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Biology Open Jun 2024Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT)....
Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, we show that ESCRTs have cell-autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. We also identify key heterotypic roles for ESCRT in position-dependent control of Notch activation to suppress crystal cell differentiation. Our study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.
Topics: Animals; Endosomal Sorting Complexes Required for Transport; Drosophila Proteins; Cell Lineage; Cell Differentiation; Drosophila; Signal Transduction; Hematopoietic Stem Cells; Immunity
PubMed: 38828842
DOI: 10.1242/bio.060412 -
BioRxiv : the Preprint Server For... May 2024Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise...
Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of these proteins has been challenging . A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP system in fruit flies. This approach depends on the automatic reconstitution of the GFP and GFP fragments, creating a fluorescence signal. We insert the fragment into a specific genomic locus while expressing its counterpart, , through an available Gal4 driver line. The unique aspect of this system is that neither GFP nor GFP alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells. We illustrate this technique using the adhesion molecule gene () as a model, highlighting the generation and validation of GFP protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.
PubMed: 38826199
DOI: 10.1101/2024.05.06.592806 -
STAR Protocols Jun 2024The MS2-PP7 two-color live-imaging system provides insights into the spatiotemporal dynamics of nascent transcripts at tagged loci. Here, we present a protocol to...
The MS2-PP7 two-color live-imaging system provides insights into the spatiotemporal dynamics of nascent transcripts at tagged loci. Here, we present a protocol to quantitatively measure the rate of RNA polymerase II elongation for each actively transcribing nucleus in living Drosophila embryos. The elongation rate is calculated by measuring the effective distance and the time elapsed between MS2 and PP7 trajectories. We describe steps for preparing embryo samples, performing live imaging, and measuring the elongation rate. For complete details on the use and execution of this protocol, please refer to Keller et al..
Topics: Animals; RNA Polymerase II; Embryo, Nonmammalian; Drosophila; Drosophila melanogaster; Drosophila Proteins
PubMed: 38824639
DOI: 10.1016/j.xpro.2024.103099 -
Nature Communications May 2024Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding...
Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the network's regulatory outputs and a decrease in fitness. We subjected these flies to EMS mutagenesis, followed by experimental evolution. After only 8-15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level.
Topics: Animals; Drosophila Proteins; Gene Expression Regulation, Developmental; Gene Regulatory Networks; Female; Drosophila melanogaster; Gene Dosage; Homeodomain Proteins; Phenotype; Male; Embryo, Nonmammalian; Drosophila; Mutagenesis; Trans-Activators
PubMed: 38811562
DOI: 10.1038/s41467-024-48960-4 -
Nature Communications May 2024Gene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian...
Gene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian inheritance. CRISPR-based homing gene drives convert wild type alleles into drive alleles in heterozygotes with Cas9 and gRNA. It is thus desirable to identify Cas9 promoters that yield high drive conversion rates, minimize the formation rate of resistance alleles in both the germline and the early embryo, and limit somatic Cas9 expression. In Drosophila, the nanos promoter avoids leaky somatic expression, but at the cost of high embryo resistance from maternally deposited Cas9. To improve drive efficiency, we test eleven Drosophila melanogaster germline promoters. Some achieve higher drive conversion efficiency with minimal embryo resistance, but none completely avoid somatic expression. However, such somatic expression often does not carry detectable fitness costs for a rescue homing drive targeting a haplolethal gene, suggesting somatic drive conversion. Supporting a 4-gRNA suppression drive, one promoter leads to a low drive equilibrium frequency due to fitness costs from somatic expression, but the other outperforms nanos, resulting in successful suppression of the cage population. Overall, these Cas9 promoters hold advantages for homing drives in Drosophila species and may possess valuable homologs in other organisms.
Topics: Animals; Promoter Regions, Genetic; Drosophila melanogaster; Drosophila Proteins; Gene Drive Technology; CRISPR-Cas Systems; Germ Cells; RNA, Guide, CRISPR-Cas Systems; Animals, Genetically Modified; CRISPR-Associated Protein 9; Alleles; Female; Male; RNA-Binding Proteins
PubMed: 38811556
DOI: 10.1038/s41467-024-48874-1 -
Science Advances May 2024Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are...
Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are targeted to the growing LD is still unclear. Rab1b, a GTPase regulating secretory transport, was recently associated with targeting proteins to LDs in a Drosophila RNAi screen. LD formation was prevented in human hepatoma cells overexpressing dominant-negative Rab1b. We thus hypothesized that Rab1b recruits lipid-synthesizing enzymes, facilitating LD growth. Here, FRET between diacylglycerol acyltransferase 2 (DGAT2) and Rab1b and activity mutants of the latter demonstrated that Rab1b promotes DGAT2 ER to the LD surface redistribution. Last, alterations in LD metabolism and DGAT2 redistribution, consistent with Rab1b activity, were caused by mutations in the Rab1b-GTPase activating protein TBC1D20 in Warburg Micro syndrome (WARBM) model mice fibroblasts. These data contribute to our understanding of the mechanism of Rab1b in LD homeostasis and WARBM, a devastating autosomal-recessive disorder caused by mutations in TBC1D20.
Topics: Lipid Droplets; Animals; Humans; rab1 GTP-Binding Proteins; Diacylglycerol O-Acyltransferase; Mice; Endoplasmic Reticulum; Mutation; Lipid Metabolism; GTPase-Activating Proteins
PubMed: 38809969
DOI: 10.1126/sciadv.ade7753 -
PLoS Genetics May 2024Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been...
Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.
Topics: Animals; Chromatin; Circadian Rhythm; Drosophila Proteins; Drosophila melanogaster; Gene Expression Regulation; Transcription, Genetic; CLOCK Proteins; Neurons; Promoter Regions, Genetic; Period Circadian Proteins; Circadian Clocks; Drosophila; Enhancer Elements, Genetic; Basic-Leucine Zipper Transcription Factors
PubMed: 38805552
DOI: 10.1371/journal.pgen.1011278 -
PLoS Biology May 2024Despite significant progress in understanding epigenetic reprogramming of cells, the mechanistic basis of "organ reprogramming" by (epi-)gene-environment interactions...
Despite significant progress in understanding epigenetic reprogramming of cells, the mechanistic basis of "organ reprogramming" by (epi-)gene-environment interactions remained largely obscure. Here, we use the ether-induced haltere-to-wing transformations in Drosophila as a model for epigenetic "reprogramming" at the whole organism level. Our findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to haltere-to-wing transformations manifested at the larval stage and on. We show that ether interferes with protein integrity in the egg, leading to altered deployment of Hsp90 and widespread repression of Trithorax-mediated establishment of active H3K4me3 chromatin marks throughout the genome. Despite this global reduction, Ubx targets and wing development genes preferentially retain higher levels of H3K4me3 that predispose these genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the penetrance of bithorax transformations increases by genetic or chemical reduction of Hsp90 function. Moreover, joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations without exposure to ether, supporting an underlying epistasis between Hsp90 and trx loss-of-functions. These findings implicate environmental disruption of protein integrity at the onset of histone methylation with altered epigenetic regulation of developmental patterning genes. The emerging picture provides a unique example wherein the alleviation of the Hsp90 "capacitor function" by the environment drives a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ transformation by environmental cues.
Topics: Animals; Drosophila Proteins; Epigenesis, Genetic; Histones; HSP90 Heat-Shock Proteins; Wings, Animal; Drosophila melanogaster; Larva; Gene Expression Regulation, Developmental; Gene-Environment Interaction; Chromosomal Proteins, Non-Histone; Chromatin; Homeodomain Proteins; Epigenetic Memory; Transcription Factors
PubMed: 38805504
DOI: 10.1371/journal.pbio.3002629 -
ELife May 2024is a powerful model to study how lipids affect spermatogenesis. Yet, the contribution of neutral lipids, a major lipid group which resides in organelles called lipid...
is a powerful model to study how lipids affect spermatogenesis. Yet, the contribution of neutral lipids, a major lipid group which resides in organelles called lipid droplets (LD), to sperm development is largely unknown. Emerging evidence suggests LD are present in the testis and that loss of neutral lipid- and LD-associated genes causes subfertility; however, key regulators of testis neutral lipids and LD remain unclear. Here, we show LD are present in early-stage somatic and germline cells within the testis. We identified a role for triglyceride lipase () in regulating testis LD, and found that whole-body loss of leads to defects in sperm development. Importantly, these represent cell-autonomous roles for in regulating testis LD and spermatogenesis. Because lipidomic analysis of mutants revealed excess triglyceride accumulation, and spermatogenic defects in mutants were rescued by genetically blocking triglyceride synthesis, our data suggest that -mediated regulation of triglyceride influences sperm development. This identifies triglyceride as an important neutral lipid that contributes to sperm development, and reveals a key role for in regulating testis triglyceride levels during spermatogenesis.
Topics: Spermatogenesis; Animals; Male; Triglycerides; Drosophila Proteins; Testis; Drosophila melanogaster; Lipase; Lipid Droplets; Spermatozoa
PubMed: 38805376
DOI: 10.7554/eLife.87523 -
Frontiers in Immunology 2024Since infections with antibiotic-resistant bacteria cause increasing problems worldwide, the identification of alternative therapies is of great importance....
Since infections with antibiotic-resistant bacteria cause increasing problems worldwide, the identification of alternative therapies is of great importance. Plant-derived bioactives, including allyl-isothiocyanate (AITC), have received attention for their antimicrobial properties. The present study therefore investigates the impact of AITC on survival and antimicrobial peptide (AMP) levels in challenged with the fly pathogenic bacteria subsp. and . AITC, a sulfur-containing compound derived from glucosinolates, exhibits antimicrobial properties and has been suggested to modulate AMP expression. By using , we demonstrate that AITC treatment resulted in a concentration-dependent decrease of survival rates among female flies, particularly in the presence of the Gram-negative bacterium subsp. , whereas AITC did not affect survival in male flies. Despite the ability of isothiocyanates to induce AMP expression in cell culture, we did not detect significant changes in AMP mRNA levels in infected flies exposed to AITC. Our findings suggest sex-specific differences in response to AITC treatment and bacterial infections, underlining the complexity of host-pathogen interactions and potential limitations of AITC as a preventive or therapeutic compound at least in models of bacterial infections.
Topics: Animals; Isothiocyanates; Drosophila melanogaster; Female; Male; Antimicrobial Peptides; Pectobacterium carotovorum; Drosophila Proteins
PubMed: 38803500
DOI: 10.3389/fimmu.2024.1404086