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Current papers in developmental biology and gene function


Wednesday, November 23rd - Adult Physiology

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Yan, X., Chen, X., Fu, C., Jing, C., Zhao, D. and Sun, L. (2022). Ginseng oligosaccharides protect neurons from glutamate-induced oxidative damage through the Nrf2/HO-1 signaling pathway. Food Funct 13(16): 8605-8615. PubMed ID: 35894549
The effects of ginseng oligosaccharides (GSOs) on neuronal oxidative injury induced by glutamate (GLU) and the molecular mechanisms involved were investigated. Cell damage was assessed using MTT assays, and the lactate dehydrogenase (LDH) release rate and flow cytometry were used to detect the accumulation of reactive oxygen species (ROS) and mitochondrial membrane potential respectively. The levels of catalase (CAT) and glutathione (GSH) were measured in PC12 cells and Drosophila brain tissue. The climbing ability of Drosophila was observed. Levels of proteins, including Cyt C, Bcl-2/BAX, and Nrf2/HO-1-associated proteins, were determined by western blotting and immunofluorescence. It was found that GSOs reversed GLU-induced reductions in cell viability and the LDH release rate, and rescued ROS accumulation. GSOs also mitigated the deleterious effects of GLU on the mitochondrial membrane potential and Cyt C release, thus alleviating mitochondrial dysfunction, and increased GSH levels and CAT activity in both cells and Drosophila brain tissue. The climbing index in GSO-treated Drosophila was significantly higher than that in the tert-butyl-hydroperoxide-treated flies. Furthermore, GSOs protected cells against GLU-induced apoptosis by reducing the expression of the mitochondrial apoptosis-associated Bcl-2 family effector proteins and protected cells from GLU-induced oxidative damage by increasing the nuclear translocation of Nrf2 and HO-1 expression. These findings indicate that GSOs protect against GLU-induced neuronal oxidative damage through Nrf2/HO-1 activation.
Catalani, E., Zecchini, S., Giovarelli, M., Cherubini, A., Del Quondam, S., Brunetti, K., Silvestri, F., Roux-Biejat, P., Napoli, A., Casati, S. R., Ceci, M., Romano, N., Bongiorni, S., Prantera, G., Clementi, E., Perrotta, C., De Palma, C. and Cervia, D. (2022). RACK1 is evolutionary conserved in satellite stem cell activation and adult skeletal muscle regeneration. Cell Death Discov 8(1): 459. PubMed ID: 36396939
Skeletal muscle growth and regeneration involves the activity of resident adult stem cells, namely satellite cells (SC). This study demonstrated that the Receptor for Activated C-Kinase 1 (RACK1) is important in SC function. RACK1 was expressed transiently in the skeletal muscle of post-natal mice, being abundant in the early phase of muscle growth and almost disappearing in adult mature fibers. The presence of RACK1 in interstitial SC was also detected. After acute injury in muscle of both mouse and the fruit fly Drosophila melanogaster (used as alternative in vivo model) this study found that RACK1 accumulated in regenerating fibers while it declined with the progression of repair process. To note, RACK1 also localized in the active SC that populate recovering tissue. The dynamics of RACK1 levels in isolated adult SC of mice, i.e., progressively high during differentiation and low compared to proliferating conditions, and RACK1 silencing indicated that RACK1 promotes both the formation of myotubes and the accretion of nascent myotubes. In Drosophila with depleted RACK1 in all muscle cells or, specifically, in SC lineage resulted in a delayed recovery of skeletal muscle after physical damage as well as the low presence of active SC in the wound area. These results also suggest the coupling of RACK1 to muscle unfolded protein response during SC activation. Collectively, this study provides the first evidence that transient levels of the evolutionarily conserved factor RACK1 are critical for adult SC activation and proper skeletal muscle regeneration, favoring the efficient progression of SC from a committed to a fully differentiated state.
Xu, M., Ren, P., Tian, J., Xiao, L., Hu, P., Chen, P., Li, W. and Xue, L. (2022). dGLYAT modulates Gadd45-mediated JNK activation and cell invasion. Cell Div 17(1): 4. PubMed ID: 35933447
Cell invasion is a crucial step of tumor metastasis, finding new regulators of which offers potential drug targets for cancer therapy. Aberrant GLYAT expression is associated with human cancers, yet its role in cancer remains unknown. This study aims to understand the function and mechanism of Drosophila Glycine N-acyltransferase (GLYAT) in cell invasion. dGLYAT was found to regulate Gadd45-mediated JNK pathway activation and cell invasion. Firstly, loss of dGLYAT suppressed scrib depletion- or Egr overexpression-induced JNK pathway activation and invasive cell migration. Secondary, mRNA-seq analysis identified Gadd45 as a potential transcriptional target of dGLYAT, as depletion of dGLYAT decreased Gadd45 mRNA level. Finally, Gadd45 knockdown suppressed scrib depletion-induced JNK pathway activation and cell invasion. These evidences reveal the role of dGLYAT and Gadd45 in JNK-dependent cell invasion, and provide insight for the roles of their human homologs in cancers.
Rauskolb, C., Han, A., Kirichenko, E., Ibar, C. and Irvine, K. D. (2022). Analysis of the Drosophila Ajuba LIM protein defines functions for distinct LIM domains. PLoS One 17(8): e0269208. PubMed ID: 35969522
The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, this study investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.
Banreti, A., Bhattacharya, S., Wien, F., Matsuo, K., Refregiers, M., Meinert, C., Meierhenrich, U., Hudry, B., Thompson, D. and Noselli, S. (2022). Biological effects of the loss of homochirality in a multicellular organism. Nat Commun 13(1): 7059. PubMed ID: 36400783
Homochirality is a fundamental feature of all known forms of life, maintaining biomolecules (amino-acids, proteins, sugars, nucleic acids) in one specific chiral form. While this condition is central to biology, the mechanisms by which the adverse accumulation of non-L-α-amino-acids in proteins lead to pathophysiological consequences remain poorly understood. To address how heterochirality build-up impacts organism's health, this study used chiral-selective in vivo assays to detect protein-bound non-L-α-amino acids (focusing on aspartate) and assess their functional significance in Drosophila. Altering the in vivo chiral balance creates a 'heterochirality syndrome' with impaired caspase activity, increased tumour formation, and premature death. This work shows that preservation of homochirality is a key component of protein function that is essential to maintain homeostasis across the cell, tissue and organ level.
Kowalczyk, W., Romanelli, L., Atkins, M., Hillen, H., Bravo Gonzalez-Blas, C., Jacobs, J., Xie, J., Soheily, S., Verboven, E., Moya, I. M., Verhulst, S., de Waegeneer, M., Sansores-Garcia, L., van Huffel, L., Johnson, R. L., van Grunsven, L. A., Aerts, S. and Halder, G. (2022). Hippo signaling instructs ectopic but not normal organ growth. Science 378(6621): eabg3679. PubMed ID: 36395225
The Hippo signaling pathway is widely considered a master regulator of organ growth because of the prominent overgrowth phenotypes caused by experimental manipulation of its activity. Contrary to this model, this study shows that removing Hippo transcriptional output did not impair the ability of the mouse liver and Drosophila eyes to grow to their normal size. Moreover, the transcriptional activity of the Hippo pathway effectors Yap/Taz/Yki did not correlate with cell proliferation, and hyperactivation of these effectors induced gene expression programs that did not recapitulate normal development. Concordantly, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not normal growth. Thus, Hippo signaling does not instruct normal growth, and the Hippo-induced overgrowth phenotypes are caused by the activation of abnormal genetic programs.

Tuesday, November 22nd - Larval and Adult Physiology and Metabolism

Shin, S. W., Jeon, J. H., Kim, J. A., Park, D. S., Shin, Y. J. and Oh, H. W. (2022). Inducible Expression of Several Drosophila melanogaster Genes Encoding Juvenile Hormone Binding Proteins by a Plant Diterpene Secondary Metabolite, Methyl Lucidone. Insects 13(5). PubMed ID: 35621756
Juvenile hormones prevent molting and metamorphosis in the juvenile stages of insects. There are multiple genes encoding a conserved juvenile hormone binding protein (JHBP) domain in a single insect species. Although some JHBPs have been reported to serve as carriers to release hormones to target tissues, the molecular functions of the other members of the diverse JHBP family of proteins remain unclear. This study characterized 16 JHBP genes with conserved JHBP domains in Drosophila melanogaster. Among them, seven JHBP genes were induced by feeding the flies with methyl lucidone, a plant diterpene secondary metabolite (PDSM). Induction was also observed upon feeding the juvenile hormone (JH) analog methoprene. Considering that methyl lucidone and methoprene perform opposite functions in JH-mediated regulation, specifically the heterodimeric binding between a JH receptor (JHR) and steroid receptor coactivator (SRC), the induction of these seven JHBP genes is independent of JH-mediated regulation by the JHR/SRC heterodimer. Tissue-specific gene expression profiling through the FlyAtlas 2 database indicated that some JHBP genes are mainly enriched in insect guts and rectal pads, indicating their possible role during food uptake. Hence, it is proposed that JHBPs are induced by PDSMs and respond to toxic plant molecules ingested during feeding.
Dong, W., Zhang, X., Kong, Y., Zhao, Z., Mahmoud, A., Wu, L., Moussian, B. and Zhang, J. (2022). CYP311A1 in the anterior midgut is involved in lipid distribution and microvillus integrity in Drosophila melanogaster. Cell Mol Life Sci 79(5): 261. PubMed ID: 35478270
Lipids are either taken up from food sources or produced internally in specialized tissues such as the liver. Among others, both routes of lipid metabolism involve cytochrome P450 monooxygenases (CYPs). This study sought to analyze the function of Cyp311a1 that has been shown to be expressed in the midgut of the fruit fly Drosophila melanogaster. Using a GFP-tagged version of CYP311A1 that is expressed under the control of its endogenous promoter, it was shown that Cyp311a1 localizes to the endoplasmic reticulum in epithelial cells of the anterior midgut. In larvae with reduced Cyp311a1 expression in the anterior midgut, compared to control larvae, the apical plasma membrane of the respective epithelial cells contains less and shorter microvilli. In addition, reduction of neutral lipids was observed in the fat body, the insect liver, and decreased phosphatidylethanolamine (PE) and triacylglycerols (TAG) amounts in the whole body of these larvae. Probably as a consequence, they cease to grow and eventually die. The microvillus defects in larvae with reduced Cyp311a1 expression are restored by supplying PE, a major phospholipid of plasma membranes, to the food. Moreover, the growth arrest phenotype of these larvae is partially rescued. Together, these results suggest that the anterior midgut is an import hub in lipid distribution and that the midgut-specific CYP311A1 contributes to this function by participating in shaping microvilli in a PE-dependent manner.
Gautrey, S. L. and Simons, M. J. P. (2022). Amino acid availability is not essential for lifespan extension by dietary restriction in the fly. J Gerontol A Biol Sci Med Sci. PubMed ID: 35486979
Dietary restriction (DR) is one of the most potent ways to extend health- and lifespan. Key progress in understanding the mechanisms of DR, and ageing more generally, was made when dietary protein, and more specifically essential amino acids (EAA), were identified as the dietary component to restrict to obtain DR's health and lifespan benefits. This role of dietary amino acids has influenced work on ageing mechanisms, especially in nutrient sensing, e.g. Tor and insulin(-like) signalling networks. Experimental biology in Drosophila melanogaster has been instrumental in generating and confirming the hypothesis that EAA availability is important in ageing. This study expanded on previous work testing the involvement of EAA in DR through large scale (N=6,238) supplementation experiments across four diets and two genotypes in female flies. Surprisingly, it was found that EAA are not essential to DR's lifespan benefits. Importantly, the fecundity benefits of EAA supplementation were not identified suggesting the supplemented EAA were bioavailable. Furthermore, it was found that the effects of amino acids on lifespan vary by diet and genetic line studied and that, at the most restricted diet, fecundity is constrained by other nutrients than EAA. It is suggested that DR for optimal health is a concert of nutritional effects, orchestrated by genetic, dietary and other environmental interactions. These results question the universal importance of amino acid availability in the biology of ageing and DR.
Ghosh, S., Leng, W., Wilsch-Brauninger, M., Barrera-Velazquez, M., Leopold, P. and Eaton, S. (2022). A local insulin reservoir in Drosophila alpha cell homologs ensures developmental progression under nutrient shortage. Curr Biol 32(8): 1788-1797. PubMed ID: 35316653
Insulin/insulin-like growth factor (IGF) signaling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides called Dilps is produced by brain neurosecretory cells, and it regulates organismal growth and developmental timing. To accomplish these systemic functions, the Dilps are secreted into the general circulation, and they signal to peripheral tissues in an endocrine fashion. This study describes the local uptake and storage of Dilps in the corpora cardiaca (CC), an endocrine organ composed of alpha cell homologs known to produce the glucagon-like adipokinetic hormone (AKH). Dilp uptake by the CC relies on the expression of an IGF-binding protein called ImpL2. Following their uptake, immunogold staining demonstrates that Dilps are co-packaged with AKH in dense-core vesicles for secretion. In response to nutrient shortage, this specific Dilp reservoir is released and activates IIS in a paracrine manner in the prothoracic gland. This stimulates the production of the steroid hormone ecdysone and initiates entry into pupal development. This study therefore uncover a sparing mechanism whereby insulin stores in CC serve to locally activate IIS and the production of ecdysone in the PG, accelerating developmental progression in adverse food conditions.
Huang, Y., Wan, Z., Tang, Y., Xu, J., Laboret, B., Nallamothu, S., Yang, C., Liu, B., Lu, R. O., Lu, B., Feng, J., Cao, J., Hayflick, S., Wu, Z. and Zhou, B. (2022). Pantothenate kinase 2 interacts with PINK1 to regulate mitochondrial quality control via acetyl-CoA metabolism. Nat Commun 13(1): 2412. PubMed ID: 35504872
Human neurodegenerative disorders often exhibit similar pathologies, suggesting a shared aetiology. Key pathological features of Parkinson's disease (PD) are also observed in other neurodegenerative diseases. Pantothenate Kinase-Associated Neurodegeneration (PKAN) is caused by mutations in the human PANK2 gene, which catalyzes the initial step of de novo CoA synthesis. This study shows that fumble (fbl), the human PANK2 homolog in Drosophila, interacts with PINK1 genetically. fbl and PINK1 mutants display similar mitochondrial abnormalities, and overexpression of mitochondrial Fbl rescues PINK1 loss-of-function (LOF) defects. Dietary vitamin B5 derivatives effectively rescue CoA/acetyl-CoA levels and mitochondrial function, reversing the PINK1 deficiency phenotype. Mechanistically, Fbl regulates Ref(2)P (p62/SQSTM1 homolog) by acetylation to promote mitophagy, whereas PINK1 regulates fbl translation by anchoring mRNA molecules to the outer mitochondrial membrane. In conclusion, Fbl (or PANK2) acts downstream of PINK1, regulating CoA/acetyl-CoA metabolism to promote mitophagy, uncovering a potential therapeutic intervention strategy in PD treatment.
Ader, F., Russi, M., Tixier-Cardoso, L., Jullian, E., Martin, E., Richard, P., Villard, E. and Monnier, V. (2022). Drosophila CRISPR/Cas9 mutants as tools to analyse cardiac filamin function and pathogenicity of human FLNC variants. Biol Open 11(9). PubMed ID: 36066120
Filamins are large proteins with actin binding properties. Mutations in FLNC, one of the three filamin genes in humans, have recently been implicated in dominant cardiomyopathies, but the underlying mechanisms are not well understood. This study aimed to use Drosophila melanogaster as a new in vivo model to study these diseases. First, it was shown that adult-specific cardiac RNAi-induced depletion of Drosophila Filamin (dFil) induced cardiac dilatation, impaired systolic function and sarcomeric alterations, highlighting its requirement for cardiac function and maintenance of sarcomere integrity in the adult stage. Next, the cheerio gene was introduced using CRISPR/Cas9 gene editing. Three missense variants were introduced, previously identified in patients with hypertrophic cardiomyopathy. Flies carrying these variants did not exhibit cardiac defects or increased propensity to form filamin aggregates, arguing against their pathogenicity. Finally, it was shown that deletions of the C-term part of dFil carrying the last four Ig-like domains are dispensable for cardiac function. Collectively, these results highlight the relevance of this model to explore the cardiac function of filamins and increase understanding of physio-pathological mechanisms involved in FLNC-related cardiomyopathies.

Thursday, September 19th - Disease Models

Palacios-Munoz, A., de Paula Moreira, D., Silva, V., Garcia, I. E., Aboitiz, F., Zarrei, M., Campos, G., Rennie, O., Howe, J. L., Anagnostou, E., Ambrozewic, P., Scherer, S. W., Passos-Bueno, M. R. and Ewer, J. (2022). Mutations in trpgamma, the homologue of TRPC6 autism candidate gene, causes autism-like behavioral deficits in Drosophila. Mol Psychiatry. PubMed ID: 35501408
Autism Spectrum Disorder (ASD) is characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. The TRPC6 (transient receptor potential channel 6) represents an ASD candidate gene under an oligogenic/multifactorial model based on the initial description and cellular characterization of an individual with ASD bearing a de novo heterozygous mutation disrupting TRPC6, together with the enrichment of disruptive TRPC6 variants in ASD cases as compared to controls. This study performed a clinical re-evaluation of the initial non-verbal patient, and also present eight newly reported individuals ascertained for ASD and bearing predicted loss-of-function mutations in TRPC6. In order to understand the consequences of mutations in TRPC6 on nervous system function, the fruit fly, Drosophila melanogaster, was used to show that null mutations in transient receptor gamma (trpγ the fly gene most similar to TRPC6), cause a number of behavioral defects that mirror features seen in ASD patients, including deficits in social interactions (based on courtship behavior), impaired sleep homeostasis (without affecting the circadian control of sleep), hyperactivity in both young and old flies, and defects in learning and memory. Some defects, most notably in sleep, differed in severity between males and females and became normal with age. Interestingly, hyperforin, a TRPC6 agonist and the primary active component of the St. John's wort antidepressant, attenuated many of the deficits expressed by trpγ mutant flies. In summary, these results provide further evidence that the TRPC6 gene is a risk factor for ASD. In addition, they show that the behavioral defects caused by mutations in TRPC6 can be modeled in Drosophila, thereby establishing a paradigm to examine the impact of mutations in other candidate genes.
Schulz, L., Ramirez, P., Lemieux, A., Gonzalez, E., Thomson, T. and Frost, B. (2022). Tau-Induced Elevation of the Activity-Regulated Cytoskeleton Associated Protein Arc1 Causally Mediates Neurodegeneration in the Adult Drosophila Brain. Neuroscience. PubMed ID: 35487302
Alzheimer's disease and other tauopathies are neurodegenerative disorders pathologically defined by aggregated forms of tau protein in the brain. While synaptic degradation is a well-established feature of tau-induced neurotoxicity, the underlying mechanisms of how pathogenic forms of tau drive synaptic dysfunction are incompletely understood. Synaptic function and subsequent memory consolidation are dependent upon synaptic plasticity, the ability of synapses to adjust their structure and strength in response to changes in activity. The activity regulated cytoskeleton associated protein ARC acts in the nucleus and at postsynaptic densities to regulate various forms of synaptic plasticity. ARC harbors a retrovirus-like Gag domain that facilitates ARC multimerization and capsid formation. Trans-synaptic transfer of RNA-containing ARC capsids is required for synaptic plasticity. While ARC is elevated in brains of patients with Alzheimer's disease and genetic variants in ARC increase susceptibility to Alzheimer's disease, mechanistic insight into the role of ARC in Alzheimer's disease is lacking. Using a Drosophila model of tauopathy, this study found that pathogenic tau significantly increases multimeric species of the protein encoded by the Drosophila homolog of ARC, Arc1, in the adult fly brain. Arc1 is elevated within nuclei and the neuropil of tau transgenic Drosophila, but does not localize to synaptic vesicles or presynaptic terminals. Lastly, this study found that genetic manipulation of Arc1 modifies tau-induced neurotoxicity, suggesting that tau-induced Arc1 elevation mediates neurodegeneration. Taken together, these results suggest that ARC elevation in human Alzheimer's disease is a consequence of tau pathology and is a causal factor contributing to neuronal death.
Lee, Y., Kim, J., Kim, H., Han, J. E., Kim, S., Kang, K. H., Kim, D., Kim, J. M. and Koh, H. (2022). Pyruvate Dehydrogenase Kinase Protects Dopaminergic Neurons from Oxidative Stress in Drosophila DJ-1 Null Mutants. Mol Cells. PubMed ID: 35444068
DJ-1 is one of the causative genes of early-onset familial Parkinson's disease (PD). As a result, DJ-1 influences the pathogenesis of sporadic PD. DJ-1 has various physiological functions that converge to control the levels of intracellular reactive oxygen species (ROS). Based on genetic analyses that sought to investigate novel antioxidant DJ-1 downstream genes, pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to increase survival rates and decrease dopaminergic (DA) neuron loss in DJ-1 mutant flies under oxidative stress. PDK phosphorylates and inhibits the PDH complex (PDC), subsequently downregulating glucose metabolism in the mitochondria, which is a major source of intracellular ROS. A loss-of-function mutation in PDK was not found to have a significant effect on fly development and reproduction, but severely ameliorated oxidative stress resistance. Thus, PDK plays a critical role in the protection against oxidative stress. Loss of PDH phosphatase (PDP), which dephosphorylates and activates PDH, was also shown to protect DJ-1 mutants from oxidative stress, ultimately supporting these findings. Further genetic analyses suggested that DJ-1 controls PDK expression through hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator of the adaptive response to hypoxia and oxidative stress. Furthermore, CPI-613, an inhibitor of PDH, protected DJ-1 null flies from oxidative stress, suggesting that the genetic and pharmacological inhibition of PDH may be a novel treatment strategy for PD associated with DJ-1 dysfunction.
Piper, M. D. W., Zanco, B., Sgro, C. M., Adler, M. I., Mirth, C. K. and Bonduriansky, R. (2022). Dietary restriction and lifespan: adaptive reallocation or somatic sacrifice?. Febs j. PubMed ID: 35466532
Reducing overall food intake, or lowering the proportion of protein relative to other macronutrients, can extend the lifespan of diverse organisms. A number of mechanistic theories have been developed to explain this phenomenon, mostly assuming that the molecules connecting diet to lifespan are evolutionarily conserved. A recent study using Drosophila melanogaster females has pinpointed a single essential micronutrient that can explain how lifespan is changed by dietary restriction. This study proposes a likely mechanism for this observation, which involves a trade-off between lifespan and reproduction, but in a manner that is conditional on the dietary supply of an essential micronutrient - a sterol. Importantly, these observations argue against previous evolutionary theories that rely on constitutive resource reallocation or damage directly inflicted by reproduction. Instead, they are compatible with a model in which the inverse relationship between lifespan and food level is caused by the consumer suffering from varying degrees of malnutrition when maintained on lab food. The data also indicate that animals on different lab foods may suffer from different nutritional imbalances and that the mechanisms by which dietary restriction benefits the lifespan of different species may vary. This means that translating the mechanistic findings from lab animals to humans will not be simple and should be interpreted in light of the range of challenges that have shaped each organism's lifespan in the wild and the composition of the natural diets upon which they would feed.
Rajan, S., Toh, H. T., Ye, H., Wang, Z., Basil, A. H., Parnaik, T., Yoo, J. Y., Lim, K. L. and Yoon, H. S. (2022). Prostaglandin A2 Interacts with Nurr1 and Ameliorates Behavioral Deficits in Parkinson's Disease Fly Model. Neuromolecular Med. PubMed ID: 35482177
The orphan nuclear receptor Nurr1 (Drosophila ortholog: Hr38) is critical for the development, maintenance, and protection of midbrain dopaminergic neurons. Recently, it was demonstrated that prostaglandins E1 (PGE1) and PGA1 directly bind to the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional activation function. In this direction, this study reports the transcriptional activation of Nurr1 by PGA2, a dehydrated metabolite of PGE2, through physical binding ably supported by NMR titration and crystal structure. The co-crystal structure of Nurr1-LBD bound to PGA2 revealed the covalent coupling of PGA2 with Nurr1-LBD through Cys566. PGA2 binding also induces a 21° shift of the activation function 2 (AF-2) helix H12 away from the protein core, similar to that observed in the Nurr1-LBD-PGA1 complex. This study also showed that PGA2 can rescue the locomotor deficits and neuronal degeneration in LRRK2 G2019S transgenic fly models.
Mayneris-Perxachs, J., Castells-Nobau, A., ..., Maldonado, R. and Fernandez-Real, J. M. (2022). Microbiota alterations in proline metabolism impact depression. Cell Metab 34(5): 681-701.e610. PubMed ID: 35508109
The microbiota-gut-brain axis has emerged as a novel target in depression, a disorder with low treatment efficacy. This study applied a multi-omics approach combining pre-clinical models with three human cohorts including patients with mild depression. Microbial functions and metabolites converging onto glutamate/GABA metabolism, particularly proline, were linked to depression. High proline consumption was the dietary factor with the strongest impact on depression. Whole-brain dynamics revealed rich club network disruptions associated with depression and circulating proline. Proline supplementation in mice exacerbated depression along with microbial translocation. Human microbiota transplantation induced an emotionally impaired phenotype in mice and alterations in GABA-, proline-, and extracellular matrix-related prefrontal cortex genes. RNAi-mediated knockdown of proline and GABA transporters in Drosophila and mono-association with L. plantarum, a high GABA producer, conferred protection against depression-like states. Targeting the microbiome and dietary proline may open new windows for efficient depression treatment.

Wednesday, September 28th - Adult Neural Development and function

Liang, X., Holy, T. E. and Taghert, P. H. (2022). Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations. Proc Natl Acad Sci U S A 119(17): e2109969119. PubMed ID: 35446620
Circadian pacemaker neurons in the Drosophila brain display daily rhythms in the levels of intracellular calcium. These calcium rhythms are driven by molecular clocks and are required for normal circadian behavior. To study their biological basis, this study employed genetic manipulations in conjunction with improved methods of in vivo light-sheet microscopy to measure calcium dynamics in individual pacemaker neurons over complete 24-h durations at sampling frequencies as high as 5 Hz. This technological advance unexpectedly revealed cophasic daily rhythms in basal calcium levels and in high-frequency calcium fluctuations. Further, the rhythms of basal calcium levels and of fast calcium fluctuations were found to reflect the activities of two proteins that mediate distinct forms of calcium fluxes. One is the inositol trisphosphate receptor (ITPR), a channel that mediates calcium fluxes from internal endoplasmic reticulum calcium stores, and the other is a T-type voltage-gated calcium channel, which mediates extracellular calcium influx. These results suggest that Drosophila molecular clocks regulate ITPR and T-type channels to generate two distinct but coupled rhythms in basal calcium and in fast calcium fluctuations. It is proposed that both internal and external calcium fluxes are essential for circadian pacemaker neurons to provide rhythmic outputs and thereby, regulate the activities of downstream brain centers.
Castaneda-Sampedro, A., Calvin-Cejudo, L., Martin, F., Gomez-Diaz, C. and Alcorta, E. (2022). The Ntan1 gene is expressed in perineural glia and neurons of adult Drosophila. Sci Rep 12(1): 14749. PubMed ID: 36042338
The Drosophila Ntan1 gene encodes an N-terminal asparagine amidohydrolase that is highly conserved throughout evolution. Protein isoforms share more than 72% of similarity with their human counterparts. At the cellular level, this gene regulates the type of glial cell growth in Drosophila larvae by its different expression levels. The Drosophila Ntan1 gene has 4 transcripts that encode 2 protein isoforms. This study describes that although this gene is expressed at all developmental stages and adult organs tested (eye, antennae and brain) there are some transcript-dependent specificities. Therefore, both quantitative and qualitative cues could account for gene function. However, widespread developmental stage and organ-dependent expression could be masking cell-specific constraints that can be explored in Drosophila by using Gal4 drivers. A new genetic driver within this gene, Mz317-Gal4, is reported that recapitulates the Ntan1 gene expression pattern in adults. It shows specific expression for perineural glia in the olfactory organs but mixed expression with some neurons in the adult brain. Memory and social behavior disturbances in mice and cancer and schizophrenia in humans have been linked to the Ntan1 gene. Therefore, these new tools in Drosophila may contribute to greater understanding of the cellular basis of these alterations.
Dweck, H. K. M., Talross, G. J. S., Luo, Y., Ebrahim, S. A. M. and Carlson, J. R. (2022). Ir56b is an atypical ionotropic receptor that underlies appetitive salt response in Drosophila. Curr Biol 32(8): 1776-1787. PubMed ID: 35294865
Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of salt taste remains unclear in invertebrates. This study shows that the response to low, appetitive salt concentrations in Drosophila depends on Ir56b, an atypical member of the ionotropic receptor (Ir) family. Ir56b acts in concert with two coreceptors, Ir25a and Ir76b. Mutation of Ir56b virtually eliminates an appetitive behavioral response to salt. Ir56b is expressed in neurons that also sense sugars via members of the Gr (gustatory receptor) family. Misexpression of Ir56b in bitter-sensing neurons confers physiological responses to appetitive doses of salt. Ir56b is unique among tuning Irs in containing virtually no N-terminal region, a feature that is evolutionarily conserved. Moreover, Ir56b is a "pseudo-pseudogene": its coding sequence contains a premature stop codon that can be replaced with a sense codon without loss of function. This stop codon is conserved among many Drosophila species but is absent in a number of species associated with cactus in arid regions. Thus, Ir56b serves the evolutionarily ancient function of salt detection in neurons that underlie both salt and sweet taste modalities.
Fenckova, M., Muha, V., Mariappa, D., Catinozzi, M., Czajewski, I., Blok, L. E. R., Ferenbach, A. T., Storkebaum, E., Schenck, A. and van Aalten, D. M. F. (2022). Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila. PLoS Genet 18(5): e1010159. PubMed ID: 35500025
O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. This study manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrated an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG.
Fujiwara, T., Brotas, M. and Chiappe, M. E. (2022). Walking strides direct rapid and flexible recruitment of visual circuits for course control in Drosophila. Neuron. PubMed ID: 35525243
Flexible mapping between activity in sensory systems and movement parameters is a hallmark of motor control. This flexibility depends on the continuous comparison of short-term postural dynamics and the longer-term goals of an animal, thereby necessitating neural mechanisms that can operate across multiple timescales. To understand how such body-brain interactions emerge across timescales to control movement, whole-cell patch recordings were performed from visual neurons involved in course control in Drosophila. The activity of leg mechanosensory cells, propagating via specific ascending neurons, is critical for stride-by-stride steering adjustments driven by the visual circuit, and, at longer timescales, it provides information about the moving body's state to flexibly recruit the visual circuit for course control. Thus, these findings demonstrate the presence of an elegant stride-based mechanism operating at multiple timescales for context-dependent course control. It is proposed that this mechanism functions as a general basis for the adaptive control of locomotion.
Wang, Y., Lobb-Rabe, M., Ashley, J., Chatterjee, P., Anand, V., Bellen, H. J., Kanca, O. and Carrillo, R. A. (2022). Systematic expression profiling of Dpr and DIP genes reveals cell surface codes in Drosophila larval motor and sensory neurons. Development 149(10). PubMed ID: 35502740
In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell-surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. This study investigated expression of Dpr and DIP genes, two CSP subfamilies belonging to the immunoglobulin superfamily, in Drosophila larval motor neurons (MNs), muscles, glia and sensory neurons (SNs) using a collection of GAL4 driver lines. Dpr genes were found to be more broadly expressed than DIP genes in MNs and SNs, and each examined neuron expresses a unique combination of Dpr and DIP genes. Interestingly, many Dpr and DIP genes are not robustly expressed, but are found instead in gradient and temporal expression patterns. In addition, the unique expression patterns of Dpr and DIP genes revealed three uncharacterized MNs. This study sets the stage for exploring the functions of Dpr and DIP genes in Drosophila MNs and SNs and provides genetic access to subsets of neurons.

Tuesday, September 27th - Cytoskeleton and Junctions

Li, H. and Gavis, E. R. (2022). The Drosophila fragile X mental retardation protein modulates the neuronal cytoskeleton to limit dendritic arborization. Development 149(10). PubMed ID: 35502752
Dendritic arbor development is a complex, highly regulated process. Post-transcriptional regulation mediated by RNA-binding proteins plays an important role in neuronal dendrite morphogenesis by delivering on-site, on-demand protein synthesis. This study shows how the Drosophila Fragile X mental retardation protein (FMRP), a conserved RNA-binding protein, limits dendrite branching to ensure proper neuronal function during larval sensory neuron development. FMRP knockdown causes increased dendritic terminal branch growth and a resulting overelaboration defect due, in part, to altered microtubule stability and dynamics. FMRP also controls dendrite outgrowth by regulating the Drosophila profilin homolog Chickadee (Chic). FMRP colocalizes with Chic mRNA in dendritic granules and regulates its dendritic localization and protein expression. Whereas RNA-binding domains KH1 and KH2 are both crucial for FMRP-mediated dendritic regulation, KH2 specifically is required for FMRP granule formation and Chic mRNA association, suggesting a link between dendritic FMRP granules and FMRP function in dendrite elaboration. These studies implicate FMRP-mediated modulation of both the neuronal microtubule and actin cytoskeletons in multidendritic neuronal architecture, and provide molecular insight into FMRP granule formation and its relevance to FMRP function in dendritic patterning.
Qu, Y., Alves-Silva, J., Gupta, K., Hahn, I., Parkin, J., Sanchez-Soriano, N. and Prokop, A. (2022). Re-evaluating the actin-dependence of spectraplakin functions during axon growth and maintenance. Dev Neurobiol 82(4): 288-307. PubMed ID: 35333003
Axons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The growth and maintenance of axons require loose microtubule bundles that extend through their entire length. Understanding microtubule regulation is therefore an essential aspect of axon biology. Key regulators of neuronal microtubules are the spectraplakins, a well-conserved family of cytoskeletal cross-linkers that underlie neuropathies in mouse and humans. Spectraplakin deficiency in mouse or Drosophila causes severe decay of microtubule bundles and reduced axon growth. The underlying mechanisms are best understood for Drosophila's spectraplakin Short stop (Shot) and believed to involve cytoskeletal cross-linkage: Shot's binding to microtubules and Eb1 via its C-terminus has been thoroughly investigated, whereas its F-actin interaction via N-terminal calponin homology (CH) domains is little understood. New understanding was gained in this study by showing that the F-actin interaction must be finely balanced: altering the properties of F-actin networks or deleting/exchanging Shot's CH domains induces changes in Shot function-with a Lifeact-containing Shot variant causing remarkable remodeling of neuronal microtubules. In addition to actin-microtubule (MT) cross-linkage, this study found strong indications that Shot executes redundant MT bundle-promoting roles that are F-actin-independent. It is argued that these likely involve the neuronal Shot-PH isoform, which is characterized by a large, unexplored central plakin repeat region (PRR) similarly existing also in mammalian spectraplakins.
Foldi, I., Toth, K., Gombos, R., Gaszler, P., Gorog, P., Zygouras, I., Bugyi, B. and Mihaly, J. (2022). Molecular Dissection of DAAM Function during Axon Growth in Drosophila Embryonic Neurons. Cells 11(9). PubMed ID: 35563792
Axonal growth is mediated by coordinated changes of the actin and microtubule (MT) cytoskeleton. Ample evidence suggests that members of the formin protein family are involved in the coordination of these cytoskeletal rearrangements, but the molecular mechanisms of the formin-dependent actin-microtubule crosstalk remains largely elusive. Of the six Drosophila formins, DAAM was shown to play a pivotal role during axonal growth in all stages of nervous system development, while FRL was implicated in axonal development in the adult brain. This study aimed to investigate the potentially redundant function of these two formins, and attempts were made to clarify which molecular activities are important for axonal growth. A combination of genetic analyses, cellular assays and biochemical approaches was used to demonstrate that the actin-processing activity of DAAM is indispensable for axonal growth in every developmental condition. In addition, a novel MT-binding motif was identified within the FH2 domain of DAAM, which is required for proper growth and guidance of the mushroom body axons, while being dispensable during embryonic axon development. Together, these data suggest that DAAM is the predominant formin during axonal growth in Drosophila, and highlight the contribution of multiple formin-mediated mechanisms in cytoskeleton coordination during axonal growth.
Lehne, F., Pokrant, T., Parbin, S., Salinas, G., Grosshans, J., Rust, K., Faix, J. and Bogdan, S. (2022). Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure. Nat Commun 13(1): 2492. PubMed ID: 35524157
Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. This study identified the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, this study showed that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, these data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.
Akhmanova, M., Emtenani, S., Krueger, D., Gyoergy, A., Guarda, M., Vlasov, M., Vlasov, F., Akopian, A., Ratheesh, A., De Renzis, S. and Siekhaus, D. E. (2022). Cell division in tissues enables macrophage infiltration. Science 376(6591): 394-396. PubMed ID: 35446632
Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. This study found that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.
Malin, J., Rosa Birriel, C., Astigarraga, S., Treisman, J. E. and Hatini, V. (2022). Sidekick dynamically rebalances contractile and protrusive forces to control tissue morphogenesis. J Cell Biol 221(5). PubMed ID: 35258563
Contractile actomyosin and protrusive branched F-actin networks interact in a dynamic balance, repeatedly contracting and expanding apical cell contacts to organize the epithelium of the developing fly retina. Previously it was shown that the immunoglobulin superfamily protein Sidekick (Sdk) contributes to contraction by recruiting the actin binding protein Polychaetoid (Pyd) to vertices. This study shows that as tension increases during contraction, Sdk progressively accumulates at vertices, where it toggles to recruit the WAVE regulatory complex (WRC) to promote actin branching and protrusion. Sdk alternately interacts with the WRC and Pyd using the same C-terminal motif. With increasing protrusion, levels of Sdk and the WRC decrease at vertices while levels of Pyd increase paving the way for another round of contraction. Thus, by virtue of dynamic association with vertices and interchangeable associations with contractile and protrusive effectors, Sdk is central to controlling the balance between contraction and expansion that shapes this epithelium.

Friday, September 24th - Transcriptional Regulation

Zhang, K., Ramos, A. F., Wang, E. and Wang, J. (2022). The rate of thermodynamic cost against adiabatic and nonadiabatic fluctuations of a single gene circuit in Drosophila embryos. J Chem Phys 156(22): 225101. PubMed ID: 35705404
Stochastic dynamics of the externally regulating gene circuit was studied as an example of an even-skipped gene stripe in the development of Drosophila. Three gene regulation regimes are considered: an adiabatic phase when the switching rate of the gene from the OFF to ON state is faster than the rate of mRNA degradation; a nonadiabatic phase when the switching rate from the OFF to ON state is slower than that of the mRNA degradation; and a bursting phase when the gene switching is fast and transcription is very fast, while the ON state probability is very low. The rate of thermodynamic cost quantified by the entropy production rate was found to be able to suppress the fluctuations of the gene circuit. A higher (lower) rate of thermodynamic cost leads to reduced (increased) fluctuations in the number of gene products in the adiabatic (nonadiabatic) regime. This study also found that higher thermodynamic cost is often required to sustain the emergence of more gene states and, therefore, more heterogeneity was found coming from genetic mutations or epigenetics. The stability of the gene state was studied using the mean first passage time from one state to another. The monotonic decrease in time, i.e., in the stability of the state, in the transition from the nonadiabatic to adiabatic regimes. Therefore, as the higher rate of thermodynamic cost suppresses the fluctuations, higher stability requires higher
Levo, M., Raimundo, J., Bing, X. Y., Sisco, Z., Batut, P. J., Ryabichko, S., Gregor, T. and Levine, M. S. (2022). Transcriptional coupling of distant regulatory genes in living embryos. Nature 605(7911): 754-760. PubMed ID: 35508662
The prevailing view of metazoan gene regulation is that individual genes are independently regulated by their own dedicated sets of transcriptional enhancers. Past studies have reported long-range gene-gene associations, but their functional importance in regulating transcription remains unclear. This study used quantitative single-cell live imaging methods to provide a demonstration of co-dependent transcriptional dynamics of genes separated by large genomic distances were found in living Drosophila embryos. Extensive physical and functional associations of distant paralogous genes, including co-regulation by shared enhancers and co-transcriptional initiation over distances of nearly 250 kilobases. Regulatory interconnectivity depends on promoter-proximal tethering elements, and perturbations in these elements uncouple transcription and alter the bursting dynamics of distant genes, suggesting a role of genome topology in the formation and stability of co-transcriptional hubs. Transcriptional coupling is detected throughout the fly genome and encompasses a broad spectrum of conserved developmental processes, suggesting a general strategy for long-range integration of gene activity.
Pamudurti, N. R., Patop, I. L., Krishnamoorthy, A., Bartok, O., Maya, R., Lerner, N., Ashwall-Fluss, R., Konakondla, J. V. V., Beatus, T. and Kadener, S. (2022). circMbl functions in cis and in trans to regulate gene expression and physiology in a tissue-specific fashion. Cell Rep 39(4): 110740. PubMed ID: 35476987
Muscleblind (mbl) is an essential muscle and neuronal splicing regulator. Mbl hosts multiple circular RNAs (circRNAs), including circMbl, which is conserved from flies to humans. This study shows that mbl-derived circRNAs are key regulators of MBL by cis- and trans-acting mechanisms. By generating fly lines to specifically modulate the levels of all mbl RNA isoforms, including circMbl, this study demonstrated that the two major mbl protein isoforms, MBL-O/P and MBL-C, buffer their own levels by producing different types of circRNA isoforms in the eye and fly brain, respectively. Moreover, it was shown that circMbl has unique functions in trans, as knockdown of circMbl results in specific morphological and physiological phenotypes. In addition, depletion of MBL-C or circMbl results in opposite behavioral phenotypes, showing that they also regulate each other in trans. Together, these results illuminate key aspects of mbl regulation and uncover cis and trans functions of circMbl in vivo.
Bhogale, S. and Sinha, S. (2022). Thermodynamics-based modeling reveals regulatory effects of indirect transcription factor-DNA binding. iScience 25(5): 104152. PubMed ID: 35465052
Transcription factors (TFs) influence gene expression by binding to DNA, yet experimental data suggests that they also frequently bind regulatory DNA indirectly by interacting with other DNA-bound proteins. This study used a data modeling approach to test if such indirect binding by TFs plays a significant role in gene regulation. Regulatory function of indirectly bound TFs was incorporated into a thermodynamics-based model for predicting enhancer-driven expression from its sequence. The new model was fit to a rich data set comprising hundreds of enhancers and their regulatory activities during mesoderm specification in Drosophila embryogenesis and showed that the newly incorporated mechanism results in significantly better agreement with data. In the process, the first sequence-level model was derived of this extensively characterized regulatory program. It was further shown that allowing indirect binding of a TF explains its localization at enhancers more accurately than with direct binding only. This model also provided a simple explanation of how a TF may switch between activating and repressive roles depending on context.
Wang, J., Zhang, S., Lu, H. and Xu, H. (2022). Differential regulation of alternative promoters emerges from unified kinetics of enhancer-promoter interaction. Nat Commun 13(1): 2714. PubMed ID: 35581264
Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. This study applied single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, this study found that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, it was shown that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the kinetic mechanisms of complex eukaryotic gene regulation.
de Almeida, B. P., Reiter, F., Pagani, M. and Stark, A. (2022). . DeepSTARR predicts enhancer activity from DNA sequence and enables the de novo design of synthetic enhancers. Nat Genet 54(5): 613-624. PubMed ID: 35551305
Enhancer sequences control gene expression and comprise binding sites (motifs) for different transcription factors (TFs). Despite extensive genetic and computational studies, the relationship between DNA sequence and regulatory activity is poorly understood, and de novo enhancer design has been challenging. This study built a deep-learning model, DeepSTARR, to quantitatively predict the activities of thousands of developmental and housekeeping enhancers directly from DNA sequence in Drosophila melanogaster S2 cells. The model learned relevant TF motifs and higher-order syntax rules, including functionally nonequivalent instances of the same TF motif that are determined by motif-flanking sequence and intermotif distances. These rules were evaluated experimentally, and it was demonstrated that they can be generalized to humans by testing more than 40,000 wildtype and mutant Drosophila and human enhancers. Finally, synthetic enhancers with desired activities de novo were designed and functionally validated.

Thursday, September 23rd - Disease models

Lee, J., Xu, Y., Saidi, L., Xu, M., Zinsmaier, K. and Ye, Y. (2022). Abnormal triaging of misfolded proteins by adult neuronal ceroid lipofuscinosis-associated DNAJC5/CSPalpha mutants causes lipofuscin accumulation. Autophagy: 1-20. PubMed ID: 35506243
Mutations in DNAJC5/CSPα are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage materials (AFSMs) termed lipofuscin. Functionally, DNAJC5 has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how DNAJC5 dysfunction causes lipofuscinosis and neurodegeneration is unclear. This study reports two functionally distinct but coupled chaperoning activities of DNAJC5, which jointly regulate lysosomal homeostasis: While endolysosome-associated DNAJC5 promotes ESCRT-dependent microautophagy, a fraction of perinuclear and non-lysosomal DNAJC5 mediates MAPS. Functional proteomics identifies a previously unknown DNAJC5 interactor SLC3A2/CD98hc that is essential for the perinuclear DNAJC5 localization and MAPS but dispensable for microautophagy. Importantly, uncoupling these two processes, as seen in cells lacking SLC3A2 or expressing ANCL-associated DNAJC5 mutants, generates DNAJC5-containing AFSMs resembling NCL patient-derived lipofuscin and induces neurodegeneration in a Drosophila ANCL model. These findings suggest that MAPS safeguards microautophagy to avoid DNAJC5-associated lipofuscinosis and neurodegeneration.
Saoji, M., Petersen, C. E., Sen, A., Tripoli, B. A., Smyth, J. T. and Cox, R. T. (2022). Reduction of Drosophila Mitochondrial RNase P in Skeletal and Heart Muscle Causes Muscle Degeneration, Cardiomyopathy, and Heart Arrhythmia. Front Cell Dev Biol 10: 788516. PubMed ID: 35663400
This study examined the cause and progression of mitochondrial diseases linked to the loss of mtRNase P, a three-protein complex responsible for processing and cleaving mitochondrial transfer RNAs (tRNA) from their nascent transcripts. When mtRNase P function is missing, mature mitochondrial tRNA levels are decreased, resulting in mitochondrial dysfunction. mtRNase P is composed of Mitochondrial RNase P Protein (MRPP) 1, 2, and 3. MRPP1 and 2 have their own enzymatic activity separate from MRPP3, which is the endonuclease responsible for cleaving tRNA. Human mutations in all subunits cause mitochondrial disease. Orthologs of each mtRNase P subunit (Roswell/MRPP1, Scully/MRPP2, Mulder/MRPP3) have been identified. This study used Drosophila to model mtRNase P mitochondrial diseases by reducing the level of each subunit in skeletal and heart muscle using tissue-specific RNAi knockdown. mtRNase P reduction in skeletal muscle decreases adult eclosion and causes reduced muscle mass and function. Adult flies exhibit significant age-progressive locomotor defects. Cardiac-specific mtRNase P knockdowns reduce fly lifespan for Roswell and Scully, but not Mulder. Using intravital imaging, it was found that adult hearts have impaired contractility and exhibit substantial arrhythmia. This occurs for roswell and mulder knockdowns, but with little effect for scully. The phenotypes shown in this study are similar to those exhibited by patients with mitochondrial disease, including disease caused by mutations in MRPP1 and 2. These findings also suggest that skeletal and cardiac deficiencies induced by mtRNase P loss are differentially affected by the three subunits. These differences could have implications for disease progression in skeletal and heart muscle and shed light on how the enzyme complex functions in different tissues.
Shenoi, V. N., Brengdahl, M. I., Grace, J. L., Eriksson, B., Ryden, P. and Friberg, U. (2022). A genome-wide test for paternal indirect genetic effects on lifespan in Drosophila melanogaster. Proc Biol Sci 289(1974): 20212707. PubMed ID: 35538781
Exposing sires to various environmental manipulations has demonstrated that paternal effects can be non-trivial also in species where male investment in offspring is almost exclusively limited to sperm. Whether paternal effects also have a genetic component (i.e. paternal indirect genetic effects (PIGEs)) in such species is however largely unknown, primarily because of methodological difficulties separating indirect from direct effects of genes. PIGEs may nevertheless be important since they have the capacity to contribute to evolutionary change. This studyd used Drosophila genetics to construct a breeding design that allows testing nearly complete haploid genomes (more than 99%) for PIGEs. Using this technique, the variance was estimated in male lifespan due to PIGEs among four populations and compare this to the total paternal genetic variance (the sum of paternal indirect and direct genetic effects). These results indicate that a substantial part of the total paternal genetic variance results from PIGEs. A screen of 38 haploid genomes, randomly sampled from a single population, suggests that PIGEs also influence variation in lifespan within populations. Collectively, these results demonstrate that PIGEs may constitute an underappreciated source of phenotypic variation.
Shafik, A. M., Zhou, H., Lim, J., Dickinson, B. and Jin, P. (2022). Dysregulated mitochondrial and cytosolic tRNA m1A methylation in Alzheimer's disease. Hum Mol Genet 31(10): 1673-1680. PubMed ID: 34897434
RNA modifications affect many aspects of RNA metabolism and are involved in the regulation of many different biological processes. Mono-methylation of adenosine in the N1 position, N1-methyladensoine (m1A), is a reversible modification that is known to target rRNAs and tRNAs. m1A has been shown to increase tRNA structural stability and induce correct tRNA folding. Recent studies have begun to associate the dysregulation of epitranscriptomic control with age-related disorders such as Alzheimer's disease. This study applied the newly developed m1A-quant-seq approach to map the brain abundant m1A RNA modification in the cortex of an Alzheimer's disease mouse model, 5XFAD. Hypomethylation was observed in both mitochondrial and cytosolic tRNAs in 5XFAD mice compared with wild type. Furthermore, the main enzymes responsible for the addition of m1A in mitochondrial (TRMT10C, HSD17B10) and cytosolic tRNAs (TRMT61A) displayed decreased expression in 5XFAD compared with wild-type mice. Knockdown of these enzymes results in a more severe phenotype in a Drosophila tau model, and differential m1A methylation is correlated with differences in mature mitochondrial tRNA expression. Collectively, this work suggests that hypo m1A modification in tRNAs may play a role in Alzheimer's disease pathogenesis.
Pushpalatha, K. V., Solyga, M., Nakamura, A. and Besse, F. (2022). RNP components condense into repressive RNP granules in the aging brain. Nat Commun 13(1): 2782. PubMed ID: 35589695
Cytoplasmic RNP condensates enriched in mRNAs and proteins are found in various cell types and associated with both buffering and regulatory functions. While a clear link has been established between accumulation of aberrant RNP aggregates and progression of aging-related neurodegenerative diseases, the impact of physiological aging on neuronal RNP condensates has never been explored. Through high-resolution imaging, this study uncover that RNP components progressively cluster into large yet dynamic granules in the aging Drosophila brain. It was further shown that age-dependent clustering is caused by an increase in the stoichiometry of the conserved helicase Me31B/DDX6, and requires PKA kinase activity. Finally, this functional analysis reveals that mRNA species recruited to RNP condensates upon aging exhibit age-dependent translational repression, indicating that co-clustering of selected mRNAs and translation regulators into repressive condensates may contribute to the specific post-transcriptional changes in gene expression observed in the course of aging.
Lee, W. S., Al-Ramahi, I., Jeong, H. H., Jang, Y., Lin, T., Adamski, C. J., Lavery, L. A., Rath, S., Richman, R., Bondar, V. V., Alcala, E., Revelli, J. P., Orr, H. T., Liu, Z., Botas, J. and Zoghbi, H. Y. (2022). Cross-species genetic screens identify transglutaminase 5 as a regulator of polyglutamine-expanded ataxin-1. J Clin Invest 132(9). PubMed ID: 35499073
Many neurodegenerative disorders are caused by abnormal accumulation of misfolded proteins. In spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded (polyQ-expanded) ataxin-1 (ATXN1) causes neuronal toxicity. Lowering total ATXN1, especially the polyQ-expanded form, alleviates disease phenotypes in mice, but the molecular mechanism by which the mutant ATXN1 is specifically modulated is not understood. This study identified 22 mutant ATXN1 regulators by performing a cross-species screen of 7787 and 2144 genes in human cells and Drosophila eyes, respectively. Among them, TG5 (TG5) preferentially regulated mutant ATXN1 over the WT protein. TG enzymes catalyzed cross-linking of ATXN1 in a polyQ-length-dependent manner, thereby preferentially modulating mutant ATXN1 stability and oligomerization. Perturbing Tg in Drosophila SCA1 models modulated mutant ATXN1 toxicity. Moreover, TG5 was enriched in the nuclei of SCA1-affected neurons and colocalized with nuclear ATXN1 inclusions in brain tissue from patients with SCA1. This work provides a molecular insight into SCA1 pathogenesis and an opportunity for allele-specific targeting for neurodegenerative disorders.

Wednesday, September 21st - Synapse and Vesicles

Mallik, B. and Frank, C. A. (2022). Roles for Mitochondrial Complex I Subunits in Regulating Synaptic Transmission and Growth. Front Neurosci 16: 846425. PubMed ID: 35557603
A genetic screen was conducted to identify conserved components of synapse function that are also associated with human diseases. The Drosophila melanogaster neuromuscular junction (NMJ) was used as a model. RNA interference (RNAi) was employed on selected targets and synapse function and plasticity was assayed by electrophysiology. The screen focussed on genetic factors known to be conserved from human neurological or muscle functions (300 Drosophila lines screened). From this screen, knockdown of a Mitochondrial Complex I (MCI) subunit gene (ND-20L) lowered levels of NMJ neurotransmission. Due to the severity of the phenotype, MCI function was studied further. Knockdown of core MCI subunits concurrently in neurons and muscle led to impaired neurotransmission. This neurotransmission function was localized to the muscle. Pharmacology targeting MCI phenocopied the impaired neurotransmission phenotype. Finally, MCI subunit knockdowns or pharmacological inhibition led to profound cytological defects, including reduced NMJ growth and altered NMJ morphology. Mitochondria are essential for cellular bioenergetics and produce ATP through oxidative phosphorylation. Five multi-protein complexes achieve this task, and MCI is the largest. Impaired Mitochondrial Complex I subunits in humans are associated with disorders such as Parkinson's disease, Leigh syndrome, and cardiomyopathy. Together, these data present an analysis of Complex I in the context of synapse function and plasticity. It is speculated that in the context of human MCI dysfunction, similar neuronal and synaptic defects could contribute to pathogenesis.
Knodel, M. M., Dutta Roy, R. and Wittum, G. (2022). Influence of T-Bar on Calcium Concentration Impacting Release Probability. Front Comput Neurosci 16: 855746. PubMed ID: 35586479
The relation of form and function, namely the impact of the synaptic anatomy on calcium dynamics in the presynaptic bouton, is a major challenge of present (computational) neuroscience at a cellular level. The Drosophila larval neuromuscular junction (NMJ) is a simple model system, which allows studying basic effects in a rather simple way. This synapse harbors several special structures. In particular, in opposite to standard vertebrate synapses, the presynaptic boutons are rather large, and they have several presynaptic zones. In these zones, different types of anatomical structures are present. Some of the zones bear a so-called T-bar, a particular anatomical structure. The geometric form of the T-bar resembles the shape of the letter 'T' or a table with one leg. When an action potential arises, calcium influx is triggered. The probability of vesicle docking and neurotransmitter release is superlinearly proportional to the concentration of calcium close to the vesicular release site. It is tempting to assume that the T-bar causes some sort of calcium accumulation and hence triggers a higher release probability and thus enhances neurotransmitter exocytosis. In order to study this influence in a quantitative manner, a typical T-bar geometry was constructed and the calcium concentration close to active zones (AZs) with and without T-bars was compared. . Indeed, a substantial influence was found of the T-bar structure on the presynaptic calcium concentrations close to the AZs, indicating that this anatomical structure increases vesicle release probability. Therefore, this study reveals how the T-bar zone implies a strong relation between form and function. This study answers the question of experimental studies concerning the sense of the anatomical structure of the T-bar.
Ammer, G., Vieira, R. M., Fendl, S. and Borst, A. (2022). Anatomical distribution and functional roles of electrical synapses in Drosophila. Curr Biol 32(9): 2022-2036.e2024. PubMed ID: 35385694
Electrical synapses are present in almost all organisms that have a nervous system. However, their brain-wide expression patterns and the full range of contributions to neural function are unknown in most species. This study first provides a light-microscopic, immunohistochemistry-based anatomical map of all innexin gap junction proteins-the building blocks of electrical synapses-in the central nervous system of Drosophila melanogaster. Of those innexin types that are expressed in the nervous system, some localize to glial cells, whereas others are predominantly expressed in neurons, with shakB being the most widely expressed neuronal innexin. Then focus was placed on the function of shakB in VS/HS cells-a class of visual projection neurons-thereby uncovering an unexpected role for electrical synapses. Removing shakB from these neurons leads to spontaneous, cell-autonomous voltage and calcium oscillations, demonstrating that electrical synapses are required for these cells' intrinsic stability. Furthermore, the role of shakB-type electrical synapses was investigated in early visual processing. Loss of shakB from the visual circuits upstream of VS/HS cells differentially impairs ON and OFF visual motion processing pathways but is not required for the computation of direction selectivity per se. Taken together, this study demonstrates that electrical synapses are widespread across the Drosophila nervous system and that they play essential roles in neuronal function and visual information processing.
Shah, H. P. and Devergne, O. (2022). Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins during Drosophila Oogenesis. JJ Vis Exp(183). PubMed ID: 35662240
The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.
Zohar-Fux, M., Ben-Hamo-Arad, A., Arad, T., Volin, M., Shklyar, B., Hakim-Mishnaevski, K., Porat-Kuperstein, L., Kurant, E. and Toledano, H. (2022). The phagocytic cyst cells in Drosophila testis eliminate germ cell progenitors via phagoptosis. Sci Adv 8(24): eabm4937. PubMed ID: 35714186
Phagoptosis is a frequently occurring nonautonomous cell death pathway in which phagocytes eliminate viable cells. While it is thought that phosphatidylserine (PS) 'eat-me' signals on target cells initiate the process, the precise sequence of events is largely unknown. This study shows that in Drosophila testes, progenitor germ cells are spontaneously removed by neighboring cyst cells through phagoptosis. Using live imaging with multiple markers, it was demonstrated that cyst cell-derived early/late endosomes and lysosomes fused around live progenitors to acidify them, before DNA fragmentation and substantial PS exposure on the germ cell surface. Furthermore, the phagocytic receptor Draper is expressed on cyst cell membranes and is necessary for phagoptosis. Significantly, germ cell death is blocked by knockdown of either the endosomal component Rab5 or the lysosomal associated protein Lamp1, within the cyst cells. These data ascribe an active role for phagocytic cyst cells in removal of live germ cell progenitors.
Moreno, M. R., Boswell, K., Casbolt, H. L. and Bulgakova, N. A. (2022). Multifaceted control of E-cadherin dynamics by the Adaptor Protein Complex 1 during epithelial morphogenesis. Mol Biol Cell: mbcE21120598. PubMed ID: 35609212
Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargos. This study examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. A distinct pool of AP-1, which localizes at the subapical Adherens Junctions, was identified. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. Evidence is provided that E-cadherin hyperinternalization acts upstream of cell death in a potential tumour-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion.

Tuesday, September 20th - Embryonic Development

Zhang, W., Lei, X., Zhou, X., He, B., Xiao, L., Yue, H., Wang, S., Sun, Y., Wu, Y., Wang, L., Ghartey-Kwansah, G., Jones, O. D., Bryant, J. L., Xu, M., Ma, J. and Xu, X. (2022). A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo. J Vis Exp(183). PubMed ID: 35604165
Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. This work introduceS a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster, was studied. DmFKBP12 was identified in as early as the syncytial blastoderm stage, and the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.
Guo, H., Huang, S. and He, B. (2022). Evidence for a Role of the Lateral Ectoderm in Drosophila Mesoderm Invagination. Front Cell Dev Biol 10: 867438. PubMed ID: 35547820
he folding of two-dimensional epithelial sheets into specific three-dimensional structures is a fundamental tissue construction mechanism in animal development. A common mechanism that mediates epithelial folding is apical constriction, the active shrinking of cell apices driven by actomyosin contractions. It remains unclear whether cells outside of the constriction domain also contribute to folding. During Drosophila mesoderm invagination, ventrally localized mesoderm epithelium undergoes apical constriction and subsequently folds into a furrow. While the critical role of apical constriction in ventral furrow formation has been well demonstrated, it remains unclear whether, and if so, how the laterally localized ectodermal tissue adjacent to the mesoderm contributes to furrow invagination. This study combined experimental and computational approaches to test the potential function of the ectoderm in mesoderm invagination. Through laser-mediated, targeted disruption of cell formation prior to gastrulation, it was found that the presence of intact lateral ectoderm is important for the effective transition between apical constriction and furrow invagination in the mesoderm. In addition, using a laser-ablation approach widely used for probing tissue tension, this study found that the lateral ectodermal tissues exhibit signatures of tissue compression when ablation was performed shortly before the onset of mesoderm invagination. These observations led to the hypothesis that in-plane compression from the surrounding ectoderm facilitates mesoderm invagination by triggering buckling of the mesoderm epithelium. In support of this notion, it was shown that the dynamics of tissue flow during mesoderm invagination displays characteristic of elastic buckling, and this tissue dynamics can be recapitulated by combining local apical constriction and global compression in a simulated elastic monolayer. It is proposed that Drosophila mesoderm invagination is achieved through epithelial buckling jointly mediated by apical constriction in the mesoderm and compression from the neighboring ectoderm.
Lv, Z., Zhang, N., Zhang, X., Grosshans, J. and Kong, D. (2022). The Lateral Epidermis Actively Counteracts Pulling by the Amnioserosa During Dorsal Closure. Front Cell Dev Biol 10: 865397. PubMed ID: 35652100
Dorsal closure is a prominent morphogenetic process during Drosophila embryogenesis, which involves two epithelial tissues, that is, the squamous amnioserosa and the columnar lateral epidermis. Non-muscle myosin II-driven constriction in the amnioserosa leads to a decrease in the apical surface area and pulls on the adjacent lateral epidermis, which subsequently moves dorsally. The pull by the amnioserosa becomes obvious in an elongation of the epidermal cells, especially of those in the first row. The contribution of the epidermal cell elongation has remained unclear to dorsal closure. Cell elongation may be a mere passive consequence or an active response to the pulling by the amnioserosa. This study found that the lateral epidermis actively responds. Tensions within tissues and cell junctions were analyzed by laser ablation before and during dorsal closure, the elliptical and dorsal closure stages, respectively. Furthermore, chronic and acute cell contraction were genetically and optochemically induced, respectively. In this way, it was found that tension in the epidermis increased during dorsal closure. A correspondingly increased tension was not observed at individual junctions, however. Junctional tension even decreased during dorsal closure in the epidermis. A strong increase of the microtubule amount was strikingly observed in the epidermis, while non-muscle myosin II increased in both tissues. These data suggest that the epidermis actively antagonizes the pull from the amnioserosa during dorsal closure and the increased microtubules might help the epidermis bear part of the mechanical force.
Mitchell, N. P., Cislo, D. J., Shankar, S., Lin, Y., Shraiman, B. I. and Streichan, S. J. (2022). Visceral organ morphogenesis via calcium-patterned muscle constrictions. Elife 11. PubMed ID: 35593701
Organ architecture is often composed of multiple laminar tissues arranged in concentric layers. During morphogenesis, the initial geometry of visceral organs undergoes a sequence of folding, adopting a complex shape that is vital for function. Genetic signals are known to impact form, yet the dynamic and mechanical interplay of tissue layers giving rise to organs' complex shapes remains elusive. This study traced the dynamics and mechanical interactions of a developing visceral organ across tissue layers, from sub-cellular to organ scale in vivo. Combining deep tissue light-sheet microscopy for in toto live visualization with a novel computational framework for multilayer analysis of evolving complex shapes, this study found a dynamic mechanism for organ folding using the embryonic midgut of Drosophila as a model visceral organ. Hox genes, known regulators of organ shape, control the emergence of high-frequency calcium pulses. Spatiotemporally patterned calcium pulses trigger muscle contractions via myosin light chain kinase. Muscle contractions, in turn, induce cell shape change in the adjacent tissue layer. This cell shape change collectively drives a convergent extension pattern. Through tissue incompressibility and initial organ geometry, this in-plane shape change is linked to out-of-plane organ folding. This analysis follows tissue dynamics during organ shape change in vivo, tracing organ-scale folding to a high-frequency molecular mechanism. These findings offer a mechanical route for gene expression to induce organ shape change: genetic patterning in one layer triggers a physical process in the adjacent layer - revealing post-translational mechanisms that govern shape change.
Klussmann-Fricke, B. J., Martin-Bermudo, M. D. and Llimargas, M. (2022). The basement membrane controls size and integrity of the Drosophila tracheal tubes. Cell Rep 39(4): 110734. PubMed ID: 35476979
Biological tubes are fundamental units of most metazoan organs. Their defective morphogenesis can cause malformations and pathologies. An integral component of biological tubes is the extracellular matrix, present apically (aECM) and basally (BM). Studies using the Drosophila tracheal system established an essential function for the aECM in tubulogenesis. This study demonstrates that the BM also plays a critical role in this process. BM components are deposited in a spatial-temporal manner in the trachea. Laminins, core BM components, control size and shape of tracheal tubes and their topology within the embryo. At a cellular level, laminins control cell shape changes and distribution of the cortical cytoskeleton component α-spectrin. Finally, the BM and aECM act independently-yet cooperatively-to control tube elongation and together to guarantee tissue integrity. These results unravel key roles for the BM in shaping, positioning, and maintaining biological tubes.
Lusk, J. B., Chua, E. H. Z., Kaur, P., Sung, I. C. H., Lim, W. K., Lam, V. Y. M., Harmston, N. and Tolwinski, N. S. (2022). A non-canonical Raf function is required for dorsal-ventral patterning during Drosophila embryogenesis. Sci Rep 12(1): 7684. PubMed ID: 35538124
Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. This study describes a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. These results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.

Monday, September 19th - Evolution

Chahda, J. S., Ambrosi, P. and Mizutani, C. M. (2022). The nested embryonic dorsal domains of BMP-target genes are not scaled to size during the evolution of Drosophila species. J Exp Zool B Mol Dev Evol. PubMed ID: 35451554
Egg size is a fast-evolving trait among Drosophilids expected to change the spatial distribution of morphogens that pattern the embryonic axes. This study asked whether the patterning of the dorsal region of the embryo by the Decapentaplegic/Bone Morphogenetic Protein-4 (DPP/BMP-4) gradient is scaled among Drosophila species with different egg sizes. This region specifies the extra-embryonic tissue amnioserosa and the ectoderm. This study found that the entire dorsal region scales with embryo size, but the gene expression patterns regulated by DPP are not proportional, suggesting that the DPP gradient is differentially scaled during evolution. To further test whether the DPP gradient can scale or not in Drosophila melanogaster, embryos were created with expanded dorsal regions that mimic changes in scale seen in other species, and the resulting domains were measured of DPP-target genes. The proportions of these domains were found to not be maintained, suggesting that the DPP gradient is unable to scale in the embryo. These and previous findings suggest that embryonic dorso-ventral patterning lacks scaling in the ventral and dorsal sides but is robust in the lateral region where the neuroectoderm is specified and two opposing gradients, Dorsal/NFkappa-B and DPP, intersect. It is proposed that the lack of scaling of the DPP gradient may contribute to changes in the size of the amnioserosa and the number of ectodermal cells with specific cortical tensions, which are expected to generate distinct mechanical forces for gastrulating embryos of different sizes.
Wang, S., Nalley, M. J., Chatla, K., Aldaimalani, R., MacPherson, A., Wei, K. H., Corbett-Detig, R. B., Mai, D. and Bachtrog, D. (2022). Neo-sex chromosome evolution shapes sex-dependent asymmetrical introgression barrier. Proc Natl Acad Sci U S A 119(19): e2119382119. PubMed ID: 35512091
Sex chromosomes play a special role in the evolution of reproductive barriers between species. This study describes conflicting roles of nascent sex chromosomes on patterns of introgression in an experimental hybrid swarm. Drosophila nasuta and Drosophila albomicans are recently diverged, fully fertile sister species that have different sex chromosome systems. The fusion between an autosome (Muller CD) with the ancestral X and Y gave rise to neo-sex chromosomes in D. albomicans, while Muller CD remains unfused in D. nasuta. This study found that a large block containing overlapping inversions on the neo-sex chromosome stood out as the strongest barrier to introgression. Intriguingly, the neo-sex chromosome introgression barrier is asymmetrical and sex-dependent. Female hybrids showed significant D. albomicans–biased introgression on Muller CD (neo-X excess), while males showed heterosis with excessive (neo-X, D. nasuta Muller CD) genotypes. A population genetic model was used to dissect the interplay of sex chromosome drive, heterospecific pairing incompatibility between the neo-sex chromosomes and unfused Muller CD, neo-Y disadvantage, and neo-X advantage in generating the observed sex chromosome genotypes in females and males. Moderate neo-Y disadvantage and D. albomicans specific meiotic drive were shown to be required to observe female-specific D. albomicans–biased introgression in this system, together with pairing incompatibility and neo-X advantage. In conclusion, this hybrid swarm between a young species pair sheds light onto the multifaceted roles of neo-sex chromosomes in a sex-dependent asymmetrical introgression barrier at a species boundary.
Garlovsky, M. D., Holman, L., Brooks, A. L., Novicic, Z. K. and Snook, R. R. (2022). Experimental sexual selection affects the evolution of physiological and life-history traits. J Evol Biol 35(5): 742-751. PubMed ID: 35384100
Sexual selection and sexual conflict are expected to affect all aspects of the phenotype, not only traits that are directly involved in reproduction. This study shows coordinated evolution of multiple physiological and life-history traits in response to long-term experimental manipulation of the mating system in populations of Drosophila pseudoobscura. Development time was extended under polyandry relative to monogamy in both sexes, potentially due to higher investment in traits linked to sexual selection and sexual conflict. Individuals (especially males) evolving under polyandry had higher metabolic rates and locomotor activity than those evolving under monogamy. Polyandry individuals also invested more in metabolites associated with increased endurance capacity and efficient energy metabolism and regulation, namely lipids and glycogen. Finally, polyandry males were less desiccation- and starvation resistant than monogamy males, suggesting trade-offs between resistance and sexually selected traits. These results provide experimental evidence that mating systems can impose selection that influences the evolution of non-sexual phenotypes such as development, activity, metabolism and nutrient homeostasis.
Mier, P., Fontaine, J. F., Stoldt, M., Libbrecht, R., Martelli, C., Foitzik, S. and Andrade-Navarro, M. A. (2022). Annotation and Analysis of 3902 Odorant Receptor Protein Sequences from 21 Insect Species Provide Insights into the Evolution of Odorant Receptor Gene Families in Solitary and Social Insects. Genes (Basel) 13(5). PubMed ID: 35627304
The gene family of insect olfactory receptors (ORs) has expanded greatly over the course of evolution. ORs enable insects to detect volatile chemicals and therefore play an important role in social interactions, enemy and prey recognition, and foraging. The sequences of several thousand ORs are known, but their specific function or their ligands have only been identified for very few of them. To advance the functional characterization of ORs, this study has assembled, curated, and aligned the sequences of 3902 ORs from 21 insect species, which is provided as an annotated online resource. Using functionally characterized proteins from the fly Drosophila melanogaster, the mosquito Anopheles gambiae and the ant Harpegnathos saltator, amino acid positions were identified that best predict response to ligands. The conservation of these predicted relevant residues was examined in all OR subfamilies; the results showed that the subfamilies that expanded strongly in social insects had a high degree of conservation in their binding sites. This suggests that the ORs of social insect families are typically finely tuned and exhibit sensitivity to very similar odorants. The novel approach of this study provides a powerful tool to exploit functional information from a limited number of genes to study the functional evolution of large gene families.
Kaneko, G. (2022). Phylogenetic annotation of Drosophila melanogaster heat shock protein 70 genes. MicroPubl Biol 2022. PubMed ID: 35622499
The traditional classification of stress-inducible 70 kDa heat shock protein (Hsp70) and heat shock cognate (Hsc70) requires a revision because of a recent finding that neither of them constitutes a monophyletic gene family. This study inferred a phylogenetic relationship among Drosophila melanogaster Hsp70 family members. D. melanogaster Hsp70 family members were separated into four known metazoan Hsp70 lineages: cytosolic A, cytosolic B, endoplasmic reticulum, and mitochondria. Hsc70s sporadically distributed in the phylogenetic tree, indicating their paraphyletic origin. Detailed sequence inspection found several motifs that support the phylogenetic analysis. Taken together, this study proposes new aliases of D. melanogaster Hsp70 family members based on their evolutionary history.
York, R. A., Brezovec, L. E., Coughlan, J., Herbst, S., Krieger, A., Lee, S. Y., Pratt, B., Smart, A. D., Song, E., Suvorov, A., Matute, D. R., Tuthill, J. C. and Clandinin, T. R. (2022). The evolutionary trajectory of drosophilid walking. Curr Biol. PubMed ID: 35671756
Neural circuits must both execute the behavioral repertoire of individuals and account for behavioral variation across species. Understanding how this variation emerges over evolutionary time requires large-scale phylogenetic comparisons of behavioral repertoires. This study describes the evolution of walking in fruit flies by capturing high-resolution, unconstrained movement from 13 species and 15 strains of drosophilids. Walking can be captured in a universal behavior space, the structure of which is evolutionarily conserved. However, the occurrence of and transitions between specific movements have evolved rapidly, resulting in repeated convergent evolution in the temporal structure of locomotion. Moreover, a meta-analysis demonstrates that many behaviors evolve more rapidly than other traits. Thus, the architecture and physiology of locomotor circuits can execute precise individual movements in one species and simultaneously support rapid evolutionary changes in the temporal ordering of these modular elements across clades.

Friday, September 16th - RNA and Transposons

Dozier, C., Montigny, A., Viladrich, M., Culerrier, R., Combier, J. P., Besson, A. and Plaza, S. (2022). Small ORFs as New Regulators of Pri-miRNAs and miRNAs Expression in Human and Drosophila. Int J Mol Sci 23(10). PubMed ID: 35628573
MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis-regulatory elements involved in lncRNA decay. This study investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila, focusing on pri-miRNAs previously shown to contain translatable smORFs. smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster.
Fernandez-Castillo, E., Barbosa-Santillan, L. I., Falcon-Morales, L. and Sanchez-Escobar, J. J. (2022). Deep Splicer: A CNN Model for Splice Site Prediction in Genetic Sequences. Genes (Basel) 13(5). PubMed ID: 35627292
The genes of higher eukaryotic organisms contain coding sequences, known as exons and non-coding sequences, known as introns, which are removed on splice sites after the DNA is transcribed into RNA. Genome annotation is the process of identifying the location of coding regions and determining their function. This process is fundamental for understanding gene structure; however, it is time-consuming and expensive when done by biochemical methods. With technological advances, splice site detection can be done computationally. Although various software tools have been developed to predict splice sites, they need to improve accuracy and reduce false-positive rates. The main goal of this research was to generate Deep Splicer, a deep learning model to identify splice sites in the genomes of humans and other species. This model has good performance metrics and a lower false-positive rate than the currently existing tools. Deep Splicer achieved an accuracy between 93.55% and 99.66% on the genetic sequences of different organisms, while Splice2Deep, another splice site detection tool, had an accuracy between 90.52% and 98.08%. Splice2Deep surpassed Deep Splicer on the accuracy obtained after evaluating C. elegans genomic sequences (97.88% vs. 93.62%) and A. thaliana (95.40% vs. 94.93%); however, Deep Splicer's accuracy was better for H. sapiens (98.94% vs. 97.15%) and D. melanogaster (97.14% vs. 92.30%). The rate of false positives was 0.11% for human genetic sequences and 0.25% for other species' genetic sequences. Another splice prediction tool, Splice Finder, had between 1% and 3% of false positives for human sequences, while other species' sequences had around 4% and 10%.
He, J., Zhu, Y. N., Wang, B., Yang, P., Guo, W., Liang, B., Jiang, F., Wang, H., Wei, Y. and Kang, L. (2022). piRNA-guided intron removal from pre-mRNAs regulates density-dependent reproductive strategy. Cell Rep 39(4): 110593. PubMed ID: 35476998
Animal density-dependent experiences have profound effects on reproductive strategies with marked fecundity differences. Migratory locust adopts distinct population density-dependent reproductive strategies to cope with their respective life cycles, but the mechanisms remain poorly understood. This study report that Piwi-interacting RNAs (piRNAs) in the locust germline play key roles in this process. Locust Piwi protein Liwi1 and piRNAs were found to be highly expressed in early developing egg chambers in solitarious locusts, which have higher fecundity than gregarious locusts. Approximately 40% of solitarious locust-associated piRNAs map to protein-coding genes. Liwi1/piRNAs facilitate pre-mRNA splicing of oocyte development-related genes, such as oo18 RNA-binding protein (Orb), in the germline by recruiting the splicing factor U2AF35 to piRNA-targeted introns, thereby increasing fecundity. Such piRNA-guided pre-mRNA splicing is also functional in Drosophila and mouse germ cells. A piRNA-guided splicing mechanism was uncovered for processing reproduction-related mRNAs and determining animal reproductive strategies.
Allen, S. R., Stewart, R. K., Rogers, M., Ruiz, I. J., Cohen, E., Laederach, A., Counter, C. M., Sawyer, J. K. and Fox, D. T. (2022). Distinct responses to rare codons in select Drosophila tissues. JElife 11. PubMed ID: 35522036
Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. This study used codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however,the testis and brain are uniquely capable of expressing rare codon-enriched reporters. A new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index (taCAI), to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. A role was further demonstrate for rare codons in an evolutionarily young testis-specific gene, RpL10Aa. Optimizing RpL10Aa codons disrupts female fertility. This work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.
Shabar, H. and DiAngelo, J. R. (2022). The regulation of lipid and carbohydrate storage by the splicing factor gene snRNP-U1-70K in the Drosophila fat body. MicroPubl Biol 2022. PubMed ID: 35655607
Excess triglycerides from the diet are stored in structures called lipid droplets in adipose tissue. Genome-wide RNAi screens have identified mRNA splicing factors as important for lipid droplet formation; however, the full complement of splicing factors that regulate lipid storage is not known. This study characterized the role of snRNP-U1-70K, the gene encoding for a splicing protein involved in recognizing the 5' splice site in introns, in regulating lipid and carbohydrate storage in the Drosophila fat body. Decreasing snRNP-U1-70K specifically in the fly fat body resulted in less triglyceride, glycogen, and glucose in each fat body cell. Consistent with these decreased nutrient storage phenotypes, snRNP-U1-70K-RNAi flies ate less, providing a potential cause for less lipid and carbohydrate storage in these flies. These data further support the role of mRNA processing in regulating metabolic homeostasis in Drosophila.
Bornelov, S., Czech, B. and Hannon, G. J. (2022). An evolutionarily conserved stop codon enrichment at the 5' ends of mammalian piRNAs. Nat Commun 13(1): 2118. PubMed ID: 35440552
PIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5' ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. This study examined murine piRNAs and revealed a strong and specific enrichment of three sequences (UAA, UAG, UGA)-corresponding to stop codons-at piRNA 5' ends. Stop codon sequences are also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses reveal that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5' end stop codons. This observation is conserved across mammals and possibly further. This work provides new insights into Zuc-dependent cleavage and may point to a previously unrecognized connection between piRNA biogenesis and the translational machinery.

Thursday, September 15th - Stem Cells

Kapoor, A., Padmavathi, A., Madhwal, S. and Mukherjee, T. (2022). Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth. EMBO Rep 23(6): e52951. PubMed ID: 35476897
In Drosophila, definitive haematopoiesis takes place in a specialized organ termed 'lymph gland'. It harbours multi-potent stem-like blood progenitor cells whose development controls overall growth of this haematopoietic tissue and formation of mature blood cells. With respect to its development, neurotransmitters have emerged as potent regulators of blood-progenitor cell development and function. This study extended the understanding of neurotransmitters and showed that progenitors are self-sufficient with regard to synthesizing dopamine, a well-established neurotransmitter. These cells also have modules for dopamine sensing through the receptor and transporter. Modulating expression of these components in progenitor cells affected lymph gland growth, which suggested growth-promoting function of dopamine in blood-progenitor cells. Cell-cycle analysis of developing lymph glands revealed an unexpected requirement for intracellular dopamine in moderating the progression of early progenitor cells from S to G2 phase of the cell cycle, while activation of dopamine receptor signalling later in development regulated their progression from G2 and entry into mitosis. The dual capacity in which dopamine operated, first intracellularly to coordinate S/G2 transition and later extracellularly in G2/M transition, was critical for the growth of the lymph gland. Overall, the data presented highlight a novel non-canonical use of dopamine in the myeloid system that reveals an uncharacterized function of intracellular dopamine in cell-cycle phasing with outcomes on haematopoietic growth and immunity as well.
Keliinui, C. N., Doyle, S. E. and Siegrist, S. E. (2022). Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants. J Vis Exp(183). PubMed ID: 35665723
Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. This study found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, it will be used to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.
Greenspan, L. J., de Cuevas, M., Le, K. H., Viveiros, J. M. and Matunis, E. L. (2022). Activation of the EGFR/MAPK pathway drives transdifferentiation of quiescent niche cells to stem cells in the Drosophila testis niche. Elife 11. PubMed ID: 35468055
Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. This study found that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. It is proposed that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation.
Sang, R., Wu, C., Xie, S., Xu, X., Lou, Y., Ge, W., Xi, Y. and Yang, X. (2022). Mxc, a Drosophila homolog of mental retardation-associated gene NPAT, maintains neural stem cell fate. Cell Biosci 12(1): 78. PubMed ID: 35642004
Mental retardation is a complex neurodevelopmental disorder. NPAT, a component of the histone locus body (HLB), has been implicated as a candidate gene for mental retardation. This study identified that multi sex combs (mxc), the Drosophila ortholog of NPAT, is required for the development of nervous system. Knockdown of mxc resulted in a massive loss of neurons and locomotion dysfunction in adult flies. In the mxc mutant or RNAi knockdown larval brains, the neuroblast (NB, also known as neural stem cell) cell fate is prematurely terminated and its proliferation potential is impeded concurrent with the blocking of the differentiation process of ganglion mother cells (GMCs). A reduction of transcription levels of histone genes was shown in mxc knockdown larval brains, accompanied by DNA double-strand breaks (DSBs). The subsidence of histone transcription levels leads to prematurely termination of NB cell fate and blockage of the GMC differentiation process. These data also show that the increase in autophagy induced by mxc knockdown in NBs could be a defense mechanism in response to abnormal HLB assembly and premature termination of NB cell fate.
Wang, C. and Spradling, A. C. (2022). Drosophila renal stem cells enhance fitness by delayed remodeling of adult Malpighian tubules. Sci Adv 8(20): eabn7436. PubMed ID: 35594355
Drosophila renal stem cells (RSCs) contradict the common expectation that stem cells maintain tissue homeostasis. RSCs are abundant, quiescent, and confined to the peri-ureter region of the kidney-like Malpighian tubules (MTs). Although derived during pupation - as are intestinal stem cells - RSCs initially remodel the larval MTs only near the intestinal junction. However, following adult injury to the ureter by xanthine stones, RSCs remodel the damaged region in a similar manner. Thus, RSCs represent stem cells encoding a developmental redesign. The remodeled tubules have a larger luminal diameter and shorter brush border, changes linked to enhanced stone resistance. However, RSC-mediated modifications also raise salt sensitivity and reduce fecundity. These results suggest that RSCs arose by arresting developmental progenitors to preserve larval physiology until a time in adulthood when it becomes advantageous to complete the development by RSC activation.
Restrepo, L. J., DePew, A. T., Moese, E. R., Tymanskyj, S. R., Parisi, M. J., Aimino, M. A., Duhart, J. C., Fei, H. and Mosca, T. J. (2022). Gamma-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor. Dev Cell. PubMed ID: 35654038
Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. This study showed that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development.

Wednesday, September 14th - Signaling

Yuen, A. C., Prasad, A. R., Fernandes, V. M. and Amoyel, M. (2022). A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila. Biol Open 11(5). PubMed ID: 35608229
Extracellular signal-regulated kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. This study adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, this study made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active epidermal growth factor receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, the direction of migration of retinal basal glia correlated with their ERK activity levels, suggesting an explanation for the heterogeneity in ERK activity observed in fixed tissue. These results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.
Gao, Y., Chen, N., Zhang, X., Li, E. Y., Luo, W., Zhang, J., Zhang, W., Li, S., Wang, J. and Liu, S. (2022). Juvenile Hormone Membrane Signaling Enhances its Intracellular Signaling Through Phosphorylation of Met and Hsp83. Front Physiol 13: 872889. PubMed ID: 35574494
Juvenile hormone (JH) regulates insect development and reproduction through both intracellular and membrane signaling, and the two pathways might crosstalk with each other. Recent studies have reported that JH membrane signaling induces phosphorylation of the JH intracellular receptor Met, thus enhancing its transcriptional activity. To gain more insights into JH-induced Met phosphorylation, phosphoproteomics was performed to identify potential phosphorylation sites of Met and its paralog Germ-cell expressed (Gce) in Drosophila Kc cells. In vitro experiments demonstrate that JH-induced phosphorylation sites in the basic helix-loop-helix (bHLH) domain, but not in the Per-Arnt-Sim-B (PAS-B) domain, are required for maximization of Met transcriptional activity. Moreover, phosphoproteomics analysis reveals that JH also induces the phosphorylation of Hsp83, a chaperone protein involved in JH-activated Met nuclear import. The JH-induced Hsp83 phosphorylation at S219 facilitates Hsp83-Met binding, thus promoting Met nuclear import and its transcription. By using proteomics, subcellular distribution, and co-immunoprecipitation approaches, this study further characterized 14-3-3 proteins as negative regulators of Met nuclear import through physical interaction with Hsp83. These results show that JH membrane signaling induces phosphorylation of the key components in JH intracellular signaling, such as Met and Hsp83, and consequently facilitating JH intracellular signaling.
Graca, F. A., Rai, M., Hunt, L. C., Stephan, A., Wang, Y. D., Gordon, B., Wang, R., Quarato, G., Xu, B., Fan, Y., Labelle, M. and Demontis, F. (2022). The myokine Fibcd1 is an endogenous determinant of myofiber size and mitigates cancer-induced myofiber atrophy. Nat Commun 13(1): 2370. PubMed ID: 35501350
Decline in skeletal muscle cell size (myofiber atrophy) is a key feature of cancer-induced wasting (cachexia). In particular, atrophy of the diaphragm, the major muscle responsible for breathing, is an important determinant of cancer-associated mortality. However, therapeutic options are limited. This study used Drosophila transgenic screening to identify muscle-secreted factors (myokines) that act as paracrine regulators of myofiber growth. Subsequent testing in mouse myotubes revealed that mouse Fibcd1 is an evolutionary-conserved myokine that preserves myofiber size via ERK signaling. Local administration of recombinant Fibcd1 (rFibcd1) ameliorates cachexia-induced myofiber atrophy in the diaphragm of mice bearing patient-derived melanoma xenografts and LLC carcinomas. Moreover, rFibcd1 impedes cachexia-associated transcriptional changes in the diaphragm. Fibcd1-induced signaling appears to be muscle selective because rFibcd1 increases ERK activity in myotubes but not in several cancer cell lines tested. It is proposed that rFibcd1 may help reinstate myofiber size in the diaphragm of patients with cancer cachexia.
Zhao, H., Ren, X., Kong, R., Shi, L., Li, Z., Wang, R., Ma, R., Zhao, H., Liu, F., Chang, H. C., Chen, C. H. and Li, Z. (2022). Auxilin regulates intestinal stem cell proliferation through EGFR. Stem Cell Reports 17(5): 1120-1137. PubMed ID: 35427486
Adult tissue homeostasis is maintained by residential stem cells. The proliferation and differentiation of adult stem cells must be tightly balanced to avoid excessive proliferation or premature differentiation. However, how stem cell proliferation is properly controlled remains elusive. This study found that auxilin (Aux) restricts intestinal stem cell (ISC) proliferation mainly through EGFR signaling. aux depletion leads to excessive ISC proliferation and midgut homeostasis disruption, which is unlikely caused by defective Notch signaling. Aux is expressed in multiple types of intestinal cells. Interestingly, aux depletion causes a dramatic increase in EGFR signaling, with a strong accumulation of EGFR at the plasma membrane and an increased expression of EGFR ligands in response to tissue stress. Furthermore, Aux co-localizes and associates with EGFR. Finally, blocking EGFR signaling completely suppresses the defects caused by aux depletion. Together, these data demonstrate that Aux mainly safeguards EGFR activation to keep a proper ISC proliferation rate to maintain midgut homeostasis.
Cuevas-Navarro, A., Rodriguez-Munoz, L., Grego-Bessa, J., Cheng, A., Rauen, K. A., Urisman, A., McCormick, F., Jimenez, G. and Castel, P. (2022). Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs. Elife 11. PubMed ID: 35467524
RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. Previously, a novel Cullin 3 RING E3 ubiquitin ligase complex has been described, formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. This study hase analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, it was shown that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, these results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.
Chen, T. A., Lin, K. Y., Yang, S. M., Tseng, C. Y., Wang, Y. T., Lin, C. H., Luo, L., Cai, Y. and Hsu, H. J. (2022). Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation. Front Cell Dev Biol 10: 877047. PubMed ID: 35517512
Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. This study shows that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. These results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, it was demonstrated the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.

Tuesday September 13th - Adult Neural Development and Function

Chouhan, N. S. and Sehgal, A. (2022). Consolidation of Sleep-Dependent Appetitive Memory Is Mediated by a Sweet-Sensing Circuit. J Neurosci 42(18): 3856-3867. PubMed ID: 35361706
Sleep is a universally conserved physiological state which contributes toward basic organismal functions, including cognitive operations such as learning and memory. Intriguingly, organisms can sometimes form memory even without sleep, such that Drosophila display sleep-dependent and sleep-independent memory in an olfactory appetitive training paradigm. Sleep-dependent memory can be elicited by the perception of sweet taste, and this study now shows that a mixed-sex population of flies maintained on sorbitol, a tasteless but nutritive substance, do not require sleep for memory consolidation. Consistent with this, silencing sugar-sensing gustatory receptor neurons in fed flies triggers a switch to sleep-independent memory consolidation, whereas activating sugar-sensing gustatory receptor neurons results in the formation of sleep-dependent memory in starved flies. Sleep-dependent and sleep-independent memory relies on distinct subsets of reward signaling protocerebral anterior medial dopaminergic neurons (PAM DANs) such that PAM-β'2mp DANs mediate memory in fed flies whereas PAM-α1 DANs are required in starved flies. Correspondingly, a feeding-dependent calcium increase was observed in PAM-β'2mp DANs, but not in PAM-α1 DANs. Following training, the presence of sweet sugars recruits PAM-β'2mp DANs, whereas tasteless medium increases calcium in PAM-α1 DANs. Together, this work identifies mechanistic underpinnings of sleep-dependent memory consolidation, in particular demonstrating a role for the processing of sweet taste reward signals.
Dinges, G. F., Bockemuhl, T., Iacoviello, F., Shearing, P. R., Byschges, A. and Blanke, A. (2022). Ultra high-resolution biomechanics suggest that substructures within insect mechanosensors decisively affect their sensitivity. J R Soc Interface 19(190): 20220102. PubMed ID: 35506211
Insect load sensors, called campaniform sensilla (CS), measure strain changes within the cuticle of appendages. This mechanotransduction provides the neuromuscular system with feedback for posture and locomotion. Owing to their diverse morphology and arrangement, CS can encode different strain directions. Nano-computed tomography and finite-element analysis were used to investigate how different CS morphologies within one location-the femoral CS field of the leg in the fruit fly Drosophila-interact under load. By investigating the influence of CS substructures' material properties during simulated limb displacement with naturalistic forces, it was shown that CS substructures (i.e. socket and collar) influence strain distribution throughout the whole CS field. Altered socket and collar elastic moduli resulted in 5% relative differences in displacement, and the artificial removal of all sockets caused differences greater than 20% in cap displacement. Apparently, CS sockets support the distribution of distal strain to more proximal CS, while collars alter CS displacement more locally. Harder sockets can increase or decrease CS displacement depending on sensor location. Furthermore, high-resolution imaging revealed that sockets are interconnected in subcuticular rows. In summary, the sensitivity of individual CS is dependent on the configuration of other CS and their substructures.
Zatsepina, O. G., Chuvakova, L. N., Nikitina, E. A., Rezvykh, A. P., Zakluta, A. S., Sarantseva, S. V., Surina, N. V., Ksenofontov, A. L., Baratova, L. A., Shilova, V. Y. and Evgen'ev, M. B. (2022). Genes Responsible for H(2)S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster. Biomolecules 12(6). PubMed ID: 35740876
The gasotransmitter hydrogen sulfide (H(2)S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H(2)S production and metabolism. This study investigated the role of H(2)S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. The learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.
Xie, Q., Li, J., Li, H., Udeshi, N. D., Svinkina, T., Orlin, D., Kohani, S., Guajardo, R., Mani, D. R., Xu, C., Li, T., Han, S., Wei, W., Shuster, S. A., Luginbuhl, D. J., Quake, S. R., Murthy, S. E., Ting, A. Y., Carr, S. A. and Luo, L. (2022). Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code. Neuron. PubMed ID: 35613619
Transcription factors specify the fate and connectivity of developing neurons. This study investigated how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.
Xie, Q., Li, J., Li, H., Udeshi, N. D., Svinkina, T., Orlin, D., Kohani, S., Guajardo, R., Mani, D. R., Xu, C., Li, T., Han, S., Wei, W., Shuster, S. A., Luginbuhl, D. J., Quake, S. R., Murthy, S. E., Ting, A. Y., Carr, S. A. and Luo, L. (2022). Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code. Neuron. PubMed ID: 35613619
Transcription factors specify the fate and connectivity of developing neurons. This study investigated how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.
Yang, K., Liu, T., Wang, Z., Liu, J., Shen, Y., Pan, X., Wen, R., Xie, H., Ruan, Z., Tan, Z., Chen, Y., Guo, A., Liu, H., Han, H., Di, Z. and Zhang, K. (2022). Classifying Drosophila olfactory projection neuron boutons by quantitative analysis of electron microscopic reconstruction. iScience 25(5): 104180. PubMed ID: 35494235
In Drosophila melanogaster, olfactory projection neurons (PNs) convey odor information from the antenna lobe to higher brain regions. Recent transcriptomic studies reveal a large diversity of transcription factors, cell-surface molecules, neurotransmitter-coding, and neuropeptide-coding genes in PNs; however, their structural diversity remains unknown. This study achieved a volumetric reconstruction of 89 PN boutons under Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and quantitatively analyzed the internal presynaptic active zones (PAZs) and dense-core vesicles (DCVs). The ultrastructure-based cluster analysis reveals three morphological distinct bouton subtypes: complex boutons, unilobed boutons, and simple boutons. The complex boutons contain the most PAZs and DCVs, which suggests that they are of the highest capability of releasing neurotransmitters and neuromodulators. By labeling a subset of boutons under FIB-SEM, it was found that DCVs are preferentially distributed in certain GH146-positive subtypes. This study demonstrates that PN boutons display distinct morphology, which may determine their capacity of releasing neurotransmitters and neuromodulators.

Monday, September 12th - Adult Development

Zheng, H., Lou, Z., Yuan, X., Wu, H., Yang, X. and Xi, Y. (2022). Phosphatase of Regenerating Liver-1 Regulates Wing Vein Formation through TGF-β Pathway in Drosophila melanogaster. Front Biosci (Landmark Ed) 27(6): 176. PubMed ID: 35748252
Drosophila Phosphatase of Regenerating Liver-1 (PRL-1) is the only homolog of the mammalian PRLs with which it shares high sequence and structural similarities. Whilst PRLs are most notable for their high expression in malignant cancers and related promotion of cancer progression, the specific biological functions of the PRLs remain largely elusive. Using a gain-of-function approach, it was found that PRL-1 functions during wing vein development in Drosophila melanogaster. Overexpression of Drosophila PRL-1 caused dose-dependent wing vein proliferation. Genetic screening of the main TGF-;beta; signaling factors, Mad and Smox, showed that the RNAi-mediated knockdown of Mad could alleviate the extra vein phenotype caused by overexpressed PRL-1 and lead to loss of the posterior section of longitudinal veins. However, knockdown of Smox resulted in an identical phenotype with or without the overexpression of Drosophila PRL-1. Clonal analyses revealed that overexpression of PRL-1 led to decreased expressions of activated phospho-Mad protein, as measured by immunostaining. Real-time PCR showed that the transcriptional levels of Smox were significantly increased upon overexpression of the Drosophila PRL-1 in wing discs, with a dose dependent effect. This study proposed that the main function of Drosophila PRL-1 in wing development is to affect the phospho-Mad levels and Smox transcriptional levels, therefore influencing the competitive balance for Medea between Mad and Smox. This study demonstrates the novel role for Drosophila PRL-1 in regulating TGF-β signaling to influence wing vein formation which may also provide insight into the understanding of the relationship between PRLs and TGF-β signaling in mammals.
Athilingam, T., Parihar, S. S., Bhattacharya, R., Rizvi, M. S., Kumar, A. and Sinha, P. (2022). Proximate larval epidermal cell layer generates forces for Pupal thorax closure in Drosophila. Genetics 221(1). PubMed ID: 35166774
During tissue closures, such as embryonic dorsal closure in Drosophila melanogaster, a proximate extra-embryonic layer, amnioserosa, generates forces that drive migration of the flanking lateral embryonic epidermis, thereby zip-shutting the embryo. Arguably, this paradigm of tissue closure is also recapitulated in mammalian wound healing wherein proximate fibroblasts transform into contractile myofibroblasts, develop cell junctions, and form a tissue layer de novo: contraction of the latter then aids in wound closure. Given this parallelism between disparate exemplars, a general principle of tissue closure is posited via proximate cell layer-generated forces. This hypothesis was tested in pupal thorax closure wherein 2 halves of the presumptive adult thorax of Drosophila, the contralateral heminotal epithelia, migrate over an underlying larval epidermal cell layer. The proximate larval epidermal cell layer promotes thorax closure by its active contraction, orchestrated by its elaborate actomyosin network-driven epithelial cell dynamics, cell delamination, and death-the latter being prefigured by the activation of caspases. Larval epidermal cell dynamics generate contraction forces, which when relayed to the flanking heminota-via their mutual integrin-based adhesions-mediate thorax closure. Compromising any of these contraction force-generating mechanisms in the larval epidermal cell layer slows down heminotal migration, while loss of its relay to the flanking heminota abrogates the thorax closure altogether. Mathematical modeling further reconciles the biophysical underpinning of this emergent mechanism of thorax closure. Revealing mechanism of thorax closure apart, these findings show conservation of an essential principle of a proximate cell layer-driven tissue closure.
Hayashi, T., Tomomizu, T., Sushida, T., Akiyama, M., Ei, S. I. and Sato, M. (2022). Tiling mechanisms of the Drosophila compound eye through geometrical tessellation. Curr Biol 32(9): 2101-2109. PubMed ID: 35390281
Tiling patterns are observed in many biological structures. The compound eye is an interesting example of tiling and is often constructed by hexagonal arrays of ommatidia, the optical unit of the compound eye. Hexagonal tiling may be common due to mechanical restrictions such as structural robustness, minimal boundary length, and space-filling efficiency. However, some insects exhibit tetragonal facets. Some aquatic crustaceans, such as shrimp and lobsters, have evolved with tetragonal facets. Mantis shrimp is an insightful example as its compound eye has a tetragonal midband region sandwiched between hexagonal hemispheres. This casts doubt on the naive explanation that hexagonal tiles recur in nature because of their mechanical stability. Similarly, tetragonal tiling patterns are also observed in some Drosophila small-eye mutants, whereas the wild-type eyes are hexagonal, suggesting that the ommatidial tiling is not simply explained by such mechanical restrictions. If so, how are the hexagonal and tetragonal patterns controlled during development? This study demonstrates that geometrical tessellation determines the ommatidial tiling patterns. In small-eye mutants, the hexagonal pattern is transformed into a tetragonal pattern as the relative positions of neighboring ommatidia are stretched along the dorsal-ventral axis. It is proposed that the regular distribution of ommatidia and their uniform growth collectively play an essential role in the establishment of tetragonal and hexagonal tiling patterns in compound eyes.
Lerch, S., Yang, Y., Flaven-Pouchon, J., Gehring, N. and Moussian, B. (2022). Resilin is needed for wing posture in Drosophila suzukii. Arch Insect Biochem Physiol: e21913. PubMed ID: 35599599
Resilin is a protein matrix in movable regions of the cuticle conferring resistance to fatigue. The main component of Resilin is Pro-Resilin that polymerises via covalent di- and tri-tyrosine bounds (DT). Loss of Pro-Resilin is nonlethal and causes a held-down wing phenotype (hdw) in the fruit fly Drosophila melanogaster. To test whether this mild phenotype is recurrent in other insect species, resilin was analyzed in the spotted-wing fruit fly Drosophila suzukii. As quantified by DT autofluorescence by microscopy, DT intensities in the trochanter and the wing hinge are higher in D. suzukii than in D. melanogaster, while in the proboscis the DT signal is stronger in D. melanogaster compared to D. suzukii. To study the function of Pro-Resilin in D. suzukii, a mutation was generated in the proresilin gene applying the Crispr/Cas9 technique. D. suzukii pro-resilin mutant flies are flight-less and show a hdw phenotype resembling respective D. melanogaster mutants. DT signal intensity at the wing hinge is reduced but not eliminated in D. suzukii hdw flies. Either residual Pro-Resilin accounts for the remaining DT signal or, as proposed for the hdw phenotype in D. melanogaster, other DT forming proteins might be present in Resilin matrices. Interestingly, DT signal intensity reduction rates in D. suzukii and D. melanogaster are somehow different. Taken together, in general, the function of Pro-Resilin seems to be conserved in the Drosophila genus; small differences in DT quantity, however, lead to the hypothesis that Resilin matrices might be modulated during evolution probably to accommodate the species-specific lifestyle.
Ajayi, P. T., Katti, P., Zhang, Y., Willingham, T. B., Sun, Y., Bleck, C. K. E. and Glancy, B. (2022). Regulation of the evolutionarily conserved muscle myofibrillar matrix by cell type dependent and independent mechanisms. Nat Commun 13(1): 2661. PubMed ID: 35562354
Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. It has been recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. This study demonstrates the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster. Moreover, it was found that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and it was shown that sarcomere branching can be turned on in IF muscles by salm-mediated conversion to tubular muscles. Finally, it was demonstrated that neurochondrin misexpression results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms.
Velasquez, E., Gomez-Sanchez, J. A., Donier, E., Grijota-Martinez, C., Cabedo, H. and Garcia-Alonso, L. (2022). Fasciclin 2 engages EGFR in an auto-stimulatory loop to promote imaginal disc cell proliferation in Drosophila. PLoS Genet 18(6): e1010224. PubMed ID: 35666718
How cell to cell interactions control local tissue growth to attain a species-specific organ size is a central question in developmental biology. The Drosophila Neural Cell Adhesion Molecule, Fasciclin 2, is expressed during the development of neural and epithelial organs. Fasciclin 2 is a homophilic-interaction protein that shows moderate levels of expression in the proliferating epithelia and high levels in the differentiating non-proliferative cells of imaginal discs. Genetic interactions and mosaic analyses reveal a cell autonomous requirement of Fasciclin 2 to promote cell proliferation in imaginal discs. This function is mediated by the EGFR, and indirectly involves the JNK and Hippo signaling pathways. it was further shown that Fasciclin 2 physically interacts with EGFR and that, in turn, EGFR activity promotes the cell autonomous expression of Fasciclin 2 during imaginal disc growth. It is proposed that this auto-stimulatory loop between EGFR and Fasciclin 2 is at the core of a cell to cell interaction mechanism that controls the amount of intercalary growth in imaginal discs.

Friday September 9th - Adult Physiology

Rai, M., Carter, S. M., Shefali, S., Mahmoudzadeh, N. H., Pepin, R. and Tennessen, J. M. (2022). The Drosophila melanogaster enzyme glycerol-3-phosphate dehydrogenase 1 is required for oogenesis, embryonic development, and amino acid homeostasis. G3 (Bethesda). PubMed ID: 35536221
As the fruit fly, Drosophila melanogaster, progresses from one life stage to the next, many of the enzymes that compose intermediary metabolism undergo substantial changes in both expression and activity. These predictable shifts in metabolic flux allow the fly meet stage-specific requirements for energy production and biosynthesis. In this regard, the enzyme Glycerol-3-phosphate dehydrogenase (GPDH1) has been the focus of biochemical genetics studies for several decades, and as a result, is one of the most well-characterized Drosophila enzymes. Among the findings of these earlier studies is that GPDH1 acts throughout the fly lifecycle to promote mitochondrial energy production and triglyceride accumulation while also serving a key role in maintaining redox balance. This study expanded upon the known roles of GPDH1 during fly development by examining how depletion of both the maternal and zygotic pools of this enzyme influences development, metabolism, and viability. The findings not only confirm previous observations that Gpdh1 mutants exhibit defects in larval development, lifespan, and fat storage but also reveal that GPDH1 serves essential roles in oogenesis and embryogenesis. Moreover, metabolomics analysis reveals that a Gpdh1 mutant stock maintained in a homozygous state exhibits larval metabolic defects that significantly differ from those observed in the F1 mutant generation. Overall, these findings highlight unappreciated roles for GPDH1 in early development and uncover previously undescribed metabolic adaptations that could allow flies to survive loss of this key enzyme.
Zhang, Y. C., Pei, X. G., Yu, Z. T., Gao, Y., Wang, L. X., Zhang, N., Song, X. Y., Wu, S. F. and Gao, C. F. (2022). Effects of nicotinic acetylcholine receptor subunit deletion mutants on insecticide susceptibility and fitness in Drosophila melanogaster. Pest Manag Sci. PubMed ID: 35576366
Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in insects and also the target site for many insecticides. Unfortunately, the effectiveness of these insecticides is diminishing as a consequence of the evolution of insecticide resistance. Further exploration of insecticide targets is important to sustainable pest management. In order to validate the role of nAChR subunits in insecticide susceptibility and test whether the subunit's absence imposes the fitness cost on insects, the susceptibility of eight nAChR subunit deletion mutants of Drosophila melanogaster to nine insecticides was tested. These findings highlighted the specific resistance of the Dα6 deletion mutant to spinosyns. Although triflumezopyrim, dinotefuran and imidacloprid are competitive modulators of nAChRs, differences in susceptibility of the insect with different deletion mutants suggested that the target sites of these three insecticides do not overlap completely. Mutants showed decreased susceptibility to insecticides, accompanied by a reduction in fitness. The number of eggs produced by Dα1(attP), Dα2(attP), Dβ2(attP) and Dβ3(attP) females was significantly lesser than that of the vas-Cas9 strain as the control. In addition, adults of Dα2(attP) , Dα3(attP) and Dα7(attP) strains showed lower climbing performance. Meanwhile, males of Dα3(attP) , Dα5(attP) , Dβ2(attP) and Dβ3(attP) , and females of Dβ2(attP) showed significantly shorter longevity than those of the vas-Cas9 strain. This study provides new insights into the interactions of different insecticides with different nAChRs subunit in D. melanogaster as a research model, it could help better understand such interaction in agricultural pests whose genetic manipulations for toxicological research are often challenging.
Murari, A., Goparaju, N. S. V., Rhooms, S. K., Hossain, K. F. B., Liang, F. G., Garcia, C. J., Osei, C., Liu, T., Li, H., Kitsis, R. N., Patel, R. and Owusu-Ansah, E. (2022). IDH2-mediated regulation of the biogenesis of the oxidative phosphorylation system. Sci Adv 8(19): eabl8716. PubMed ID: 35544578
Several subunits in the matrix domain of mitochondrial complex I (CI) have been posited to be redox sensors for CI, but how elevated levels of reactive oxygen species (ROS) impinge on CI assembly is unknown. This study reports that genetic disruption of the mitochondrial NADPH-generating enzyme, isocitrate dehydrogenase 2 (IDH2), in Drosophila flight muscles results in elevated ROS levels and impairment of assembly of the oxidative phosphorylation system (OXPHOS). Mechanistically, this begins with an inhibition of biosynthesis of the matrix domain of CI and progresses to involve multiple OXPHOS complexes. Despite activation of multiple compensatory mechanisms, including enhanced coenzyme Q biosynthesis and the mitochondrial unfolded protein response, ferroptotic cell death ensues. Disruption of enzymes that eliminate hydrogen peroxide, but not those that eliminate the superoxide radical, recapitulates the phenotype, thereby implicating hydrogen peroxide as the signaling molecule involved. Thus, IDH2 modulates the assembly of the matrix domain of CI and ultimately that of the entire OXPHOS.
Rose, S., Beckwith, E. J., Burmester, C., May, R. C., Dionne, M. S. and Rezaval, C. (2022). Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria. Proc Biol Sci 289(1974): 20220492. PubMed ID: 35538789
The activation of the immune system upon infection exerts a huge energetic demand on an individual, likely decreasing available resources for other vital processes, like reproduction. The factors that determine the trade-off between defensive and reproductive traits remain poorly understood. This stydt exploited the experimental tractability of the fruit fly Drosophila melanogaster to systematically assess the impact of immune system activation on pre-copulatory reproductive . Contrary to expectations, it was found that male flies undergoing an immune activation continue to display high levels of courtship and mating success. Similarly, immune-challenged female flies remain highly sexually receptive. By combining behavioural paradigms, a diverse panel of pathogens and genetic strategies to induce the fly immune system, this study shows that pre-copulatory reproductive behaviours are preserved in infected flies, despite the significant metabolic cost of infection.
Santalla, M., García, A., Mattiazzi, A., Valverde, C. A., Schiemann, R., Paululat, A., Hernandez, G., Meyer, H. and Ferrero, P. (2022). Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart. PLoS One 17(5): e0267156. PubMed ID: 35588119
Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. This study gives evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, it was demonstrated that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads to a proposal that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity.
Aryal, B. and Lee, Y. (2022). Histamine avoidance through three gustatory receptors in Drosophila melanogaster. Insect Biochem Mol Biol 144: 103760. PubMed ID: 35346814
Histamine is a fermented food product that exerts adverse health effects on animals when consumed in high amounts. This biogenic amine is fermented by microorganisms from histidine through the activity of histidine decarboxylase. Drosophila melanogaster can discriminate histidine and histamine using GR22e and IR76b in bitter-sensing gustatory receptor neurons (GRNs). In this study, RNA interference screens were conducted to examine 28 uncharacterized gustatory receptor genes using electrophysiology and behavioral experiments, including the binary food choice and proboscis extension response assays. GR9a and GR98a were first identified as specific histamine receptors by evaluating newly generated null mutants and recovery experiments by expressing their wild-type cDNA in the bitter-sensing GRNs. It was further determined that histamine sensation was mainly mediated by the labellum but not by the legs, as demonstrated by the proboscis extension response assay. These findings indicated that toxic histamine directly activates bitter-sensing GRNs in S-type sensilla, and this response is mediated by the GR9a, GR22e, and GR98a gustatory receptors.

Thursday, September 8th - Chromatin

Clay, D. E., Jezuit, E. A., Montague, R. A. and Fox, D. T. (2022). Conserved Function Of Drosophila Fancd2 Monoubiquitination In Response To Double-Strand DNA Breaks. G3 (Bethesda). PubMed ID: 35595243
Fanconi Anemia (FA) genes play key roles in metazoan DNA damage responses, and human FA mutations cause numerous disease phenotypes. In human cells, activating monoubiquitination of the FA protein Fancd2 occurs following diverse DNA damage stimuli. Monoubiquitinated Fancd2 forms nuclear foci to recruit additional repair factors. Fancd2 animal models to date have focused on molecular nulls or whole gene knockdown, leaving the specific in vivo role of monoubiquitination unclear. Using a point mutant in a conserved residue, Drosophila Fancd2 monoubiquitination has been linked to a mitosis-specific DNA double-strand break (DSB) response. In this context, CRISPR/Cas9 was used to generate the first animal model of an endogenous mutation in the conserved monoubiquitination site (fancd2K595R). This study expands on the characterization of fancd2K595R. Additional Drosophila tools were introduced and characterized to study fancd2, including new mutant alleles and GFP-tagged rescue transgenes. Using these new reagents, the impact was shown of Drosophila Fancd2 on organismal and cell viability, as well as on repair protein localization, in the presence or absence of DSBs. These findings expand understanding of FA gene function in vivo and provide useful reagents for DNA repair research.
Cheng, Q., Xie, H., Zhang, X. Y., Wang, M. Y., Bi, C. L., Wang, Q., Wang, R. and Fang, M. (2022). An essential role for PTIP in mediating Hox gene regulation along PcG and trxG pathways. Febs J. PubMed ID: 35655387
During Drosophila development, Polycomb-group and Trithorax group proteins function to ensure correct maintenance of transcription patterns by epigenetically repressing or activating target gene expression. To get a deep insight into the PcG and trxG pathways, this study investigated a BRCT domain-containing protein called PTIP, which was generally identified as a transcriptional coactivator and belongs to the TRR complex. At the genome scale, given PTIP-binding peaks were sorted into two groups: PTIP/TRR-cobound and PTIP/PC-cobound peaks. In particular, it was found that PTIP mediates the molecular switch between H3K4me3/H3K27ac and H3K27me3 histone modifications at TRR or PC occupied regions. Thus, it is suggested that PTIP is a mediator rather than a dedicated co-activator along PcG and trxG pathways. This hypothesis is further supported by the genetic assay: PTIP interacts genetically with either PcG or TrxG in a dosage-dependent manner, suggesting that PTIP functions as a co-factor of PcG/TrxG proteins. In addition, in accordance with the analysis of ChIP-seq, these genetic interactions correlate with modified ectopic HOX protein levels in imaginal discs, which reveals an essential role for PTIP in PcG-mediated Hox gene repression. Hence, this study revealed a novel role for PTIP in the epigenetic regulation of gene expression along PcG and trxG pathways.
Kaushal, A., Dorier, J., Wang, B., Mohana, G., Taschner, M., Cousin, P., Waridel, P., Iseli, C., Semenova, A., Restrepo, S., Guex, N., Aiden, E. L. and Gambetta, M. C. (2022). Essential role of Cp190 in physical and regulatory boundary formation. Sci Adv 8(19): eabl8834. PubMed ID: 35559678
Boundaries in animal genomes delimit contact domains with enhanced internal contact frequencies and have debated functions in limiting regulatory cross-talk between domains and guiding enhancers to target promoters. Most mammalian boundaries form by stalling of chromosomal loop-extruding cohesin by CTCF, but most Drosophila boundaries form CTCF independently. However, how CTCF-independent boundaries form and function remains largely unexplored. This study assessed genome folding and developmental gene expression in fly embryos lacking the ubiquitous boundary-associated factor Cp190. Sequence-specific DNA binding proteins such as CTCF and Su(Hw) directly interact with and recruit Cp190 to form most promoter-distal boundaries. Cp190 is essential for early development and prevents regulatory cross-talk between specific gene loci that pattern the embryo. Cp190 was, in contrast, dispensable for long-range enhancer-promoter communication at tested loci. Cp190 is thus currently the major player in fly boundary formation and function, revealing that diverse mechanisms evolved to partition genomes into independent regulatory domains.
Gaultier, C., Foppolo, S. and Maurange, C. (2022). Regulation of developmental hierarchy in Drosophila neural stem cell tumors by COMPASS and Polycomb complexes. Sci Adv 8(19): eabi4529. PubMed ID: 35544555
COMPASS and Polycomb complexes are antagonistic chromatin complexes that are frequently inactivated in cancers, but how these events affect the cellular hierarchy, composition, and growth of tumors is unclear. These characteristics can be systematically investigated in Drosophila neuroblast tumors in which cooption of temporal patterning induces a developmental hierarchy that confers cancer stem cell (CSC) properties to a subset of neuroblasts retaining an early larval temporal identity. Using single-cell transcriptomics, this study reveal thats the trithorax/MLL1/2-COMPASS-like complex guides the developmental trajectory at the top of the tumor hierarchy. Consequently, trithorax knockdown drives larval-to-embryonic temporal reversion and the marked expansion of CSCs that remain locked in a spectrum of early temporal states. Unexpectedly, this phenotype is amplified by concomitant inactivation of Polycomb repressive complex 2 genes, unleashing tumor growth. This study illustrates how inactivation of specific COMPASS and Polycomb complexes cooperates to impair tumor hierarchies, inducing CSC plasticity, heterogeneity, and expansion.
Zhu, Y., Dong, L., Wang, C., Hao, K., Wang, J., Zhao, L., Xu, L., Xia, Y., Jiang, Q. and Qin, J. (2022). Functional redundancy among Polycomb complexes in maintaining the pluripotent state of embryonic stem cells. Stem Cell Reports 17(5): 1198-1214. PubMed ID: 35364009
Polycomb group proteins assemble into multi-protein complexes, known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), that guide cell fate decisions during embryonic development. PRC1 forms an array of biochemically distinct canonical PRC1 (cPRC1) or non-canonical PRC1 (ncPRC1) complexes characterized by the mutually exclusive presence of PCGF (PCGF1-PCGF6) paralog subunit; however, whether each one of these subcomplexes fulfills a distinct role remains largely controversial. By performing a CRISPR-based loss-of-function screen in embryonic stem cells (ESCs), this study uncovered a previously unappreciated functional redundancy among PRC1 subcomplexes. Disruption of ncPRC1, but not cPRC1, displayed severe defects in ESC pluripotency. Remarkably, coablation of non-canonical and canonical PRC1 in ESCs resulted in exacerbation of the phenotype observed in the non-canonical PRC1-null ESCs, highlighting the importance of functional redundancy among PRC1 subcomplexes. Together, these studies demonstrate that PRC1 subcomplexes act redundantly to silence lineage-specific genes and ensure robust maintenance of ESC identity.
Ullah, I., Tholken, C., Zhong, Y., John, M., Rossbach, O., Lenz, J., Gossringer, M., Nist, A., Albert, L., Stiewe, T., Hartmann, R., Vazquez, O., Chung, H. R., Mackay, J. P. and Brehm, A. (2022). RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodeling. Cell Rep 39(9): 110895. PubMed ID: 35649367
The ATP-dependent nucleosome remodeler Mi-2/CHD4 broadly modulates chromatin landscapes to repress transcription and to maintain genome integrity. This study used individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to show that Drosophila Mi-2 associates with thousands of mRNA molecules in vivo. Biochemical data reveal that recombinant dMi-2 preferentially binds to G-rich RNA molecules using two intrinsically disordered regions of unclear function. Pharmacological inhibition of transcription and RNase digestion approaches establish that RNA inhibits the association of dMi-2 with chromatin. It was also shown that RNA inhibits dMi-2-mediated nucleosome mobilization by competing with the nucleosome substrate. Importantly, this activity is shared by CHD4, the human homolog of dMi-2, strongly suggesting that RNA-mediated regulation of remodeler activity is an evolutionary conserved mechanism. These data support a model in which RNA serves to protect actively transcribed regions of the genome from dMi-2/CHD4-mediated establishment of repressive chromatin structures.

Wednesday, September 7th - Disease Models

Morozova, T. V., Shankar, V., MacPherson, R. A., Mackay, T. F. C. and Anholt, R. R. H. (2022). Modulation of the Drosophila transcriptome by developmental exposure to alcohol. BMC Genomics 23(1): 347. PubMed ID: 35524193
Prenatal exposure to ethanol can cause fetal alcohol spectrum disorder (FASD), a prevalent, preventable pediatric disorder. Identifying genetic risk alleles for FASD is challenging since time, dose, and frequency of exposure are often unknown, and manifestations of FASD are diverse and evident long after exposure. Drosophila melanogaster is an excellent model to study the genetic basis of the effects of developmental alcohol exposure since many individuals of the same genotype can be reared under controlled environmental conditions. This study used 96 sequenced, wild-derived inbred lines from the Drosophila melanogaster Genetic Reference Panel (DGRP) to profile genome-wide transcript abundances in young adult flies that developed on ethanol-supplemented medium or standard culture medium. Substantial genetic variation was found in gene expression in response to ethanol with extensive sexual dimorphism. Sex-specific genetic networks associated with alcohol-dependent modulation of gene expression were constructed that include the following: protein-coding genes, Novel Transcribed Regions (NTRs, postulated to encode long non-coding RNAs) and female-specific coordinated regulation of snoRNAs that regulate pseudouridylation of ribosomal RNA. DGRP lines which showed extreme upregulation or downregulation of snoRNA expression during developmental alcohol exposure were reared on standard or ethanol supplemented medium ; it was demonstrated that developmental exposure to ethanol has genotype-specific effects on adult locomotor activity and sleep. There is significant and sex-specific natural genetic variation in the transcriptional response to developmental exposure to ethanol in Drosophila that comprises networks of genes affecting nervous system development and ethanol metabolism as well as networks of regulatory non-coding RNAs.
Nisha and Sarkar, S. (2022). Downregulation of glob1 mitigates human tau mediated neurotoxicity by restricting heterochromatin loss and elevating the autophagic response in Drosophila. Mol Biol Rep. PubMed ID: 35633418
Human neuronal tauopathies are typically characterized by the accumulation of hyperphosphorylated tau in the forms of paired helical filaments and/or neurofibrillary tangles in the brain neurons. Tau-mediated heterochromatin loss and subsequent global transcriptional upsurge have been demonstrated as one of the key factors that promotes tau toxicity. It has been reported earlier that expression of human tau-transgene in Drosophila induces the expression of glob1, and its restored level restricts tau etiology by regulating tau hyperphosphorylation and ROS generation via GSK-3β/p-Akt and Nrf2-keap1-ARE pathways, respectively. In view of this noted capability of glob1 in regulation of oxidative stress, and involvement of ROS in chromatin remodeling; this study investigated if downregulation of glob1 restores tau-mediated heterochromatin loss in order to alleviate neurotoxicity. The tauV337M transgene was expressed in Drosophila eye by utilizing GAL4/UAS system. Expression of glob1 was depleted in tauV337M expressing tissues by co-expressing an UAS-glob1RNAi transgene by GMR-Gal4 driver. Immunostaining and wstern blot analysis suggested that tissue-specific downregulation of glob1 restores the cellular level of CBP and minimizes tau-mediated heterochromatin loss. It also assists in mounting an improved protective autophagic response to alleviate the human tau-induced neurotoxicity in Drosophila tauopathy models. This study unfolds a novel aspect of the multitasking globin protein in restricting the pathogenesis of neuronal tauopathies. Interestingly, due to notable similarities between Drosophila glob1 and human globin gene(s), these findings may be helpful in developing novel therapeutic approaches against tauopathies.
Neves, P. F. R., Milanesi, B. B., Paz, L. V., de Miranda Monteiro, V. A. C., Neves, L. T., da Veiga, L. C., da Silva, R. B., Sulzbach, J. H., Knijkik, G. P., de Revoredo Ribeiro, E. C., de Souza Silva, E. L., Vieira, M. Q., Bagatini, P. B., Wieck, A., Mestriner, R. G. and Xavier, L. L. (2022). Age-related tolerance to paraquat-induced parkinsonism in Drosophila melanogaster. Toxicol Lett 361: 43-53. PubMed ID: 35367327
Paraquat (PQ) is a widely used herbicide that can cross the dopaminergic neuronal membrane, accumulate in mitochondria and damage complex I of the electron transport chain, leading to neuronal death. In Drosophila melanogaster, PQ exposure leads to the development of parkinsonism and is a classical model for studying Parkinson's Disease (PD). Muscle mitochondrial dysfunction, affecting survival and locomotion, is described in familial PD in D. melanogaster mutants. However, no study has shown the effects of PQ-induced parkinsonism in D. melanogaster regarding muscle ultrastructure and locomotor behavior at different ages. Thus, this study evaluated survival, locomotion, and morphological parameters of mitochondria and myofibrils using transmission electron microscopy in 2 and 15-day-old D. melanogaster, treated with different PQ doses: control, 10, 50, 100, 150, and 200 mM. PQ100mM presented 100% lethality in 15-day-old D. melanogaster, while in 2-day-old animals PQ150mM produced 20% lethality. Bradykinesia was only observed in 15-day-old D. melanogaster treated with PQ10 mM and PQ50 mM. However, these results are unlikely to be associated with changes to morphology. Taken together, these data indicate pathophysiological differences between PQ-induced parkinsonism and familial parkinsonism in D. melanogaster (resultant from gene mutations), demonstrating for the first time a differential susceptibility to PQ in two developmental stages.
François-Moutal, L., Scott, D. D., Ambrose, A. J., Zerio, C. J., Rodriguez-Sanchez, M., Dissanayake, K., May, D. G., Carlson, J. M., Barbieri, E., Moutal, A., Roux, K. J., Shorter, J., Khanna, R., Barmada, S. J., McGurk, L. and Khanna, M. (2022). Heat shock protein Grp78/BiP/HspA5 binds directly to TDP-43 and mitigates toxicity associated with disease pathology. Sci Rep 12(1): 8140. PubMed ID: 35581326
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, this study investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. It was further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression, the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly this study observed upregulation of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.
Chiereghin, C., Robusto, M., Massa, V., Castorina, P., Ambrosetti, U., Asselta, R. and Solda, G. (2022). Role of Cytoskeletal Diaphanous-Related Formins in Hearing Loss. Cells 11(11). PubMed ID: 35681420
Hearing relies on the proper functioning of auditory hair cells and on actin-based cytoskeletal structures. Diaphanous-related formins (DRFs) are evolutionarily conserved cytoskeletal proteins that regulate the nucleation of linear unbranched actin filaments. They play key roles during metazoan development, and they seem particularly pivotal for the correct physiology of the reproductive and auditory systems. Indeed, in Drosophila melanogaster, a single diaphanous (dia) gene is present, and mutants show sterility and impaired response to sound. Vertebrates, instead, have three orthologs of the diaphanous gene: DIAPH1, DIAPH2, and DIAPH3. In humans, defects in DIAPH1 and DIAPH3 have been associated with different types of hearing loss. In particular, heterozygous mutations in DIAPH1 are responsible for autosomal dominant deafness with or without thrombocytopenia (DFNA1, MIM #124900), whereas regulatory mutations inducing the overexpression of DIAPH3 cause autosomal dominant auditory neuropathy 1 (AUNA1, MIM #609129). This study provides an overview of the expression and function of DRFs in normal hearing and deafness.
Palu, R. A. S., Owings, K. G., Garces, J. G. and Nicol, A. (2022). A natural genetic variation screen identifies insulin signaling, neuronal communication, and innate immunity as modifiers of hyperglycemia in the absence of Sirt1. G3 (Bethesda) 12(6). PubMed ID: 35435227
Variation in the onset, progression, and severity of symptoms associated with metabolic disorders such as diabetes impairs the diagnosis and treatment of at-risk patients. Diabetes symptoms, and patient variation in these symptoms, are attributed to a combination of genetic and environmental factors, but identifying the genes and pathways that modify diabetes in humans has proven difficult. A greater understanding of genetic modifiers and the ways in which they interact with metabolic pathways could improve the ability to predict a patient's risk for severe symptoms, as well as enhance the development of individualized therapeutic approaches. This study usef the Drosophila Genetic Reference Panel to identify genetic variation influencing hyperglycemia associated with loss of Sirt1 function. Through analysis of individual candidate functions, physical interaction networks, and gene set enrichment analysis, this study identified not only modifiers involved in canonical glucose metabolism and insulin signaling, but also genes important for neuronal signaling and the innate immune response. Furthermore, reducing the expression of several of these candidates suppressed hyperglycemia, making them potential candidate therapeutic targets. These analyses showcase the diverse processes contributing to glucose homeostasis and open up several avenues of future investigation.

Tuesday, September 7 - Disease models

Adashev, V. E., Bazylev, S. S., Potashnikova, D. M., Godneeva, B. K., Shatskikh, A. S., Olenkina, O. M., Olenina, L. V. and Kotov, A. A. (2022). Comparative transcriptional analysis uncovers molecular processes in early and mature somatic cyst cells of Drosophila testes. Eur J Cell Biol 101(3): 151246. PubMed ID: 35667338
The tight interaction between somatic and germline cells is conserved in animal spermatogenesis. The testes of Drosophila melanogaster are the model of choice to identify processes responsible for mature gamete production. However, processes of differentiation and soma-germline interactions occurring in somatic cyst cells are currently understudied. This study focused on the comparison of transcriptome expression patterns of early and mature somatic cyst cells to find out the developmental changes taking place in them. A FACS-based approach was employed for the isolation of early and mature somatic cyst cells from fly testes, subsequent preparation of RNA-Seq libraries, and analysis of gene differential expression in the sorted cells. Increased expression was found of genes involved in cell cycle-related processes in early cyst cells, which is necessary for the proliferation and self-renewal of a crucial population of early cyst cells, cyst stem cells. Genes proposedly required for lamellipodium-like projection organization for proper cyst formation were also detected among the upregulated ones in early cyst cells. Gene Ontology and interactome analyses of upregulated genes in mature cyst cells revealed a striking over-representation of gene categories responsible for metabolic and catabolic cellular processes, as well as genes supporting the energetic state of the cells provided by oxidative phosphorylation that is carried out in mitochondria. This comparative analyses of differentially expressed genes revealed major peculiarities in early and mature cyst cells and provide novel insight into their regulation, which is important for male fertility.
Kaur, R., Leigh, B. A., Ritchie, I. T. and Bordenstein, S. R. (2022). The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility. PLoS Biol 20(5): e3001584. PubMed ID: 35609042
Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally transmitted Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. This study used cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild-type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers; however, Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, these results strongly support the Host modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue.
Butsch, T. J., Dubuisson, O., Johnson, A. E. and Bohnert, K. A. (2022). A meiotic switch in lysosome activity supports spermatocyte development in young flies but collapses with age. iScience 25(6): 104382. PubMed ID: 35620438
Gamete development ultimately influences animal fertility. Identifying mechanisms that direct gametogenesis, and how they deteriorate with age, may inform ways to combat infertility. Recentl work has shown that lysosomes acidify during oocyte maturation in Caenorhabditis elegans, suggesting that a meiotic switch in lysosome activity promotes female germ-cell health. Using Drosophila melanogaster, this study reports that lysosomes likewise acidify in male germ cells during meiosis. Inhibiting lysosomes in young-male testes causes E-cadherin accumulation and loss of germ-cell partitioning membranes. Notably, analogous changes occur naturally during aging; in older testes, a reduction in lysosome acidity precedes E-cadherin accumulation and membrane dissolution, suggesting one potential cause of age-related spermatocyte abnormalities. Consistent with lysosomes governing the production of mature sperm, germ cells with homozygous-null mutations in lysosome-acidifying machinery fail to survive through meiosis. Thus, lysosome activation is entrained to meiotic progression in developing sperm, as in oocytes, and lysosomal dysfunction may instigate male reproductive aging.
Varga, V. B., Schuller, D., Szikszai, F., Szinykovics, J., Puska, G., Vellai, T. and Kovacs, T. (2022). Autophagy is required for spermatogonial differentiation in the Drosophila testis. Biol Futur 73(2): 187-204. PubMed ID: 35672498
Autophagy is a conserved, lysosome-dependent catabolic process of eukaryotic cells which is involved in cellular differentiation. Its specific role in the differentiation of spermatogonial cells in the Drosophila testis was studied. In the apical part of the Drosophila testis, there is a niche of germline stem cells (GSCs), which are connected to hub cells. Hub cells emit a ligand for bone morhphogenetic protein (BMP)-mediated signalling that represses Bam (bag of marbles) expression in GSCs to maintain them in an undifferentiated state. GSCs divide asymmetrically, and one of the daughter cells differentiates into a gonialblast, which eventually generates a cluster of spermatogonia (SG) by mitoses. Bam is active in SG, and defects in Bam function arrest these cells at mitosis. This study shows that BMP signalling represses autophagy in GSCs, but upregulates the process in SG. Inhibiting autophagy in SG results in an overproliferating phenotype similar to that caused by bam mutations. Furthermore, Bam deficiency leads to a failure in downstream mechanisms of the autophagic breakdown. These results suggest that the BMP-Bam signalling axis regulates developmental autophagy in the Drosophila testis, and that acidic breakdown of cellular materials is required for spermatogonial differentiation.
Wu, Z. and Liu, J. L. (2022). CTP synthase does not form cytoophidia in Drosophila interfollicular stalks. Exp Cell Res 418(1): 113250. PubMed ID: 35691380
CTP synthase (CTPS) catalyzes the final step of de novo synthesis of the nucleotide CTP. In 2010, CTPS has been found to form filamentous structures termed cytoophidia in Drosophila follicle cells and germline cells. Subsequently, cytoophidia have been reported in many species across three domains of life: bacteria, eukaryotes and archaea. Forming cytoophidia appears to be a highly conserved and ancient property of CTPS. Surprisingly, this study found that polar cells and stalk cells, two specialized types of cells composing Drosophila interfollicular stalks, do not possess obvious cytoophidia. Myc level is low in these two types of cells. Treatment with a glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), increases cytoophidium assembly in main follicle cells, but not in polar cells or stalk cells. Moreover, overexpressing Myc induces cytoophidium formation in stalk cells. When CTPS is overexpressed, cytoophidia can be observed both in stalk cells and polar cells. These findings provide an interesting paradigm for the in vivo study of cytoophidium assembly and disassembly among different populations of follicle cells.
Topfer, U., Guerra Santillan, K. Y., Fischer-Friedrich, E. and Dahmann, C. (2022). Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila. Development 149(10). PubMed ID: 35575071
The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. This study systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. The Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, this study showed that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy it was shown that basement membrane stiffness mainly depends on Collagen IV. This analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Friday September 2nd - Signaling

Qu, C., Yang, W., Kan, Y., Zuo, H., Wu, M., Zhang, Q., Wang, H., Wang, D. and Chen, J. (2022). RhoA/ROCK Signaling Regulates Drp1-Mediated Mitochondrial Fission During Collective Cell Migration. Front Cell Dev Biol 10: 882581. PubMed ID: 35712666
Collective migration plays critical roles in developmental, physiological and pathological processes, and re,quires a dynamic actomyosin network for cell shape change, cell adhesion and cell-cell communication. The dynamic network of mitochondria in individual cells is regulated by mitochondrial fission and fusion, and is required for cellular processes including cell metabolism, apoptosis and cell division. But whether mitochondrial dynamics interplays with and regulates actomyosin dynamics during collective migration is not clear. This study demonstrated that proper regulation of mitochondrial dynamics is critical for collective migration of Drosophila border cells during oogenesis, and misregulation of fission or fusion results in reduction of ATP levels. Specifically, Drp1 is genetically required for border cell migration, and Drp1-mediated mitochondrial fission promotes formation of leading protrusion, likely through its regulation of ATP levels. Reduction of ATP levels by drug treatment also affects protrusion formation as well as actomyosin dynamics. Importantly, this study found that RhoA/ROCK signaling, which is essential for actin and myosin dynamics during border cell migration, could exert its effect on mitochondrial fission through regulating Drp1's recruitment to mitochondria. These findings suggest that RhoA/ROCK signaling may couple or coordinate actomyosin dynamics with mitochondrial dynamics to achieve optimal actomyosin function, leading to protrusive and migratory behavior.
Peterson, A. J., Murphy, S. J., Mundt, M. G., Shimell, M., Leof, E. B. and O'Connor, M. B. (2022). A juxtamembrane basolateral targeting motif regulates signaling through a TGF-beta pathway receptor in Drosophila. PLoS Biol 20(5): e3001660. PubMed ID: 35594316
In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. This study explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-β family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. The Dpp Type II receptor Punt was found to be enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. This study found that basolateral presentation of Punt is required for optimal signaling. Finally, evidence is presented that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.
Terakawa, A., Hu, Y., Kokaji, T., Yugi, K., Morita, K., Ohno, S., Pan, Y., Bai, Y., Parkhitko, A. A., Ni, X., Asara, J. M., Bulyk, M. L., Perrimon, N. and Kuroda, S. (2022). Trans-omics analysis of insulin action reveals a cell growth subnetwork which co-regulates anabolic processes. iScience 25(5): 104231. PubMed ID: 35494245
Insulin signaling promotes anabolic metabolism to regulate cell growth through multi-omic interactions. To obtain a comprehensive view of the cellular responses to insulin, a trans-omic network of insulin action was constructed in Drosophila cells that involves the integration of multi-omic data sets. In this network, 14 transcription factors, including Myc, coordinately upregulate the gene expression of anabolic processes such as nucleotide synthesis, transcription, and translation, consistent with decreases in metabolites such as nucleotide triphosphates and proteinogenic amino acids required for transcription and translation. Next, as cell growth is required for cell proliferation and insulin can stimulate proliferation in a context-dependent manner, the trans-omic network was integrated with results from a CRISPR functional screen for cell proliferation. This analysis validates the role of a Myc-mediated subnetwork that coordinates the activation of genes involved in anabolic processes required for cell growth.
Sriskanthadevan-Pirahas, S., Turingan, M. J., Chahal, J. S., Thorson, E., Khan, S., Tinwala, A. Q. and Grewal, S. S. (2022). Adipose mitochondrial metabolism controls body growth by modulating systemic cytokine and insulin signaling. Cell Rep 39(6): 110802. PubMed ID: 35545043
Animals must adapt their growth to fluctuations in nutrient availability to ensure proper development. These adaptations often rely on specific nutrient-sensing tissues that control whole-body physiology through inter-organ communication. While the signaling mechanisms that underlie this communication are well studied, the contributions of metabolic alterations in nutrient-sensing tissues are less clear. This study show how the reprogramming of adipose mitochondria controls whole-body growth in Drosophila larvae. Dietary nutrients alter fat-body mitochondrial morphology to lower their bioenergetic activity, leading to rewiring of fat-body glucose metabolism. Strikingly, it was found that genetic reduction of mitochondrial bioenergetics just in the fat body is sufficient to accelerate body growth and development. These growth effects are caused by inhibition of the fat-derived secreted peptides ImpL2 and tumor necrosis factor alpha (TNF-α)/Eiger, leading to enhanced systemic insulin signaling. This work reveals how reprogramming of mitochondrial metabolism in one nutrient-sensing tissue can couple nutrient availability to whole-body growth.
Niklas, B., Lapied, B. and Nowak, W. (2022). In Search of Synergistic Insect Repellents: Modeling of Muscarinic GPCR Interactions with Classical and Bitopic Photoactive Ligands. Molecules 27(10). PubMed ID: 35630759
Insect vector-borne diseases pose serious health problems, so there is a high demand for efficient molecules that could reduce transmission. Using molecular docking and molecular dynamics (MD) simulation, a series was studied of compounds acting on human and insect muscarinic acetylcholine receptors (mAChRs), a novel target of synergistic agents in pest control. Early conformational changes were characterized of human M1 and fruit fly type-A mAChR G protein-coupled receptors (GPCRs) in response to DEET, IR3535, and muscarine binding based on the MD analysis of the activation microswitches known to form the signal transduction pathway in class A GPCRs. Groups of microswitches were indicated that are the most affected by the presence of a ligand. Moreover, to increase selectivity towards insects, a new, bitopic, photoswitchable mAChR ligand-BQCA-azo-IR353 was proposed and its interactions with both receptors was studied. Modeling data showed that using a bitopic ligand may be a promising strategy in the search for better insect control.
Phillips, L. A., Atienza, M. L., Ryu, J. R., Svendsen, P. C., Kelemen, L. K. and Brook, W. J. (2022). midline represses Dpp signaling and target gene expression in Drosophila ventral leg development. Biol Open 11(5). PubMed ID: 35608103
Ventral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. This study shows that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus T-box binding element (TBE) site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. It was also demonstrated that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, mid does not affect all Dpp-target genes as it was demonstrated that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid.

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