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


Wednesday May 31st, 2023 - Enhancers and Transcriptional Regulation

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Farfan-Pira, K. J., Martínez-Cuevas, T. I., Evans, T. A. and Nahmad, M. (2023). A cis-regulatory sequence of the selector gene, vestigial, drives the evolution of wing scaling in Drosophila species. J Exp Biol. PubMed ID: 37078652ID:
Scaling between specific organs and overall body size has long fascinated biologists, being a primary mechanism by which organ shapes evolve. Yet, the genetic mechanisms that underlie the evolution of scaling relationships remain elusive. This study compared wing and fore tibia lengths (the latter as a proxy of body size) in Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, and Drosophila virilis, and showed that the first three of these species have roughly a similar wing-to-tibia scaling behavior. In contrast, D. virilis exhibits much smaller wings relative their body size compared to the other species and this is reflected in the intercept of the wing-to-tibia allometry. It was then asked whether the evolution of this relationship could be explained by changes in a specific cis-regulatory region or enhancer that drives expression of the wing selector gene, vestigial (vg), whose function is broadly conserved in insects and contributes to wing size. To test this hypothesis directly, CRISPR/Cas9 was used to replace the DNA sequence of the predicted Quadrant Enhancer (vgQE) from D. virilis for the corresponding vgQE sequence in the genome of D. melanogaster. Strikingly, it was discovered that D. melanogaster flies carrying the D. virilis vgQE sequence have wings that are significantly smaller with respect to controls, partially shifting the intercept of the wing-to-tibia scaling relationship towards that observed in D. virilis. It is concluded that a single cis-regulatory element in D. virilis contributes to constraining wing size in this species, supporting the hypothesis that scaling could evolve through genetic variations in cis-regulatory elements.
Kapil, S., Sobti, R. C. and Kaur, T. (2023). Prediction and analysis of cis-regulatory elements in Dorsal and Ventral patterning genes of Tribolium castaneum and its comparison with Drosophila melanogaster. Mol Cell Biochem. PubMed ID: 37004638
Insect embryonic development and morphology are characterized by their anterior-posterior and dorsal-ventral (DV) patterning. In Drosophila embryos, DV patterning is mediated by a Dorsal protein gradient which activates twist and snail genes, the important regulators of DV patterning. To activate or repress gene expression, some regulatory proteins bind in clusters to their target gene at sites known as cis-regulatory elements or enhancers. To understand how variations in gene expression in different lineages might lead to different phenotypes, it is necessary to understand enhancers and their evolution. Drosophila melanogaster has been widely studied to understand the interactions between transcription factors and the transcription factor binding sites. Tribolium castaneum is an upcoming model animal which is catching the interest of biologists and the research on the enhancer mechanisms in the insect's axes patterning is still in infancy. Therefore, the current study was designed to compare the enhancers of DV patterning in the two insect species. The sequences of ten proteins involved in DV patterning of D. melanogaster were obtained from Flybase. The protein sequences of T. castaneum orthologous to those obtained from D. melanogaster were acquired from NCBI BLAST, and these were then converted to DNA sequences which were modified by adding 20 kb sequences both upstream and downstream to the gene. These modified sequences were used for further analysis. Bioinformatics tools (Cluster-Buster and MCAST) were used to search for clusters of binding sites (enhancers) in the modified DV genes. The results obtained showed that the transcription factors in Drosophila melanogaster and Tribolium castaneum are nearly identical; however, the number of binding sites varies between the two species, indicating transcription factor binding site evolution, as predicted by two different computational tools. It was observed that Dorsal, Twist, Snail, Zelda, and Supressor of Hairless are the transcription factors responsible for the regulation of DV patterning in the two insect species
Li, X. C., Fuqua, T., van Breugel, M. E. and Crocker, J. (2023). Mutational scans reveal differential evolvability of Drosophila promoters and enhancers. Philos Trans R Soc Lond B Biol Sci 378(1877): 20220054. PubMed ID: 37004721
Rapid enhancer and slow promoter evolution have been demonstrated through comparative genomics. However, it is not clear how this information is encoded genetically and if this can be used to place evolution in a predictive context. Part of the challenge is that our understanding of the potential for regulatory evolution is biased primarily toward natural variation or limited experimental perturbations. To explore the evolutionary capacity of promoter variation, this study surveyed an unbiased mutation library for three promoters in Drosophila melanogaster. It was found that mutations in promoters had limited to no effect on spatial patterns of gene expression. Compared to developmental enhancers, promoters are more robust to mutations and have more access to mutations that can increase gene expression, suggesting that their low activity might be a result of selection. Consistent with these observations, increasing the promoter activity at the endogenous locus of shavenbaby led to increased transcription yet limited phenotypic changes. Taken together, developmental promoters may encode robust transcriptional outputs allowing evolvability through the integration of diverse developmental enhancers. This article is part of the theme issue 'Interdisciplinary approaches to predicting evolutionary biology'.
Percival-Smith, A., Cheng, S. and Ouellette, K. (2023). The frequency and differential pleiotropy of phenotypic non-specificity in Drosophila melanogaster. Genetics. PubMed ID: 36999545:
The regulation of the initiation of transcription by transcription factors (TFs) is often assumed to be dependent on specific recognition of DNA-binding sites and non-redundant. However, the redundant induction or rescue of a phenotype by TFs, phenotypic non-specificity, challenges this assumption. To assess the frequency of phenotypic non-specificity in the rescue of TF phenotypes, seven TF phenotypes labial (lab), Deformed (Dfd), Sex combs reduced (Scr), Ultrabithorax (Ubx), fruitless (fru), doublesex (dsx) and apterous (ap)) were screened for rescue by the expression of twelve, or more, non-resident TFs. From three hundred and eight assessments of rescue by non-resident TFs, eighteen rescues were identified for six of the seven TF phenotypes. Seventeen of the eighteen rescues were with TFs that recognize distinct DNA binding sites relative to the resident TF. All rescues were non-uniform across pleiotropic TF phenotypes suggesting extensive differential pleiotropy of the rescue. Primarily using RNAi to knockdown expression, and with the exceptions of the requirement of Bric a Brac 1 (BAB1) for female abdominal pigmentation and Myb oncogene like (MYB) for wing development, no evidence was found for a role of the other sixteen non-resident TFs in the TF phenotypes assessed. Therefore, these sixteen rescues are likely due to functional complementation and not due to the expression of an epistatic function in the developmental/behavioral pathway. Phenotypic non-specificity is both differentially pleiotropic and frequent, as on average 1 in 10-20 non-resident TFs rescue a phenotype. These observations will be important in future considerations of TF function.
Harden, T. T., Vincent, B. J. and DePace, A. H. (2023). Transcriptional activators in the early Drosophila embryo perform different kinetic roles. Cell Syst 14(4): 258-272. PubMed ID: 37080162
Combinatorial regulation of gene expression by transcription factors (TFs) may in part arise from kinetic synergy-wherein TFs regulate different steps in the transcription cycle. Kinetic synergy requires that TFs play distinguishable kinetic roles. This study used live imaging to determine the kinetic roles of three TFs that activate transcription in the Drosophila embryo-Zelda, Bicoid, and Stat92E-by introducing their binding sites into the even-skipped stripe 2 enhancer. These TFs influence different sets of kinetic parameters, and their influence can change over time. All three TFs increased the fraction of transcriptionally active nuclei; Zelda also shortened the first-passage time into transcription and regulated the interval between transcription events. Stat92E also increased the lifetimes of active transcription. Different TFs can therefore play distinct kinetic roles in activating the transcription. This has consequences for understanding the composition and flexibility of regulatory DNA sequences and the biochemical function of TFs.
Forbes Beadle, L., Zhou, H., Rattray, M. and Ashe, H. L. (2023). Modulation of transcription burst amplitude underpins dosage compensation in the Drosophila embryo. Cell Rep 42(4): 112382. PubMed ID: 37060568
Dosage compensation, the balancing of X-linked gene expression between sexes and to the autosomes, is critical to an organism's fitness and survival. In Drosophila, dosage compensation involves hypertranscription of the male X chromosome. This study used quantitative live imaging and modeling at single-cell resolution to study X chromosome dosage compensation in Drosophila. The four X chromosome genes studied undergo transcriptional bursting in male and female embryos. Mechanistically, the data reveal that transcriptional upregulation of male X chromosome genes is primarily mediated by a higher RNA polymerase II initiation rate and burst amplitude across the expression domain. In contrast, burst frequency is spatially modulated in nuclei within the expression domain in response to different transcription factor concentrations to tune the transcriptional response. Together, these data show how the local and global regulation of distinct burst parameters can establish the complex transcriptional outputs underpinning developmental patterning.

Tuesday May 30th - Immune Response

Elguero, J. E., Liu, G., Tiemeyer, K., Gandevia, H., Duro, L. and McCall, K. (2023). Defective phagocytosis leads to neurodegeneration through systemic increased innate immune signaling. bioRxiv. PubMed ID: 36711924
In nervous system development, disease and injury, neurons undergo programmed cell death, leaving behind cell corpses that are removed by phagocytic glia. Altered glial phagocytosis has been implicated in several neurological diseases including Alzheimer's disease, Parkinson's disease, and traumatic brain injury. To untangle the links between glial phagocytosis and neurodegeneration, this study investigated Drosophila mutants lacking the phagocytic receptor Draper. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. This study investigated whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration. A major immune response in Drosophila is the activation of two NFκB signaling pathways that produce antimicrobial peptides, primarily in the fat body. This study found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration, indicating that immune activation promotes neurodegeneration in draper mutants. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain.
Perlmutter, J. I., Chapman, J. R., Wilkinson, M. C., Nevarez-Saenz, I. and Unckless, R. L. (2023). A single amino acid polymorphism in natural Metchnikowin alleles of Drosophila results in systemic immunity and life history tradeoffs. bioRxiv. PubMed ID: 36712113
Previous work demonstrated that one Antimicrobial peptide, Metchikowin (Mtk), has a single residue that segregates as either proline (P) or arginine (R) in populations of four different Drosophila species, some of which diverged more than 10 million years ago. The recurrent finding of this polymorphism regardless of geography or host species, coupled with evidence of balancing selection in Drosophila AMPs, suggest there is a distinct functional importance to each allele. To assess their functional differences, D. melanogaster lines were created with the P allele, R allele, or Mtk null mutation using CRISPR/Cas9 genome editing. This study report results from experiments assessing the two hypotheses using these lines. In males, testing of systemic immune responses to a repertoire of bacteria and fungi demonstrated that the R allele performs as well or better than the P and null alleles with most infections. With some pathogens, however, females show results in contrast with males where Mtk alleles either do not contribute to survival or where the P allele outperforms the R allele. In addition, measurements of life history traits demonstrate that the R allele is more costly in the absence of infection for both sexes. These results provide strong in vivo evidence that differential fitness with or without infection and sex-based functional differences in alleles may be adaptive mechanisms of maintaining immune gene polymorphisms in contrast with expectations of rapid evolution. Therefore, a complex interplay of forces including pathogen species and host sex may lead to balancing selection for immune genotypes. Strikingly, this selection may act on even a single amino acid polymorphism in an AMP.
Parks, S. C., Okakpu, O. K., Azizpor, P., Nguyen, S., Martinez-Beltran, S., Claudio, I., Anesko, K., Bhatia, A., Dhillon, H. S. and Dillman, A. R. (2023). Parasitic nematode secreted phospholipase A(2) suppresses cellular and humoral immunity by targeting hemocytes in Drosophila melanogaster. Front Immunol 14: 1122451. PubMed ID: 37006283
A key aspect of parasitic nematode infection is the nematodes' ability to evade and/or suppress host immunity. This immunomodulatory ability is likely driven by the release of hundreds of excretory/secretory proteins (ESPs) during infection. While ESPs have been shown to display immunosuppressive effects on various hosts, understanding of the molecular interactions between individual proteins released and host immunity requires further study. A recently discovered secreted phospholipase A2 (sPLA(2)) released from the entomopathogenic nematode (EPN) Steinernema carpocapsae was named Sc-sPLA(2). This study reports that Sc-sPLA(2) increased mortality of Drosophila melanogaster infected with Streptococcus pneumoniae and promoted increased bacterial growth. Furthermore, the data showed that Sc-sPLA(2) was able to downregulate both Toll and Imd pathway-associated antimicrobial peptides (AMPs) including drosomycin and defensin, in addition to suppressing phagocytosis in the hemolymph. Sc-sPLA(2) was also found to be toxic to D. melanogaster with the severity being both dose- and time-dependent. Collectively, these data highlighted that Sc-sPLA(2) possessed both toxic and immunosuppressive capabilities.
Kato, D., Miura, K. and Yokoi, K. (2023). Analysis of the Toll and Spaetzle Genes Involved in Toll Pathway-Dependent Antimicrobial Gene Induction in the Red Flour Beetle, Tribolium castaneum (Coleoptera; Tenebrionidae). Int J Mol Sci 24(2). PubMed ID: 36675034
Insects rely only on their innate immune system to protect themselves from pathogens. Antimicrobial peptide (AMP) production is the main immune reaction in insects. In Drosophila melanogaster, the reaction is regulated mainly by the Toll and immune deficiency (IMD) pathways. Spaetzle proteins, activated by immune signals from upstream components, bind to Toll proteins, thus, activating the Toll pathway, which in turn, induces AMP genes. Previous studies have shown the difference in immune systems related to Toll and IMD pathways between D. melanogaster and Tribolium castaneum. In T. castaneum, nine Toll and seven spaetzle (spz) genes were identified. To extend understanding of AMP production by T. castaneum, functional assays of Toll and spaetzle genes related to Toll-pathway-dependent AMP gene expression were conducted in T. castaneum under challenge with bacteria or budding yeast. The results revealed that Toll3 and Toll4 double-knockdown and spz7 knockdown strongly and moderately reduced the Toll-pathway-dependent expression of AMP genes, respectively. Moreover, Toll3 and Toll4 double-knockdown pupae more rapidly succumbed to entomopathogenic bacteria than the control pupae, but spz7 knockdown pupae did not. The results suggest that Toll3 and Toll4 play a large role in Toll-pathway-dependent immune reactions, whereas spz7 plays a small part.
Nukala, K. M., Lilienthal, A. J., Lye, S. H., Bassuk, A. G., Chtarbanova, S. and Manak, J. R. (2023). Downregulation of oxidative stress-mediated glial innate immune response suppresses seizures in a fly epilepsy model. Cell Rep 42(1): 112004. PubMed ID: 36641750
Previous work has shown that mutations in prickle (pk) cause myoclonic-like seizures and ataxia in Drosophila, similar to what is observed in humans carrying mutations in orthologous PRICKLE genes. This study shows that pk mutant brains show elevated, sustained neuronal cell death that correlates with increasing seizure penetrance, as well as an upregulation of mitochondrial oxidative stress and innate immune response (IIR) genes. Moreover, flies exhibiting more robust seizures show increased levels of IIR-associated target gene expression suggesting they may be linked. Genetic knockdown in glia of either arm of the IIR (Immune Deficiency [Imd] or Toll) leads to a reduction in neuronal death, which in turn suppresses seizure activity, with oxidative stress acting upstream of IIR. These data provide direct genetic evidence that oxidative stress in combination with glial-mediated IIR leads to progression of an epilepsy disorder.
Zhu, Y., Liu, L., Zhang, C., Zhang, C., Han, T., Duan, R., Jin, Y., Guo, H., She, K., Xiao, Y., Goto, A., Cai, Q. and Ji, S. (2022). Endoplasmic reticulum-associated protein degradation contributes to Toll innate immune defense in Drosophila melanogaster. Front Immunol 13: 1099637. PubMed ID: 36741393
In Drosophila, the endoplasmic reticulum-associated protein degradation (ERAD) is engaged in regulating pleiotropic biological processes, with regard to retinal degeneration, intestinal homeostasis, and organismal development. The extent to which it functions in controlling the fly innate immune defense, however, remains largely unknown. This study shows that blockade of the ERAD in fat bodies antagonizes the Toll but not the IMD innate immune defense in Drosophila. Genetic approaches further suggest a functional role of Me31B in the ERAD-mediated fly innate immunity. Moreover, evidence is provided that silence of Xbp1 other than PERK or Atf6 partially rescues the immune defects by the dysregulated ERAD in fat bodies. Collectively, this study uncovers an essential function of the ERAD in mediating the Toll innate immune reaction in Drosophila.

Friday May 26th - Cancer, Oncogenes, and Tumor Supressors

Rebelo, A. R. and Homem, C. C. F. (2023). dMyc-dependent upregulation of CD98 amino acid transporters is required for Drosophila brain tumor growth. Cell Mol Life Sci 80(1): 30. PubMed ID: 36609617
Tumor cells have an increased demand for nutrients to sustain their growth, but how these increased metabolic needs are ensured or how this influences tumor formation and progression remains unclear. To unravel tumor metabolic dependencies, particularly from extracellular metabolites, this study has analyzed the role of plasma membrane metabolic transporters in Drosophila brain tumors. Using a well-established neural stem cell-derived tumor model, caused by brat knockdown, this study has found that 13 plasma membrane metabolic transporters, including amino acid, carbohydrate and monocarboxylate transporters, are upregulated in tumors and are required for tumor growth. CD98hc, and several of the light chains with which it can form heterodimeric amino acid transporters, were identified as crucial players in brat RNAi (brat (IR)) tumor progression. Knockdown of these components of CD98 heterodimers caused a dramatic reduction in tumor growth. The data also reveal that the oncogene dMyc is required and sufficient for the upregulation of CD98 transporter subunits in these tumors. Furthermore, tumor-upregulated dmyc and CD98 transporters orchestrate the overactivation of the growth-promoting signaling pathway TOR, forming a core growth regulatory network to support brat (IR) tumor progression. These findings highlight the important link between oncogenes, metabolism, and signaling pathways in the regulation of tumor growth and allow for a better understanding of the mechanisms necessary for tumor progression.
Negishi, T., Xing, F., Koike, R., Iwasaki, M., Wakasugi, M. and Matsunaga, T. (2023). UVA causes specific mutagenic DNA damage through ROS production, rather than CPD formation, in Drosophila larvae. Mutat Res Genet Toxicol Environ Mutagen 887: 503616. PubMed ID: 37003653
Evidence is accumulating that ultraviolet A (UVA) plays an important role in photo-carcinogenesis. However, the types of DNA damage involved in the resulting mutations remain unclear. Previously, using Drosophila, it was found that UVA from light-emitting diode (LED-UVA) induces double-strand breaks in DNA through oxidative damage in an oxidative damage-sensitive (urate-null) strain. Recently, it was proposed that cyclobutane pyrimidine dimers (CPDs), which also are induced by UVA irradiation, might play a significant role in the induction of mutations. The present study investigated whether reactive oxygen species (ROS) and CPDs are produced in larval bodies following LED-UVA irradiation. In addition, this study assessed the somatic cell mutation rate in urate-null Drosophila induced by monochromatic UVA irradiation. The production of ROS through LED-UVA irradiation was markedly higher in the urate-null strain than in the wild-type Drosophila. CPDs were detected in the DNA of both of UVA- and UVB-irradiated larvae. The level of CPDs was unexpectedly higher in the wild-type strain than in urate-null flies following UVA irradiation, whereas this parameter was expectedly similar between the urate-null and wild-type Drosophila following UVB irradiation. The somatic cell mutation rate induced by UVA irradiation was higher in the urate-null strain than in the wild-type strain. These results suggest that mutations induced by UVA-specific pathways occur through ROS production, rather than via CPD formation.
Bharti, M., Bajpai, A., Rautela, U., Manzar, N., Ateeq, B. and Sinha, P. (2023). Human ERG oncoprotein represses a Drosophila LIM domain binding protein-coding gene Chip. Proc Natl Acad Sci U S A 120(2): e2211189119. PubMed ID: 36595681
Human ETS Related Gene, ERG, a master transcription factor, turns oncogenic upon its out-of-context activation in diverse developmental lineages. However, the mechanism underlying its lineage-specific activation of Notch (N), Wnt, or EZH2-three well-characterized oncogenic targets of ERG-remains elusive. It was reasoned that deep homology in genetic tool kits might help uncover such elusive cancer mechanisms in Drosophila. By heterologous gain of human ERG in Drosophila, this study revealed that Chip, which codes for a transcriptional coactivator, LIM-domain-binding (LDB) protein, as its novel target. ERG represses Drosophila Chip via its direct binding and, indirectly, via E(z)-mediated silencing of its promoter. Downregulation of Chip disrupts LIM-HD complex formed between Chip and Tailup (Tup)-a LIM-HD transcription factor-in the developing notum. A consequent activation of N-driven Wg signaling leads to notum-to-wing transdetermination. These fallouts of ERG gain are arrested upon a simultaneous gain of Chip, sequestration of Wg ligand, and, alternatively, loss of N signaling or E(z) activity. Finally, this study showed that the human LDB1, a homolog of Drosophila Chip, is repressed in ERG-positive prostate cancer cells. Besides identifying an elusive target of human ERG, this study unravels an underpinning of its lineage-specific carcinogenesis.
Sollazzo, M., Paglia, S., Di Giacomo, S. and Grifoni, D. (2022). Apoptosis inhibition restrains primary malignant traits in different Drosophila cancer models. Front Cell Dev Biol 10: 1043630. PubMed ID: 36704198
Tumor cells exploit multiple mechanisms to evade apoptosis, hence the strategies aimed at reactivating cell death in cancer. However, recent studies are revealing that dying cells play remarkable pro-oncogenic roles. Among the mechanisms promoting cell death, cell competition, elicited by disparities in MYC activity in confronting cells, plays the primary role of assuring tissue robustness during development from Drosophila to mammals: cells with high MYC levels (winners) overproliferate while killing suboptimal neighbors (losers), whose death is essential to process completion. This mechanism is coopted by tumor cells in cancer initiation, where host cells succumb to high-MYC-expressing precancerous neighbors. Also in this case, inhibition of cell death restrains aberrant cell competition and rescues tissue structure. Inhibition of apoptosis may thus emerge as a good strategy to counteract cancer progression in competitive contexts; of note, a positive correlation was recently found between cell death amount at the tumor/stroma interface and MYC levels in human cancers. This study used Drosophila to investigate the functional role of competition-dependent apoptosis in advanced cancers, observing dramatic changes in mass dimensions and composition following a boost in cell competition, rescued by apoptosis inhibition. This suggests the role of competition-dependent apoptosis be not confined to the early stages of tumorigenesis. This study also showed that apoptosis inhibition, beside restricting cancer mass, is sufficient to rescue tissue architecture and counteract cell migration in various cancer contexts, suggesting that a strong activation of the apoptotic pathways intensifies cancer burden by affecting distinct phenotypic traits at different stages of the disease.
Chatterjee, D., Cong, F., Wang, X. F., Machado Costa, C. A., Huang, Y. C. and Deng, W. M. (2023). Cell polarity opposes Jak/STAT-mediated Escargot activation that drives intratumor heterogeneity in a Drosophila tumor model. Cell Rep 42(2): 112061. PubMed ID: 36709425
In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, the expression of Snail family transcription factor Escargot (Esg) was detected and further validated. A similar relationship was ascertained between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, these results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity.
Lewerentz, J., Johansson, A. M. and Stenberg, P. (2023). The path to immortalization of cells starts by managing stress through gene duplications. Exp Cell Res 422(1): 113431. PubMed ID: 36423660
The genomes of immortalized cell lines (and cancer cells) are characterized by multiple types of aberrations, ranging from single nucleotide polymorphisms (SNPs) to structural rearrangements that have accumulated over time. Consequently, it is difficult to estimate the relative impact of different aberrations, the order of events, and which gene functions were under selective pressure at the early stage towards cellular immortalization. This study has established novel cell cultures derived from Drosophila melanogaster embryos that were sampled at multiple time points over a one-year period. Using short-read DNA sequencing, we show that copy-number gain in preferentially stress-related genes were acquired in a dominant fraction of cells in 300-days old cultures. Furthermore, transposable elements were active in cells of all cultures. Only a few (<1%) SNPs could be followed over time, and these showed no trend to increase or decrease. It is concluded that the early cellular responses of a novel culture comprise sequence duplication and transposable element activity. During immortalization, positive selection first occurs on genes that are related to stress response before shifting to genes that are related to growth.

Thursday May 25th - Disease Models

Castelli, L. M., Lin, Y. H., Sanchez-Martinez, A., Gül, A., Mohd Imran, K., Higginbottom, A., Upadhyay, S. K., Márkus, N. M., Rua Martins, R., Cooper-Knock, J., Montmasson, C., Cohen, R., Walton, A., Bauer, C. S., De Vos, K. J., Mead, R. J., Azzouz, M., Dominguez, C., Ferraiuolo, L., Shaw, P. J., Whitworth, A. J. and Hautbergue, G. M. (2023). A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export reduces the neurotoxic effects of dipeptide repeat proteins. Sci Transl Med 15(685): eabo3823. PubMed ID: 36857431
Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. A cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.
Koza, Z., Ayajuddin, M., Das, A., Chaurasia, R., Phom, L. and Yenisetti, S. C. (2023). Sexual dysfunction precedes motor defects, dopaminergic neuronal degeneration, and impaired dopamine metabolism: Insights from Drosophila model of Parkinson's disease. Front Neurosci 17: 1143793. PubMed ID: 37025374
Sexual dysfunction (SD) is one of the most common non-motor symptoms of Parkinson's disease (PD) and remains the most neglected, under-reported, and under-recognized aspect of PD. Studies have shown that Dopamine (DA) in the hypothalamus plays a role in regulating sexual behavior. But the detailed mechanism of SD in PD is not known. Drosophila melanogaster shares several genes and signaling pathways with humans which makes it an ideal model for the study of a neurodegenerative disorder such as PD. Courtship behavior of Drosophila is one such behavior that is closely related to human sexual behavior and so plays an important role in understanding sexual behavior in diseased conditions as well. In the present study, a sporadic SD model of PD using Drosophila was developed and SD phenotype was observed based on abnormalities in courtship behavior markers. The Drosophila SD model was developed in such a way that at the window of neurotoxin paraquat (PQ) treatment [PQ is considered a crucial risk factor for PD due to its structural similarity with 1-methyl-4-phenyl pyridinium (MPP+), the active form of PD-inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)], it does not exhibit mobility defects but shows SD. The whole brain tyrosine hydroxylase immunostaining showed no observable dopaminergic (DAergic) degeneration (number of DA neurons and fluorescence intensity of fluorescently labeled secondary antibodies that target anti-TH primary antibody) of the SD model. Similarly, there was no significant depletion of brain DA and its metabolite levels (HVA and DOPAC) as determined using HPLC-ECD (High-Performance Liquid Chromatography using Electrochemical Detector). The present study illustrates that the traits associated with courtship and sexual activity provide sensitive markers at the earlier stage of PD onset. This PQ-induced SD fly model throws an opportunity to decipher the molecular basis of SD under PD conditions and to screen nutraceuticals/potential therapeutic molecules to rescue SD phenotype and further to DAergic neuroprotection.
Messelodi, D., Strocchi, S., Bertuccio, S. N., Baden, P., Indio, V., Giorgi, F. M., Taddia, A., Serravalle, S., Valente, S., di Fonzo, A., Frattini, E., Bernardoni, R., Pession, A., Grifoni, D., Deleidi, M., Astolfi, A. and Pession, A. (2023). Neuronopathic Gaucher disease models reveal defects in cell growth promoted by Hippo pathway activation. Commun Biol 6(1): 431. PubMed ID: 37076591
Gaucher Disease (GD), the most common lysosomal disorder, arises from mutations in the GBA1 gene and is characterized by a wide spectrum of phenotypes, ranging from mild hematological and visceral involvement to severe neurological disease. Neuronopathic patients display dramatic neuronal loss and increased neuroinflammation, whose molecular basis are still unclear. Using a combination of Drosophila dGBA1b loss-of-function models and GD patient-derived iPSCs differentiated towards neuronal precursors and mature neurons this study showed that different GD- tissues and neuronal cells display an impairment of growth mechanisms with an increased cell death and reduced proliferation. These phenotypes are coupled with the downregulation of several Hippo transcriptional targets, mainly involved in cells and tissue growth, and YAP exclusion from nuclei. Interestingly, Hippo knock-down in the GBA-KO flies rescues the proliferative defect, suggesting that targeting the Hippo pathway can be a promising therapeutic approach to neuronopathic GD.
Liu, H., Caballero-Floran, R. N., Hergenreder, T., Yang, T., Hull, J. M., Pan, G., Li, R., Veling, M. W., Isom, L. L., Kwan, K. Y., Huang, Z. J., Fuerst, P. G., Jenkins, P. M. and Ye, B. (2023). DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models. PLoS Biol 21(4): e3002078. PubMed ID: 37079499
Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. This study shows that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.
Hao, Y., Shao, L., Hou, J., Zhang, Y., Ma, Y., Liu, J., Xu, C., Chen, F., Cao, L. H. and Ping, Y. (2023). Resveratrol and Sir2 Reverse Sleep and Memory Defects Induced by Amyloid Precursor Protein. Neurosci Bull. PubMed ID: 37041405
Resveratrol (RES), a natural polyphenolic phytochemical, has been suggested as a putative anti-aging molecule for the prevention and treatment of Alzheimer's disease (AD) by the activation of sirtuin 1 (Sirt1/Sir2). This study tested the effects of RES and Sirt1/Sir2 on sleep and courtship memory in a Drosophila model by overexpression of amyloid precursor protein (APP), whose duplications and mutations cause familial AD. A mild but significant transcriptional increase was found of Drosophila Sir2 (dSir2) by RES supplementation for up to 17 days in APP flies, but not for 7 days. RES and dSir2 almost completely reversed the sleep and memory deficits in APP flies. It was further demonstrated that dSir2 acts as a sleep promotor in Drosophila neurons. Interestingly, RES increased sleep in the absence of dSir2 in dSir2-null mutants, and RES further enhanced sleep when dSir2 was either overexpressed or knocked down in APP flies. Finally, it was shown that Aβ aggregates in APP flies were reduced by RES and dSir2, probably via inhibiting Drosophila β-secretase (dBACE). The data suggest that RES rescues the APP-induced behavioral deficits and Aβ burden largely, but not exclusively, via dSir2.
Kim, S. H., Nichols, K. D., Anderson, E. N., Liu, Y., Ramesh, N., Jia, W., Kuerbis, C. J., Scalf, M., Smith, L. M., Pandey, U. B. and Tibbetts, R. S. (2023). Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2. Elife 12. PubMed ID: 37039476
Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. This study shows that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2(ALS)) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2(ALS) alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2(ALS) knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2(ALS) alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

Wednesday, May 24th - Chromatin

Hill, H., Bonser, D. and Golic, K. G. (2023). Dicentric chromosome breakage in Drosophila melanogaster is influenced by pericentric heterochromatin and occurs in non-conserved hotspots. Genetics. PubMed ID: 37010100
Chromosome breakage plays an important role in the evolution of karyotypes, and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster this study found that breakage under tension also tends to occur in specific hotspots. The experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. The breakage patterns were analyzed of three different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all three chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, it was found that the hotspot locations are not conserved between the three chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment varies significantly between the three chromosomes and is correlated with the origin of the centromere and the amount of pericentric heterochromatin. It is suggested that different centromere strengths could account for this.
Kim, L. H., Kim, J. Y., Xu, Y. Y., Lim, M. A., Koo, B. S., Kim, J. H., Yoon, S. E., Kim, Y. J., Choi, K. W., Chang, J. W. and Hong, S. T. (2023). Tctp, a unique Ing5-binding partner, inhibits the chromatin binding of Enok in Drosophila. Proc Natl Acad Sci U S A 120(15): e2218361120. PubMed ID: 37014852
The MOZ/MORF histone acetyltransferase complex is highly conserved in eukaryotes and controls transcription, development, and tumorigenesis. However, little is known about how its chromatin localization is regulated. Inhibitor of growth 5 (ING5) tumor suppressor is a subunit of the MOZ/MORF complex. Nevertheless, the in vivo function of ING5 remains unclear. This study reports an antagonistic interaction between Drosophila Translationally controlled tumor protein (TCTP) (Tctp) and ING5 (Ing5) required for chromatin localization of the MOZ/MORF (Enok) complex and H3K23 acetylation. Yeast two-hybrid screening using Tctp identified Ing5 as a unique binding partner. In vivo, Ing5 controlled differentiation and down-regulated epidermal growth factor receptor signaling, whereas it is required in the Yorkie (Yki) pathway to determine organ size. Ing5 and Enok mutants promoted tumor-like tissue overgrowth when combined with uncontrolled Yki activity. Tctp depletion rescued the abnormal phenotypes of the Ing5 mutation and increased the nuclear translocation of Ing5 and chromatin binding of Enok. Nonfunctional Enok promoted the nuclear translocation of Ing5 by reducing Tctp, indicating a feedback mechanism between Tctp, Ing5, and Enok to regulate histone acetylation. Therefore, Tctp is essential for H3K23 acetylation by controlling the nuclear translocation of Ing5 and chromatin localization of Enok, providing insights into the roles of human TCTP and ING5-MOZ/MORF in tumorigenesis.
Amin, A., Kadam, S., Mieczkowski, J., Ahmed, I., Bhat, Y. A., Shah, F., Tolstorukov, M. Y., Kingston, R. E., Padinhateeri, R. and Wani, A. H. (2023). Disruption of polyhomeotic polymerization decreases nucleosome occupancy and alters genome accessibility. Life Sci Alliance 6(5). PubMed ID: 36849253
Chromatin attains its three-dimensional (3D) conformation by establishing contacts between different noncontiguous regions. Sterile Alpha Motif (SAM)-mediated polymerization of the Polyhomeotic (PH) protein regulates subnuclear clustering of Polycomb Repressive Complex 1 (PRC1) and chromatin topology. The mutations that perturb the ability of the PH to polymerize, disrupt long-range chromatin contacts, alter Hox gene expression, and lead to developmental defects. To understand the underlying mechanism, this study combined the experiments and theory to investigate the effect of this SAM domain mutation on nucleosome occupancy and accessibility on a genome wide scale. The data show that disruption of PH polymerization because of SAM domain mutation decreases nucleosome occupancy and alters accessibility. Polymer simulations investigating the interplay between distant chromatin contacts and nucleosome occupancy, both of which are regulated by PH polymerization, suggest that nucleosome density increases when contacts between different regions of chromatin are established. Taken together, it appears that SAM domain-mediated PH polymerization biomechanically regulates the organization of chromatin at multiple scales from nucleosomes to chromosomes and it is suggested that higher order organization can have a top-down causation effect on nucleosome occupancy.
Ball, M. L., Koestler, S. A., Muresan, L., Rehman, S. A., O'Holleran, K. and White, R. (2023). The anatomy of transcriptionally active chromatin loops in Drosophila primary spermatocytes using super-resolution microscopy. PLoS Genet 19(3): e1010654. PubMed ID: 36867662
While the biochemistry of gene transcription has been well studied, understanding of how this process is organised in 3D within the intact nucleus is less well understood. This study we investigated the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, super-resolution microscopy was used to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. It was found that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. Foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops. These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription.
Gibson, T. J. and Harrison, M. M. (2023). Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function. bioRxiv. PubMed ID: 37066406
Chromatin is a barrier to the binding of many transcription factors. By contrast, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type specific binding and activity. The mechanisms governing pioneer-factor occupancy and the relationship between chromatin occupancy and opening remain unclear. Yhis work studied three Drosophila transcription factors with distinct DNA-binding domains and biological functions: Zelda, Grainy head, and Twist. It was demonstrated that the level of chromatin occupancy is a key determinant of pioneering activity. Multiple factors regulate occupancy, including motif content, local chromatin, and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. These results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features.
Li, X., Tang, X., Bing, X., Catalano, C., Li, T., Dolsten, G., Wu, C. and Levine, M. (2023). GAGA-associated factor fosters loop formation in the Drosophila genome. Mol Cell. PubMed ID: 37003261
The impact of genome organization on the control of gene expression persists as a major challenge in regulatory biology. Most efforts have focused on the role of CTCF-enriched boundary elements and TADs, which enable long-range DNA-DNA associations via loop extrusion processes. However, there is increasing evidence for long-range chromatin loops between promoters and distal enhancers formed through specific DNA sequences, including tethering elements, which bind the GAGA-associated factor (GAF). Previous studies showed that GAF possesses amyloid properties in vitro, bridging separate DNA molecules. This study investigated whether GAF functions as a looping factor in Drosophila development. Micro-C assays were employed to examine the impact of defined GAF mutants on genome topology. These studies suggest that the N-terminal POZ/BTB oligomerization domain is important for long-range associations of distant GAGA-rich tethering elements, particularly those responsible for promoter-promoter interactions that coordinate the activities of distant paralogous genes.

Tuesday May 23rd - Evolutionary Homologs of Drosophila Gene

Deng, Y., Peng, D., Xiao, J., Zhao, Y., Ding, W., Yuan, S., Sun, L., Ding, J., Zhou, Z. and Zhan, M. (2022). Inhibition of the transcription factor ZNF281 by SUFU to suppress tumor cell migration. Cell Death Differ. PubMed ID: 36220888
Although the Hedgehog (Hh) pathway plays an evolutionarily conserved role from Drosophila to mammals, some divergences also exist. Loss of Sufu, an important component of the Hh pathway, does not lead to an obvious developmental defect in Drosophila. However, in mammals, loss of SUFU results in serious disorder, even various cancers. This divergence suggests that SUFU plays additional roles in mammalian cells, besides regulating the Hh pathway. This study identified that the transcription factor ZNF281 is a novel binding partner of SUFU. Intriguingly, the Drosophila genome does not encode any homologs of ZNF281. SUFU is able to suppress ZNF281-induced tumor cell migration and DNA damage repair by inhibiting ZNF281 activity. Mechanistically, SUFU binds ZNF281 to mask the nuclear localization signal of ZNF281, culminating in ZNF281 cytoplasmic retention. In addition, SUFU also hampers the interactions between ZNF281 and promoters of target genes. Finally, we show that SUFU is able to inhibit ZNF281-induced tumor cell migration using an in vivo model. Taken together, these results uncover a Hh-independent mechanism of SUFU exerting the anti-tumor role, in which SUFU suppresses tumor cell migration through antagonizing ZNF281. Therefore, this study provides a possible explanation for the functional divergence of SUFU in mammals and Drosophila.
Wu, L., Huang, J., Trivedi, P., Sun, X., Yu, H., He, Z. and Zhang, X. (2022). Zinc finger myeloid Nervy DEAF-1 type (ZMYND) domain containing proteins exert molecular interactions to implicate in carcinogenesis. Discov Oncol 13(1): 139. PubMed ID: 36520265
Morphogenesis and organogenesis in the low organisms have been found to be modulated by a number of proteins, and one of such factor, deformed epidermal auto-regulatory factor-1 (DEAF-1) has been initially identified in Drosophila. The mammalian homologue of DEAF-1 and structurally related proteins have been identified, and they formed a family with over 20 members. The factors regulate gene expression through association with co-repressors, recognition of genomic marker, to exert histone modification by catalyze addition of some chemical groups to certain amino acid residues on histone and non-histone proteins, and degradation host proteins, so as to regulate cell cycle progression and execution of cell death. The formation of fused genes during chromosomal translocation, exemplified with myeloid transforming gene on chromosome 8 (MTG8)/eight-to-twenty one translocation (ETO) /ZMYND2, MTG receptor 1 (MTGR1)/ZMYND3, MTG on chromosome 16/MTGR2/ZMYND4 and BS69/ZMYND11 contributes to malignant transformation. Other anomaly like copy number variation (CNV) of BS69/ZMYND11 and promoter hyper methylation of BLU/ZMYND10 has been noted in malignancies. It has been reported that when fusing with Runt-related transcription factor 1 (RUNX1), the binding of MTG8/ZMYND2 with co-repressors is disturbed, and silencing of BLU/ZMYND10 abrogates its ability to inhibition of cell cycle and promotion of apoptotic death. Further characterization of the implication of ZMYND proteins in carcinogenesis would enhance understanding of the mechanisms of occurrence and early diagnosis of tumors, and effective antitumor efficacy.
Nazar, A. P., Delgado, M. J. and Lavore, A. (2022). Empty-spiracles is maternally expressed and essential for neurodevelopment and early embryo determination in Rhodnius prolixus. Dev Biol 490: 144-154. PubMed ID: 35988717
Since empty-spiracles (ems) was identified and characterized in Drosophila melanogaster as a head-gap gene, several studies have been carried out in other insect orders to confirm its evolutionary conserved function. Using the blood-sucking bug Rhodnius prolixus as biological model, this study found an ems transcript with three highly conserved regions: Box-A, Box-B, and the homeodomain. R. prolixus embryos silenced by parental RNAi for two of these ems conserved regions showed both maternal and zygotic defects. Rp-emsB fragment results in early lethal embryogenesis, with eggs without any embryonic structure inside. Rp-emsB expression pattern is only maternally expressed and localized in the ovary tropharium, follicular cells, and in the unfertilized female pronucleus. Rp-emsA fragment is zygotically expressed during early blastoderm formation until late developmental stages in two main patterns: anterior in the antennal segment, and in a segmentary in the neuroblast and tracheal pits. R. prolixus knockdown embryos for Rp-emsA showed an incomplete larval hatching, reduced heads, and severe neuromotor defects. Furthermore, in situ hybridization revealed a spatial and temporal expression pattern that highly correlates with Rp-ems observed function. Here, Rp-ems function in R. prolixus development was validated, showing that empty-spiracles does not act as a true head-gap gene, but it is necessary for proper head development and crucial for early embryo determination and neurodevelopment.
Damkham, N., Lorthongpanich, C., Klaihmon, P., Lueangamornnara, U., Kheolamai, P., Trakarnsanga, K. and Issaragrisil, S. (2022). YAP and TAZ play a crucial role in human erythrocyte maturation and enucleation. Stem Cell Res Ther 13(1): 467. PubMed ID: 36076260
Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ), see Drosophila Yorkie, are two key transcription co-activators of the Hippo pathway. Both were originally characterized as organ size and cell proliferation regulators. Later studies demonstrated that the Hippo pathway may play a role in Drosophila and mammal hematopoiesis. However, the role of the Hippo pathway in human erythropoiesis has not yet been fully elucidated. The role of YAP and TAZ was studied in human erythropoiesis and hematopoietic stem cell (HSC) lineage determination by using mobilized peripheral blood (PB) and cord blood (CB)-derived HSC as a model. HSCs were isolated and cultured in an erythroid differentiation medium for erythroid differentiation and culture in methylcellulose assay for HSC lineage determination study. YAP and TAZ were barely detectable in human HSCs, but became highly expressed in pro-erythroblasts and erythroblasts. Depletion or knockdown of YAP and/or TAZ did not affect the ability of HSC lineage specification to erythroid lineage in either methylcellulose assay or liquid culture. However, depletion of YAP and TAZ did impair erythroblast terminal differentiation to erythrocytes and their enucleation. Moreover, ectopic expression of YAP and TAZ in pro-erythroblasts did not exert an apparent effect on erythroid differentiation, expansion, or morphology. This study demonstrated that YAP/TAZ plays important role in erythroid maturation and enucleation but is dispensable for lineage determination of human HSCs.
Ravindran, P. and Puschel, A. W. (2023). An isoform-specific function of Cdc42 in regulating mammalian Exo70 during axon formation. Life Sci Alliance 6(3). PubMed ID: 36543541
The highly conserved GTPase Cdc42 is an essential regulator of cell polarity and promotes exocytosis through the exocyst complex in budding yeast and Drosophila In mammals, this function is performed by the closely related GTPase TC10, whereas mammalian Cdc42 does not interact with the exocyst. Axon formation is facilitated by the exocyst complex that tethers vesicles before their fusion to expand the plasma membrane. This function depends on the recruitment of the Exo70 subunit to the plasma membrane. Alternative splicing generates two Cdc42 isoforms that differ in their C-terminal 10 amino acids. These results identify an isoform-specific function of Cdc42 in neurons.The brain-specific Cdc42b isoform, in contrast to the ubiquitous isoform Cdc42u, can interact with Exo70. Inactivation of Arhgef7 or Cdc42b interferes with the exocytosis of post-Golgi vesicles in the growth cone. Cdc42b regulates exocytosis and axon formation downstream of its activator Arhgef7. Thus, the function of Cdc42 in regulating exocytosis is conserved in mammals but specific to one isoform.
Li, J., Kong, Y., Sun, L., Tang, Y., Sun, X., Qin, S. and Li, M. (2023). Overexpression of Ultrabithorax Changes the Development of Silk Gland and the Expression of Fibroin Genes in Bombyx mori. Int J Mol Sci 24(7). PubMed ID: 37047645
Ultrabithorax (Ubx) is a member of the Hox gene group involved in cell fate decisions, cell proliferation and organ identity. Its function has been extensively researched in Drosophila melanogaster but little is known about it in Lepidoptera. To uncover the function of Ubx in the development of lepidopterans, this study constructed the Ubx overexpression (Ubx(OE)) strain based on the Nistari strain of Bombyx mori. The Ubx(OE) strain showed a small body size, transparent intersegmental membrane and abnormal posterior silk gland (PSG). The current study focused on the effect of Ubx overexpression on the posterior silk gland. As the major protein product of PSG, the mRNA expression of fibroin heavy chain (Fib-H) and fibroin light chain (Fib-L) was upregulated three fold in Ubx(OE), but the protein expression of Fib-H and Fib-L was not significantly different. It is speculated that the overexpression of Ubx downregulated the expression of Myc and further caused abnormal synthesis of the spliceosome and ribosome. Abnormalities of the spliceosome and ribosome affected the synthesis of protein in the PSG and changed its morphology.

Monday May 22nd - Cytoskeleton and Junctions

Ashour, D. J., Durney, C. H., Planelles-Herrero, V. J., Stevens, T. J., Feng, J. J. and Roper, K. (2023). Zasp52 strengthens whole embryo tissue integrity through supracellular actomyosin networks. Development 150(7). PubMed ID: 36897564
During morphogenesis, large-scale changes of tissue primordia are coordinated across an embryo. In Drosophila, several tissue primordia and embryonic regions are bordered or encircled by supracellular actomyosin cables, junctional actomyosin enrichments networked between many neighbouring cells. This study shows that the single Drosophila Alp/Enigma-family protein Zasp52, which is most prominently found in Z-discs of muscles, is a component of many supracellular actomyosin structures during embryogenesis, including the ventral midline and the boundary of the salivary gland placode. This study reveals that Zasp52 contains within its central coiled-coil region a type of actin-binding motif usually found in CapZbeta proteins, and this domain displays actin-binding activity. Using endogenously-tagged lines, it was identified that Zasp52 interacts with junctional components, including APC2, Polychaetoid and Sidekick, and actomyosin regulators. Analysis of zasp52 mutant embryos reveals that the severity of the embryonic defects observed scales inversely with the amount of functional protein left. Large tissue deformations occur where actomyosin cables are found during embryogenesis, and in vivo and in silico analyses suggest a model whereby supracellular Zasp52-containing cables aid to insulate morphogenetic changes from one another.
Nanda, S., Bhattacharjee, S., Cox, D. N. and Ascoli, G. A. (2023). Local Microtubule and F-Actin Distributions Fully Constrain the Spatial Geometry of Drosophila Sensory Dendritic Arbors. Int J Mol Sci 24(7). PubMed ID: 37047715
Dendritic morphology underlies the source and processing of neuronal signal inputs. Morphology can be broadly described by two types of geometric characteristics. The first is dendrogram topology, defined by the length and frequency of the arbor branches; the second is spatial embedding, mainly determined by branch angles and straightness. It has been previously demonstrated that microtubules and actin filaments are associated with arbor elongation and branching, fully constraining dendrogram topology. This study relates the local distribution of these two primary cytoskeletal components with dendritic spatial embedding. 167 sensory neurons from the Drosophila larva encompassing multiple cell classes and genotypes were reconstructed and analyzed. It was observe that branches with a higher microtubule concentration tend to deviate less from the direction of their parent branch across all neuron types. Higher microtubule branches are also overall straighter. F-actin displays a similar effect on angular deviation and branch straightness, but not as consistently across all neuron types as microtubule. These observations raise the question as to whether the associations between cytoskeletal distributions and arbor geometry are sufficient constraints to reproduce type-specific dendritic architecture. Therefore, this study create a computational model of dendritic morphology purely constrained by the cytoskeletal composition measured from real neurons. The model quantitatively captures both spatial embedding and dendrogram topology across all tested neuron groups. These results suggest a common developmental mechanism regulating diverse morphologies, where the local cytoskeletal distribution can fully specify the overall emergent geometry of dendritic arbors.
Xie, P. (2023). Determinant factors for residence time of kinesin motors at microtubule ends. J Biol Phys. PubMed ID: 36645568
Kinesins constitute a superfamily of microtubule (MT)-based motor proteins, which can perform diverse biological functions in cells such as transporting vesicle, regulating MT dynamics, and segregating chromosome. Some motors such as kinesin-1, kinesin-2, and kinesin-3 do the activity mainly on the MT lattice, while others such as kinesin-7 and kinesin-8 do the activity mainly at the MT plus end. To perform the different functions, it is required that the former motors can reside on the MT lattice for longer times than at the end, while the latter motors can reside at the MT plus end for long times. This study presents a simple but general theory of the MT-end residence time of the kinesin motor is presented, with which the factors dictating the residence time are determined. The theory is further used to study specifically the MT-end residence times of Drosophila kinesin-1, kinesin-2/KIF3AB, kinesin-3/Unc104, kinesin-5/Eg5, kinesin-7/CENP-E, and kinesin-8/Kip3 motors, with the theoretical results being in agreement with the available experimental data.
Kraus, J., Travis, S. M., King, M. R. and Petry, S. (2023). Augmin is a Ran-regulated spindle assembly factor. J Biol Chem: 104736. PubMed ID: 37086784
Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αβ and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. In Xenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is non-essential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. This study used in vitro pulldowns and TIRF microscopy to show that augmin is a Ran-regulated SAF. Augmin was shown to directly interact with both importin-α and importin-&beta; through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT binding site. Moreover, Ran was shown to control localization of augmin to MTs in both Xenopus egg extract and in vitro. These results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2.
Galenza, A., Moreno-Roman, P., Su, Y. H., Acosta-Alvarez, L., Debec, A., Guichet, A., Knapp, J. M., Kizilyaprak, C., Humbel, B. M., Kolotuev, I. and O'Brien, L. E. (2023). Basal stem cell progeny establish their apical surface in a junctional niche during turnover of an adult barrier epithelium. Nat Cell Biol. PubMed ID: 36997641
Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. New replacement cells-the progeny of basal stem cells-are born without barrier-forming structures such as a specialized apical membrane and occluding junctions. This study investigated how new progeny acquire barrier structures as they integrate into the intestinal epithelium of adult Drosophila. They were found gestate their future apical membrane in a sublumenal niche created by a transitional occluding junction that envelops the differentiating cell and enables it to form a deep, microvilli-lined apical pit. The transitional junction seals the pit from the intestinal lumen until differentiation-driven, basal-to-apical remodelling of the niche opens the pit and integrates the now-mature cell into the barrier. By coordinating junctional remodelling with terminal differentiation, stem cell progeny integrate into a functional, adult epithelium without jeopardizing barrier integrity.
Wang, J., Michel, M., Bialas, L., Pierini, G. and Dahmann, C. (2023). Preferential recruitment and stabilization of Myosin II at compartment boundaries in Drosophila. J Cell Sci. PubMed ID: 36718636
The regulation of mechanical tension exerted at cell junctions guides cell behavior during tissue formation and homeostasis. Cell junctions along compartment boundaries, which are lineage restrictions separating cells with different fates and functions within tissues, are characterized by increased mechanical tension compared to cell junctions in the bulk of the tissue. Mechanical tension depends on the actomyosin cytoskeleton, however, the mechanisms by which mechanical tension is locally increased at cell junctions along compartment boundaries remain elusive. This study show that non-muscle Myosin II and F-actin transiently accumulate, and mechanical tension is increased, at cell junctions along the forming anteroposterior compartment boundary in the Drosophila pupal abdominal epidermis. Fluorescence recovery after photobleaching experiments show that Myosin II accumulation correlates with its increased stabilization at these junctions. Moreover, photoconversion indicates that Myosin II is preferentially recruited within cells to junctions along the compartment boundary. These results indicate that the preferential recruitment and stabilization of Myosin II contribute to the initial build-up of mechanical tension at compartment boundaries.

Friday May 19th - Gonads

Laursen, W. J., Busby, R., Sarkissian, T., Chang, E. C. and Garrity, P. A. (2023). DMKPs provide a generalizable strategy for studying genes required for reproduction or viability in non-traditional model organisms. Genetics. PubMed ID: 37036394
The advent of CRISPR/Cas9-mediated genome editing has expanded the range of animals amenable to targeted genetic analysis. This has accelerated research in animals not traditionally studied using molecular genetics. However, studying genes essential for reproduction or survival in such animals remains challenging, as they lack the tools that aid genetic analysis in traditional genetic model organisms. The use of distinguishably marked knock-in pairs (DMKPs) was recently introduced as a strategy for rapid and reliable genotyping in such species. This study shows that DMKPs also facilitate the maintenance and study of mutations that cannot be maintained in a homozygous state, a group which includes recessive lethal and sterile mutations. Using DMKPs, the zero population growth (zpg) locus was disrupted in Drosophila melanogaster and in the dengue vector mosquito Aedes aegypti. In both species, DMKPs enable the maintenance of zpg mutant strains and the reliable recovery of zpg mutant animals. Male and female gonad development is disrupted in fly and mosquito zpg mutants, rendering both sexes sterile. In Aedes aegypti,zpg mutant males remain capable of inducing a mating refractory period in wild-type females and of competing with wild-type males for mates, properties compatible with zpg serving as a target in mosquito population suppression strategies. DMKP is readily generalizable to other species amenable to CRISPR/Cas9-mediated gene targeting, and should facilitate the study of sterile and lethal mutations in multiple organisms not traditionally studied using molecular genetics.
Brown, N. C., Gordon, B., McDonough-Goldstein, C. E., Misra, S., Findlay, G. D., Clark, A. G. and Wolfner, M. F. (2023). The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster. bioRxiv. PubMed ID: 36798169
In Drosophila and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology and behavior. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). This study used RNAi and CRISPR/Cas9 generated mutants to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). Obp56g was found to be required for male fertility and the induction of the PMR, whereas the other six genes had no effect on fertility when mutated individually. Obp56g is expressed in the male's ejaculatory bulb, an important tissue in the reproductive tract that synthesizes components of the mating plug. This study found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage. The evolutionary history of these seminal Obp genes was examined, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression only in species of the melanogaster and obscura groups, though conserved head expression in all species tested. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.
You, D. D., Zhou, X. L., Wang, Q. Q. and Liu, J. L. (2023). Cytoophidia safeguard binucleation of Drosophila male accessory gland cells. Exp Cell Res 422(1): 113433. PubMed ID: 36423659
Although most cells are mononuclear, the nucleus can exist in the form of binucleate or even multinucleate to respond to different physiological processes. The male accessory gland of Drosophila is the organ that produces semen, and its main cells are binucleate. This study observe that CTP synthase (CTPS) forms filamentous cytoophidia in binuclear main cells, primarily located at the cell boundary. In CTPSH355A, a point mutation that destroys the formation of cytoophidia, it was found that the nucleation mode of the main cells changes, including mononucleates and vertical distribution of binucleates. Although the overexpression of CTPSH355A can restore the level of CTPS protein, it will neither form cytoophidia nor eliminate the abnormal nucleation pattern. Therefore, these data indicate that there is an unexpected functional link between the formation of cytoophidia and the maintenance of binucleation in Drosophila main cells.
Hakes, A. C. and Gavis, E. R. (2023). Plasticity of Drosophila germ granules during germ cell development. PubMed ID: PLoS Biol 21(4): e3002069. PubMed ID: 37053289
Compartmentalization of RNAs and proteins into membraneless structures called granules is a ubiquitous mechanism for organizing and regulating cohorts of RNAs. Germ granules are ribonucleoprotein (RNP) assemblies required for germline development across the animal kingdom, but their regulatory roles in germ cells are not fully understood. This study shows that after germ cell specification, Drosophila germ granules enlarge through fusion and this growth is accompanied by a shift in function. Whereas germ granules initially protect their constituent mRNAs from degradation, they subsequently target a subset of these mRNAs for degradation while maintaining protection of others. This functional shift occurs through the recruitment of decapping and degradation factors to the germ granules, which is promoted by decapping activators and renders these structures P body-like. Disrupting either the mRNA protection or degradation function results in germ cell migration defects. OThese findings reveal plasticity in germ granule function that allows them to be repurposed at different stages of development to ensure population of the gonad by germ cells. Additionally, these results reveal an unexpected level of functional complexity whereby constituent RNAs within the same granule type can be differentially regulated.
Dutta, S., Farhadifar, R., Lu, W., Kabacaoaylu, G., Blackwell, R., Stein, D. B., Lakonishok, M., Gelfand, V. I., Shvartsman, S. Y. and Shelley, M. J. (2023). Self-organized intracellular twisters. bioRxiv. PubMed ID: 37066165
Life in complex systems, such as cities and organisms, comes to a standstill when global coordination of mass, energy, and information flows is disrupted. Global coordination is no less important in single cells, especially in large oocytes and newly formed embryos, which commonly use fast fluid flows for dynamic reorganization of their cytoplasm. This study combined theory, computing, and imaging to investigate such flows in the Drosophila oocyte, where streaming has been proposed to spontaneously arise from hydrodynamic interactions among cortically anchored microtubules loaded with cargo-carrying molecular motors. A fast, accurate, and scalable numerical approach was used to investigate fluid-structure interactions of 1000s of flexible fibers, and the robust emergence and evolution of cell-spanning vortices, or twisters was demonstrated. Dominated by a rigid body rotation and secondary toroidal components, these flows are likely involved in rapid mixing and transport of ooplasmic components.
Galasso, A., Xu, D. C., Hill, C., Iakovleva, D., Stefana, M. I. and Baena-Lopez, L. A. (2023). Non-apoptotic caspase activation ensures the homeostasis of ovarian somatic stem cells. EMBO Rep: e51716. PubMed ID: 37039000
Current evidence has associated caspase activation with the regulation of basic cellular functions without causing apoptosis. Malfunction of non-apoptotic caspase activities may contribute to specific neurological disorders, metabolic diseases, autoimmune conditions and cancers. However, understanding of non-apoptotic caspase functions remains limited. This study showed that non-apoptotic caspase activation prevents the intracellular accumulation of the Patched receptor in autophagosomes and the subsequent Patched-dependent induction of autophagy in Drosophila follicular stem cells. These events ultimately sustain Hedgehog signalling and the physiological properties of ovarian somatic stem cells and their progeny under moderate thermal stress. Importantly, the key findings are partially conserved in ovarian somatic cells of human origin. These observations attribute to caspases a pro-survival role under certain cellular conditions.

Thursday May 18th - Larval and Adult Physiology and Metabolism

Jacobs, H. T., Tuomela, T. and Lillsunde, P. (2023). Nuclear genetic background influences the phenotype of the Drosophila tko25t mitochondrial protein-synthesis mutant. G3 (Bethesda). PubMed ID: 37017029
The Drosophila A mutation in the Drosophila gene technical knockout (tko25t), encoding mitoribosomal protein S12, phenocopies human mitochondrial disease. Three spontaneous X-dominant suppressors of tko25t (designated Weeble), exhibiting almost wild-type phenotype and containing overlapping segmental duplications including the mutant allele, plus a second mitoribosomal protein gene, mRpL14. Ectopic, expressed copies of tko25t and mRpL14 conferred no phenotypic suppression. When placed over a null allele of tko, Weeble retained the mutant phenotype, even in the presence of additional transgenic copies of tko25t. Increased mutant gene dosage can thus compensate the mutant phenotype, but only when located in its normal chromosomal context.
Bradlaugh, A. A., Fedele, G., Munro, A. L., Hansen, C. N., Hares, J. M., Patel, S., Kyriacou, C. P., Jones, A. R., Rosato, E. and Baines, R. A. (2023). Essential elements of radical pair magnetosensitivity in Drosophila. Nature 615(7950): 111-116. PubMed ID: 36813962
Many animals use Earth's magnetic field (also known as the geomagnetic field) for navigation. The favoured mechanism for magnetosensitivity involves a blue-light-activated electron-transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan residues within the photoreceptor protein CRYPTOCHROME (CRY). The spin-state of the resultant radical pair, and therefore the concentration of CRY in its active state, is influenced by the geomagnetic field. However, the canonical CRY-centric radical-pair mechanism does not explain many physiological and behavioural observations. Using electrophysiology and behavioural analyses, this study assayed magnetic-field responses at the single-neuron and organismal levels. The 52 C-terminal amino acid residues of Drosophila melanogaster CRY, lacking the canonical FAD-binding domain and tryptophan chain, was shown to be sufficient to facilitate magnetoreception. It was also shown that increasing intracellular FAD potentiates both blue-light-induced and magnetic-field-dependent effects on the activity mediated by the C terminus. High levels of FAD alone are sufficient to cause blue-light neuronal sensitivity and, notably, the potentiation of this response in the co-presence of a magnetic field. These results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (that is, non-CRY-dependent) radical pairs can elicit magnetic-field responses in cells.
Borchardt, L. A., Scharenbrock, A. R., Olufs, Z. P. G., Wassarman, D. A. and Perouansky, M. (2023). Mutations in Complex I of the Mitochondrial Electron-Transport Chain Sensitize the Fruit Fly (Drosophila melanogaster) to Ether and Non-Ether Volatile Anesthetics. Int J Mol Sci 24(3). PubMed ID: 36768163
The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-2360114 and mt:ND2del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-2360114 flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II-V of the mETC cause AiN. It was found that (1) ND-2360114 and mt:ND2del1 were susceptible to toxicity from halothane; (2) in wild-type flies, halothane was toxic under anoxic conditions; (3) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II-V did not result in an AiN phenotype. It is concluded that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I. 5$agAllon
Borchardt, L. A., Scharenbrock, A. R., Olufs, Z. P. G., Wassarman, D. A. and Perouansky, M. (2023). Mutations in Complex I of the Mitochondrial Electron-Transport Chain Sensitize the Fruit Fly (Drosophila melanogaster) to Ether and Non-Ether Volatile Anesthetics. Int J Mol Sci 24(3). PubMed ID: 36768163
The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-2360114 and mt:ND2del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-2360114 flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II-V of the mETC cause AiN. It was found that (1) ND-2360114 and mt:ND2del1 were susceptible to toxicity from halothane; (2) in wild-type flies, halothane was toxic under anoxic conditions; (3) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II-V did not result in an AiN phenotype. It is concluded that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I.
Bressan, G. N., Cardoso, P. M., Reckziegel, J. and Fachinetto, R. (2023). Reserpine and PCPA reduce heat tolerance in Drosophila melanogaster. Life Sci 318: 121497. PubMed ID: 36780938
Drosophila melanogaster is a model organism to study molecular mechanisms and the role of the genes and proteins involved in thermal nociception. Monoamines (i.e. dopamine) have been involved in temperature preference behavior in D. melanogaster. Therefore, this study investigated whether the monoamines, particularly dopamine and serotonin, participate in the response to thermal nociceptive stimuli in D. melanogaster. Flies were treated with reserpine (an inhibitor of vesicular monoamines transporter, 3-300 μM), 3-Iodo-L-tyrosine (3-I-T, an inhibitor of tyrosine hydroxylase, 16.28-65.13 mM), and para-Chloro-DL-phenylalanine (PCPA, an inhibitor of tryptophan hydroxylase, 20-80 mM); then, the flies were subjected to tests of thermal tolerance and avoidance of noxious heat. Climbing behavior was used as a test to evaluate locomotor activity. Reserpine reduces the thermal tolerance profile of the D. melanogaster, as well as the avoidance of noxious heat and locomotor activity depending on the concentration. PCPA, but not 3-I-T, decreased heat tolerance and avoidance of noxious heat. These data suggest that monoamines, particularly serotonin, are associated with the impaired avoidance of noxious heat which could be related to the reduction of heat tolerance in D. melanogaster.
Karpati, Z., Deutsch, F., Kiss, B. and Schmitt, T. (2023). Seasonal changes in photoperiod and temperature lead to changes in cuticular hydrocarbon profiles and affect mating success in Drosophila suzukii. Sci Rep 13(1): 5649. PubMed ID: 37024537
Seasonal plasticity in insects is often triggered by temperature and photoperiod changes. When climatic conditions become sub-optimal, insects might undergo reproductive diapause, a form of seasonal plasticity delaying the development of reproductive organs and activities. During the reproductive diapause, the cuticular hydrocarbon (CHC) profile, which covers the insect body surface, might also change to protect insects from desiccation and cold temperature. However, CHCs are often important cues and signals for mate recognition and changes in CHC composition might affect mate recognition. This study investigated the CHC profile composition and the mating success of Drosophila suzukii in 1- and 5-day-old males and females of summer and winter morphs. CHC compositions differed with age and morphs. However, no significant differences were found between the sexes of the same age and morph. The results of the behavioral assays show that summer morph pairs start to mate earlier in their life, have a shorter mating duration, and have more offspring compared to winter morph pairs. It is hypothesized that CHC profiles of winter morphs are adapted to survive winter conditions, potentially at the cost of reduced mate recognition cues.

Wednesday May 17th - Adult Neural Development and Function

Ahmed, M., Rajagopalan, A. E., Pan, Y., Li, Y., Williams, D. L., Pedersen, E. A., Thakral, M., Previero, A., Close, K. C., Christoforou, C. P., Cai, D., Turner, G. C. and Clowney, E. J. (2023). Hacking brain development to test models of sensory coding. bioRxiv. PubMed ID: 36747712
Animals can discriminate myriad sensory stimuli but can also generalize from learned experience. One can probably distinguish the favorite teas of your colleagues while still recognizing that all tea pales in comparison to coffee. Tradeoffs between detection, discrimination, and generalization are inherent at every layer of sensory processing. During development, specific quantitative parameters are wired into perceptual circuits and set the playing field on which plasticity mechanisms play out. A primary goal of systems neuroscience is to understand how material properties of a circuit define the logical operations- computations--that it makes, and what good these computations are for survival. A cardinal method in biology-and the mechanism of evolution--is to change a unit or variable within a system and ask how this affects organismal function. This study used of knowledge of developmental wiring mechanisms to modify hard-wired circuit parameters in the Drosophila melanogaster mushroom body and assess the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, it was found that input number, but not cell number, tunes odor selectivity. Simple odor discrimination performance is maintained when Kenyon cell number is reduced and augmented by Kenyon cell expansion.
Lez-Segarra, A. J. G. Pontes, G., Jourjine, N., Toro, A. D. and Scott, K. (2023). Hunger- and thirst-sensing neurons modulate a neuroendocrine network to coordinate sugar and water ingestion. bioRxiv. PubMed ID: 37066363
Consumption of food and water is tightly regulated by the nervous system to maintain internal nutrient homeostasis. Although generally considered independently, interactions between hunger and thirst drives are important to coordinate competing needs. In Drosophila, four neurons called the Interoceptive Subesophageal zone Neurons (ISNs) respond to intrinsic hunger and thirst signals to oppositely regulate sucrose and water ingestion. This study investigated the neural circuit downstream of the ISNs to examine how ingestion is regulated based on internal needs. Utilizing the recently available fly brain connectome, this study found that the ISNs synapse with a novel cell type Bilateral T-shaped neuron (BiT) that projects to neuroendocrine centers. In vivo neural manipulations revealed that BiT oppositely regulates sugar and water ingestion. Neuroendocrine cells downstream of ISNs include several peptide-releasing and peptide-sensing neurons, including insulin producing cells (IPC), crustacean cardioactive peptide (CCAP) neurons, and CCHamide-2 receptor isoform RA (CCHa2R-RA) neurons. These neurons contribute differentially to ingestion of sugar and water, with IPCs and CCAP neurons oppositely regulating sugar and water ingestion, and CCHa2R-RA neurons modulating only water ingestion. Thus, the decision to consume sugar or water occurs via regulation of a broad peptidergic network that integrates internal signals of nutritional state to generate nutrient-specific ingestion.
Chang, N., Huang, H. P. and Lo, C. C. (2023). Global inhibition in head-direction neural circuits: a systematic comparison between connectome-based spiking neural circuit models. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 36781446
The recent discovery of the head-direction (HD) system in fruit flies has provided unprecedented insights into the neural mechanisms of spatial orientation. Despite the progress, the neural substance of global inhibition, an essential component of the HD circuits, remains controversial. Some studies suggested that the ring neurons provide global inhibition, while others suggested the Δ7 neurons. The present study provides evaluations from the theoretical perspective by performing systematic analyses on the computational models based on the ring-neuron (R models) and Δ7-neurons (Delta models) hypotheses with modifications according to the latest connectomic data. Four tests were performed: robustness, persistency, speed, and dynamical characteristics. It was discovered that the two models led to a comparable performance in general, but each excelled in different tests. The R Models were more robust, while the Delta models were better in the persistency test. A hybrid model was tested that combines both inhibitory mechanisms. While the performances of the R and Delta models in each test are highly parameter-dependent, the Hybrid model performed well in all tests with the same set of parameters. These results suggest the possibility of combined inhibitory mechanisms in the HD circuits of fruit flies.
Frighetto, G. and Frye, M. A. (2023). Columnar neurons support saccadic bar tracking in Drosophila. Elife 12. PubMed ID: 37014060
Tracking visual objects while maintaining stable gaze is complicated by the different computational requirements for figure-ground discrimination, and the distinct behaviors that these computations coordinate. Drosophila melanogaster uses smooth optomotor head and body movements to stabilize gaze, and impulsive saccades to pursue elongated vertical bars. Directionally selective motion detectors T4 and T5 cells provide inputs to large-field neurons in the lobula plate, which control optomotor gaze stabilization behavior. This study hypothesized that an anatomically parallel pathway represented by T3 cells, which provide inputs to the lobula, drives bar tracking body saccades. Physiological and behavioral experiments were combined to show that T3 neurons respond omnidirectionally to the same visual stimuli that elicit bar tracking saccades, silencing T3 reduced the frequency of tracking saccades, and optogenetic manipulation of T3 acted on the saccade rate in a push-pull manner. Manipulating T3 did not affect smooth optomotor responses to large-field motion. Our results show that parallel neural pathways coordinate smooth gaze stabilization and saccadic bar tracking behavior during flight.
Habenstein, J., Grubel, K., Pfeiffer, K. and Rossler, W. (2023). 3D atlas of cerebral neuropils with previously unknown demarcations in the honey bee brain. J Comp Neurol. PubMed ID: 37070301
Honey bees (Apis mellifera) express remarkable social interactions and cognitive capabilities that have been studied extensively. In many cases, behavioral studies were accompanied by neurophysiological and neuroanatomical investigations. While most studies have focused on primary sensory neuropils, such as the optic lobes or antennal lobes, and major integration centers, such as the mushroom bodies or the central complex, many regions of the cerebrum (the central brain without the optic lobes) of the honey bee are only poorly explored so far, both anatomically and physiologically. To promote studies of these brain regions, this study used anti-synapsin immunolabeling and neuronal tract tracings followed by confocal imaging and 3D reconstructions to demarcate all neuropils in the honey bee cerebrum and close this gap at the anatomical level. 35 neuropils and 25 fiber tracts were demarcated in the honey bee cerebrum, most of which have counterparts in the fly (Drosophila melanogaster) and other insect species that have been investigated so far at this level of detail. The role of cerebral neuropils in multisensory integration in the insect brain is discussed, the importance of this brain atlas for comparative studies is emphasized, and specific architectural features of the honey bee cerebrum are discussed.
Jang, H., Goodman, D. P., Ausborn, J. and von Reyn, C. R. (2023). Azimuthal invariance to looming stimuli in the Drosophila giant fiber escape circuit. J Exp Biol 226(8). PubMed ID: 37066993
Spatially invariant feature detection is a property of many visual systems that rely on visual information provided by two eyes. However, how information across both eyes is integrated for invariant feature detection is not fully understood. This study investigated spatial invariance of looming responses in descending neurons (DNs) of Drosophila melanogaster. It was found that multiple looming responsive DNs integrate looming information across both eyes, even though their dendrites are restricted to a single visual hemisphere. One DN, the giant fiber (GF), responds invariantly to looming stimuli across tested azimuthal locations. It was confirmed that visual information propagates to the GF from the contralateral eye, through an unidentified pathway, and it was demonstrated that the absence of this pathway alters GF responses to looming stimuli presented to the ipsilateral eye. The data highlight a role for bilateral visual integration in generating consistent, looming-evoked escape responses that are robust across different stimulus locations and parameters.

Monday May 15th - Disease Models

Chalmers, M. R., Kim, J. and Kim, N. C. (2023). Eip74EF is a dominant modifier for ALS-FTD-linked VCP(R152H) phenotypes in the Drosophila eye model. BMC Res Notes 16(1): 30. PubMed ID: 36879317
miR-34 is an age-related miRNA regulating age-associated events and long-term brain integrity in Drosophila. Modulating miR-34 and its downstream target, Eip74EF, showed beneficial effects on an age-related disease using a Drosophila model of Spinocerebellar ataxia type 3 expressing SCA3trQ78. These results imply that miR-34 could be a general genetic modifier and therapeutic candidate for age-related diseases. Thus, the goal of this study was to examine the effect of miR-34 and Eip47EF on another age-related Drosophila disease model. Using a Drosophila eye model expressing mutant Drosophila VCP (dVCP) that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or multisystem proteinopathy (MSP), this study demonstrated that abnormal eye phenotypes generated by dVCPR152H were rescued by Eip74EF siRNA expression. Contrary to expectations, miR-34 overexpression alone in the eyes with GMR-GAL4 resulted in complete lethality due to the leaky expression of GMR-GAL4 in other tissues. Interestingly, when miR-34 was co-expressed with dVCPR152H, a few survivors were produced; however, their eye degeneration was greatly exacerbated. These data indicate that, while confirming that the downregulation of Eip74EF is beneficial to the dVCPR152H Drosophila eye model, the high expression level of miR-34 is actually toxic to the developing flies and the role of miR-34 in dVCP(R152H)-mediated pathogenesis is inconclusive in the GMR-GAL4 eye model. Identifying the transcriptional targets of Eip74EF might provide valuable insights into diseases caused by mutations in VCP such as ALS, FTD, and MSP.
Pereira, P. E., ..., Dermaut, B. (2023). C-terminal frameshift variant of TDP-43 with pronounced aggregation-propensity causes rimmed vacuole myopathy but not ALS/FTD. Acta Neuropathol. PubMed ID: 37000196
Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. This study identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43(p.Trp385IlefsTer10)) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43(Trp385IlefsTer10) does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43(p.Trp385IlefsTer10) behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43(p.Trp385IlefsTer10) showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP-43(p.Trp385IlefsTer10) is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. This study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology.
Geng, J., Khaket, T. P., Pan, J., Li, W., Zhang, Y., Ping, Y., Cobos Sillero, M. I. and Lu, B. (2023). Deregulation of ER-mitochondria contact formation and mitochondrial calcium homeostasis mediated by VDAC in fragile X syndrome. Dev Cell 58(7): 597-615.e510. PubMed ID: 37040696
Loss of fragile X messenger ribonucleoprotein (FMRP) causes fragile X syndrome (FXS), the most prevalent form of inherited intellectual disability. This study shows that FMRP interacts with the voltage-dependent anion channel (VDAC) to regulate the formation and function of endoplasmic reticulum (ER)-mitochondria contact sites (ERMCSs), structures that are critical for mitochondrial calcium (mito-Ca(2+)) homeostasis. FMRP-deficient cells feature excessive ERMCS formation and ER-to-mitochondria Ca(2+) transfer. Genetic and pharmacological inhibition of VDAC or other ERMCS components restored synaptic structure, function, and plasticity and rescued locomotion and cognitive deficits of the Drosophila dFmr1 mutant. Expressing FMRP C-terminal domain (FMRP-C), which confers FMRP-VDAC interaction, rescued the ERMCS formation and mito-Ca(2+) homeostasis defects in FXS patient iPSC-derived neurons and locomotion and cognitive deficits in Fmr1 knockout mice. These results identify altered ERMCS formation and mito-Ca(2+) homeostasis as contributors to FXS and offer potential therapeutic targets.
Chang, Y. H. and Dubnau, J. (2023). Endogenous retroviruses and TDP-43 proteinopathy form a sustaining feedback driving intercellular spread of Drosophila neurodegeneration. Nat Commun 14(1): 966. PubMed ID: 36810738
Inter-cellular movement of "prion-like" proteins is thought to explain propagation of neurodegeneration between cells. For example, propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) is proposed to underlie progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases, ALS and FTD are not infectious and injection of aggregated TDP-43 is not sufficient to cause disease. This suggests a missing component of a positive feedback necessary to sustain disease progression. This study demonstrated that endogenous retrovirus (ERV) expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either Drosophila mdg4-ERV (gypsy) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Viral ERV transmission also triggers TDP-43 pathology in recipient cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism potentially underlies the TDP-43 proteinopathy-caused neurodegenerative propagation through neuronal tissue.
Du, S., Zeng, S., Song, L., Ma, H., Chen, R., Luo, J., Wang, X., Ma, T., Xu, X., Sun, H., Yi, P., Guo, J., Huang, Y., Liu, M., Wang, T., Liao, W. P., Zhang, L., Liu, J. Y. and Tang, B. (2023). Functional characterization of novel NPRL3 mutations identified in three families with focal epilepsy. Sci China Life Sci. PubMed ID: 37071290
Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study, three novel mutations in NPRL3 (nitrogen permease regulator-like 3), c.937_945del, c.1514dupC and 6,706-bp genomic DNA (gDNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing (WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of mTOR signaling. These mutations led to truncation of the NPRL3 protein and hampered the binding between NPRL3 and DEPDC5, which is another component of the GATOR1 complex. Consequently, the mutant proteins enhanced mTOR signaling in cultured cells, possibly due to impaired inhibition of mTORC1 by GATOR1. Knockdown of nprl3 in Drosophila resulted in epilepsy-like behavior and abnormal synaptic development. Taken together, these findings expand the genotypic spectrum of NPRL3-associated focal epilepsy and provide further insight into how NPRL3 mutations lead to epilepsy.
Hao, Y., Shao, L., Hou, J., Zhang, Y., Ma, Y., Liu, J., Xu, C., Chen, F., Cao, L. H. and Ping, Y. (2023). Resveratrol and Sir2 Reverse Sleep and Memory Defects Induced by Amyloid Precursor Protein. Neurosci Bull. PubMed ID: 37041405
=Resveratrol (RES), a natural polyphenolic phytochemical, has been suggested as a putative anti-aging molecule for the prevention and treatment of Alzheimer's disease (AD) by the activation of sirtuin 1 (Sirt1/Sir2). This study, tested the effects of RES and Sirt1/Sir2 on sleep and courtship memory in a Drosophila model by overexpression of amyloid precursor protein (APP), whose duplications and mutations cause familial AD. A mild but significant transcriptional increase of Drosophila Sir2 (dSir2) was found by RES supplementation for up to 17 days in APP flies, but not for 7 days. RES and dSir2 almost completely reversed the sleep and memory deficits in APP flies. It was further demonstrated that dSir2 acts as a sleep promotor in Drosophila neurons. Interestingly, RES increased sleep in the absence of dSir2 in dSir2-null mutants, and RES further enhanced sleep when dSir2 was either overexpressed or knocked down in APP flies. Finally, this study showed that A&betal aggregates in APP flies were reduced by RES and dSir2, probably via inhibiting Drosophila β-secretase (dBACE). These data suggest that RES rescues the APP-induced behavioral deficits and Aβ burden largely, but not exclusively, via dSir2.

Friday May 12th - Disease Models

Baisgaard, A. E., Koldby, K. M., Kristensen, T. N., Nyegaard, M. and Rohde, P. D. (2023). Functionally Validating Evolutionary Conserved Risk Genes for Parkinson's Disease in Drosophila melanogaster. Insects 14(2). PubMed ID: 36835737
Parkinson's disease (PD) is a heterogeneous and complex neurodegenerative disorder and large-scale genetic studies have identified >130 genes associated with PD. Although genomic studies have been decisive for understanding of the genetic contributions underlying PD, these associations remain as statistical associations. Lack of functional validation limits the biological interpretation; however, it is labour extensive, expensive, and time consuming. Therefore, the ideal biological system for functionally validating genetic findings must be simple. This study's aim was to assess systematically evolutionary conserved PD-associated genes using Drosophila melanogaster. From a literature review, a total of 136 genes have found to be associated with PD in GWAS studies, of which 11 are strongly evolutionary conserved between Homo sapiens and D. melanogaster. By ubiquitous gene expression knockdown of the PD-genes in D. melanogaster, the flies' escape response was investigated by assessing their negative geotaxis response, a phenotype that has previously been used to investigate PD in D. melanogaster. Gene expression knockdown was successful in 9/11 lines, and phenotypic consequences were observed in 8/9 lines. The results provide evidence that genetically modifying expression levels of PD genes in D. melanogaster caused reduced climbing ability of the flies, potentially supporting their role in dysfunctional locomotion, a hallmark of PD.
Bhatnagar, A., Krick, K., Karisetty, B. C., Armour, E. M., Heller, E. A. and Elefant, F. (2023). Tip60's Novel RNA-Binding Function Modulates Alternative Splicing of Pre-mRNA Targets Implicated in Alzheimer's Disease. J Neurosci 43(13): 2398-2423. PubMed ID: 36849418
The severity of Alzheimer's disease (AD) progression involves a complex interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT)-mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. This study reports a novel RNA binding function for Tip60 in addition to its HAT function. Tip60 preferentially interacts with pre-mRNAs emanating from its chromatin neural gene targets in the Drosophila brain and this RNA binding function is conserved in human hippocampus and disrupted in Drosophila brains that model AD pathology and in AD patient hippocampus of either sex. Since RNA splicing occurs co-transcriptionally and alternative splicing (AS) defects are implicated in AD, this study investigated whether Tip60-RNA targeting modulates splicing decisions and whether this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq datasets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs are identified as bona-fide Tip60-RNA targets that are enriched for in the AD-gene curated database, with some of these AS alterations prevented against by increasing Tip60 in the fly brain. Further, human orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60's splicing function in AD pathogenesis. These results support a novel RNA interaction and splicing regulatory function for Tip60 that may underly AS impairments that hallmark AD etiology.
Agostini, F., Bubacco, L., Chakrabarti, S. and Bisaglia, M. (2023). α-Synuclein Toxicity in Drosophila melanogaster Is Enhanced by the Presence of Iron: Implications for Parkinson's Disease. Antioxidants (Basel) 12(2). PubMed ID: 36829820
Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the preferential loss of dopaminergic neurons and by the accumulation of intracellular inclusions mainly composed of α-synuclein (&alpha-Syn). While the etiopathogenesis of the disorder is still elusive, recent experimental evidence supports the involvement of ferroptosis, an iron-dependent cell death pathway, in the pathogenesis of PD. Using different ferroptosis inducers and inhibitors, this study evaluated, in vivo, the involvement of iron in the α-Syn-mediated toxicity. Using a Drosophila melanogaster model of PD based on the selective over-expression of α-Syn within dopaminergic neurons, this study demonstrated that the over-expression of α-Syn promotes the accumulation of protein aggregates, which is accompanied by dopaminergic neurodegeneration, locomotor impairment, and lifespan reduction. These pathological phenotypes were further exacerbated by reduced intracellular levels of glutathione or increased concentrations of iron. Coherently, both the use of an iron chelator and the presence of the antioxidant compound N-acetylcysteine exerted protective effects. Overall, these results support the involvement of ferroptosis in the α-Syn-mediated toxicity.
Bademosi, A. T., Decet, M., Kuenen, S., Calatayud, C., Swerts, J., Gallego, S. F., Schoovaerts, N., Karamanou, S., Louros, N., Martin, E., Sibarita, J. B., Vints, K., Gounko, N. V., Meunier, F. A., Economou, A., Versees, W., Rousseau, F., Schymkowitz, J., Soukup, S. F. and Verstreken, P. (2023). EndophilinA-dependent coupling between activity-induced calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation. Neuron. PubMed ID: 36827984
Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. This study shows in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, this study revealed a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival.
Beckmann, A., Ramirez, P., Gamez, M., Gonzalez, E., De Mange, J., Bieniek, K. F., Ray, W. J. and Frost, B. (2023). Moesin is an effector of tau-induced actin overstabilization, cell cycle activation, and neurotoxicity in Alzheimer's disease. iScience 26(3): 106152. PubMed ID: 36879821
In Alzheimer's disease, neurons acquire phenotypes that are also present in various cancers, including aberrant activation of the cell cycle. Unlike cancer, cell cycle activation in post-mitotic neurons is sufficient to induce cell death. Multiple lines of evidence suggest that abortive cell cycle activation is a consequence of pathogenic forms of tau, a protein that drives neurodegeneration in Alzheimer's disease and related "tauopathies." This study combined network analyses of human Alzheimer's disease and mouse models of Alzheimer's disease and primary tauopathy with studies in Drosophila to discover that pathogenic forms of tau drive cell cycle activation by disrupting a cellular program involved in cancer and the epithelial-mesenchymal transition (EMT). Moesin, an EMT driver, is elevated in cells harboring disease-associated phosphotau, over-stabilized actin, and ectopic cell cycle activation. It was further found that genetic manipulation of Moesin mediates tau-induced neurodegeneration. Taken together, this study identifies novel parallels between tauopathy and cancer.
Carvajal-Oliveros, A., Dominguez-Baleon, C., Sanchez-Diaz, I., Zambrano-Tipan, D., Hernandez-Vargas, R., Campusano, J. M., Narváez-Padilla, V. and Reynaud, E. (2023). Parkinsonian phenotypes induced by Synphilin-1 expression are differentially contributed by serotonergic and dopaminergic circuits and suppressed by nicotine treatment. PLoS One 18(3): e0282348. PubMed ID: 36857384
Synphilin-1 is a protein encoded by the human SNCAIP gene whose function has yet to be fully understood. However, it has been linked to familial Parkinson's disease (PD). Synphilin-1 is a major component of the Lewy bodies found in neurons in the substantia nigra pars compacta of PD patients. Synphilin-1 expression in serotonergic and/or dopaminergic neurons of Drosophila melanogaster induces neurodegeneration, as well as motor and non-motor PD like symptoms. This work examined the contribution of the serotonergic and dopaminergic circuits in the development of PD-like phenotypes. It was found that olfactory and visual symptoms are majorly contributed by the Parkinson's disease (PD). serotonergic system, and that motor symptoms and reduction in survival are mainly contributed by the dopaminergic system. Chronic nicotine treatment was able to suppress several of these symptoms. These results indicate that both the serotonergic and dopaminergic systems contribute to different aspects of PD symptomatology and that nicotine has beneficial effects on specific symptoms.

Thursday May 11th - Evolution

Benton, R. and Himmel, N. J. (2023). Structural screens identify candidate human homologs of insect chemoreceptors and cryptic Drosophila gustatory receptor-like proteins. Elife 12. PubMed ID: 36803935
Insect odorant receptors and gustatory receptors define a superfamily of seven transmembrane domain ion channels (referred to here as 7TMICs), with homologs identified across Animalia except Chordata. Previously, sequence-based screening methods were used to reveal conservation of this family in unicellular eukaryotes and plants (DUF3537 proteins). This study combined three-dimensional structure-based screening, ab initio protein folding predictions, phylogenetics, and expression analyses to characterize additional candidate homologs with tertiary but little or no primary structural similarity to known 7TMICs, including proteins in disease-causing Trypanosoma. Unexpectedly, structural similarity was identified between 7TMICs and PHTF proteins, a deeply conserved family of unknown function, whose human orthologs display enriched expression in testis, cerebellum, and muscle. Divergent groups of 7TMICs were found in insects, which was termed the gustatory receptor-like (Grl) proteins. Several Drosophila melanogaster Grls display selective expression in subsets of taste neurons, suggesting that they are previously unrecognized insect chemoreceptors. Although the possibility of remarkable structural convergence cannot be excluded, these findings support the origin of 7TMICs in a eukaryotic common ancestor, counter previous assumptions of complete loss of 7TMICs in Chordata, and highlight the extreme evolvability of this protein fold, which likely underlies its functional diversification in different cellular contexts.
Cridland, J. M., Contino, C. E. and Begun, D. J. (2023). Selection and Geography Shape Male Reproductive Tract Transcriptomes in Drosophila Melanogaster. Genetics. PubMed ID: 36869688
Transcriptome analysis of several animal clades suggests that male reproductive tract gene expression evolves quickly. However, the factors influencing the abundance and distribution of within-species variation, the ultimate source of interspecific divergence, are poorly known. Drosophila melanogaster, an ancestrally African species that has recently spread throughout the world and colonized the Americas in the last roughly 100 years, exhibits phenotypic and genetic latitudinal clines on multiple continents, consistent with a role for spatially varying selection in shaping its biology. Nevertheless, geographic expression variation in the Americas is poorly described, as is its relationship to African expression variation. This study investigate these issues through analysis of two male reproductive tissue transcriptomes (testis and accessory gland) in samples from Maine (USA), Panama, and Zambia. Dramatic differences were found between these tissues in differential expression between Maine and Panama, with the accessory glands exhibiting abundant expression differentiation and the testis exhibiting very little. Latitudinal expression differentiation appears to be influenced by selection on Panama expression phenotypes. While the testis shows little latitudinal expression differentiation, it exhibits much greater differentiation than the accessory gland in Zambia vs. American population comparisons. Expression differentiation for both tissues is non-randomly distributed across the genome on a chromosome arm scale. Interspecific expression divergence between D. melanogaster and D. simulans is discordant with rates of differentiation between D. melanogaster populations. Strongly heterogeneous expression differentiation across tissues and timescales suggests a complex evolutionary process involving major temporal changes in the way selection influences expression evolution in these organs. /td>
Lollar, M. J., Biewer-Heisler, T. J., Danen, C. E. and Pool, J. E. (2023). Hybrid Breakdown in Male Reproduction Between Recently-Diverged Drosophila melanogaster Populations Has a Complex and Variable Genetic Architecture. Evolution. PubMed ID: 37071601
Secondary contact between formerly isolated populations may result in hybrid breakdown, in which untested allelic combinations in hybrids are maladaptive and limit genetic exchange. Studying early-stage reproductive isolation may yield key insights into the genetic architectures and evolutionary forces underlying the first steps toward speciation. This study leveraged the recent worldwide expansion of Drosophila melanogaster to test for hybrid breakdown between populations that diverged within the last 13,000 years. Clear evidence was found for hybrid breakdown in male reproduction, but not female reproduction or viability, supporting the prediction that hybrid breakdown affects the heterogametic sex first. The frequency of non-reproducing F2 males varied among different crosses involving the same southern African and European populations, as did the qualitative effect of cross direction, implying a genetically variable basis of hybrid breakdown and a role for uniparentally inherited factors. The levels of breakdown observed in F2 males were not recapitulated in backcrossed individuals, consistent with the existence of incompatibilities with at least three partners. Thus, some of the very first steps toward reproductive isolation could involve incompatibilities with complex and variable genetic architectures. Collectively, these findings emphasize this system's potential for future studies on the genetic and organismal basis of early-stage reproductive isolation.
Chang, C. H., Mejia Natividad, I. and Malik, H. S. (2023). Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes. Elife 12. PubMed ID: 36763410
Many animal species employ sperm nuclear basic proteins (SNBPs; see Protamine A) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. This study used a phylogenomic approach to investigate SNBP diversification in Drosophila species. It was found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. Nineteen independent SNBP gene amplification events were found that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. It is hypothesized that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species.
Forejt, J. and Jansa, P. (2023). Meiotic Recognition of Evolutionarily Diverged Homologs: Chromosomal Hybrid Sterility Revisited. Mol Biol Evol. PubMed ID: 37030001
Hybrid sterility (HS) is an early postzygotic reproductive isolation mechanism observed in all sexually reproducing species. Infertility of hybrids prevents gene flow between incipient species and leads to speciation. While Drosophila studies have focused almost exclusively on the genic control of HS, two other model species, Mus musculus and budding yeast provided the first experimental evidence of hybrid sterility governed by the nongenic effects of DNA sequence divergence. This study proposes that the nongenic effect of increasing DNA divergence between closely related species may impair mutual recognition of homologous chromosomes and disrupt their synapsis. Unsynapsed or mispaired homologs can induce early meiotic arrest, or their random segregation can cause aneuploidy of spermatids and sperm cells. Impaired recognition of homologs may thus act as a universal chromosomal checkpoint contributing to the complexity of genetic control of HS. Chromosomal HS controlled by the Prdm9 gene in mice and HS driven by the mismatch repair machinery in yeast are currently the most advanced examples of chromosomal homology search-based HS. More focus on the cellular and molecular phenotypes of meiosis will be needed to further validate the role of homolog recognition in hybrid sterility and speciation.
Flynn, J. M., Hu, K. B. and Clark, A. G. (2023). Three recent sex chromosome-to-autosome fusions in a Drosophila virilis strain with high satellite DNA content. Genetics. PubMed ID: 37052958
The karyotype, or number and arrangement of chromosomes, has varying levels of stability across both evolution and disease. Karyotype changes often originate from DNA breaks near the centromeres of chromosomes, which generally contain long arrays of tandem repeats or satellite DNA. Drosophila virilis possesses among the highest relative satellite abundances of studied species, with almost half its genome composed of three related 7 bp satellites. We discovered a strain of D. virilis that is inferred to have recently undergone three independent chromosome fusion events involving the X and Y chromosomes, in addition to one subsequent fission event. This study isolated and characterized the four different karyotypes discovered in this strain which is believed to demonstrate remarkable genome instability. One of the substrains with an X-autosome fusion has a X-to-Y chromosome nondisjunction rate 20x higher than the D. virilis reference strain (21% vs. 1%). Finally, an overall higher rate of DNA breakage was found in the substrain with higher satellite DNA compared to a genetically similar substrain with less satellite DNA. This suggests satellite DNA abundance may play a role in the risk of genome instability. Overall, this study introduces a novel system consisting of a single strain with four different karyotypes, which will be useful for future studies of genome instability, centromere function, and sex chromosome evolution.

Wednesday May 10th - Signaling

Zhang, Y., Ting, C. Y., Yang, S., Reich, J., Fru, K. and Lilly, M. A. (2023). Wdr59 promotes or inhibits TORC1 activity depending on cellular context. Proc Natl Acad Sci U S A 120(1): e2212330120. PubMed ID: 36577058
Target of Rapamycin Complex I (TORC1) is a central regulator of metabolism in eukaryotes that responds to a wide array of negative and positive inputs. The GTPase-activating protein toward Rags (GATOR) signaling pathway acts upstream of TORC1 and is comprised of two subcomplexes. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation by serving as a GTPase-activating protein (GAP) for the TORC1 activator RagA/B, a component of the lysosomally located Rag GTPase. The multi-protein GATOR2 complex inhibits the activity of GATOR1 and thus promotes TORC1 activation. This study reports that Wdr59, originally assigned to the GATOR2 complex based on studies performed in tissue culture cells, unexpectedly has a dual function in TORC1 regulation in Drosophila. <../aimorph/oocyte.htm">ovary and the eye imaginal disc brain complex, Wdr59 inhibits TORC1 activity by opposing the GATOR2-dependent inhibition of GATOR1. Conversely, in the Drosophila fat body, Wdr59 promotes the accumulation of the GATOR2 component Mio and is required for TORC1 activation. Similarly, in mammalian HeLa cells, Wdr59 prevents the proteolytic destruction of GATOR2 proteins Mio and Wdr24. Consistent with the reduced levels of the TORC1-activating GATOR2 complex, Wdr59KOs HeLa cells have reduced TORC1 activity which is restored along with GATOR2 protein levels upon proteasome inhibition. Taken together, our data support the model that the Wdr59 component of the GATOR2 complex functions to promote or inhibit TORC1 activity depending on cellular context.
Bu, S., Lau, S. S. Y., Yong, W. L., Zhang, H., Thiagarajan, S., Bashirullah, A. and Yu, F. (2023). Polycomb group genes are required for neuronal pruning in Drosophila. BMC Biol 21(1): 33. PubMed ID: 36793038
Pruning that selectively eliminates unnecessary or incorrect neurites is required for proper wiring of the mature nervous system. During Drosophila metamorphosis, dendritic arbourization sensory neurons (ddaCs) and mushroom body (MB) γ neurons can selectively prune their larval dendrites and/or axons in response to the steroid hormone ecdysone. An ecdysone-induced transcriptional cascade plays a key role in initiating neuronal pruning. However, how downstream components of ecdysone signalling are induced remains not entirely understood. This study identified that Scm, a component of Polycomb group (PcG) complexes, is required for dendrite pruning of ddaC neurons. Two PcG complexes, PRC1 and PRC2, are important for dendrite pruning. Interestingly, depletion of PRC1 strongly enhances ectopic expression of Abdominal B (Abd-B) and Sex combs reduced, whereas loss of PRC2 causes mild upregulation of Ultrabithorax and Abdominal A in ddaC neurons. Among these Hox genes, overexpression of Abd-B causes the most severe pruning defects, suggesting its dominant effect. Knockdown of the core PRC1 component Polyhomeotic (Ph) or Abd-B overexpression selectively downregulates Mical expression, thereby inhibiting ecdysone signalling. Finally, Ph is also required for axon pruning and Abd-B silencing in MB γ neurons, indicating a conserved function of PRC1 in two types of pruning. This study demonstrates important roles of PcG and Hox genes in regulating ecdysone signalling and neuronal pruning in Drosophila. Moreover, our findings suggest a non-canonical and PRC2-independent role of PRC1 in Hox gene silencing during neuronal pruning.
Ayoub, M., David, L. M., Shklyar, B., Hakim-Mishnaevski, K. and Kurant, E. (2023). Drosophila FGFR/Htl signaling shapes embryonic glia to phagocytose apoptotic neurons. Cell Death Discov 9(1): 90. PubMed ID: 36898998
Glial phagocytosis of apoptotic neurons is crucial for development and proper function of the central nervous system. Relying on transmembrane receptors located on their protrusions, phagocytic glia recognize and engulf apoptotic debris. Like vertebrate microglia, Drosophila phagocytic glial cells form an elaborate network in the developing brain to reach and remove apoptotic neurons. However, the mechanisms controlling creation of the branched morphology of these glial cells critical for their phagocytic ability remain unknown. This study demonstrated that during early embryogenesis, the Drosophila fibroblast growth factor receptor (FGFR) Heartless (Htl) and its ligand Pyramus are essential in glial cells for the formation of glial extensions, the presence of which strongly affects glial phagocytosis of apoptotic neurons during later stages of embryonic development. Reduction in Htl pathway activity results in shorter lengths and lower complexity of glial branches, thereby disrupting the glial network. This work thus illuminates the important role Htl signaling plays in glial subcellular morphogenesis and in establishing glial phagocytic ability.
Borkowsky, S., Gass, M., Alavizargar, A., Hanewinkel, J., Hallstein, I., Nedvetsky, P., Heuer, A. and Krahn, M. P. (2023). Phosphorylation of LKB1 by PDK1 Inhibits Cell Proliferation and Organ Growth by Decreased Activation of AMPK. Cells 12(5). PubMed ID: 36899949
The master kinase LKB1 is a key regulator of several cellular processes, including cell proliferation, cell polarity and cellular metabolism. It phosphorylates and activates several downstream kinases, including AMP-dependent kinase, AMPK. Activation of AMPK by low energy supply and phosphorylation of LKB1 results in an inhibition of mTOR, thus decreasing energy-consuming processes, in particular translation and, thus, cell growth. LKB1 itself is a constitutively active kinase, which is regulated by posttranslational modifications and direct binding to phospholipids of the plasma membrane. This study reports that LKB1 binds to Phosphoinositide-dependent kinase (PDK1) by a conserved binding motif. Furthermore, a PDK1-consensus motif is located within the kinase domain of LKB1 and LKB1 gets phosphorylated by PDK1 in vitro. In Drosophila, knockin of phosphorylation-deficient LKB1 results in normal survival of the flies, but an increased activation of LKB1, whereas a phospho-mimetic LKB1 variant displays decreased AMPK activation. As a functional consequence, cell growth as well as organism size is decreased in phosphorylation-deficient LKB1. Molecular dynamics simulations of PDK1-mediated LKB1 phosphorylation revealed changes in the ATP binding pocket, suggesting a conformational change upon phosphorylation, which in turn can alter LKB1's kinase activity. Thus, phosphorylation of LKB1 by PDK1 results in an inhibition of LKB1, decreased activation of AMPK and enhanced cell growth.
Bonello, T. T., Cai, D., Fletcher, G. C., Wiengartner, K., Pengilly, V., Lange, K. S., Liu, Z., Lippincott-Schwartz, J., Kavran, J. M. and Thompson, B. J. (2023). Phase separation of Hippo signalling complexes. Embo j 42(6): e112863. PubMed ID: 36807601
The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs-Expanded (Crb-Ex) and/or Merlin-Kibra (Mer-Kib) proteins at the apical domain of epithelial cells. This study shows that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6-hexanediol treatment. Overexpressing Ex or Kib induces formation of micron-scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low-complexity domains and purified Hpo-Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. It is proposed that apical Hpo kinase activation occurs in phase separated "signalosomes" induced by clustering of upstream pathway components.
Chen, D., Lan, X., Huang, X., Huang, J., Zhou, X., Miao, Z., Ma, Y., Goto, A., Ji, S. and Hoffmann, J. A. (2023). Single Cell Analysis of the Fate of Injected Oncogenic RasV12 Cells in Adult Wild Type Drosophila. J Innate Immun 15(1): 442-467. PubMed ID: 36996781
=Dish-cultured oncogenic RasV12 cells into adult male flies and single cell transcriptomics was used to examine their destiny within the host after 11 days. The preinjection samples were identified in the 11-day postinjection samples in all 16 clusters of cells, of which 5 disappeared during the experiment in the host. The other cell clusters expanded and expressed genes involved in the regulation of cell cycle, metabolism, and development. In addition, three clusters expressed genes related to inflammation and defense. Predominant among these were genes coding for phagocytosis and/or characteristic for a plasmatocytes (the fly equivalent of macrophages). A pilot experiment indicated that the injection into flies of oncogenic cells, in which two of most strongly expressed genes had been previously silenced by RNA interference, into flies resulted in a dramatic reduction of their proliferation in the host flies as compared to controls. As has been shown earlier, the proliferation of the injected oncogenic cells in the adult flies is a hallmark of the disease and induces a wave of transcriptions in the experimental flies. We hypothesize that this results from a bitter dialogue between the injected cells and the host, while the experiments presented here should contribute to deciphering this dialogue.

Monday May 8th - Disease Models

Wan, R. P., Liu, Z. G., Huang, X. F., Kwan, P., Li, Y. P., Qu, X. C., Ye, X. G., Chen, F. Y., Zhang, D. W., He, M. F., Wang, J., Mao, Y. L. and Qiao, J. D. (2023). YWHAZ variation causes intellectual disability and global developmental delay with brain malformation. Hum Mol Genet 32(3): 462-472. PubMed ID: 36001342
YWHAZ encodes an adapter protein 14-3-3ζ, which is involved in many signaling pathways that control cellular proliferation, migration and differentiation. It has not been definitely correlated to any phenotype in OMIM. To investigate the role of YWHAZ gene in intellectual disability and global developmental delay, whole-exon sequencing was conducted in all of the available members from a large three-generation family, and it was discovered that a novel variant of the YWHAZ gene was associated with intellectual disability and global developmental delay. This variant is a missense mutation of YWHAZ, p.Lys49Asn/c.147A > T, which was found in all affected members but not found in other unaffected members. Computational modeling and knockdown/knockin was conducted with Drosophila to confirm the role of the YWHAZ variant in intellectual disability. Computational modeling showed that the binding energy was increased in the mutated protein combining with the ligand indicating that the c147A > T variation was a loss-of-function variant. Cognitive defects and mushroom body morphological abnormalities were observed in YWHAZ c.147A > T knockin flies. The YWHAZ knockdown flies also manifested serious cognitive defects with hyperactivity behaviors, which is consistent with the clinical features. These clinical and experimental results consistently suggested that YWHAZ was a novel intellectual disability pathogenic gene.
Kasture, A. S., Fischer, F. P., Kunert, L., Burger, M. L., Burgstaller, A. C., El-Kasaby, A., Hummel, T. and Sucic, S. (2022). Drosophila melanogaster as a model for unraveling unique molecular features of epilepsy elicited by human GABA transporter 1 variants. Front Neurosci 16: 1074427. PubMed ID: 36741049
Mutations in the human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1: see Drosophila VGAT) can instigate myoclonic-atonic and other generalized epilepsies in the afflicted individuals. This study systematically examined fifteen hGAT-1 disease variants, all of which dramatically reduced or completely abolished GABA uptake activity. Many of these loss-of-function variants were absent from their regular site of action at the cell surface, due to protein misfolding and/or impaired trafficking machinery (as verified by confocal microscopy and de-glycosylation experiments). A modest fraction of the mutants displayed correct targeting to the plasma membrane, but nonetheless rendered the mutated proteins devoid of GABA transport, possibly due to structural alterations in the GABA binding site/translocation pathway. This study focused on a folding-deficient A288V variant. In flies, A288V reiterated its impeded expression pattern, closely mimicking the ER-retention demonstrated in transfected HEK293 cells. Functionally, A288V presented a temperature-sensitive seizure phenotype in fruit flies. We employed diverse small molecules to restore the expression and activity of folding-deficient hGAT-1 epilepsy variants, in vitro (in HEK293 cells) and in vivo (in flies). This study identified three compounds (chemical and pharmacological chaperones) conferring moderate rescue capacity for several variants. These data grant crucial new insights into: (1) the molecular basis of epilepsy in patients harboring hGAT-1 mutations, and (2) a proof-of-principle that protein folding deficits in disease-associated hGAT-1 variants can be corrected using the pharmacochaperoning approach. Such innovative pharmaco-therapeutic prospects inspire the rational design of novel drugs for alleviating the clinical symptoms triggered by the numerous emerging pathogenic mutations in hGAT-1.
Andrews, J. C., Mok, J. W., Kanca, O., Jangam, S., Tifft, C., Macnamara, E. F., Russell, B. E., Wang, L. K., Nelson, S. F., Bellen, H. J., Yamamoto, S., Malicdan, M. C. V. and Wangler, M. F. (2023). De Novo Variants in MRTFB have gain of function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features. Genet Med: 100833. PubMed ID: 37013900
Myocardin-Related Transcription Factor B (MRTFB: Drosophila Myocardin-related transcription factor) is an important transcriptional regulator which promotes the activity of an estimated 300 genes but is not known to underlie a Mendelian disorder. Probands were identified through the efforts of the Undiagnosed Disease Network. As the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, this study generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Actin binding assays were used to validate the effect of the variants on MRTFB. This study reports two pediatric probands with de novo variants in MRTFB (p.R104G and p.A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. Expression of the variants within wing tissues of a fruit fly model resulted in changes in wing morphology. The MRTFB(R104G) and MRTFB(A91P) variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the Actin cytoskeleton. The MRTFB(R104G) and MRTFB(A91P) variants affect the regulation of the protein and underlie a novel neurodevelopmental disorder. Overall, these data suggests these variants act as a gain of function.
Witt, E., Langer, C. B., Svetec, N. and Zhao, L. (2023). Transcriptional and mutational signatures of the Drosophila ageing germline. Nat Ecol Evol. PubMed ID: 36635344
Ageing is a complex biological process that is accompanied by changes in gene expression and mutational load. In many species, including humans, older fathers pass on more paternally derived de novo mutations; however, the cellular basis and cell types driving this pattern are still unclear. To explore the root causes of this phenomenon, this study performed single-cell RNA sequencing on testes from young and old male Drosophila and genomic sequencing (DNA sequencing) on somatic tissues from the same flies. Early germ cells from old and young flies were found to enter spermatogenesis with similar mutational loads but older flies are less able to remove mutations during spermatogenesis. Mutations in old cells may also increase during spermatogenesis. These data reveal that old and young flies have distinct mutational biases. Many classes of genes show increased postmeiotic expression in the germlines of older flies. Late spermatogenesis-biased genes have higher dN/dS (ratio of non-synonymous to synonymous substitutions) than early spermatogenesis-biased genes, supporting the hypothesis that late spermatogenesis is a source of evolutionary innovation. Surprisingly, genes biased in young germ cells show higher dN/dS than genes biased in old germ cells. These results provide new insights into the role of the germline in de novo mutation.
Wilson, A., Periandri, E. M., Sievers, M. and Petruccelli, E. (2023). Drosophila Stat92E Signaling Following Pre-exposure to Ethanol. Neurosci Insights 18: 26331055221146755. PubMed ID: 36643884
Repeated exposure to alcohol alters neuromolecular signaling that influences acute and long-lasting behaviors underlying Alcohol Use Disorder (AUD). Recent animal model research has implicated changes in the conserved JAK/STAT pathway, a signaling pathway classically associated with development and the innate immune system. How ethanol exposure impacts STAT signaling within neural cells is currently unclear. This study investigated the role of Drosophila Stat92E in ethanol-induced locomotion, signaling activity, and downstream transcriptional responses. Findings suggest that expressing Stat92E-RNAi causes enhanced ethanol-induced hyperactivity in flies previously exposed to ethanol. Furthermore, alternative splicing of Stat92E itself was detected after repeated ethanol exposure, although no changes were found in downstream transcriptional activity. This work adds to our growing understanding of altered neuromolecular signaling following ethanol exposure and suggests that STAT signaling may be a relevant target to consider for AUD treatment.
Chang, Y. H. and Dubnau, J. (2023). Endogenous retroviruses and TDP-43 proteinopathy form a sustaining feedback driving intercellular spread of Drosophila neurodegeneration. Nat Commun 14(1): 966. PubMed ID: 36810738
Inter-cellular movement of "prion-like" proteins is thought to explain propagation of neurodegeneration between cells. For example, propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) is proposed to underlie progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases, ALS and FTD are not infectious and injection of aggregated TDP-43 is not sufficient to cause disease. This suggests a missing component of a positive feedback necessary to sustain disease progression. This study demonstrates that endogenous retrovirus (ERV) expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either Drosophila mdg4-ERV (gypsy) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Viral ERV transmission also triggers TDP-43 pathology in recipient cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism potentially underlies the TDP-43 proteinopathy-caused neurodegenerative propagation through neuronal tissue.

Thursday May 4th - Evolution

Ohashi, T. S., Ishikawa, Y., Awasaki, T., Su, M. P., Yoneyama, Y., Morimoto, N. and Kamikouchi, A. (2023). Evolutionary conservation and diversification of auditory neural circuits that process courtship songs in Drosophila. Sci Rep 13(1): 383. PubMed ID: 36611081
Acoustic communication signals diversify even on short evolutionary time scales. To understand how the auditory system underlying acoustic communication could evolve, this study conducted a systematic comparison of the early stages of the auditory neural circuit involved in song information processing between closely-related fruit-fly species. Male Drosophila melanogaster and D. simulans produce different sound signals during mating rituals, known as courtship songs. Female flies from these species selectively increase their receptivity when they hear songs with conspecific temporal patterns. This study firstly confirmed interspecific differences in temporal pattern preferences; D. simulans preferred pulse songs with longer intervals than D. melanogaster. Primary and secondary song-relay neurons, JO neurons and AMMC-B1 neurons, shared similar morphology and neurotransmitters between species. The temporal pattern preferences of AMMC-B1 neurons were also relatively similar between species, with slight but significant differences in their band-pass properties. Although the shift direction of the response property matched that of the behavior, these differences are not large enough to explain behavioral differences in song preferences. This study enhances understanding of the conservation and diversification of the architecture of the early-stage neural circuit which processes acoustic communication signals.
Lu, G. A., Zhang, J., Zhao, Y., Chen, Q., Lin, P., Tang, T., Tang, Z., Wen, H., Liufu, Z. and Wu, C. I. (2023). Canalization of Phenotypes-When the Transcriptome is Constantly but Weakly Perturbed. Mol Biol Evol 40(1). PubMed ID: 36617265
Recent studies have increasingly pointed to microRNAs (miRNAs) as the agent of gene regulatory network (GRN) stabilization as well as developmental canalization against constant but small environmental perturbations. To analyze mild perturbations, this study constructed a Dicer-1 knockdown line (dcr-1 KD) in Drosophila that modestly reduces all miRNAs by, on average, ~20%. The defining characteristic of stabilizers is that, when their capacity is compromised, GRNs do not change their short-term behaviors. Indeed, even with such broad reductions across all miRNAs, the changes in the transcriptome are very modest during development in stable environment. By comparison, broad knockdowns of other regulatory genes (esp. transcription factors) by the same method should lead to drastic changes in the GRNs. The consequence of destabilization may thus be in long-term development as postulated by the theory of canalization. Flies with modest miRNA reductions may gradually deviate from the developmental norm, resulting in late-stage failures such as shortened longevity. In the optimal culture condition, the survival to adulthood is indeed normal in the dcr-1 KD line but, importantly, adult longevity is reduced by ~90%. When flies are stressed by high temperature, dcr-1 KD induces lethality earlier in late pupation and, as the perturbations are shifted earlier, the affected stages are shifted correspondingly. Hence, in late stages of development with deviations piling up, GRN would be increasingly in need of stabilization. In conclusion, miRNAs appear to be a solution to weak but constant environmental perturbations.
Casier, K., Autaa, J., Gueguen, N., Delmarre, V., Marie, P. P., Ronsseray, S., Carre, C., Brasset, E., Teysset, L. and Boivin, A. (2023). The histone demethylase Kdm3 prevents auto-immune piRNAs production in Drosophila. Sci Adv 9(14): eade3872. PubMed ID: 37027460
Genome integrity of the animal germline is protected from transposable element activity by PIWI-interacting RNAs (piRNAs). While piRNA biogenesis is intensively explored, little is known about the genetical determination of piRNA clusters, the genomic sources of piRNAs. Using a bimodal epigenetic state piRNA cluster (BX2), this study identified the histone demethylase Kdm3 as being able to prevent a cryptic piRNA production. In the absence of Kdm3, dozens of coding gene-containing regions become genuine germline dual-strand piRNA clusters. Eggs laid by Kdm3 mutant females show developmental defects phenocopying loss of function of genes embedded into the additional piRNA clusters, suggesting an inheritance of functional ovarian "auto-immune" piRNAs. Antagonizing piRNA cluster determination through chromatin modifications appears crucial to prevent auto-immune genic piRNAs production.
Forbes Beadle, L., Love, J. C., Shapovalova, Y., Artemev, A., Rattray, M. and Ashe, H. L. (2023). Combined modelling of mRNA decay dynamics and single-molecule imaging in the Drosophila embryo uncovers a role for P-bodies in 5' to 3' degradation. PLoS Biol 21(1): e3001956. PubMed ID: 36649329
Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. This study used a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. This study showed how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, this study provided evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, this study detect an enrichment of mRNA 3' ends in P-bodies in the early embryo, consistent with 5' to 3' degradation occurring in P-bodies for at least a subset of mRNAs. These findings are discussedin relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.
Itai, T., Sugie, A., Nitta, Y., Maki, R., Suzuki, T., Shinkai, Y., Watanabe, Y., Nakano, Y., Ichikawa, K., Okamoto, N., Utsuno, Y., Koshimizu, E., Fujita, A., Hamanaka, K., Uchiyama, Y., Tsuchida, N., Miyake, N., Misawa, K., Mizuguchi, T., Miyatake, S. and Matsumoto, N. (2023). A novel NONO variant that causes developmental delay and cardiac phenotypes. Sci Rep 13(1): 975. PubMed ID: 36653413
The Drosophila behavior/human splicing protein family is involved in numerous steps of gene regulation. In humans, this family consists of three proteins: SFPQ, PSPC1, and NONO. Hemizygous loss-of-function (LoF) variants in NONO cause a developmental delay with several complications (e.g., distinctive facial features, cardiac symptoms, and skeletal symptoms) in an X-linked recessive manner. Most of the reported variants have been LoF variants, and two missense variants have been reported as likely deleterious but with no functional validation. This study reports three individuals from two families harboring an identical missense variant that is located in the nuclear localization signal, NONO: NM_001145408.2:c.1375C > G p.(Pro459Ala). All of them were male and the variant was inherited from their asymptomatic mothers. Individual 1 was diagnosed with developmental delay and cardiac phenotypes (ventricular tachycardia and dilated cardiomyopathy), which overlapped with the features of reported individuals having NONO LoF variants. Individuals 2 and 3 were monozygotic twins. Unlike in Individual 1, developmental delay with autistic features was the only symptom found in them. A fly experiment and cell localization experiment showed that the NONO variant impaired its proper intranuclear localization, leading to mild LoF. These findings suggest that deleterious NONO missense variants should be taken into consideration when whole-exome sequencing is performed on male individuals with developmental delay with or without cardiac symptoms.
Layana, C., Vilardo, E. S., Corujo, G., Hernandez, G. and Rivera-Pomar, R. (2023). Drosophila Me31B is a Dual eIF4E-Interacting Protein. J Mol Biol 435(5): 167949. PubMed ID: 36638908
Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). This study showed that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. This study shows that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.

Wednesday May 3rd- Adult Neural Development and Function

Yamada, D., Bushey, D., Li, F., Hibbard, K. L., Sammons, M., Funke, J., Litwin-Kumar, A., Hige, T. and Aso, Y. (2023). Hierarchical architecture of dopaminergic circuits enables second-order conditioning in Drosophila. Elife 12. PubMed ID: 36692262
Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. This study identified a feedforward circuit formed between dopamine subsystems and showed that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. A slow and stable memory compartment can serve as an effective 'teacher' by instructing other faster and transient memory compartments via a single key interneuron, which we identify by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the 'student' compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists.
Bhattacharjee, S., Iyer, E. P. R., Iyer, S. C., Nanda, S., Rubaharan, M., Ascoli, G. A. and Cox, D. N. (2023). The Zinc-BED Transcription Factor Bedwarfed Promotes Proportional Dendritic Growth and Branching through Transcriptional and Translational Regulation in Drosophila. Int J Mol Sci 24(7). PubMed ID: 37047316
This study dissected the functional roles of a previously uncharacterized gene, CG3995, in cell type-specific Dendritic development in Drosophila melanogaster. CG3995, which was named bedwarfed (bdwf), encodes a zinc-finger BED-type protein that is required for proportional growth and branching of dendritic arbors. It also exhibits nucleocytoplasmic expression and functions in both transcriptional and translational cellular pathways. At the transcriptional level, a reciprocal regulatory relationship was demonstrated between Bdwf and the homeodomain transcription factor (TF) Cut. Cut positively regulates Bdwf expression and that Bdwf acts as a downstream effector of Cut-mediated dendritic development, whereas overexpression of Bdwf negatively regulates Cut expression in multidendritic sensory neurons. Proteomic analyses revealed that Bdwf interacts with ribosomal proteins and disruption of these proteins resulted in phenotypically similar dendritic hypotrophy defects as observed in bdwf mutant neurons. We further demonstrate that Bdwf and its ribosomal protein interactors are required for normal microtubule and F-actin cytoskeletal architecture. Finally, these findings reveal that Bdwf is required to promote protein translation and ribosome trafficking along the dendritic arbor. These findings shed light on the complex, combinatorial, and multi-functional roles of transcription factors (TFs) in directing the diversification of cell type-specific dendritic development.
Aimon, S., Cheng, K. Y., Gjorgjieva, J. and Grunwald Kadow, I. C. (2023). Global change in brain state during spontaneous and forced walk in Drosophila is composed of combined activity patterns of different neuron classes. Elife 12. PubMed ID: 37067152
Movement-correlated brain activity has been found across species and brain regions. This study used fast whole-brain lightfield imaging in adult Drosophila to investigate the relationship between walking and brain-wide neuronal activity. This study observed a global change in activity that tightly correlated with spontaneous bouts of walking. While imaging specific sets of excitatory, inhibitory, and neuromodulatory neurons highlighted their joint contribution, spatial heterogeneity in walk- and turning-induced activity allowed parsing unique responses from subregions and sometimes individual candidate neurons. For example, previously uncharacterized serotonergic neurons were inhibited during walk. While activity onset in some areas preceded walk onset exclusively in spontaneously walking animals, spontaneous and forced walk elicited similar activity in most brain regions. These data suggest a major contribution of walk and walk-related sensory or proprioceptive information to global activity of all major neuronal classes.
Zeng, J., Li, X., Zhang, R., Lv, M., Wang, Y., Tan, K., Xia, X., Wan, J., Jing, M., Zhang, X., Li, Y., Yang, Y., Wang, L., Chu, J., Li, Y. and Li, Y. (2023). Local 5-HT signaling bi-directionally regulates the coincidence time window for associative learning. Neuron. PubMed ID: 36706757
The coincidence between conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning; however, the mechanism regulating the duration of this temporal window remains unclear. This study found that serotonin (5-HT) bi-directionally regulates the coincidence time window of olfactory learning in Drosophila and affects synaptic plasticity of Kenyon cells (KCs) in the mushroom body (MB). Utilizing GPCR-activation-based (GRAB) neurotransmitter sensors, this study found that Kenyon cell (KC)-released acetylcholine (ACh) activates a serotonergic dorsal paired medial (DPM) neuron, which in turn provides inhibitory feedback to KCs. Physiological stimuli induce spatially heterogeneous 5-HT signals, which proportionally gate the intrinsic coincidence time windows of different MB compartments. Artificially reducing or increasing the DPM neuron-released 5-HT shortens or prolongs the coincidence window, respectively. In a sequential trace conditioning paradigm, this serotonergic neuromodulation helps to bridge the CS-US temporal gap. Altogether, we report a model circuitry for perceiving the temporal coincidence and determining the causal relationship between environmental events.
Yu, J., Guo, X., Zheng, S. and Zhang, W. (2023). A dedicate sensorimotor circuit enables fine texture discrimination by active touch. PLoS Genet 19(1): e1010562. PubMed ID: 36649336
Active touch facilitates environments exploration by voluntary, self-generated movements. However, the neural mechanisms underlying sensorimotor control for active touch are poorly understood. During foraging and feeding, Drosophila gather information on the properties of food (texture, hardness, taste) by constant probing with their proboscis. This study identified a group of neurons (sd-L neurons) on the fly labellum< that are mechanosensitive to labellum displacement and synapse onto the sugar-sensing neurons via axo-axonal synapses to induce preference to harder food. These neurons also feed onto the motor circuits that control proboscis extension and labellum spreading to provide on-line sensory feedback critical for controlling the probing processes, thus facilitating ingestion of less liquified food. Intriguingly, this preference was eliminated in mated female flies, reflecting an elevated need for softer food. These results propose a sensorimotor circuit composed of mechanosensory, gustatory and motor neurons that enables the flies to select ripe yet not over-rotten food by active touch.
Anthoney, N., Tainton-Heap, L. A. L., Luong, H., Notaras, E., Zhao, Q., Perry, T., Batterham, P., Shaw, P. J. and van Swinderen, B. (2023). Experimentally induced active and quiet sleep engage non-overlapping transcriptomes in Drosophila. bioRxiv. PubMed ID: 37066182
Sleep in mammals is broadly classified into two different categories: rapid eye movement (REM) sleep and slow wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. This study compared two commonly used approaches for studying sleep experimentally in Drosophila : optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, Gaboxadol. These different sleep-induction methods were found to have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.

Tuesday May 2nd - Evolution

Raicu, A. M., Kadiyala, D., Niblock, M., Jain, A., Yang, Y., Bird, K. M., Bertholf, K., Seenivasan, A., Siddiq, M. and Arnosti, D. N. (2023). The cynosure of CtBP: evolution of a bilaterian transcriptional corepressor. Mol Biol Evol. PubMed ID: 36625090
Evolution of sequence-specific transcription factors clearly drives lineage-specific innovations, but less is known about how changes in the central transcriptional machinery may contribute to evolutionary transformations. In particular, transcriptional regulators are rich in intrinsically disordered regions that appear to be magnets for evolutionary innovation. The C-terminal Binding Protein (CtBP) is a transcriptional corepressor derived from an ancestral lineage of alpha hydroxyacid dehydrogenases; it is found in mammals and invertebrates, and features a core NAD-binding domain as well as an unstructured C-terminus (CTD) of unknown function. CtBP can act on promoters and enhancers to repress transcription through chromatin-linked mechanisms. Comparative phylogenetic study shows that CtBP is a bilaterian innovation whose CTD of about 100 residues is present in almost all orthologs. CtBP CTDs contain conserved blocks of residues and retain a predicted disordered property, despite having variations in the primary sequence. Interestingly, the structure of the C-terminus has undergone radical transformation independently in certain lineages including flatworms and nematodes. Also contributing to CTD diversity is the production of myriad alternative RNA splicing products, including the production of "short" tailless forms of CtBP in Drosophila. Additional diversity stems from multiple gene duplications in vertebrates, where up to five CtBP orthologs have been observed. Vertebrate lineages show fewer major modifications in the unstructured CTD, possibly because gene regulatory constraints of the vertebrate body plan place specific constraints on this domain. This study highlights the rich regulatory potential of this previously unstudied domain of a central transcriptional regulator.
Pal, S., Oliver, B. and Przytycka, T. M. (2023). Stochastic Modeling of Gene Expression Evolution Uncovers Tissue- and Sex-Specific Properties of Expression Evolution in the Drosophila Genus. J Comput Biol 30(1): 21-40. PubMed ID: 36037023
Gene expression evolution is typically modeled with the stochastic Ornstein-Uhlenbeck (OU) process. It has been suggested that the estimation of within-species variations using replicated data can increase the predictive power of such models, but this hypothesis has not been fully tested. This study developed EvoGeneX, a computationally efficient implementation of the OU-based method that models within-species variation. Using extensive simulations, this study showed that modeling within-species variations and appropriate selection of species improve the performance of the model. Further, to facilitate a comparative analysis of expression evolution, a formal measure of evolutionary expression divergence was introduced for a group of genes using the rate and the asymptotic level of divergence. With these tools in hand, this study performed the first-ever analysis of the evolution of gene expression across different body-parts, species, and sexes of the Drosophila genus. It was observed that genes with adaptive expression evolution tend to be body-part specific, whereas the genes with constrained evolution tend to be shared across body-parts. Among the neutrally evolving gene expression patterns, gonads in both sexes have higher expression divergence relative to other tissues and the male gonads have even higher divergence than the female gonads. Among the evolutionarily constrained genes, the gonads show different divergence patterns, where the male gonads, and not the female gonads, show less constrained divergence than other body-parts. Finally, interesting examples were shown of adaptive expression evolution, including adaptation of odor binding proteins.
Reisenman, C. E., Wong, J., Vedagarbha, N., Livelo, C. and Scott, K. (2023). Taste adaptations associated with host-specialization in the specialist Drosophila sechellia. J Exp Biol. PubMed ID: 36637369
Chemosensory-driven hostplant specialization is a major force mediating insect ecological adaptation and speciation. Drosophila sechellia, a species endemic to the Seychelles islands, feeds and oviposits on Morinda citrifolia almost exclusively. This fruit is harmless to D. sechellia but toxic to other Drosophilidae, including the closely related generalists D. simulans and D. melanogaster, due to its high content of fatty acids. While several olfactory adaptations mediating D. sechellia's preference for its host have been uncovered, the role of taste has been much less examined. This study found that D. sechellia has reduced taste and feeding aversion to bitter compounds and host fatty acids that are aversive to D. melanogaster and D. simulans. The loss of aversion to canavanine, coumarin, and fatty acids arose in the D. sechellia lineage, as its sister species D. simulans showed responses akin to those of D. melanogaster. D. sechellia has increased taste and feeding responses towards M. citrifolia. These results are in line with D. sechellia's loss of genes encoding bitter gustatory receptors (GRs) in D. melanogaster. It was found that two gustatory receptor (GR) genes which are lost in D. sechellia, GR39a.a and GR28b.a, influence the reduction of aversive responses to some bitter compounds. Also, D. sechellia has increased appetite for a prominent host fatty acid compound that is toxic to its relatives. These results support the hypothesis that changes in the taste system, specifically a reduction of sensitivity to bitter compounds that deter generalist ancestors, contribute to the specialization of D. sechellia for its host.
Yukilevich, R. and LaGraff, J. T. (2023). Divergence of a speciation trait through artificial selection: Insights into constraints, by-product effects and sexual isolation.. J Evol Biol 36(2): 355-367. PubMed ID: 36576691
Speciation and a href="../aimain/7evolution2.htm#Isolation2">sexual isolation often occur when divergent female mating preferences target male secondary sexual traits. Despite the importance of such male signals, little is known about their evolvability and genetic linkage to other traits during speciation. To answer these questions, divergent artificial selection was imposed for 10 non-overlapping generations on the Inter-Pulse-Interval (IPI) of male courtship songs; which has been previously shown to be a major species recognition trait for females in the Drosophila athabasca species complex. Focusing on one of the species, Drosophila mahican (previously known as EA race), IPI's were examined: (1) rate of divergence, (2) response to selection in different directions, (3) genetic architecture of divergence and (4) by-product effects on other traits that have diverged in the species complex. Rapid and consistent response was found for for higher IPI but less response to lower IPI; implying asymmetrical constraints. Genetic divergence in IPI differed from natural species in X versus autosome contribution and in dominance, suggesting that evolution may take different paths. Finally, selection on IPI did not alter other components of male songs, or other ecological traits, and did not cause divergence in female preferences, as evidenced by lack of sexual isolation. This suggests that divergence of male courtship song IPI is unconstrained by genetic linkage with other traits in this system. This lack of linkage between male signals and other traits implies that female preferences or ecological selection can co-opt and mould specific male signals for species recognition free of genetic constraints from other traits.
Anastasio, O. E., Sinclair, C. S. and Pischedda, A. (2023). Cryptic male mate choice for high-quality females reduces male postcopulatory success in future matings. Evolution. PubMed ID: 37073992
Cryptic male mate choice occurs when males differentially allocate resources to females during or after copulation. When male resources are limited, males may benefit by strategically allocating more resources toward higher-quality females. In the fruit fly, Drosophila melanogaster, males mate for longer and may transfer more sperm and more seminal proteins when they mate with larger females compared to smaller females. It is unclear, however, whether this increased investment in large females has any impact on the males' later matings. D. melanogaster males were mated sequentially to females of large or small body size in all possible combinations to test whether cryptic male mate choice for large females is costly to the males' subsequent matings. Second matings were shorter for males compared to their first matings, but there were no differences in fecundity between females mated first or second by a male. Interestingly, male success at defensive sperm competition declined between his first and second matings only when his first mating had been with a large female. This suggests that the higher initial investment in large females reduced male postcopulatory success in their subsequent matings. Cryptic male mate choice may carry underappreciated costs to males that could limit their reproductive potential.
Moreyra, N. N., Almeida, F. C., Allan, C., Frankel, N., Matzkin, L. M. and Hasson, E. (2023). Phylogenomics provides insights into the evolution of cactophily and host plant shifts in Drosophila. Mol Phylogenet Evol 178: 107653. PubMed ID: 36404461
Cactophilic species of the Drosophila buzzatii cluster (repleta group) comprise an excellent model group to investigate genomic changes underlying adaptation to extreme climate conditions and host plants. In particular, these species form a tractable system to study the transition from chemically simpler breeding sites (like prickly pears of the genus Opuntia) to chemically more complex hosts (columnar cacti). This study reports four highly contiguous genome assemblies of three species of the buzzatii cluster. Based on this genomic data and inferred phylogenetic relationships, candidate taxonomically restricted genes (TRGs) likely involved in the evolution of cactophily and cactus host specialization were identified. Functional enrichment analyses of TRGs within the buzzatii cluster identified genes involved in detoxification, water preservation, immune system response, anatomical structure development, and morphogenesis. In contrast, processes that regulate responses to stress, as well as the metabolism of nitrogen compounds, transport, and secretion were found in the set of species that are columnar cacti dwellers. These findings are in line with the hypothesis that those genomic changes brought about key mechanisms underlying the adaptation of the buzzatii cluster species to arid regions in South America.
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