Genetic analysis of rab7 mutants in zebrafish

Vascular network formation requires the fusion of newly formed blood vessels and the emergence of a patent lumen between the newly established connections so that blood flow can start. Lumen formation has been shown to depend on the late endosomal/lysosomal pathway in various organs of animal tubular systems. Here, we identified a late endosomal/lysosomal vesicular fraction (Rab7/Lamp2) in early zebrafish angiogenic sprouts, which appears to contribute to apical membrane growth during lumen formation. To study the effect of the late endocytic pathway on vascular development, we generated mutant alleles for all three rab7 genes in zebrafish ( rab7a, rab7ba, rab7bb ). All rab7 genes are expressed in wild-type zebrafish and we did not detect any compensatory effects by the other rab7 isoforms in single knockout mutants, which were all viable. Only the triple mutant was lethal suggesting some functional redundancy. However, the different rab7 isoforms fulfil also at least partially independent functions because eggs laid from mothers lacking two rab7 ( rab7a and/or rab7bb ). showed reduced survival and contained enlarged yolk granules, suggesting maternal contribution of these two rab7 . Finally, we observed minor effects on lumen formation in embryos which still express one copy of rab7 . Our results support the notion that the late endocytic/lysosomal compartment contributes to lumen expansion

Probing the Effects of the FGFR-Inhibitor Derazantinib on Vascular Development in Zebrafish Embryos

Angiogenesis is a fundamental developmental process and a hallmark of cancer progression. Receptor tyrosine kinases (RTK) are targets for cancer therapy which may include their action as anti-angiogenic agents. Derazantinib (DZB) is an inhibitor of the fibroblast growth factor receptors (FGFRs) 1-3 as well as other kinase targets including vascular endothelial growth factor receptor 2 (VEGFR2), colony stimulating factor-1 receptor (CSF1R) and platelet-derived growth factor beta receptor (PDGFRbeta). This study aimed to investigate the effect of DZB on blood vessel morphogenesis and to compare its activity to known specific FGFR and VEGFR inhibitors. For this purpose, we used the developing vasculature in the zebrafish embryo as a model system for angiogenesis in vivo. We show that DZB interferes with multiple angiogenic processes that are linked to FGF and VEGF signalling, revealing a potential dual role for DZB as a potent anti-angiogenic treatment

Escape Mutations, Ganciclovir Resistance, and Teratoma Formation in Human iPSCs Expressing an HSVtk Suicide Gene

Human pluripotent stem cells (hPSCs) hold great promise for cell therapy. However, a major concern is the risk of tumor formation by residual undifferentiated cells contaminating the hPSC-derived cell product. Suicide genes could safeguard against such adverse events by enabling elimination of cells gone astray, but the efficacy of this approach has not yet been thoroughly tested. Here, we engineered a lentivirally encoded herpes simplex virus thymidine kinase (HSVtk) with expression restricted to undifferentiated hPSCs through regulation by the let7 family of miRNAs. We show that induced pluripotent stem cells (iPSCs) expressing a let7-regulated HSVtk transgene are selectively killed by ganciclovir (GCV), whereas differentiated cells are fully protected. However, in contrast to previous studies, we find that in vivo GCV administration results in longer latency but does not prevent teratoma formation by iPSCs expressing either a constitutive or a let7-regulated HSVtk, without evidence of silencing of the HSVtk. Clonal analyses of iPSCs expressing HSVtk revealed frequent emergence of GCV resistance which, at least in some cases, could be attributed to preexisting inactivating mutations in the HSVtk coding sequence, selected for upon GCV treatment. Our findings have important consequences for the future use of suicide genes in hPSC-based cell therapies

Vinculin strengthens the endothelial barrier during vascular development

Remodelling of cell-cell junctions is crucial for proper tissue development and barrier function. The Cadherin-based adherens junctions anchor via β-Catenin and α-Catenin to the actomyosin cytoskeleton, together forming a junctional mechanotransduction complex. Tension-induced conformational changes in the mechanosensitive α-Catenin protein induce junctional Vinculin recruitment. In endothelial cells, Vinculin protects the remodelling VE-Cadherin junctions. In this study, we have addressed the role of Vinculin in endothelial barrier function in the developing vasculature. In vitro experiments, using endothelial cells in which α-Catenin was replaced by a Vinculin-binding deficient mutant, showed that junctional recruitment of Vinculin promotes endothelial barrier function. To assess the role of Vinculin within blood vessels in vivo, we next investigated barrier function in the vasculature of Vinculin knockout zebrafish. In the absence of Vinculin, sprouting angiogenesis and vessel perfusion still occurred. Intriguingly, the absence of Vinculin made the blood vessels more permeable for 10 kDa dextran molecules, but not for larger tracers. Taken together, our findings demonstrate that Vinculin strengthens the endothelial barrier and prevents vascular leakage in developing vessels

Vinculin controls endothelial cell junction dynamics during vascular lumen formation

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts in vivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis

Vinculin controls endothelial cell junction dynamics during vascular lumen formation

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts in vivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis

Vinculin controls endothelial cell junction dynamics during vascular lumen formation

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors, which depend on dynamic cell-cell interactions. Remodeling of endothelial cell-cell junctions promote morphogenetic cellular events while preserving vascular integrity. Here, we have analyzed the dynamics of endothelial cell-cell junctions during lumen formation in angiogenic sprouts. By live-imaging of the formation of intersegmental blood vessels in zebrafish, we demonstrate that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Formation and maintenance of these junctional fingers are positively regulated by blood pressure whereas inhibition of blood flow prevents their formation. Using fluorescent reporters, we show that the tension-sensor Vinculin localizes to junctional fingers. Furthermore, loss of vinculin function, in vinculin a and -b double knockouts, prevents junctional finger formation in angiogenic sprouts, whereas endothelial expression of a vinculin transgene is sufficient to restore junctional fingers. Taken together, our findings suggest a mechanism in which lumen expansion during angiogenesis leads to an increase in junctional tension, which triggers recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells may employ force-dependent junctional remodeling to react to changes in external forces to protect cell-cell contacts and to maintain vascular integrity during sprouting angiogenesis

Dissecting the Contributions of Cooperating Gene Mutations to Cancer Phenotypes and Drug Responses with Patient-Derived iPSCs

Summary: Connecting specific cancer genotypes with phenotypes and drug responses constitutes the central premise of precision oncology but is hindered by the genetic complexity and heterogeneity of primary cancer cells. Here, we use patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing to dissect the individual contributions of two recurrent genetic lesions, the splicing factor SRSF2 P95L mutation and the chromosome 7q deletion, to the development of myeloid malignancy. Using a comprehensive panel of isogenic iPSCs—with none, one, or both genetic lesions—we characterize their relative phenotypic contributions and identify drug sensitivities specific to each one through a candidate drug approach and an unbiased large-scale small-molecule screen. To facilitate drug testing and discovery, we also derive SRSF2-mutant and isogenic normal expandable hematopoietic progenitor cells. We thus describe here an approach to dissect the individual effects of two cooperating mutations to clinically relevant features of malignant diseases. : Papapetrou and colleagues develop a comprehensive panel of isogenic iPSC lines with SRSF2 P95L mutation and chr7q deletion. They use these cells to identify cellular phenotypes contributed by each genetic lesion and therapeutic vulnerabilities specific to each one and develop expandable hematopoietic progenitor cell lines to facilitate drug discovery. Keywords: induced pluripotent stem cells, myelodysplastic syndrome, CRISPR/Cas9, gene editing, mutational cooperation, splicing factor mutations, spliceosomal mutations, SRSF2, chr7q deletio

Interferon-γ drives macrophage reprogramming, cerebrovascular remodeling, and cognitive dysfunction in a zebrafish and a mouse model of ion imbalance and pressure overload

Dysregulated immune response contributes to inefficiency of treatment strategies to control hypertension and reduce the risk of end-organ damage. Uncovering the immune pathways driving the transition from the onset of hypertensive stimulus to the manifestation of multi-organ dysfunction are much-needed insights for immune targeted therapy.; To aid visualization of cellular events orchestrating multi-organ pathogenesis, we modeled hypertensive cardiovascular remodeling in zebrafish. Zebrafish larvae exposed to ion-poor environment exhibited rapid angiotensinogen upregulation, followed by manifestation of arterial hypertension and cardiac remodeling that recapitulates key characteristics of incipient Heart Failure with preserved Ejection Fraction. In the brain, time-lapse imaging revealed the occurrence of cerebrovascular regression through endothelial retraction and migration in response to the ion-poor treatment. This phenomenon is associated with macrophage/microglia-endothelial contacts and endothelial junctional retraction. Cytokine and transcriptomic profiling identified systemic upregulation of interferon-γ and interleukin 1β, and revealed altered macrophage/microglia transcriptional program characterized by suppression of innate immunity and vasculo/neuroprotective gene expression. Both zebrafish and a murine model of pressure overload-induced brain damage demonstrated that the brain pathology and macrophage/microglia phenotypic alteration are dependent on interferon-γ signaling. In zebrafish, interferon-γ receptor 1 mutation prevents cerebrovascular remodeling and dysregulation of macrophage/microglia transcriptomic profile. Supplementation of bone morphogenetic protein 5, identified from the transcriptomic approach as a downregulated gene in ion-poor-treated macrophages/microglia that is rescued by interferon-γ blockage, mitigated cerebral microvessel loss. In mice subjected to transverse aortic constriction-induced pressure overload, typically developing cerebrovascular injury, neuroinflammation and cognitive dysfunction, interferon-γ neutralization protected them from blood-brain-barrier disruption, cerebrovascular rarefaction, and cognitive decline.; These findings uncover cellular and molecular players of an immune pathway communicating hypertensive stimulus to structural and functional remodeling of the brain and identify anti-interferon-γ treatment as a promising intervention strategy capable of preventing pressure overload-induced damage of the cerebrovascular and nervous systems.; Hypertension is a major risk factor for damages of the vasculature, heart, and brain, and thereby a major healthcare burden. Inadequate cerebral blood supply due to altered cerebrovascular structure and vasoregulatory disruption upon hypertension render the brain highly susceptible to stroke and cognitive decline. We envision that the cellular and molecular mechanisms uncovered here linking immune dysregulation to cerebrovascular remodeling and functional impairment of the brain will inform future development of immunomodulatory therapeutic strategies for counteracting derangement of macrophage/microglia activation and their vasculo/neuroprotective function in response to systemic inflammation in hypertension

Acute Myeloid Leukemia iPSCs Reveal a Role for RUNX1 in the Maintenance of Human Leukemia Stem Cells

Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs. [Display omitted] •AML-iPSC-derived hematopoietic cells recapitulate a LSC hierarchy•iLSCs can be easily prospectively isolated•A LSC 16-gene set correlates with AML patient survival•The RUNX1 TF is critical for the maintenance of LSCs across AML genetic subgroups Wesely et al. report that AML-iPSC-derived hematopoietic cells are hierarchically organized and contain cells with hallmark features of LSCs (iLSCs). Through integrative genomic studies of bulk and single-cell transcriptomes and chromatin accessibility, they derive a LSC gene signature and identify RUNX1 as an AML LSC dependency with therapeutic implications