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


Friday, March 24th - Larval and Adult Development

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Ding, Y., Lv, Y., Pan, Y., Li, J., Yan, K., Yu, Z., Shang, Q. (2023). A masked gene concealed hand in glove in the forkhead protein crocodile regulates the predominant detoxification CYP6DA1 in Aphis gossypii Glover. International journal of biological macromolecules, 253(Pt 3):126824 PubMed ID: 37690634
Cytochrome P450-mediated metabolism is an important mechanism of insecticide resistance, most studies show upregulated transcript levels of P450s in resistant insect strains. Previous studies illustrated that some upregulated P450s were associated with resistance to insecticide cyantraniliprole, and it is more comprehensive to use the tissue specificity of transcriptomes to compare resistant (CyR) and susceptible (SS) strains. In this study, the expression profiles of P450s in a CyR strain compared with a SS strain in remaining carcass or midgut were investigated by RNA sequencing, and candidate genes were selected for functional study. Drosophila melanogaster bioassays suggested that ectopic overexpression of cytochromes CYP4CK1, CYP6CY5, CYP6CY9, CYP6CY19, CYP6CZ1 and CYP6DA1 in flies was sufficient to confer cyantraniliprole resistance, among which CYP6DA1 was the predominant contributor to resistance (12.24-fold). RNAi suppression of CYP4CK1, CYP6CY5, CYP6CY9 and CYP6DA1 significantly increased CyR aphid sensitivity to cyantraniliprole. The CYP6DA1 promoter had two predicted binding sites for Crocodile (CROC), an intron-free ORF with bidirectional transcription yielding CROC (+) and CROC (-). Y1H, RNAi and EMSA found that CROC (-) was a transcription factor directly regulating CYP6DA1 expression. In conclusion, P450 genes contribute to cyantraniliprole resistance, and the transcription factor CROC (-) regulates the expression of CYP6DA1 in A. gossypii.
Rodrigues, N. R., Macedo, G. E., Martins, I. K., Vieira, P. B., Kich, K. G., Posser, T., Franco, J. L. (2023). Sleep disturbance induces a modulation of clock gene expression and alters metabolism regulation in Drosophila. Physiology & behavior, 271:114334 PubMed ID: 37595818
Sleep disorders are catching attention worldwide as they can induce dyshomeostasis and health issues in all animals, including humans. Circadian rhythms are biological 24-hour cycles that influence physiology and behavior in all living organisms. Sleep is a crucial resting state for survival and is under the control of circadian rhythms. Studies have shown the influence of sleep on various pathological conditions, including metabolic diseases; however, the biological mechanisms involving the circadian clock, sleep, and metabolism regulation are not well understood. Previous work standardized a sleep disturbance protocol and, observed that short-time sleep deprivation and sleep-pattern alteration induce homeostatic sleep regulation, locomotor deficits, and increase oxidative stress. This study investigated the relationship between these alterations with the circadian clock and energetic metabolism. In this study, the expression of the circadian clock and drosophila insulin-like peptides (DILPs) genes and metabolic markers glucose, triglycerides, and glycogen were examined in fruit flies subjected to short-term sleep disruption protocols. The sleep disturbance altered the expression of clock genes and DILPs genes expression, and modulated glucose, triglycerides, and glycogen levels. Moreover, this study demonstrated changes in mTor/dFoxo genes, AKT phosphorylation, and dopamine levels in nocturnal light-exposed flies. Thus, these results suggest a connection between clock genes and metabolism disruption as a consequence of sleep disruption, demonstrating the importance of sleep quality in health maintenance.
Lee, J., Song, X., Hyun, B., Jeon, C. O., Hyun, S. (2023). Drosophila Gut Immune Pathway Suppresses Host Development-Promoting Effects of Acetic Acid Bacteria. Molecules and cells, 46(10):637-653 PubMed ID: 37853687
The physiology of most organisms, including Drosophila, is heavily influenced by their interactions with certain types of commensal bacteria. Acetobacter and Lactobacillus, two of the most representative Drosophila commensal bacteria, have stimulatory effects on host larval development and growth. However, how these effects are related to host immune activity remains largely unknown. This study shows that the Drosophila development-promoting effects of commensal bacteria are suppressed by host immune activity. Mono-association of germ-free Drosophila larvae with Acetobacter pomorum stimulated larval development, which was accelerated when host immune deficiency (IMD) pathway genes were mutated. This phenomenon was not observed in the case of mono-association with Lactobacillus plantarum. Moreover, the mutation of Toll pathway, which constitutes the other branch of the Drosophila immune pathway, did not accelerate A. pomorum-stimulated larval development. The mechanism of action of the IMD pathway-dependent effects of A. pomorum did not appear to involve previously known host mechanisms and bacterial metabolites such as gut peptidase expression, acetic acid, and thiamine, but appeared to involve larval serum proteins. These findings may shed light on the interaction between the beneficial effects of commensal bacteria and host immune activity.
Axelrod, S., Li, X., Sun, Y., Lincoln, S., Terceros, A., O'Neil, J., Wang, Z., Nguyen, A., Vora, A., Spicer, C., Shapiro, B., Young, M. W. (2023). The Drosophila blood-brain barrier regulates sleep via Moody G protein-coupled receptor signaling. Proc Natl Acad Sci U S A, 120(42):e2309331120 PubMed ID: 37831742
Sleep is vital for most animals, yet its mechanism and function remain unclear. This study found that permeability of the BBB (blood-brain barrier)-the organ required for the maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain-is modulated by sleep deprivation (SD) and can cell-autonomously effect sleep changes. Increased BBB permeability was observed in known sleep mutants as well as in acutely sleep-deprived animals. In addition to molecular tracers, SD-induced BBB changes also increased the penetration of drugs used in the treatment of brain pathologies. After chronic/genetic or acute SD, rebound sleep or administration of the sleeping aid gaboxadol normalized BBB permeability, showing that SD effects on the BBB are reversible. Along with BBB permeability, RNA levels of the BBB master regulator moody are modulated by sleep. Conversely, altering BBB permeability alone through glia-specific modulation of moody, gαo, loco, lachesin, or neuroglian-each a well-studied regulator of BBB function-was sufficient to induce robust sleep phenotypes. These studies demonstrate a tight link between BBB permeability and sleep and indicate a unique role for the BBB in the regulation of sleep.
Corra, S., Checchetto, V., Brischigliaro, M., Rampazzo, C., Bottani, E., Gagliani, C., Cortese, K., De Pitta, C., Roverso, M., De Stefani, D., Bogialli, S., Zeviani, M., Viscomi, C., Szabo, I., Costa, R. (2023). Drosophila Mpv17 forms an ion channel and regulates energy metabolism. iScience, 26(10):107955 PubMed ID: 37810222
Mutations in MPV17 are a major contributor to mitochondrial DNA (mtDNA) depletion syndromes, a group of inherited genetic conditions due to mtDNA instability. To investigate the role of MPV17 in mtDNA maintenance, this study generated and characterized a Drosophila melanogaster Mpv17 (dMpv17) KO model showing that the absence of dMpv17 caused profound mtDNA depletion in the fat body but not in other tissues, increased glycolytic flux and reduced lifespan in starvation. Accordingly, the expression of key genes of glycogenolysis and glycolysis was upregulated in dMpv17 KO flies. In addition, it was demonstrated that dMpv17 formed a channel in planar lipid bilayers at physiological ionic conditions, and its electrophysiological hallmarks were affected by pathological mutations. Importantly, the reconstituted channel translocated uridine but not orotate across the membrane. These results indicate that dMpv17 forms a channel involved in translocation of key metabolites and highlight the importance of dMpv17 in energy homeostasis and mitochondrial function.
Sun, X., Shen, J., Perrimon, N., Kong, X., Wang, D. (202x3). The endoribonuclease Arlr is required to maintain lipid homeostasis by downregulating lipolytic genes during aging. Nat Commun, 14(1):6254 PubMed ID: 37803019
While disorders in lipid metabolism have been associated with aging and age-related diseases, how lipid metabolism is regulated during aging is poorly understood. This study characterize the Drosophila endoribonuclease CG2145, an ortholog of mammalian EndoU that this study named Age-related lipid regulator (Arlr), as a regulator of lipid homeostasis during aging. In adult adipose tissues, Arlr is necessary for maintenance of lipid storage in lipid droplets (LDs) as flies age, a phenotype that can be rescued by either high-fat or high-glucose diet. Interestingly, RNA-seq of arlr mutant adipose tissues and RIP-seq suggest that Arlr affects lipid metabolism through the degradation of the mRNAs of lipolysis genes - a model further supported by the observation that knockdown of Lsd-1, regucalcin, yip2 (Acetyl-CoA acyltransferase) or CG5162, which encode genes involved in lipolysis, rescue the LD defects of arlr mutants. In addition, DendoU as a functional paralog of Arlr was characterize, and human ENDOU was able to rescue arlr mutants. Altogether, this study reveals a role of ENDOU-like endonucleases as negative regulator of lipolysis.

Wednesday, March 27th - Disease Models

Ismael, S., Colvin, R. A., Lee, D. (2024). Activation of cyclic AMP signaling pathway in dopaminergic neurons rescues locomotion defects in a Drosophila larval model of Parkinson's disease. Brain Res, 1822:148641 PubMed ID: 37866407
Parkinson's disease (PD) is a neurodegenerative disease showing uncontrollable motor symptoms that are primarily caused by the progressive loss of dopaminergic neurons in the brain. Currently no treatment exists to prevent PD progression. Therefore, discovery of new neuroprotective strategies still has great potential to benefit PD patients. A handful of studies show that activation of cAMP pathways is neuroprotective against PD progression. However, the neuroprotective role of this signaling cascade specifically in DA neurons has not been explored. In this study, fruit fly Drosophila melanogaster was used because of its sophisticated and powerful genetic approaches, especially with related to cAMP signaling pathway. This study investigated molecular mechanisms of neuroprotection in a fly larval model of PD by administering an environmental PD toxin rotenone. Increased cAMP signaling in the dunce mutant fly carrying defects in phosphodiesterase (PDE) gene, is neuroprotective against rotenone-induced locomotion deficits. Furthermore, the neuroprotective role of cAMP signaling specifically in DA neurons has been studied as it has not been explored. By using transgenic flies expressing designer receptors exclusively activated by designer drugs (DREADDs), an increase of cAMP levels in DA neurons was shown to rescues rotenone-induced locomotion deficits. We also showed that this neuroprotectio n is mediated by activation of Gαs and PKA-C1 subunits. The results provide novel findings that expand knowledge of neuroprotective mechanisms in DA neurons affecting PD progression, which could contribute to the development of new therapeutic treatments against PD. An important future study will explore downstream targets of cAMP-PKA signaling.

Moron-Oset, J., Fischer, L. K. S., Jaure, N., Zhang, P., Jahn, A. J., Super, T., Pahl, A., Isaacs, A. M., Gronke, S., Partridge, L. (2023). Repeat length of C9orf72-associated glycine-alanine polypeptides affects their toxicity. Acta neuropathologica communications, 11(1):140 PubMed ID: 37644512
G(4)C(2) hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G(4)C(2) insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPRs) translated from G(4)C(2) transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPRs have shown that arginine-rich DPRs are most toxic, while polyGlycine-Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length or tagging affect the toxicity of GA has not been systematically assessed. Therefore, this study generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, these data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in patients.
Yi, S., Wang, L., Ho, M. S., Zhang, S. (2024). The autophagy protein Atg9 functions in glia and contributes to parkinsonian symptoms in a Drosophila model of Parkinson's disease. Neural Regen Res, 19(5):1150-1155 PubMed ID: 37862221
Parkinson's disease is a progressive neurodegenerative disease characterized by motor deficits, dopaminergic neuron loss, and brain accumulation of α-synuclein aggregates called Lewy bodies. Dysfunction in protein degradation pathways, such as autophagy, has been demonstrated in neurons as a critical mechanism for eliminating protein aggregates in Parkinson's disease. However, it is less well understood how protein aggregates are eliminated in glia, the other cell type in the brain. The present study shows that autophagy-related gene 9 (Atg9), the only transmembrane protein in the autophagy machinery, is highly expressed in Drosophila glia from adult brain. Results from immunostaining and live cell imaging analysis reveal that a portion of Atg9 localizes to the trans-Golgi network, autophagosomes, and lysosomes in glia. Atg9 is persistently in contact with these organelles. Lacking glial atg9 reduces the number of omegasomes and autophagosomes, and impairs autophagic substrate degradation. This suggests that glial Atg9 participates in the early steps of autophagy, and hence the control of autophagic degradation. Importantly, loss of glial atg9 induces parkinsonian symptoms in Drosophila including progressive loss of dopaminergic neurons, locomotion deficits, and glial activation. These findings identify a functional role of Atg9 in glial autophagy and establish a potential link between glial autophagy and Parkinson's disease. These results may provide new insights on the underlying mechanism of Parkinson's disease.
Oyarce-Pezoa, S., Rucatti, G. G., Muñoz-Carvajal, F., Sanhueza, N., Gomez, W., Espinoza, S., Leiva, M., García, N., Ponce, D. P., SanMartín, C. D., Rojas-Rivera, D., Salvadores, N., Behrens, M. I., Woehlbier, U., Calegaro-Nassif, M., Sanhueza, M. (2023). The autophagy protein Def8 is altered in Alzheimer's disease and Aβ42-expressing Drosophila brains. Sci Rep, 13(1):17137 PubMed ID: 37816871
Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by protein accumulation in the brain as a main neuropathological hallmark. Among them, Aβ42 peptides (see Drosophila APPL) tend to aggregate and create oligomers and plaques. Macroautophagy, a form of autophagy characterized by a double-membrane vesicle, plays a crucial role in maintaining neuronal homeostasis by degrading protein aggregates and dysfunctional organelles as a quality control process. Recently, DEF8, a relatively uncharacterized protein, has been proposed as a participant in vesicular traffic and autophagy pathways. We have reported increased DEF8 levels in lymphocytes from mild cognitive impairment (MCI) and early-stage AD patients and a neuronal profile in a murine transgenic AD model. This study analyzed DEF8 localization and levels in the postmortem frontal cortex of AD patients, finding increased levels compared to healthy controls. To evaluate the potential function of DEF8 in the nervous system, an in silico assessment wzs performed of its expression and network profiles, followed by an in vivo evaluation of a neuronal Def8 deficient model using a Drosophila melanogaster model of AD based on Aβ42 expression. These findings show that DEF8 is an essential protein for maintaining cellular homeostasis in the nervous system, and it is upregulated under stress conditions generated by Aβ42 aggregation. This study suggests DEF8 as a novel actor in the physiopathology of AD, and its exploration may lead to new treatment avenues.
Moore, M. C., Taylor, D. T. (2023). Effects of valproate on seizure-like activity in Drosophila melanogaster with a knockdown of Ube3a in different neuronal populations as a model of Angelman Syndrome. Epilepsy & behavior reports, 24:100622 PubMed ID: 37842098
Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. This study combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, a highly migratory and immune-responsive pupal cell population was identified expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, this study presents the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis.
Mirzoyan, Z., Valenza, A., Zola, S., Bonfanti, C., Arnaboldi, L., Ferrari, N., Pollard, J., Lupi, V., Cassinelli, M., Frattaroli, M., Sahin, M., Pasini, M. E., Bellosta, P. (2023). A Drosophila model targets Eiger/TNFα to alleviate obesity-related insulin resistance and macrophage infiltration. Disease models & mechanisms, 16(11) PubMed ID: 37828911
Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a new Drosophila model to investigate the mechanisms underlying these obesity-related pathologies. Genetic manipulation was employed to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy and high circulating glucose levels. Moreover, significant infiltration of immune cells (hemocytes) into the fat bodies, accompanied by insulin resistance. Attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidants anthocyanins, both phenotypes were ameliorated. The data highlight evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. This model can also provide a platform to perform genetic screens or test the efficacy of therapeutic interventions for diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease.

Friday, March 22th - Developmental Biology

Hirschhauser, A., Molitor, D., Salinas, G., Grosshans, J., Rust, K., Bogdan, S. (2023). Single-cell transcriptomics identifies new blood cell populations in Drosophila released at the onset of metamorphosis. Development, 150(18) PubMed ID: . PubMed ID: 37681301
Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. This study combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, a highly migratory and immune-responsive pupal cell population was identified expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, this study presents the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis.
Clark, J. M., Gibbs, A. G. (2023). Starvation selection reduces and delays larval ecdysone production and signaling. The Journal of experimental biology, 226(18) PubMed ID: . PubMed ID: 37671530
Previous studies have shown that selection for starvation resistance in Drosophila melanogaster results in delayed eclosion and increased adult fat stores. It is assumed that these traits are caused by the starvation selection pressure, but its mechanism is unknown. This study found that starvation-selected (SS) population stores more fat during larval development and has extended larval development and pupal development time. Developmental checkpoints in the third instar associated with ecdysteroid hormone pulses are increasingly delayed. The delay in the late larval period seen in the SS population is indicative of reduced and delayed ecdysone signaling. An enzyme immunoassay for ecdysteroids (with greatest affinity to the metabolically active 20-hydroxyecdysone and the α-ecdysone precursor) confirmed that the SS population had reduced and delayed hormone production compared with that of fed control (FC) flies. Feeding third instar larvae on food supplemented with α-ecdysone partially rescued the developmental delay and reduced subsequent adult starvation resistance. This work suggests that starvation selection causes reduced and delayed production of ecdysteroids in the larval stage and affects the developmental delay phenotype that contributes to subsequent adult fat storage and starvation resistance.
Bernasek, S. M., Hur, S. S. J., Pelaez-Restrepo, N., Lachance, J. B., Bakker, R., Navarro, H. T., Sanchez-Luege, N., Amaral, L. A. N., Bagheri, N., Rebay, I. and Carthew, R. W. (2023). Ratiometric sensing of Pnt and Yan transcription factor levels confers ultrasensitivity to photoreceptor fate transitions in Drosophila. Development. PubMed ID: 36942737
Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. This study investigated how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein despite expressing both proteins with pulsatile dynamics. A rapid and sustained two-fold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a two-fold shift in the ratio. A biophysical model based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how two-fold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program (Bernasek, 2023).
Liu, B. P., Hua, B. Z. (2024). Distinct roles of the Hox genes Ultrabithorax and abdominal-A in scorpionfly embryonic proleg development. Insect Mol Biol, 33(1):69-80 PubMed ID: 37792400
The abdominal appendages of larval insects have a complex evolutionary history of gain and loss, but the regulatory mechanisms underlying the abdominal appendage development remain largely unclear. This study investigated the embryogenesis of abdominal prolegs in the scorpionfly Panorpa liui Hua (Mecoptera: Panorpidae) using in situ hybridization and parental RNA interference. The results show that RNAi-mediated knockdown of Ultrabithorax (Ubx) led to a homeotic transformation of the first abdominal segment (A1) into the third thoracic segment (T3) and changed the distributions of the downstream target Distal-less (Dll) expression but did not affect the expression levels of Dll. Knockdown of abdominal-A (abd-A) resulted in malformed segments, abnormal prolegs and disrupted Dll expression. The results demonstrate that the gene Ubx maintains an ancestral role of modulating A1 appendage fate without preventing Dll initiation, and a secondary adaptation of abd-A evolves the ability to specify abdominal segments and proleg identity. It is concluded that changes in abdominal Hox gene expression and their target genes regulate abdominal appendage morphology during the evolutionary course of holometabolous larvae.
Tsuboi, A., Fujimoto, K., Kondo, T. (2023). Spatiotemporal remodeling of extracellular matrix orients epithelial sheet folding. Sci Adv, 9(35):eadh2154 PubMed ID: 37656799
Biological systems are inherently noisy; however, they produce highly stereotyped tissue morphology. Drosophila pupal wings show a highly stereotypic folding through uniform expansion and subsequent buckling of wing epithelium within a surrounding cuticle sac. The folding pattern produced by buckling is generally stochastic; it is thus unclear how buckling leads to stereotypic tissue folding of the wings. This study found that the extracellular matrix (ECM) protein, Dumpy, guides the position and direction of buckling-induced folds. Dumpy anchors the wing epithelium to the overlying cuticle at specific tissue positions. Tissue-wide alterations of Dumpy deposition and degradation yielded different buckling patterns. In summary, it is proposed that spatiotemporal ECM remodeling shapes stereotyped tissue folding through dynamic interactions between the epithelium and its external structures.
Perez-Mockus, G., Cocconi, L., Alexandre, C., Aerne, B., Salbreux, G., Vincent, J. P. (2023). The Drosophila ecdysone receptor promotes or suppresses proliferation according to ligand level. Dev Cell, 58(20):2128-2139 PubMed ID: 37769663
The steroid hormone 20-hydroxy-ecdysone (20E) promotes proliferation in Drosophila wing precursors at low titer but triggers proliferation arrest at high doses. Remarkably, wing precursors proliferate normally in the complete absence of the 20E receptor, suggesting that low-level 20E promotes proliferation by overriding the default anti-proliferative activity of the receptor. By contrast, 20E needs its receptor to arrest proliferation. Dose-response RNA sequencing (RNA-seq) analysis of ex vivo cultured wing precursors identifies genes that are quantitatively activated by 20E across the physiological range, likely comprising positive modulators of proliferation and other genes that are only activated at high doses. It is suggested that some of these 'high-threshold' genes dominantly suppress the activity of the pro-proliferation genes. It was then shown mathematically and with synthetic reporters that combinations of basic regulatory elements can recapitulate the behavior of both types of target genes. Thus, a relatively simple genetic circuit can account for the bimodal activity of this hormone.

Thursday, March 21th - RNAs and Transposons

Courret, C., Larracuente, A. M. (2023). High levels of intra-strain structural variation in Drosophila simulans X pericentric heterochromatin. Genetics, 225(4) PubMed ID: 37768175
Large genome structural variations can impact genome regulation and integrity. Repeat-rich regions like pericentric heterochromatin are vulnerable to structural rearrangements although we know little about how often these rearrangements occur over evolutionary time. Repetitive genome regions are particularly difficult to study with genomic approaches, as they are missing from most genome assemblies. However, cytogenetic approaches offer a direct way to detect large rearrangements involving pericentric heterochromatin. This study used a cytogenetic approach to reveal large structural rearrangements associated with the X pericentromeric region of Drosophila simulans. These rearrangements involve large blocks of satellite DNA-the 500-bp and Rsp-like satellites-which colocalize in the X pericentromeric heterochromatin. This region is polymorphic not only among different strains, but between isolates of the same strain from different labs, and even within individual isolates. On the one hand, these observations raise questions regarding the potential impact of such variation at the phenotypic level and the ability to control for such genetic variability. This highlights the very rapid turnover of the pericentric heterochromatin most likely associated with genomic instability of the X pericentromere. It represents a unique opportunity to study the dynamics of pericentric heterochromatin, the evolution of associated satellites on a very short time scale, and to better understand how structural variation arises.
Gerve, M. P., Sanchez, J. A., Ingaramo, M. C., Dekanty, A. (2023). Myc-regulated miRNAs modulate p53 expression and impact animal survival under nutrient deprivation. PLoS Genet, 19(8):e1010721 PubMed ID: 37639481
The conserved transcription factor Myc regulates cell growth, proliferation and apoptosis, and its deregulation has been associated with human pathologies. Although specific miRNAs have been identified as fundamental components of the Myc tumorigenic program, how Myc regulates miRNA biogenesis remains controversial. This study shows that Myc functions as an important regulator of miRNA biogenesis in Drosophila by influencing both miRNA gene expression and processing. Through the analysis of ChIP-Seq datasets, it was discovered that nearly 56% of Drosophila miRNA genes show dMyc binding, exhibiting either the canonical or non-canonical E-box sequences within the peak region. Consistently, reduction of dMyc levels resulted in widespread downregulation of miRNAs gene expression. dMyc also modulates miRNA processing and activity by controlling Drosha and AGO1 levels through direct transcriptional regulation. By using in vivo miRNA activity sensors this study demonstrated that dMyc promotes miRNA-mediated silencing in different tissues, including the wing primordium and the fat body. It was also shown that dMyc-dependent expression of miR-305 in the fat body modulates Dmp53 levels depending on nutrient availability, having a profound impact on the ability of the organism to respond to nutrient stress. Indeed, dMyc depletion in the fat body resulted in extended survival to nutrient deprivation which was reverted by expression of either miR-305 or a dominant negative version of Dmp53. This study reveals a previously unrecognized function of dMyc as an important regulator of miRNA biogenesis and suggests that Myc-dependent expression of specific miRNAs may have important tissue-specific functions.
Kiuchi, T., Shoji, K., Izumi, N., Tomari, Y., Katsuma, S. (2023). Non-gonadal somatic piRNA pathways ensure sexual differentiation, larval growth, and wing development in silkworms. PLoS Genet, 19(9):e1010912 PubMed ID: 37733654
PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. This study generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. These results highlight the roles of non-gonadal somatic piRNAs in B. mori development.
Bodelon, A., Fablet, M., Siqueira de Oliveira, D., Vieira, C., García Guerreiro, M. P. (2023). Impact of Heat Stress on Transposable Element Expression and Derived Small RNAs in Drosophila subobscura. Genome biology and evolution, 15(11) PubMed ID: 37847062
Global warming is forcing insect populations to move and adapt, triggering adaptive genetic responses. Thermal stress is known to alter gene expression, repressing the transcription of active genes, and inducing others, such as those encoding heat shock proteins. It has also been related to the activation of some specific transposable element (TE) families. However, the actual magnitude of this stress on the whole genome and the factors involved in these genomic changes are still unclear. mRNAs and small RNAs were studied in gonads of two Drosophila subobscura populations, considered a good model to study adaptation to temperature changes. In control conditions, it was found that a few genes and TE families were differentially expressed between populations, pointing out their putative involvement in the adaptation of populations to their different environments. Under heat stress, sex-specific changes in gene expression together with a trend toward overexpression, mainly of heat shock response-related genes, were observed. No large changes of TE expression nor small RNA production due to stress were observed. Only population and sex-specific expression changes of some TE families (mainly retrotransposons), or the amounts of siRNAs and piRNAs, derived from specific TE families were observed, as well as the piRNA production from some piRNA clusters. Changes in small RNA amounts and TE expression could not be clearly correlated, indicating that other factors as chromatin modulation could also be involved. This work provides the first whole transcriptomic study including genes, TEs, and small RNAs after a heat stress in D. subobscura.
Fablet, M., Salces-Ortiz, J., Jacquet, A., Menezes, B. F., Dechaud, C., Veber, P., Rebollo, R., Vieira, C. (2023). A Quantitative, Genome-Wide Analysis in Drosophila Reveals Transposable Elements' Influence on Gene Expression is Species-Specific. Genome biology and evolution, 15(9) PubMed ID: 37652057
Transposable elements (TEs) are parasite DNA sequences that are able to move and multiply along the chromosomes of all genomes. They can be controlled by the host through the targeting of silencing epigenetic marks, which may affect the chromatin structure of neighboring sequences, including genes. This study used transcriptomic and epigenomic high-throughput data produced from ovarian samples of several Drosophila melanogaster and Drosophila simulans wild-type strains, in order to finely quantify the influence of TE insertions on gene RNA levels and histone marks (H3K9me3 and H3K4me3). Thw results reveal a stronger epigenetic effect of TEs on ortholog genes in D. simulans compared with D. melanogaster. At the same time, a larger contribution of TEs to gene H3K9me3 variance within genomes in D. melanogaster, which is evidenced by a stronger correlation of TE numbers around genes with the levels of this chromatin mark in D. melanogaster. Overall, this work contributes to the understanding of species-specific influence of TEs within genomes. It provides a new light on the considerable natural variability provided by TEs, which may be associated with contrasted adaptive and evolutionary potentials.
Wierzbicki, F., Kofler, R. (2023). The composition of piRNA clusters in Drosophila melanogaster deviates from expectations under the trap model. BMC Biol, 21(1):224 PubMed ID: 37858221
It is widely assumed that the invasion of a transposable element (TE) in mammals and invertebrates is stopped when a copy of the TE jumps into a piRNA cluster (i.e., the trap model). However, recent works, which for example showed that deletion of three major piRNA clusters has no effect on TE activity, cast doubt on the trap model. This study tested the trap model from a population genetics perspective. Simulations show that the composition of regions that act as transposon traps (i.e., potentially piRNA clusters) ought to deviate from regions that have no effect on TE activity. TEs were investigated in five Drosophila melanogaster strains using three complementary approaches to test whether the composition of piRNA clusters matches these expectations. It was found that the abundance of TE families inside and outside of piRNA clusters is highly correlated, although this is not expected under the trap model. Furthermore, the distribution of the number of TE insertions in piRNA clusters is also much broader than expected. This study has found that the observed composition of piRNA clusters is not in agreement with expectations under the simple trap model. Dispersed piRNA producing TE insertions and temporal as well as spatial heterogeneity of piRNA clusters may account for these deviations.

Wednesday, March 20th - Signaling

Meng, Q., Li, Y., Xu, Y., Wang, Y. (2024). Acetobacter and lactobacillus alleviate the symptom of insulin resistance by blocking the JNK-JAK/STAT pathway in Drosophila melanogaster. Biochimica et biophysica acta Molecular basis of disease, 1870(1):166901 PubMed ID: 37774935
The dysregulation of intestinal microbiota is well-known to be one of the main causes of insulin resistance in both vertebrates and invertebrates. Specially, the acetobacter and lactobacillus have been identified as potentially capable of alleviating insulin resistance. However, the molecular mechanism underlying this effect requires further elucidation. In this study, Drosophila was used to delineate how intestinal microbiota disrupts the host intestinal signaling pathway, contributing to insulin resistance. These findings demonstrate that a long-term high-sugar diet leads to a reduction in the general diversity of intestinal microbiota in flies, as well as a marked decrease in the abundances of acetobacter and lactobacillus. Furthermore, symptoms of insulin resistance were alleviated by feeding flies with acetobacter or lactobacillus, indicating that these microorganisms play an essential role in maintaining blood sugar homeostasis in flies. Conversely, when all intestinal microbiota was removed, flies show severe symptoms of insulin resistance, confirming that the critical role of intestinal microbiota in maintaining host blood sugar homeostasis. These studies suggested that the intestinal but not fat body JNK pathway mediates the communication of intestinal microbiota and host insulin pathway. In flies, downregulation of JNK activity alleviates symptoms of insulin resistance by decreasing the activity of the JAK/STAT pathway. However, this offsets the therapeutic effects of supplying flies with acetobacter or lactobacillus, suggesting that the therapeutic function of these microorganisms is based on their interaction with JNK-JAK/STAT axis. Taken together, this study reveals that acetobacter and lactobacillus alleviate insulin resistance symptoms in a JNK-JAK/STAT pathway-dependent manner, indicating the therapeutic potential of probiotic supplementation and regulation of the activities of JNK-JAK/STAT pathway for diabetes control.
DeGroot, M. S., Williams, B., Chang, T. Y., Maas Gamboa, M. L., Larus, I. M., Hong, G., Fromme, J. C., Liu, J. (2023). SMOC-1 interacts with both BMP and glypican to regulate BMP signaling in C. elegans. PLoS Biol, 21(8):e3002272 PubMed ID: 37590248
Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium-binding (EC) domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. This study used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. Both in vitro biochemical and in vivo functional evidence is provided demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. It was further shown that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. This work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling.
Rader, A. E., Bayarmagnai, B., Frolov, M. V. (2023). Combined inactivation of RB and Hippo converts differentiating Drosophila photoreceptors into eye progenitor cells through derepression of homothorax. Dev Cell, 58(21):2261-2274 PubMed ID: 37848027
The retinoblastoma (RB) and Hippo pathways interact to regulate cell proliferation and differentiation. However, the mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain their neuronal identity and dedifferentiate. Single-cell RNA sequencing was performed to elucidate the cause of dedifferentiation and to determine the fate of these cells. Dedifferentiated cells were found to adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in the activation of a distinct Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. Rbf physically interacts with Yki and, together with the GAGA factor, inhibits the hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differentiating photoreceptors. This work highlights the importance of both RB and Hippo pathway activities for maintaining the state of terminal differentiation.
Bare, Y., Matusek, T., Vriz, S., Deffieu, M. S., Therond, P. P., Gaudin, R. (2023). TMED10 mediates the loading of neosynthesised Sonic Hedgehog in COPII vesicles for efficient secretion and signalling. Cell Mol Life Sci, 80(9):266 PubMed ID: 37624561
The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. This study employed the retention using selective hooks (RUSH) system to show that newly-synthesized SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, the Drosophila wing imaginal disc model to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, this work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesized SHH secretion with potential impact on embryonic development.
Juarez-Carreno, S., Geissmann, F. (2023). The macrophage genetic cassette inr/dtor/pvf2 is a nutritional status checkpoint for developmental timing. Sci Adv, 9(38):eadh0589 PubMed ID: 37729406
A small number of signaling molecules, used reiteratively, control differentiation programs, but the mechanisms that adapt developmental timing to environmental cues are less understood. This study reports that a macrophage inr/dtor/pvf2 genetic cassette is a developmental timing checkpoint in Drosophila, which either licenses or delays biosynthesis of the steroid hormone in the endocrine gland and metamorphosis according to the larval nutritional status. Insulin receptor/dTor signaling in macrophages is required and sufficient for production of the PDGF/VEGF family growth factor Pvf2, which turns on transcription of the sterol biosynthesis Halloween genes in the prothoracic gland via its receptor Pvr. In response to a starvation event or genetic manipulation, low Pvf2 signal delays steroid biosynthesis until it becomes Pvr-independent, thereby prolonging larval growth before pupariation. The significance of this developmental timing checkpoint for host fitness is illustrated by the observation that it regulates the size of the pupae and adult flies.
Wilcockson, S. G., Guglielmi, L., Araguas Rodriguez, P., Amoyel, M., Hill, C. S. (2023). An improved Erk biosensor detects oscillatory Erk dynamics driven by mitotic erasure during early development. Dev Cell, 58(23):2802-2818.e2805 PubMed ID: 37714159
Extracellular signal-regulated kinase (Erk) signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics in vivo, as a gradient of activated diphosphorylated Erk (P-Erk) is induced by fibroblast growth factor (Fgf) signaling at the blastula margin. This study describes an improved Erk-specific biosensor, which we term modified Erk kinase translocation reporter (modErk-KTR). The utility of this biosensor was demonstrated in vitro and in developing zebrafish and Drosophila embryos. Moreover, it was shown that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Erk activity is rapidly extinguished just prior to mitosis, which is referred to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. These modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics in vivo.

Tuesday March 19th - Adult Neural Structure, Development and Function

Lin, Z., Green, E. W., Webster, S. G., Hastings, M. H., Wilcockson, D. C., Kyriacou, C. P. (2023). The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach). PLoS Genet, 19(10):e1011011 PubMed ID: 37856540
Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 (see Drosophila Cryptochrome) and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. This study shows that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. DsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 was performed in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. A neurogenetic model is suggested whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.
Mabuchi, Y., Cui, X., Xie, L., Kim, H., Jiang, T., Yapici, N. (2023). Visual feedback neurons fine-tune Drosophila male courtship via GABA-mediated inhibition. Curr Biol, 33(18):3896-3910.e3897 PubMed ID: 37673068
Many species of animals use vision to regulate their social behaviors. However, the molecular and circuit mechanisms underlying visually guided social interactions remain largely unknown. This study shows that the Drosophila ortholog of the human GABA(A)-receptor-associated protein (GABARAP), Autophagy-related 8a is required in a class of visual feedback neurons, lamina tangential (Lat) cells, to fine-tune male courtship. GABARAP is a ubiquitin-like protein that maintains cell-surface levels of GABA(A) receptors. Knocking down GABARAP or GABA(A)receptors in Lat neurons or hyperactivating them induces male courtship toward other males. Inhibiting Lat neurons, on the other hand, delays copulation by impairing the ability of males to follow females. Remarkably, the fly GABARAP protein and its human ortholog share a strong sequence identity, and the fly GABARAP function in Lat neurons can be rescued by its human ortholog. Using in vivo two-photon imaging and optogenetics, it was revealed that Lat neurons are functionally connected to neural circuits that mediate visually guided courtship pursuits in males. This work identifies a novel physiological function for GABARAP in regulating visually guided courtship pursuits in Drosophila males. Reduced GABA(A) signaling has been linked to social deficits observed in the autism spectrum and bipolar disorders. The functional similarity between the human and the fly GABARAP raises the possibility of a conserved role for this gene in regulating social behaviors across insects and mammals.
Bustillo, M. E., Douthit, J., Astigarraga, S., Treisman, J. E. (2023). Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis. bioRxiv, PubMed ID: 37609142
Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. This study found that in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of Semaphorin function indicates that Semaphorin 1a, provided by cells that include Tm5 neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A does not disrupt the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles. It is concluded that the axon guidance molecule Plexin A has two functions in Drosophila medulla development; morphogenesis of the neuropil requires its cytoplasmic domain, but establishing synaptic layers through Semaphorin 1a does not.
Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology 61(1):487-497 PubMed ID: 37626270
Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. The current study made an attempt to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. It was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia.
Chen, C. C., Lin, H. W., Feng, K. L., Tseng, D. W., de Belle, J. S., Chiang, A. S. (2023). A subset of cholinergic mushroom body neurons blocks long-term memory formation in Drosophila. Cell Rep, 42(8):112974 PubMed ID: 37590142
Long-term memory (LTM) requires learning-induced synthesis of new proteins allocated to specific neurons and synapses in a neural circuit. Not all learned information, however, becomes permanent memory. How the brain gates relevant information into LTM remains unclear. In Drosophila adults, weak learning after a single training session in an olfactory aversive task typically does not induce protein-synthesis-dependent LTM. Instead, strong learning after multiple spaced training sessions is required. This study reports that pre-synaptic active-zone protein synthesis and cholinergic signaling from the early α/β subset of mushroom body (MB) neurons produce a downstream inhibitory effect on LTM formation. When inhibitory signaling was eliminated from these neurons, weak learning was then sufficient to form LTM. This bidirectional circuit mechanism modulates the transition between distinct memory phase functions in different subpopulations of MB neurons in the olfactory memory circuit.
Wu, M. S., Liao, T. W., Wu, C. Y., Hsieh, T. H., Kuo, P. C., Li, Y. C., Cheng, K. C., Chiang, H. C. (2023). Aversive conditioning information transmission in Drosophila. Cell Rep, 42(10):113207 PubMed ID: 37782557
Animals rapidly acquire surrounding information to perform the appropriate behavior. Although social learning is more efficient and accessible than self-learning for animals, the detailed regulatory mechanism of social learning remains unknown, mainly because of the complicated information transfer between animals, especially for aversive conditioning information transmission. The current study revealed that, during social learning, the neural circuit in observer flies used to process acquired aversive conditioning information from demonstrator flies differs from the circuit used for self-learned classic aversive conditioning. This aversive information transfer is species dependent. Solitary flies cannot learn this information through social learning, suggesting that this ability is not an innate behavior. Neurons used to process and execute avoidance behavior to escape from electrically shocked flies are all in the same brain region, indicating that the fly brain has a common center for integrating external stimuli with internal states to generate flight behavior.

Monday March 18th - Chromatin

DiVito Evans, A., Fairbanks, R. A., Schmidt, P., Levine, M. T. (2023). Histone methylation regulates reproductive diapause in Drosophila melanogaster. PLoS Genet, 19(9):e1010906 PubMed ID: 37703303
Fluctuating environments threaten fertility and viability. To better match the immediate, local environment, many organisms adopt alternative phenotypic states, a phenomenon called "phenotypic plasticity." Natural populations that predictably encounter fluctuating environments tend to be more plastic than conspecific populations that encounter a constant environment, suggesting that phenotypic plasticity can be adaptive. Despite pervasive evidence of such "adaptive phenotypic plasticity," gene regulatory mechanisms underlying plasticity remains poorly understood. This study tested the hypothesis that environment-dependent phenotypic plasticity is mediated by epigenetic factors. To test this hypothesis, the adaptive reproductive arrest of Drosophila melanogaster females, called diapause, was exploited. Using an inbred line from a natural population with high diapause plasticity, it was demonstrated that diapause is determined epigenetically: only a subset of genetically identical individuals enter diapause and this diapause plasticity is epigenetically transmitted for at least three generations. Upon screening a suite of epigenetic marks, it was discovered that the active histone marks H3K4me3 and H3K36me1 are depleted in diapausing ovaries. Using ovary-specific knockdown of histone mark writers and erasers, it was demonstrated that H3K4me3 and H3K36me1 depletion promotes diapause. Given that diapause is highly polygenic, that is, distinct suites of alleles mediate diapause plasticity across distinct genotypes, the potential for genetic variation in diapause-determining epigenetic marks was also investigated. Specifically, it was asked if these histone marks were similarly depleted in diapause of a genotypically distinct line. Evidence was found of divergence in both the gene expression program and histone mark abundance. This study reveals chromatin determinants of phenotypic plasticity and suggests that these determinants may be genotype-dependent, offering new insight into how organisms may exploit and evolve epigenetic mechanisms to persist in fluctuating environments.
Godneeva, B., Ninova, M., Fejes Tóth, K., Aravin, A. A. (2023). SUMOylation of Bonus, the Drosophila homolog of Transcription Intermediary Factor 1, safeguards germline identity by recruiting repressive chromatin complexes to silence tissue-specific genes. bioRxiv, PubMed ID: 37645991
The conserved family of Transcription Intermediary Factors (TIF1) proteins consists of key transcriptional regulators that control transcription of target genes by modulating chromatin state. Unlike mammals that have four TIF1 members, Drosophila only encodes one member of the family, Bonus. Bonus has been implicated in embryonic development and organogenesis and shown to regulate several signaling pathways, however, its targets and mechanism of action remained poorly understood. This study found that knockdown of Bonus in early oogenesis results in severe defects in ovarian development and in ectopic expression of genes that are normally repressed in the germline, demonstrating its essential function in the ovary. Recruitment of Bonus to chromatin leads to silencing associated with accumulation of the repressive H3K9me3 mark. Bonus associates with the histone methyltransferase SetDB1 and the chromatin remodeler NuRD and depletion of either component releases Bonus-induced repression. This study further established that Bonus is SUMOylated at a single site at its N-terminus that is conserved among insects and this modification is indispensable for Bonus's repressive activity. SUMOylation influences Bonus's subnuclear localization, its association with chromatin and interaction with SetDB1. Finally, Bonus SUMOylation was shown to be mediated by the SUMO E3-ligase Su(var)2-10, revealing that although SUMOylation of TIF1 proteins is conserved between insects and mammals, both the mechanism and specific site of modification is different in the two taxa. Together, this work identified Bonus as a regulator of tissue-specific gene expression and revealed the importance of SUMOylation as a regulator of complex formation in the context of transcriptional repression.
Rajshekar, S., Adame-Arana, O., Bajpai, G., Lin, K., Colmenares, S., Safran, S., Karpen, G. H. (2023). Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates. bioRxiv, PubMed ID: 37808710
Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, it is proposed that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.
Soukar, I., Mitra, A., Pile, L. A. (2023). Analysis of the chromatin landscape and RNA polymerase II binding at SIN3-regulated genes. Biol Open, 12(11) PubMed ID: 37850739
The chromatin environment has a significant impact on gene expression. Chromatin structure is highly regulated by histone modifications and RNA polymerase II binding dynamics. The SIN3 histone modifying complex regulates the chromatin environment leading to changes in gene expression. In Drosophila melanogaster, the Sin3A gene is alternatively spliced to produce different protein isoforms, two of which include SIN3 220 and SIN3 187. Both SIN3 isoforms are scaffolding proteins that interact with several other factors to regulate the chromatin landscape. The mechanism through which the SIN3 isoforms regulate chromatin is not well understood. This study analyze publicly available data sets to facilitate specific questions on how SIN3 isoforms regulate chromatin and gene activity. It was determined that genes repressed by the SIN3 isoforms exhibited enrichment in histone H3K4me2, H3K4me3, H3K14ac and H3K27ac near the transcription start site. An increase was observed in the amount of paused RNA polymerase II on the promoter of genes repressed by the isoforms as compared to genes that require SIN3 for maximum activation. Furthermore, a subset of genes was analyzed regulated by SIN3 187 that suggest a mechanism in which SIN3 187 might exhibit hard regulation as well as soft regulation. Data presented in this study expand knowledge of how the SIN3 isoforms regulate the chromatin environment and RNA polymerase II binding dynamics.
Zraly, C. B., Schultz, R., Diaz, M. O., Dingwall, A. K. (2023). New twists of a TAIL: novel insights into the histone binding properties of a highly conserved PHD finger cluster within the MLR family of H3K4 mono-methyltransferases. Nucleic Acids Res, 51(18):9672-9689 PubMed ID: 37638761
Enhancer activation by the MLR family of H3K4 mono-methyltransferases requires proper recognition of histones for the deposition of the mono-methyl mark. MLR proteins contain two clusters of PHD zinc finger domains implicated in chromatin regulation. The second cluster is the most highly conserved, preserved as an ancient three finger functional unit throughout evolution. Studies of the isolated 3rd PHD finger within this cluster suggested specificity for the H4 [aa16-20] tail region. The histone binding properties were determined of the full three PHD finger cluster b module (PHDb) from the Drosophila Cmi protein which revealed unexpected recognition of an extended region of H3. Importantly, the zinc finger spacer separating the first two PHDb fingers from the third is critical for proper alignment and coordination among fingers for maximal histone engagement. Human homologs, MLL3 and MLL4, also show conservation of H3 binding, expanding current views of histone recognition for this class of proteins. Chromatin remodeling was further implicated by the SWI/SNF complex as a possible mechanism for the accessibility of PHDb to globular regions of histone H3 beyond the tail region. These results suggest a two-tail histone recognition mechanism by the conserved PHDb domain involving a flexible hinge to promote interdomain coordination.
Messina, O., Raynal, F., Gurgo, J., Fiche, J. B., Pancaldi, V., Nollmann, M. (2023). 3D chromatin interactions involving Drosophila insulators are infrequent but preferential and arise before TADs and transcription. Nat Commun, 14(1):6678 PubMed ID: 37865700
In mammals, insulators contribute to the regulation of loop extrusion to organize chromatin into topologically associating domains. In Drosophila the role of insulators in 3D genome organization is, however, under current debate. This study addressed this question by combining bioinformatics analysis and multiplexed chromatin imaging. A class of Drosophila insulators enriched at regions forming preferential chromatin interactions genome-wide. Notably, most of these 3D interactions do not involve TAD borders. Multiplexed imaging shows that these interactions occur infrequently, and only rarely involve multiple genomic regions coalescing together in space in single cells. Finally, it was shown that non-border preferential 3D interactions enriched in this class of insulators are present before TADs and transcription during Drosophila development. These results are inconsistent with insulators forming stable hubs in single cells, and instead suggest that they fine-tune existing 3D chromatin interactions, providing an additional regulatory layer for transcriptional regulation. are present before TADs and transcription during Drosophila development. These results are inconsistent with insulators forming stable hubs in single cells, and instead suggest that they fine-tune existing 3D chromatin interactions, providing an additional regulatory layer for transcriptional regulation.

Friday March 15th - Embryonic Development

Cheikh, M. I., Tchoufag, J., Osterfield, M., Dean, K., Bhaduri, S., Zhang, C., Mandadapu, K. K., Doubrovinski, K. (2023). A comprehensive model of Drosophila epithelium reveals the role of embryo geometry and cell topology in mechanical responses. Elife, 12 PubMed ID: 37782009
In order to understand morphogenesis, it is necessary to know the material properties or forces shaping the living tissue. In spite of this need, very few in vivo measurements are currently available. Using the early Drosophila embryo as a model, this study describes a novel cantilever-based technique which allows for the simultaneous quantification of applied force and tissue displacement in a living embryo. By analyzing data from a series of experiments in which embryonic epithelium is subjected to developmentally relevant perturbations, it is concluded that the response to applied force is adiabatic and is dominated by elastic forces and geometric constraints, or system size effects. Crucially, computational modeling of the experimental data indicated that the apical surface of the epithelium must be softer than the basal surface, a result which was confirmed experimentally. Further, the combination of experimental data and comprehensive computational model was used to estimate the elastic modulus of the apical surface and set a lower bound on the elastic modulus of the basal surface. More generally, these investigations revealed important general features that should be more widely addressed when quantitatively modeling tissue mechanics in any system. Specifically, different compartments of the same cell can have very different mechanical properties; when they do, they can contribute differently to different mechanical stimuli and cannot be merely averaged together. Additionally, tissue geometry can play a substantial role in mechanical response, and cannot be neglected.
Dey, B., Mitra, D., Das, T., Sherlekar, A., Balaji, R., Rikhy, R. (2023). Adhesion and Polarity protein distribution-regulates hexagon dominated plasma membrane organization in Drosophila blastoderm embryos. Genetics, 225(4) PubMed ID: 37804533
Epithelial cells contain polarity complexes on the lateral membrane and are organized in a hexagon-dominated polygonal array. The mechanisms regulating the organization of polygonal architecture in metazoan embryogenesis are not completely understood. Drosophila embryogenesis enables mechanistic analysis of epithelial polarity formation and its impact on polygonal organization. The plasma membrane (PM) of syncytial Drosophila blastoderm embryos is organized as a polygonal array with pseudocleavage furrow formation during the almost synchronous cortical division cycles. Polygonal (PM) organization arises in the metaphase (MP) of division cycle 11, and hexagon dominance occurs with an increase in furrow length in the metaphase of cycle 12. There is a decrease in cell shape index in metaphase from cycles 11 to 13. This coincides with Drosophila E-cad (DE-cadherin) and Bazooka enrichment at the edges and the septin, Peanut at the vertices of the furrow. The role of polarity and adhesion proteins in pseudocleavage furrow formation and its organization as a polygonal array was assessed. DE-cadherin depletion leads to decreased furrow length, loss of hexagon dominance, and increased cell shape index. Bazooka and Peanut depletion lead to decreased furrow length, delay in onset of hexagon dominance from cycle 12 to 13, and increased cell shape index. Hexagon dominance occurs with an increase in furrow length in cycle 13 and increased DE-cadherin, possibly due to the inhibition of endocytosis. It is concluded that polarity protein recruitment and regulation of endocytic pathways enable pseudocleavage furrow stability and the formation of a hexagon-dominated polygon array.
Smits, C. M., Dutta, S., Jain-Sharma, V., Streichan, S. J. and Shvartsman, S. Y. (2023). Maintaining symmetry during body axis elongation. Curr Biol 33(16): 3536-3543. PubMed ID: 37562404
Bilateral symmetry defines much of the animal kingdom and is crucial for numerous functions of bilaterian organisms. Genetic approaches have discovered highly conserved patterning networks that establish bilateral symmetry in early embryos,(1) but how this symmetry is maintained throughout subsequent morphogenetic events remains largely unknown.(2) This study shows that the terminal patterning system-which relies on Ras/ERK signaling through activation of the Torso receptor by its ligand Trunk(3)-is critical for preserving bilateral symmetry during Drosophila body axis elongation, a process driven by cell rearrangements in the two identical lateral regions of the embryo and specified by the dorsal-ventral and anterior-posterior patterning systems.(4)Fluctuating asymmetries were detected in this rapid convergent-extension process are attenuated in normal embryos over time, possibly through noise-dissipating forces from the posterior midgut invagination and movement. However, when Torso signaling is attenuated via mutation of Trunk or RNAi directed against downstream Ras/ERK pathway components, body axis elongation results in a characteristic corkscrew phenotype,(5) which reflects dramatic reorganization of global tissue flow and is incompatible with viability. Our results reveal a new function downstream of the Drosophila terminal patterning system in potentially active control of bilateral symmetry and should motivate systematic search for similar symmetry-preserving regulatory mechanisms in other bilaterians.
Frasch, M., Ismat, A., Reim, I., Raufer, J. (2023). The RNF220 domain nuclear factor Teyrha-Meyrha (Tey) regulates the migration and differentiation of specific visceral and somatic muscles in Drosophila. Development, 150(18) PubMed ID: 37642089
Development of the visceral musculature of the Drosophila midgut encompasses a closely coordinated sequence of migration events of cells from the trunk and caudal visceral mesoderm that underlies the formation of the stereotypic orthogonal pattern of circular and longitudinal midgut muscles. This study focuses on the last step of migration and morphogenesis of longitudinal visceral muscle precursors and shows that these multinucleated precursors utilize dynamic filopodial extensions to migrate in dorsal and ventral directions over the forming midgut tube. The establishment of maximal dorsoventral distances from one another, and anteroposterior alignments, lead to the equidistant coverage of the midgut with longitudinal muscle fibers. Teyrha-Meyhra (Tey), a tissue-specific nuclear factor related to the RNF220 domain protein family, as a crucial regulator of this process of muscle migration and morphogenesis that is further required for proper differentiation of longitudinal visceral muscles. In addition, Tey is expressed in a single somatic muscle founder cell in each hemisegment, regulates the migration of this founder cell, and is required for proper pathfinding of its developing myotube to specific myotendinous attachment sites.
Sakaguchi, S., Mizuno, S., Okochi, Y., Tanegashima, C., Nishimura, O., Uemura, T., Kadota, M., Naoki, H. and Kondo, T. (2023). Single-cell transcriptome atlas of Drosophila gastrula 2.0. Cell Rep 42(7): 112707. PubMed ID: 37433294
During development, positional information directs cells to specific fates, leading them to differentiate with their own transcriptomes and express specific behaviors and functions. However, the mechanisms underlying these processes in a genome-wide view remain ambiguous, partly because the single-cell transcriptomic data of early developing embryos containing accurate spatial and lineage information are still lacking. This study reports a single-cell transcriptome atlas of Drosophila gastrulae, divided into 77 transcriptomically distinct clusters. The expression profiles of plasma-membrane-related genes, but not those of transcription-factor genes, represent each germ layer, supporting the nonequivalent contribution of each transcription-factor mRNA level to effector gene expression profiles at the transcriptome level. The spatial expression patterns of all genes were resolved at the single-cell stripe level as the smallest unit. This atlas is an important resource for the genome-wide understanding of the mechanisms by which genes cooperatively orchestrate Drosophila gastrulation.
Carrasco-Rando, M., Culi, J., Campuzano, S., Ruiz-Gomez, M. (2023). An acytokinetic cell division creates PIP2-enriched membrane asymmetries leading to slit diaphragm assembly in Drosophila nephrocytes. Development, 150(18) PubMed ID: 37681291
Vertebrate podocytes and Drosophila nephrocytes display slit diaphragms, specialised cell junctions that are essential for the execution of the basic excretory function of ultrafiltration. To elucidate the mechanisms of slit diaphragm assembly, their formation was studied in Drosophila embryonic garland nephrocytes. These cells of mesenchymal origin lack overt apical-basal polarity. Their initial membrane symmetry is broken by an acytokinetic cell division that generates PIP2 (Pipe)-enriched domains at their equator. The PIP2-enriched equatorial cortex becomes a favourable domain for hosting slit diaphragm proteins and the assembly of the first slit diaphragms. Indeed, when this division is either prevented or forced to complete cytokinesis, the formation of diaphragms is delayed to larval stages. Furthermore, although apical polarity determinants also accumulate at the equatorial cortex, they do not appear to participate in the recruitment of slit diaphragm proteins. The mechanisms described in this study allow the acquisition of functional nephrocytes in embryos, which may confer on them a biological advantage similar to the formation of the first vertebrate kidney, the pronephros.

Thursday, March 14th - Evolution

Signor, S., Vedanayagam, J., Kim, B. Y., Wierzbicki, F., Kofler, R., Lai, E. C. (2023). Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species. PLoS Genet, 19(8):e1010914 PubMed ID: 37643184
Suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. In D. melanogaster, the flamenco locus is a master suppressor of TEs, preventing the mobilization of certain endogenous retrovirus-like TEs from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, and metabolized into ~24-32 nt Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity. flamenco is thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, it was found that flamenco has undergone radical transformations in sequence content and even copy number across simulans clade Drosophilid species. Drosophila simulans flamenco has duplicated and diverged, and neither copy exhibits synteny with D. melanogaster beyond the core promoter. Moreover, flamenco organization is highly variable across D. simulans individuals. Next, it was found that D. simulans and D. mauritiana flamenco display signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual-stranded clusters. Overall, the physical and functional diversity of flamenco orthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals.
Mullinax, S. R., Darby, A. M., Gupta, A., Chan, P., Smith, B. R., Unckless, R. L. (2023). A suite of selective pressures supports the maintenance of alleles of a Drosophila immune peptide. bioRxiv, PubMed ID: 37662279
The innate immune system provides hosts with a crucial first line of defense against pathogens. While immune genes are often among the fastest evolving genes in the genome, in Drosophila, antimicrobial peptides (AMPs) are notable exceptions. Instead, AMPs may be under balancing selection, such that over evolutionary timescales multiple alleles are maintained in populations. This study focused on the Drosophila antimicrobial peptide Diptericin A, which has a segregating amino acid polymorphism associated with differential survival after infection with the Gram-negative bacteria Providencia rettgeri. Diptericin A also helps control opportunistic gut infections by common Drosophila gut microbes, especially those of Lactobacillus plantarum. In addition to genotypic effects on gut immunity, strong sex-specific effects are also seen that are most prominent in flies without functional Diptericin A. To further characterize differences in microbiomes between different diptericin genotypes, 16S metagenomics was use to look at the microbiome composition. Both lab reared and wild caught flies were used for sequencing and overall composition was looked at as well as the differential abundance of individual bacterial families. Overall, flies were found that are homozygous serine for diptericin A are better equipped to survive a systemic infection from P. rettgeri, but in general homozygous arginine flies have a longer lifespan after being fed common gut commensals. These results suggest a possible mechanism for the maintenance of genetic variation of diptericin A through the complex interactions of sex, systemic immunity, and the maintenance of the gut microbiome.
Vea, I. M., Wilcox, A. S., Frankino, W. A., Shingleton, A. W. (2023). Genetic Variation in Sexual Size Dimorphism Is Associated with Variation in Sex-Specific Plasticity in Drosophila. The American naturalist, 202(3):368-381 PubMed ID: 37606943
The difference in body size between females and males, or sexual size dimorphism (SSD), is ubiquitous, yet we have a poor understanding of the developmental genetic mechanisms that generate it and how these mechanisms may vary within and among species. Such an understanding of the genetic architecture of SSD is important if we are to evaluate alternative models of SSD evolution, but the genetic architecture is difficult to describe because SSD is a characteristic of populations, not individuals. Here, we overcome this challenge by using isogenic lineages of Drosophila to measure SSD for 196 genotypes. We demonstrate extensive genetic variation for SSD, primarily driven by higher levels of genetic variation for body size among females than among males. While a general increase is observed in SSD with sex-averaged body size (pooling for sex) among lineages, most of the variation in SSD is independent of sex-averaged body size and shows a strong genetic correlation with sex-specific plasticity, such that increased female-biased SSD is associated with increased body size plasticity in females. Our data are consistent with the condition dependence hypothesis of sexual dimorphism and suggest that SSD in Drosophila is a consequence of selection on the developmental genetic mechanisms that regulate the plasticity of body size.
Molina-Gil, S., Sotillos, S., Espinosa-Vazquez, J. M., Almudi, I., Hombría, J. C. (2023). Interlocking of co-opted developmental gene networks in Drosophila and the evolution of pre-adaptive novelty. Nat Commun, 14(1):5730 PubMed ID: 37714829
The re-use of genes in new organs forms the base of many evolutionary novelties. A well-characterised case is the recruitment of the posterior spiracle. gene network to the Drosophila male genitalia. This study found that this network has also been co-opted to the testis mesoderm where is required for sperm liberation, providing an example of sequentially repeated developmental co-options. Associated to this co-option event, an evolutionary expression novelty appeared, the activation of the posterior segment determinant Engrailed to the anterior A8 segment controlled by common testis and spiracle regulatory elements. Enhancer deletion shows that A8 anterior Engrailed activation is not required for spiracle development but only necessary in the testis. This study presents an example of pre-adaptive developmental novelty: the activation of the Engrailed transcription factor in the anterior compartment of the A8 segment where, despite having no specific function, opens the possibility of this developmental factor acquiring one. It is proposed that recently co-opted networks become interlocked, so that any change to the network because of its function in one organ, will be mirrored by other organs even if it provides no selective advantage to them.
Doucet, D., Friesen, N., Derksen, N., Mulder, M., Ingram, S., Malagon, J. N. (2023). Homeotic transformations suggest mechanisms for rapid evolution diversification in Drosophila sex combs. microPublication biology, 2023 PubMed ID: 37645232
Evolutionary innovations refer to the emergence of new traits, functions, or behaviors in organisms and lineages over time. Although research has demonstrated that such innovations can arise gradually or through small step, the mechanisms by which rapid morphological diversification takes place remain poorly understood. To explore this question, this study used the evolution of sex combs, as a system. This male-specific row of leg bristles, comprising sex combs as a system, because it displays spectacular morphological diversification in a short time. Homeotic mutations in the fruit fly, Drosophila melanogaster, are those which create modifications in one part of a fly to resemble another region. This study described effects of some of these mutations which transform the D. melanogaster fly sex comb morphology to closely resemble sex comb morphology in other species. These findings support previous research indicating that minor alterations to regulatory elements can play a significant role in explaining morphological evolution. Thus, our results suggest that rapid diversification may not require starting from scratch, but rather may require minor modifications to the sex comb ground plan, which may account for its rapid morphological evolution.
Grill, S., Riley, A., Selvaraj, M., Lehmann, R. (2023). HP6/Umbrea is dispensable for viability and fertility, suggesting essentiality of newly evolved genes is rare. Proc Natl Acad Sci U S A, 120(39):e2309478120 PubMed ID: 37725638
The newly evolved gene Heterochromatin Protein 6 (HP6), which has been previously classified as essential, challenged the dogma that functions required for viability are only seen in genes with a long evolutionary history. Based on previous RNA-sequencing analysis in Drosophila germ cells, it was asked whether HP6 might play a role in germline development. Surprisingly, it was found that CRISPR-generated HP6 mutants are viable and fertile. Using previously generated mutants, an independent lethal allele and an RNAi off-target effect were identified that prevented accurate interpretation of HP6 essentiality. By reviewing existing data, it was found that the vast majority of young genes that were previously classified as essential were indeed viable when tested with orthologous methods. Together, these data call into question the frequency with which newly evolved genes gain essential functions and suggest that using multiple independent genetic methods is essential when probing the functions of young genes.

Thursday, March 15th - Gonads

Cridland, J. M., Begun, D. J. (2023). Male-derived transcripts isolated from the mated female reproductive tract in Drosophila melanogaster. G3 (Bethesda), 13(11) PubMed ID: 37725947
In species with internal fertilization, sperm, and seminal fluid are transferred from male to female during mating. While both sperm and seminal fluid contain various types of molecules, including RNA, the role of most of these molecules in the coordination of fertilization or in other possible functions is poorly understood. In Drosophila, exosomes from the accessory gland, which produces seminal fluid, are transferred to females, but their potential cargoes have not been described. Moreover, while the RNA composition of sperm has been described in several mammalian species, little work on this problem has occurred in Drosophila. This study used single nucleotide polymorphism differences between males and females from a set of highly inbred lines of D. melanogaster, and transcriptome data from the female reproductive tract, sperm, testis, and accessory gland, to investigate the potential origin, male vs female, RNA molecules isolated from 3 female reproductive tract organs, the seminal receptacle and spermatheca, which store sperm, and the parovaria, which does not. Mated females were found to carry male-derived transcripts from many genes, including those that are markers of the accessory gland and known seminal fluid proteins. These observations also support the idea that intact sperm transcripts can be isolated from the female sperm storage organs.
Zeender, V., Pfammatter, S., Roschitzki, B., Dorus, S., Lupold, S. (2023). Genotype-by-environment interactions influence the composition of the Drosophila seminal proteome. Proceedings Biological sciences, 290(2006):20231313 PubMed ID: 37700651
Ejaculate proteins are key mediators of post-mating sexual selection and sexual conflict, as they can influence both male fertilization success and female reproductive physiology. However, the extent and sources of genetic variation and condition dependence of the ejaculate proteome are largely unknown. Such knowledge could reveal the targets and mechanisms of post-mating selection and inform about the relative costs and allocation of different ejaculate components, each with its own potential fitness consequences. This study used liquid chromatography coupled with tandem mass spectrometry to characterize the whole-ejaculate protein composition across 12 isogenic lines of Drosophila melanogaster that were reared on a high- or low-quality diet. New proteins were discovered in the transferred ejaculate, and their origin in the male reproductive system was inferred. It was further found that the ejaculate composition was mainly determined by genotype identity and genotype-specific responses to larval diet, with no clear overall diet effect. Nutrient restriction increased proteolytic protein activity and shifted the balance between reproductive function and RNA metabolism. These results open new avenues for exploring the intricate role of genotypes and their environment in shaping ejaculate composition, or for studying the functional dynamics and evolutionary potential of the ejaculate in its multivariate complexity.
Wang, Z. H., Zhao, W., Combs, C. A., Zhang, F., Knutson, J. R., Lilly, M. A., Xu, H. (2023). Mechanical stimulation from the surrounding tissue activates mitochondrial energy metabolism in Drosophila differentiating germ cells. Dev Cell, 58(21):2249-2260.e2249 PubMed ID: 37647895
In multicellular lives, the differentiation of stem cells and progenitor cells is often accompanied by a transition from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). However, the underlying mechanism of this metabolic transition remains largely unknown. In this study, we investigate the role of mechanical stress in activating OXPHOS during differentiation of the female germline cyst in Drosophila. This study demonstrates that the surrounding somatic cells flatten the 16-cell differentiating cyst, resulting in an increase of the membrane tension of germ cells inside the cyst. This mechanical stress is necessary to maintain cytosolic Ca(2+) concentration in germ cells through a mechanically activated channel, transmembrane channel-like. The sustained cytosolic Ca(2+) triggers a CaMKI-Fray-JNK signaling relay, leading to the transcriptional activation of OXPHOS in differentiating cysts. These findings demonstrate a molecular link between cell mechanics and mitochondrial energy metabolism, with implications for other developmentally orchestrated metabolic transitions in mammals.
Penfield, L., Montell, D. J. (2023). Nuclear lamin facilitates collective border cell invasion into confined spaces in vivo. J Cell Biol, 222(11) PubMed ID: 37695420
Cells migrate collectively through confined environments during development and cancer metastasis. The nucleus, a stiff organelle, impedes single cells from squeezing into narrow channels within artificial environments. However, how nuclei affect collective migration into compact tissues is unknown. This study used border cells in the fly ovary to study nuclear dynamics in collective, confined in vivo migration. Border cells delaminate from the follicular epithelium and squeeze into tiny spaces between cells called nurse cells. The lead cell nucleus transiently deforms within the lead cell protrusion, which then widens. The nuclei of follower cells deform less. Depletion of the Drosophila B-type lamin, Lam, compromises nuclear integrity, hinders expansion of leading protrusions, and impedes border cell movement. In wildtype, cortical myosin II accumulates behind the nucleus and pushes it into the protrusion, whereas in Lam-depleted cells, myosin accumulates but does not move the nucleus. These data suggest that the nucleus stabilizes lead cell protrusions, helping to wedge open spaces between nurse cells.
Sun, Z., Nystul, T. G., Zhong, G. (2023). Single-cell RNA sequencing identifies eggplant as a regulator of germ cell development in Drosophila. EMBO reports, 24(10):e56475 PubMed ID: 37603128
Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. This study used single-cell RNA sequencing (scRNA-seq), an RNAi screen and bioinformatic analysis, to identify genes involved in germ cell differentiation, including 34 genes with upregulated expression during early germ cell development and 19 genes that may regulate germ cell differentiation. Among these, a gene we have named eggplant (eggpl) is highly expressed in GSCs and downregulated in early daughter cells. RNAi knockdown of eggpl causes germ cell proliferation and differentiation defects. In flies fed a rich yeast diet, the expression of eggpl is significantly lower and knockdown or knockout of eggpl phenocopies a rich diet. In addition, eggpl knockdown suppresses the reduction in germ cell proliferation caused by inhibition of the insulin effector PI3K. These findings suggest that downregulation of eggpl links nutritional status to germ cell proliferation and differentiation. Collectively, this study provides new insights into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.
Deshpande, G., Ng, C., Jourjine, N., Chiew, J. W., Dasilva, J., Schedl, P. (2023). Hedgehog signaling guides migration of primordial germ cells to the Drosophila somatic gonad. Genetics, 225(3) PubMed ID: 37708366
In addition to inducing nonautonomous specification of cell fate in both Drosophila and vertebrates, the Hedgehog pathway guides cell migration in a variety of different tissues. Although its role in axon guidance in the vertebrate nervous system is widely recognized, its role in guiding the migratory path of primordial germ cells (PGCs) from the outside surface of the Drosophila embryo through the midgut and mesoderm to the SGPs (somatic gonadal precursors) has been controversial. This study presents new experiments demonstrating (1) that Hh produced by mesodermal cells guides PGC migration, (2) that HMG CoenzymeA reductase (Hmgcr) potentiates guidance signals emanating from the SGPs, functioning upstream of hh and of 2 Hh pathway genes important for Hh-containing cytonemes, and (3) that factors required in Hh receiving cells in other contexts function in PGCs to help direct migration toward the SGPs. The data reported by 4 different laboratories that have studied the role of the Hh pathway in guiding PGC migration were compared.

Tuesday, March 12 - Adult Neural Development, Structure and Development

Aso, Y., Yamada, D., Bushey, D., Hibbard, K. L., Sammons, M., Otsuna, H., Shuai, Y., Hige, T. (2023). Neural circuit mechanisms for transforming learned olfactory valences into wind-oriented movement. Elife, 12 PubMed ID: 37721371
How memories are used by the brain to guide future action is poorly understood. In olfactory associative learning in Drosophila, multiple compartments of the mushroom body act in parallel to assign a valence to a stimulus. This study shows that appetitive memories stored in different compartments induce different levels of upwind locomotion. Using a photoactivation screen of a new collection of split-GAL4 drivers and EM connectomics, this study identified a cluster of neurons postsynaptic to the mushroom body output neurons (MBONs) that can trigger robust upwind steering. These UpWind Neurons (UpWiNs) integrate inhibitory and excitatory synaptic inputs from MBONs of appetitive and aversive memory compartments, respectively. After formation of appetitive memory, UpWiNs acquire enhanced response to reward-predicting odors as the response of the inhibitory presynaptic MBON undergoes depression. Blocking UpWiNs impaired appetitive memory and reduced upwind locomotion during retrieval. Photoactivation of UpWiNs also increased the chance of returning to a location where activation was terminated, suggesting an additional role in olfactory navigation. Thus, these results provide insight into how learned abstract valences are gradually transformed into concrete memory-driven actions through divergent and convergent networks, a neuronal architecture that is commonly found in the vertebrate and invertebrate brains.
Coleman, R. T., Morantte, I., Koreman, G. T., Cheng, M. L., Ding, Y., Ruta, V. (2023). A modular circuit architecture coordinates the diversification of courtship strategies in Drosophila. bioRxiv, PubMed ID: 37745588
This study leveraged the rapid evolution of female pheromones across the Drosophila genus to gain insight into how males coordinately adapt their detection and interpretation of these chemical cues to hone their mating strategies. While in some Drosophila species females produce unique pheromones that act to attract and arouse their conspecific males, the pheromones of most species are sexually monomorphic such that females possess no distinguishing chemosensory signatures that males can use for mate recognition. By comparing several close and distantly-related Drosophila species, it was revealed that D. yakuba males have evolved the distinct ability to use a sexually-monomorphic pheromone, 7-tricosene (7-T), as an excitatory cue to promote courtship, a sensory innovation that enables D. yakuba males to court in the dark thereby expanding their reproductive opportunities. To gain insight into the neural adaptations that enable 7-T to act as an excitatory cue, the functional properties were compared of two key nodes within the pheromone circuits of D. yakuba and a subset of its closest relatives. The instructive role of 7-T in D. yakuba arises from concurrent peripheral and central circuit changes: a distinct subpopulation of sensory neurons has acquired sensitivity to 7-T which in turn selectively signals to a distinct subset of P1 neurons in the central brain that trigger courtship behaviors. Such a modular circuit organization, in which different sensory inputs can independently couple to multiple parallel courtship control nodes, may facilitate the evolution of mate recognition systems by allowing males to take advantage of novel sensory modalities to become aroused. Together, these findings suggest how peripheral and central circuit adaptations can be flexibly linked to underlie the rapid evolution of mate recognition and courtship strategies across species.
Metaxakis, A., Pavlidis, M., Tavernarakis, N. (2023). Neuronal atg1 Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling. Cells, 12(16) PubMed ID: 37626835
The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. This study shows that mTORC1 inhibition acutely perturbs serotonergic neurotransmission via proteostatic alterations evoked by the autophagy inducer Atg1. Neuronal ATG1 alters the intracellular localization of the serotonin transporter, which increases the extracellular serotonin and stimulates the 5HTR7 postsynaptic receptor. 5HTR7 enhances food-searching behaviour and ecdysone-induced catabolism in Drosophila. Along similar lines, the pharmacological inhibition of mTORC1 in zebrafish also stimulates food-searching behaviour via serotonergic activity. These effects occur in parallel with neuronal autophagy induction, irrespective of the autophagic activity and the protein synthesis reduction. In addition, ectopic neuronal atg1 expression enhances catabolism via insulin pathway downregulation, impedes peptidergic secretion, and activates non-cell autonomous cAMP/PKA. The above exert diverse systemic effects on organismal metabolism, development, melanisation, and longevity. It is concluded that neuronal atg1 aligns neuronal autophagy induction with distinct physiological modulations, to orchestrate a coordinated physiological response against reduced mTORC1 activity.
Mano, O., Choi, M., Tanaka, R., Creamer, M. S., Matos, N. C. B., Shomar, J. W., Badwan, B. A., Clandinin, T. R., Clark, D. A. (2023). Long-timescale anti-directional rotation in Drosophila optomotor behavior. Elife, 12 PubMed ID: 37751469
Locomotor movements cause visual images to be displaced across the eye, a retinal slip that is counteracted by stabilizing reflexes in many animals. In insects, optomotor turning causes the animal to turn in the direction of rotating visual stimuli, thereby reducing retinal slip and stabilizing trajectories through the world. This behavior has formed the basis for extensive dissections of motion vision. This study reports that under certain stimulus conditions, two Drosophila species, including the widely studied Drosophila melanogaster, can suppress and even reverse the optomotor turning response over several seconds. Such 'anti-directional turning' is most strongly evoked by long-lasting, high-contrast, slow-moving visual stimuli that are distinct from those that promote syn-directional optomotor turning. Anti-directional turning, like the syn-directional optomotor response, requires the local motion detecting neurons T4 and T5. A subset of lobula plate tangential cells, CH cells, show involvement in these responses. Imaging from a variety of direction-selective cells in the lobula plate shows no evidence of dynamics that match the behavior, suggesting that the observed inversion in turning direction emerges downstream of the lobula plate. Further, anti-directional turning declines with age and exposure to light. These results show that Drosophila optomotor turning behaviors contain rich, stimulus-dependent dynamics that are inconsistent with simple reflexive stabilization responses.
Nobel, S., Danchin, E., Isabel, G. (2023). Mate copying requires the coincidence detector Rutabaga in the mushroom bodies of Drosophila melanogaster. iScience, 26(9):107682 PubMed ID: 37694137
Mate choice constitutes a major fitness-affecting decision often involving social learning leading to copying the preference of other individuals (i.e., mate copying). While mate copying exists in many taxa, its underlying neurobiological mechanisms remain virtually unknown. This study shows in Drosophila melanogaster that the rutabaga gene is necessary to support mate copying. Rutabaga encodes an adenylyl cyclase (AC-Rut(+)) acting as a coincidence detector in associative learning. Since the brain localization requirements for AC-Rut(+) expression differ in classical and operant learning, this study determine the functional localization of AC-Rut(+) for mate copying by artificially rescuing the expression of AC-Rut(+) in neural subsets of a rutabaga mutant. It was found that AC-Rut(+) has to be expressed in the mushroom bodies' Kenyon cells (KCs), specifically in the γ-KCs subset. Thus, this form of discriminative social learning requires the same KCs as non-social Pavlovian learning, suggesting that pathways of social and asocial learning overlap significantly.
Jelen, M., Musso, P. Y., Junca, P., Gordon, M. D. (2023). Optogenetic induction of appetitive and aversive taste memories in Drosophila. Elife, 12 PubMed ID: 37750673
Tastes typically evoke innate behavioral responses that can be broadly categorized as acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses also exhibit plasticity through experience-dependent changes in mushroom body circuits. This study developed a novel taste learning paradigm using closed-loop optogenetics. Appetitive and aversive taste memories can be formed by pairing gustatory stimuli with optogenetic activation of sensory neurons or dopaminergic neurons encoding reward or punishment. As with olfactory memories, distinct dopaminergic subpopulations drive the parallel formation of short- and long-term appetitive memories. Long-term memories are protein synthesis-dependent and have energetic requirements that are satisfied by a variety of caloric food sources or by direct stimulation of MB-MP1 dopaminergic neurons. This paradigm affords new opportunities to probe plasticity mechanisms within the taste system and understand the extent to which taste responses depend on experience.

Monday, March 11th - Disease Models

Zhang, W., Ye, Y., Sun, Y., Li, Y., Ge, M., Chen, K., Yang, L., Chen, G., Zhou, J. (2023). Protein Restriction Effects on Healthspan and Lifespan in Drosophila melanogaster Are Additive With a Longevity-Promoting Diet. The journals of gerontology Series A, Biological sciences and medical sciences, 78(12):2251-2259 PubMed ID: 37738989
Aging of the organism is associated diminished response to external stimuli including weakened immune function, resulting in diseases that impair health and lifespan. Several dietary restriction modalities have been reported to improve health and lifespan in different animal models, but it is unknown whether any of the lifespan-extending dietary treatments could be combined to achieve an additive effect. This study investigated the effects of halving amino acids components in the HUNTaa diet, a synthetic medium known to extend lifespan in Drosophila. Dietary restriction by halving the entire amino acid components (DR group) could further extend lifespan and improve resistance to oxidative stress, desiccation stress, and starvation than flies on HUNTaa diet alone (wt group). Transcriptome analysis of Drosophila at 40, 60, and 80 days of age revealed that genes related to cell proliferation and metabolism decreased with age in the wt group, whereas background stimulus response and amino acid metabolism increased with age. However, these trends differed in the DR group, that is, the DR flies had downregulated stress response genes, including reduced background immune activation. Infection experiments demonstrated that these flies survived longer after feeding infection with Serratia marcescens and Enterococcus faecalis, suggesting that these flies had stronger immune function, and therefore reduced immune senescence. These results demonstrated that halving the entire amino acid components in the HUNTaa diet further extended health and lifespan and suggested that lifespan-extending diet and dietary restriction treatment could be combined to achieve additive beneficial results.
Hsieh, T. C., Chiang, H. C. (2023). IMD signaling in the gut and the brain modulates Amyloid-beta-induced deficits in Drosophila. Life sciences, 332:122118 PubMed ID: 37741318
Evidence indicates accumulating Aβ peptides (see Drosophila Appl) in brain activates immune responses in neuronal and peripheral system, which may collaboratively influence pathogenesis of Alzheimer's disease (AD). This study aimed to investigate whether regulating intestinal innate immune signaling ameliorates Aβ-induced impairments in Drosophila melanogaster. Quantitative polymerase chain reaction (qPCR) was used to observe expression changes of innate immune responses related genes in brain and in gut under the circumstance of Aβ overexpressing in nerve system. Aversive olfactory conditioning and survival assay were used to investigate effects of modulating Attacin-A (AttA) and Dpitercin-A (DptA). Fluorometric assays of respiratory burst activity was introduced to explore whether reducing oxidative stress enables overexpressing intestinal AttA and DptA to reverse Aβ-induced deficits. In vivo genetic analysis revealed that accumulating Aβ42 in neurons modulates innate immune signaling of the IMD pathway both in the brain and in the gut. Increased expression levels of the intestinal AttA and DptA improved learning performance and extended the lifespan of Aβ42 flies. The administration of apramycin led to alleviations of Aβ-induced behavioral changes, indicating that gram-negative bacteria are associated with the development of Aβ-induced pathologies. Further analysis showed that the neural expression of Aβ42 increased oxidative stress in the gut, which disrupted intestinal integrity and decreased learning performance. In addition, increased levels of AMPs targeting gram-negative bacteria and antioxidants reduced oxidative stress in the gut and reversed Aβ-induced behavioral damage. These findings suggest that innate immune responses in the gut play a pivotal role in modulating Aβ-induced pathologies.
Liu, X., Yang, M., Liu, R., Zhou, F., Zhu, H., Wang, X. (2023). The impact of Parkinson's disease-associated gut microbiota on the transcriptome in Drosophila. Microbiology spectrum, 11(5):e0017623 PubMed ID: 37754772
Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people, and many studies have confirmed that the disorder of gut microbiota is involved in the pathophysiological process of PD. However, the molecular mechanism of gut microbiota in regulating the pathogenesis of PD is still lacking. To investigate the impact of PD-associated gut microbiota on host transcriptome, this study established various PD models with fecal microbiota transplantation (FMT) in the model organism Drosophila followed by integrative data analysis of microbiome and transcriptome. First, rotenone-induced PD models were established in Drosophila followed by FMT in different groups. Microbial analysis by 16S rDNA sequencing showed that gut microbiota from PD Drosophila could affect bacterial structure of normal Drosophila, and gut microbiota from normal Drosophila could affect bacterial structure of PD Drosophila. Transcriptome analysis revealed that PD-associated gut microbiota influenced expression patterns of genes enriched in neuroactive ligand-receptor interaction, lysosome, and diverse metabolic pathways. Importantly, to verify these findings, Drosophila were transplanted with fecal samples from clinical PD patients. Compared to the control, Drosophila transplanted with fecal samples from PD patients had reduced microbiota Acetobacter and Lactobacillus, and differentially expressed genes enriched in diverse metabolic pathways. In summary, these results reveal the influence of PD-associated gut microbiota on host gene expression, and this study can help better understand the link between gut microbiota and PD pathogenesis through gut-brain axis.
Mariano, V., Kanellopoulos, A. K., Ricci, C., Di Marino, D., Borrie, S. C., Dupraz, S., Bradke, F., Achsel, T., Legius, E., Odent, S., Billuart, P., Bienvenu, T., Bagni, C. (2024). Intellectual Disability and Behavioral Deficits Linked to CYFIP1 Missense Variants Disrupting Actin Polymerization. Biological psychiatry, 95(2):161-174 PubMed ID: 37704042
15q11.2 deletions and duplications have been linked to autism spectrum disorder, schizophrenia, and intellectual disability. Recent evidence suggests that dysfunctional CYFIP1 (cytoplasmic FMR1 interacting protein 1) contributes to the clinical phenotypes observed in individuals with 15q11.2 deletion/duplication syndrome. CYFIP1 plays crucial roles in neuronal development and brain connectivity, promoting actin polymerization and regulating local protein synthesis. However, information about the impact of single nucleotide variants in CYFIP1 on neurodevelopmental disorders is limited. This study reports a family with 2 probands exhibiting intellectual disability, autism spectrum disorder, spastic tetraparesis, and brain morphology defects and who carry biallelic missense point mutations in the CYFIP1 gene.This study used skin fibroblasts from one of the probands, the parents, and typically developing individuals to investigate the effect of the variants on the functionality of CYFIP1. In addition,Drosophila knockin mutants were created to address the effect of the variants in vivo and gain insight into the molecular mechanism that underlies the clinical phenotype. This study revealed that the 2 missense variants are in protein domains responsible for maintaining the interaction within the wave regulatory complex. Molecular and cellular analyses in skin fibroblasts from one proband showed deficits in actin polymerization. The fly model for these mutations exhibited abnormal brain morphology and F-actin loss and recapitulated the core behavioral symptoms, such as deficits in social interaction and motor coordination. These findings suggest that the 2 CYFIP1 variants contribute to the clinical phenotype in the probands that reflects deficits in actin-mediated brain development processes.
Xu, D., Vincent, A., Gonzalez-Gutierrez, A., Aleyakpo, B., Anoar, S., Giblin, A., Atilano, M. L., Adams, M., Shen, D., Thoeng, A., Tsintzas, E., Maeland, M., Isaacs, A. M., Sierralta, J., Niccoli, T. (2023). A monocarboxylate transporter rescues frontotemporal dementia and Alzheimer's disease models. PLoS Genet, 19(9):e1010893 PubMed ID: 37733679
Brains are highly metabolically active organs, consuming 20% of a person's energy at resting state. A decline in glucose metabolism is a common feature across a number of neurodegenerative diseases. Another common feature is the progressive accumulation of insoluble protein deposits, it's unclear if the two are linked. Glucose metabolism in the brain is highly coupled between neurons and glia, with glucose taken up by glia and metabolised to lactate, which is then shuttled via transporters to neurons, where it is converted back to pyruvate and fed into the TCA cycle for ATP production. Monocarboxylates are also involved in signalling, and play broad ranging roles in brain homeostasis and metabolic reprogramming. However, the role of monocarboxylates in dementia has not been tested. This study found that increasing pyruvate import in Drosophila neurons by over-expression of the transporter bumpel, leads to a rescue of lifespan and behavioural phenotypes in fly models of both frontotemporal dementia and Alzheimer's disease. The rescue is linked to a clearance of late stage autolysosomes, leading to degradation of toxic peptides associated with disease. It is proposed that upregulation of pyruvate import into neurons as potentially a broad-scope therapeutic approach to increase neuronal autophagy, which could be beneficial for multiple dementias.
Richardson, K., Wessells, R. (2023). A novel panel of Drosophila TAFAZZIN mutants in distinct genetic backgrounds as a resource for therapeutic testing. PLoS One, 18(9):e0286380 PubMed ID: 37756350
Barth Syndrome is a rare, X-linked disorder caused by mutation of the gene TAFAZZIN (TAZ). The corresponding Tafazzin protein is involved in the remodeling of cardiolipin, a phospholipid with critical roles in mitochondrial function. While recent clinical trials have been promising, there is still no cure for Barth Syndrome. Because TAZ is highly conserved, multiple animal and cell culture models exist for pre-clinical testing of therapeutics. However, since the same mutation in different patients can lead to different symptoms and responses to treatment, isogenized experimental models can't fully account for human disease conditions. On the other hand, isogenized animal models allow for sufficient numbers to thoroughly establish efficacy for a given genetic background. Therefore, a combined method for testing treatments in a panel of isogenized cohorts that are genetically distinct from each other would be transformative for testing emerging pre-clinical therapies. To aid in this effort, this study has created a novel panel of 10 Drosophila lines, each with the same TAZ mutation in highly diverse genetic backgrounds, to serve as a helpful resource to represent natural variation in background genetics in pre-clinical studies. As a proof of principle, the panel was tested using nicotinamide riboside (NR), a treatment with established therapeutic value, to evaluate how robust this therapy is across the 10 genetic backgrounds in this novel reference panel. Substantial variation was found in the response to NR across backgrounds. This resource is expected to be valuable in pre-clinical testing of emerging therapies for Barth Syndrome.

Friday, March 8th - Stem Cells

Gujar, M. R., Gao, Y., Teng, X., Ding, W. Y., Lin, J., Tan, Y. S., Chew, L. Y., Toyama, Y. and Wang, H. (2023). Patronin/CAMSAP promotes reactivation and regeneration of Drosophila quiescent neural stem cells. EMBO Rep: e56624. PubMed ID: 37440685
The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. Drosophila quiescent neural stem cells (qNSCs) extend a primary protrusion that is enriched in acentrosomal microtubules and can be regenerated upon injury. Arf1 promotes microtubule growth, reactivation (exit from quiescence), and regeneration of qNSC protrusions upon injury. However, how Arf1 is regulated in qNSCs remains elusive. This study shows that the microtubule minus-end binding protein Patronin/CAMSAP promotes acentrosomal microtubule growth and quiescent NSC reactivation. Patronin is important for the localization of Arf1 at Golgi and physically associates with Arf1, preferentially with its GDP-bound form. Patronin is also required for the regeneration of qNSC protrusion, likely via the regulation of microtubule growth. Finally, Patronin functions upstream of Arf1 and its effector Msps/XMAP215 to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. These findings reveal a novel link between Patronin/CAMSAP and Arf1 in the regulation of microtubule growth and NSC reactivation. A similar mechanism might apply to various microtubule-dependent systems in mammals.
Li, Y., Chaurasia, B., Rahman, M. M., Kaddai, V., Maschek, J. A., Berg, J. A., Wilkerson, J. L., Mahmassani, Z. S., Cox, J., Wei, P., Meikle, P. J., Atkinson, D., Wang, L., Poβ, A. M., Playdon, M. C., Tippetts, T. S., Mousa, E. M., Nittayaboon, K., Anandh Babu, P. V., Drummond, M. J., Clevers, H., Shayman, J. A., Hirabayashi, Y., Holland, W. L., Rutter, J., Edgar, B. and Summers, S. A. (2023). Ceramides increase fatty acid utilization in intestinal progenitors to enhance stemness and increase tumor risk. Gastroenterology. PubMed ID: 37541526
Cancers of the alimentary tract are common comorbidities of obesity. Prolonged, excesssive delivery of macronutrients to the cells lining the gut can increase one's risk for these cancers by inducing imbalances in the rate of intestinal stem cell proliferation vs. differentiation. This study investigated whether ceramides, which are sphingolipids that serve as a signals of nutritional excess, alter stem cell behaviors to influence cancer risk. This study profiled sphingolipids and sphingolipid-synthesizing enzymes in human adenomas and tumors. Thereafter, expression of sphingolipid-producing enzymes, including serine palmitoyltransferase (SPT), was examined in intestinal progenitors of mice, cultured organoids, and Drosophila to discern whether sphingolipids altered stem cell proliferation and metabolism. SPT, which diverts dietary fatty- and amino-acids into the biosynthetic pathway that produces ceramides and other sphingolipids, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the sphingolipid biosynthesis pathway are upregulated in human intestinal adenomas. They produce ceramides which serve as pro-stemness signals that stimulate peroxisome-proliferator activated receptor alpha and induce fatty acid binding protein-1. These actions lead to increased lipid utilization and enhanced proliferation of intestinal progenitors. Ceramides serve as critical links between dietary macronutrients, epithelial regeneration, and cancer risk.
Breznak, S. M., Peng, Y., Deng, L., Kotb, N. M., Flamholz, Z., Rapisarda, I. T., Martin, E. T., LaBarge, K. A., Fabris, D., Gavis, E. R. and Rangan, P. (2023). H/ACA snRNP-dependent ribosome biogenesis regulates translation of polyglutamine proteins. Sci Adv 9(25): eade5492. PubMed ID: 37343092
Stem cells in many systems, including Drosophila germline stem cells (GSCs), increase ribosome biogenesis and translation during terminal differentiation. This study showns that the H/ACA small nuclear ribonucleoprotein (snRNP) complex that promotes pseudouridylation of ribosomal RNA (rRNA) and ribosome biogenesis is required for oocyte specification. Reducing ribosome levels during differentiation decreased the translation of a subset of messenger RNAs that are enriched for CAG trinucleotide repeats and encode polyglutamine-containing proteins, including differentiation factors such as RNA-binding Fox protein 1. Moreover, ribosomes were enriched at CAG repeats within transcripts during oogenesis. Increasing target of rapamycin (TOR) activity to elevate ribosome levels in H/ACA snRNP complex-depleted germlines suppressed the GSC differentiation defects, whereas germlines treated with the TOR inhibitor rapamycin had reduced levels of polyglutamine-containing proteins. Thus, ribosome biogenesis and ribosome levels can control stem cell differentiation via selective translation of CAG repeat-containing transcripts.
Keegan, S. E., Haskins, J., Simmonds, A. J. and Hughes, S. C. (2023).. A chromatin remodelling SWI/SNF subunit, Snr1, regulates neural stem cell determination and differentiation. Development 150(13). PubMed ID: 37294080
Coordinated spatio-temporal regulation of the determination and differentiation of neural stem cells is essential for brain development. Failure to integrate multiple factors leads to defective brain structures or tumour formation. Previous studies suggest changes of chromatin state are needed to direct neural stem cell differentiation, but the mechanisms are unclear. Analysis of Snr1, the Drosophila orthologue of SMARCB1, an ATP-dependent chromatin remodelling protein, identified a key role in regulating the transition of neuroepithelial cells into neural stem cells and subsequent differentiation of neural stem cells into the cells needed to build the brain. loss of Snr1 in neuroepithelial cells leads to premature neural stem cell formation. Additionally, loss of Snr1 in neural stem cells results in inappropriate perdurance of neural stem cells into adulthood. Snr1 reduction in neuroepithelial or neural stem cells leads to the differential expression of target genes. Snr1 is associated with the actively transcribed chromatin region of these target genes. Thus, Snr1 likely regulates the chromatin state in neuroepithelial cells and maintains chromatin state in neural stem cells for proper brain development.
Gong, J., Nirala, N. K., Chen, J., Wang, F., Gu, P., Wen, Q., Ip, Y. T. and Xiang, Y. (2023). TrpA1 is a shear stress mechanosensing channel regulating intestinal stem cell proliferation in Drosophila. Sci Adv 9(21): eadc9660. PubMed ID: 37224252
Adult stem cells are essential for tissue maintenance and repair. Although genetic pathways for controlling adult stem cells are extensively investigated in various tissues, much less is known about how mechanosensing could regulate adult stem cells and tissue growth. This study demonstrated that shear stress sensing regulates intestine stem cell proliferation and epithelial cell number in adult Drosophila. Ca(2+) imaging in ex vivo midguts shows that shear stress, but not other mechanical forces, specifically activates enteroendocrine cells among all epithelial cell types. This activation is mediated by transient receptor potential A1 (TrpA1), a Ca(2+)-permeable channel expressed in enteroendocrine cells. Furthermore, specific disruption of shear stress, but not chemical, sensitivity of TrpA1 markedly reduces proliferation of intestinal stem cells and midgut cell number. Therefore, it is proposed that shear stress may act as a natural mechanical stimulation to activate TrpA1 in enteroendocrine cells, which, in turn, regulates intestine stem cell behavior.
Nagai, H., Nagai, L. A. E., Tasaki, S., Nakato, R., Umetsu, D., Kuranaga, E., Miura, M., Nakajima, Y. (2023). Nutrient-driven dedifferentiation of enteroendocrine cells promotes adaptive intestinal growth in Drosophila. Dev Cell, 58(18):1764-1781.e1710 PubMed ID: 37689060
Post-developmental organ resizing improves organismal fitness under constantly changing nutrient environments. Although stem cell abundance is a fundamental determinant of adaptive resizing, understanding of its underlying mechanisms remains primarily limited to the regulation of stem cell division. This study demonstrates that nutrient fluctuation induces dedifferentiation in the Drosophila adult midgut to drive adaptive intestinal growth. From lineage tracing and single-cell RNA sequencing, a subpopulation of enteroendocrine (EE) cells was identified that convert into functional intestinal stem cells (ISCs) in response to dietary glucose and amino acids by activating the JAK-STAT pathway. Genetic ablation of EE-derived ISCs severely impairs ISC expansion and midgut growth despite the retention of resident ISCs, and in silico modeling further indicates that EE dedifferentiation enables an efficient increase in the midgut cell number while maintaining epithelial cell composition. These findings identify a physiologically induced dedifferentiation that ensures ISC expansion during adaptive organ growth in concert with nutrient conditions.

Thursday, March 7th - Adult Physiology and Metabolism

Chen, J., Lewis, O. T. (2023). Experimental heatwaves facilitate invasion and alter species interactions and composition in a tropical host-parasitoid community. Global change biology, 29(22):6261-6275 PubMed ID: 37733768
As mean temperatures increase and heatwaves become more frequent, species are expanding their distributions to colonise new habitats. The resulting novel species interactions will simultaneously shape the temperature-driven reorganization of resident communities. The interactive effects of climate change and climate change-facilitated invasion have rarely been studied in multi-trophic communities, and are likely to differ depending on the nature of the climatic driver (i.e., climate extremes or constant warming). This study re-created under laboratory conditions a host-parasitoid community typical of high-elevation rainforest sites in Queensland, Australia, comprising four Drosophila species and two associated parasitoid species. These communities were subjected to an equivalent increase in average temperature in the form of periodic heatwaves or constant warming, in combination with an invasion treatment involving a novel host species from lower-elevation habitats. The two parasitoid species were sensitive to both warming and heatwaves, while the demographic responses of Drosophila species were highly idiosyncratic, reflecting the combined effects of thermal tolerance, parasitism, competition, and facilitation. After multiple generations, the heatwave treatment promoted the establishment of low-elevation species in upland communities. Invasion of the low-elevation species correlated negatively with the abundance of one of the parasitoid species, leading to cascading effects on its hosts and their competitors. This study, therefore, reveals differing, sometimes contrasting, impacts of extreme temperatures and constant warming on community composition. It also highlights how the scale and direction of climate impacts could be further modified by invading species within a bi-trophic community network.
Czarnoleski, M., Szlachcic, E., Privalova, V., Maria Labecka, A., Sikorska, A., Sobczyk, Ł., VandenBrooks, J., Angilletta, M. J., Jr. (2023). Oxygen and temperature affect cell sizes differently among tissues and between sexes of Drosophila melanogaster. J Insect Physiol, 150:104559 PubMed ID: 37640139
Spatio-temporal gradients in thermal and oxygen conditions trigger evolutionary and developmental responses in ectotherms' body size and cell size, which are commonly interpreted as adaptive. However, the evidence for cell-size responses is fragmentary, as cell size is typically assessed in single tissues. In a laboratory experiment, genotypes of Drosophila melanogaster were raised at all combinations of two temperatures (16 °C or 25 °C) and two oxygen levels (10% or 22%), and body size and the sizes of cells were measured in different tissues. For each sex,epidermal cells in a wing and a leg and ommatidial cells of an eye were measured. For males, epithelial cells of a Malpighian tubule and muscle cells of a flight muscle were measured. On average, females emerged at a larger body size than did males, having larger cells in all tissues. Flies of either sex emerged at a smaller body size when raised under warm or hypoxic conditions. Development at 25 °C resulted in smaller cells in most tissues. Development under hypoxia resulted in smaller cells in some tissues, especially among females. Altogether, these results show thermal and oxygen conditions trigger shifts in adult size, coupled with the systemic orchestration of cell sizes throughout the body of a fly. The nature of these patterns supports a model in which an ectotherm adjusts its life-history traits and cellular composition to prevent severe hypoxia at the cellular level. However, the results revealed some inconsistencies linked to sex, cell type, and environmental parameters, which suggest caution in translating information obtained for single type of cells to the organism as a whole.
Liska, O., Boross, G., Rocabert, C., Szappanos, B., Tengolics, R., Papp, B. (2023). Principles of metabolome conservation in animals. Proc Natl Acad Sci U S A, 120(35):e2302147120 PubMed ID: 37603743
Metabolite levels shape cellular physiology and disease susceptibility, yet the general principles governing metabolome evolution are largely unknown. This study introduce a measure of conservation of individual metabolite levels among related species. By analyzing multispecies tissue metabolome datasets in phylogenetically diverse mammals and fruit flies, this study shows that conservation varies extensively across metabolites. Three major functional properties, metabolite abundance, essentiality, and association with human diseases predict conservation, highlighting a striking parallel between the evolutionary forces driving metabolome and protein sequence conservation. Metabolic network simulations recapitulated these general patterns and revealed that abundant metabolites are highly conserved due to their strong coupling to key metabolic fluxes in the network. Finally, this study shows that biomarkers of metabolic diseases can be distinguished from other metabolites simply based on evolutionary conservation, without requiring any prior clinical knowledge. Overall, this study uncovers simple rules that govern metabolic evolution in animals and implies that most tissue metabolome differences between species are permitted, rather than favored by natural selection. More broadly, this work paves the way toward using evolutionary information to identify biomarkers, as well as to detect pathogenic metabolome alterations in individual patients.
Cobb, T., Hwang, I., Soukar, M., Namkoong, S., Cho, U. S., Safdar, M., Kim, M., Wessells, R. J., Lee, J. H. (2023). Iditarod, a Drosophila homolog of the Irisin precursor FNDC5, is critical for exercise performance and cardiac autophagy. Proc Natl Acad Sci U S A, 120(39):e2220556120 PubMed ID: 37722048
Mammalian FNDC5 encodes a protein precursor of Irisin, which is important for exercise-dependent regulation of whole-body metabolism. In a genetic screen in Drosophila, this study identified Iditarod (Idit), which shows substantial protein homology to mouse and human FNDC5, as a regulator of autophagy acting downstream of Atg1/Atg13. Physiologically, Idit-deficient flies showed reduced exercise performance and defective cold resistance, which were rescued by exogenous expression of Idit. Exercise training increased endurance in wild-type flies, but not in Idit-deficient flies. Conversely, Idit is induced upon exercise training, and transgenic expression of Idit in wild-type flies increased endurance to the level of exercise trained flies. Finally, Idit deficiency prevented both exercise-induced increase in cardiac Atg8 and exercise-induced cardiac stress resistance, suggesting that cardiac autophagy may be an additional mechanism by which Idit is involved in the adaptive response to exercise. This work suggests an ancient role of an Iditarod/Irisin/FNDC5 family of proteins in autophagy, exercise physiology, and cold adaptation, conserved throughout metazoan species.
Venkatasubramani, A. V., Ichinose, T., Kanno, M., Forne, I., Tanimoto, H., Peleg, S., Imhof, A. (2023). The fruit fly acetyltransferase chameau promotes starvation resilience at the expense of longevity. EMBO reports, 24(10):e57023 PubMed ID: 37724628
Proteins involved in cellular metabolism and molecular regulation can extend lifespan of various organisms in the laboratory. However, any improvement in aging would only provide an evolutionary benefit if the organisms were able to survive under non-ideal conditions. Previous work has shown that Drosophila melanogaster carrying a loss-of-function allele of the acetyltransferase chameau (chm) has an increased healthy lifespan when fed ad libitum. This study shows that loss of chm and reduction in its activity results in a substantial reduction in weight and a decrease in starvation resistance. This phenotype is caused by failure to properly regulate the genes and proteins required for energy storage and expenditure. The previously observed increase in survival time thus comes with the inability to prepare for and cope with nutrient stress. As the ability to survive in environments with restricted food availability is likely a stronger evolutionary driver than the ability to live a long life, chm is still present in the organism's genome despite its apparent negative effect on lifespan.
Liu, J., Zhang, Y., Wang, Q. Q., Zhou, Y., Liu, J. L. (2023). Fat body-specific reduction of CTPS alleviates HFD-induced obesity. Elife, 12 PubMed ID: 37695169
Obesity induced by high-fat diet (HFD) is a multi-factorial disease including genetic, physiological, behavioral, and environmental components. Drosophila has emerged as an effective metabolic disease model. Cytidine 5'-triphosphate synthase (CTPS) is an important enzyme for the de novo synthesis of CTP, governing the cellular level of CTP and the rate of phospholipid synthesis. CTPS is known to form filamentous structures called cytoophidia, which are found in bacteria, archaea, and eukaryotes. This study demonstrates that CTPS is crucial in regulating body weight and starvation resistance in Drosophila by functioning in the fat body. HFD-induced obesity leads to increased transcription of CTPS and elongates cytoophidia in larval adipocytes. Depleting CTPS in the fat body prevented HFD-induced obesity, including body weight gain, adipocyte expansion, and lipid accumulation, by inhibiting the PI3K-Akt-SREBP axis. Furthermore, a dominant-negative form of CTPS also prevented adipocyte expansion and downregulated lipogenic genes. These findings not only establish a functional link between CTPS and lipid homeostasis but also highlight the potential role of CTPS manipulation in the treatment of HFD-induced obesity.

Wednesday, March 6th - Signaling

Abdullah, A., Herdenberg, C., Hedman, H. (2023). Ligand-specific regulation of transforming growth factor beta superfamily factors by leucine-rich repeats and immunoglobulin-like domains proteins. PLoS One, 18(8):e0289726 PubMed ID: 37603563
Leucine-rich repeats and immunoglobulin-like domains (LRIG) are transmembrane proteins shown to promote bone morphogenetic protein (BMP) signaling in Caenorhabditis elegans, Drosophila melanogaster, and mammals. BMPs comprise a subfamily of the transforming growth factor beta (TGFβ) superfamily, or TGFβ family, of ligands. In mammals, LRIG1 and LRIG3 promote BMP4 signaling. BMP6 signaling, but not BMP9 signaling, is also regulated by LRIG proteins, although the specific contributions of LRIG1 (see Drosophila lambik), LRIG2, and LRIG3 have not been investigated, nor is it known whether other mammalian TGFβ family members are regulated by LRIG proteins. To address these questions, advantage was taken of Lrig-null mouse embryonic fibroblasts (MEFs) with doxycycline-inducible LRIG1, LRIG2, and LRIG3 alleles, which were stimulated with ligands representing all the major TGFβ family subgroups. By analyzing the signal mediators pSmad1/5 and pSmad3, as well as the induction of Id1 expression, it was shown that LRIG1 promoted BMP2, BMP4, and BMP6 signaling and suppressed GDF7 signaling; LRIG2 promoted BMP2 and BMP4 signaling; and LRIG3 promoted BMP2, BMP4, BMP6, and GDF7 signaling. BMP9 and BMP10 signaling was not regulated by individual LRIG proteins, however, it was enhanced in Lrig-null cells. LRIG proteins did not regulate TGFβ1-induced pSmad1/5 signaling, or GDF11- or TGFβ1-induced pSmad3 signaling. Taken together, these results show that some, but not all, TGFβ family ligands are regulated by LRIG proteins and that the three LRIG proteins display differential regulatory effects. LRIG proteins thereby provide regulatory means for the cell to further diversify the signaling outcomes generated by a limited number of TGFβ family ligands and receptors.
Parra, A. S., Moezzi, C. A., Johnston, C. A. (2023). Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex. Frontiers in cell and developmental biology, 11:1220529 PubMed ID: 37655159
Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In Drosophila neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). This study identified Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (Hts(HEAD)) to bind a short Mud coiled-coil domain (Mud(CC)) that is adjacent to the Pins-binding domain (Mud(PBD)). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. In vitro analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, this study found that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via Mud(CC) oligomerization) and protein disorder (via an intrinsically disordered region in Hts; Hts(IDR)) in phase separation of the Hts/Mud/Pins complex. This study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output.
Ho, E. K., Oatman, H. R., McFann, S. E., Yang, L., Johnson, H. E., Shvartsman, S. Y., Toettcher, J. E. (2023). Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo. Development, 150(17) PubMed ID: 37602510
Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. This study used optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. High or low doses of ERK signaling were found to produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, this study provides a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.
Chen, X., Chen, X., Chao, R., Wang, Y., Mao, Y., Fan, B., Zhang, Y., Xu, W., Qin, A., Zhang, S. (2023). Dlk2 interacts with Syap1 to activate Akt signaling pathway during osteoclast formation. Cell Death Dis, 14(9):589 PubMed ID: 37669921
Excessive osteoclast formation and bone resorption are related to osteolytic diseases. Delta drosophila homolog-like 2 (Dlk2; see Drosophia Delta), a member of the epidermal growth factor (EGF)-like superfamily, reportedly regulates adipocyte differentiation, but its roles in bone homeostasis are unclear. This study demonstrated that Dlk2 deletion in osteoclasts significantly inhibited osteoclast formation in vitro and contributed to a high-bone-mass phenotype in vivo. Importantly, Dlk2 was shown to interact with synapse-associated protein 1 (Syap1), which regulates Akt phosphorylation at Ser473 (see Drosophila Akt1). Dlk2 deletion inhibited Syap1 (see Drosophila Sap47)-mediated activation of the Akt(Ser473), ERK1/2 and p38 signaling cascades. Additionally, Dlk2 deficiency exhibits increased bone mass in ovariectomized mice. These results reveal the important roles of the Dlk2-Syap1 signaling pathway in osteoclast differentiation and osteoclast-related bone disorders.
Song, S., Ma, X. (2023). E2 enzyme Bruce negatively regulates Hippo signaling through POSH-mediated expanded degradation. Cell Death Dis, 14(9):602 PubMed ID: 37699871
The Hippo pathway is a master regulator of organ growth, stem cell renewal, and tumorigenesis, its activation is tightly controlled by various post-translational modifications, including ubiquitination. While several E3 ubiquitin ligases have been identified as regulators of Hippo pathway, the corresponding E2 ubiquitin-conjugating enzymes (E2s) remain unknown. This study performed a screen in Drosophila to identify E2s involved in regulating wing overgrowth caused by the overexpression of Crumbs (Crb) intracellular domain and identified Bruce as a critical regulator. Loss of Bruce downregulates Hippo target gene expression and suppresses Hippo signaling inactivation induced tissue growth. Unexpectedly, the genetic data indicate that Bruce acts upstream of Expanded (Ex) but in parallel with the canonical Hippo (Hpo) -Warts (Wts) cascade to regulate Yorkie (Yki), the downstream effector of Hippo pathway. Mechanistically, Bruce synergizes with E3 ligase POSH to regulate growth and ubiquitination-mediated Ex degradation. Moreover, it was demonstrated that Bruce is required for Hippo-mediated malignant tumor progression. Altogether, these findings unveil Bruce as a crucial E2 enzyme that bridges the signal from the cell surface to regulate Hippo pathway activation in Drosophila.
Zhao, Z., Brooks, D., Guo, Y., Geisbrecht, E. R. (2023). Identification of CryAB as a target of NUAK kinase activity in Drosophila muscle tissue. Genetics, 225(3) PubMed ID: 37713608
Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. This study characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. It was further shown that a phospho-mimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis.

Tuesday, March 5th - Disease Models

Cembran, A., Fernandez-Funez, P. (2023). Intrinsic determinants of prion protein neurotoxicity in Drosophila: from sequence to (dys)function. Frontiers in molecular neuroscience, 16:1231079 PubMed ID: 37645703
Prion diseases are fatal brain disorders characterized by deposition of insoluble isoforms of the prion protein (PrP). The normal and pathogenic structures of PrP are relatively well known after decades of studies. Yet current understanding of the intrinsic determinants regulating PrP misfolding are largely missing. A 3D subdomain of PrP comprising the β2-&alpah2 loop and helix 3 contains high sequence and structural variability among animals and has been proposed as a key domain regulating PrP misfolding. This study combined in vivo work in Drosophila with molecular dynamics (MD) simulations, which provide additional insight to assess the impact of candidate substitutions in PrP from conformational dynamics. MD simulations revealed that in human PrP WT the β2-α2 loop explores multiple β-turn conformations, whereas the Y225A (rabbit PrP-like) substitution strongly favors a 3(10)-turn conformation, a short right-handed helix. This shift in conformational diversity correlates with lower neurotoxicity in flies. This study has identified additional conformational features and candidate amino acids regulating the high toxicity of human PrP and propose a new strategy for testing candidate modifiers first in MD simulations followed by functional experiments in flies. This review expands on these new results to provide additional insight into the structural and functional biology of PrP through the prism of the conformational dynamics of a 3D domain in the C-terminus. It is proposed that the conformational dynamics of this domain is a sensitive measure of the propensity of PrP to misfold and cause toxicity. This provides renewed opportunities to identify the intrinsic determinants of PrP misfolding through the contribution of key amino acids to different conformational states by MD simulations followed by experimental validation in transgenic flies.
Pragati, Sarkar, S. (2023). Reinstated Activity of Human Tau-induced Enhanced Insulin Signaling Restricts Disease Pathogenesis by Regulating the Functioning of Kinases/Phosphatases and Tau Hyperphosphorylation in Drosophila. Molecular neurobiology, PubMed ID: 37674037
Tauopathies such as Alzheimer's disease (AD), Frontotemporal dementia, and parkinsonism linked to chromosome 17 (FTDP-17), etc. are characterized by tau hyperphosphorylation and distinguished accumulation of paired helical filaments (PHFs)/or neurofibrillary tangles (NFTs) in a specific-neuronal subset of the brain. Among different reported risk factors, type 2 diabetes (T2D) has gained attention due to its correlation with tau pathogenesis. However, mechanistic details and the precise contribution of insulin pathway in tau etiology is still debatable. This study demonstrated that expression of human tau causes overactivation of insulin pathway in Drosophila disease models. It was subsequently noted that tissue-specific downregulation of insulin signaling or even exclusive reduction of its growth-promoting sub-branch effectively reinstates the overactivated insulin signaling pathway in human tau expressing cells, which in turn restricts pathogenic tau hyperphosphorylation and aggregate formation. It was further noted that restored tau phosphorylation was achieved due to a reestablished balance between the levels of different kinase(s) (GSK3β and ERK/P38 MAP kinase) and phosphatase (PP2A). Taken together, this study demonstrates a precise involvement of the insulin pathway and associated molecular events in the pathogenesis of human tauopathies in Drosophila, which will be immensely helpful in developing novel therapeutic options against these devastating human brain disorders. Moreover, this study reveals an interesting link between tau etiology and aberrant insulin signaling, which is a characteristic feature of Type 2 Diabetes.
Lovato, T. L., Blotz, B., Bileckyj, C., Johnston, C. A., Cripps, R. M. (2023).. Modeling a variant of unknown significance in the Drosophila ortholog of the human cardiogenic gene NKX2.5. Disease models & mechanisms, 16(9) PubMed ID: 37691628
Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. This study used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene NKX2.5 (also known as NKX2-5). An R321N allele of the NKX2.5 ortholog tinman (tin) was generated to model a human K158N variant, and its function was tested in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila T-box cardiac factor named Dorsocross1. A tinR321N allele was generated using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. It is proposed that the human K158N variant is pathogenic through causing a deficiency in DNA binding and a reduced ability to interact with a cardiac co-factor, and that cardiac defects might arise later in development or adult life.
Thackray, A. M., McNulty, E. E., Nalls, A. V., Cardova, A., Tran, L., Telling, G., Benestad, S. L., Gilch, S., Mathiason, C. K., Bujdoso, R. (2023). Genetic modulation of CWD prion propagation in cervid PrP Drosophila. Biochem J, 480(19):1485-1501 PubMed ID: 37747806
Chronic wasting disease is a fatal prion condition of cervids such as deer, elk, moose and reindeer. Secretion and excretion of prion infectivity from North American cervids with this condition causes environmental contamination and subsequent efficient lateral transmission in free-ranging and farmed cervids. Variants of cervid PrP exist that affect host susceptibility to chronic wasting disease. This study generated cervid PrP transgenic Drosophila to fulfil this purpose. Drosophila transgenic for S138 wild type cervid PrP, or the N138 variant were associated with resistance to chronic wasting disease. Cervid PrP Drosophila accumulate bona fide prion infectivity after exposure to cervid prions. Furthermore, S138 and N138 PrP fly lines are susceptible to cervid prion isolates from either North America or Europe when assessed phenotypically by accelerated loss of locomotor ability or survival, or biochemically by accumulation of prion seeding activity. However, after exposure to European reindeer prions, N138 PrP Drosophila accumulated prion seeding activity with slower kinetics than the S138 fly line. These novel data show that prion susceptibility characteristics of cervid PrP variants are maintained when expressed in Drosophila, which highlights this novel invertebrate host in modelling chronic wasting disease.
Dunn, E., Steinert, J. R., Stone, A., Sahota, V., Williams, R. S. B., Snowden, S., Augustin, H. (2023). Medium-Chain Fatty Acids Rescue Motor Function and Neuromuscular Junction Degeneration in a Drosophila Model of Amyotrophic Lateral Sclerosis. Cells, 12(17) PubMed ID: 37681895
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterised by progressive degeneration of the motor neurones. An expanded GGGGCC (G4C2) hexanucleotide repeat in C9orf72 is the most common genetic cause of ALS and frontotemporal dementia (FTD); therefore, the resulting disease is known as C9ALS/FTD. This study employed a Drosophila melanogaster model of C9ALS/FTD (C9 model) to investigate a role for specific medium-chain fatty acids (MCFAs) in reversing pathogenic outcomes. Drosophila larvae overexpressing the ALS-associated dipeptide repeats (DPRs) in the nervous system exhibit reduced motor function and neuromuscular junction (NMJ) defects. Two MCFAs, nonanoic acid (NA) and 4-methyloctanoic acid (4-MOA), can ameliorate impaired motor function in C9 larvae and improve NMJ degeneration, although their mechanisms of action are not identical. NA modified postsynaptic glutamate receptor density, whereas 4-MOA restored defects in the presynaptic vesicular release. It was also demonstrated the effects of NA and 4-MOA on metabolism in C9 larvae and implicate various metabolic pathways as dysregulated in the ALS model. These findings pave the way to identifying novel therapeutic targets and potential treatments for ALS.
Dehn, A. S., Duhaime, L., Gogna, N., Nishina, P. M., Kelley, K., Losick, V. P. (2023). Epithelial mechanics are maintained by inhibiting cell fusion with age in Drosophila. J Cell Sci, 136(20) PubMed ID: 37732459
A characteristic of normal aging and age-related diseases is the remodeling of the cellular organization of a tissue through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. This study found that abdominal epithelium of the adult fruit fly becomes polyploid with age through generation of multinucleated cells by cell fusion. Inhibition of fusion does not improve the lifespan of the fly, but does enhance its biomechanical fitness, a measure of the healthspan of the animal. Remarkably, Drosophila can maintain their epithelial tension and abdominal movements with age when cell fusion is inhibited. Epithelial cell fusion also appears to be dependent on a mechanical cue, as knockdown of Rho kinase, E-cadherin or α-catenin is sufficient to induce multinucleation in young animals. Interestingly, mutations in α-catenin in mice result in retina pigment epithelial multinucleation associated with macular disease. Therefore, this study has discovered that polyploid cells arise by cell fusion and contribute to the decline in the biomechanical fitness of the animal with age.

Monday, March 4th - Immune Response

Yao, X., Ni, J., Lin, L., Jin, P. and Ma, F. (2023). The NF-kappaB/Relish Activates miR-308 to Negatively Regulate Imd Pathway Immune Signaling in Drosophila. J Immunol 211(4): 591-600. PubMed ID: 37358278
The strength and duration of the NF-κB signaling response must be tightly modulated to avoid an inadequate or excessive immune response. Relish, a core NF-7kappa;B transcription factor of the Drosophila Imd pathway, can control the expression of antimicrobial peptides, including Dpt and AttA, to defend against Gram-negative bacterial infections, but whether Relish may regulate miRNA expression to participate in the immune response remains unclear. In this study, taking advantage of Drosophila S2 cells and different overexpression/knockout/knockdown flies, it was first found that Relish could directly activate the expression of miR-308 to negatively regulate the immune response and promote the survival of Drosophila during Enterobacter cloacae infection. Second, the results demonstrated that Relish-mediated expression of miR-308 could suppress target gene Tab2 to attenuate the Drosophila Imd pathway signal during the middle and late stages of the immune response. Third, the dynamic expression patterns of Dpt, AttA, Relish, miR-308, and Tab2 was detected in wild-type flies after E. coli infection, which further revealed that the feedback regulatory loop of Relish-miR-308-Tab2 plays a crucial role in the immune response and homeostasis maintenance of the Drosophila Imd pathway. Overall, this study not only illustrates an important mechanism by which this Relish-miR-308-Tab2 regulatory axis can negatively control the Drosophila immune response and participate in homeostasis maintenance but also provides new insights into the dynamic regulation of the NF-κB/miRNA expression network of animal innate immunity.
Odnokoz, O., Earland, N., Badinloo, M., Klichko, V. I., Benes, J., Orr, W. C., Radyuk, S. N. (2023). Peroxiredoxins Play an Important Role in the Regulation of Immunity and Aging in Drosophila. Antioxidants (Basel, Switzerland), 12(8) PubMed ID: 37627611
Aberrant immune responses and chronic inflammation can impose significant health risks and promote premature aging. Pro-inflammatory responses are largely mediated via reactive oxygen species (ROS) and reduction-oxidation reactions. A pivotal role in maintaining cellular redox homeostasis and the proper control of redox-sensitive signaling belongs to a family of antioxidant and redox-regulating thiol-related peroxidases designated as peroxiredoxins (Prx). Recent studies in Drosophila have shown that Prxs play a critical role in aging and immunity. This study identified two important 'hubs', the endoplasmic reticulum (ER) and mitochondria, where extracellular and intracellular stress signals are transformed into pro-inflammatory responses that are modulated by the activity of the Prxs residing in these cellular organelles. This study found that mitochondrial Prx activity in the intestinal epithelium is required to prevent the development of intestinal barrier dysfunction, which can drive systemic inflammation and premature aging. Using a redox-negative mutant, this study demonstrated that Prx acts in a redox-dependent manner in regulating the age-related immune response. The hyperactive immune response observed in flies under-expressing mitochondrial Prxs is due to a response to abiotic signals but not to changes in the bacterial content. This hyperactive response, but not reduced lifespan phenotype, can be rescued by the ER-localized Prx.
Hastings, C. J., Keledjian, M. V., Musselman, L. P., Marques, C. N. H. (2023). Delayed host mortality and immune response upon infection with P. aeruginosa persister cells. Infection and immunity, 91(10):e0024623 PubMed ID: 37732789
Chronic infections are a heavy burden on healthcare systems worldwide. Persister cells are thought to be largely responsible for chronic infection due to their tolerance to antimicrobials and recalcitrance to innate immunity factors. Pseudomonas aeruginosa is a common and clinically relevant pathogen that contains stereotypical persister cells. Despite their importance in chronic infection, there have been limited efforts to study persister cell infections in vivo. Drosophila melanogaster has a well-described innate immune response similar to that of vertebrates and is a good candidate for the development of an in vivo model of infection for persister cells. Similar to what is observed in other bacterial strains, in this work it was found that infection with P. aeruginosa persister cells resulted in a delayed mortality phenotype in Caenorhabditis elegans, Arabidopsis thaliana, and D. melanogaster compared to infection with regular cells. An in-depth characterization of infected D. melanogaster found that bacterial loads differed between persister and regular cells' infections during the early stages. Furthermore, hemocyte activation and antimicrobial peptide expression were delayed/reduced in persister infections over the same time course, indicating an initial suppression of, or inability to elicit, the fly immune response. Overall, these findings support the use of D. melanogaster as a model in which to study persister cells in vivo, where this bacterial subpopulation exhibits delayed virulence and an attenuated immune response.
Smith, B. R., Patch, K. B., Gupta, A., Knoles, E. M. and Unckless, R. L. (2023). The genetic basis of variation in immune defense against Lysinibacillus fusiformis infection in Drosophila melanogaster. PLoS Pathog 19(8): e1010934. PubMed ID: 37549163
The genetic causes of phenotypic variation often differ depending on the population examined, particularly if the populations were founded by relatively small numbers of genotypes. Similarly, the genetic causes of phenotypic variation among similar traits (resistance to different xenobiotic compounds or pathogens) may also be completely different or only partially overlapping. Differences in genetic causes for variation in the same trait among populations suggests context dependence for how selection acts on those traits. Similarities in the genetic causes of variation for different traits, on the other hand, suggests pleiotropy which would also influence how natural selection shapes variation in a trait. This study characterized immune defense against a natural Drosophila pathogen, the Gram-positive bacterium Lysinibacillus fusiformis, in three different populations and found almost no overlap in the genetic architecture of variation in survival post infection. However, when comparing these results to a similar experiment with the fungal pathogen, B. bassiana, a convincing shared QTL peak was found for both pathogens. This peak contains the Bomanin cluster of Drosophila immune effectors. Loss of function mutants and RNAi knockdown experiments confirms a role of some of these genes in immune defense against both pathogens. This suggests that natural selection may act on the entire cluster of Bomanin genes (and the linked region under the QTL) or specific peptides for specific pathogens.
Shu, S., Jiang, M., Deng, X., Yue, W., Cao, X., Zhang, K., Wang, Z., He, H., Cui, J., Wang, Q., Qu, K. and Fang, Y. (2023). Heterochromatic silencing of immune-related genes in glia is required for BBB integrity and normal lifespan in Drosophila. Aging Cell: e13947. PubMed ID: 37594178
Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. This study reports that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, a series of lifespan rescue experiments indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, GLAD encoded heterochromatin-associating protein bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, these findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan.
Cattabriga, G., Giordani, G., Gargiulo, G., Cavaliere, V. (2023). Effect of aminergic signaling on the humoral innate immunity response of Drosophila. Frontiers in physiology, 14:1249205 PubMed ID: 37693001
Biogenic amines are crucial signaling molecules that modulate various physiological life functions both in vertebrates and invertebrates. In humans, these neurotransmitters influence the innate and adaptive immunity systems. This work analyzed whether the aminergic neurotransmission of dopamine, serotonin, and octopamine could have an impact on the humoral innate immune response of Drosophila melanogaster. This is a powerful model system widely used to uncover the insect innate immunity mechanisms which are also conserved in mammals. The neurotransmission of all these amines positively modulates the Toll-responsive antimicrobial peptide (AMP) drosomycin (drs) gene in adult flies infected with the Micrococcus luteus bacterium. Indeed, either blocking the neurotransmission in their specific aminergic neurons by expressing shibire(ts) (Shi(ts)) or silencing the vesicular monoamine transporter gene (dVMAT) by RNAi caused a significantly reduced expression of the Toll-responsive drs gene. However, upon M. luteus infection, the block of aminergic transmission did not alter the expression of AMP attacin genes responding to the immune deficiency (Imd) and Toll pathways. Overall, the results not only reveal a neuroimmune function for biogenic amines in humoral immunity but also further highlight the complexity of the network controlling AMP gene regulation.

Friday, March 1st - Protein expression, evolution, structure, and function

Kolonko-Adamska, M., Zawadzka-Kazimierczuk, A., Bartosinska-Marzec, P., Kozminski, W., Popowicz, G., Kręzel, A., Ozyhar, A., Greb-Markiewicz, B. (2023). Interaction patterns of methoprene-tolerant and germ cell-expressed Drosophila JH receptors suggest significant differences in their functioning. Front Mol Biosci, 10:1215550 PubMed ID: 37654797
Methoprene-tolerant (Met) and germ cell-expressed (Gce) proteins were shown to be juvenile hormone (JH) receptors of Drosophila melanogaster with partially redundant functions. This study raised the question of where the functional differentiation of paralogs comes from. Therefore, Met and Gce interaction patterns with selected partners was tested. The ability of Gce and its C-terminus (GceC) to interact with 14-3-3 in the absence of JH was tested. In contrast, Met or Met C-terminus (MetC) interactions with 14-3-3 were not observed. A detailed structural analysis was carried out of Met/Gce interactions with the nuclear receptor Fushi tarazu factor-1 (Ftz-F1) ligand-binding domain. GceC comprising an Ftz-F1-binding site and full-length protein interacts with Ftz-F1. In contrast to Gce, only MetC (not full-length Met) can interact with Ftz-F1 in the absence of JH. It is proposed that the described differences result from the distinct tertiary structure and accessibility of binding sites in the full-length Met/Gce. Moreover, it was hypothesize that each interacting partner can force disordered MetC and GceC to change the structure in a partner-specific manner. The observed interactions seem to determine the subcellular localization of Met/Gce by forcing their translocation between the nucleus and the cytoplasm, which may affect the activity of the proteins. The presented differences between Met and Gce can be crucial for their functional differentiation during D. melanogaster development and indicate Gce as a more universal and more active paralog. It is consistent with the theory indicating gce as an ancestor gene.
Singh, J., Karunaraj, P., Luf, M., Pfleger, C. M. (2023). Lysines K117 and K147 play conserved roles in Ras activation from Drosophila to mammals. G3 (Bethesda), 13(11) PubMed ID: 37665961
Ras signaling plays an important role in growth, proliferation, and developmental patterning. Maintaining appropriate levels of Ras signaling is important to establish patterning in development and to prevent diseases such as cancer in mature organisms. The Ras protein is represented by Ras85D in Drosophila and by HRas, NRas, and KRas in mammals. In the past dozen years, multiple reports have characterized both inhibitory and activating ubiquitination events regulating Ras proteins. Inhibitory Ras ubiquitination mediated by Rabex-5 or Lztr1 is highly conserved between flies and mammals. Activating ubiquitination events at K117 and K147 have been reported in mammalian HRas, NRas, and KRas, but it is unclear if these activating roles of K117 and K147 are conserved in flies. Addressing a potential conserved role for these lysines in Drosophila Ras activation requires phenotypes strong enough to assess suppression. Therefore, oncogenic Ras, RasG12V, which biases Ras to the GTP-loaded active conformation, was used. Double mutants RasG12V,K117R and RasG12V,K147R and triple mutant RasG12V,K117R,K147R were created to prevent lysine-specific post-translational modification of K117, K147, or both, respectively. Their phenotypes were compared to RasG12V in the wing to reveal the roles of these lysines. Although RasG12V,K147R did not show compelling or quantifiable differences from RasG12V, RasG12V,K117R showed visible and quantifiable suppression compared to RasG12V, and triple mutant RasG12V,K117R,K147R showed dramatic suppression compared to RasG12V and increased suppression compared to RasG12V,K117R. These data are consistent with highly conserved roles for K117 and K147 in Ras activation from flies to mammals.
Li, H., Aboudhiaf, S., Parrot, S., Scote-Blachon, C., Benetollo, C., Lin, J. S., Seugnet, L. (2023). Pallidin function in Drosophila surface glia regulates sleep and is dependent on amino acid availability. Cell Rep, 42(9):113025 PubMed ID: 37682712
The Pallidin protein is a central subunit of a multimeric complex called biogenesis of lysosome-related organelles complex 1 (BLOC1) that regulates specific endosomal functions and has been linked to schizophrenia. Downregulation of Pallidin and other members of BLOC1 in the surface glia, the Drosophila equivalent of the blood-brain barrier, reduces and delays nighttime sleep in a circadian-clock-dependent manner. In agreement with BLOC1 involvement in amino acid transport, downregulation of the large neutral amino acid transporter 1 (LAT1)-like transporters JhI-21 and mnd, as well as of TOR (target of rapamycin) amino acid signaling, phenocopy Pallidin knockdown. Furthermore, supplementing food with leucine normalizes the sleep/wake phenotypes of Pallidin downregulation, and this study identified a role for Pallidin in the subcellular trafficking of JhI-21. Finally, evidence is provided that Pallidin in surface glia is required for GABAergic neuronal activity. These data identify a BLOC1 function linking essential amino acid availability and GABAergic sleep/wake regulation.
Pandey, A., Galeone, A., Han, S. Y., Story, B. A., Consonni, G., Mueller, W. F., Steinmetz, L. M., Vaccari, T., Jafar-Nejad, H. (2023). Gut barrier defects, intestinal immune hyperactivation and enhanced lipid catabolism drive lethality in NGLY1-deficient Drosophila. Nat Commun, 14(1):5667 PubMed ID: 37704604
Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of gut bacteria versus non-bacterial insults on animal health in the context of barrier dysfunction is not well understood. This study establish that loss of Drosophila N-glycanase 1 (Pngl) in a specific intestinal cell type leads to gut barrier defects, causing starvation and JNK overactivation. These abnormalities, along with loss of Pngl in enterocytes and fat body, result in Foxo overactivation, leading to hyperactive innate immune response and lipid catabolism and thereby contributing to lethality. Germ-free rearing of Pngl mutants rescued their developmental delay but not lethality. However, raising Pngl mutants on isocaloric, fat-rich diets partially rescued lethality. These data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the lethality caused by loss of Pngl is primarily mediated through non-bacterial induction of immune and metabolic abnormalities.
Nithianantham, S., Iwanski, M. K., Gaska, I., Pandey, H., Bodrug, T., Inagaki, S., Major, J., Brouhard, G. J., Gheber, L., Rosenfeld, S. S., Forth, S., Hendricks, A. G., Al-Bassam, J. (2023). The kinesin-5 tail and bipolar minifilament domains are the origin of its microtubule crosslinking and sliding activity. Mol Biol Cell, 34(11):ar111 PubMed ID: 37610838
Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, a minimal kinesin-5 tetramer (mini-tetramer) was constructed. The x-ray structure of the extended, 34-nm BASS domain was determined. Guided by these structural studies, A active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues were constructed which are half the length of native kinesin-5. These kinesin-5 mini-tetramers were used to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, this study found that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.
Mattedi, F., Lloyd-Morris, E., Hirth, F., Vagnoni, A. (2023). Optogenetic cleavage of the Miro GTPase reveals the direct consequences of real-time loss of function in Drosophila. PLoS Biol, 21(8):e3002273 PubMed ID: 37590319
Miro GTPases control mitochondrial morphology, calcium homeostasis, and regulate mitochondrial distribution by mediating their attachment to the kinesin and dynein motor complex. It is not clear, however, how Miro proteins spatially and temporally integrate their function as acute disruption of protein function has not been performed. To address this issue, this study has developed an optogenetic loss of function "Split-Miro" allele for precise control of Miro-dependent mitochondrial functions in Drosophila. Rapid optogenetic cleavage of Split-Miro leads to a striking rearrangement of the mitochondrial network, which is mediated by mitochondrial interaction with the microtubules. Unexpectedly, this treatment did not impact the ability of mitochondria to buffer calcium or their association with the endoplasmic reticulum. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows that Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. In adult fly neurons in vivo, Split-Miro photocleavage affects both mitochondrial trafficking and neuronal activity. Furthermore, functional replacement of endogenous Miro with Split-Miro identifies its essential role in the regulation of locomotor activity in adult flies, demonstrating the feasibility of tuning animal behaviour by real-time loss of protein function.
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