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Tuesday, June 30th, 2021 - Chromatin

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Nguyen, A. H. and Bachtrog, D. (2021). Toxic Y chromosome: Increased repeat expression and age-associated heterochromatin loss in male Drosophila with a young Y chromosome. PLoS Genet 17(4): e1009438. PubMed ID: 33886541
Summary:
Sex-specific differences in lifespan are prevalent across the tree of life and influenced by heteromorphic sex chromosomes. In species with XY sex chromosomes, females often outlive males. Males and females can differ in their overall repeat content due to the repetitive Y chromosome, and repeats on the Y might lower survival of the heterogametic sex (toxic Y effect). This study takes advantage of the well-assembled young Y chromosome of Drosophila miranda to study the sex-specific dynamics of chromatin structure and repeat expression during aging in male and female flies. Male D. miranda have about twice as much repetitive DNA compared to females, and live shorter than females. Heterochromatin is crucial for silencing of repetitive elements, yet old D. miranda flies lose H3K9me3 modifications in their pericentromere, with heterochromatin loss being more severe during aging in males than females. Satellite DNA becomes de-repressed more rapidly in old vs. young male flies relative to females. In contrast to what is observed in D. melanogaster, it was fond that transposable elements (TEs) are expressed at higher levels in male D. miranda throughout their life. Epigenetic silencing via heterochromatin formation is ineffective on the TE-rich neo-Y chromosome, presumably due to active transcription of a large number of neo-Y linked genes, resulting in up-regulation of Y-linked TEs already in young males. This is consistent with an interaction between the evolutionary age of the Y chromosome and the genomic effects of aging. These data support growing evidence that "toxic Y chromosomes" can diminish male fitness and a reduction in heterochromatin can contribute to sex-specific aging.
Ngian, Z. K., Lin, W. Q. and Ong, C. T. (2021). NELF-A controls Drosophila healthspan by regulating heat-shock protein-mediated cellular protection and heterochromatin maintenance. Aging Cell: e13348. PubMed ID: 33788376
Summary:
NELF-mediated pausing of RNA polymerase II (RNAPII) constitutes a crucial step in transcription regulation. However, it remains unclear how control release of RNAPII pausing can affect the epigenome and regulate important aspects of animal physiology like aging. This study found that NELF-A dosage regulates Drosophila healthspan: Halving NELF-A level in the heterozygous mutants or via neuronal-specific RNAi depletion improves their locomotor activity, stress resistance, and lifespan significantly. Conversely, NELF-A overexpression shortens fly lifespan drastically. Mechanistically, lowering NELF-A level facilitates the release of paused RNAPII for productive transcription of the heat-shock protein (Hsp) genes. The elevated HSPs expression in turn attenuates the accumulation of insoluble protein aggregates, reactive oxidative species, DNA damage and systemic inflammation in the brains of aging NELF-A depleted flies as compared to their control siblings. This pro-longevity effect is unique to NELF-A due to its higher expression level and more efficient pausing of RNAPII than other NELF subunits. Importantly, enhanced resistance to oxidative stress in NELF-A heterozygous mutants is highly conserved such that knocking down its level in human SH-SY5Y cells attenuates hydrogen peroxide-induced DNA damage and apoptosis. Depleting NELF-A reconfigures the epigenome through the maintenance of H3K9me2-enriched heterochromatin during aging, leading to the repression of specific retrotransposons like Gypsy-1 in the brains of NELF-A mutants. Taken together, this study showed that the dosage of neuronal NELF-A affects multiple aspects of aging in Drosophila by regulating transcription of Hsp genes in the brains, suggesting that targeting transcription elongation might be a viable therapeutic strategy against age-onset diseases like neurodegeneration.
Li, M., Zhao, Q., Belloli, R., Duffy, C. R. and Cai, H. N. (2021). Insulator foci distance correlates with cellular and nuclear morphology in early Drosophila embryos. Dev Biol 476: 189-199. PubMed ID: 33844976
Summary:
The three-dimensional (3D) organization of the genome is highly dynamic, changing during development and varying across different tissues and cell types. Recent studies indicate that these changes alter regulatory interactions, leading to changes in gene expression. Despite its importance, the mechanisms that influence genomic organization remain poorly understood. A network of chromatin boundary elements, or insulators, has ben identified in the Drosophila Antennapedia homeotic complex (ANT-C). These genomic elements interact with one another to tether chromatin loops that could block or promote enhancer-promoter interactions. To understand the function of these insulators, their interactions were assessed by measuring their 3D nuclear distance in developing animal tissues. The data suggest that the ANT-C Hox complex might be in a folded or looped configuration rather than in a random or extended form. The architecture of the ANT-C complex, as read out by the pair-wise distance between insulators, undergoes a strong compression during late embryogenesis, coinciding with the reduction of cell and nuclear diameters due to continued cell divisions in post-cleavage cells. These results suggest that genomic architecture and gene regulation may be influenced by cellular morphology and movement during development.
Rozenwald, M. B., Galitsyna, A. A., Sapunov, G. V., Khrameeva, E. E. and Gelfand, M. S. (2020). A machine learning framework for the prediction of chromatin folding in Drosophila using epigenetic features. PeerJ Comput Sci 6: e307. PubMed ID: 33816958
Summary:
Technological advances have lead to the creation of large epigenetic datasets, including information about DNA binding proteins and DNA spatial structure. Hi-C experiments have revealed that chromosomes are subdivided into sets of self-interacting domains called Topologically Associating Domains (TADs). TADs are involved in the regulation of gene expression activity, but the mechanisms of their formation are not yet fully understood. This study focussed on machine learning methods to characterize DNA folding patterns in Drosophila based on chromatin marks across three cell lines. This study presents linear regression models with four types of regularization, gradient boosting, and recurrent neural networks (RNN) as tools to study chromatin folding characteristics associated with TADs given epigenetic chromatin immunoprecipitation data. The bidirectional long short-term memory recurrent neural network architecture produced the best prediction scores and identified biologically relevant features. Distribution of protein Chriz (Chromator) and histone modification H3K4me3 were selected as the most informative features for the prediction of TADs characteristics. This approach may be adapted to any similar biological dataset of chromatin features across various cell lines and species. The code for the implemented pipeline, Hi-ChiP-ML, is publicly available.
Prozzillo, Y., Cuticone, S., Ferreri, D., Fattorini, G., Messina, G. and Dimitri, P. (2021). In Vivo Silencing of Genes Coding for dTip60 Chromatin Remodeling Complex Subunits Affects Polytene Chromosome Organization and Proper Development in Drosophila melanogaster. Int J Mol Sci 22(9). PubMed ID: 33926075
Summary:
Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. This work combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.
Pooryasin, A., Maglione, M., Schubert, M., Matkovic-Rachid, T., Hasheminasab, S. M., Pech, U., Fiala, A., Mielke, T. and Sigrist, S. J. (2021). Unc13A and Unc13B contribute to the decoding of distinct sensory information in Drosophila. Nat Commun 12(1): 1932. PubMed ID: 33771998
Summary:
The physical distance between presynaptic Ca(2+) channels and the Ca(2+) sensors triggering the release of neurotransmitter-containing vesicles regulates short-term plasticity (STP). While STP is highly diversified across synapse types, the computational and behavioral relevance of this diversity remains unclear. In the Drosophila brain, at nanoscale level, distinct coupling distances between Ca(2+) channels and the (m)unc13 family priming factors, Unc13A and Unc13B can be distinguished. Importantly, coupling distance defines release components with distinct STP characteristics. Here, this study shows that while Unc13A and Unc13B both contribute to synaptic signalling, they play distinct roles in neural decoding of olfactory information at excitatory projection neuron (ePN) output synapses. Unc13A clusters closer to Ca(2+) channels than Unc13B, specifically promoting fast phasic signal transfer. Reduction of Unc13A in ePNs attenuates responses to both aversive and appetitive stimuli, while reduction of Unc13B provokes a general shift towards appetitive values. Collectively, this study provides direct genetic evidence that release components of distinct nanoscopic coupling distances differentially control STP to play distinct roles in neural decoding of sensory information.

Monday, June 29th - Disease Models

Madi, J. R., Outa, A. A., Ghannam, M., Hussein, H. M., Shehab, M., Hasan, Z., Fayad, A. A., Shirinian, M. and Rahal, E. A. (2021). Drosophila melanogaster as a Model System to Assess the Effect of Epstein-Barr Virus DNA on Inflammatory Gut Diseases. Front Immunol 12: 586930. PubMed ID: 33828545
Summary:
The Epstein-Barr virus (EBV) commonly infects humans and is highly associated with different types of cancers and autoimmune diseases. EBV has also been detected in inflamed gastrointestinal mucosa of patients suffering from prolonged inflammation of the digestive tract such as inflammatory bowel disease (IBD) with no clear role identified yet for EBV in the pathology of such diseases. Since immune-stimulating capabilities of EBV DNA has been reported in various models, this study investigated whether EBV DNA may play a role in exacerbating intestinal inflammation through innate immune and regeneration responses using the Drosophila melanogaster model. Inflamed gastrointestinal tracts were generated in adult fruit flies through the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide that causes human ulcerative colitis- like pathologies due to its toxicity to intestinal cells. Intestinal damage induced by inflammation recruited plasmatocytes to the ileum in fly hindguts. EBV DNA aggravated inflammation by enhancing the immune deficiency (IMD) pathway as well as further increasing the cellular inflammatory responses manifested upon the administration of DSS. The study at hand proposes a possible immunostimulatory role of the viral DNA exerted specifically in the fly hindgut hence further developing understanding of immune responses mounted against EBV DNA in the latter intestinal segment of the D. melanogaster gut. These findings suggest that EBV DNA may perpetuate proinflammatory processes initiated in an inflamed digestive system. These findings indicate that D. melanogaster can serve as a model to further understand EBV-associated gastroinflammatory pathologies. Further studies employing mammalian models may validate the immunogenicity of EBV DNA in an IBD context and its role in exacerbating the disease through inflammatory mediators.
Lubojemska, A., Stefana, M. I., Sorge, S., Bailey, A. P., Lampe, L., Yoshimura, A., Burrell, A., Collinson, L. and Gould, A. P. (2021). Adipose triglyceride lipase protects renal cell endocytosis in a Drosophila dietary model of chronic kidney disease. PLoS Biol 19(5): e3001230. PubMed ID: 33945525
Summary:
Obesity-related renal lipotoxicity and chronic kidney disease (CKD) are prevalent pathologies with complex aetiologies. One hallmark of renal lipotoxicity is the ectopic accumulation of lipid droplets in kidney podocytes and in proximal tubule cells. Renal lipid droplets are observed in human CKD patients and in high-fat diet (HFD) rodent models, but their precise role remains unclear. This study establish a HFD model in Drosophila that recapitulates renal lipid droplets and several other aspects of mammalian CKD. Cell type-specific genetic manipulations show that lipid can overflow from adipose tissue and is taken up by renal cells called nephrocytes. A HFD drives nephrocyte lipid uptake via the multiligand receptor Cubilin (Cubn), leading to the ectopic accumulation of lipid droplets. These nephrocyte lipid droplets correlate with endoplasmic reticulum (ER) and mitochondrial deficits, as well as with impaired macromolecular endocytosis, a key conserved function of renal cells. Nephrocyte knockdown of diglyceride acyltransferase 1 (DGAT1), overexpression of adipose triglyceride lipase (ATGL; brummer), and epistasis tests together reveal that fatty acid flux through the lipid droplet triglyceride compartment protects the ER, mitochondria, and endocytosis of renal cells. Strikingly, boosting nephrocyte expression of the lipid droplet resident enzyme ATGL is sufficient to rescue HFD-induced defects in renal endocytosis. Moreover, endocytic rescue requires a conserved mitochondrial regulator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α). This study demonstrates that lipid droplet lipolysis counteracts the harmful effects of a HFD via a mitochondrial pathway that protects renal endocytosis. It also provides a genetic strategy for determining whether lipid droplets in different biological contexts function primarily to release beneficial or to sequester toxic lipids.
Martin, E., Heidari, R., Monnier, V. and Tricoire, H. (2021). Genetic Screen in Adult Drosophila Reveals That dCBP Depletion in Glial Cells Mitigates Huntington Disease Pathology through a Foxo-Dependent Pathway. Int J Mol Sci 22(8). PubMed ID: 33918672
Summary:
Huntington's disease (HD) is a progressive and fatal autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the first exon of the huntingtin gene (HTT). In spite of considerable efforts, there is currently no treatment to stop or delay the disease. Although HTT is expressed ubiquitously, most of the current knowledge has been obtained on neurons. More recently, the impact of mutant huntingtin (mHTT) on other cell types, including glial cells, has received growing interest. It is currently unclear whether new pathological pathways could be identified in these cells compared to neurons. To address this question, an in vivo screen was performed for modifiers of mutant huntingtin (HTT-548-128Q) induced pathology in Drosophila adult glial cells and several putative therapeutic targets were identified. Among them, it was discovered that partial nej/dCBP depletion in these cells was protective, as revealed by strongly increased lifespan and restored locomotor activity. Thus, dCBP promotes the HD pathology in glial cells, in contrast to previous opposite findings in neurons. Further investigations implicated the transcriptional activator Foxo as a critical downstream player in this glial protective pathway. These data suggest that combinatorial approaches combined to specific tissue targeting may be required to uncover efficient therapies in HD.
Muraoka, Y., Nikaido, A., Kowada, R., Kimura, H., Yamaguchi, M. and Yoshida, H. (2021). Identification of Rpd3 as a novel epigenetic regulator of Drosophila FIG 4, a Charcot-Marie-Tooth disease-causing gene. Neuroreport 32(7): 562-568. PubMed ID: 33850086
Summary:
Mutations in the factor-induced-gene 4 (FIG 4) gene are associated with multiple disorders, including Charcot-Marie-Tooth disease (CMT), epilepsy with polymicrogyria, Yunis-Varon syndrome and amyotrophic lateral sclerosis. The wide spectrum of disorders associated with FIG 4 may be related to the dysregulated epigenetics. Using Gene Expression Omnibus, this study found that HDAC1 binds to the FIG 4 gene locus in the genome of human CD4+ T cells. Rpd3 is a well-known Drosophila homolog of human HDAC1. Previous work established Drosophila models targeting Drosophila FIG 4 (FIG 4) that exhibited defective locomotive ability, abnormal synapse morphology at neuromuscular junctions, enlarged vacuoles in the fat body and aberrant compound eye morphology. Genetic crossing experiments followed by physiological and immunocytochemical analyses revealed that Rpd3 mutations suppressed these defects induced by dFIG 4 knockdown. This demonstrated that Rpd3 is an important epigenetic regulator of dFIG 4, suggesting that the inhibition of HDAC1 represses the pathogenesis of FIG 4-associated disorders, including CMT. Defects in epigenetic regulators, such as HDAC1, may also explain the diverse symptoms of FIG 4-associated disorders.
Liang, Y., Piao, C., Beuschel, C. B., Toppe, D., Kollipara, L., Bogdanow, B., Maglione, M., Lutzkendorf, J., See, J. C. K., Huang, S., Conrad, T. O. F., Kintscher, U., Madeo, F., Liu, F., Sickmann, A. and Sigrist, S. J. (2021). eIF5A hypusination, boosted by dietary spermidine, protects from premature brain aging and mitochondrial dysfunction. Cell Rep 35(2): 108941. PubMed ID: 33852845
Summary:
Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (N(ε)-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). This study shows that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline.
Lin, X., Wen, X., Wei, Z., Guo, K., Shi, F., Huang, T., Wang, W. and Zheng, J. (2021). Vitamin K2 protects against Abeta42-induced neurotoxicity by activating autophagy and improving mitochondrial function in Drosophila. Neuroreport 32(6): 431-437. PubMed ID: 33788812
Summary:
Alzheimer disease is characterized by progressive decline in cognitive function due to neurodegeneration induced by accumulation of Aβ and hyperphosphorylated tau protein. This study was conducted to explore the protective effect of vitamin K2 against Aβ42-induced neurotoxicity. Alzheimer disease transgenic Drosophila model used in this study was amyloid beta with the arctic mutation expressed in neurons. Alzheimer disease flies were treated with vitamin K2 for 28 days after eclosion. Aβ42 level in brain was detected by ELISA. Autophagy-related genes and NDUFS3, the core subunit of mitochondrial complex I, were examined using real-Time PCR (RT-PCR) and western blot analysis. Vitamin K2 improved climbing ability, prolonged lifespan and decreased Aβ42 levels, upregulated the expression of LC3 and Beclin1, increased the conversion of LC3I to LC3II and decreased p62 level. in Alzheimer disease flies. In addition, vitamin K2 upregulated the expression of NDUFS3 and increased ATP production in Alzheimer disease flies. It seems that vitamin K2 protect against Aβ42-induced neurotoxicity by activation of autophagy and rescue mitochondrial dysfunction, which suggests that it may be a potential valuable therapeutic approach for Alzheimer disease (Lin, 2021).

Thursday, June 24th - Stem Cells

Madi, J. R., Outa, A. A., Ghannam, M., Hussein, H. M., Shehab, M., Hasan, Z., Fayad, A. A., Shirinian, M. and Rahal, E. A. (2021). Drosophila melanogaster as a Model System to Assess the Effect of Epstein-Barr Virus DNA on Inflammatory Gut Diseases. Front Immunol 12: 586930. PubMed ID: 33828545
Summary:
The Epstein-Barr virus (EBV) commonly infects humans and is highly associated with different types of cancers and autoimmune diseases. EBV has also been detected in inflamed gastrointestinal mucosa of patients suffering from prolonged inflammation of the digestive tract such as inflammatory bowel disease (IBD) with no clear role identified yet for EBV in the pathology of such diseases. Since immune-stimulating capabilities of EBV DNA has been reported in various models, this study investigated whether EBV DNA may play a role in exacerbating intestinal inflammation through innate immune and regeneration responses using the Drosophila melanogaster model. Inflamed gastrointestinal tracts were generated in adult fruit flies through the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide that causes human ulcerative colitis- like pathologies due to its toxicity to intestinal cells. Intestinal damage induced by inflammation recruited plasmatocytes to the ileum in fly hindguts. EBV DNA aggravated inflammation by enhancing the immune deficiency (IMD) pathway as well as further increasing the cellular inflammatory responses manifested upon the administration of DSS. The study at hand proposes a possible immunostimulatory role of the viral DNA exerted specifically in the fly hindgut hence further developing understanding of immune responses mounted against EBV DNA in the latter intestinal segment of the D. melanogaster gut. These findings suggest that EBV DNA may perpetuate proinflammatory processes initiated in an inflamed digestive system. These findings indicate that D. melanogaster can serve as a model to further understand EBV-associated gastroinflammatory pathologies. Further studies employing mammalian models may validate the immunogenicity of EBV DNA in an IBD context and its role in exacerbating the disease through inflammatory mediators.
Lubojemska, A., Stefana, M. I., Sorge, S., Bailey, A. P., Lampe, L., Yoshimura, A., Burrell, A., Collinson, L. and Gould, A. P. (2021). Adipose triglyceride lipase protects renal cell endocytosis in a Drosophila dietary model of chronic kidney disease. PLoS Biol 19(5): e3001230. PubMed ID: 33945525
Summary:
Obesity-related renal lipotoxicity and chronic kidney disease (CKD) are prevalent pathologies with complex aetiologies. One hallmark of renal lipotoxicity is the ectopic accumulation of lipid droplets in kidney podocytes and in proximal tubule cells. Renal lipid droplets are observed in human CKD patients and in high-fat diet (HFD) rodent models, but their precise role remains unclear. This study establish a HFD model in Drosophila that recapitulates renal lipid droplets and several other aspects of mammalian CKD. Cell type-specific genetic manipulations show that lipid can overflow from adipose tissue and is taken up by renal cells called nephrocytes. A HFD drives nephrocyte lipid uptake via the multiligand receptor Cubilin (Cubn), leading to the ectopic accumulation of lipid droplets. These nephrocyte lipid droplets correlate with endoplasmic reticulum (ER) and mitochondrial deficits, as well as with impaired macromolecular endocytosis, a key conserved function of renal cells. Nephrocyte knockdown of diglyceride acyltransferase 1 (DGAT1), overexpression of adipose triglyceride lipase (ATGL; brummer), and epistasis tests together reveal that fatty acid flux through the lipid droplet triglyceride compartment protects the ER, mitochondria, and endocytosis of renal cells. Strikingly, boosting nephrocyte expression of the lipid droplet resident enzyme ATGL is sufficient to rescue HFD-induced defects in renal endocytosis. Moreover, endocytic rescue requires a conserved mitochondrial regulator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α). This study demonstrates that lipid droplet lipolysis counteracts the harmful effects of a HFD via a mitochondrial pathway that protects renal endocytosis. It also provides a genetic strategy for determining whether lipid droplets in different biological contexts function primarily to release beneficial or to sequester toxic lipids.
Martin, E., Heidari, R., Monnier, V. and Tricoire, H. (2021). Genetic Screen in Adult Drosophila Reveals That dCBP Depletion in Glial Cells Mitigates Huntington Disease Pathology through a Foxo-Dependent Pathway. Int J Mol Sci 22(8). PubMed ID: 33918672
Summary:
Huntington's disease (HD) is a progressive and fatal autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the first exon of the huntingtin gene (HTT). In spite of considerable efforts, there is currently no treatment to stop or delay the disease. Although HTT is expressed ubiquitously, most of the current knowledge has been obtained on neurons. More recently, the impact of mutant huntingtin (mHTT) on other cell types, including glial cells, has received growing interest. It is currently unclear whether new pathological pathways could be identified in these cells compared to neurons. To address this question, an in vivo screen was performed for modifiers of mutant huntingtin (HTT-548-128Q) induced pathology in Drosophila adult glial cells and several putative therapeutic targets were identified. Among them, it was discovered that partial nej/dCBP depletion in these cells was protective, as revealed by strongly increased lifespan and restored locomotor activity. Thus, dCBP promotes the HD pathology in glial cells, in contrast to previous opposite findings in neurons. Further investigations implicated the transcriptional activator Foxo as a critical downstream player in this glial protective pathway. These data suggest that combinatorial approaches combined to specific tissue targeting may be required to uncover efficient therapies in HD.
Muraoka, Y., Nikaido, A., Kowada, R., Kimura, H., Yamaguchi, M. and Yoshida, H. (2021). Identification of Rpd3 as a novel epigenetic regulator of Drosophila FIG 4, a Charcot-Marie-Tooth disease-causing gene. Neuroreport 32(7): 562-568. PubMed ID: 33850086
Summary:
Mutations in the factor-induced-gene 4 (FIG 4) gene are associated with multiple disorders, including Charcot-Marie-Tooth disease (CMT), epilepsy with polymicrogyria, Yunis-Varon syndrome and amyotrophic lateral sclerosis. The wide spectrum of disorders associated with FIG 4 may be related to the dysregulated epigenetics. Using Gene Expression Omnibus, this study found that HDAC1 binds to the FIG 4 gene locus in the genome of human CD4+ T cells. Rpd3 is a well-known Drosophila homolog of human HDAC1. Previous work established Drosophila models targeting Drosophila FIG 4 (FIG 4) that exhibited defective locomotive ability, abnormal synapse morphology at neuromuscular junctions, enlarged vacuoles in the fat body and aberrant compound eye morphology. Genetic crossing experiments followed by physiological and immunocytochemical analyses revealed that Rpd3 mutations suppressed these defects induced by dFIG 4 knockdown. This demonstrated that Rpd3 is an important epigenetic regulator of dFIG 4, suggesting that the inhibition of HDAC1 represses the pathogenesis of FIG 4-associated disorders, including CMT. Defects in epigenetic regulators, such as HDAC1, may also explain the diverse symptoms of FIG 4-associated disorders.
Liang, Y., Piao, C., Beuschel, C. B., Toppe, D., Kollipara, L., Bogdanow, B., Maglione, M., Lützkendorf, J., See, J. C. K., Huang, S., Conrad, T. O. F., Kintscher, U., Madeo, F., Liu, F., Sickmann, A. and Sigrist, S. J. (2021). eIF5A hypusination, boosted by dietary spermidine, protects from premature brain aging and mitochondrial dysfunction. Cell Rep 35(2): 108941. PubMed ID: 33852845
Summary:
Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (N(ε)-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). This study shows that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline.
Lin, X., Wen, X., Wei, Z., Guo, K., Shi, F., Huang, T., Wang, W. and Zheng, J. (2021). Vitamin K2 protects against Abeta42-induced neurotoxicity by activating autophagy and improving mitochondrial function in Drosophila. Neuroreport 32(6): 431-437. PubMed ID: 33788812
Summary:
Alzheimer disease is characterized by progressive decline in cognitive function due to neurodegeneration induced by accumulation of Aβ and hyperphosphorylated tau protein. This study was conducted to explore the protective effect of vitamin K2 against Aβ42-induced neurotoxicity. Alzheimer disease transgenic Drosophila model used in this study was amyloid beta with the arctic mutation expressed in neurons. Alzheimer disease flies were treated with vitamin K2 for 28 days after eclosion. Aβ42 level in brain was detected by ELISA. Autophagy-related genes and NDUFS3, the core subunit of mitochondrial complex I, were examined using real-Time PCR (RT-PCR) and western blot analysis. Vitamin K2 improved climbing ability, prolonged lifespan and decreased Aβ42 levels, upregulated the expression of LC3 and Beclin1, increased the conversion of LC3I to LC3II and decreased p62 level. in Alzheimer disease flies. In addition, vitamin K2 upregulated the expression of NDUFS3 and increased ATP production in Alzheimer disease flies. It seems that vitamin K2 protect against Aβ42-induced neurotoxicity by activation of autophagy and rescue mitochondrial dysfunction, which suggests that it may be a potential valuable therapeutic approach for Alzheimer disease (Lin, 2021).

Thursday, June 24th - Stem Cells

Rackley, B., Seong, C. S., Kiely, E., Parker, R. E., Rupji, M., Dwivedi, B., Heddleston, J. M., Giang, W., Anthony, N., Chew, T. L. and Gilbert-Ross, M. (2021). The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo. Commun Biol 4(1): 142. PubMed ID: 33514834
Summary:
The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. This study generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. Low-level expression of oncogenic Ras (RasLow) was shown to promote the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, this study has characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. This molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS were shown to exhibit worse overall survival and increased AMPK activation. These results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.
Manikowski, D., Kastl, P., Schurmann, S., Ehring, K., Steffes, G., Jakobs, P. and Grobe, K. (2020). C-Terminal Peptide Modifications Reveal Direct and Indirect Roles of Hedgehog Morphogen Cholesteroylation. Front Cell Dev Biol 8: 615698. PubMed ID: 33511123
Summary:
Hedgehog (Hh) morphogens are involved in embryonic development and stem cell biology and, if misregulated, can contribute to cancer. One important post-translational modification with profound impact on Hh biofunction is its C-terminal cholesteroylation during biosynthesis. The current hypothesis is that the cholesterol moiety is a decisive factor in Hh association with the outer plasma membrane leaflet of producing cells, cell-surface Hh multimerization, and its transport and signaling. Yet, it is not decided whether the cholesterol moiety is directly involved in all of these processes, because their functional interdependency raises the alternative possibility that the cholesterol initiates early processes directly and that these processes can then steer later stages of Hh signaling independent of the lipid. This study generated variants of the C-terminal Hh peptide and observed that these cholesteroylated peptides variably impaired several post-translational processes in producing cells and Hh biofunction in Drosophila melanogaster eye and wing development. This study also found that substantial Hh amounts separated from cholesteroylated peptide tags in vitro and in vivo and that tagged and untagged Hh variants lacking their C-cholesterol moieties remained bioactive. This approach thus confirms that Hh cholesteroylation is essential during the early steps of Hh production and maturation but also suggests that it is dispensable for Hh signal reception at receiving cells.
Pathak, H., Maya, A. V., Tanari, A. B., Sarkar, S. and Varghese, J. (2021). Lint, a transmembrane serine protease, regulates growth and metabolism in Drosophila. Genetics. PubMed ID: 33693655
Summary:
Insulin signaling in Drosophila has a significant role in regulating growth, metabolism, fecundity, stress response, and longevity. The molecular mechanism by which insulin signaling regulates these vital processes is dependent on the nutrient status and oxygen availability of the organism. In a genetic screen to identify novel genes that regulate Drosophila insulin signalling, Lumens interrupted (lint), a gene that has previously been shown to act in tracheal development, was discovered. The knockdown of lint gene expression using a Dilp2Gal4 driver which expresses in the neuronal insulin-producing cells (IPCs), led to defects in systemic insulin signaling, metabolic status and growth. However, this analysis of lint knockdown phenotypes revealed that downregulation of lint in the trachea and not IPCs was responsible for the growth phenotypes, as the Gal4 driver is also expressed in the tracheal system. Various tracheal terminal branch defects were found, including reduction in the length as well as number of branches in the lint knockdown background. This study reveals that substantial effects of lint downregulation arose because of tracheal defects, which induced tissue hypoxia, altered systemic insulin/TOR signaling, and resulted in effects on developmental growth regulation.
Huang, J., Gujar, M. R., Deng, Q., S, Y. C., Li, S., Tan, P., Sung, W. K. and Wang, H. (2021). Histone lysine methyltransferase Pr-set7/SETD8 promotes neural stem cell reactivation. EMBO Rep: e50994. PubMed ID: 33565211
Summary:
The ability of neural stem cells (NSCs) to switch between quiescence and proliferation is crucial for brain development and homeostasis. Increasing evidence suggests that variants of histone lysine methyltransferases including KMT5A are associated with neurodevelopmental disorders. However, the function of KMT5A/Pr-set7/SETD8 in the central nervous system is not well established. This study shows that Drosophila Pr-Set7 is a novel regulator of NSC reactivation. Loss of function of pr-set7 causes a delay in NSC reactivation and loss of H4K20 monomethylation in the brain. Through NSC-specific in vivo profiling, this study demonstrated that Pr-set7 binds to the promoter region of cyclin-dependent kinase 1 (cdk1) and Wnt pathway transcriptional co-activator earthbound1/jerky (ebd1). Further validation indicates that Pr-set7 is required for the expression of cdk1 and ebd1 in the brain. Similar to Pr-set7, Cdk1 and Ebd1 promote NSC reactivation. Finally, overexpression of Cdk1 and Ebd1 significantly suppressed NSC reactivation defects observed in pr-set7-depleted brains. Therefore, Pr-set7 promotes NSC reactivation by regulating Wnt signaling and cell cycle progression. These findings may contribute to the understanding of mammalian KMT5A/PR-SET7/SETD8 during brain development.
Bras, R., Monteiro, A., Sunkel, C. E. and Resende, L. P. (2021). Aneuploidy facilitates dysplastic and tumorigenic phenotypes in the Drosophila gut. Biol Open. PubMed ID: 33948620
Summary:
Aneuploidy has been strongly linked to cancer development, and published evidence has suggested that aneuploidy can have an oncogenic or a tumor suppressor role depending on the tissue context. Using the Drosophila midgut as a model, it was recently described that adult intestinal stem cells (ISCs), do not activate programmed cell death upon aneuploidy induction, leading to an increase in ISC proliferation rate, and tissue dysplasia.  How aneuploidy impacts ISCs in intestinal tumorigenic models remains to be investigated, and it represents a very important biological question to address since data from multiple in vivo models suggests that the cellular impact of aneuploidy is highly dependent on the cellular and tissue context. Using manipulation of different genetic pathways such as EGFR, JAK-STAT and Notch that cause dysplastic phenotypes in the Drosophila gut, this study found that concomitant aneuploidy induction by impairment of the Spindle Assembly Checkpoint (SAC) consistently leads to a more severe progression of intestinal dysplasia or tumorigenesis. This is characterized by an accumulation of progenitor cells, high tissue cell density and higher stem cell proliferation rates, revealing an additive or synergistic effect depending on the misregulated pathway in which aneuploidy was induced. Thus, these data suggests that in the Drosophila gut, both dysplasia and tumorigenic phenotypes can be fueled by inducing genomic instability of resident stem cells.
Yatsenko, A. S. and Shcherbata, H. R. (2021). Distant activation of Notch signaling induces stem cell niche assembly. PLoS Genet 17(3): e1009489. PubMed ID: 33780456
Summary:
This study shows that multiple modes of Notch signaling activation specify the complexity of spatial cellular interactions necessary for stem cell niche assembly. In particular, the formation was studied of the germline stem cell niche in Drosophila ovaries, which is a two-step process whereby terminal filaments are formed first. Then, terminal filaments signal to the adjacent cap cell precursors, resulting in Notch signaling activation, which is necessary for the lifelong acquisition of stem cell niche cell fate. The genetic data suggest that in order to initiate the process of stem cell niche assembly, Notch signaling is activated among non-equipotent cells via distant induction, where germline Delta is delivered to somatic cells located several diameters away via cellular projections generated by primordial germ cells. At the same time, to ensure the robustness of niche formation, terminal filament cell fate can also be induced by somatic Delta via cis- or trans-inhibition. This exemplifies a double security mechanism that guarantees that the germline stem cell niche is formed, since it is indispensable for the adjacent germline precursor cells to acquire and maintain stemness necessary for successful reproduction. These findings contribute to understanding of the formation of stem cell niches in their natural environment, which is important for stem cell biology and regenerative medicine.

Wednesday, June 23rd - Cytoskeleton and Junctions

Mysh, M. and Poulton, J. S. (2021). The Basolateral Polarity Module Promotes Slit Diaphragm Formation in Drosophila Nephrocytes, a Model of Vertebrate Podocytes. J Am Soc Nephrol. PubMed ID: 33795424

Podocyte slit diaphragms (SDs) are intercellular junctions that function as size-selective filters, excluding most proteins from urine. Abnormalities in SDs cause proteinuria and nephrotic syndrome. Podocytes exhibit apicobasal polarity, which can affect fundamental aspects of cell biology, including morphology, intercellular junction formation, and asymmetric protein distribution along the plasma membrane. Apical polarity protein mutations cause nephrotic syndrome, and data suggest apical polarity proteins regulate SD formation. However, there is no evidence that basolateral polarity proteins regulate SDs. Thus, the role of apicobasal polarity in podocytes remains unclear. Genetic manipulations and transgenic reporters determined the effects of disrupting apicobasal polarity proteins in Drosophila nephrocytes, which have SDs similar to those of mammalian podocytes. Confocal and electron microscopy were used to characterize SD integrity after loss of basolateral polarity proteins, and genetic-interaction studies illuminated relationships among apicobasal polarity proteins. The study identified four novel regulators of nephrocyte SDs: Dlg, Lgl, Scrib, and Par-1. These proteins comprise the basolateral polarity module and its effector kinase. The data suggest these proteins work together, with apical polarity proteins, to regulate SDs by promoting normal endocytosis and trafficking of SD proteins. Given the recognized importance of apical polarity proteins and SD protein trafficking in podocytopathies, the findings connecting basolateral polarity proteins to these processes significantly advance understanding of SD regulation.

Durney, C. H. and Feng, J. J. (2021). A three-dimensional vertex model for Drosophila salivary gland invagination. Phys Biol. PubMed ID: 33882465
Summary:
During epithelial morphogenesis, force generation at the cellular level not only causes cell deformation, but may also produce coordinated cell movement and rearrangement on the tissue level. This study used a novel three-dimensional vertex model to explore the roles of cellular forces during the formation of the salivary gland in the Drosophila embryo. Representing the placode as an epithelial sheet of initially columnar cells, focus was placed on the spatial and temporal patterning of contractile forces due to three actomyosin pools: the apicomedial actomyosin in the pit of the placode, junctional actomyosin arcs outside the pit, and a supracellular actomyosin cable along the circumference of the placode. In an in silico "wild type" model, these pools are activated at different times according to experimental data. To identify the role of each myosin pool, various in silico "mutants" were also simulated in which only one or two of the myosin pools are activated. It was found that the apicomedial myosin initiates a small dimple in the pit, but this is not essential for the overall invagination of the placode. The myosin arcs are the main driver of invagination and are responsible for the internalization of the apical surface. The circumferential actomyosin cable acts to constrict the opening of the developing tube, and is responsible for forming a properly shaped lumen. Cell intercalation tends to facilitate the invagination, but the geometric constraints of this model only allow a small number of intercalations, and their effect is minor. The placode invagination predicted by the model is in general agreement with experimental observations. It confirms some features of the current "belt-and-braces" model for the process, and provides new insights on the separate roles of the various myosin pools and their spatio-temporal coordination.
Denk-Lobnig, M., Totz, J. F., Heer, N. C., Dunkel, J. and Martin, A. C. (2021). Combinatorial patterns of graded RhoA activation and uniform F-actin depletion promote tissue curvature. Development. PubMed ID: 34124762
Summary:
During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Using quantitative imaging in Drosophila melanogaster, this study investigated how patterned cell shape changes promote tissue bending during early embryogenesis. The transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch, or maintain their shape. The myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. These experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature.
Thumecke, S. and Schroder, R. (2021). The odd-skipped related gene drumstick is required for leg development in the beetle Tribolium castaneum. Dev Dyn. PubMed ID: 33871128
Summary:
The evolutionarily conserved odd-skipped related genes odd-skipped (odd), drumstick (drm), sister of odd and bowel (sob), and brother-of-odd-with-entrails-limited (bwl) act downstream of the Notch pathway in various insect tissues including the appendages and the gut. While the function of some of these genes have been analyzed in the adult Tribolium beetle, the expression during and their requirement for embryonic development is not known. This study describes the embryonic expression patterns of drm, sob, and bwl and analyze the RNAi knockdown phenotypes with emphasize on the appendages and the hindgut. In Tribolium, drm acts independently of other odd-family members in the formation of legs, hindgut, and the dorsal epidermis. Moreover, drm and sob were establishee as further markers for segment borders in the appendages that include the gnathobasic mandibles. It is concluded that the regulatory interrelationship among the odd genes differs between Tribolium and Drosophila, where odd and drm seem to act redundantly. In Tribolium, the genes drm and sob uncover the relic of a precoxal joint incorporated in the lateral body wall.
Blatt, P., Wong-Deyrup, S. W., McCarthy, A., Breznak, S., Hurton, M. D., Upadhyay, M., Bennink, B., Camacho, J., Lee, M. T. and Rangan, P. (2021). RNA degradation is required for the germ-cell to maternal transition in Drosophila. Curr Biol. PubMed ID: 33989522
Summary:
In sexually reproducing animals, the oocyte contributes a large supply of RNAs that are essential to launch development upon fertilization. The mechanisms that regulate the composition of the maternal RNA contribution during oogenesis are unclear. This study shows that a subset of RNAs expressed during the early stages of oogenesis is subjected to regulated degradation during oocyte specification. Failure to remove these RNAs results in oocyte dysfunction and death. The RNA-degrading Super Killer complex and No-Go Decay factor Pelota were identified as key regulators of oogenesis via targeted degradation of specific RNAs expressed in undifferentiated germ cells. These regulators target RNAs enriched for cytidine sequences that are bound by the polypyrimidine tract binding protein Half pint. Thus, RNA degradation helps orchestrate a germ cell-to-maternal transition that gives rise to the maternal contribution to the zygote
Dye, N. A., Popovic, M., Iyer, K. V., Fuhrmann, J., Piscitello-Gomez, R., Eaton, S. and Julicher, F. (2021). Self-organized patterning of cell morphology via mechanosensitive feedback. Elife 10. PubMed ID: 33769281
Summary:
Tissue organization is often characterized by specific patterns of cell morphology. How such patterns emerge in developing tissues is a fundamental open question. This study investigated the emergence of tissue-scale patterns of cell shape and mechanical tissue stress in the Drosophila wing imaginal disc during larval development. Using quantitative analysis of the cellular dynamics, a pattern of radially oriented cell rearrangements was revealee that is coupled to the buildup of tangential cell elongation. Developing a laser ablation method, tissue stresses were mapped, and key parameters of tissue mechanics were extract. A continuum theory is presented showing that this pattern of cell morphology and tissue stress can arise via self-organization of a mechanical feedback that couples cell polarity to active cell rearrangements. The predictions of this model are supported by knockdown of MyoVI, a component of mechanosensitive feedback. This work reveals a mechanism for the emergence of cellular patterns in morphogenesis.

Tuesday, June 22 - Adult neural development and function

Muria, A., Musso, P. Y., Durrieu, M., Portugal, F. R., Ronsin, B., Gordon, M. D., Jeanson, R. and Isabel, G. (2021). Social facilitation of long-lasting memory is mediated by CO(2) in Drosophila. Curr Biol. PubMed ID: 33740428
Summary:
How social interactions influence cognition is a fundamental question, yet rarely addressed at the neurobiological level. It is well established that the presence of conspecifics affects learning and memory performance, but the neural basis of this process has only recently begun to be investigated. In the fruit fly Drosophila melanogaster, the presence of other flies improves retrieval of a long-lasting olfactory memory. This study demonstrates that this is a composite memory composed of two distinct elements. One is an individual memory that depends on outputs from the α'β' Kenyon cells (KCs) of the mushroom bodies (MBs), the memory center in the insect brain. The other is a group memory requiring output from the αβ KCs, a distinct sub-part of the MBs. Social facilitation of memory increases with group size and is triggered by CO(2) released by group members. Among the different known neurons carrying CO(2) information in the brain, this study established that the bilateral ventral projection neuron (biVPN), which projects onto the MBs, is necessary for social facilitation. Moreover, it was demonstrated that CO(2)-evoked memory engages a serotoninergic pathway involving the dorsal-paired medial (DPM) neurons, revealing a new role for this pair of serotonergic neurons. Overall, this study identified both the sensorial cue and the neural circuit (biVPNαβ>DPM>αβ) governing social facilitation of memory in flies. This study provides demonstration that being in a group recruits the expression of a cryptic memory and that variations in CO(2) concentration can affect cognitive processes in insects.
Springer, M. and Nawrot, M. P. (2021). A mechanistic model for reward prediction and extinction learning in the fruit fly. eNeuro. PubMed ID: 33785523
Summary:
Extinction learning, the ability to update previously learned information by integrating novel contradictory information, is of high clinical relevance for therapeutic approaches to the modulation of maladaptive memories. Insect models have been instrumental in uncovering fundamental processes of memory formation and memory update. Recent experimental results in Drosophila melanogaster suggest that, after the behavioral extinction of a memory, two parallel but opposing memory traces coexist, residing at different sites within the mushroom body. This study proposes a minimalistic circuit model of the Drosophila mushroom body that supports classical appetitive and aversive conditioning and memory extinction. The model is tailored to the existing anatomical data and involves two circuit motives of central functional importance. It employs plastic synaptic connections between Kenyon cells and mushroom body output neurons (MBONs) in separate and mutually inhibiting appetitive and aversive learning pathways. Recurrent modulation of plasticity through projections from MBONs to reinforcement-mediating dopaminergic neurons implements a simple reward prediction mechanism. A distinct set of four MBONs encodes odor valence and predicts behavioral model output. Subjecting this model to learning and extinction protocols reproduced experimental results from recent behavioral and imaging studies. Simulating the experimental blocking of synaptic output of individual neurons or neuron groups in the model circuit confirmed experimental results and allowed formulation of testable predictions. In the temporal domain, this model achieves rapid learning with a step-like increase in the encoded odor value after a single pairing of the conditioned stimulus with a reward or punishment, facilitating single-trial learning.
Mark, B., Lai, S. L., Zarin, A. A., Manning, L., Pollington, H. Q., Litwin-Kumar, A., Cardona, A., Truman, J. W. and Doe, C. Q. (2021). A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS. Elife 10. PubMed ID: 33973523
Summary:
The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, the developmental origin was mapped of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and they were identified in a synapse-scale TEM reconstruction of the Drosophila larval CNS. Lineages were found to concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, this stuy showed that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, this study demonstrated that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.
Nave, C., Roberts, L., Hwu, P., Estrella, J. D., Vo, T. C., Nguyen, T. H., Bui, T. T., Rindner, D. J., Pervolarakis, N., Shaw, P. J., Leise, T. L. and Holmes, T. C. (2021). Weekend light shifts evoke persistent Drosophila circadian neural network desynchrony. J Neurosci. PubMed ID: 33931552
Summary:
This study developed a method for single-cell resolution longitudinal bioluminescence imaging of PERIOD (PER) protein and TIMELESS (TIM) oscillations in cultured male adult Drosophila brains that captures circadian circuit-wide cycling under simulated day/night cycles. Light input analysis confirms that CRYPTOCHROME (CRY) is the primary circadian photoreceptor and mediates clock disruption by constant light, and that eye light input is redundant to CRY. 3hr light phase delays (Friday) followed by 3hr light phase advances (Monday morning) simulate the common practice of staying up later at night on weekends, sleeping in later on weekend days then returning to standard schedule Monday morning (weekend light shift, WLS). WLS significantly dampens PER oscillator synchrony and rhythmicity in most circadian neurons during and after exposure. Lateral ventral neuron (LNv) oscillations are the first to desynchronize in WLS and the last to resynchronize in WLS. Surprisingly, the dorsal neuron group-3 (DN3s), increase their within-group synchrony in response to WLS. In vivo, WLS induces transient defects in sleep stability, learning, and memory that temporally coincide with circuit desynchrony. Our findings suggest that WLS schedules disrupt circuit-wide circadian neuronal oscillator synchrony for much of the week, thus leading to observed behavioral defects in sleep, learning, and memory.
Michki, N. S., Li, Y., Sanjasaz, K., Zhao, Y., Shen, F. Y., Walker, L. A., Cao, W., Lee, C. Y. and Cai, D. (2021). The molecular landscape of neural differentiation in the developing Drosophila brain revealed by targeted scRNA-seq and multi-informatic analysis. Cell Rep 35(4): 109039. PubMed ID: 33909998
Summary:
The Drosophila type II neuroblast lineages present an attractive model to investigate the neurogenesis and differentiation process as they adapt to a process similar to that in the human outer subventricular zone. Targeted single-cell mRNA sequencing in third instar larval brains was performed to study this process of the type II NB lineage. Combining prior knowledge, in silico analyses, and in situ validation, this multi-informatic investigation describes the molecular landscape from a single developmental snapshot. 17 markers are identified to differentiate distinct maturation stages. 30 markers are identified to specify the stem cell origin and/or cell division numbers of INPs, and at least 12 neuronal subtypes are identified. To foster future discoveries, annotated tables are provided of pairwise gene-gene correlation in single cells and MiCV, a web tool for interactively analyzing scRNA-seq datasets. Taken together, these resources advance understanding of the neural differentiation process at the molecular level.
Mitchell, J., Smith, C. S., Titlow, J., Otto, N., van Velde, P., Booth, M., Davis, I. and Waddell, S. (2021). Selective dendritic localization of mRNA in Drosophila mushroom body output neurons. Elife 10. PubMed ID: 33724180
Summary:
Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process requires the visualization of the relevant mRNAs within these neuronal compartments. This study used single-molecule fluorescence in situ hybridization to localize mRNAs at subcellular resolution in the adult Drosophila brain. mRNAs for subunits of nicotinic acetylcholine receptors and kinases could be detected within the dendrites of co-labeled mushroom body output neurons (MBONs) and their relative abundance showed cell specificity. Moreover, aversive olfactory learning produced a transient increase in the level of CaMKII mRNA within the dendritic compartments of the γ5β'2a MBONs. Localization of specific mRNAs in MBONs before and after learning represents a critical step towards deciphering the role of dendritic translation in the neuronal plasticity underlying behavioral change in Drosophila.

Monday, June 21st - RNA

McGurk, M. P., Dion-Cote, A. M. and Barbash, D. A. (2021). Rapid evolution at the Drosophila telomere: transposable element dynamics at an intrinsically unstable locus. Genetics 217(2). PubMed ID: 33724410
Summary:
Drosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE, and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. This study leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85  Drosophila melanogaster genomes and compared their variation to other more typical TE families. Occasional large-scale copy number expansions of both HTTs and other TE families occured, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, it is instead suggested that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. This study has extended previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than one-third do not appear to have been healed with an HTT insertion. It is also reported that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. It is further suggested that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. It is proposed that HTT evolution is driven by multiple processes, with niche specialization and telomere instability being previously underappreciated and likely predominant.
Lee, S., Wei, L., Zhang, B., Goering, R., Majumdar, S., Wen, J., Taliaferro, J. M. and Lai, E. C. (2021). ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing. PLoS Genet 17(4): e1009439. PubMed ID: 33826609
Summary:
ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, this study addressed their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. This study corroborates the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, evidence is provided for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons.
Kingston, E. R. and Bartel, D. P. (2021). Ago2 protects Drosophila siRNAs and microRNAs from target-directed degradation, even in the absence of 2'-O-methylation. RNA. PubMed ID: 33853897
Summary:
Target-directed microRNA (miRNA) degradation (TDMD), which is mediated by the protein ZSWIM8, plays a widespread role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger TDMD, raising the question as to whether they too might undergo such regulation by Dorado, the Drosophila ZSWIM8 homolog. This study finds that some of these siRNAs are indeed sensitive to Dora when loaded into Ago1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into Ago1, these siRNAs are not detectably regulated by target-directed degradation because most molecules are loaded into Ago2, the Argonaute paralog that preferentially associates with siRNAs, and this study found that siRNAs and miRNAs loaded into Ago2 are insensitive to Dora. One explanation for the protection of these small RNAs loaded into Ago2 is that these small RNAs are 2'-O-methylated at their 3' termini. However, 2'-O-methylation does not protect these RNAs from Dora-mediated target-directed degradation, which indicates that their protection is instead conferred by features of the Ago2 protein itself. Together, these observations clarify the requirements for regulation by target-directed degradation and expand understanding of the role of 2'-O-methylation in small-RNA biology.
Maurya, B., Surabhi, S., Das, R., Pandey, P., Mukherjee, A. and Mutsuddi, M. (2021). Maheshvara regulates JAK/STAT signaling by interacting and stabilizing hopscotch transcripts which leads to apoptosis in Drosophila melanogaster. Cell Death Dis 12(4): 363. PubMed ID: 33824299
Summary:
Maheshvara (mahe), an RNA helicase that is widely conserved across taxa, regulates Notch signaling and neuronal development in Drosophila. In order to identify novel components regulated by mahe, transcriptome profiling of ectopic mahe was carried out and this revealed striking upregulation of JAK/STAT pathway components like upd1, upd2, upd3, and socs36E. Further, significant downregulation of the pathway components in mahe loss-of-function mutant as well as upon lowering the level of mahe by RNAi, supported and strengthened the transcriptome data. Parallelly, it was observed that mahe, induced caspase-dependent apoptosis in photoreceptor neurons, and this phenotype was significantly modulated by JAK/STAT pathway components. RNA immunoprecipitation unveiled the presence of JAK/STAT tyrosine kinase hopscotch (hop) transcripts in the complex immunoprecipitated with Mahe, which ultimately resulted in stabilization and elevation of hop transcripts. Additionally, the surge in activity of downstream transcription factor Stat92E, which is indicative of activation of the JAK/STAT signaling, was also observed, and this in turn led to apoptosis via upregulation of hid. Taken together, these data provide a novel regulation of JAK/STAT pathway by RNA helicase Maheshvara, which ultimately promotes apoptosis.
Mohr, S., Kenny, A., Lam, S. T. Y., Morgan, M. B., Smibert, C. A., Lipshitz, H. D. and Macdonald, P. M. (2021). Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of oskar mRNA in the Drosophila oocyte. PLoS Genet 17(4): e1009500. PubMed ID: 33798193
Summary:
Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3' UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. This study shows that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. This study also showed that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3' UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.
Montigny, A., Tavormina, P., Duboe, C., San Clemente, H., Aguilar, M., Valenti, P., Lauressergues, D., Combier, J. P. and Plaza, S. (2021). Drosophila primary microRNA-8 encodes a microRNA-encoded peptide acting in parallel of miR-8. Genome Biol 22(1): 118. PubMed ID: 33892772
Summary:
Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, it was recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. This study investigated whether this regulatory feedback loop is present in Drosophila melanogaster. A survey of ribosome profiling data was performed and it was revealed that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, this study has shown that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, this study shows that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.

Friday, June 18th - Disease models

Ingles-Prieto, A., Furthmann, N., Crossman, S. H., Tichy, A. M., Hoyer, N., Petersen, M., Zheden, V., Biebl, J., Reichhart, E., Gyoergy, A., Siekhaus, D. E., Soba, P., Winklhofer, K. F. and Janovjak, H. (2021). Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease. PLoS Genet 17(4): e1009479. PubMed ID: 33857132
Summary:
Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. This study reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-kappaB pathway. These results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.
Li, W. H., Gan, L. H., Ma, F. F., Feng, R. L., Wang, J., Li, Y. H., Sun, Y. Y., Wang, Y. J., Diao, X., Qian, F. Y. and Wen, T. Q. (2021). Deletion of Dcf1 Reduces Amyloid-beta Aggregation and Mitigates Memory Deficits. J Alzheimers Dis. PubMed ID: 33896839
Summary:
Alzheimer's disease (AD). is a progressive neurodegenerative disease. One of the pathologies of AD is the accumulation of amyloid-β (Aβ) to form senile plaques, leading to a decline in cognitive ability and a lack of learning and memory. However, the cause leading to Aβ aggregation is not well understood. Dendritic cell factor 1 (Dcf1) shows a high expression in the entorhinal cortex neurons and neurofibrillary tangles in AD patients. This study investigated the effect of Dcf1 on Aβ aggregation and memory deficits in AD development. The mouse and Drosophila AD model were used to test the expression and aggregation of Aβ, senile plaque formation, and pathological changes in cognitive behavior during dcf1 knockout and expression. Possible drug target effects were explored through intracerebroventricular delivery of Dcf1 antibodies. Deletion of Dcf1 resulted in decreased Aβ42 level and deposition, and rescued AMPA Receptor (GluA2) levels in the hippocampus of APP-PS1-AD mice. In Aβ42 AD Drosophila, the expression of Dcf1 in Aβ42 AD flies aggravated the formation and accumulation of senile plaques, significantly reduced its climbing ability and learning-memory. Data analysis from all 20 donors with and without AD patients aged between 80 and 90 indicated a high-level expression of Dcf1 in the temporal neocortex. Dcf1 contributed to Aβ aggregation by UV spectroscopy assay. Intracerebroventricular delivery of Dcf1 antibodies in the hippocampus reduced the area of senile plaques and reversed learning and memory deficits in APP-PS1-AD mice. Dcf1 causes Aβ-plaque accumulation, inhibiting dcf1 expression could potentially offer therapeutic avenues.
Lehmkuhl, E. M., Loganathan, S., Alsop, E., Blythe, A. D., Kovalik, T., Mortimore, N. P., Barrameda, D., Kueth, C., Eck, R. J., Siddegowda, B. B., Joardar, A., Ball, H., Macias, M. E., Bowser, R., Van Keuren-Jensen, K. and Zarnescu, D. C. (2021). TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6. Acta Neuropathol Commun 9(1): 52. PubMed ID: 33762006
Summary:
Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, TDP-43 dependent translational alterations in motor neurons were identified impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.
Kim, E. S., Chung, C. G., Park, J. H., Ko, B. S., Park, S. S., Kim, Y. H., Cha, I. J., Kim, J., Ha, C. M., Kim, H. J. and Lee, S. B. (2021). C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons. Hum Mol Genet 30(12): 1084-1100. PubMed ID: 33783499
Summary:
RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). This study report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. In Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS-but not with mutated endogenous NLS-potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/-), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/- is protective. Stau+/- also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, these data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.
Jiao, J., Kavdia, K., Pagala, V., Palmer, L., Finkelstein, D., Fan, Y., Peng, J. and Demontis, F. (2021). An age-downregulated ribosomal RpS28 protein variant regulates the muscle proteome. G3 (Bethesda). PubMed ID: 33974070
Summary:
Recent evidence indicates that the composition of the ribosome is heterogeneous and that multiple types of specialized ribosomes regulate the synthesis of specific protein subsets. In Drosophila, expression of the ribosomal RpS28 protein variants RpS28a and RpS28-like were found to preferentially occurs in the germline, a tissue resistant to aging; it significantly declines in skeletal muscle during aging. Muscle-specific overexpression of RpS28a at levels similar to those seen in the germline decreases early mortality and promotes the synthesis of a subset of proteins with known anti-aging roles, some of which have preferential expression in the germline. These findings indicate a contribution of specialized ribosomal proteins to the regulation of the muscle proteome during aging.
Han, Y., Zhuang, N. and Wang, T. (2021). Roles of PINK1 in regulation of systemic growth inhibition induced by mutations of PTEN in Drosophila. Cell Rep 34(12): 108875. PubMed ID: 33761355
Summary:
The maintenance of mitochondrial homeostasis requires PTEN-induced kinase 1 (PINK1)-dependent mitophagy, and mutations in PINK1 are associated with Parkinson's disease (PD). PINK1 is also downregulated in tumor cells with PTEN mutations. However, there is limited information concerning the role of PINK1 in tissue growth and tumorigenesis. This study shows that the loss of pink1 caused multiple growth defects independent of its pathological target, Parkin. Moreover, knocking down pink1 in muscle cells induced hyperglycemia and limited systemic organismal growth by the induction of Imaginal morphogenesis protein-Late 2 (ImpL2). Similarly, disrupting PTEN activity in multiple tissues impaired systemic growth by reducing pink1 expression, resembling wasting-like syndrome in cancer patients. Furthermore, the re-expression of PINK1 fully rescued defects in carbohydrate metabolism and systemic growth induced by the tissue-specific pten mutations. These data suggest a function for PINK1 in regulating systemic growth in Drosophila and shed light on its role in wasting in the context of PTEN mutations.

Thursday, June 16th - Adult neural development and function

Mann, K., Deny, S., Ganguli, S. and Clandinin, T. R. (2021). Coupling of activity, metabolism and behaviour across the Drosophila brain. Nature 593(7858): 244-248. PubMed ID: 33911283
Summary:
Coordinated activity across networks of neurons is a hallmark of both resting and active behavioural states in many species. These global patterns alter energy metabolism over seconds to hours, which underpins the widespread use of oxygen consumption and glucose uptake as proxies of neural activity. However, whether changes in neural activity are causally related to metabolic flux in intact circuits on the timescales associated with behaviour is unclear. This study combine two-photon microscopy of the fly brain with sensors that enable the simultaneous measurement of neural activity and metabolic flux, across both resting and active behavioural states. Neural activity was demonstrated to drive changes in metabolic flux, creating a tight coupling between these signals that can be measured across brain networks. Using local optogenetic perturbation, it was demonstrated that even transient increases in neural activity result in rapid and persistent increases in cytosolic ATP, which suggests that neuronal metabolism predictively allocates resources to anticipate the energy demands of future activity. Finally, these studies reveal that the initiation of even minimal behavioural movements causes large-scale changes in the pattern of neural activity and energy metabolism, which reveals a widespread engagement of the brain. As the relationship between neural activity and energy metabolism is probably evolutionarily ancient and highly conserved, these studies provide a critical foundation for using metabolic proxies to capture changes in neural activity.
Main, P., Tan, W. J., Wheeler, D. and Fitzsimons, H. L. (2021). Increased Abundance of Nuclear HDAC4 Impairs Neuronal Development and Long-Term Memory. Front Mol Neurosci 14: 616642. PubMed ID: 33859551
Summary:
Dysregulation of mammalian histone deacetylase HDAC4 is associated with both neurodevelopmental and neurodegenerative disorders. HDAC4 shuttles between the nucleus and cytoplasm in both vertebrates and invertebrates and alterations in the amounts of nuclear and/or cytoplasmic HDAC4 have been implicated in these diseases. In Drosophila, HDAC4 also plays a critical role in the regulation of memory. Nuclear and cytoplasmically-restricted HDAC4 mutants were expressed in the Drosophila brain to investigate a mechanistic link between HDAC4 subcellular distribution, transcriptional changes and neuronal dysfunction. Deficits in mushroom body morphogenesis, eye development and long-term memory correlated with increased abundance of nuclear HDAC4 but were associated with minimal transcriptional changes. Although HDAC4 sequesters MEF2 into punctate foci within neuronal nuclei, no alteration in MEF2 activity was observed on overexpression of HDAC4, and knockdown of MEF2 had no impact on long-term memory, indicating that HDAC4 is likely not acting through MEF2. In support of this, mutation of the MEF2 binding site within HDAC4 also had no impact on nuclear HDAC4-induced impairments in long-term memory or eye development. In contrast, the defects in mushroom body morphogenesis were ameliorated by mutation of the MEF2 binding site, as well as by co-expression of MEF2 RNAi, thus nuclear HDAC4 acts through MEF2 to disrupt mushroom body development. These data provide insight into the mechanisms through which dysregulation of HDAC4 subcellular distribution impairs neurological function and provides new avenues for further investigation.
Li, D., et al. (2021). Pathogenic variants in SMARCA5, a chromatin remodeler, cause a range of syndromic neurodevelopmental features. Sci Adv 7(20). PubMed ID: 33980485
Summary:
Intellectual disability encompasses a wide spectrum of neurodevelopmental disorders, with many linked genetic loci. However, the underlying molecular mechanism for more than 50% of the patients remains elusive. This study describes pathogenic variants in SMARCA5, encoding the ATPase motor of the ISWI chromatin remodeler, as a cause of a previously unidentified neurodevelopmental disorder, identifying 12 individuals with de novo or dominantly segregating rare heterozygous variants. Accompanying phenotypes include mild developmental delay, frequent postnatal short stature and microcephaly, and recurrent dysmorphic features. Loss of function of the SMARCA5 Drosophila ortholog Iswi led to smaller body size, reduced sensory dendrite complexity, and tiling defects in larvae. In adult flies, Iswi neural knockdown caused decreased brain size, aberrant mushroom body morphology, and abnormal locomotor function. Iswi loss of function was rescued by wild-type but not mutant SMARCA5. These results demonstrate that SMARCA5 pathogenic variants cause a neurodevelopmental syndrome with mild facial dysmorphia.
Lee, S. S. and Adams, M. E. (2021). Regulation of Drosophila Long-Term Courtship Memory by Ecdysis Triggering Hormone. Front Neurosci 15: 670322. PubMed ID: 33967686
Summary:
Endocrine state is an important determinant of learning and memory in animals. In Drosophila, rejection of male courtship overtures by mated females leads to an aversive response manifested as courtship memory. This study reports that ecdysis triggering hormone (ETH) is an obligatory enabler of long-term courtship memory (LTM). ETH deficiency suppresses LTM, whereas augmented ETH release reduces the minimum training period required for LTM induction. ETH receptor knockdown either in the mushroom body (MB) γ lobe or in octopaminergic dorsal-anterior-lateral (DAL) neurons impairs memory performance, indicating its direct action in these brain areas. Consistent with these findings, brain exposure to ETH mobilizes calcium in MB γ lobe neuropils and DAL neurons. ETH receptor (ETHR) knockdown in the corpus allatum (CA) to create juvenile hormone (JH) deficiency also suppresses LTM, as does knockdown of the JH receptor Met in the MB γ lobe, indicating a convergence of ETH and JH signaling in this region of the brain. These findings identify endocrine-enabled neural circuit components in the brain that are critical for persistent behavioral changes resulting from aversive social experience.
Leinwand, S. G. and Scott, K. (2021). Juvenile hormone drives the maturation of spontaneous mushroom body neural activity and learned behavior. Neuron. PubMed ID: 33915110
Summary:
Mature behaviors emerge from neural circuits sculpted by genetic programs and spontaneous and evoked neural activity. However, how neural activity is refined to drive maturation of learned behavior remains poorly understood. This study explored how transient hormonal signaling coordinates a neural activity state transition and maturation of associative learning. Spontaneous, asynchronous activity was identified in a Drosophila learning and memory brain region, the mushroom body. This activity declines significantly over the first week of adulthood. Moreover, this activity is generated cell-autonomously via Cacophony voltage-gated calcium channels in a single cell type, α'/β' Kenyon cells. Juvenile hormone, a crucial developmental regulator, acts transiently in α'/β' Kenyon cells during a young adult sensitive period to downregulate spontaneous activity and enable subsequent enhanced learning. Hormone signaling in young animals therefore controls a neural activity state transition and is required for improved associative learning, providing insight into the maturation of circuits and behavior.
Lau, C. K. S., Jelen, M. and Gordon, M. D. (2021). A closed-loop optogenetic screen for neurons controlling feeding in Drosophila. G3 (Bethesda) 11(5). PubMed ID: 33714999
Summary:
Feeding is an essential part of animal life that is greatly impacted by the sense of taste. Although the characterization of taste-detection at the periphery has been extensive, higher order taste and feeding circuits are still being elucidated. This study used an automated closed-loop optogenetic activation screen to detect novel taste and feeding neurons in Drosophila melanogaster. Out of 122 Janelia FlyLight Project GAL4 lines preselected based on expression pattern, this study identified six lines that acutely promote feeding and 35 lines that inhibit it. As proof of principle, R70C07-GAL4, which labels neurons that strongly inhibit feeding, was analyzed. Using split-GAL4 lines to isolate subsets of the R70C07-GAL4 population, both appetitive and aversive neurons were found. Furthermore, this study shows that R70C07-GAL4 labels putative second-order taste interneurons in the subesophageal zone that contact both sweet and bitter sensory neurons. These results serve as a resource for further functional dissection of fly feeding circuits (Lau, 2021).

Wednesday, June 16th - Signaling

Kon, N., Wang, H. T., Kato, Y. S., Uemoto, K., Kawamoto, N., Kawasaki, K., Enoki, R., Kurosawa, G., Nakane, T., Sugiyama, Y., Tagashira, H., Endo, M., Iwasaki, H., Iwamoto, T., Kume, K. and Fukada, Y. (2021). Na(+)/Ca(2+) exchanger mediates cold Ca(2+) signaling conserved for temperature-compensated circadian rhythms. Sci Adv 7(18). PubMed ID: 33931447
Summary:
Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. This study shows that Na(+)/Ca(2+) exchanger (NCX) mediates cold-responsive Ca(2+) signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca(2+), which activates Ca(2+)/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca(2+) signaling is conserved among mice, Drosophila, and Arabidopsis. The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. These results suggest that NCX-mediated Ca(2+) signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.
Frankenreiter, L., Gahr, B. M., Schmid, H., Zimmermann, M., Deichsel, S., Hoffmeister, P., Turkiewicz, A., Borggrefe, T., Oswald, F. and Nagel, A. C. (2021). Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila. Front Cell Dev Biol 9: 658820. PubMed ID: 33937259
Summary:
Notch signaling controls developmental processes including hematopoiesis. A phospho-mimetic mutation of the Drosophila CSL ortholog Suppressor of Hairless [Su(H)] at Ser(269) has been shown to impede DNA-binding. By genome-engineering, this study introduced phospho-specific Su(H) mutants at the endogenous Su(H) locus, encoding either a phospho-deficient [Su(H) (S269A) ] or a phospho-mimetic [Su(H) (S269D) ] isoform. Su(H) (S269D) mutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficient Su(H) (S269A) mutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressor Hairless acting otherwise as a general Notch antagonist in Drosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, these studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJ (S221D) was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJ (S221A) rather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.
Iyer, K. V., Taubenberger, A., Zeidan, S. A., Dye, N. A., Eaton, S. and Julicher, F. (2021). Apico-basal cell compression regulates Lamin A/C levels in epithelial tissues. Nat Commun 12(1): 1756. PubMed ID: 33767161
Summary:
The levels of nuclear protein Lamin A/C (see Drosophila LamC) are crucial for nuclear mechanotransduction. Lamin A/C levels are known to scale with tissue stiffness and extracellular matrix levels in mesenchymal tissues. But in epithelial tissues, where cells lack a strong interaction with the extracellular matrix, it is unclear how Lamin A/C is regulated. This study shows in epithelial tissues that Lamin A/C levels scale with apico-basal cell compression, independent of tissue stiffness. Using genetic perturbations in Drosophila epithelial tissues, it was shown that apico-basal cell compression regulates the levels of Lamin A/C by deforming the nucleus. Further, in mammalian epithelial cells, this study show that nuclear deformation regulates Lamin A/C levels by modulating the levels of phosphorylation of Lamin A/C at Serine 22, a target for Lamin A/C degradation. Taken together, these results reveal a mechanism of Lamin A/C regulation which could provide key insights for understanding nuclear mechanotransduction in epithelial tissues.
Kong, D., Lu, J. Y., Li, X., Zhao, S., Xu, W., Fang, J., Wang, X. and Ma, X. (2021). Misshapen Disruption Cooperates with Ras(V12) to Drive Tumorigenesis. Cells 10(4). PubMed ID: 33919765
Summary:
Although RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. A large-scale genetic screen in Drosophila eye imaginal discs identified Misshapen (Msn) as a tumor suppressor that synergizes with oncogenic Ras (Ras(V12)) to induce c-Jun N-terminal kinase (JNK) activation and Hippo inactivation, then subsequently leads to tumor overgrowth and invasion. Moreover, ectopic Msn expression activates Hippo signaling pathway and suppresses Hippo signaling disruption-induced overgrowth. Importantly, it was further found that Msn acts downstream of protocadherin Fat (Ft) to regulate Hippo signaling. Finally, msn as a Yki/Sd target gene that regulates Hippo pathway in a negative feedback manner. Together, these findings identified Msn as a tumor suppressor and provide a novel insight into RAS-related tumorigenesis that may be relevant to human cancer biology.
Lin, Y. H., Maaroufi, H. O., Kucerova, L., Rouhova, L., Filip, T. and Zurovec, M. (2021). Adenosine Receptor and Its Downstream Targets, Mod(mdg4) and Hsp70, Work as a Signaling Pathway Modulating Cytotoxic Damage in Drosophila. Front Cell Dev Biol 9: 651367. PubMed ID: 33777958
Summary:
Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mammalian models have shown that Ado regulates signaling mechanisms involved in "danger-sensing" and tissue-protection. Yet, little is known about the role of Ado signaling in Drosophila. This study observed lower extracellular Ado concentration and suppressed expression of Ado transporters in flies expressing mutant huntingtin protein (mHTT). Ado signaling was altered using genetic tools; the overexpression of Ado metabolic enzymes, as well as the suppression of Ado receptor (AdoR) and transporters (ENTs) were found to minimize mHTT-induced mortality. The downstream targets of the AdoR pathway were identified, the modifier of mdg4 (Mod(mdg4)) and heat-shock protein 70 (Hsp70), which modulated the formation of mHTT aggregates. Finally, a decrease in Ado signaling affects other Drosophila stress reactions, including paraquat and heat-shock treatments. This study provides important insights into how Ado regulates stress responses in Drosophila.
Lavalou, J., Mao, Q., Harmansa, S., Kerridge, S., Lellouch, A. C., Philippe, J. M., Audebert, S., Camoin, L. and Lecuit, T. (2021). Formation of polarized contractile interfaces by self-organized Toll-8/Cirl GPCR asymmetry. Dev Cell. PubMed ID: 33932333
Summary:
Interfaces between cells with distinct genetic identities elicit signals to organize local cell behaviors driving tissue morphogenesis. The Drosophila embryonic axis extension requires planar polarized enrichment of myosin-II powering oriented cell intercalations. Myosin-II levels are quantitatively controlled by GPCR signaling, whereas myosin-II polarity requires patterned expression of several Toll receptors. How Toll receptors polarize myosin-II and how this involves GPCRs remain unknown. This study reports that differential expression of a single Toll receptor, Toll-8, polarizes myosin-II through binding to the adhesion GPCR Cirl/latrophilin. Asymmetric expression of Cirl is sufficient to enrich myosin-II, and Cirl localization is asymmetric at Toll-8 expression boundaries. Exploring the process dynamically, this study revealed that Toll-8 and Cirl exhibit mutually dependent planar polarity in response to quantitative differences in Toll-8 expression between neighboring cells. Collectively, it is proposed that the cell surface protein complex Toll-8/Cirl self-organizes to generate local asymmetric interfaces essential for planar polarization of contractility.

Tuesday, June 15th - Signaling

Kapoor, T., Dubey, P., Shirolikar, S. and Ray, K. (2021). An actomyosin clamp assembled by the Amphiphysin-Rho1-Dia/DAAM-Rok pathway reinforces somatic cell membrane folded around spermatid heads. Cell Rep 34(13): 108918. PubMed ID: 33789114
Summary:
Membrane curvature recruits Bin-Amphiphysin-Rvs (BAR)-domain proteins and induces local F-actin assembly, which further modifies the membrane curvature and dynamics. The downstream molecular pathway in vivo is still unclear. This study shows that a tubular endomembrane scaffold supported by contractile actomyosin stabilizes the somatic cyst cell membrane folded around rigid spermatid heads during the final stages of sperm maturation in Drosophila testis. The structure resembles an actin "basket" covering the bundle of spermatid heads. Genetic analyses suggest that the actomyosin organization is nucleated exclusively by the formins - Diaphanous and Dishevelled Associated Activator of Morphogenesis (DAAM) - downstream of Rho1, which is recruited by the BAR-domain protein Amphiphysin. Actomyosin activity at the actin basket gathers the spermatid heads into a compact bundle and resists the somatic cell invasion by intruding spermatids. These observations reveal a distinct response mechanism of actin-membrane interactions, which generates a cell-adhesion-like strategy through active clamping.
Krishnan, R. K., Baskar, R., Anna, B., Elia, N., Boermel, M., Bausch, A. R. and Abdu, U. (2021). Recapitulating Actin Module Organization in the Drosophila Oocyte Reveals New Roles for Bristle-Actin-Modulating Proteins. Int J Mol Sci 22(8). PubMed ID: 33924532
Summary:
The generation of F-actin bundles is controlled by the action of actin-binding proteins. In Drosophila bristle development, two major actin-bundling proteins-Forked and Fascin-were identified, but still the molecular mechanism by which these actin-bundling proteins and other proteins generate bristle actin bundles is unknown. This study developed a technique that allows recapitulation of bristle actin module organization using the Drosophila ovary by a combination of confocal microscopy, super-resolution structured illumination microscopy, and correlative light and electron microscope analysis. Since Forked generated a distinct ectopic network of actin bundles in the oocyte, the additive effect of two other actin-associated proteins, namely, Fascin and Javelin (Jv), was studied. Co-expression of Fascin and Forked demonstrated that the number of actin filaments within the actin bundles dramatically increased, and in their geometric organization, they resembled bristle-like actin bundles. On the other hand, co-expression of Jv with Forked increased the length and density of the actin bundles. When all three proteins co-expressed, the actin bundles were longer and denser, and contained a high number of actin filaments in the bundle. Thus, these results demonstrate that the Drosophila oocyte could serve as a test tube for actin bundle analysis.
John, A. and Rauzi, M. (2021). A two-tier junctional mechanism drives simultaneous tissue folding and extension. Dev Cell. PubMed ID: 33891900
Summary:
During embryo development, tissues often undergo multiple concomitant changes in shape. It is unclear which signaling pathways and cellular mechanisms are responsible for multiple simultaneous tissue shape transformations. This study focussed on the process of concomitant tissue folding and extension that is key during gastrulation and neurulation. The Drosophila embryo was used as model system and focus was placed on the process of mesoderm invagination. This study shows that the prospective mesoderm simultaneously folds and extends. Mesoderm cells, under the control of anterior-posterior and dorsal-ventral gene patterning synergy, establish two sets of adherens junctions at different apical-basal positions with specialized functions: while apical junctions drive apical constriction initiating tissue bending, lateral junctions concomitantly drive polarized cell intercalation, resulting in tissue convergence-extension. Thus, epithelial cells devise multiple specialized junctional sets that drive composite morphogenetic processes under the synergistic control of apparently orthogonal signaling sources.
Lattao, R., Rangone, H., Llamazares, S. and Glover, D. M. (2021). Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins. Dev Cell 56(7): 1000-1013.e1006. PubMed ID: 33725482
Summary:
Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. This study showed that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients.
Dobramysl, U., Jarsch, I. K., Inoue, Y., Shimo, H., Richier, B., Gadsby, J. R., Mason, J., Szalapak, A., Ioannou, P. S., Correia, G. P., Walrant, A., Butler, R., Hannezo, E., Simons, B. D. and Gallop, J. L. (2021). Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation. J Cell Biol 220(4). PubMed ID: 33740033
Summary:
Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, the localization was analyzed of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). The composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. The data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. An explanation is provided for the observed heterogeneity and it is suggested that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.
Feng, C., Cleary, J. M., Kothe, G. O., Stone, M. C., Weiner, A. T., Hertzler, J. I., Hancock, W. O. and Rolls, M. M. (2021). Trim9 and Klp61F promote polymerization of new dendritic microtubules along parallel microtubules. J Cell Sci. PubMed ID: 33988240
Summary:
Axons and dendrites are distinguished by microtubule polarity. In Drosophila, dendrites are dominated by minus-end-out microtubules while axons contain plus-end-out microtubules. Local nucleation in dendrites generates microtubules in both orientations. To understand why dendritic nucleation does not disrupt polarity, this study used live imaging to analyze the fate of microtubules generated at branch points. It was found that they had different rates of success exiting the branch based on orientation: correctly oriented minus-end-out microtubules succeeded in leaving about twice as often as incorrectly oriented microtubules. Increased success relied on other microtubules in a parallel orientation. From a candidate screen, Trim9 and kinesin-5 (Klp61F) were identified as machinery that promoted growth of new microtubules. In S2 cells, EB1 recruited Trim9 to microtubules. Klp61F promoted microtubule growth in vitro and in vivo, and could recruit Trim9 in S2 cells. In summary, the data argue that Trim9 and kinesin-5 act together at microtubule plus ends to help polymerizing microtubules parallel to pre-existing ones resist catastrophe.

Monday June 14th - Chromatin

Fursova, N. A., Turberfield, A. H., Blackledge, N. P., Findlater, E. L., Lastuvkova, A., Huseyin, M. K., Dobrinic, P. and Klose, R. J. (2021). BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome. Genes Dev 35(9-10): 749-770. PubMed ID: 33888563
Summary:
Histone-modifying systems play fundamental roles in gene regulation and the development of multicellular organisms. Histone modifications that are enriched at gene regulatory elements have been heavily studied, but the function of modifications found more broadly throughout the genome remains poorly understood. This is exemplified by histone H2A monoubiquitylation (H2AK119ub1), which is enriched at Polycomb-repressed gene promoters but also covers the genome at lower levels. Using inducible genetic perturbations and quantitative genomics, this study found that the BAP1 deubiquitylase plays an essential role in constraining H2AK119ub1 throughout the genome. Removal of BAP1 leads to pervasive genome-wide accumulation of H2AK119ub1, which causes widespread reductions in gene expression. Elevated H2AK119ub1 preferentially counteracts Ser5 phosphorylation on the C-terminal domain of RNA polymerase II at gene regulatory elements and causes reductions in transcription and transcription-associated histone modifications. Furthermore, failure to constrain pervasive H2AK119ub1 compromises Polycomb complex occupancy at a subset of Polycomb target genes, which leads to their derepression, providing a potential molecular rationale for why the BAP1 ortholog in Drosophila has been characterized as a Polycomb group gene. Together, these observations reveal that the transcriptional potential of the genome can be modulated by regulating the levels of a pervasive histone modification.
Khan, M. H. F., Akhtar, J., Umer, Z., Shaheen, N., Shaukat, A., Munir, M. S., Mithani, A., Anwar, S. and Tariq, M. (2021). Kinome-Wide RNAi Screen Uncovers Role of Ballchen in Maintenance of Gene Activation by Trithorax Group in Drosophila. Front Cell Dev Biol 9: 637873. PubMed ID: 33748127
Summary:
Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionary conserved factors that contribute to cell fate determination and maintenance of cellular identities during development of multicellular organisms. The PcG maintains heritable patterns of gene silencing while trxG acts as anti-silencing factors by conserving activation of cell type specific genes. Genetic and molecular analysis has revealed extensive details about how different PcG and trxG complexes antagonize each other to maintain cell fates, however, the cellular signaling components that contribute to the preservation of gene expression by PcG/trxG remain elusive. This study reports an ex vivo kinome-wide RNAi screen in Drosophila aimed at identifying cell signaling genes that facilitate trxG in counteracting PcG mediated repression. From the list of trxG candidates, Ballchen (BALL), a histone kinase known to phosphorylate histone H2A at threonine 119 (H2AT119p), was characterized as a trxG regulator. The ball mutant exhibits strong genetic interactions with Polycomb (Pc) and trithorax (trx) mutants and loss of BALL affects expression of trxG target genes. BALL co-localizes with Trithorax on chromatin and depletion of BALL results in increased H2AK118 ubiquitination, a histone mark central to PcG mediated gene silencing. Moreover, BALL was found to substantially associate with known TRX binding sites across the genome. Genome wide distribution of BALL also overlaps with H3K4me3 and H3K27ac at actively transcribed genes. It is proposed that BALL mediated signaling positively contributes to the maintenance of gene activation by trxG in counteracting the repressive effect of PcG.
Hu, X., Li, M., Hao, X., Lu, Y., Zhang, L. and Wu, G. (2021). The Osa-Containing SWI/SNF Chromatin-Remodeling Complex Is Required in the Germline Differentiation Niche for Germline Stem Cell Progeny Differentiation. Genes (Basel) 12(3). PubMed ID: 33806269
Summary:
The Drosophila ovary is recognized as a powerful model to study stem cell self-renewal and differentiation. Decapentaplegic (Dpp) is secreted from the germline stem cell (GSC) niche to activate Bone Morphogenic Protein (BMP) signaling in GSCs for their self-renewal and is restricted in the differentiation niche for daughter cell differentiation. This study reports that Switch/sucrose non-fermentable (SWI/SNF) component Osa depletion in escort cells (ECs) results in a blockage of GSC progeny differentiation. Further molecular and genetic analyses suggest that the defective germline differentiation is partially attributed to the elevated dpp transcription in ECs. Moreover, ectopic Engrailed (En) expression in osa-depleted ECs partially contributes to upregulated dpp transcription. Furthermore, it was shown that Osa regulates germline differentiation in a Brahma (Brm)-associated protein (BAP)-complex-dependent manner. Additionally, the loss of EC long cellular processes upon osa depletion may also partly contribute to the germline differentiation defect. Taken together, these data suggest that the epigenetic factor Osa plays an important role in controlling EC characteristics and germline lineage differentiation.
Kemp, J. P., Jr., Yang, X. C., Dominski, Z., Marzluff, W. F. and Duronio, R. J. (2021). Superresolution light microscopy of the Drosophila histone locus body reveals a core-shell organization associated with expression of replication-dependent histone genes. Mol Biol Cell 32(9): 942-955. PubMed ID: 33788585
Summary:
The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. This study demonstrates, using structured illumination microscopy, that Drosophila HLBs have a "core-shell" organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that cotranscriptionally cleaves RD histone pre-mRNA. Consistent with these results, this study shows biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.
Gaskill, M. M., Gibson, T. J., Larson, E. D. and Harrison, M. M. (2021). GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo. Elife 10. PubMed ID: 33720012
Summary:
Following fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are essential for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal-to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were required for this transition. This study identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is necessary to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. This study has demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer-like factors, and it is proposed that as development proceeds control is gradually transferred from Zelda to GAF.
Kursel, L. E., McConnell, H., de la Cruz, A. F. A. and Malik, H. S. (2021). Gametic specialization of centromeric histone paralogs in Drosophila virilis. Life Sci Alliance 4(7). PubMed ID: 33986021
Summary:
In most eukaryotes, centromeric histone (CenH3) proteins mediate mitosis and meiosis and ensure epigenetic inheritance of centromere identity. It was hypothesized that disparate chromatin environments in soma versus germline might impose divergent functional requirements on single CenH3 genes, which could be ameliorated by gene duplications and subsequent specialization. This study analyzed the cytological localization of two recently identified CenH3 paralogs, Cid1 and Cid5, in Drosophila virilis using specific antibodies and epitope-tagged transgenic strains. Only ancestral Cid1 (Cid in Drosophila melanogaster) was found to be present in somatic cells, whereas both Cid1 and Cid5 are expressed in testes and ovaries. However, Cid1 is lost in male meiosis but retained throughout oogenesis, whereas Cid5 is lost during female meiosis but retained in mature sperm. Following fertilization, only Cid1 is detectable in the early embryo, suggesting that maternally deposited Cid1 is rapidly loaded onto paternal centromeres during the protamine-to-histone transition. These studies reveal mutually exclusive gametic specialization of divergent CenH3 paralogs. Duplication and divergence might allow essential centromeric genes to resolve an intralocus conflict between maternal and paternal centromeric requirements in many animal species.

Friday, June 11th - Methods

Cicconi, A., Micheli, E., Raffa, G. D. and Cacchione, S. (2021). Atomic Force Microscopy Reveals that the Drosophila Telomere-Capping Protein Verrocchio Is a Single-Stranded DNA-Binding Protein. Methods Mol Biol 2281: 241-263. PubMed ID: 33847963
Summary:
Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of biological samples with a nanometric resolution. Determination of the physical properties of biological molecules at a single-molecule level is achieved through topographic analysis of the sample adsorbed on a flat and smooth surface. AFM has been widely used for the structural analysis of nucleic acid-protein interactions, providing insights on binding specificity and stoichiometry of proteins forming complexes with DNA substrates. Analysis of single-stranded DNA-binding proteins by AFM requires specific single-stranded/double-stranded hybrid DNA molecules as substrates for protein binding. This paper describes the protocol for AFM characterization of binding properties of Drosophila telomeric protein Ver using DNA constructs that mimic the structure of chromosome ends. Details are provided on the methodology used, including the procedures for the generation of DNA substrates, the preparation of samples for AFM visualization, and the data analysis of AFM images. The presented procedure can be adapted for the structural studies of any single-stranded DNA-binding protein.
Droujinine, I. A., Meyer, A. S., Wang, D., Udeshi, N. D., Hu, Y., Rocco, D., McMahon, J. A., Yang, R., Guo, J., Mu, L., Carey, D. K., Svinkina, T., Zeng, R., Branon, T., Tabatabai, A., Bosch, J. A., Asara, J. M., Ting, A. Y., Carr, S. A., McMahon, A. P. and Perrimon, N. (2021). Proteomics of protein trafficking by in vivo tissue-specific labeling. Nat Commun 12(1): 2382. PubMed ID: 33888706
Summary:
Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. A platform was established to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, 51 muscle-secreted proteins were identifed from heads and 269 fat body-secreted proteins were identified from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas were identified, including low-abundance proteins with hormonal properties. These findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.
Hines, A. D. and van Swinderen, B. (2021). Tracking single molecule dynamics in the adult Drosophila brain. eNeuro. PubMed ID: 33875453
Summary:
Super-resolution microscopy provides valuable insight for understanding the nanoscale organization within living tissue, although this method is typically restricted to cultured or dissociated cells. A method was developed to track the mobility of individual proteins in ex vivo adult Drosophila melanogaster brains, focusing on a key component of the presynaptic release machinery, syntaxin1A. Individual Syntaxin1A dynamics can be reliably tracked within neurons in the whole fly brain; the mobility of Syntaxin1A molecules increases following conditional neural stimulation. This preparation was applied to the problem of general anesthesia, to address how different anesthetics might affect single molecule dynamics in intact brain synapses. Propofol, etomidate, and isoflurane significantly impair Syntaxin1A mobility, while ketamine and sevoflurane have little effect. Resolving single molecule dynamics in intact fly brains provides a novel approach to link localized molecular effects with systems-level phenomena such as general anesthesia.
Gamez, S., Vesga, L. C., Mendez-Sanchez, S. C. and Akbari, O. S. (2021). Spatial control of gene expression in flies using bacterially derived binary transactivation systems. Insect Mol Biol. PubMed ID: 33963794
Summary:
Controlling gene expression is an instrumental tool for biotechnology, as it enables the dissection of gene function, affording precise spatial-temporal resolution. To generate this control, binary transactivational systems have been used employing a modular activator consisting of a DNA binding domain(s) fused to activation domain(s). For fly genetics, many binary transactivational systems have been exploited in vivo; however as the study of complex problems often requires multiple systems that can be used in parallel, there is a need to identify additional bipartite genetic systems. To expand this molecular genetic toolbox, multiple bacterially-derived binary transactivational systems were tested in Drosophila melanogaster including the p-CymR operon from Pseudomonas putida, PipR operon from Streptomyces coelicolor, TtgR operon from Pseudomonas putida, and the VanR operon from Caulobacter crescentus. This work provides the first characterization of these systems in an animal model in vivo. For each system robust tissue-specific spatial transactivation of reporter gene expression was demonstrated, enabling future studies to exploit these transactivational systems for molecular genetic studies.
Klima, R., Romano, G., Gbadamosi, M., Megighian, A. and Feiguin, F. (2021). Immuno-electrophysiology on Neuromuscular Junctions of Drosophila Third Instar Larva. Bio Protoc 11(3): e3913. PubMed ID: 33732800
Summary:
Alterations in synaptic transmission are critical early events in neuromuscular disorders. However, reliable methodologies to analyze the functional organization of the neuromuscular synapses are still needed. This manuscript provides a detailed protocol to analyze the molecular assembly of the neuromuscular synapses through immune-electrophysiology in Drosophila melanogaster. This technique allows the quantification of the molecular behavior of the neuromuscular synapses by correlating the structural configuration of the synaptic boutons with their electrical activity.
Feng, S., Lu, S., Grueber, W. B. and Mann, R. S. (2021). Scarless engineering of the Drosophila genome near any site-specific integration site. Genetics 217(3). PubMed ID: 33772309
Summary:
This paper describes a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

Thursday, June 10th - Adult Neural Development and Function

Klose, M. K., Bruchez, M. P., Deitcher, D. L. and Levitan, E. S. (2021). Temporally and spatially partitioned neuropeptide release from individual clock neurons. Proc Natl Acad Sci U S A 118(17). PubMed ID: 33875606
Summary:
Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca(2+) data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity-triggered synaptic transmission, terminals require Ca(2+) influx, while somatic neuropeptide release is triggered by the biochemical signal IP(3) Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep-wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron-dependent behaviors. Thus, compartment-specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors.
Guzman-Palma, P., Contreras, E. G., Mora, N., Smith, M., Gonzalez-Ramirez, M. C., Campusano, J. M., Sierralta, J., Hassan, B. A. and Oliva, C. (2021). Slit/Robo Signaling Regulates Multiple Stages of the Development of the Drosophila Motion Detection System. Front Cell Dev Biol 9: 612645. PubMed ID: 33968921
Summary:
Neurogenesis is achieved through a sequence of steps that include specification and differentiation of progenitors into mature neurons. Frequently, precursors migrate to distinct positions before terminal differentiation. The Slit-Robo pathway, formed by the secreted ligand Slit and its membrane bound receptor Robo, was first discovered as a regulator of axonal growth. However, today, it is accepted that this pathway can regulate different cellular processes even outside the nervous system. This study describes the participation of the Slit-Robo pathway in the development of motion sensitive neurons of the Drosophila visual system. Slit and Robo receptors are expressed in different stages during the neurogenesis of motion sensitive neurons. Furthermore, it was found that Slit and Robo regulate multiple aspects of their development including neuronal precursor migration, cell segregation between neural stem cells and daughter cells and formation of their connectivity pattern. Specifically, loss of function of slit or robo receptors in differentiated motion sensitive neurons impairs dendritic targeting, while knocking down robo receptors in migratory progenitors or neural stem cells leads to structural defects in the adult optic lobe neuropil, caused by migration and cell segregation defects during larval development. Thus, this work reveals the co-option of the Slit-Robo signaling pathway in distinct developmental stages of a neural lineage.
Kim, S., Kim, J., Park, S., Park, J. J. and Lee, S. (2021). Drosophila Graf regulates mushroom body beta-axon extension and olfactory long-term memory. Mol Brain 14(1): 73. PubMed ID: 33892766
Summary:
Loss-of-function mutations in the human oligophrenin-1 (OPHN1) gene cause intellectual disability, a prevailing neurodevelopmental condition. However, the role OPHN1 plays during neuronal development is not well understood. This study investigated the role of the Drosophila OPHN1 ortholog Graf in the development of the mushroom body (MB), a key brain structure for learning and memory in insects. Loss of Graf causes abnormal crossing of the MB β lobe over the brain midline during metamorphosis. This defect in Graf mutants is rescued by MB-specific expression of Graf and OPHN1. Furthermore, MB α/β neuron-specific RNA interference experiments and mosaic analyses indicate that Graf acts via a cell-autonomous mechanism. Consistent with the negative regulation of epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) signaling by Graf, activation of this pathway is required for the β-lobe midline-crossing phenotype of Graf mutants. Finally, Graf mutants have impaired olfactory long-term memory. These findings reveal a role for Graf in MB axon development and suggest potential neurodevelopmental functions of human OPHN1.
Jin, X., Tian, Y., Zhang, Z. C., Gu, P., Liu, C. and Han, J. (2021). A subset of DN1p neurons integrates thermosensory inputs to promote wakefulness via CNMa signaling. Curr Biol. PubMed ID: 33740429
Summary:
Sleep is an essential and evolutionarily conserved behavior that is modulated by many environmental factors. Ambient temperature shifting usually occurs during climatic or seasonal change or travel from high-latitude area to low-latitude area that affects animal physiology. Increasing ambient temperature modulates sleep in both humans and Drosophila. Although several thermosensory molecules and neurons have been identified, the neural mechanisms that integrate temperature sensation into the sleep neural circuit remain poorly understood. This study reveals that prolonged increasing of ambient temperature induces a reversible sleep reduction and impaired sleep consolidation in Drosophila via activating the internal thermosensory anterior cells (ACs). ACs form synaptic contacts with a subset of posterior dorsal neuron 1 (DN1p) neurons and release acetylcholine to promote wakefulness. Furthermore, this study identified that this subset of DN1ps promotes wakefulness by releasing CNMamide (CNMa) neuropeptides to inhibit the Dh44-positive pars intercerebralis (PI) neurons through CNMa receptors. This study demonstrates that the AC-DN1p-PI neural circuit is responsible for integrating thermosensory inputs into the sleep neural circuit. Moreover, the CNMa signaling pathway was identified as a newly recognized wakefulness-promoting DN1 pathway.
Hardcastle, B. J., Omoto, J. J., Kandimalla, P., Nguyen, B. M., Keleş, M. F., Boyd, N. K., Hartenstein, V. and Frye, M. A. (2021). A visual pathway for skylight polarization processing in Drosophila. Elife 10. PubMed ID: 33755020
Summary:
Many insects use patterns of polarized light in the sky to orient and navigate. This study functionally characterized neural circuitry in the fruit fly, Drosophila melanogaster, that conveys polarized light signals from the eye to the central complex, a brain region essential for the fly's sense of direction. Neurons tuned to the angle of polarization of ultraviolet light are found throughout the anterior visual pathway, connecting the optic lobes with the central complex via the anterior optic tubercle and bulb, in a homologous organization to the 'sky compass' pathways described in other insects. This study detailed how a consistent, map-like organization of neural tunings in the peripheral visual system is transformed into a reduced representation suited to flexible processing in the central brain. This study identifies computational motifs of the transformation, enabling mechanistic comparisons of multisensory integration and central processing for navigation in the brains of insects.
Hatch, H. A. M., Belalcazar, H. M., Marshall, O. J. and Secombe, J. (2021). A KDM5-Prospero transcriptional axis functions during early neurodevelopment to regulate mushroom body formation. Elife 10. PubMed ID: 33729157
Summary:
Mutations in the lysine demethylase 5 (KDM5) family of transcriptional regulators are associated with intellectual disability, yet little is known regarding their spatiotemporal requirements or neurodevelopmental contributions. Utilizing the mushroom body (MB), a major learning and memory center within the Drosophila brain, this study demonstrates that KDM5 is required within ganglion mother cells and immature neurons for proper axogenesis. Moreover, the mechanism by which KDM5 functions in this context is independent of its canonical histone demethylase activity. Using in vivo transcriptional and binding analyses, a network of genes directly regulated by KDM5 was identified that are critical modulators of neurodevelopment. KDM5 directly regulates the expression of prospero, a transcription factor that is demonstrated is essential for MB morphogenesis. Prospero functions downstream of KDM5 and binds to approximately half of KDM5-regulated genes. Together, these data provide evidence for a KDM5-Prospero transcriptional axis that is essential for proper MB development.

Wednesday, June 9th - RNA and Transposons

Keegan, R. M., Talbot, L. R., Chang, Y. H., Metzger, M. J. and Dubnau, J. (2021). Intercellular viral spread and intracellular transposition of Drosophila gypsy. PLoS Genet 17(4): e1009535. PubMed ID: 33886543
Summary:
It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. This study demonstrates that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, the ability of gypsy to move between cells was demonstrated to be dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, it was also found that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome.
Kenny, A., Morgan, M. B. and Macdonald, P. M. (2021). Different roles for the adjoining and structurally similar A-rich and poly(A) domains of oskar mRNA: Only the A-rich domain is required for oskar noncoding RNA function, which includes MTOC positioning. Dev Biol 476: 117-127. PubMed ID: 33798537
Summary:
Drosophila oskar (osk) mRNA has both coding and noncoding functions, with the latter required for progression through oogenesis. Noncoding activity is mediated by the osk 3' UTR. Three types of cis elements act most directly and are clustered within the final ~120 nucleotides of the 3' UTR: multiple binding sites for the Bru1 protein, a short highly conserved region, and A-rich sequences abutting the poly(A) tail. All three elements were shown to be required for correct positioning of the microtubule organizing center (MTOC). Normally, the MTOC is located at the posterior of the oocyte during previtellogenic stages of oogenesis, and this distribution underlies the strong posterior enrichment of many mRNAs transported into the oocyte from the nurse cells. When osk noncoding function was disrupted the MTOC was dispersed in the oocyte and osk mRNA failed to be enriched at the posterior, although transport to the oocyte was not affected. Further characterization of the cis elements required for osk noncoding function included completion of saturation mutagenesis of the most highly conserved region, providing critical information for evaluating the possible contribution of candidate binding factors. The 3'-most cis element is a cluster of A-rich sequences, the ARS. The close juxtaposition and structural similarity of the ARS and poly(A) tail raised the possibility that they comprise an extended A-rich element required for osk noncoding function. This study found that absence of the poly(A) tail did not mimic the effects of mutation of the ARS, causing neither arrest of oogenesis nor mispositioning of osk mRNA in previtellogenic stage oocytes. Thus, the ARS and the poly(A) tail are not interchangeable for osk noncoding RNA function, suggesting that the role of the ARS is not in recruitment of Poly(A) binding protein (PABP), the protein that binds the poly(A) tail. Furthermore, although PABP has been implicated in transport of osk mRNA from the nurse cells to the oocyte, mutation of the ARS in combination with loss of the poly(A) tail did not disrupt transport of osk mRNA into the oocyte. It is concluded that PABP acts indirectly in osk mRNA transport, or is associated with osk mRNA independent of an A-rich binding site. Although the poly(A) tail was not required for osk mRNA transport into the oocyte, its absence was associated with a novel osk mRNA localization defect later in oogenesis, potentially revealing a previously unrecognized step in the localization process.
Chen, P., Kotov, A. A., Godneeva, B. K., Bazylev, S. S., Olenina, L. V. and Aravin, A. A. (2021). piRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D. melanogaster male germline. Genes Dev. PubMed ID: 33985970
Summary:
Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. This study comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, new piRNA clusters were identified, and complex satellites were identified as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. Two piRNA clusters were identified that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene.
Dominguez, A. (2021). Interrogating the 5'UTR tandem repeats of retrotransposon roo of Drosophila about horizontal transfer. Genetica. PubMed ID: 33900494
Summary:
Horizontal transfer in Drosophila has been inferred for several families of transposable elements. Specifically, the retroelement roo has been suggested to have been horizontally transferred between the species D. melanogaster, D. simulans, D. sechellia and D. yakuba. The inferences were based on the observation that divergence between transposable elements in different species was lower than the divergence found in typical nuclear genes and in the incongruence of phylogenies of the species and their TEs. This study addressed the question of the possible horizontal transfer of roo between species of the Drosophila genus by studying the presence absence of a duplication of 99 bp in the 5'UTR of the transposon, as well as comparing the sequences of the paralogous and orthologous duplicated repeats within and between species. First, the repeats were only found in five species of the melanogaster subgroup. Second, the date of occurrence of the duplication event originating the repeats was posterior to the split of the subgroup. The duplication date suggests an origin previous to the split of D. simulans and D. sechellia and close to the divergence of D. melanogaster from the D. simulans complex. These data point to horizontal transfer to the afrotropical species D. yakuba and D. erecta from one of the cosmopolitan species D. melanogaster or D. simulans. It is proposed that the parasitoid wasp Leptopilina could have been the vector of horizontal transfer after the observation that a sequence of 845 bp with high homology to a fragment of roo was isolated from this wasp.
Formicola, N., Heim, M., Dufourt, J., Lancelot, A. S., Nakamura, A., Lagha, M. and Besse, F. (2021). Tyramine induces dynamic RNP granule remodeling and translation activation in the Drosophila brain. Elife 10. PubMed ID: 33890854
Summary:
Ribonucleoprotein (RNP) granules are dynamic condensates enriched in regulatory RNA binding proteins (RBPs) and RNAs under tight spatiotemporal control. Extensive recent work has investigated the molecular principles underlying RNP granule assembly, unraveling that they form through the self-association of RNP components into dynamic networks of interactions. How endogenous RNP granules respond to external stimuli to regulate RNA fate is still largely unknown. This study demonstrated through high-resolution imaging of intact Drosophila brains that Tyramine induces a reversible remodeling of somatic RNP granules characterized by the decondensation of granule-enriched RBPs (e.g. Imp/ZBP1/IGF2BP) and helicases (e.g. Me31B/DDX-6/Rck). Furthermore, functional analysis reveals that Tyramine signals both through its receptor TyrR and through the calcium-activated kinase CamkII to trigger RNP component decondensation. Finally, it was uncover that RNP granule remodeling is accompanied by the rapid and specific translational activation of associated mRNAs. Thus, this work sheds new light on the mechanisms controlling cue-induced rearrangement of physiological RNP condensates.
Clerbaux, L. A., Schultz, H., Roman-Holba, S., Ruan, D. F., Yu, R., Lamb, A. M., Bommer, G. T. and Kennell, J. A. (2021). The microRNA miR-33 is a pleiotropic regulator of metabolic and developmental processes in Drosophila melanogaster. Dev Dyn. PubMed ID: 33840153
Summary:
miR-33 family members are well characterized regulators of cellular lipid levels in mammals. Previous studies have shown that overexpression of miR-33 in Drosophila melanogaster leads to elevated triacylglycerol (TAG) levels in certain contexts. Although loss of miR-33 in flies causes subtle defects in larval and adult ovaries, the effects of miR-33 deficiency on lipid metabolism and other phenotypes impacted by metabolic state have not yet been characterized. This study found that loss of miR-33 predisposes flies to elevated TAG levels, and genes involved in TAG synthesis were identified as direct targets of miR-33, including atpcl, midway, and Akt1. miR-33 mutants survived longer upon starvation but showed greater sensitivity to an oxidative stressor. Evidence was found that miR-33 is a negative regulator of cuticle pigmentation and that miR-33 mutants show a reduction in interfollicular stalk cells during oogenesis. These data suggest that miR-33 is a conserved regulator of lipid homeostasis, and its targets are involved in both degradation and synthesis of fatty acids and TAG. The constellation of phenotypes involving tissues that are highly sensitive to metabolic state suggests that miR-33 serves to prevent extreme fluctuations in metabolically sensitive tissues.

Tuesday, June 8th - Behavior

Cheng, K. Y. and Frye, M. A. (2021). Odour boosts visual object approach in flies. Biol Lett 17(3): 20200770. PubMed ID: 33726562
Summary:
Multisensory integration is synergistic-input from one sensory modality might modulate the behavioural response to another. Work in flies has shown that a small visual object presented in the periphery elicits innate aversive steering responses in flight, likely representing an approaching threat. Object aversion is switched to approach when paired with a plume of food odour. The 'open-loop' design of prior work facilitated the observation of changing valence. How does odour influence visual object responses when an animal has naturally active control over its visual experience? This study used closed-loop feedback conditions, in which a fly's steering effort is coupled to the angular velocity of the visual stimulus, to confirm that flies steer toward or 'fixate' a long vertical stripe on the visual midline. They tend either to steer away from or 'antifixate' a small object or to disengage active visual control, which manifests as uncontrolled object 'spinning' within this experimental paradigm. Adding a plume of apple cider vinegar decreases the probability of both antifixation and spinning, while increasing the probability of frontal fixation for objects of any size, including a normally typically aversive small object.
De Nardo, A. N., Roy, J., Sbilordo, S. H. and Lupold, S. (2021). Condition-dependent interaction between mating success and competitive fertilization success in Drosophila melanogaster. Evolution. PubMed ID: 33834478
Summary:
In many species, larger (high-condition) males gain higher mating success through male-male competition and female choice, and female condition can affect the extent of both female mate choice and male investment in courtship or ejaculates. This study manipulated the larval diet of male and female Drosophila melanogaster to study how body size variation in both sexes biases competitive outcomes at different reproductive stages, from mating to paternity. Not difference was found in mate preference or mating latency between females of different conditions, nor any interaction between male and female conditions. However, large males were more successful in gaining matings, but only when in direct competition, whereas mating latencies were shorter for low-condition males in noncompetitive settings. Small males also transferred more sperm to nonvirgin females, displaced a larger proportion of resident sperm, and achieved higher paternity shares per mating than large males. In agreement with existing theory, it is suggested that small males might partially compensate for their low mating success by strategically investing in larger sperm numbers and potentially other, unmeasured ejaculate traits, when they do have a mating opportunity.
Ebrahim, S. A. M., Talross, G. J. S. and Carlson, J. R. (2021). Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila. Nat Commun 12(1): 2453. PubMed ID: 33907186
Summary:
Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasitoid wasp species kill a vast range of insect species. Insects have evolved defensive responses to the threat of wasps, some cellular and some behavioral. This study found an unexpected response of adult Drosophila to the presence of certain parasitoid wasps: accelerated mating behavior. Flies exposed to certain wasp species begin mating more quickly. The effect is mediated via changes in the behavior of the female fly and depends on visual perception. The sight of wasps induces the dramatic upregulation in the fly nervous system of a gene that encodes a 41-amino acid micropeptide. Mutational analysis reveals that the gene is essential to the behavioral response of the fly. This work provides a foundation for further exploration of how the activation of visual circuits by the sight of a wasp alters both sexual behavior and gene expression.
Borne, F., Prigent, S. R., Molet, M. and Courtier-Orgogozo, V. (2021). Drosophila glue protects from predation. Proc Biol Sci 288(1947): 20210088. PubMed ID: 33726597
Summary:
Animals can be permanently attached to a substrate in terrestrial environments at certain stages of their development. Pupa adhesion has evolved multiple times in insects and is thought to maintain the animal in a place where it is not detectable by predators. This study investigated whether pupa adhesion in Drosophila can also protect the animal by preventing potential predators from detaching the pupa. The adhesion was measured of Drosophila species sampled from the same area, and pupa adhesion was found to vary among species, which can be explained by different glue production strategies. Then, attached and manually detached pupae were compared in both field and laboratory assays to investigate the role of pupa adhesion to prevent predation. First, it was found that attached pupae remain onsite 30% more than detached pupae in the field after 3 days, probably because they are less predated. Second, it was observed that attached pupae are less efficiently predated by ants in the laboratory: they are not carried back to the ant nest and more ants are needed to consume them onsite. These results show that pupa adhesion can prevent the animal from being taken away by predators and is crucial for Drosophila fly survival.
Ikarashi, M. and Tanimoto, H. (2021). Drosophila acquires seconds-scale rhythmic behavior. J Exp Biol. PubMed ID: 33795422
Summary:
Detection of the temporal structure of stimuli is crucial for prediction. While perception of interval timing is relevant for immediate behavioral adaptations, it has been scarcely investigated, especially in invertebrates. This study examined if the fruit fly, Drosophila melanogaster, can acquire rhythmic behavior in the range of seconds. To this end, a novel temporal conditioning paradigm utilizing repeated electric shocks was developed. Combined automatic behavioral annotation and time-frequency analysis revealed that behavioral rhythms continued after cessation of the shocks. Furthermore, it was found that aging impaired interval timing. This study thus not only demonstrated the ability of insects to acquire behavioral rhythms of a few seconds, but highlighted a life-course decline of temporal coordination, that is common also in mammals.
Jia, Y., Jin, S., Hu, K., Geng, L., Han, C., Kang, R., Pang, Y., Ling, E., Tan, E. K., Pan, Y. and Liu, W. (2021). Gut microbiome modulates Drosophila aggression through octopamine signaling. Nat Commun 12(1): 2698. PubMed ID: 33976215
Summary:
Gut microbiome profoundly affects many aspects of host physiology and behaviors. This study reports that gut microbiome modulates aggressive behaviors in Drosophila. Germ-free males showed substantial decrease in inter-male aggression, which could be rescued by microbial re-colonization. These germ-free males are not as competitive as wild-type males for mating with females, although they displayed regular levels of locomotor and courtship behaviors. it was further found that Drosophila microbiome interacted with diet during a critical developmental period for the proper expression of octopamine and manifestation of aggression in adult males. These findings provide insights into how gut microbiome modulates specific host behaviors through interaction with diet during development.

Monday, June 7th - Adult Development

Zhou, D., Stobdan, T., Visk, D., Xue, J. and Haddad, G. G. (2021). Genetic interactions regulate hypoxia tolerance conferred by activating Notch in excitatory amino acid transporter 1-positive glial cells in Drosophila melanogaster. G3 (Bethesda) 11(2). PubMed ID: 33576765
Summary:
Hypoxia is a critical pathological element in many human diseases, including ischemic stroke, myocardial infarction, and solid tumors. Of particular significance and interest are the cellular and molecular mechanisms that underlie susceptibility or tolerance to low O2. Previous studies have demonstrated that Notch signaling pathway regulates hypoxia tolerance in both Drosophila melanogaster and humans. However, the mechanisms mediating Notch-conferred hypoxia tolerance are largely unknown. This study delineates the evolutionarily conserved mechanisms underlying this hypoxia tolerant phenotype. The role of a group of conserved genes was determined that were obtained from a comparative genomic analysis of hypoxia-tolerant D.melanogaster populations and human highlanders living at the high-altitude regions of the world (Tibetans, Ethiopians, and Andeans). A novel dual-UAS/Gal4 system was developed that allows activation of Notch signaling in the Eaat1-positive glial cells, which remarkably enhances hypoxia tolerance in D.melanogaster, and, simultaneously, knock down a candidate gene in the same set of glial cells. Using this system, it was discovered that the interactions between Notch signaling and bnl (fibroblast growth factor), croc (forkhead transcription factor C), or Mkk4 (mitogen-activated protein kinase kinase 4) are important for hypoxia tolerance, at least in part, through regulating neuronal development and survival under hypoxic conditions. Because these genetic mechanisms are evolutionarily conserved, this group of genes may serve as novel targets for developing therapeutic strategies and have a strong potential to be translated to humans to treat/prevent hypoxia-related diseases.
Goh, G. H., Blache, D., Mark, P. J., Kennington, W. J. and Maloney, S. K. (2021). Daily temperature cycles prolong lifespan and have sex-specific effects on peripheral clock gene expression in Drosophila melanogaster. J Exp Biol. PubMed ID: 33758022
Summary:
Circadian rhythms optimize health by coordinating the timing of physiological processes to match predictable daily environmental challenges. The circadian rhythm of body temperature is thought to be an important modulator of molecular clocks in peripheral tissues, but how daily temperature cycles impact physiological function is unclear. This study examined the effect of constant (25°C, T(CON)) and cycling (28°C/22°C during light/dark, T(CYC)) temperature paradigms on lifespan of Drosophila melanogaster, and the expression of clock genes, Heat shock protein 83 (Hsp83), Frost (Fst), and Senescence-associated protein 30 (smp-30). Male and female Drosophila housed at T(CYC) had longer median lifespans than those housed at T(CON) T(CYC) induced robust Hsp83 rhythms and rescued the age-related decrease in smp-30 expression that was observed in flies at T(CON), potentially indicating an increased capacity to cope with age-related cellular stress. Ageing under T(CON) led to a decrease in the amplitude of expression of all clock genes in the bodies of male flies, except for cyc, which was non-rhythmic, and for per and cry in female flies. Strikingly, housing under T(CYC) conditions rescued the age-related decrease in amplitude of all clock genes, and generated rhythmicity in cyc expression, in the male flies, but not the female flies. The results suggest that ambient temperature rhythms modulate Drosophila lifespan, and that the amplitude of clock gene expression in peripheral body clocks may be a potential link between temperature rhythms and longevity in male Drosophila Longevity due to T(CYC) appeared predominantly independent of clock gene amplitude in female Drosophila.
Almeida-Oliveira, F., Tuthill, B. F., Gondim, K. C., Majerowicz, D. and Musselman, L. P. (2021). dHNF4 regulates lipid homeostasis and oogenesis in Drosophila melanogaster. Insect Biochem Mol Biol: 103569. PubMed ID: 33753225
Summary:
The fly genome contains a single ortholog of the evolutionarily conserved transcription factor hepatocyte nuclear factor 4 (HNF4), a broadly and constitutively expressed member of the nuclear receptor superfamily. Like its mammalian orthologs, Drosophila HNF4 (dHNF4) acts as a critical regulator of fatty acid and glucose homeostasis. Because of its role in energy storage and catabolism, the insect fat body controls non-autonomous organs including the ovaries, where lipid metabolism is essential for oogenesis. This study used dHNF4 overexpression (OE) in the fat bodies and ovaries to investigate its potential roles in lipid homeostasis and oogenesis. When the developing fat body overexpressed dHNF4, animals exhibited reduced size and failed to pupariate, but no changes in body composition were observed. Conditional OE of dHNF4 in the adult fat body produced a reduction in triacylglycerol content and reduced oogenesis. Ovary-specific dHNF4 OE increased oogenesis and egg-laying, but reduced the number of adult offspring. The phenotypic effects on oogenesis that arise upon dHNF4 OE in the fat body or ovary may be due to its function in controlling lipid utilization.
Akhund-Zade, J., Lall, S., Gajda, E., Yoon, D., Ayroles, J. F. and de Bivort, B. L. (2021). Genetic basis of offspring number-body weight tradeoff in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 33871609
Summary:
Drosophila melanogaster egg production, a proxy for fecundity, is an extensively studied life-history trait with a strong genetic basis. As eggs develop into larvae and adults, space and resource constraints can put pressure on the developing offspring, leading to a decrease in viability, body size, and lifespan. The goal of this study was to map the genetic basis of offspring number and weight under the restriction of a standard laboratory vial. 143 lines from the Drosophila Genetic Reference Panel were screened for offspring numbers and weights to create an 'offspring index' that captured the number vs. weight trade-off. 18 genes containing 30 variants were found associated with variation in the offspring index. Validation of hid, Sox21b, CG8312, and mub candidate genes using gene disruption mutants demonstrated a role in adult stage viability, while mutations in Ih and Rbp increased offspring number and increased weight, respectively. The polygenic basis of offspring number and weight, with many variants of small effect, as well as the involvement of genes with varied functional roles, support the notion of Fisher's "infinitesimal model" for this life-history trait.
Blumrich, A., Vogler, G., Dresen, S., Diop, S. B., Jaeger, C., Leberer, S., Grune, J., Wirth, E. K., Hoeft, B., Renko, K., Foryst-Ludwig, A., Spranger, J., Sigrist, S., Bodmer, R. and Kintscher, U. (2021). Fat-body brummer lipase determines survival and cardiac function during starvation in Drosophila melanogaster. iScience 24(4): 102288. PubMed ID: 33889813
Summary:
The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. Evidence is provided that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, it is suggested that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, this study identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress.
Ji, Z., Chuen, J., Kiparaki, M. and Baker, N. (2021). Cell competition removes segmental aneuploid cells from Drosophila imaginal disc-derived tissues based on ribosomal protein gene dose. Elife 10. PubMed ID: 33847264
Summary:
Aneuploidy causes birth defects and miscarriages, occurs in nearly all cancers and is a hallmark of aging. Individual aneuploid cells can be eliminated from developing tissues by unknown mechanisms. Cells with ribosomal protein (Rp) gene mutations are also eliminated, by cell competition with normal cells. Because Rp genes are spread across the genome, their copy number is a potential marker for aneuploidy. Elimination of imaginal disc cells with irradiation-induced genome damage often required cell competition genes. Segmentally aneuploid cells derived from targeted chromosome excisions were eliminated by the RpS12-Xrp1 cell competition pathway if they differed from neighboring cells in Rp gene dose, whereas cells with normal doses of the Rp and eIF2γ genes survived and differentiated adult tissues. Thus, cell competition, triggered by differences in Rp gene dose between cells, is a significant mechanism for the elimination of aneuploid somatic cells, likely to contribute to preventing cancer.

Friday, June 4th - Disease Models

Dinh, E., Rival, T., Carrier, A., Asfogo, N., Corti, O., Melon, C., Salin, P., Lortet, S. and Kerkerian-Le Goff, L. (2021). TP53INP1 exerts neuroprotection under ageing and Parkinson's disease-related stress condition. Cell Death Dis 12(5): 460. PubMed ID: 33966044
Summary:
TP53INP1 is a stress-induced protein, which acts as a dual positive regulator of transcription and of autophagy and whose deficiency has been linked with cancer and metabolic syndrome. This study addressed the unexplored role of TP53INP1 and of its Drosophila homolog dDOR in the maintenance of neuronal homeostasis under chronic stress, focusing on dopamine (DA) neurons under normal ageing- and Parkinson's disease (PD)-related context. Trp53inp1(-/-) mice displayed additional loss of DA neurons in the substantia nigra compared to wild-type (WT) mice, both with ageing and in a PD model based on targeted overexpression of α-synuclein. Nigral Trp53inp1 expression of WT mice was not significantly modified with ageing but was markedly increased in the PD model. Trp53inp2 expression showed similar evolution and did not differ between WT and Trp53inp1(-/-) mice. In Drosophila, pan-neuronal dDOR overexpression improved survival under paraquat exposure and mitigated the progressive locomotor decline and the loss of DA neurons caused by the human α-synuclein A30P variant. dDOR overexpression in DA neurons also rescued the locomotor deficit in flies with RNAi-induced downregulation of dPINK1 or dParkin. Live imaging, confocal and electron microscopy in fat bodies, neurons, and indirect flight muscles showed that dDOR acts as a positive regulator of basal autophagy and mitophagy independently of the PINK1-mediated pathway. Analyses in a mammalian cell model confirmed that modulating TP53INP1 levels does not impact mitochondrial stress-induced PINK1/Parkin-dependent mitophagy. These data provide the first evidence for a neuroprotective role of TP53INP1/dDOR and highlight its involvement in the regulation of autophagy and mitophagy in neurons.
Formica, M., Storaci, A. M., Bertolini, I., Carminati, F., Knævelsrud, H., Vaira, V. and Vaccari, T. (2021). V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis. Autophagy: 1-11. PubMed ID: 33978540
Summary:
Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, an established Drosophila melanogaster model of glioma was used based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H(+)-ATPase (V-ATPase) subunits and ref(2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, these data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM.
Bhat, S., Guthrie, D. A., Kasture, A., El-Kasaby, A., Cao, J., Bonifazi, A., Ku, T., Giancola, J. B., Hummel, T., Freissmuth, M. and Newman, A. H. (2021). Tropane-Based Ibogaine Analog Rescues Folding-Deficient Serotonin and Dopamine Transporters. ACS Pharmacol Transl Sci 4(2): 503-516. PubMed ID: 33860180
Summary:
Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutant proteins, which give rise to infantile Parkinsonism and dystonia. In this study, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild-type transporters, as well as for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG(601,602)AA and DAT-PG(584,585)AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo in Drosophila harboring the DAT-PG(584,585)AA mutation and rescued 6 out of 13 disease-associated human DAT mutant proteins in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutations.
Carvajal-Oliveros, A., Domínguez-Baleon, C., Zarate, R. V., Campusano, J. M., Narvaez-Padilla, V. and Reynaud, E. (2021). Nicotine suppresses Parkinson's disease like phenotypes induced by Synphilin-1 overexpression in Drosophila melanogaster by increasing tyrosine hydroxylase and dopamine levels. Sci Rep 11(1): 9579. PubMed ID: 33953275
Summary:
It has been observed that there is a lower Parkinson's disease (PD) incidence in tobacco users. Nicotine is a cholinergic agonist and is the principal psychoactive compound in tobacco linked to cigarette addiction. Different studies have shown that nicotine has beneficial effects on sporadic and genetic models of PD. This work evaluated nicotine's protective effect in a Drosophila melanogaster model for PD where Synphilin-1 (Sph-1) is expressed in dopaminergic neurons. Nicotine has a moderate effect on dopaminergic neuron survival that becomes more evident as flies age. Nicotine is beneficial on fly survival and motility increasing tyrosine hydroxylase and dopamine levels, suggesting that cholinergic agonists may promote survival and metabolic function of the dopaminergic neurons that express Sph-1. The Sph-1 expressing fly is a good model for the study of early-onset phenotypes such as olfaction loss one of the main non-motor symptom related to PD. The data suggest that nicotine is an interesting therapeutic molecule whose properties should be explored in future research on the phenotypic modulators of the disease and for the development of new treatments.
Das, T. K., Gatto, J., Mirmira, R., Hourizadeh, E., Kaufman, D., Gelb, B. D. and Cagan, R. (2021). Drosophila RASopathy models identify disease subtype differences and biomarkers of drug efficacy. iScience 24(4): 102306. PubMed ID: 33855281
Summary:
RASopathies represent a family of mostly autosomal dominant diseases that are caused by missense variants in the rat sarcoma viral oncogene/mitogen activated protein kinase (RAS/MAPK) pathway including KRAS, NRAS, BRAF, RAF1, and SHP2. These variants are associated with overlapping but distinct phenotypes that affect the heart, craniofacial, skeletal, lymphatic, and nervous systems. This study reports an analysis of 13 Drosophila transgenic lines, each expressing a different human RASopathy isoform. Similar to their human counterparts, each Drosophila line displayed common aspects but also important differences including distinct signaling pathways such as the Hippo and SAPK/JNK signaling networks. Multiple classes of clinically relevant drugs-including statins and histone deacetylase inhibitors-were identified that improved viability across most RASopathy lines; in contrast, several canonical RAS pathway inhibitors proved less broadly effective. Overall, this study compares and contrasts a large number of RASopathy-associated variants including their therapeutic responses.
Fumagalli, L., et al. (2021). C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility. Sci Adv 7(15). PubMed ID: 33837088
Summary:
A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. This study shows using patient stem cell-derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, this study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

Thursday, June 3rd - Gonads

Zhang, M. J., Shi, X. X., Wang, N., Zhang, C., Zhang, C., Quais, M. K., Ali, S. A., Zhou, W., Mao, C. and Zhu, Z. R. (2021). Transcriptional changes revealed genes and pathways involved in the deficient testis caused by the inhibition of alkaline ceramidase (Dacer) in Drosophila melanogaster. Arch Insect Biochem Physiol 106(3): e21765. PubMed ID: 33590535
Summary:
Sphingolipids are ubiquitous structural components of eukaryotic cell membranes which are vital for maintaining the integrity of cells. Alkaline ceramidase is a key enzyme in sphingolipid biosynthesis pathway; however, little is known about the role of the enzyme in the male reproductive system of Drosophila melanogaster. To investigate the impact of alkaline ceramidase (Dacer) on male Drosophila, Dacer deficiency mutants (MUs) were obtained and were found to displayed apparent defects in the testis's phenotype. To profile the molecular changes associated with this abnormal phenotype, de novo transcriptome analyses of the MU and wildtype (WT) testes was performed; 1239 upregulated genes and 1102 downregulated genes were obtained. Then, six upregulated genes (papilin [Ppn], croquemort [Crq], terribly reduced optic lobes [Trol], Laminin, Wunen-2, collagen type IV alpha 1 [Cg25C]) and three downregulated genes (mucin related 18B [Mur18B], rhomboid-7 [Rho-7], CG3168) were confirmed through quantitative real-time polymerase chain reaction in WT and MU samples. The differentially expressed genes were mainly associated with catalytic activity, oxidoreductase activity and transmembrane transporter activity, which significantly contributed to extracellular matrix-receptor interaction, fatty acids biosynthesis as well as glycine, serine, and threonine metabolism. The results highlight the importance of Dacer in the reproductive system of D. melanogaster and provide valuable resources to dig out the specific biological functions of Dacer in insect reproduction.
Fang, Y., Zong, Q., He, Z., Liu, C. and Wang, Y. F. (2021). Knockdown of RpL36 in testes impairs spermatogenesis in Drosophila melanogaster. J Exp Zool B Mol Dev Evol. PubMed ID: 33734578
Summary:
Many ribosomal proteins (RPs) not only play essential roles in ribosome biogenesis, but also have "extraribosomal" functions in various cellular processes. RpL36 encodes ribosomal protein L36, a component of the 60S subunit of ribosomes in Drosophila melanogaster. This paper reports RpL36 is required for spermatogenesis in D. melanogaster. After showing the evolutionary conservation of RpL36 sequences in animals, it was shown that RpL36 expression level in fly testes was significantly higher than in ovaries. Knockdown RpL36 in fly testes resulted in a significantly decreased egg hatch rate when these males mated with wild-type females. Furthermore, 76.67% of the RpL36 knockdown fly testes were much smaller in comparison to controls. Immunofluorescence staining showed that in the RpL36 knockdown testis hub cell cluster was enlarged, while the number of germ cells, including germ stem cells, was reduced. Knockdown of RpL36 in fly testis caused much fewer or no mature sperms in seminal vesicles. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) signal was stronger in RpL36 knockdown fly testes than in the control testes, but the TUNEL-positive cells could not be stained by Vasa antibody, indicating that apoptotic cells are not germ cells. The percentage of pH3-positive cells among the Vasa-positive cells was significantly reduced. The expression of genes involved in cell death, cell cycle progression, and JAK/STAT signaling pathway was significantly changed by RpL36 knockdown in fly testes. These results suggest that RpL36 plays an important role in spermatogenesis, likely through JAK/STAT pathway, thus resulting in defects in cell-cycle progression and cell death in D. melanogaster testes.
Goldman, C. H., Neiswender, H., Baker, F., Veeranan-Karmegam, R., Misra, S. and Gonsalvez, G. B. (2021). Optimal RNA binding by Egalitarian, a Dynein cargo adaptor, is critical for maintaining oocyte fate in Drosophila. RNA Biol: 1-14. PubMed ID: 33904382
Summary:
The Dynein motor is responsible for the localization of numerous mRNAs within Drosophila oocytes and embryos. The RNA binding protein, Egalitarian (Egl), is thought to link these various RNA cargoes with Dynein. Although numerous studies have shown that Egl is able to specifically associate with these RNAs, the nature of these interactions has remained elusive. Egl contains a central RNA binding domain that shares limited homology with an exonuclease, yet Egl binds to RNA without degrading it. Mutations have been identified within Egl that disrupt its association with its protein interaction partners, BicaudalD (BicD) and Dynein light chain (Dlc), but no mutants have been described that are specifically defective for RNA binding. This report identified a series of positively charged residues within Egl that are required for RNA binding. Using corresponding RNA binding mutants, it was demonstrated that specific RNA cargoes are more reliant on maximal Egl RNA biding activity for their correct localization in comparison to others. It was also demonstrated that specification and maintenance of oocyte fate requires maximal Egl RNA binding activity. Even a subtle reduction in Egl's RNA binding activity completely disrupts this process. These results show that efficient RNA localization at the earliest stages of oogenesis is required for specification of the oocyte and restriction of meiosis to a single cell.
Grmai, L., Harsh, S., Lu, S., Korman, A., Deb, I. B. and Bach, E. A. (2021). Transcriptomic analysis of feminizing somatic stem cells in the Drosophila testis reveals putative downstream effectors of the transcription factor Chinmo. G3 (Bethesda) 11(4). PubMed ID: 33751104
Summary:
One of the best examples of sexual dimorphism is the development and function of the gonads, ovaries and testes, which produce sex-specific gametes, oocytes, and spermatids, respectively. The development of these specialized germ cells requires sex-matched somatic support cells. The sexual identity of somatic gonadal cells is specified during development and must be actively maintained during adulthood. Previous work showed that the transcription factor Chinmo is required to ensure the male sexual identity of somatic support cells in the Drosophila melanogaster testis. Loss of chinmo from male somatic gonadal cells results in feminization: they transform from squamous to epithelial-like cells that resemble somatic cells in the female gonad but fail to properly ensheath the male germline, causing infertility. To identify potential target genes of Chinmo, somatic cells deficient for chinmo were purified from the adult Drosophila testis, and next-generation sequencing was performed to compare their transcriptome to that of control somatic cells. Bioinformatics revealed 304 and 1549 differentially upregulated and downregulated genes, respectively, upon loss of chinmo in early somatic cells. Using a combination of methods, several differentially expressed genes were validated. These data sets will be useful resources to the community.
Ali-Murthy, Z., Fetter, R. D., Wang, W., Yang, B., Royer, L. A. and Kornberg, T. B. (2021). Elimination of nurse cell nuclei that shuttle into oocytes during oogenesis. J Cell Biol 220(7). PubMed ID: 33950159
Summary:
Drosophila oocytes develop together with 15 sister germline nurse cells (NCs), which pass products to the oocyte through intercellular bridges. The NCs are completely eliminated during stages 12-14, but this study discovered that at stage 10B, two specific NCs fuse with the oocyte and extrude their nuclei through a channel that opens in the anterior face of the oocyte. These nuclei extinguish in the ooplasm, leaving 2 enucleated and 13 nucleated NCs. At stage 11, the cell boundaries of the oocyte are mostly restored. Oocytes in egg chambers that fail to eliminate NC nuclei at stage 10B develop with abnormal morphology. These findings show that stage 10B NCs are distinguished by position and identity, and that NC elimination proceeds in two stages: first at stage 10B and later at stages 12-14.
Immarigeon, C., Frei, Y., Delbare, S. Y. N., Gligorov, D., Machado Almeida, P., Grey, J., Fabbro, L., Nagoshi, E., Billeter, J. C., Wolfner, M. F., Karch, F. and Maeda, R. K. (2021). Identification of a micropeptide and multiple secondary cell genes that modulate Drosophila male reproductive success. Proc Natl Acad Sci U S A 118(15). PubMed ID: 33876742
Summary:
In the process of characterizing the ribosome-bound transcriptome of an important cell type of the seminal fluid-producing accessory gland of Drosophila melanogaster, an RNA, previously thought to be noncoding, called male-specific abdominal (msa), was identified. Notably, msa is nested in the HOX gene cluster of the Bithorax complex and is known to contain a micro-RNA within one of its introns. This study found that this RNA encodes a "micropeptide" (9 or 20 amino acids, MSAmiP) that is expressed exclusively in the secondary cells of the male accessory gland, where it seems to accumulate in nuclei. Importantly, loss of function of this micropeptide causes defects in sperm competition. This work underlines the importance of small peptides, a class of molecules that is now emerging as important actors in complex biological processes.

Wednesday, May 2nd - Adult neural development and function

Buck, S. A., Steinkellner, T., Aslanoglou, D., Villeneuve, M., Bhatte, S. H., Childers, V. C., Rubin, S. A., De Miranda, B. R., O'Leary, E. I., Neureiter, E. G., Fogle, K. J., Palladino, M. J., Logan, R. W., Glausier, J. R., Fish, K. N., Lewis, D. A., Greenamyre, J. T., McCabe, B. D., Cheetham, C. E. J., Hnasko, T. S. and Freyberg, Z. (2021). Vesicular glutamate transporter modulates sex differences in dopamine neuron vulnerability to age-related neurodegeneration.. Aging Cell: e13365. PubMed ID: 33909313
Summary:
Age is the greatest risk factor for Parkinson's disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. Intriguingly, some DA neurons are more resilient to degeneration than others. Increasing evidence suggests that vesicular glutamate transporter (VGLUT) expression in DA neurons plays a role in this selective vulnerability. We investigated the role of DA neuron VGLUT in sex- and age-related differences in DA neuron vulnerability using the genetically tractable Drosophila model. This study found sex differences in age-related DA neurodegeneration and its associated locomotor behavior, where males exhibit significantly greater decreases in both DA neuron number and locomotion during aging compared with females. Dynamic changes in DA neuron VGLUT expression mediate these age- and sex-related differences, as a potential compensatory mechanism for diminished DA neurotransmission during aging. Importantly, female Drosophila possess higher levels of VGLUT expression in DA neurons compared with males, and this finding is conserved across flies, rodents, and humans. Moreover, this study showed that diminishing VGLUT expression in DA neurons eliminates females' greater resilience to DA neuron loss across aging. This offers a new mechanism for sex differences in selective DA neuron vulnerability to age-related DA neurodegeneration. Finally, in mice, it was shown that the ability of DA neurons to achieve optimal control over VGLUT expression is essential for DA neuron survival. These findings lay the groundwork for the manipulation of DA neuron VGLUT expression as a novel therapeutic strategy to boost DA neuron resilience to age- and PD-related neurodegeneration.
Brown, E. B., Shah, K. D., Palermo, J., Dey, M., Dahanukar, A. and Keene, A. C. (2021). Ir56d-dependent fatty acid responses in Drosophila uncovers taste discrimination between different classes of fatty acids. Elife 10. PubMed ID: 33949306
Summary:
Chemosensory systems are critical for evaluating the caloric value and potential toxicity of food prior to ingestion. While animals can discriminate between 1000's of odors, much less is known about the discriminative capabilities of taste systems. Fats and sugars represent calorically potent and innately attractive food sources that contribute to hedonic feeding. Despite the differences in nutritional value between fats and sugars, the ability of the taste system to discriminate between different rewarding tastants is thought to be limited. In Drosophila, sweet taste neurons expressing the Ionotropic Receptor 56d (IR56d) are required for reflexive behavioral responses to the medium-chain fatty acid, hexanoic acid. Further, it was found that flies can discriminate between a fatty acid and a sugar in aversive memory assays, establishing a foundation to investigate the capacity of the Drosophila gustatory system to differentiate between various appetitive tastants. This study tested whether flies can discriminate between different classes of fatty acids using an aversive memory assay. The results indicate that flies are able to discriminate medium-chain fatty acids from both short- and long-chain fatty acids, but not from other medium-chain fatty acids. While IR56d neurons are broadly responsive to short-, medium-, and long-chain fatty acids, genetic deletion of IR56d selectively disrupts response to medium-chain fatty acids. Further, IR56d+GR64f+ neurons are necessary for proboscis extension response (PER) to medium-chain fatty acids, but both IR56d and GR64f neurons are dispensable for PER to short- and long-chain fatty acids, indicating the involvement of one or more other classes of neurons. Together, these findings reveal that IR56d is selectively required for medium-chain fatty acid taste, and discrimination of fatty acids occurs through differential receptor activation in shared populations of neurons. Our study uncovers a capacity for the taste system to encode tastant identity within a taste category.
Damrau, C., Colomb, J. and Brembs, B. (2021). Sensitivity to expression levels underlies differential dominance of a putative null allele of the Drosophila tbetah gene in behavioral phenotypes. PLoS Biol 19(5): e3001228. PubMed ID: 33970909
Summary:
The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-β-hydroxylase (tβh) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. This study reports differential dominance (from recessive to overdominant) of the putative null hnM18 allele in 2 behavioral measures in Buridan's paradigm (walking speed and stripe deviation) and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tβh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. This analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tβh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics.
Dapergola, E., Menegazzi, P., Raabe, T. and Hovhanyan, A. (2021). Light Stimuli and Circadian Clock Affect Neural Development in Drosophila melanogaster. Front Cell Dev Biol 9: 595754. PubMed ID: 33763414
Summary:
Endogenous clocks enable organisms to adapt cellular processes, physiology, and behavior to daily variation in environmental conditions. Metabolic processes in cyanobacteria to humans are under the influence of the circadian clock, and dysregulation of the circadian clock causes metabolic disorders. In mouse and Drosophila, the circadian clock influences translation of factors involved in ribosome biogenesis and synchronizes protein synthesis. Notably, nutrition signals are mediated by the insulin receptor/target of rapamycin (InR/TOR) pathways to regulate cellular metabolism and growth. However, the role of the circadian clock in Drosophila brain development and the potential impact of clock impairment on neural circuit formation and function is less understood. This study demonstrates that changes in light stimuli or disruption of the molecular circadian clock cause a defect in neural stem cell growth and proliferation. Moreover, this study shows that disturbed cell growth and proliferation are accompanied by reduced nucleolar size indicative of impaired ribosomal biogenesis. Further, this study defines that light and clock independently affect the InR/TOR growth regulatory pathway due to the effect on regulators of protein biosynthesis. Altogether, these data suggest that alterations in InR/TOR signaling induced by changes in light conditions or disruption of the molecular clock have an impact on growth and proliferation properties of neural stem cells in the developing Drosophila brain.
Boulanger, A., Thinat, C., Zuchner, S., Fradkin, L. G., Lortat-Jacob, H. and Dura, J. M. (2021). Axonal chemokine-like Orion induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling. Nat Commun 12(1): 1849. PubMed ID: 33758182
Summary:
The remodeling of neurons is a conserved fundamental mechanism underlying nervous system maturation and function. Astrocytes can clear neuronal debris and they have an active role in neuronal remodeling. Developmental axon pruning of Drosophila memory center neurons occurs via a degenerative process mediated by infiltrating astrocytes. However, how astrocytes are recruited to the axons during brain development is unclear. Using an unbiased screen, the gene requirement of orion/CG2206, encoding for a chemokine-like protein, was identified in the developing mushroom bodies. Functional analysis shows that Orion is necessary for both axonal pruning and removal of axonal debris. Orion performs its functions extracellularly and bears some features common to chemokines, a family of chemoattractant cytokines. It is proposed that Orion is a neuronal signal that elicits astrocyte infiltration and astrocyte-driven axonal engulfment required during neuronal remodeling in the Drosophila developing brain.
Georganta, E. M., Moressis, A. and Skoulakis, E. M. (2021). Associative learning requires Neurofibromin to modulate GABAergic inputs to Drosophila Mushroom Bodies. J Neurosci. PubMed ID: 33972401
Summary:
Cognitive dysfunction, is among the hallmark symptoms of Neurofibromatosis 1, and accordingly, loss of the Drosophila melanogaster ortholog of Neurofibromin 1 (dNf1), precipitates associative learning deficits. However, the affected circuitry in the adult CNS remained unclear and the compromised mechanisms debatable. Although the main evolutionarily conserved function attributed to Nf1 is to inactivate Ras, decreased cAMP signalling upon its loss has been thought to underlie impaired learning. Using mixed sex populations, it was determined that dNf1 loss results in excess GABAergic signaling to the central for associative learning Mushroom Body (MB) neurons, apparently suppressing learning. dNf1 is necessary and sufficient for learning within these non-MB neurons, as a dAlk and Ras1-dependent, but PKA-independent modulator of GABAergic neurotransmission. Surprisingly, this study also uncovered and discuss a postsynaptic Ras1-dependent, but dNf1-independnet signaling within the MBs that apparently responds to presynaptic GABA levels and contributes to the learning deficit on the mutants.

Tuesday May 1st - Enhancers and gene regulation

Fujioka, M., Nezdyur, A. and Jaynes, J. B. (2021). An insulator blocks access to enhancers by an illegitimate promoter, preventing repression by transcriptional interference. PLoS Genet 17(4): e1009536. PubMed ID: 33901190
Summary:
Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. This paper describes a novel mechanism of functional separation whereby an insulator prevents gene repression. When the homie insulator is deleted from the end of a Drosophila even skipped (eve) locus, a flanking P-element promoter is activated in a partial eve pattern, causing expression driven by enhancers in the 3' region to be repressed. The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even when homie is in place. Furthermore, when the flanking promoter is turned around, repression is minimal. Transcriptional read-through that does not produce anti-sense RNA can still repress expression, ruling out RNAi as the mechanism in this case. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses eve promoter-driven expression. We also show that enhancer-promoter specificity and processivity of transcription can have decisive effects on the consequences of insulator removal. First, a core heat shock 70 promoter that is not activated well by eve enhancers did not cause read-through sufficient to repress the eve promoter. Second, these transcripts are less processive than those initiated at the P-promoter, measured by how far they extend through the eve locus, and so are less disruptive. These results highlight the importance of considering transcriptional read-through when assessing the effects of insulators on gene expression.
Witt, E., Svetec, N., Benjamin, S. and Zhao, L. (2021). Transcription factors drive opposite relationships between gene age and tissue specificity in male and female Drosophila gonads. Mol Biol Evol. PubMed ID: 33481021
Summary:
Evolutionarily young genes are usually preferentially expressed in the testis across species. While it is known that older genes are generally more broadly expressed than younger genes, the properties that shaped this pattern are unknown. Older genes may gain expression across other tissues uniformly, or faster in certain tissues than others. Using Drosophila gene expression data, this study confirmed previous findings that younger genes are disproportionately testis-biased and older genes are disproportionately ovary-biased. The relationship between gene age and expression is stronger in the ovary than any other tissue, and weakest in testis. This study performed ATAC-seq on Drosophila testis and found that while genes of all ages are more likely to have open promoter chromatin in testis than in ovary, promoter chromatin alone does not explain the ovary-bias of older genes. Instead, it was found that upstream transcription factor (TF) expression is highly predictive of gene expression in ovary, but not in testis. In ovary, TF expression is more predictive of gene expression than open promoter chromatin, whereas testis gene expression is similarly influenced by both TF expression and open promoter chromatin. It is proposed that the testis is uniquely able to expresses younger genes controlled by relatively few TFs, while older genes with more TF partners are broadly expressed with peak expression most likely in ovary. The testis allows widespread baseline expression that is relatively unresponsive to regulatory changes, whereas the ovary transcriptome is more responsive to trans-regulation and has a higher ceiling for gene expression.
Coates, J. A., Brooks, E., Brittle, A. L., Armitage, E. L., Zeidler, M. P. and Evans, I. R. (2021). Identification of functionally distinct macrophage subpopulations in Drosophila. Elife 10. PubMed ID: 33885361
Summary:
Vertebrate macrophages are a highly heterogeneous cell population, but while Drosophila blood is dominated by a macrophage-like lineage (plasmatocytes), until very recently these cells were considered to represent a homogeneous population. This study presents an identification of enhancer elements labelling plasmatocyte subpopulations, which vary in abundance across development. These subpopulations exhibit functional differences compared to the overall population, including more potent injury responses and differential localisation and dynamics in pupae and adults. This enhancer analysis identified candidate genes regulating plasmatocyte behaviour: pan-plasmatocyte expression of one such gene (Calnexin14D) improves wound responses, causing the overall population to resemble more closely the subpopulation marked by the Calnexin14D-associated enhancer. Finally, this study shows that exposure to increased levels of apoptotic cell death modulates subpopulation cell numbers. Taken together this demonstrates macrophage heterogeneity in Drosophila, identifies mechanisms involved in subpopulation specification and function and facilitates the use of Drosophila to study macrophage heterogeneity in vivo.
Fukaya, T. (2021). Dynamic regulation of anterior-posterior patterning genes in living Drosophila embryos. Curr Biol. PubMed ID: 33761316
Summary:
Expression of the gap and pair-rule genes plays an essential role in body segmentation during Drosophila embryogenesis. However, it remains unclear how precise expression patterns of these key developmental genes arise from stochastic transcriptional activation at the single-cell level. This study employed genome-editing and live-imaging approaches to comprehensively visualize regulation of the gap and pair-rule genes at the endogenous loci. Quantitative image analysis revealed that the total duration of active transcription (transcription period) is a major determinant of spatial patterning of gene expression in early embryos. The length of the transcription period is determined by the continuity of bursting activities in individual nuclei, with the core expression domain producing more bursts than boundary regions. Each gene exhibits a distinct rate of nascent RNA production during transcriptional bursting, which contributes to gene-to-gene variability in the total output. Evidence is provided for "enhancer interference," wherein a distal weak enhancer interferes with transcriptional activation by a strong proximal enhancer to downregulate the length of the transcription period without changing the transcription rate. Analysis of the endogenous hunchback (hb) locus revealed that the removal of the distal shadow enhancer induces strong ectopic transcriptional activation, which suppresses refinement of the initial broad expression domain into narrower stripe patterns at the anterior part of embryos. This study provides key insights into the link between transcriptional bursting, enhancer-promoter interaction, and spatiotemporal patterning of gene expression during animal development.
Cofer, E. M., Raimundo, J., Tadych, A., Yamazaki, Y., Wong, A. K., Theesfeld, C. L., Levine, M. S. and Troyanskaya, O. G. (2021). Modeling transcriptional regulation of model species with deep learning. Genome Res. PubMed ID: 33888512
Summary:
DeepArk was developed to enable large-scale analyses of regulatory logic in model species. The program consists of a set of deep learning models of the cis-regulatory codes of four widely-studied species: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, and Mus musculus. DeepArk accurately predicts the presence of thousands of different context-specific regulatory features, including chromatin states, histone marks, and transcription factors. In vivo studies show that DeepArk can predict the regulatory impact of any genomic variant (including rare or not previously observed), and enables the regulatory annotation of understudied model species.
Espinola, S. M., Gotz, M., Bellec, M., Messina, O., Fiche, J. B., Houbron, C., Dejean, M., Reim, I., Cardozo Gizzi, A. M., Lagha, M. and Nollmann, M. (2021). Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development. Nat Genet 53(4): 477-486. PubMed ID: 33795867
Summary:
This study employed Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, it was shown that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. This approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.
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