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Proceedings of the National Academy of... Jun 2023Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how...
Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how quiescent cells are reactivated using a zebrafish model, in which a population of differentiated epithelial cells are reactivated under a physiological context. A robust and sustained increase in mitochondrial membrane potential was observed in the reactivated cells. Genetic and pharmacological perturbations show that elevated mitochondrial metabolism and ATP synthesis are critical for cell reactivation. Further analyses showed that elevated mitochondrial metabolism increases mitochondrial ROS levels, which induces Sgk1 expression in the mitochondria. Genetic deletion and inhibition of Sgk1 in zebrafish abolished epithelial cell reactivation. Similarly, ROS-dependent mitochondrial expression of SGK1 promotes S phase entry in human breast cancer cells. Mechanistically, SGK1 coordinates mitochondrial activity with ATP synthesis by phosphorylating FF-ATP synthase. These findings suggest a conserved intramitochondrial signaling loop regulating epithelial cell renewal.
Topics: Animals; Humans; Reactive Oxygen Species; Zebrafish; Mitochondria; Epithelial Cells; Adenosine Triphosphate
PubMed: 37276417
DOI: 10.1073/pnas.2216310120 -
Developmental Cell Jun 2023The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with...
The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.
Topics: Animals; Stem Cell Niche; Transcription Factors; Endothelial Cells; Zebrafish; Gene Expression Regulation
PubMed: 37119815
DOI: 10.1016/j.devcel.2023.04.007 -
Nucleic Acids Research Jul 2023Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal...
Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ∼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.
Topics: Animals; Humans; Binding Sites; Cold Temperature; Cold-Shock Response; CRISPR-Cas Systems; Gene Editing; Mammals; Ribonucleoproteins; Zebrafish
PubMed: 37246708
DOI: 10.1093/nar/gkad456 -
Nature Jan 2024Chemical signalling is the primary means by which cells communicate in the embryo. The underlying principle refers to a group of ligand-producing cells and a group of...
Chemical signalling is the primary means by which cells communicate in the embryo. The underlying principle refers to a group of ligand-producing cells and a group of cells that respond to this signal because they express the appropriate receptors. In the zebrafish embryo, Wnt5b binds to the receptor Ror2 to trigger the Wnt-planar cell polarity (PCP) signalling pathway to regulate tissue polarity and cell migration. However, it remains unclear how this lipophilic ligand is transported from the source cells through the aqueous extracellular space to the target tissue. In this study, we provide evidence that Wnt5b, together with Ror2, is loaded on long protrusions called cytonemes. Our data further suggest that the active Wnt5b-Ror2 complexes form in the producing cell and are handed over from these cytonemes to the receiving cell. Then, the receiving cell has the capacity to initiate Wnt-PCP signalling, irrespective of its functional Ror2 receptor status. On the tissue level, we further show that cytoneme-dependent spreading of active Wnt5b-Ror2 affects convergence and extension in the zebrafish gastrula. We suggest that cytoneme-mediated transfer of ligand-receptor complexes is a vital mechanism for paracrine signalling. This may prompt a reevaluation of the conventional concept of characterizing responsive and non-responsive tissues solely on the basis of the expression of receptors.
Topics: Animals; Gastrula; Ligands; Wnt Proteins; Wnt Signaling Pathway; Zebrafish; Cell Polarity; Cell Movement; Pseudopodia; Receptor Tyrosine Kinase-like Orphan Receptors; Paracrine Communication
PubMed: 38123680
DOI: 10.1038/s41586-023-06850-7 -
Genetics Jan 2024The fibrillinopathies represent a group of diseases in which the 10-12 nm extracellular microfibrils are disrupted by genetic variants in one of the genes encoding... (Review)
Review
The fibrillinopathies represent a group of diseases in which the 10-12 nm extracellular microfibrils are disrupted by genetic variants in one of the genes encoding fibrillin molecules, large glycoproteins of the extracellular matrix. The best-known fibrillinopathy is Marfan syndrome, an autosomal dominant condition affecting the cardiovascular, ocular, skeletal, and other systems, with a prevalence of around 1 in 3,000 across all ethnic groups. It is caused by variants of the FBN1 gene, encoding fibrillin-1, which interacts with elastin to provide strength and elasticity to connective tissues. A number of mouse models have been created in an attempt to replicate the human phenotype, although all have limitations. There are also natural bovine models and engineered models in pig and rabbit. Variants in FBN2 encoding fibrillin-2 cause congenital contractural arachnodactyly and mouse models for this condition have also been produced. In most animals, including birds, reptiles, and amphibians, there is a third fibrillin, fibrillin-3 (FBN3 gene) for which the creation of models has been difficult as the gene is degenerate and nonfunctional in mice and rats. Other eukaryotes such as the nematode C. elegans and zebrafish D. rerio have a gene with some homology to fibrillins and models have been used to discover more about the function of this family of proteins. This review looks at the phenotype, inheritance, and relevance of the various animal models for the different fibrillinopathies.
Topics: Animals; Cattle; Humans; Mice; Rats; Rabbits; Swine; Mutation; Caenorhabditis elegans; Models, Genetic; Zebrafish; Fibrillins
PubMed: 37972149
DOI: 10.1093/genetics/iyad189 -
ELife May 2023Single-cell transcriptome analysis of zebrafish cells clarifies the signalling pathways controlling skin formation and reveals that some cells produce proteins required...
Single-cell transcriptome analysis of zebrafish cells clarifies the signalling pathways controlling skin formation and reveals that some cells produce proteins required for human teeth to acquire their enamel.
Topics: Animals; Humans; Ameloblasts; Zebrafish; Tooth
PubMed: 37218526
DOI: 10.7554/eLife.88597 -
Disease Models & Mechanisms Aug 2023Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation...
Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation tools have been reported in zebrafish, which have significantly impacted regeneration studies. However, NTR1.0-based tools are not appropriate for modeling chronic cell loss as prolonged application of the required MTZ dose (10 mM) is deleterious to zebrafish health. We established that this dose corresponds to the median lethal dose (LD50) of MTZ in larval and adult zebrafish and that it induced intestinal pathology. NTR2.0 is a more active nitroreductase engineered from Vibrio vulnificus NfsB that requires substantially less MTZ to induce cell ablation. Here, we report on the generation of two new NTR2.0-based zebrafish lines in which acute β-cell ablation can be achieved without MTZ-associated intestinal pathology. For the first time, we were able to sustain β-cell loss and maintain elevated glucose levels (chronic hyperglycemia) in larvae and adults. Adult fish showed significant weight loss, consistent with the induction of a diabetic state, indicating that this paradigm will allow the modeling of diabetes and associated pathologies.
Topics: Animals; Zebrafish; Hyperglycemia; Metronidazole; Diabetes Mellitus; Nitroreductases; Animals, Genetically Modified
PubMed: 37401381
DOI: 10.1242/dmm.050215 -
Genetics in Medicine : Official Journal... Nov 2023Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental...
PURPOSE
Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype but with limited neuroradiological data and insufficient evidence for causality of the variants.
METHODS
Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays and a zebrafish model.
RESULTS
We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs, and increased blood lactate. In vitro studies showed that variants within the TARS2 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model.
CONCLUSION
We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity.
Topics: Animals; Humans; Mutation; Zebrafish; RNA, Transfer; Mechanistic Target of Rapamycin Complex 1; Ligases; Phenotype
PubMed: 37454282
DOI: 10.1016/j.gim.2023.100938 -
Development (Cambridge, England) Jun 2023Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains...
Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines.
Topics: Animals; CRISPR-Cas Systems; Zebrafish; Gene Knock-In Techniques; Genome; Gene Editing
PubMed: 37309812
DOI: 10.1242/dev.201323 -
Nature Nov 2023The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of these data,...
The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of these data, however, has been collected from wild-type embryos without an appreciation for the latent variation that is present in development. Here we present the 'zebrafish single-cell atlas of perturbed embryos': single-cell transcriptomic data from 1,812 individually resolved developing zebrafish embryos, encompassing 19 timepoints, 23 genetic perturbations and a total of 3.2 million cells. The high degree of replication in our study (eight or more embryos per condition) enables us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild-type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about early origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single-cell data from large numbers of individual embryos will enable mapping of the genetic dependencies of zebrafish cell types, while also addressing longstanding challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.
Topics: Animals; Embryo, Mammalian; Gene Expression Profiling; Gene Expression Regulation, Developmental; Reverse Genetics; Transcriptome; Zebrafish; Mutation; Single-Cell Analysis; Notochord
PubMed: 37968389
DOI: 10.1038/s41586-023-06720-2