarkar, A., Wang, B. and Han, C. (2022). Phagocytosis and self-destruction break down dendrites of Drosophila sensory neurons at distinct steps of Wallerian degeneration. Proc Natl Acad Sci U S A 119(4). PubMed ID: 35058357
After injury, severed dendrites and axons expose the "eat-me" signal phosphatidylserine (PS) on their surface while they break down. The degeneration of injured axons is controlled by a conserved Wallerian degeneration (WD) pathway, which is thought to activate neurite self-destruction through Sarm-mediated nicotinamide adenine dinucleotide (NAD(+)) depletion. While neurite PS exposure is known to be affected by genetic manipulations of NAD(+), how the WD pathway coordinates both neurite PS exposure and self-destruction and whether PS-induced phagocytosis contributes to neurite breakdown in vivo remain unknown. This study shows that in Drosophila sensory dendrites, PS exposure and self-destruction are two sequential steps of WD resulting from Sarm activation. Surprisingly, phagocytosis is the main driver of dendrite degeneration induced by both genetic NAD(+) disruptions and injury. However, unlike neuronal Nmnat loss, which triggers PS exposure only and results in phagocytosis-dependent dendrite degeneration, injury activates both PS exposure and self-destruction as two redundant means of dendrite degeneration. Furthermore, the axon-death factor Axed is only partially required for self-destruction of injured dendrites, acting in parallel with PS-induced phagocytosis. Lastly, injured dendrites exhibit a unique rhythmic calcium-flashing that correlates with WD. Therefore, both NAD(+)-related general mechanisms and dendrite-specific programs govern PS exposure and self-destruction in injury-induced dendrite degeneration in vivo.
Hao, Y., Pan, J., Chen, Q., Gu, H., Ji, G., Yue, G. and Yang, S. (2022). Jumu is required for the activation of JAK/STAT in Drosophila lymph gland development and epidermal wounds. Biochem Biophys Res Commun 591: 68-75. PubMed ID: 34999256
The regulatory mechanism of hematopoiesis and innate immunity in Drosophila is highly similar to that in mammals, and Drosophila has become a suitable model to understand vertebrate hematopoiesis and the immune response. JAK-STAT signaling pathway components are widely conserved during evolution, and contribute to hematopoiesis and multiple tissue damage and immune responses. This study demonstrates that Stat92E is widely expressed in the lymph gland, and the loss of jumu inhibits the maintenance of the JAK/STAT pathway in the CZ and MZ but not in the PSC of the lymph gland. Furthermore, this study found that clean puncture wounding of the larval epidermis can lead to the activation of JAK/STAT signaling and the generation of lamellocytes, and Jumu is required for the activation of JAK/STAT in response to epidermal wounds.
Jarvela-Stolting, M., Vesala, L., Maasdorp, M. K., Ciantar, J., Ramet, M. and Valanne, S. (2021). Proteasome α6 Subunit Negatively Regulates the JAK/STAT Pathway and Blood Cell Activation in Drosophila melanogaster. Front Immunol 12: 729631. PubMed ID: 35003057
JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. This study identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. These results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo.

Friday, December 28th - Evolution

Sprengelmeyer, Q. D. and Pool, J. E. (2021). Ethanol resistance in Drosophila melanogaster has increased in parallel cold-adapted populations and shows a variable genetic architecture within and between populations. Ecol Evol 11(21): 15364-15376. PubMed ID: 34765183
Understanding the genetic properties of adaptive trait evolution is a fundamental crux of biological inquiry that links molecular processes to biological diversity. Important uncertainties persist regarding the genetic predictability of adaptive trait change, the role of standing variation, and whether adaptation tends to result in the fixation of favored variants. This study used the recurrent evolution of enhanced ethanol resistance in Drosophila melanogaster during this species' worldwide expansion as a promising system to add to understanding of the genetics of adaptation. Elevated ethanol resistance was found to hav evolved at least three times in different cooler regions of the species' modern range-not only at high latitude but also in two African high-altitude regions. Applying a bulk segregant mapping framework, it was found that the genetic architecture of ethanol resistance evolution differs substantially not only between the three resistant populations, but also between two crosses involving the same European population. Population genetic scans were applied for local adaptation within quantitative trait locus regions, and potential contributions were found of genes with annotated roles in spindle localization, membrane composition, sterol and alcohol metabolism, and other processes. Simulation-based analyses were applied that confirm the variable genetic basis of ethanol resistance and hint at a moderately polygenic architecture. However, these simulations indicate that larger-scale studies will be needed to more clearly quantify the genetic architecture of adaptive evolution and to firmly connect trait evolution to specific causative loci.
Syed, Z. A., Dallai, R., Nasirzadeh, N., Brill, J. A., O'Grady, P. M., Cong, S., Leef, E. M., Rice, S., Asif, A., Nguyen, S., Hansen, M. M., Dorus, S. and Pitnick, S. (2021). Sperm Cyst "Looping": A Developmental Novelty Enabling Extreme Male Ornament Evolution. Cells 10(10). PubMed ID: 34685746
Postcopulatory sexual selection is credited as a principal force behind the rapid evolution of reproductive characters, often generating a pattern of correlated evolution between interacting, sex-specific traits. Because the female reproductive tract is the selective environment for sperm, one taxonomically widespread example of this pattern is the co-diversification of sperm length and female sperm-storage organ dimension. In Drosophila, having testes that are longer than the sperm they manufacture was believed to be a universal physiological constraint. Further, the energetic and time costs of developing long testes have been credited with underlying the steep evolutionary allometry of sperm length and constraining sperm length evolution in Drosophila. This report on the discovery of a novel spermatogenic mechanism-sperm cyst looping-that enables males to produce relatively long sperm in short testis. This phenomenon (restricted to members of the saltans and willistoni species groups) begins early during spermatogenesis and is potentially attributable to heterochronic evolution, resulting in growth asynchrony between spermatid tails and the surrounding spermatid and somatic cyst cell membranes. By removing the allometric constraint on sperm length, this evolutionary innovation appears to have enabled males to evolve extremely long sperm for their body mass while evading delays in reproductive maturation time. On the other hand, sperm cyst looping was found to exact a cost by requiring greater total energetic investment in testes and a pronounced reduction in male lifespan. The ecological selection pressures underlying the evolutionary origin and maintenance of this unique adaptation are discussed.
Vedelek, B., Kovacs, Á. and Boros, I. M. (2021). Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins. Open Biol 11(11): 210261. PubMed ID: 34784790
DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon this study investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on the results and on homology-based three-dimensional models for Ver and Moi, it is concluded that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. It is proposed that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. This study also provides the first evidence on Moi DNA binding.
Chang, C. H., Gregory, L. E., Gordon, K. E., Meiklejohn, C. D. and Larracuente, A. M. (2022). Unique structure and positive selection promote the rapid divergence of Drosophila Y chromosomes. Elife 11. PubMed ID: 34989337
Y chromosomes across diverse species convergently evolve a gene-poor, heterochromatic organization enriched for duplicated genes, LTR retrotransposons, and satellite DNA. Sexual antagonism and a loss of recombination play major roles in the degeneration of young Y chromosomes. However, the processes shaping the evolution of mature, already degenerated Y chromosomes are less well-understood. Because Y chromosomes evolve rapidly, comparisons between closely related species are particularly useful. De novo long read assemblies, complemented with cytological validation, were generated to reveal Y chromosome organization in three closely related species of the Drosophila simulans complex, which diverged only 250,000 years ago and share >98% sequence identity. These Y chromosomes were found to be divergent in their organization and repetitive DNA composition, and new Y-linked gene families were discovered whose evolution is driven by both positive selection and gene conversion. These Y chromosomes are also enriched for large deletions, suggesting that the repair of double-strand breaks on Y chromosomes may be biased toward microhomology-mediated end joining over canonical non-homologous end-joining. It is proposed that this repair mechanism contributes to the convergent evolution of Y chromosome organization across organisms.
Currea, J. P., Frazer, R., Wasserman, S. M. and Theobald, J. (2022). Acuity and summation strategies differ in vinegar and desert fruit flies. iScience 25(1): 103637. PubMed ID: 35028530
An animal's vision depends on terrain features that limit the amount and distribution of available light. Approximately 10,000 years ago, vinegar flies (Drosophila melanogaster) transitioned from a single plant specialist into a cosmopolitan generalist. Much earlier, desert flies (D. mojavensis) colonized the New World, specializing on rotting cactuses in southwest North America. Their desert habitats are characteristically flat, bright, and barren, implying environmental differences in light availability. This study demonstrated differences in eye morphology and visual motion perception under three ambient light levels. Reducing ambient light from 35 to 18 cd/m(2) causes sensitivity loss in desert but not vinegar flies. However, at 3 cd/m(2), desert flies sacrifice spatial and temporal acuity more severely than vinegar flies to maintain contrast sensitivity. These visual differences help vinegar flies navigate under variably lit habitats around the world and desert flies brave the harsh desert while accommodating their crepuscular lifestyle.
Suvorov, A., Kim, B. Y., Wang, J., Armstrong, E. E., Peede, D., D'Agostino, E. R. R., Price, D. K., Waddell, P., Lang, M., Courtier-Orgogozo, V., David, J. R., Petrov, D., Matute, D. R., Schrider, D. R. and Comeault, A. A. (2021). Widespread introgression across a phylogeny of 155 Drosophila genomes. Curr Biol. PubMed ID: 34788634
Genome-scale sequence data have invigorated the study of hybridization and introgression, particularly in animals. However, outside of a few notable cases, systematic tests for introgression at a larger phylogenetic scale across entire clades are lacking. This study leverage 155 genome assemblies from 149 species to generate a fossil-calibrated phylogeny and conduct multilocus tests for introgression across 9 monophyletic radiations within the genus Drosophila. Using complementary phylogenomic approaches, widespread introgression was identified across the evolutionary history of Drosophila. Mapping gene-tree discordance onto the phylogeny revealed that both ancient and recent introgression has occurred across most of the 9 clades that were examined. These results provide the first evidence of introgression occurring across the evolutionary history of Drosophila and highlight the need to continue to study the evolutionary consequences of hybridization and introgression in this genus and across the tree of life.

Thursday, January 27th - Disease Models

De Filippis, C., Napoli, B., Rigon, L., Guarato, G., Bauer, R., Tomanin, R. and Orso, G. (2021). Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes. Cells 11(1). PubMed ID: 35011691
Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. This paper reports a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). This study showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. These results provide new starting points for future investigations on the disease's pathogenic mechanisms and possible pharmacological manipulations.
Gabrawy, M. M., Khosravian, N., Morcos, G. S., Morozova, T. V., Jezek, M., Walston, J. D., Huang, W., Abadir, P. M. and Leips, J. (2022). Genome-Wide Analysis in Drosophila Reveals the Genetic Basis of Variation in Age-Specific Physical Performance and Response to ACE Inhibition. Genes (Basel) 13(1). PubMed ID: 35052483
Despite impressive results in restoring physical performance in rodent models, treatment with renin-angiotensin system (RAS) inhibitors, such as Lisinopril, have highly mixed results in humans, likely, in part, due to genetic variation in human populations. To date, the genetic determinants of responses to drugs, such as RAS inhibitors, remain unknown. Given the complexity of the relationship between physical traits and genetic background, genomic studies which predict genotype- and age-specific responses to drug treatments in humans or vertebrate animals are difficult. Using 126 genetically distinct lines of Drosophila melanogaster, this study tested the effects of Lisinopril on age-specific climbing speed and endurance. The data show that functional response and sensitivity to Lisinopril treatment ranges from significant protection against physical decline to increased weakness depending on genotype and age. Furthermore, genome-wide analyses led to identification of evolutionarily conserved genes in the WNT signaling pathway as being significantly associated with variations in physical performance traits and sensitivity to Lisinopril treatment. Genetic knockdown of genes in the WNT signaling pathway, Axin, frizzled, nemo, and wingless, diminished or abolished the effects of Lisinopril treatment on climbing speed traits. These results implicate these genes as contributors to the genotype- and age-specific effects of Lisinopril treatment and because they have orthologs in humans, they are potential therapeutic targets for improvement of resiliency. This approach should be widely applicable for identifying genomic variants that predict age- and sex-dependent responses to any type of pharmaceutical treatment.
Cortot, J., Farine, J. P., Ferveur, J. F. and Everaerts, C. (2022). Aging-Related Variation of Cuticular Hydrocarbons in Wild Type and Variant Drosophila melanogaster. J Chem Ecol. PubMed ID: 35022940
The cuticle of all insects is covered with hydrocarbons which have multiple functions. Cuticular hydrocarbons (CHCs) basically serve to protect insects against environmental harm and reduce dehydration. In many species, some CHCs also act as pheromones. CHCs have been intensively studied in Drosophila species and more especially in D. melanogaster. In this species, flies produce about 40 CHCs forming a complex sex- and species-specific bouquet. The quantitative and qualitative pattern of the CHC bouquet was characterized during the first days of adult life but remains unexplored in aging flies. This study characterized CHCs during the whole-or a large period of-adult life in males and females of several wild type and transgenic lines. Both types of lines included standard and variant CHC profiles. Some of the genotypes tested in this study showed very dramatic and unexpected aging-related variation based on their early days' profile. This study provides a concrete dataset to better understand the mechanisms underlying the establishment and maintenance of CHCs on the fly cuticle. It could be useful to determine physiological parameters, including age and response to climate variation, in insects collected in the wild.
De Groef, S., Wilms, T., Balmand, S., Calevro, F. and Callaerts, P. (2021). Sexual Dimorphism in Metabolic Responses to Western Diet in Drosophila melanogaster. Biomolecules 12(1). PubMed ID: 35053181
Obesity is a chronic disease affecting millions of people worldwide. The fruit fly (Drosophila melanogaster) is an interesting research model to study metabolic and transcriptomic responses to obesogenic diets. However, the sex-specific differences in these responses are still understudied and perhaps underestimated. This study exposed adult male and female Dahomey fruit flies to a standard diet supplemented with sugar, fat, or a combination of both. The exposure to a diet supplemented with 10% sugar and 10% fat efficiently induced an increase in the lipid content in flies, a hallmark for obesity. This increase in lipid content was more prominent in males, while females displayed significant changes in glycogen content. A strong effect of the diets on the ovarian size and number of mature oocytes was also present in females exposed to diets supplemented with fat and a combination of fat and sugar. In both males and females, fat body morphology changed and was associated with an increase in lipid content of fat cells in response to the diets. The expression of metabolism-related genes also displayed a strong sexually dimorphic response under normal condi-tions and in response to sugar and/or fat-supplemented diets. This study shows that the exposure of adult fruit flies to an obesogenic diet containing both sugar and fat allowed studying sexual dimorphism in metabolism and the expression of genes regulating metabolism.
Casale, A. M., Liguori, F., Ansaloni, F., Cappucci, U., Finaurini, S., Spirito, G., Persichetti, F., Sanges, R., Gustincich, S. and Piacentini, L. (2022). Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease. iScience 25(1): 103702. PubMed ID: 35036881
Huntington's disease (HD) is an autosomal dominant disorder with progressive motor dysfunction and cognitive decline. The disease is caused by a CAG repeat expansion in the IT15 gene, which elongates a polyglutamine stretch of the HD protein, Huntingtin. No therapeutic treatments are available, and new pharmacological targets are needed. Retrotransposons are transposable elements (TEs) that represent 40% and 30% of the human and Drosophila genomes and replicate through an RNA intermediate. Mounting evidence suggests that mammalian TEs are active during neurogenesis and may be involved in diseases of the nervous system. This study shows that TE expression and mobilization are increased in a Drosophila melanogaster HD model. By inhibiting TE mobilization with Reverse Transcriptase inhibitors, polyQ-dependent eye neurodegeneration and genome instability in larval brains are rescued and fly lifespan is increased. These results suggest that TE activation may be involved in polyQ-induced neurotoxicity and a potential pharmacological target.
Cheng, X., Xie, M., Luo, L., Tian, Y., Yu, G., Wu, Q., Fan, X., Yang, D., Mao, X., Gaur, U. and Yang, M. (2022). Inhibitor GSK690693 extends Drosophila lifespan via reduce AKT signaling pathway. Mech Ageing Dev 202: 111633. PubMed ID: 35065134
Aging is a process involving physiological changes that lead to the decline of biological functions of various tissues and organs of the body. Therefore, it is crucial to find anti-aging drugs that can intervene with the changes induced because of aging and slow down the degeneration of the biological functions. Among many signaling pathways linked with aging and aging-related diseases, PI3K-AKT signaling pathway has attracted major attention in aging biology. This paper demonstrates that AKT inhibitor GSK690693 can extend lifespan in Drosophila irrespective of start of the treatment from the beginning of life or the mid-life. Effect of GSK690693 for lifespan extension has been primarily related to the improvements in oxidative resistance, intestinal integrity and increased autophagy, but not in physical activity or starvation resistance. Furthermore, GSK690693 treatment reduced the activation of AKT and ERK, consequently activating FOXO, GSK-3β and apoptosis to modulate longevity of flies. Remarkably, GSK690693 did not induce hyperglycemia after treatment. The results indicate that GSK690693 may become an effective compound for anti-aging intervention.

Wednesday, January 26th - Enzyme and Protein Function

Shi, F., Mendrola, J. M., Sheetz, J. B., Wu, N., Sommer, A., Speer, K. F., Noordermeer, J. N., Kan, Z. Y., Perry, K., Englander, S. W., Stayrook, S. E., Fradkin, L. G. and Lemmon, M. A. (2021). ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes. Cell Rep 37(3): 109834. PubMed ID: 34686333
WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases (RTKs).This study describes crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. The crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. It was also found that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, these structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.
Zhang, X., Yu, H., Liu, X. and Song, C. (2021). The Impact of Mutation L138F/L210F on the Orai Channel: A Molecular Dynamics Simulation Study. Front Mol Biosci 8: 755247. PubMed ID: 34796201
The calcium release-activated calcium channel, composed of the Orai channel and the STIM protein, plays a crucial role in maintaining the Ca(2+) concentration in cells. Previous studies showed that the L138F mutation in the human Orai1 creates a constitutively open channel independent of STIM, causing severe myopathy, but how the L138F mutation activates Orai1 is still unclear. Based on the crystal structure of Drosophila melanogaster Orai (dOrai), molecular dynamics simulations for the wild-type (WT) and the L210F (corresponding to L138F in the human Orai1) mutant were conducted to investigate their structural and dynamical properties. The results showed that the L210F dOrai mutant tends to have a more hydrated hydrophobic region (V174 to F171), as well as more dilated basic region (K163 to R155) and selectivity filter (E178). Sodium ions were located deeper in the mutant than in the wild-type. Further analysis revealed two local but essential conformational changes that may be the key to the activation. A rotation of F210, a previously unobserved feature, was found to result in the opening of the K163 gate through hydrophobic interactions. At the same time, a counter-clockwise rotation of F171 occurred more frequently in the mutant, resulting in a wider hydrophobic gate with more hydration. Ultimately, the opening of the two gates may facilitate the opening of the Orai channel independent of STIM.
Zhou, X., Guo, C. J., Chang, C. C., Zhong, J., Hu, H. H., Lu, G. M. and Liu, J. L. (2021). Structural basis for ligand binding modes of CTP synthase. Proc Natl Acad Sci U S A 118(30). PubMed ID: 34301892
Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. This study reports the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, it was observed the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. These findings not only delineate the structure of CTPS in the presence of all substrates but also complete an understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.
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
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 cinAwNo expression in the female germ line. The proteins behave similarly in yeast. CinBwNo 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.
Margreiter, M. A., et al. (2022). Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death. Comput Struct Biotechnol J 20:443-458. PubMed ID: 35070167
Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. Previous work identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. This work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.
Okada, A. K., Teranishi, K., Ambroso, M. R., Isas, J. M., Vazquez-Sarandeses, E., Lee, J. Y., Melo, A. A., Pandey, P., Merken, D., Berndt, L., Lammers, M., Daumke, O., Chang, K., Haworth, I. S. and Langen, R. (2021). Lysine acetylation regulates the interaction between proteins and membranes. Nat Commun 12(1): 6466. PubMed ID: 34753925
Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Thia study use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. These data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

Tuesday, January 25th - RNA

Rounds, J. C., Corgiat, E. B., Ye, C., Behnke, J. A., Kelly, S. M., Corbett, A. H. and Moberg, K. H. (2021). The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology. Genetics. PubMed ID: 34791182
Nab2 encodes the Drosophila melanogaster member of a conserved family of zinc finger polyadenosine RNA-binding proteins (RBPs) linked to multiple steps in post-transcriptional regulation. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships remain unidentified. This study presents evidence that Nab2 genetically interacts with Ataxin-2 (Atx2), which encodes a neuronal translational regulator, and that these factors coordinately regulate neuronal morphology, circadian behavior, and adult viability. The first high-throughput identifications are presented of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using RNA immunoprecipitation-sequencing (RIP-Seq). Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g., drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g., Arpc2 and tea) promise insight into neuronal functions of, and genetic interactions between, each RBP. Consistent with prior biochemical studies, Nab2-associated neuronal RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. These data support a model where Nab2 functionally opposes Atx2 in neurons, demonstrate Nab2 shares associated neuronal RNAs with Atx2, and reveal Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.
Kao, S. Y., Nikonova, E., Chaabane, S., Sabani, A., Martitz, A., Wittner, A., Heemken, J., Straub, T. and Spletter, M. L. (2021). A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle. Cells 10(10). PubMed ID: 34685485
The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing, including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently, only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. This study demonstrate sthe utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. This study found that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. Knockdown was performed with 105 RNAi hairpins targeting 35 RBPs, and associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu are reported. Knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. It was further shown that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of kettin to the Z-disc. These results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.
Castro Alvarez, J. J., Revel, M., Carrasco, J., Cleard, F., Pauli, D., Hilgers, V., Karch, F. and Maeda, R. K. (2021). Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference. PLoS Genet 17(11): e1009843. PubMed ID: 34780465
Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. Here, this study investigates the iab-8 ncRNA from the Drosophila Bithorax Complex and shows that this RNA is able to repress the transcription of genes located at its 3' end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, this study found that its repressive activity is limited to the CNS, where, in wild-type embryos, it acts on the Hox gene, abd-A, located immediately downstream of it. The CNS specificity is achieved through a 3' extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3' extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote (Castro Alvarez, 2021).
Joseph, B., Scala, C., Kondo, S. and Lai, E. C. (2022). Molecular and genetic dissection of recursive splicing. Life Sci Alliance 5(1). PubMed ID: 34759052
Intronic ratchet points (RPs) are abundant within long introns in the Drosophila genome and consist of juxtaposed splice acceptor and splice donor (SD) sites. Although they appear to encompass zero-nucleotide exons, it was recently clarified that intronic recursive splicing (RS) requires a cryptic exon at the RP (an RS-exon), which is subsequently always skipped and thus absent from mRNA. In addition, Drosophila encodes a smaller set of expressed exons bearing features of RS. This study investigated mechanisms that regulate the choice between RP and RS-exon SDs. First, analysis of Drosophila RP SD mutants demonstrates that SD competition suppresses inclusion of cryptic exons in endogenous contexts. Second, characterization of RS-exon reporters implicates exonic sequences as influencing choice of RS-exon usage. Using RS-exon swap and mutagenesis assays, it was shown that exonic sequences can determine RS-exon inclusion. Finally, evidence is provided that splicing can suppress utilization of RP SDs to enable RS-exon expression. Overall, multiple factors can influence splicing of Drosophila RS-exons, which usually result in their complete suppression as zero-nucleotide RPs, but occasionally yield translated RS-exons.
Mirza, S., Kalluchi, A., Raza, M., Saleem, I., Mohapatra, B., Pal, D., Ouellette, M. M., Qiu, F., Yu, L., Lobanov, A., Zheng, Z. M., Zhang, Y., Alsaleem, M. A., Rakha, E. A., Band, H., Rowley, M. J. and Band, V. (2021). Ecdysoneless Protein Regulates Viral and Cellular mRNA Splicing to Promote Cervical Oncogenesis. Mol Cancer Res. PubMed ID: 34670863
High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, was identified as a novel HPV16 E6-interacting protein. This study shows that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, it was shown that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, these results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.
Zhang, B., Ding, Z., Li, L., Xie, L. K., Fan, Y. J. and Xu, Y. Z. (2021). Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila. PLoS Genet 17(11): e1009861. PubMed ID: 34723968
SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, strains were constructed carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and-H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system.

Monday, January 24th - Enhancers and Transcriptional Regulation

Hermann, A., Kosman, D., McGinnis, W. and Tour, E. (2021). The expression of Drosophila melanogaster Hox gene Ultrabithorax is not overtly regulated by the intronic long non-coding RNA lncRNA: PS4 in a wild type genetic background. G3 (Bethesda). PubMed ID: 34791185
Long non-coding RNAs (lncRNAs) have been implicated in a variety of processes in development, differentiation, and disease. In Drosophila melanogaster, the bithorax Hox cluster (BX-C) contains three Hox genes (Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)), along with a number of lncRNAs, most with unknown functions. This study investigated the function of a long non-coding RNA, lncRNA: PS4 that originates in the second intron of Ubx and is transcribed in the antisense orientation to Ubx. The expression pattern of lncRNA: PS4 is complementary to Ubx in the thoracic primordia, and the lncRNA: PS4 coding region overlaps the location of the large insertion that causes the dominant homeotic mutation Contrabithorax-1 (UbxCbx-1), which partially transforms Drosophila wings into halteres via ectopic activation of Ubx. This led the authors to investigate the potential role of this lncRNA in regulation of Ubx expression. The UbxCbx-1 mutation dramatically changes the pattern of lncRNA: PS4, eliminating the expression of most lncRNA: PS4 sequences from parasegment 4 (where Ubx protein is normally absent) and ectopically activating lncRNA: PS4 at high levels in the abdomen (where Ubx is normally expressed). These changes, however, did not lead to changes in the Ubx embryonic transcription pattern. Targeted deletion of the two promoters of lncRNA: PS4 did not result in the change of Ubx expression in the embryos. In the genetic background of a UbxCbx-1 mutation, the lncRNA: PS4 mutation does slightly enhance the ectopic activation of Ubx protein expression in wing discs and also slightly enhances the wing phenotype seen in UbxCbx-1 heterozygotes.
Dibaeinia, P. and Sinha, S. (2021). Deciphering enhancer sequence using thermodynamics-based models and convolutional neural networks. Nucleic Acids Res 49(18): 10309-10327. PubMed ID: 34508359
Deciphering the sequence-function relationship encoded in enhancers holds the key to interpreting non-coding variants and understanding mechanisms of transcriptomic variation. Several quantitative models exist for predicting enhancer function and underlying mechanisms; however, there has been no systematic comparison of these models characterizing their relative strengths and shortcomings. This study interrogated a rich data set of neuroectodermal enhancers in Drosophila, representing cis- and trans- sources of expression variation, with a suite of biophysical and machine learning models. Rigorous comparisons were performed of thermodynamics-based models implementing different mechanisms of activation, repression and cooperativity. Moreover, a convolutional neural network (CNN) model, called CoNSEPT, was developed that learns enhancer 'grammar' in an unbiased manner. CoNSEPT is the first general-purpose CNN tool for predicting enhancer function in varying conditions, such as different cell types and experimental conditions, and such complex models can suggest interpretable mechanisms. Model-based evidence was found for mechanisms previously established for the studied system, including cooperative activation and short-range repression. The data also favored one hypothesized activation mechanism over another and suggested an intriguing role for a direct, distance-independent repression mechanism. This modeling shows that while fundamentally different models can yield similar fits to data, they vary in their utility for mechanistic inference.
Hildebrandt, K., Kubel, S., Minet, M., Furst, N., Kloppel, C., Steinmetz, E. and Walldorf, U. (2021). Enhancer analysis of the Drosophila zinc finger transcription factor Earmuff by gene targeting. Hereditas 158(1): 41. PubMed ID: 34732265
Many transcription factors are involved in the formation of the brain during the development of Drosophila melanogaster. The transcription factor Earmuff (Erm), a member of the forebrain embryonic zinc finger family (Fezf), is one of these important factors for brain development. One major function of Earmuff is the regulation of proliferation within type II neuroblast lineages in the brain; here, Earmuff is expressed in intermediate neural progenitor cells (INPs) and balances neuronal differentiation versus stem cell maintenance. Erm expression during development is regulated by several enhancers. This work shows a functional analysis of erm and some of its enhancers. A new erm mutant allele was generated by gene targeting and reintegrated Gal4 to make an erm enhancer trap strain that could also be used on an erm mutant background. The deletion of three of the previously analysed enhancers showing the most prominent expression patterns of erm by gene targeting resulted in specific temporal and spatial defects in defined brain structures. These defects were already known but could be assigned to specific enhancer regions in this study. This analysis is the first systematic analysis of several large enhancer deletions of a Drosophila gene by gene targeting and will enable deeper analysis of erm enhancer functions in the future.
Kloppel, C., Hildebrandt, K., Kolb, D., Furst, N., Bley, I., Karlowatz, R. J. and Walldorf, U. (2021). Functional analysis of enhancer elements regulating the expression of the Drosophila homeodomain transcription factor DRx by gene targeting. Hereditas 158(1): 42. PubMed ID: 34736520
The Drosophila brain is an ideal model system to study stem cells, called neuroblasts, and the generation of neural lineages. Many transcriptional activators are involved in formation of the brain during the development of Drosophila melanogaster. The transcription factor Drosophila Retinal homeobox (DRx), a member of the 57B homeobox gene cluster, is also one of these factors for brain development. In this study a detailed expression analysis of DRx in different developmental stages was conducted. DRx is expressed in the embryonic brain in the protocerebrum, in the larval brain in the DM and DL lineages, the medulla and the lobula complex and in the central complex of the adult brain. A DRx enhancer trap strain was generated by gene targeting and reintegration of Gal4 that mimics the endogenous expression of DRx. With the help of eight existing enhancer-Gal4 strains and one made by in this study, various enhancers necessary for the expression of DRx were mapped during all stages of brain development from the embryo to the adult. An analysis was made of some larger enhancer regions by gene targeting. Deletion of three of these enhancers showing the most prominent expression patterns in the brain resulted in specific temporal and spatial loss of DRx expression in defined brain structures. These data show that DRx is expressed in specific neuroblasts and defined neural lineages and suggest that DRx is another important factor for Drosophila brain development.
Pu, J., Wang, Z., Cong, H., Chin, J. S. R., Justen, J., Finet, C., Yew, J. Y. and Chung, H. (2021). Repression precedes independent evolutionary gains of a highly specific gene expression pattern. Cell Rep 37(4): 109896. PubMed ID: 34706247
Highly specific expression patterns can be caused by the overlapping activities of activator and repressor sequences in enhancers. However, few studies illuminate how these sequences evolve in the origin of new enhancers. This study shows that expression of the bond gene in the semicircular wall epithelium (swe) of the Drosophila melanogaster male ejaculatory bulb (EB) is controlled by an enhancer consisting of an activator region that requires Abdominal-B driving expression in the entire EB and a repressor region that restricts this expression to the EB swe. Although this expression pattern is independently gained in the distantly related Scaptodrosophila lebanonensis and does not require Abdominal-B, this study shows that functionally similar repressor sequences are present in Scaptodrosophila and also in species that do not express bond in the EB. It is suggested that during enhancer evolution, repressor sequences can precede the evolution of activator sequences and may lead to similar but independently evolved expression patterns.
Yazar, V., Kang, S. U., Ha, S., Dawson, V. L. and Dawson, T. M. (2021). Integrative genome-wide analysis of dopaminergic neuron-specific PARIS expression in Drosophila dissects recognition of multiple PPAR-γ associated gene regulation. Sci Rep 11(1): 21500. PubMed ID: 34728675
The transcriptional repressor called parkin interacting substrate (PARIS; ZNF746) was initially identified as a novel co-substrate of parkin and PINK1 that leads to Parkinson's disease (PD) by disrupting mitochondrial biogenesis through peroxisome proliferator-activated receptor gamma (PPARγ) coactivator -1α (PGC-1α) suppression. Since its initial discovery, growing evidence has linked PARIS to defective mitochondrial biogenesis observed in PD pathogenesis. This study employed conditional translating ribosome affinity purification (TRAP) followed by RNA sequencing (TRAP-seq) for transcriptome profiling of DA neurons in transgenic Drosophila lines expressing human PARIS wild type (WT) or mutant (C571A). The results demonstrated that PPARγ functions as a master regulator of PARIS-induced molecular changes at the transcriptome level, confirming that PARIS acts primarily on PGC-1α to lead to neurodegeneration in PD. Moreover, this study identified that PARIS actively modulates expression of PPARγ target genes by physically binding to the promoter regions. Together, this work revealed how PARIS drives adverse effects on modulation of PPAR-γ associated gene clusters in DA neurons.

Friday, December 21 - Disease Models

Xiao, G., Zhao, M., Liu, Z., Du, F. and Zhou, B. (2021). Zinc antagonizes iron-regulation of tyrosine hydroxylase activity and dopamine production in Drosophila melanogaster. BMC Biol 19(1): 236. PubMed ID: 34732185
Dopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson's disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe(2+)) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson's disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. This study analyzed the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels. Catsup was found to be a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and the increased dopamine production was shown to be due to a reduction in zinc levels in the cytosol. Zinc ion (Zn(2+)) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe(2+) binding to TH, thus rendering the enzyme ineffective or non-functional. These findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe(2+) and Zn(2+) ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson's disease and other dopamine-related diseases.
Yap, Z. Y., Efthymiou, S., Seiffert, S., ..., Houlden, H. and Yoon, W. H. (2021). Bi-allelic variants in OGDHL cause a neurodevelopmental spectrum disease featuring epilepsy, hearing loss, visual impairment, and ataxia. Am J Hum Genet. PubMed ID: 34800363
The 2-oxoglutarate dehydrogenase-like (OGDHL) protein is a rate-limiting enzyme in the Krebs cycle that plays a pivotal role in mitochondrial metabolism. OGDHL expression is restricted mainly to the brain in humans. This study reports nine individuals from eight unrelated families carrying bi-allelic variants in OGDHL with a range of neurological and neurodevelopmental phenotypes including epilepsy, hearing loss, visual impairment, gait ataxia, microcephaly, and hypoplastic corpus callosum. The variants include three homozygous missense variants (p.Pro852Ala, p.Arg244Trp, and p.Arg299Gly), three compound heterozygous single-nucleotide variants (p.Arg673Gln/p.Val488Val, p.Phe734Ser/p.Ala327Val, and p.Trp220Cys/p.Asp491Val), one homozygous frameshift variant (p.Cys553Leufs(∗)16), and one homozygous stop-gain variant (p.Arg440Ter). To support the pathogenicity of the variants, a novel CRISPR-Cas9-mediated tissue-specific knockout was developed with cDNA rescue system for dOgdh, the Drosophila ortholog of human OGDHL. Pan-neuronal knockout of dOgdh led to developmental lethality as well as defects in Krebs cycle metabolism, which was fully rescued by expression of wild-type dOgdh. Studies using the Drosophila system indicate that p.Arg673Gln, p.Phe734Ser, and p.Arg299Gly are severe loss-of-function alleles, leading to developmental lethality, whereas p.Pro852Ala, p.Ala327Val, p.Trp220Cys, p.Asp491Val, and p.Arg244Trp are hypomorphic alleles, causing behavioral defects. Transcript analysis from fibroblasts obtained from the individual carrying the synonymous variant (c.1464T>C [p.Val488Val]) in family 2 showed that the synonymous variant affects splicing of exon 11 in OGDHL. Human neuronal cells with OGDHL knockout exhibited defects in mitochondrial respiration, indicating the essential role of OGDHL in mitochondrial metabolism in humans. Together, these data establish that the bi-allelic variants in OGDHL are pathogenic, leading to a Mendelian neurodevelopmental disease in humans.
Yamazoe, T., Nakahara, Y., Katsube, H. and Inoue, Y. H. (2021). Expression of Human Mutant Preproinsulins Induced Unfolded Protein Response, Gadd45 Expression, JAK-STAT Activation, and Growth Inhibition in Drosophila. Int J Mol Sci 22(21). PubMed ID: 34769468
Mutations in the insulin gene (INS) are frequently associated with human permanent neonatal diabetes mellitus. However, the mechanisms underlying the onset of this genetic disease is not sufficiently decoded. This study induced expression of two types of human mutant INSs in Drosophila using its ectopic expression system and investigated the resultant responses in development. Expression of the wild-type preproinsulin in the insulin-producing cells (IPCs) throughout the larval stage led to a stimulation of the overall and wing growth. However, ectopic expression of human mutant preproinsulins, hINS(C96Y) and hINS(LB15YB16delinsH), neither of which secreted from the β-cells, could not stimulate the Drosophila growth. Furthermore, neither of the mutant polypeptides induced caspase activation leading to apoptosis. Instead, they induced expression of several markers indicating the activation of unfolded protein response, such as ER stress-dependent Xbp1 mRNA splicing and ER chaperone induction. The mutant polypeptides were found to induce the expression of Growth arrest and DNA-damage-inducible 45 (Gadd45) in imaginal disc cells. ER stress induced by hINS(C96Y) also activated the JAK-STAT signaling, involved in inflammatory responses. Collectively, it is speculated that the diabetes-like growth defects appeared as a consequence of the human mutant preproinsulin expression was involved in dysfunction of the IPCs, rather than apoptosis.
Wang, Y. and Westermark, G. T. (2021). The Amyloid Forming Peptides Islet Amyloid Polypeptide and Amyloid beta Interact at the Molecular Level. Int J Mol Sci 22(20). PubMed ID: 34681811
Epidemiological studies support a connection between the two common disorders, type-2 diabetes and Alzheimer's disease. Both conditions have local amyloid formation in their pathogenesis, and cross-seeding between islet amyloid polypeptide (IAPP) and amyloid β (Aβ) could constitute the link. The bimolecular fluorescence complementation (BiFC) assay was used to investigate the occurrence of heterologous interactions between IAPP and Aβ and to compare the potential toxic effects of IAPP/Aβ, IAPP/IAPP, and Aβ/Aβ expression in living cells. Microscopy was used to confirm the fluorescence and determine the lysosomal, mitochondrial areas and mitochondrial membrane potential, and a FACS analysis was used to determine ROS production and the role for autophagy. Drosophila melanogaster expressing IAPP and Aβ was used to study their co-deposition and effects on longevity. Co-expression of IAPP and Aβ resulted in fluorophore reconstitution to the same extent as determined for homologous IAPP/IAPP or Aβ/Aβ expression. The BiFC(+)/BiFC(-) ratio of lysosomal area calculations increased in transfected cells independent of the vector combinations, while only Aβ/Aβ expression increased mitochondrial membrane potential. Expression combinations containing Aβ were necessary for the formation of a congophilic amyloid. In Drosophila melanogaster expressing IAPP/Aβ, co-deposition of the amyloid-forming peptides caused reduced longevity. The BiFC results confirmed a heterologous interaction between IAPP and Aβ, while co-deposits in the brain of Drosophila suggest mixed amyloid aggregates.
Proske, A., Bossen, J., von Frieling, J. and Roeder, T. (2021). Low-protein diet applied as part of combination therapy or stand-alone normalizes lifespan and tumor proliferation in a model of intestinal cancer. Aging (Albany NY) 13:. PubMed ID: 34766923
Tumors of the intestinal tract are among the most common tumor diseases in humans, but, like many other tumor entities, show an unsatisfactory prognosis with a need for effective therapies. To test whether nutritional interventions and a combination with a targeted therapy can effectively cure these cancers, the fruit fly Drosophila was used as a model. In this system, tumors were introduced by EGFR overexpression in intestinal stem cells. Limiting the amount of protein in the diet restored life span to that of control animals. In combination with a specific EGFR inhibitor, all major tumor-associated phenotypes could be rescued. This form of treatment was also successful in a real treatment scenario, which means when they started after the full tumor phenotype was expressed. In conclusion, reduced protein administration can be a very promising form of adjuvant cancer therapy.
Scharenbrock, A. R., Katzenberger, R. J., Fischer, M. C., Ganetzky, B. and Wassarman, D. A. (2021). Beta-blockers reduce intestinal permeability and early mortality following traumatic brain injury in Drosophila. MicroPubl Biol 2021. PubMed ID: 34723144
Traumatic brain injury (TBI) frequently leads to non-neurological consequences such as intestinal permeability. The beta-blocker drug labetalol, which inhibits binding of catecholamine neurotransmitters to adrenergic receptors, reduces intestinal permeability in a rat TBI model. Using a Drosophila melanogaster TBI model, previous studies found a strong positive correlation between intestinal permeability and mortality within 24 hours of TBI in a standard laboratory line (w1118) and across genetically diverse inbred lines from the Drosophila Genetic Reference Panel (DGRP). This study reports that feeding injured w1118 flies the beta-blockers labetalol and metoprolol reduced intestinal permeability and mortality. Additionally, metoprolol reduced intestinal permeability when 18 DGRP fly lines were analyzed in aggregate, but neither beta-blocker affected mortality. These data indicate that the mechanism underlying disruption of the intestinal barrier by adrenergic signaling following TBI is conserved between humans and flies and that mortality following TBI in flies is not strictly dependent on disruption of the intestinal barrier. Thus, the fly TBI model is useful for shedding light on the mechanism and consequences of adrenergic signaling between the brain and intestine following TBI in humans.

Thursday, January 20th - Adult neural development and function

Schwarz, J. E., King, A. N., Hsu, C. T., Barber, A. F. and Sehgal, A. (2021). Hugin (+) neurons provide a link between sleep homeostat and circadian clock neurons. Proc Natl Acad Sci U S A 118(47). PubMed ID: 34782479
Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. These studies show that hugin (+) neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin (+) neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin (+) neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin (+) locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin (+) neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide-dependent fashion. It is proposed that hugin (+) neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.
Shrestha, B., Nhuchhen Pradhan, R., Nath, D. K. and Lee, Y. (2021). Cellular and molecular basis of IR3535 perception in Drosophila. Pest Manag Sci. PubMed ID: 34708523
IR3535 is among the most widely used synthetic insect repellents, particularly for the mitigation of mosquito-borne diseases such as malaria, yellow fever, dengue and Zika, as well as to control flies, ticks, fleas, lice and mites. These insects are well-known vectors of deadly diseases that affect humans, livestock and crops. Moreover, global warming could increase the populations of these vectors. This study performed IR3535 dose-response analyses on Drosophila melanogaster, a well-known insect model organism, using electrophysiology and binary food choice assays. The findings indicated that bitter-sensing gustatory receptor neurons (GRNs) are indispensable to detect IR3535. Further, potential candidate gustatory receptors were screened, among which GR47a was identified as a key molecular sensor. IR3535 concentrations in the range 0.1-0.4% affected larval development and mortality. In addition, N,N-diethyl-m-toluamide (DEET, another commonly used insecticide) was found to exert synergistic effects when co-administered with IR3535. These findings confirmed that IR3535 directly activates bitter-sensing GRNs, which are mediated by GR47a. This relatively safe and highly potent insecticide can be largely used in combination with DEET to increase its efficiency to protect livestock and crops. Collectively, these findings suggest that the molecular sensors elucidated herein could be used as targets for the development of alternative insecticides.
Tomita, J., Ban, G., Kato, Y. S. and Kume, K. (2021). Protocerebral Bridge Neurons That Regulate Sleep in Drosophila melanogaster. Front Neurosci 15: 647117. PubMed ID: 34720844
The central complex is one of the major brain regions that control sleep in Drosophila. However, the circuitry details of sleep regulation have not been elucidated yet. This study shows a novel sleep-regulating neuronal circuit in the protocerebral bridge (PB) of the central complex. Activation of the PB interneurons labeled by the R59E08-Gal4 and the PB columnar neurons with R52B10-Gal4 promoted sleep and wakefulness, respectively. A targeted GFP reconstitution across synaptic partners (t-GRASP) analysis demonstrated synaptic contact between these two groups of sleep-regulating PB neurons. Furthermore, it was found that activation of a pair of dopaminergic (DA) neurons projecting to the PB (T1 DA neurons) decreased sleep. The wake-promoting T1 DA neurons and the sleep-promoting PB interneurons formed close associations. Dopamine 2-like receptor (Dop2R) knockdown in the sleep-promoting PB interneurons increased sleep. These results indicated that the neuronal circuit in the PB, regulated by dopamine signaling, mediates sleep-wakefulness.
Shrestha, B. and Lee, Y. (2021). Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster. Mol Cells. PubMed ID: 34711686
Sour is one of the fundamental taste modalities that enable taste perception in animals. Chemoreceptors embedded in taste organs are pivotal to discriminate between different chemicals to ensure survival. Animals generally prefer slightly acidic food and avoid highly acidic alternatives. It was recently proposed that all acids are aversive at high concentrations, a response that is mediated by low pH as well as specific anions in Drosophila melanogaster. Particularly, some carboxylic acids such as glycolic acid, citric acid, and lactic acid are highly attractive to Drosophila compared with acetic acid. The present study determined that attractive carboxylic acids were mediated by broadly expressed Ir25a and Ir76b, as demonstrated by a candidate mutant library screen. The mutant deficits were completely recovered via wild-type cDNA expression in sweet-sensing gustatory receptor neurons. Furthermore, sweet gustatory receptors such as Gr5a, Gr61a, and Gr64a-f modulate attractive responses. These genetic defects were confirmed using binary food choice assays as well as electrophysiology in the labellum. Taken together, these findings demonstrate that at least two different kinds of receptors are required to discriminate attractive carboxylic acids from other acids.
Silva, V., Palacios-Munoz, A., Volonte, M., Frenkel, L., Ewer, J. and Ons, S. (2021). Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster. Insect Biochem Mol Biol 139: 103676. PubMed ID: 34742859
In animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. The present work made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, these results suggest an important role in reproductive success.
Yang, T., Yuan, Z., Liu, C., Liu, T. and Zhang, W. (2021). A neural circuit integrates pharyngeal sensation to control feeding. Cell Rep 37(6): 109983. PubMed ID: 34758309
Swallowing is an essential step of eating and drinking. However, how the quality of a food bolus is sensed by pharyngeal neurons is largely unknown. This study finds that mechanical receptors along the Drosophila pharynx are required for control of meal size, especially for food of high viscosity. The mechanical force exerted by the bolus passing across the pharynx is detected by neurons expressing the mechanotransduction channel NOMPC (no mechanoreceptor potential C) and is relayed, together with gustatory information, to IN1 neurons in the subesophageal zone (SEZ) of the brain. IN1 (ingestion neurons) neurons act directly upstream of a group of peptidergic neurons that encode satiety. Prolonged activation of IN1 neurons suppresses feeding. IN1 neurons receive inhibition from DSOG1 (descending subesophageal neurons) neurons, a group of GABAergic neurons that non-selectively suppress feeding. These results reveal the function of pharyngeal mechanoreceptors and their downstream neural circuits in the control of food ingestion.

Wednesday, January 19th - Embryonic development

Cheatle Jarvela, A. M., Trelstad, C. S. and Pick, L. (2021). Anterior-posterior patterning of segments in Anopheles stephensi offers insights into the transition from sequential to simultaneous segmentation in holometabolous insects. J Exp Zool B Mol Dev Evol. PubMed ID: 34734470
The gene regulatory network for segmentation in arthropods offers valuable insights into how networks evolve owing to the breadth of species examined and the extremely detailed knowledge gained in the model organism Drosophila melanogaster. These studies have shown that Drosophila's network represents a derived state that acquired changes to accelerate segment patterning, whereas most insects specify segments gradually as the embryo elongates. Such heterochronic shifts in segmentation have potentially emerged multiple times within holometabolous insects, resulting in many mechanistic variants and difficulties in isolating underlying commonalities that permit such shifts. Recent studies identified regulatory genes that work as timing factors, coordinating gene expression transitions during segmentation. These studies predict that changes in timing factor deployment explain shifts in segment patterning relative to other developmental events. This study tested this hypothesis by characterizing the temporal and spatial expression of the pair-rule patterning genes in the malaria vector mosquito, Anopheles stephensi. This insect is a Dipteran (fly), like Drosophila, but represents an ancient divergence within this clade, offering a useful counterpart for evo-devo studies. In mosquito embryos, this study observed anterior to posterior sequential addition of stripes for many pair-rule genes and a wave of broad timer gene expression across this axis. Segment polarity gene stripes are added sequentially in the wake of the timer gene wave and the full pattern is not complete until the embryo is fully elongated. This "progressive segmentation" mode in Anopheles displays commonalities with both Drosophila's rapid segmentation mechanism and sequential modes used by more distantly related insects (Cheatle Jarvela, 2021).
Sanchez-Corrales, Y. E., Blanchard, G. B. and Roper, K. (2021). Correct regionalization of a tissue primordium is essential for coordinated morphogenesis. Elife 10. PubMed ID: 34723792
During organ development, tubular organs often form from flat epithelial primordia. In the placodes of the forming tubes of the salivary glands in the Drosophila embryo, previous work has identified spatially defined cell behaviors of cell wedging, tilting, and cell intercalation that are key to the initial stages of tube formation. This study addresses what the requirements are that ensure the continuous formation of a narrow symmetrical tube from an initially asymmetrical primordium whilst overall tissue geometry is constantly changing. Live-imaging and quantitative methods were used to compare wild-type placodes and mutants that either show disrupted cell behaviors or an initial symmetrical placode organization, with both resulting in severe impairment of the invagination. Early transcriptional patterning of key morphogenetic transcription factors were found to drive the selective activation of downstream morphogenetic modules, such as GPCR signaling that activates apical-medial actomyosin activity to drive cell wedging at the future asymmetrically placed invagination point. Over time, transcription of key factors expands across the rest of the placode and cells switch their behavior from predominantly intercalating to predominantly apically constricting as their position approaches the invagination pit. Misplacement or enlargement of the initial invagination pit leads to early problems in cell behaviors that eventually result in a defective organ shape. This work illustrates that the dynamic patterning of the expression of transcription factors and downstream morphogenetic effectors ensures positionally fixed areas of cell behavior with regards to the invagination point. This patterning in combination with the asymmetric geometrical setup ensures functional organ formation.
Cao, W. X., Karaiskakis, A., Lin, S., Angers, S. and Lipshitz, H. D. (2021). The F-box protein Bard (CG14317) targets the Smaug RNA-binding protein for destruction during the Drosophila maternal-to-zygotic transition. Genetics. PubMed ID: 34757425
During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug (SMG) plays an essential role in progression through the MZT by translationally repressing and destabilizing a large number of maternal mRNAs. The SMG protein itself is rapidly cleared at the end of the MZT by a Skp/Cullin/F-box (SCF) E3-ligase complex. Clearance of SMG requires zygotic transcription and is required for an orderly MZT. This study shows that an F-box protein, which was named Bard (encoded by CG14317), is required for degradation of SMG. Bard is expressed zygotically and physically interacts with SMG at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of SMG. shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of SMG protein, a phenotype that is rescued by transgenes expressing Bard. Bard thus times the clearance of SMG at the end of the MZT (Cao, 2021).
Wu, X., Kong, K., Xiao, W. and Liu, F. (2021). Attractive internuclear force drives the collective behavior of nuclear arrays in Drosophila embryos. PLoS Comput Biol 17(11): e1009605. PubMed ID: 34797833
The collective behavior of the nuclear array in Drosophila embryos during nuclear cycle (NC) 11 to NC14 is crucial in controlling cell size, establishing developmental patterns, and coordinating morphogenesis. After live imaging on Drosophila embryos with light sheet microscopy, the nuclear trajectory, speed, and internuclear distance were extract with an automatic nuclear tracing method. The nuclear speed showed a period of standing waves along the anterior-posterior (AP) axis after each metaphase as the nuclei collectively migrate towards the embryo poles and partially move back. And the maximum nuclear speed dampens by 28%-45% in the second half of the standing wave. Moreover, the nuclear density is 22-42% lower in the pole region than the middle of the embryo during the interphase of NC12-NC14. To find mechanical rules controlling the collective motion and packing patterns of the nuclear array, a deep neural network (DNN) was used to learn the underlying force field from data. The learned spatiotemporal attractive force field was applied in the simulations with a particle-based model. The simulations recapitulated nearly all the observed characteristic collective behaviors of nuclear arrays in Drosophila embryos
Girard, J. R., Goins, L. M., Vuu, D. M., Sharpley, M. S., Spratford, C. M., Mantri, S. R. and Banerjee, U. (2021). Paths and pathways that generate cell-type heterogeneity and developmental progression in hematopoiesis. Elife 10. PubMed ID: 34713801
Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single cell RNA-Seq, and genetic dissection, this study identified new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. Metabolism is highlighted as a driver of development, graded Pointed expression is shown to allow distinct roles in successive developmental steps, and mature crystal cells are shown to specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, and inhibits canonical, Notch signaling. Broadly, it was found that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.
Reed, S., Chen, W., Bergstein, V. and He, B. (2021). Toll-Dorsal signaling regulates the spatiotemporal dynamics of yolk granule tubulation during Drosophila cleavage. Dev Biol 481: 64-74. PubMed ID: 34627795
The Toll-Dorsal signaling pathway controls dorsal-ventral (DV) patterning in early Drosophila embryos, which defines specific cell fates along the DV axis and controls morphogenetic behavior of cells during gastrulation and beyond. The extent by which DV patterning information regulates subcellular organization in pre-gastrulation embryos remains unclear. This study found that during Drosophila cleavage, the late endosome marker Rab7 is increasingly recruited to the yolk granules and promotes the formation of dynamic membrane tubules. The biogenesis of yolk granule tubules is positively regulated by active Rab7 and its effector complex HOPS, but negatively regulated by the Rab7 effector retromer. The occurrence of tubules is strongly biased towards the ventral side of the embryo, which shows is controlled by the Toll-Dorsal signaling pathway. This work provides the first evidence for the formation and regulation of yolk granule tubulation in oviparous embryos and elucidates an unexpected role of Toll-Dorsal signaling in regulating this process (Reed, 2021).y

Tuesday, January 18th - Signal Transduction

Tsai, C. R., Wang, Y., Jacobson, A., Sankoorikkal, N., Chirinos, J. D., Burra, S., Makthal, N., Kumaraswami, M. and Galko, M. J. (2021). Pvr and distinct downstream signaling factors are required for hemocyte spreading and epidermal wound closure at Drosophila larval wound sites. G3 (Bethesda). PubMed ID: 34751396
Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. This study discoverd a role for the Platelet-derived growth factor (PDGF)/Vascular endothelial growth factor (VEGF)-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, a genetic screen was performed in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure, another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. These data reveals that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal wound closure or hemocyte spreading.
Soundarrajan, D. K., Huizar, F. J., Paravitorghabeh, R., Robinett, T. and Zartman, J. J. (2021). From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control. PLoS Comput Biol 17(11): e1009543. PubMed ID: 34723960
Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. This study used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. These two subpopulations were termed "initiator cells," defined by elevated levels of Phospholipase C (PLC) activity, and "standby cells," which exhibit baseline activity. The type and strength of hormonal stimulation and extent of gap junctional communication were found to jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. A computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, perturbations to Ca2+ signaling tuned the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states.
Kizhedathu, A., Chhajed, P., Yeramala, L., Sain Basu, D., Mukherjee, T., Vinothkumar, K. R. and Guha, A. (2021). Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts. Elife 10. PubMed ID: 34622778
Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. This study looks into in the mechanisms that activate ATR/Chk1. Levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H2O2 generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/Claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of H2O2 in minutes. The findings presented reveal that H2O2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism (Kizhedathu, 2021).
Sang, Q., Wang, G., Morton, D. B., Wu, H. and Xie, B. (2021). The ZO-1 protein Polychaetoid as an upstream regulator of the Hippo pathway in Drosophila. PLoS Genet 17(11): e1009894. PubMed ID: 34748546
The generation of a diversity of photoreceptor (PR) subtypes with different spectral sensitivities is essential for color vision in animals. In the Drosophila eye, the Hippo pathway has been implicated in blue- and green-sensitive PR subtype fate specification. Specifically, Hippo pathway activation promotes green-sensitive PR fate at the expense of blue-sensitive PRs. In this study, using a sensitized triple heterozygote-based genetic screening approach, the identification of the single Drosophila zonula occludens-1 (ZO-1) protein Polychaetoid (Pyd) was identified as a new regulator of the Hippo pathway during the blue- and green-sensitive PR subtype binary fate choice. Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway. Furthermore, Pyd was found to repress the activity of Su(dx), a E3 ligase that negatively regulates Pez and can physically interact with Pyd, during PR subtype fate specification. Together, these results identify a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.
Ryan, S. M., Almassey, M., Burch, A. M., Ngo, G., Martin, J. M., Myers, D., Compton, D., Archie, S., Cross, M., Naeger, L., Salzman, A., Virola-Iarussi, A., Barbee, S. A., Mortimer, N. T., Sanyal, S. and Vrailas-Mortimer, A. D. (2021). Drosophila p38 MAPK interacts with BAG-3/starvin to regulate age-dependent protein homeostasis. Aging Cell 20(11): e13481. PubMed ID: 34674371
As organisms age, they often accumulate protein aggregates that are thought to be toxic, potentially leading to age-related diseases. This accumulation of protein aggregates is partially attributed to a failure to maintain protein homeostasis. A variety of genetic factors have been linked to longevity, but how these factors also contribute to protein homeostasis is not completely understood. In order to understand the relationship between aging and protein aggregation, this study tested how a gene that regulates lifespan and age-dependent locomotor behaviors, p38 MAPK (p38Kb), influences protein homeostasis as an organism ages. p38Kb was found to regulates age-dependent protein aggregation through an interaction with starvin, a regulator of muscle protein homeostasis. Furthermore, Lamin was identified as an age-dependent target of p38Kb and starvin.
Scepanovic, G., Hunter, M. V., Kafri, R. and Fernandez-Gonzalez, R. (2021). p38-mediated cell growth and survival drive rapid embryonic wound repair. Cell Rep 37(3): 109874. PubMed ID: 34686334
Embryos repair wounds rapidly, with no inflammation or scarring, in a process that involves polarization of the actomyosin cytoskeleton. Actomyosin polarization results in the assembly of a contractile cable around the wound that drives wound closure. This study demonstrates that a contractile actomyosin cable is not sufficient for rapid wound repair in Drosophila embryos. It was shown that wounding causes activation of the serine/threonine kinase p38 mitogen-activated protein kinase (MAPK) in the cells adjacent to the wound. p38 activation reduces the levels of wound-induced reactive oxygen species in the cells around the wound, limiting wound size. In addition, p38 promotes an increase in volume in the cells around the wound, thus facilitating the collective cell movements that drive rapid wound healing. These data indicate that p38 regulates cell volumes through the sodium-potassium-chloride cotransporter NKCC1. This work reveals cell growth and cell survival as cell behaviors critical for embryonic wound repair.

Monday, January 17th - Adult development

Jang, S., Chen, J., Choi, J., Lim, S. Y., Song, H., Choi, H., Kwon, H. W., Choi, M. S. and Kwon, J. Y. (2021). Spatiotemporal organization of enteroendocrine peptide expression in Drosophila. J Neurogenet: 1-12. PubMed ID: 34670462
The digestion of food and absorption of nutrients occurs in the gut. The nutritional value of food and its nutrients is detected by enteroendocrine cells, and peptide hormones produced by the enteroendocrine cells are thought to be involved in metabolic homeostasis, but the specific mechanisms are still elusive. The enteroendocrine cells are scattered over the entire gastrointestinal tract and can be classified according to the hormones they produce. This study followed the changes in combinatorial expression of regulatory peptides in the enteroendocrine cells during metamorphosis from the larva to the adult fruit fly, and re-confirmed the diverse composition of enteroendocrine cell populations. Drosophila enteroendocrine cells appear to differentially regulate peptide expression spatially and temporally depending on midgut region and developmental stage. In the late pupa, Notch activity is known to determine which peptides are expressed in mature enteroendocrine cells of the posterior midgut; it was found that the loss of Notch activity in the anterior midgut results in classes of enteroendocrine cells distinct from the posterior midgut. These results suggest that enteroendocrine cells that populate the fly midgut can differentiate into distinct subtypes that express different combinations of peptides, which likely leads to functional variety depending on specific needs.
Boulet, M., Renaud, Y., Lapraz, F., Benmimoun, B., Vandel, L. and Waltzer, L. (2021). Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential. Front Cell Dev Biol 9: 739357. PubMed ID: 34722521
While many studies have described Drosophila embryonic and larval blood cells, the hematopoietic system of the imago remains poorly characterized and conflicting data have been published concerning adult hematopoiesis. Using a combination of blood cell markers, This study shows that the adult hematopoietic system is essentially composed of a few distinct mature blood cell types. In addition, the transcriptomics results indicate that adult and larval blood cells have both common and specific features and it appears that adult hemocytes reactivate many genes expressed in embryonic blood cells. Interestingly, this study identify a small set of blood cells that does not express differentiation markers but rather maintains the expression of the progenitor marker domeMeso. Yet, this study shows that these cells are derived from the posterior signaling center, a specialized population of cells present in the larval lymph gland, rather than from larval blood cell progenitors, and that their maintenance depends on the EBF transcription factor Collier. Furthermore, while these cells are normally quiescent, this study found that some of them can differentiate and proliferate in response to bacterial infection. In sum, the results indicate that adult flies harbor a small population of specialized cells with limited hematopoietic potential and further support the idea that no substantial hematopoiesis takes place during adulthood (Boulet, 2021).
Fraire-Zamora, J. J., Tosi, S., Solon, J. and Casanova, J. (2021). Control of hormone-driven organ disassembly by ECM remodeling and Yorkie-dependent apoptosis. Curr Biol. PubMed ID: 34666006.
Epithelia grow and shape into functional structures during organogenesis. Although most of the focus on organogenesis has been drawn to the building of biological structures, the disassembly of pre-existing structures is also an important event to reach a functional adult organ. Examples of disassembly processes include the regression of the Mullerian or Wolffian ducts during gonad development and mammary gland involution during the post-lactational period in adult females. To date, it is unclear how organ disassembly is controlled at the cellular level. This study follows the Drosophila larval trachea through metamorphosis and shows that its disassembly is a hormone-driven and precisely orchestrated process. It occurs in two phases: first, remodeling of the apical extracellular matrix (aECM), mediated by matrix metalloproteases and independent of the actomyosin cytoskeleton, results in a progressive shortening of the entire trachea and a nuclear-to-cytoplasmic relocalization of the Hippo effector Yorkie (Yki). Second, a decreased transcription of the Yki target, Diap1, in the posterior metameres and the activation of caspases result in the apoptotic loss of the posterior half of the trachea while the anterior half escapes cell death. Thus, this work unravels a mechanism by which hormone-driven ECM remodeling controls sequential tissue shortening and apoptotic cell removal through the transcriptional activity of Yki, leading to organ disassembly during animal development.
Togel, M., Pass, G. and Paululat, A. (2021). Wing Hearts in Four-Winged Ultrabithorax-mutant Flies-the role of Hox genes in wing heart specification. Genetics. PubMed ID: 34791231
Wings are probably the most advanced evolutionary novelty in insects. The development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginal ecdysis, these accessory circulatory organs remove haemolymph and apoptotic epidermal cells from the premature wing through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant Drosophila that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later thoracic segments T2 and T3. However, the adult dipterian fly harbors only one pair of wing hearts and also only one pair of wings located in thoracic segment T2. This study shows, that the specification of wing heart progenitors depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, the development of four wing hearts was analyzed in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment (T3) is transformed into a second thoracic segment (HT2). This results in a second pair of wings instead of the club-shaped halteres normally formed by T3. This study shows that a second pair of wild-type wing hearts is formed in the four-winged fly and that all wing hearts originate from the wild-type progenitor cells.
Graca, F. A., Sheffield, N., Puppa, M., Finkelstein, D., Hunt, L. C. and Demontis, F. (2021). A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila. PLoS Genet 17(11): e1009926. PubMed ID: 34780463
Myofiber atrophy occurs with aging and in many diseases but the underlying mechanisms are incompletely understood. This study used >1,100 muscle-targeted RNAi interventions to comprehensively assess the function of 447 transcription factors in the developmental growth of body wall skeletal muscles in Drosophila. This screen identifies new regulators of myofiber atrophy and hypertrophy, including the transcription factor Deaf1. Deaf1 RNAi increases myofiber size whereas Deaf1 overexpression induces atrophy. Consistent with its annotation as a Gsk3 phosphorylation substrate, Deaf1 and Gsk3 induce largely overlapping transcriptional changes that are opposed by Deaf1 RNAi. The top category of Deaf1-regulated genes consists of glycolytic enzymes, which are suppressed by Deaf1 and Gsk3 but are upregulated by Deaf1 RNAi. Similar to Deaf1 and Gsk3 overexpression, RNAi for glycolytic enzymes reduces myofiber growth. Altogether, this study defines the repertoire of transcription factors that regulate developmental myofiber growth and the role of Gsk3/Deaf1/glycolysis in this process.
Marcetteau, J., Matusek, T., Luton, F. and Therond, P. P. (2021). Arf6 is necessary for senseless expression in response to Wingless signalling during Drosophila wing development. Biol Open. PubMed ID: 34779478
Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, this study analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. A dominant loss of wing margin bristles and Senseless expression was observed in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, this study showa that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. These data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. These findings represent a novel regulatory role for Arf6 in Wingless signalling.

Friday, January 14th - Methods

Chin, A. and Lecuyer, E. (2021). Puromycin Labeling Coupled with Proximity Ligation Assays to Define Sites of mRNA Translation in Drosophila Embryos and Human Cells. Methods Mol Biol 2381: 267-284. PubMed ID: 34590282
Genetic mutations, whether they occur within protein-coding or noncoding regions of the genome, can affect various aspects of gene expression by influencing the complex network of intra- and intermolecular interactions that occur between cellular nucleic acids and proteins. One aspect of gene expression control that can be impacted is the intracellular trafficking and translation of mRNA molecules. To study the occurrence and dynamics of translational regulation, researchers have developed approaches such as genome-wide ribosome profiling and artificial reporters that enable single molecule imaging. This paper describes a complementary and optimized approach that combines puromycin labeling with a proximity ligation assay (Puro-PLA) to define sites of translation of specific mRNAs in tissues or cells. This method can be used to study the mechanisms driving the translation of select mRNAs and to access the impact of genetic mutations on local protein synthesis. This approach involves the treatment of cell or tissue specimens with puromycin to label nascently translated peptides, rapid fixation, followed by immunolabeling with appropriate primary and secondary antibodies coupled to PLA oligonucleotide probes, ligation, amplification, and signal detection via fluorescence microscopy. Puro-PLA can be performed at small scale in individual tubes or in chambered slides, or in a high-throughput setup with 96-well plate, for both in situ and in vitro experimentation.
Jay, T. R., Kang, Y., Jefferson, A. and Freeman, M. R. (2021). An ELISA-based method for rapid genetic screens in Drosophila. Proc Natl Acad Sci U S A 118(43). PubMed ID: 34686600
Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines, which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. This study developed a GFP-based enzyme-linked immunosorbent assay (ELISA) that can detect GFP in Drosophila lysates collected from whole animals and dissected tissues across all stages of Drosophila development. This assay can detect membrane-localized GFP in a variety of neuronal and glial populations and validates that it can identify genes that change the morphology of these cells, as well as changes in STAT and JNK transcriptional activity. This assay can detect endogenously GFP-tagged proteins, including Draper, Cryptochrome, and the synaptic marker Brp. This approach is able to detect changes in Brp-GFP signal during developmental synaptic remodeling, and known genetic regulators of glial synaptic engulfment could be identified using this ELISA method. Finally, the assay was used to perform a small-scale screen, which identified Syntaxins as potential regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy, and quantitative in vivo screening method that can be used to assay a wide breadth of fundamental biological questions.
Li, T. and Luo, L. (2021). An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila. J Vis Exp(176). PubMed ID: 34723938
Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.
Niu, M., Zhang, X., Li, W., Wang, J. and Li, Y. (2021). dFRAME: A Video Recording-Based Analytical Method for Studying Feeding Rhythm in Drosophila. Front Genet 12: 763200. PubMed ID: 34721548
Animals, from insects to humans, exhibit obvious diurnal rhythmicity of feeding behavior. Serving as a genetic animal model, Drosophila has been reported to display feeding rhythms; however, related investigations are limited due to the lack of suitable and practical methods. This study presents a video recording-based analytical method, namely, Drosophila Feeding Rhythm Analysis Method (dFRAME). Using this newly developed computer program, FlyFeeding, the movement track of individual flies was extracted, and their food-approaching behavior was characterized. To distinguish feeding and no-feeding events, high-magnification video recording was used to optimize the method by setting cut-off thresholds to eliminate the interference of no-feeding events. Furthermore, it was verified that this method is applicable to both female and male flies and for all periods of the day. Using this method, long-term feeding status of wild-type and period mutant flies was analyzed. The results recaptured previously reported feeding rhythms and revealed detailed profiles of feeding patterns in these flies under either light/dark cycles or constant dark environments. Together, the dFRAME method enables a long-term, stable, reliable, and subtle analysis of feeding behavior in Drosophila. High-throughput studies in this powerful genetic animal model will gain great insights into the molecular and neural mechanisms of feeding rhythms.
ElMaghraby, M. F., Tirian, L., Senti, K. A., Meixner, K. and Brennecke, J. (2021). A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary. Genetics 220(1):iyab179. PubMed ID: 34718559.
Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. This study establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. Constitutive, pan-germline RNAi was combined with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows investigation of the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. This work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.
Crocker, K. L., Ahern-Djamali, S. and Boekhoff-Falk, G. (2021). Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain. J Vis Exp(176). PubMed ID: 34694294
The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described it this study now make adult Drosophila brain a robust model for neural regeneration research. Provided hin this paper are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Thursday, December 13th - Evolution

Nozawa, M., Minakuchi, Y., Satomura, K., Kondo, S., Toyoda, A. and Tamura, K. (2021). Shared evolutionary trajectories of three independent neo-sex chromosomes in Drosophila. Genome Res 31(11): 2069-2079. PubMed ID: 34675069
Dosage compensation (DC) on the X Chromosome counteracts the deleterious effects of gene loss on the Y Chromosome. However, DC is not efficient if the X Chromosome also degenerates. This indeed occurs in Drosophila miranda, in which both the neo-Y and the neo-X are under accelerated pseudogenization. To examine the generality of this pattern, this study investigated the evolution of two additional neo-sex chromosomes that emerged independently in D. albomicans and D. americana, and neo-sex chromosome evolution in D. miranda Comparative genomic and transcriptomic analyses revealed that the pseudogenization rate on the neo-X is also accelerated in D. albomicans and D. americana although to a lesser extent than in D. miranda. In males, neo-X-linked genes whose neo-Y-linked homologs are pseudogenized tended to be up-regulated more than those whose neo-Y-linked homologs remain functional. Moreover, genes under strong functional constraint and genes highly expressed in the testis tended to remain functional on the neo-X and neo-Y, respectively. Focusing on the D. miranda and D. albomicans neo-sex chromosomes that emerged independently from the same autosome, it was further found that the same genes tend to become pseudogenized in parallel on the neo-Y. These genes include Idgf6 and JhI-26, which may be unnecessary or even harmful in males. These results indicate that neo-sex chromosomes in Drosophila share a common evolutionary trajectory after their emergence, which may prevent sex chromosomes from being an evolutionary dead end.
Sprengelmeyer, Q. D. and Pool, J. E. (2021). Ethanol resistance in Drosophila melanogaster has increased in parallel cold-adapted populations and shows a variable genetic architecture within and between populations. Ecol Evol 11(21): 15364-15376. PubMed ID: 34765183
Understanding the genetic properties of adaptive trait evolution is a fundamental crux of biological inquiry that links molecular processes to biological diversity. Important uncertainties persist regarding the genetic predictability of adaptive trait change, the role of standing variation, and whether adaptation tends to result in the fixation of favored variants. This study used the recurrent evolution of enhanced ethanol resistance in Drosophila melanogaster during this species' worldwide expansion as a promising system to add to understanding of the genetics of adaptation. Elevated ethanol resistance was found to have evolved at least three times in different cooler regions of the species' modern range-not only at high latitude but also in two African high-altitude regions. Applying a bulk segregant mapping framework, this study found that the genetic architecture of ethanol resistance evolution differs substantially not only between the three resistant populations, but also between two crosses involving the same European population. Population genetic scans were applied for local adaptation within the quantitative trait locus regions, and potential contributions were found of genes with annotated roles in spindle localization, membrane composition, sterol and alcohol metabolism, and other processes. Simulation-based analyses were appleid that confirm the variable genetic basis of ethanol resistance and hint at a moderately polygenic architecture. However, these simulations indicate that larger-scale studies will be needed to more clearly quantify the genetic architecture of adaptive evolution and to firmly connect trait evolution to specific causative loci.
Rosser, N., Edelman, N. B., Queste, L. M., Nelson, M., Seixas, F., Dasmahapatra, K. K. and Mallet, J. (2021). Complex basis of hybrid female sterility and Haldane's rule in Heliconius butterflies: Z-linkage and epistasis. Mol Ecol. PubMed ID: 34779079
Hybrids between species are often sterile or inviable. Hybrid unfitness usually evolves first in the heterogametic sex - a pattern known as Haldane's rule. The genetics of Haldane's Rule have been extensively studied in species where the male is the heterogametic (XX/XY) sex, but its basis in taxa where the female is heterogametic (ZW/ZZ), such as Lepidoptera and birds, is largely unknown. This study analysed a new case of female hybrid sterility between geographic subspecies of Heliconius pardalinus. The two subspecies mate freely in captivity, but female F1 hybrids in both directions of cross are sterile. Sterility is due to arrested development of oocytes after they become differentiated from nurse cells, but before yolk deposition. Fertile male F1 hybrids were crossed to parental females, and quantitative trait loci (QTLs) were mapped for female sterility. Genes differentially expressed in the ovary and as a function of oocyte development were also identified. The Z chromosome has a major effect, similar to the "large X effect" in Drosophila, with strong epistatic interactions between loci at either end of the Z chromosome, and between the Z chromosome and auto somal loci on chromosomes 8 and 20. By intersecting the list of genes within these QTLs with those differentially expressed in sterile and fertile hybrids, three candidate genes were identified with relevant phenotypes. This study is the first to characterize hybrid sterility using genome mapping in the Lepidoptera, and shows that it is produced by multiple complex epistastic interactions often involving the sex chromosome, as predicted by the dominance theory of Haldane's Rule.
Ruzicka, F., Connallon, T. and Reuter, M. (2021). Sex differences in deleterious mutational effects in Drosophila melanogaster: combining quantitative and population genetic insights. Genetics 219(3). PubMed ID: 34740242
Fitness effects of deleterious mutations can differ between females and males due to (1) sex differences in the strength of purifying selection, and (2) sex differences in ploidy. Although sex differences in fitness effects have important broader implications (e.g., for the evolution of sex and lifespan), few studies have quantified their scope. Those that have belong to one of two distinct empirical traditions: (1) quantitative genetics, which focusses on multi-locus genetic variances in each sex, but is largely agnostic about their genetic basis, and (2) molecular population genetics, which focusses on comparing autosomal and X-linked polymorphism, but is poorly suited for inferring contemporary sex differences. This study combined both traditions to present a comprehensive analysis of female and male adult reproductive fitness among 202 outbred, laboratory-adapted, hemiclonal genomes of Drosophila melanogaster. While no clear evidence was found for sex differences in the strength of purifying selection, sex differences in ploidy generate multiple signals of enhanced purifying selection for X-linked loci. These signals are present in quantitative genetic metrics: i.e., a disproportionate contribution of the X to male (but not female) fitness variation and population genetic metrics (i.e., steeper regressions of an allele's average fitness effect on its frequency, and proportionally less nonsynonymous polymorphism on the X than autosomes). Fitting these data to models for both sets of metrics, it was inferred that deleterious alleles are partially recessive. Given the often large gap between quantitative and population genetic estimates of evolutionary parameters, this study showcases the benefits of combining genomic and fitness data when estimating such parameters.
Prieto-Godino, L. L., Schmidt, H. R. and Benton, R. (2021). Molecular reconstruction of recurrent evolutionary switching in olfactory receptor specificity. Elife 10. PubMed ID: 34677122
Olfactory receptor repertoires exhibit remarkable functional diversity, but how these proteins have evolved is poorly understood. Through analysis of extant and ancestrally reconstructed drosophilid olfactory receptors from the Ionotropic receptor (Ir) family, this study investigated evolution of two organic acid-sensing receptors, Ir75a and Ir75b. Despite their low amino acid identity, this study identified a common 'hotspot' in their ligand-binding pocket that has a major effect on changing the specificity of both Irs, as well as at least two distinct functional transitions in Ir75a during evolution. Moreover, this study showed that odor specificity is refined by changes in additional, receptor-specific sites, including those outside the ligand-binding pocket. This work reveals how a core, common determinant of ligand-tuning acts within epistatic and allosteric networks of substitutions to lead to functional evolution of olfactory receptors.
Montino, A., Balakrishnan, K., Dippel, S., Trebels, B., Neumann, P. and Wimmer, E. A. (2021). Mutually Exclusive Expression of Closely Related Odorant-Binding Proteins 9A and 9B in the Antenna of the Red Flour Beetle Tribolium castaneum. Biomolecules 11(10). PubMed ID: 34680135
Olfaction is crucial for insects to find food sources, mates, and oviposition sites. One of the initial steps in olfaction is facilitated by odorant-binding proteins (OBPs) that translocate hydrophobic odorants through the aqueous olfactory sensilla lymph to the odorant receptor complexes embedded in the dendritic membrane of olfactory sensory neurons. The Tribolium OBPs encoded by the gene pair TcasOBP9A and TcasOBP9B represent the closest homologs to Drosophila OBP Lush, which mediates pheromone reception. These two OBPs were shown not to be pheromone-specific but rather enhance the detection of a broad spectrum of organic volatiles. Both OBPs are expressed in the antenna but in a mutually exclusive pattern. A phylogenetic analysis indicates that this gene pair arose at the base of the Cucujiformia, which dates the gene duplication event to about 200 million years ago. Therefore, the high sequence conservation in spite of their expression in different sensilla is potentially the result of a common function as co-OBPs.

Wednesday January 12th - Adult physiology and metabolism

Ma, R., Haji-Ghassemi, O., Ma, D., Jiang, H., Lin, L., Xu, T., Murayama, T., Moussian, B., Van Petegem, F. and Yuchi, Z. (2022). Structural basis for diamide modulation of ryanodine receptor. J Gen Physiol 154(9). PubMed ID: 34766989
Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, the cryo-electron microscopy (cryo-EM) structure of RYR was solved in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo-voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. It was also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, the crystal structures of several RYR domains were solved from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, it was found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. These structures provide a foundation for developing novel pesticides to overcome the resistance crisis.
Ramnarine, T. J. S., Grath, S. and Parsch, J. (2021). Natural variation in the transcriptional response of Drosophila melanogaster to oxidative stress. G3 (Bethesda). PubMed ID: 34747443
Broadly distributed species must cope with diverse and changing environmental conditions, including various forms of stress. Cosmopolitan populations of Drosophila melanogaster are more tolerant to oxidative stress than those from the species' ancestral range in sub-Saharan Africa, and the degree of tolerance is associated with an insertion/deletion polymorphism in the 3' untranslated region of the Metallothionein A (MtnA) gene that varies clinally in frequency. Oxidative stress tolerance and the transcriptional response to oxidative stress were examined in cosmopolitan and sub-Saharan African populations of D. melanogaster, including paired samples with allelic differences at the MtnA locus. The effect of the MtnA polymorphism on oxidative stress tolerance was found to be dependent on the genomic background, with the deletion allele increasing tolerance only in a northern, temperate population. Genes that were differentially expressed under oxidative stress included MtnA and other metallothioneins, as well as those involved in glutathione metabolism and other genes known to be part of the oxidative stress response or the general stress response. A gene coexpression analysis revealed further genes and pathways that respond to oxidative stress including those involved in additional metabolic processes, autophagy, and apoptosis. There was a significant overlap among the genes induced by oxidative and cold stress, which suggests a shared response pathway to these two stresses. Interestingly, the MtnA deletion was associated with consistent changes in the expression of many genes across all genomic backgrounds, regardless of the expression level of the MtnA gene itself. It is hypothesize that this is an indirect effect driven by the loss of microRNA binding sites within the MtnA 3' untranslated region.
Manenti, T., Kjærsgaard, A., Schou, T. M., Pertoldi, C., Moghadam, N. N. and Loeschcke, V. (2021). Responses to Developmental Temperature Fluctuation in Life History Traits of Five Drosophila Species (Diptera: Drosophilidae) from Different Thermal Niches. Insects 12(10). PubMed ID: 34680694
Temperature has profound effects on biochemical processes as suggested by the extensive variation in performance of organisms across temperatures. Nonetheless, the use of fluctuating temperature (FT) regimes in laboratory experiments compared to constant temperature (CT) regimes is still mainly applied in studies of model organisms. This study investigated how two amplitudes of developmental temperature fluctuation (22.5/27.5 °C and 20/30 °C, 12/12 h) affected several fitness-related traits in five Drosophila species with markedly different thermal resistance. Egg-to-adult viability did not change much with temperature except in the cold-adapted D. immigrans. Developmental time increased with FT among all species compared to the same mean CT. The impact of FT on wing size was quite diverse among species. Whereas wing size decreased quasi-linearly with CT in all species, there were large qualitative differences with FT. Changes in wing aspect ratio due to FT were large compared to the other traits and presumably a consequence of thermal stress. These results demonstrate that species of the same genus but with different thermal resistance can show substantial differences in responses to fluctuating developmental temperatures not predictable by constant developmental temperatures. Testing multiple traits facilitated the interpretation of responses to FT in a broader context.
Rivera, M. J., Contreras, A., Nguyen, L. T., Eldon, E. D. and Klig, L. S. (2021). Regulated inositol synthesis is critical for balanced metabolism and development in Drosophila melanogaster. Biol Open 10(10). PubMed ID: 34710213
Myo-inositol is a precursor of the membrane phospholipid, phosphatidylinositol (PI). It is involved in many essential cellular processes including signal transduction, energy metabolism, endoplasmic reticulum stress, and osmoregulation. Inositol is synthesized from glucose-6-phosphate by myo-inositol-3-phosphate synthase (MIPSp). The Drosophila melanogaster Inos gene encodes MIPSp. Abnormalities in myo-inositol metabolism have been implicated in type 2 diabetes, cancer, and neurodegenerative disorders. Obesity and high blood (hemolymph) glucose are two hallmarks of diabetes, which can be induced in Drosophila melanogaster third-instar larvae by high-sucrose diets. This study shows that dietary inositol reduces the obese-like and high-hemolymph glucose phenotypes of third-instar larvae fed high-sucrose diets. Furthermore, this study demonstrates Inos mRNA regulation by dietary inositol; when more inositol is provided there is less Inos mRNA. Third-instar larvae with dysregulated high levels of Inos mRNA and MIPSp show dramatic reductions of the obese-like and high-hemolymph glucose phenotypes. These strains, however, also display developmental defects and pupal lethality. The few individuals that eclose die within two days with striking defects: structural alterations of the wings and legs, and heads lacking proboscises. This study is an exciting extension of the use of Drosophila melanogaster as a model organism for exploring the junction of development and metabolism.
de Brito Sanchez, G., Exposito Munoz, A., Chen, L., Huang, W., Su, S. and Giurfa, M. (2021). Adipokinetic hormone (AKH), energy budget and their effect on feeding and gustatory processes of foraging honey bees. Sci Rep 11(1): 18311. PubMed ID: 34526585
The adipokinetic hormone (AKH) of insects is considered an equivalent of the mammalian hormone glucagon as it induces fast mobilization of carbohydrates and lipids from the fat body upon starvation. Yet, in foraging honey bees, which lack fat body storage for carbohydrates, it was suggested that AKH may have lost its original function. This study manipulated the energy budget of bee foragers to determine the effect of AKH on appetitive responses. As AKH participates in a cascade leading to acceptance of unpalatable substances in starved Drosophila, its effect on foragers presented with sucrose solution spiked with salicin was also assessed. Starved and partially-fed bees were topically exposed with different doses of AKH to determine if this hormone modifies food ingestion and sucrose responsiveness. A significant effect of the energy budget (i.e. starved vs. partially-fed) was found on the decision to ingest or respond to both pure sucrose solution and sucrose solution spiked with salicin, but no effect of AKH per se. These results are consistent with a loss of function of AKH in honey bee foragers, in accordance with a social life that implies storing energy resources in the hive, in amounts that exceed individual needs.
Lee, S., Jeon, Y. M., Jo, M. and Kim, H. J. (2021). Overexpression of SIRT3 Suppresses Oxidative Stress-induced Neurotoxicity and Mitochondrial Dysfunction in Dopaminergic Neuronal Cells. Exp Neurobiol 30(5): 341-355. PubMed ID: 34737239
Sirtuin 3 (SIRT3), a well-known mitochondrial deacetylase, is involved in mitochondrial function and metabolism under various stress conditions. This study found that the expression of SIRT3 was markedly increased by oxidative stress in dopaminergic neuronal cells. In addition, SIRT3 overexpression enhanced mitochondrial activity in differentiated SH-SY5Y cells. SIRT3 overexpression was shown to attenuated rotenoneor H(2)O(2)-induced toxicity in differentiated SH-SY5Y cells (human dopaminergic cell line). It wa further found that knockdown of SIRT3 enhanced rotenone- or H(2)O(2)-induced toxicity in differentiated SH-SY5Y cells. Moreover, overexpression of SIRT3 mitigated cell death caused by LPS/IFN-γ stimulation in astrocytes. The rotenone treatment was found to increase the level of SIRT3 in Drosophila brain. Downregulation of sirt2 (Drosophila homologue of SIRT3) significantly accelerated the rotenone-induced toxicity in flies. Taken together, these findings suggest that the overexpression of SIRT3 mitigates oxidative stress-induced cell death and mitochondrial dysfunction in dopaminergic neurons and astrocytes.

Tuesday, January 11th - Cytoskeleton and Junctions

Ready, D. F. and Chang, H. C. (2021). Calcium waves facilitate and coordinate the contraction of endfeet actin stress fibers in Drosophila interommatidial cells. Development. PubMed ID: 34698814
Actomyosin contraction shapes the Drosophila eye's panoramic view. The convex curvature of the retinal epithelium, organized in ∼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. This study discovered a novel phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC waves are independent of phototransduction, but require inositol 1,4,5-triphosphate receptor (IP3R), suggesting these waves are mediated by Ca2+ releases from ER stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by ∼40%, linking IOC waves to facilitating stress fiber contraction and floor morphogenesis. Further, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating ECM and its interaction with stress fibers in eye morphogenesis. It is proposed that coordinated cytosolic Ca2+ increases in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor.
Lamb, M. C., Kaluarachchi, C. P., Lansakara, T. I., Mellentine, S. Q., Lan, Y., Tivanski, A. V. and Tootle, T. L. (2021). Fascin limits Myosin activity within Drosophila border cells to control substrate stiffness and promote migration. Elife 10. PubMed ID: 34698017
A key regulator of collective cell migrations, which drive development and cancer metastasis, is substrate stiffness. Increased substrate stiffness promotes migration and is controlled by Myosin. Using Drosophila border cell migration as a model of collective cell migration, this study identified that the actin bundling protein Fascin limits Myosin activity in vivo. Loss of Fascin results in increased activated Myosin on the border cells and their substrate, the nurse cells; decreased border cell Myosin dynamics; and increased nurse cell stiffness as measured by atomic force microscopy. Reducing Myosin restores on-time border cell migration in fascin mutant follicles. Further, Fascin's actin bundling activity is required to limit Myosin activation. Surprisingly, this study found that Fascin regulates Myosin activity in the border cells to control nurse cell stiffness to promote migration. Thus, these data shift the paradigm from a substrate stiffness-centric model of regulating migration, to reveal that collectively migrating cells play a critical role in controlling the mechanical properties of their substrate in order to promote their own migration. This understudied means of mechanical regulation of migration is likely conserved across contexts and organisms, as Fascin and Myosin are common regulators of cell migration.
Collins, M. A., Coon, L. A., Thomas, R., Mandigo, T. R., Wynn, E. and Folker, E. S. (2021). Ensconsin-dependent changes in microtubule organization and LINC complex-dependent changes in nucleus-nucleus interactions result in quantitatively distinct myonuclear positioning defects. Mol Biol Cell: mbcE21060324. PubMed ID: 34524872
Nuclear movement is a fundamental process of eukaryotic cell biology. Skeletal muscle presents an intriguing model to study nuclear movement because its development requires the precise positioning of multiple nuclei within a single cytoplasm. Furthermore, there is a high correlation between aberrant nuclear positioning and poor muscle function. Although many genes that regulate nuclear movement have been identified, the mechanisms by which these genes act is not known. Using Drosophila melanogaster muscle development as a model system, and a combination of live-embryo microscopy and laser ablation of nuclei, this study found that clustered nuclei encompass at least two phenotypes that are caused by distinct mechanisms. Specifically, Ensconsin is necessary for productive force production to drive any movement of nuclei whereas Bocksbeutel and Klarsicht are necessary to form distinct populations of nuclei that move to different cellular locations. Mechanistically, Ensconsin regulates the number of growing microtubules that are used to move nuclei whereas Bocksbeutel and Klarsicht regulate interactions between nuclei.
Kim, J. Y., Tsogtbaatar, O. and Cho, K. O. (2021). Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster. J Dev Biol 9(4). PubMed ID: 34698231
Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. This study identified Dhc64Cm115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. It is proposed that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development.
Oon, C. H. and Prehoda, K. E. (2021). Phases of cortical actomyosin dynamics coupled to the neuroblast polarity cycle. Elife 10. PubMed ID: 34779402
The Par complex dynamically polarizes to the apical cortex of asymmetrically dividing Drosophila neuroblasts where it directs fate determinant segregation. Previously it was shown that apically directed cortical movements that polarize the Par complex require F-actin. This paper report the discovery of cortical actomyosin dynamics that begin in interphase when the Par complex is cytoplasmic but ultimately become tightly coupled to cortical Par dynamics. Interphase cortical actomyosin dynamics are unoriented and pulsatile but rapidly become sustained and apically-directed in early mitosis when the Par protein aPKC accumulates on the cortex. Apical actomyosin flows drive the coalescence of aPKC into an apical cap that is depolarized in anaphase when the flow reverses direction. Together with the previously characterized role of anaphase flows in specifying daughter cell size asymmetry, the results indicate that multiple phases of cortical actomyosin dynamics regulate asymmetric cell division.
Perez-Vale, K. Z., Yow, K. D., Johnson, R. I., Byrnes, A. E., Finegan, T. M., Slep, K. C. and Peifer, M. (2021). Multivalent interactions make adherens junction-cytoskeletal linkage robust during morphogenesis. J Cell Biol 220(12). PubMed ID: 34762121
Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, this study defined molecular mechanisms mediating junction-cytoskeletal linkage and explores the role of mechanosensing. Focus was placed on the junction-cytoskeletal linker Canoe, a multidomain protein. The canoe locus was engineered to define how its domains mediate its mechanism of action. Surprising, the PDZ and FAB domains, which were thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

Monday, January 10th - Adult neural development and function

Liu, Y., Li, Q., Tang, C., Qin, S. and Tu, Y. (2021). Short-Term Plasticity Regulates Both Divisive Normalization and Adaptive Responses in Drosophila Olfactory System.. Front Comput Neurosci 15: 730431. PubMed ID: 34744674
In Drosophila, olfactory information received by olfactory receptor neurons (ORNs) is first processed by an incoherent feed forward neural circuit in the antennal lobe (AL) that consists of ORNs (input), inhibitory local neurons (LNs), and projection neurons (PNs). This "early" olfactory information processing has two important characteristics. First, response of a PN to its cognate ORN is normalized by the overall activity of other ORNs, a phenomenon termed "divisive normalization." Second, PNs respond strongly to the onset of ORN activities, but they adapt to prolonged or continuously varying inputs. Despite the importance of these characteristics for learning and memory, their underlying mechanisms are not fully understood. This study developed a circuit model for describing the ORN-LN-PN dynamics by including key neuron-neuron interactions such as short-term plasticity (STP) and presynaptic inhibition (PI). By fitting this model to experimental data quantitatively, it was shown that a strong STP balanced between short-term facilitation (STF) and short-term depression (STD) is responsible for the observed nonlinear divisive normalization in Drosophila. This circuit model suggests that either STP or PI alone can lead to adaptive response. However, by comparing the model results with experimental data, it was found that both STP and PI work together to achieve a strong and robust adaptive response. This model not only helps reveal the mechanisms underlying two main characteristics of the early olfactory process, it can also be used to predict PN responses to arbitrary time-dependent signals and to infer microscopic properties of the circuit (such as the strengths of STF and STD) from the measured input-output relation. This circuit model may be useful for understanding the role of STP in other sensory systems.
Ormerod, K. G., Scibelli, A. E. and Littleton, J. T. (2021). Regulation of excitation-contraction coupling at the Drosophila neuromuscular junction. J Physiol.. PubMed ID: 34788476
Larval muscle contraction force increases with stimulation frequency and duration, revealing substantial plasticity between 5 and 40 Hz. Fictive contraction recordings demonstrate endogenous motoneuron burst frequencies consistent with the neuromuscular system operating within the range of greatest plasticity. Genetic and pharmacological manipulation of critical components of pre- and post-synaptic Ca(2+) regulation significantly impact the strength and time-course of muscle contractions. A screen for modulators of the excitation-contraction machinery identified a FMRFa peptide, TPAEDFMRFa, and its associated signaling pathway that dramatically increases muscle performance. Drosophila serves as an excellent model for dissecting components of the excitation-contraction coupling machinery. This study developed and used a force transducer system to characterize excitation-contraction coupling at Drosophila larval neuromuscular junctions (NMJs), examining how specific neuronal and muscle manipulations disrupt muscle contractility. Muscle contraction force increased with motoneuron stimulation frequency and duration, showing considerable plasticity between 5-40 Hz and saturating above 50 Hz. Endogenous recordings of fictive contractions revealed average motoneuron burst frequencies of 20-30 Hz, consistent with the system operating within this plastic range of contractility. Temperature was also a key factor in muscle contractility, as force was enhanced at lower temperatures and dramatically reduced with increasing temperatures. Pharmacological and genetic manipulations of critical components of Ca(2+) regulation in both pre- and post-synaptic compartments impacted the strength and time-course of muscle contractions. A screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation, and modulation, to alterations in excitation-contraction coupling.
Manzanero-Ortiz, S., de Torres-Jurado, A., Hernandez-Rojas, R. and Carmena, A. (2021). Pilot RNAi Screen in Drosophila Neural Stem Cell Lineages to Identify Novel Tumor Suppressor Genes Involved in Asymmetric Cell Division. Int J Mol Sci 22(21). PubMed ID: 34768763
A connection between compromised asymmetric cell division (ACD) and tumorigenesis was proven some years ago using Drosophila larval brain neural stem cells, called neuroblasts (NBs), as a model system. Since then, it has been learned that compromised ACD does not always promote tumorigenesis, as ACD is an extremely well-regulated process in which redundancy substantially overcomes potential ACD failures. Considering this, a pilot RNAi screen was performed in Drosophila larval brain NB lineages using Ras(V)(12) scribble (scrib) mutant clones as a sensitized genetic background, in which ACD is affected but does not cause tumoral growth. First, as a proof of concept, this study has tested known ACD regulators in this sensitized background, such as lethal (2) giant larvae and warts. Although the downregulation of these ACD modulators in NB clones does not induce tumorigenesis, their downregulation along with Ras(V)(12) scrib does cause tumor-like overgrowth. Based on these results, 79 RNAi lines randomly screened detecting 15 potential novel ACD regulators/tumor suppressor genes. It is concluded that Ras(V)(12) scrib is a good sensitized genetic background in which to identify tumor suppressor genes involved in NB ACD, whose function could otherwise be masked by the high redundancy of the ACD process.
Dulac, A., Issa, A. R., Sun, J., Matassi, G., Jonas, C., Cherif-Zahar, B., Cattaert, D. and Birman, S. (2021). A Novel Neuron-Specific Regulator of the V-ATPase in Drosophila. eNeuro 8(5). PubMed ID: 34620624
The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. This study examined CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, it was observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. In addition, it was observed that CG31030 is essential for fly survival and that this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, this study showed that CG31030 knock-down induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, these data indicate that CG31030, which was renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release.
Marzano, M., Herzmann, S., Elsbroek, L., Sanal, N., Tarbashevich, K., Raz, E., Krahn, M. P. and Rumpf, S. (2021). AMPK adapts metabolism to developmental energy requirement during dendrite pruning in Drosophila. Cell Rep 37(7): 110024. PubMed ID: 34788610
To reshape neuronal connectivity in adult stages, Drosophila sensory neurons prune their dendrites during metamorphosis using a genetic degeneration program that is induced by the steroid hormone ecdysone. Metamorphosis is a nonfeeding stage that imposes metabolic constraints on development. AMP-activated protein kinase (AMPK), a regulator of energy homeostasis, is cell-autonomously required for dendrite pruning. AMPK is activated by ecdysone and promotes oxidative phosphorylation and pyruvate usage, likely to enable neurons to use noncarbohydrate metabolites such as amino acids for energy production. Loss of AMPK or mitochondrial deficiency causes specific defects in pruning factor translation and the ubiquitin-proteasome system. These findings distinguish pruning from pathological neurite degeneration, which is often induced by defects in energy production, and highlight how metabolism is adapted to fit energy-costly developmental transitions.
Pogodalla, N., Kranenburg, H., Rey, S., Rodrigues, S., Cardona, A. and Klambt, C. (2021). Drosophila Beta(Heavy)-Spectrin is required in polarized ensheathing glia that form a diffusion-barrier around the neuropil. Nat Commun 12(1): 6357. PubMed ID: 34737284
In the central nervous system (CNS), functional tasks are often allocated to distinct compartments. This is also evident in the Drosophila CNS where synapses and dendrites are clustered in distinct neuropil regions. The neuropil is separated from neuronal cell bodies by ensheathing glia, which as was shown using dye injection experiments, contribute to the formation of an internal diffusion barrier. Ensheathing glia are polarized with a basolateral plasma membrane rich in phosphatidylinositol-(3,4,5)-triphosphate (PIP(3)) and the Na(+)/K(+)-ATPase Nervana2 (Nrv2) that abuts an extracellular matrix formed at neuropil-cortex interface. The apical plasma membrane is facing the neuropil and is rich in phosphatidylinositol-(4,5)-bisphosphate (PIP(2)) that is supported by a sub-membranous Beta(Heavy)-Spectrin (Karst) cytoskeleton. Β(Heavy)-spectrin mutant larvae affect ensheathing glial cell polarity with delocalized PIP(2) and Nrv2 and exhibit an abnormal locomotion which is similarly shown by ensheathing glia ablated larvae. Thus, polarized glia compartmentalizes the brain and is essential for proper nervous system function.

Friday, January 7th - Evolution

Borne, F., Kulathinal, R. J. and Courtier-Orgogozo, V. (2021). Glue genes are subjected to diverse selective forces during Drosophila development. Genome Biol Evol. PubMed ID: 34788814
Molecular evolutionary studies usually focus on genes with clear roles in adult fitness or on developmental genes expressed at multiple time points during the life of the organism. This study examined the evolutionary dynamics of Drosophila glue genes, a set of eight genes tasked with a singular primary function during a specific developmental stage: the production of glue that allows animal pupa to attach to a substrate for several days during metamorphosis. Using phenotypic assays and available data from transcriptomics, PacBio genomes, and genetic variation from global populations, the selective forces acting on the glue genes within the cosmopolitan D. melanogaster species and its five closely related species, D. simulans, D. sechellia, D. mauritiana, D. yakuba, and D. teissieri were explored. This study observe a three-fold difference in glue adhesion between the least and the most adhesive D. melanogaster strain, indicating a strong genetic component to phenotypic variation. These eight glue genes are among the most highly expressed genes in salivary glands yet they display no notable codon bias. New copies of Sgs3 and Sgs7 are found in D. yakuba and D. teissieri with the Sgs3 coding sequence evolving rapidly after duplication in the D. yakuba branch. Multiple sites along the various glue genes appear to be constrained. Population genetics analysis in D. melanogaster suggests signs of local adaptive evolution for Sgs3, Sgs5 and Sgs5bis and traces of selective sweeps for Sgs1, Sgs3, Sgs7 and Sgs8. This study shows that stage-specific genes can be subjected to various dynamic evolutionary forces (Borne, 2021).
Kubick, N., Klimovich, P., Bienkowska, I., Poznanski, P., Lazarczyk, M., Sacharczuk, M. and Mickael, M. E. (2021). Investigation of Evolutionary History and Origin of the Tre1 Family Suggests a Role in Regulating Hemocytes Cells Infiltration of the Blood-Brain Barrier. Insects 12(10). PubMed ID: 34680651
In vertebrates, immune cells follow either a paracellular or a transcellular pathway to infiltrate the BBB. In Drosophila, glial cells form the blood-brain barrier (BBB) that regulates the access of hemocytes to the brain. In vertebrates, paracellular migration is dependent on PECAM1, while transcellular migration is dependent on the expression of CAV1. Interestingly Drosophila genome lacks both genes. Tre1 family (Tre1, moody, and Dmel_CG4313) play a diverse role in regulating transepithelial migration in Drosophila. A phylogenetic analysis, together ancestral reconstruction, were perfomed to investigate the Tre1 family. Tre1 was found to exist in Mollusca, Arthropoda, Ambulacraria, and Scalidophora. moody is shown to be a more ancient protein and it has existed since Cnidaria emergence and has a homolog (e.g., GPCR84) in mammals. The third family member (Dmel_CG4313) seems to only exist in insects. The origin of the family seems to be related to the rhodopsin-like family and in particular family α. The positive selection of the Tre1 family was investigated using PAML. Tre1 seems to have evolved under negative selection, whereas moody has evolved during positive selection. An SH3 motif was identified in Tre1, moody and Dmel_CG4313. SH3 is known to play a fundamental role in regulating actin movement in a Rho-dependent manner in PECAM1. These results suggest that the Tre1 family could be playing an important role in paracellular diapedesis in Drosophila.
Ruel, D. M., Vainer, Y., Yakir, E. and Bohbot, J. D. (2021). Identification and functional characterization of olfactory indolergic receptors in Drosophila melanogaster. Insect Biochem Mol Biol 139: 103651. PubMed ID: 34582989
Indole-sensitive odorant receptors or indolORs belong to a mosquito-specific expansion as ancient as the Culicidae lineage. Brachyceran flies appeared to lack representative members of this group despite the importance of indolics in this important group of dipterans. To explore whether indolORs occur in other brachyceran species, This study searched for candidate indolORs in Drosophila melanogaster. Using phylogenetic tools, this shows that D. melanogaster OR30a, OR43a, and OR49b form a distinct monophyletic lineage with mosquito indolORs. To explore a potential functional orthology with indolORs, this study expressed these three Drosophila ORs in Xenopus laevis oocytes and measured their responses to a panel of indolic compounds. This provide evidence that OR30a, OR43a, and OR49b exhibit high sensitivity to indoles. Along with the recent discovery of indolORs in the housefly Musca domestica, the findings suggest that indolORs are a widespread feature of the peripheral olfactory systems of Diptera (Ruel, 2021).
Dziedziech, A. and Theopold, U. (2021). Proto-pyroptosis: An ancestral origin for mammalian inflammatory cell death mechanism in Drosophila melanogaster. J Mol Biol: 167333. PubMed ID: 34756921
Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines and immune effectors upon cell bursting. This form of cell death has been increasingly well-characterized in mammals and can occur using alternative routes however, across phyla, there has been little evidence for the existence of pyroptosis. This study provide evidence for an ancient origin of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to wounds and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory-based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects and relies on ancient apoptotic machinery to induce proto-pyroptosis. Further, this study unveiled key players upstream in the activation of cell death in these cells including the apoptosome which may play an alternative role akin to the inflammasome in proto-pyroptosis. Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system.
Lyu, Y., Liufu, Z., Xiao, J. and Tang, T. (2021). A Rapid Evolving microRNA Cluster Rewires Its Target Regulatory Networks in Drosophila. Front Genet 12: 760530. PubMed ID: 34777478
New miRNAs are evolutionarily important but their functional evolution remains unclear. This study reports that the evolution of a microRNA cluster, mir-972C rewires its downstream regulatory networks in Drosophila. Genomic analysis reveals that mir-972C originated in the common ancestor of Drosophila where it comprises six old miRNAs. It has subsequently recruited six new members in the melanogaster subgroup after evolving for at least 50 million years. Both the young and the old mir-972C members evolved rapidly in seed and non-seed regions. Combining target prediction and cell transfection experiments, this study found that the seed and non-seed changes in individual mir-972C members cause extensive target divergence among D. melanogaster, D. simulans, and D. virilis, consistent with the functional evolution of mir-972C reported recently. Intriguingly, the target pool of the cluster as a whole remains relatively conserved. These results suggest that clustering of young and old miRNAs broadens the target repertoires by acquiring new targets without losing many old ones. This may facilitate the establishment of new miRNAs in existing regulatory networks.
Lai, W. Y. and Schlotterer, C. (2021). Evolution of phenotypic variance in response to a novel hot environment. Mol Ecol. PubMed ID: 34775658
Shifts in trait means are widely considered as evidence for adaptive responses, but the impact on phenotypic variance remains largely unexplored. Classic quantitative genetics provides a theoretical framework to predict how selection on phenotypic mean affects the variance. In addition to this indirect effect, it is also possible that the variance of the trait is the direct target of selection, but experimentally characterized cases are rare. Gene expression variance of Drosophila simulans males was studied before and after 100 generations of adaptation to a novel hot laboratory environment. In each of the two independently evolved populations, the variance of 125 and 97 genes was significantly reduced. It is proposed that the drastic loss in environmental complexity from nature to the lab may have triggered selection for reduced variance. The observation that selection could drive changes in the variance of gene expression could have important implications for studies of adaptation processes in natural and experimental populations.

Thursday, January 6th - Signaling

Lee, J., Lim, C., Han, T. H., Andreani, T., Moye, M., Curran, J., Johnson, E., Kath, W. L., Diekman, C. O., Lear, B. C. and Allada, R. (2021). The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms. Proc Natl Acad Sci U S A 118(47). PubMed ID: 34799448
Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. This study revealed a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, mutants of Transport and Golgi organization 10 (Tango10) were identified with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless. Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. It is proposed that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.
De Munck, S., Provost, M., Kurikawa, M., Omori, I., Mukohyama, J., Felix, J., Bloch, Y., Abdel-Wahab, O., Bazan, J. F., Yoshimi, A. and Savvides, S. N. (2021). Structural basis of cytokine-mediated activation of ALK family receptors. Nature. PubMed ID: 34646012
Anaplastic lymphoma kinase (ALK) and the related leukocyte tyrosine kinase (LTK) are recently deorphanized receptor tyrosine kinases. Together with their activating cytokines, ALKAL1 and ALKAL2(4-6) (also called FAM150A and FAM150B or AUGβ and AUGα, respectively), they are involved in neural development, cancer and autoimmune diseases. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain, consistent with a metabolic role for Drosophila ALK. Despite such functional pleiotropy and growing therapeutic relevance, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. This study shows that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. The membrane-proximal EGF-like domain was shown to dictate the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, a structural and mechanistic blueprint is proposed for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.
Hirschhauser, A., van Cann, M. and Bogdan, S. (2021). CK1alpha protects WAVE from degradation to regulate cell shape and motility in immune response. J Cell Sci. PubMed ID: 34730182
The WAVE regulatory complex (WRC) is the major Arp2/3 activator, promoting lamellipodial protrusions in migrating cells. The WRC is basally inactive but can be activated by Rac1 and phospholipids, and phosphorylation. However, the in vivo relevance of phosphorylation of WAVE remains largely unknown. This study identified the kinase CK1α as a novel regulator of WAVE controlling cell shape and cell motility in Drosophila macrophages. CK1α binds and phosphorylates WAVE in vitro. Phosphorylation of WAVE by CK1α appears not to be required for activation but rather regulates its stability. Pharmacologic inhibition of CK1α promotes ubiquitin-dependent degradation of WAVE. Consistently, loss of ck1α but not ck2 function phenocopies WAVE depletion. Phosphorylation-deficient mutations in the CK1α consensus sequences within the VCA domain of WAVE can neither rescue mutant lethality nor lamellipodia defects. By contrast, phosphomimetic mutations rescue all cellular and developmental defects. Finally, RNAi-mediated suppression of 26S proteasome or E3 ligase complexes substantially rescues lamellipodia defects in CK1α depleted macrophages. Thus, it is concluded that the basal phosphorylation of WAVE by CK1α protects it from premature ubiquitin-dependent degradation, thus promoting WAVE function in vivo.
Kramer, J., Neves, J., Koniikusic, M., Jasper, H. and Lamba, D. A. (2021). Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina. PLoS One 16(10): e0258872. PubMed ID: 34699550
Retinal homeostasis relies on intricate coordination of cell death and survival in response to stress and damage. Signaling mechanisms that coordinate this process in the adult retina remain poorly understood. This study identified Decapentaplegic (Dpp) signaling in Drosophila and its mammalian homologue Transforming Growth Factor-beta (TGFβ) superfamily, that includes TGFβ and Bone Morphogenetic Protein (BMP) signaling arms, as central mediators of retinal neuronal death and tissue survival following acute damage. Using a Drosophila model for UV-induced retinal damage, this study showed that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly, a dynamic response of retinal cells was found to this signal: in an early phase, Dpp-mediated stimulation of Saxophone/Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins/Mad axis promotes tissue repair and survival. This dual role is conserved in the mammalian retina through the TGFβ/BMP signaling, as supplementation of BMP4 or inhibition of TGFβ using small molecules promotes retinal cell survival, while inhibition of BMP negatively affects cell survival after light-induced photoreceptor damage and NMDA induced inner retinal neuronal damage. These data identify key evolutionarily conserved mechanisms by which retinal homeostasis is maintained.
Mehta, A. S., Deshpande, P., Chimata, A. V., Tsonis, P. A. and Singh, A. (2021). Newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye. iScience 24(10): 103166. PubMed ID: 34746690
Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools poses challenges to determine the function of such genes. Therefore, this study used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L (2) mutant and GMR-hid, GMR-GAL4 eye. L (2) exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly, newt genes significantly restore missing photoreceptor cells both in L (2) and GMR>lhid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. These data highlight that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.
Matsuda, S., Schaefer, J. V., Mii, Y., Hori, Y., Bieli, D., Taira, M., Pluckthun, A. and Affolter, M. (2021). Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc. Nat Commun 12(1): 6435. PubMed ID: 34750371
How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. This study generated two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. One is "HA trap" based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, the other is "Dpp trap" based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, it was found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. dpp transcriptional refinement is shown from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

Wednesday, January 5th - Disease Models

Li, J., Lim, R. G., Kaye, J. A., Dardov, V., Coyne, A. N., Wu, J., Milani, P., Cheng, A., Thompson, T. G., Ornelas, L., Frank, A., Adam, M., Banuelos, M. G., Casale, M., Cox, V., Escalante-Chong, R., Daigle, J. G., Gomez, E., Hayes, L., Holewenski, R., Lei, S., Lenail, A., Lima, L., Mandefro, B., Matlock, A., Panther, L., Patel-Murray, N. L., Pham, J., Ramamoorthy, D., Sachs, K., Shelley, B., Stocksdale, J., Trost, H., Wilhelm, M., Venkatraman, V., Wassie, B. T., Wyman, S., Yang, S., Van Eyk, J. E., Lloyd, T. E., Finkbeiner, S., Fraenkel, E., Rothstein, J. D., Sareen, D., Svendsen, C. N. and Thompson, L. M. (2021). An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients. iScience 24(11): 103221. PubMed ID: 34746695
Neurodegenerative diseases are challenging for systems biology because of the lack of reliable animal models or patient samples at early disease stages. Induced pluripotent stem cells (iPSCs) could address these challenges. This study investigated DNA, RNA, epigenetics, and proteins in iPSC-derived motor neurons from patients with ALS carrying hexanucleotide expansions in C9ORF72. Using integrative computational methods combining all omics datasets, this study identified novel and known dysregulated pathways. A C9ORF72 Drosophila model was used to distinguish pathways contributing to disease phenotypes from compensatory ones, and alterations in some pathways were confirmed in postmortem spinal cord tissue of patients with ALS. A different differentiation protocol was used to derive a separate set of C9ORF72 and control motor neurons. Many individual -omics differed by protocol, but some core dysregulated pathways were consistent. This strategy of analyzing patient-specific neurons provides disease-related outcomes with small numbers of heterogeneous lines and reduces variation from single-omics to elucidate network-based signatures.
Manivannan, S. N., Roovers, J., Smal, N., Myers, C. T., Turkdogan, D., Roelens, F., Kanca, O., Chung, H. L., Scholz, T., Hermann, K., Bierhals, T., Caglayan, H. S., Stamberger, H., Mefford, H., de Jonghe, P., Yamamoto, S., Weckhuysen, S. and Bellen, H. J. (2021). De novo FZR1 loss-of-function variants cause developmental and epileptic encephalopathies. Brain. PubMed ID: 34788397
FZR1, which encodes the Cdh1 subunit of the Anaphase Promoting Complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homolog of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype Myoclonic Atonic Epilepsy. Functional evidence is provided that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homolog fzr and overexpression studies, it was shown that patient variants can affect proper neurodevelopment. This study consolidates the relationship between FZR1 and developmental epileptic encephalopathy, and expands the associated phenotype. It is concluded that heterozygous loss-of-function of FZR1 leads to developmental epileptic encephalopathies associated with a spectrum of neonatal to childhood onset seizure types, developmental delay and mild ataxia. In summary, this approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental epileptic encephalopathy cases.
Pant, C., Chakrabarti, M., Mendonza, J. J., Ganganna, B., Pabbaraja, S. and Pal Bhadra, M. (2021). Aza-Flavanone Diminishes Parkinsonism in the Drosophila melanogaster Parkin Mutant. ACS Chem Neurosci. PubMed ID: 34763419
Parkinson's disease is a chronic and progressive neurodegenerative disease, induced by slow and progressive death of the dopaminergic (DA) neurons from the midbrain region called substantia nigra (SNc) leading to difficulty in locomotion. At present, very few potential therapeutic drugs are available for treatment, necessitating an urgent need for development. In this current study, the parkin transgenic Drosophila melanogaster model that induces selective loss in dopaminergic neurons and impairment of locomotory functions has been used to see the effect of the aza-flavanone molecule. D. melanogaster serves as an amazing in vivo model making valuable contribution in the development of promising treatment strategies. In-silico study showed spontaneous binding of this molecule to the D2 receptor making it a potential dopamine agonist. PARKIN protein is well conserved, and it has been reported that Drosophila PARKIN is 42% identical to human PARKIN. Interestingly, this molecule enhances the motor coordination and survivability rate of the transgenic flies along with an increase in expression of the master regulator of Dopamine synthesis, that is, tyrosine hydroxylase (TH), in the substantia nigra region of the fly brain. Moreover, it plays a significant effect on mitochondrial health and biogenesis via modulation of a conserved mitochondrial protein PHB2. Therefore, this molecule could lead to the development of an effective therapeutic approach for the treatment of PD.
Kowada, R., Kodani, A., Ida, H., Yamaguchi, M., Lee, I. S., Okada, Y. and Yoshida, H. (2021). The function of Scox in glial cells is essential for locomotive ability in Drosophila. Sci Rep 11(1): 21207. PubMed ID: 34707123
Synthesis of cytochrome c oxidase (Scox) is a Drosophila homolog of human SCO2 encoding a metallochaperone that transports copper to cytochrome c, and is an essential protein for the assembly of cytochrome c oxidase in the mitochondrial respiratory chain complex. SCO2 is highly conserved in a wide variety of species across prokaryotes and eukaryotes, and mutations in SCO2 are known to cause mitochondrial diseases such as fatal infantile cardioencephalomyopathy, Leigh syndrome, and Charcot-Marie-Tooth disease, a neurodegenerative disorder. These diseases have a common symptom of locomotive dysfunction. However, the mechanisms of their pathogenesis remain unknown, and no fundamental medications or therapies have been established for these diseases. This study demonstrated that the glial cell-specific knockdown of Scox perturbs the mitochondrial morphology and function, and locomotive behavior in Drosophila. In addition, the morphology and function of synapses were impaired in the glial cell-specific Scox knockdown. Furthermore, Scox knockdown in ensheathing glia, one type of glial cell in Drosophila, resulted in larval and adult locomotive dysfunction. This study suggests that the impairment of Scox in glial cells in the Drosophila CNS mimics the pathological phenotypes observed by mutations in the SCO2 gene in humans.
Licata, N. V., Cristofani, R., Salomonsson, S., Wilson, K. M., Kempthorne, L., Vaizoglu, D., D'Agostino, V. G., Pollini, D., Loffredo, R., Pancher, M., Adami, V., Bellosta, P., Ratti, A., Viero, G., Quattrone, A., Isaacs, A. M., Poletti, A. and Provenzani, A. (2021). C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation. EMBO J: e105026. PubMed ID: 34791698
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). This study developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone were found to reduce DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, these results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.
Lee, H., Lee, J. J., Park, N. Y., Dubey, S. K., Kim, T., Ruan, K., Lim, S. B., Park, S. H., Ha, S., Kovlyagina, I., Kim, K. T., Kim, S., Oh, Y., Kim, H., Kang, S. U., Song, M. R., Lloyd, T. E., Maragakis, N. J., Hong, Y. B., Eoh, H. and Lee, G. (2021). Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS. Nat Neurosci. PubMed ID: 34782793
Amyotrophic lateral sclerosis (ALS) is a devastating disorder in which motor neurons degenerate, the causes of which remain unclear. In particular, the basis for selective vulnerability of spinal motor neurons (sMNs) and resistance of ocular motor neurons to degeneration in ALS has yet to be elucidated. This study applied comparative multi-omics analysis of human induced pluripotent stem cell-derived sMNs and ocular motor neurons to identify shared metabolic perturbations in inherited and sporadic ALS sMNs, revealing dysregulation in lipid metabolism and its related genes. Targeted metabolomics studies confirmed such findings in sMNs of 17 ALS (SOD1, C9ORF72, TDP43 (TARDBP) and sporadic) human induced pluripotent stem cell lines, identifying elevated levels of arachidonic acid. Pharmacological reduction of arachidonic acid levels was sufficient to reverse ALS-related phenotypes in both human sMNs and in vivo in Drosophila and SOD1(G93A) mouse models. Collectively, these findings pinpoint a catalytic step of lipid metabolism as a potential therapeutic target for ALS.

Tuesday, January 4th - Adult Neural Function

Driscoll, M., Buchert, S. N., Coleman, V., McLaughlin, M., Nguyen, A. and Sitaraman, D. (2021). Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling.. Sci Rep 11(1): 20067. PubMed ID: 34625611
Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. As a highly conserved neurobehavioral state, sleep has been described in species ranging from jellyfish to humans. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. PAM-DANs were found to be GABA responsive and required GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, this study has identified and characterized a dopamine modulated sleep microcircuit within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave way for understanding how flies balance sleep, wakefulness and arousal.
Fulgham, C. V., Dreyer, A. P., Nasseri, A., Miller, A. N., Love, J., Martin, M. M., Jabr, D. A., Saurabh, S. and Cavanaugh, D. J. (2021). Central and Peripheral Clock Control of Circadian Feeding Rhythms. J Biol Rhythms: 7487304211045835. PubMed ID: 34547954
Many behaviors exhibit ~24-h oscillations under control of an endogenous circadian timing system that tracks time of day via a molecular biology degree circadian clock. In the fruit fly, Drosophila melanogaster, most circadian research has focused on the generation of locomotor activity rhythms, but a fundamental question is how the circadian clock orchestrates multiple distinct behavioral outputs. This study investigated the cells and circuits mediating circadian control of feeding behavior. Using an array of genetic tools, it was shown that, as is the case for locomotor activity rhythms, the presence of feeding rhythms requires molecular clock function in the ventrolateral clock neurons of the central brain. It was further demonstrated that the speed of molecular clock oscillations in these neurons dictates the free-running period length of feeding rhythms. In contrast to the effects observed with central clock cell manipulations, it was shown that genetic abrogation of the molecular clock in the fat body, a peripheral metabolic tissue, is without effect on feeding behavior. Interestingly, it was found that molecular clocks in the brain and fat body of control flies gradually grow out of phase with one another under free-running conditions, likely due to a long endogenous period of the fat body clock. Under these conditions, the period of feeding rhythms tracks with molecular oscillations in central brain clock cells, consistent with a primary role of the brain clock in dictating the timing of feeding behavior. Finally, despite a lack of effect of fat body selective manipulations, it was found that flies with simultaneous disruption of molecular clocks in multiple peripheral tissues (but with intact central clocks) exhibit decreased feeding rhythm strength and reduced overall food intake. It is concluded that both central and peripheral clocks contribute to the regulation of feeding rhythms, with a particularly dominant, pacemaker role for specific populations of central brain clock cells.
Imambocus, B. N., Zhou, F., Formozov, A., Wittich, A., Tenedini, F. M., Hu, C., Sauter, K., Macarenhas Varela, E., Heredia, F., Casimiro, A. P., Macedo, A., Schlegel, P., Yang, C. H., Miguel-Aliaga, I., Wiegert, J. S., Pankratz, M. J., Gontijo, A. M., Cardona, A. and Soba, P. (2021). A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae. Curr Biol. PubMed ID: 34798050
Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, this study uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. Acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. This work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.
French, A. S., Geissmann, Q., Beckwith, E. J. and Gilestro, G. F. (2021). Sensory processing during sleep in Drosophila melanogaster. Nature 598(7881): 479-482. PubMed ID: 34588694
During sleep, most animal species enter a state of reduced consciousness characterized by a marked sensory disconnect. Yet some processing of the external world must remain intact, given that a sleeping animal can be awoken by intense stimuli (for example, a loud noise or a bright light) or by soft but qualitatively salient stimuli (for example, the sound of a baby cooing or hearing one's own name. How does a sleeping brain retain the ability to process the quality of sensory information? This study presents a paradigm to study the functional underpinnings of sensory discrimination during sleep in Drosophila melanogaster. Sleeping vinegar flies, like humans, were shown to discern the quality of sensory stimuli and are more likely to wake up in response to salient stimuli. It ws also shown that the salience of a stimulus during sleep can be modulated by internal states. A prototypical blueprint is offered detailing a circuit involved in this process and its modulation as evidence that the system can be used to explore the cellular underpinnings of how a sleeping brain experiences the world.
French, A. S., Geissmann, Q., Beckwith, E. J. and Gilestro, G. F. (2021). Sensory processing during sleep in Drosophila melanogaster. Nature 598(7881): 479-482. PubMed ID: 34588694
During sleep, most animal species enter a state of reduced consciousness characterized by a marked sensory disconnect. Yet some processing of the external world must remain intact, given that a sleeping animal can be awoken by intense stimuli (for example, a loud noise or a bright light) or by soft but qualitatively salient stimuli (for example, the sound of a baby cooing or hearing one's own name. How does a sleeping brain retain the ability to process the quality of sensory information? This study presents a paradigm to study the functional underpinnings of sensory discrimination during sleep in Drosophila melanogaster. Sleeping vinegar flies, like humans, were shown to discern the quality of sensory stimuli and are more likely to wake up in response to salient stimuli. It ws also shown that the salience of a stimulus during sleep can be modulated by internal states. A prototypical blueprint is offered detailing a circuit involved in this process and its modulation as evidence that the system can be used to explore the cellular underpinnings of how a sleeping brain experiences the world.
Ji, W., Wu, L. F. and Altschuler, S. J. (2021). Analysis of growth cone extension in standardized coordinates highlights self-organization rules during wiring of the Drosophila visual system. PLoS Genet 17(11): e1009857. PubMed ID: 34731164
A fascinating question in neuroscience is how ensembles of neurons, originating from different locations, extend to the proper place and by the right time to create precise circuits. This study investigated this question in the Drosophila visual system, where photoreceptors re-sort in the lamina to form the crystalline-like neural superposition circuit. The repeated nature of this circuit allowed establishment of a data-driven, standardized coordinate system for quantitative comparison of sparsely perturbed growth cones within and across specimens. Using this common frame of reference, the extension was investigated of the R3 and R4 photoreceptors, which is the only pair of symmetrically arranged photoreceptors with asymmetric target choices. Specifically, it was found that extension speeds of the R3 and R4 growth cones are inherent to their cell identities. The ability to parameterize local regularity in tissue organization facilitated the characterization of ensemble cellular behaviors and dissection of mechanisms governing neural circuit formation.

Monday, January 3rd - Disease Models

Jaramillo-Martinez, V., Sivaprakasam, S., Ganapathy, V. and Urbatsch, I. L. (2021). Drosophila INDY and Mammalian INDY: Major Differences in Transport Mechanism and Structural Features despite Mostly Similar Biological Functions. Metabolites 11(10). PubMed ID: 34677384
INDY (I'm Not Dead Yet) is a plasma membrane transporter for citrate, first identified in Drosophila. Partial deficiency of INDY extends lifespan in this organism in a manner similar to that of caloric restriction. The mammalian counterpart (NaCT/SLC13A5) also transports citrate. In mice, it is the total, not partial, absence of the transporter that leads to a metabolic phenotype similar to that caloric restriction; however, there is evidence for subtle neurological dysfunction. Loss-of-function mutations in SLC13A5 (solute carrier gene family 13, member A5) occur in humans, causing a recessive disease, with severe clinical symptoms manifested by neonatal seizures and marked disruption in neurological development. Though both Drosophila INDY and mammalian INDY transport citrate, the translocation mechanism differs, the former being a dicarboxylate exchanger for the influx of citrate(2-) in exchange for other dicarboxylates, and the latter being a Na(+)-coupled uniporter for citrate(2-). Their structures also differ as evident from only ~35% identity in amino acid sequence and from theoretically modeled 3D structures. The varied biological consequences of INDY deficiency across species, with the beneficial effects predominating in lower organisms and detrimental effects overwhelming in higher organisms, are probably reflective of species-specific differences in tissue expression and also in relative contribution of extracellular citrate to metabolic pathways in different tissues.
Zhang, L., Buhr, S., Voigt, A. and Methner, A. (2021). The Evolutionary Conserved Transmembrane BAX Inhibitor Motif (TMBIM) Containing Protein Family Members 5 and 6 Are Essential for the Development and Survival of Drosophila melanogaster. Front Cell Dev Biol 9: 666484. PubMed ID: 34540824
The mammalian Transmembrane BAX Inhibitor Motif (TMBIM) protein family consists of six evolutionarily conserved hydrophobic proteins that affect programmed cell death and the regulation of intracellular calcium levels. The bacterial ortholog BsYetJ is a pH-dependent calcium channel. This study identified seven TMBIM family members in Drosophila melanogaster and describes their expression levels in diverse tissues and developmental stages. A phylogenetic analysis revealed that CG30379 represents the ortholog of human TMBIM4 although these two proteins are much less related than TMBIM5 (CG2076 and CG1287/Mics1) and TMBIM6 (CG7188/Bi-1) to their respective orthologs. For TMBIM1-3 the assignment is more dubious because the fly and the human proteins cluster together. A functional analysis was conducted based on expression levels and the availability of RNAi lines. This revealed that the ubiquitous knockdown of CG3798/Nmda1 and CG3814/Lfg had no effect on development while knockdown of CG2076/dTmbim5 resulted in death at the pupa stage and knockdown of CG7188/dTmbim6 in death at the embryonic stage. Ubiquitous knockdown of the second TMBIM5 paralog CG1287/Mics1 ensued in male sterility. Knockdown of dTmbim5 and 6 in muscle and neural tissue also greatly reduced lifespan through different mechanisms. Knockdown of the mitochondrial family member dTmbim5 resulted in reduced ATP production and a pro-apoptotic expression profile while knockdown of the ER protein dTmbim6 increased the ER calcium levels similar to findings in mammalian cells. These data demonstrate that dTmbim5 and 6 are essential for fly development and survival but affect cell survival through different mechanisms.
Gu, W., Luo, Z., Vonrhein, C., Jia, X., Ve, T., Nanson, J. D. and Kobe, B. (2021). Crystal structure determination of the armadillo repeat domain of Drosophila SARM1 using MIRAS phasing. Acta Crystallogr F Struct Biol Commun 77(Pt 10): 364-373. PubMed ID: 34605441
The crystal structure determination of the armadillo repeat motif (ARM) domain of Drosophila SARM1 (dSARM1(ARM)) is described, that required the combination of a number of sources of phase information in order to obtain interpretable electron-density maps. SARM1 is a central executioner of programmed axon degeneration, a common feature of the early phase of many neurodegenerative diseases. SARM1 is held in the inactive state in healthy axons by its N-terminal auto-inhibitory ARM domain, and is activated to cleave NAD upon injury, triggering subsequent axon degeneration. To characterize the molecular mechanism of SARM1 activation, it was sought to determine the crystal structure of the SARM1 ARM domain. Here, the recombinant production and crystallization of dSARM1(ARM) is described, as well as the unconventional process used for structure determination. Crystals were obtained in the presence of NMN, a precursor of NAD and a potential activator of SARM1, only after in situ proteolysis of the N-terminal 63 residues. After molecular-replacement attempts failed, the crystal structure of dSARM1(ARM) was determined at 1.65Å resolution using the MIRAS phasing technique with autoSHARP, combining data from native, selenomethionine-labelled and bromide-soaked crystals. The structure will further the understanding of SARM1 regulation (Gu, 2021).
Ulgherait, M., Midoun, A. M., Park, S. J., Gatto, J. A., Tener, S. J., Siewert, J., Klickstein, N., Canman, J. C., Ja, W. W. and Shirasu-Hiza, M. (2021). Circadian autophagy drives iTRF-mediated longevity. Nature 598(7880): 353-358. PubMed ID: 34588695
Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. To exploit the genetic tools and well-characterized ageing markers of Drosophila, this study developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. iTRF enhanced circadian-regulated transcription, and iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.
Klemm, J., Stinchfield, M. J. and Harris, R. E. (2021). Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs. Genetics 219(3). PubMed ID: 34740246
Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death, such as necrosis, have yet to be fully explored. This study has developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. Necrosis is shown to stimulate regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly, however, necrosis also results in significant apoptosis far from the site of ablation, which this study terms necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, it was found that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration.
Hung, Y. C., Huang, K. L., Chen, P. L., Li, J. L., Lu, S. H., Chang, J. C., Lin, H. Y., Lo, W. C., Huang, S. Y., Lee, T. T., Lin, T. Y., Imai, Y., Hattori, N., Liu, C. S., Tsai, S. Y., Chen, C. H., Lin, C. H. and Chan, C. C. (2021). UQCRC1 engages cytochrome c for neuronal apoptotic cell death. Cell Rep 36(12): 109729. PubMed ID: 34551295
Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. This study recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. This study investigates the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases Cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. The findings identified a role for UQCRC1 in regulating cyt-c-induced apoptosis (Hung, 2021). Home page: The Interactive Fly© 2020 Thomas B. Brody, Ph.D.

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