The homeobox transcription factor DUXBL controls exit from totipotency.

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.

Inhibition of fatty acid oxidation enables heart regeneration in adult mice.

Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration1,2. Here, to explore whether metabolic reprogramming can overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b. We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia-reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b-mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref. 3). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering their transcription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts.

ADPGK-AS1 long noncoding RNA switches macrophage metabolic and phenotypic state to promote lung cancer growth.

Long noncoding RNAs (lncRNAs) influence the transcription of gene networks in many cell types, but their role in tumor-associated macrophages (TAMs) is still largely unknown. We found that the lncRNA ADPGK-AS1 was substantially upregulated in artificially induced M2-like human macrophages, macrophages exposed to lung cancer cells in vitro, and TAMs from human lung cancer tissue. ADPGK-AS1 is partly located within mitochondria and binds to the mitochondrial ribosomal protein MRPL35. Overexpression of ADPGK-AS1 in macrophages upregulates the tricarboxylic acid cycle and promotes mitochondrial fission, suggesting a phenotypic switch toward an M2-like, tumor-promoting cytokine release profile. Macrophage-specific knockdown of ADPGK-AS1 induces a metabolic and phenotypic switch (as judged by cytokine profile and production of reactive oxygen species) to a pro-inflammatory tumor-suppressive M1-like state, inhibiting lung tumor growth in vitro in tumor cell-macrophage cocultures, ex vivo in human tumor precision-cut lung slices, and in vivo in mice. Silencing ADPGK-AS1 in TAMs may thus offer a novel therapeutic strategy for lung cancer.

Time-resolved Small-RNA Sequencing Identifies MicroRNAs Critical for Formation of Embryonic Stem Cells from the Inner Cell Mass of Mouse Embryos.

Cells of the inner cell mass (ICM) acquire a unique ability for unlimited self-renewal during transition into embryonic stem cells (ESCs) in vitro, while preserving their natural multi-lineage differentiation potential. Several different pathways have been identified to play roles in ESC formation but the function of non-coding RNAs in this process is poorly understood. Here, we describe several microRNAs (miRNAs) that are crucial for efficient generation of mouse ESCs from ICMs. Using small-RNA sequencing, we characterize dynamic changes in miRNA expression profiles during outgrowth of ICMs in a high-resolution, time-course dependent manner. We report several waves of miRNA transcription during ESC formation, to which miRNAs from the imprinted Dlk1-Dio3 locus contribute extensively. In silico analyses followed by functional investigations reveal that Dlk1-Dio3 locus-embedded miRNAs (miR-541-5p, miR-410-3p, and miR-381-3p), miR-183-5p, and miR-302b-3p promote, while miR-212-5p and let-7d-3p inhibit ESC formation. Collectively, these findings offer new mechanistic insights into the role of miRNAs during ESC derivation.

The HDAC2-SP1 Axis Orchestrates Protumor Macrophage Polarization.

Tumor-associated macrophages (TAM), including antitumor M1-like TAMs and protumor M2-like TAMs, are transcriptionally dynamic innate immune cells with diverse roles in lung cancer development. Epigenetic regulators are key in controlling macrophage fate in the heterogeneous tumor microenvironment. Here, we demonstrate that the spatial proximity of HDAC2-overexpressing M2-like TAMs to tumor cells significantly correlates with poor overall survival of lung cancer patients. Suppression of HDAC2 in TAMs altered macrophage phenotype, migration, and signaling pathways related to interleukins, chemokines, cytokines, and T-cell activation. In coculture systems of TAMs and cancer cells, suppressing HDAC2 in TAMs resulted in reduced proliferation and migration, increased apoptosis of cancer cell lines and primary lung cancer cells, and attenuated endothelial cell tube formation. HDAC2 regulated the M2-like TAM phenotype via acetylation of histone H3 and transcription factor SP1. Myeloid cell-specific deletion of Hdac2 and pharmacologic inhibition of class I HDACs in four different murine lung cancer models induced the switch from M2-like to M1-like TAMs, altered infiltration of CD4+ and CD8+ T cells, and reduced tumor growth and angiogenesis. TAM-specific HDAC2 expression may provide a biomarker for lung cancer stratification and a target for developing improved therapeutic approaches. SIGNIFICANCE: HDAC2 inhibition reverses the protumor phenotype of macrophages mediated by epigenetic modulation induced by the HDAC2-SP1 axis, indicating a therapeutic option to modify the immunosuppressive tumor microenvironment.

Targeting Wnt-ß-Catenin-FOSL Signaling Ameliorates Right Ventricular Remodeling.

BACKGROUND: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. METHODS: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. RESULTS: Wnt/ß-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/ß-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (ß-catenin-activated transgene driving expression of nuclear ß-galactosidase) reporter mice RVs exhibited an increase in ß-catenin expression compared with their respective controls. Genetic inhibition of ß-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. CONCLUSIONS: Wnt- ß-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.

Spurious transcription causing innate immune responses is prevented by 5-hydroxymethylcytosine.

Generation of functional transcripts requires transcriptional initiation at regular start sites, avoiding production of aberrant and potentially hazardous aberrant RNAs. The mechanisms maintaining transcriptional fidelity and the impact of spurious transcripts on cellular physiology and organ function have not been fully elucidated. Here we show that TET3, which successively oxidizes 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other derivatives, prevents aberrant intragenic entry of RNA polymerase II pSer5 into highly expressed genes of airway smooth muscle cells, assuring faithful transcriptional initiation at canonical start sites. Loss of TET3-dependent 5hmC production in SMCs results in accumulation of spurious transcripts, which stimulate the endosomal nucleic-acid-sensing TLR7/8 signaling pathway, thereby provoking massive inflammation and airway remodeling resembling human bronchial asthma. Furthermore, we found that 5hmC levels are substantially lower in human asthma airways compared with control samples. Suppression of spurious transcription might be important to prevent chronic inflammation in asthma.

TF-COMB - Discovering grammar of transcription factor binding sites.

Cooperativity between transcription factors is important to regulate target gene expression. In particular, the binding grammar of TFs in relation to each other, as well as in the context of other genomic elements, is crucial for TF functionality. However, tools to easily uncover co-occurrence between DNA-binding proteins, and investigate the regulatory modules of TFs, are limited. Here we present TF-COMB (Transcription Factor Co-Occurrence using Market Basket analysis) - a tool to investigate co-occurring TFs and binding grammar within regulatory regions. We found that TF-COMB can accurately identify known co-occurring TFs from ChIP-seq data, as well as uncover preferential localization to other genomic elements. With the use of ATAC-seq footprinting and TF motif locations, we found that TFs exhibit both preferred orientation and distance in relation to each other, and that these are biologically significant. Finally, we extended the analysis to not only investigate individual TF pairs, but also TF pairs in the context of networks, which enabled the investigation of TF complexes and TF hubs. In conclusion, TF-COMB is a flexible tool to investigate various aspects of TF binding grammar.

Epigenetic reactivation of transcriptional programs orchestrating fetal lung development in human pulmonary hypertension.

Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.

i2dash: Creation of Flexible, Interactive, and Web-based Dashboards for Visualization of Omics Data.

Data visualization and interactive data exploration are important aspects of illustrating complex concepts and results from analyses of omics data. A suitable visualization has to be intuitive and accessible. Web-based dashboards have become popular tools for the arrangement, consolidation, and display of such visualizations. However, the combination of automated data processing pipelines handling omics data and dynamically generated, interactive dashboards is poorly solved. Here, we present i2dash, an R package intended to encapsulate functionality for the programmatic creation of customized dashboards. It supports interactive and responsive (linked) visualizations across a set of predefined graphical layouts. i2dash addresses the needs of data analysts/software developers for a tool that is compatible and attachable to any R-based analysis pipeline, thereby fostering the separation of data visualization on one hand and data analysis tasks on the other hand. In addition, the generic design of i2dash enables the development of modular extensions for specific needs. As a proof of principle, we provide an extension of i2dash optimized for single-cell RNA sequencing analysis, supporting the creation of dashboards for the visualization needs of such experiments. Equipped with these features, i2dash is suitable for extensive use in large-scale sequencing/bioinformatics facilities. Along this line, we provide i2dash as a containerized solution, enabling a straightforward large-scale deployment and sharing of dashboards using cloud services. i2dash is freely available via the R package archive CRAN (https://CRAN.R-project.org/package=i2dash).

Epigenetic Regulation by Suv4-20h1 in Cardiopulmonary Progenitor Cells Is Required to Prevent Pulmonary Hypertension and Chronic Obstructive Pulmonary Disease.

BACKGROUND: The pathogenesis of life-threatening cardiopulmonary diseases such as pulmonary hypertension (PH) and chronic obstructive pulmonary disease (COPD) originates from a complex interplay of environmental factors and genetic predispositions that is not fully understood. Likewise, little is known about developmental abnormalities or epigenetic dysregulations that might predispose for PH or COPD in adult individuals. METHODS: To identify pathology-associated epigenetic alteration in diseased lung tissues, we screened a cohort of human patients with PH and COPD for changes of histone modifications by immunofluorescence staining. To analyze the function of H4K20me2/3 in lung pathogenesis, we developed a series of Suv4-20h1 knockout mouse lines targeting cardiopulmonary progenitor cells and different heart and lung cell types, followed by hemodynamic studies and morphometric assessment of tissue samples. Molecular, cellular, and biochemical techniques were applied to analyze the function of Suv4-20h1-dependent epigenetic processes in cardiopulmonary progenitor cells and their derivatives. RESULTS: We discovered a strong reduction of the histone modifications of H4K20me2/3 in human patients with COPD but not patients with PH that depend on the activity of the H4K20 di-methyltransferase SUV4-20H1. Loss of Suv4-20h1 in cardiopulmonary progenitor cells caused a COPD-like/PH phenotype in mice including the formation of perivascular tertiary lymphoid tissue and goblet cell hyperplasia, hyperproliferation of smooth muscle cells/myofibroblasts, impaired alveolarization and maturation defects of the microvasculature leading to massive right ventricular dilatation and premature death. Mechanistically, SUV4-20H1 binds directly to the 5'-upstream regulatory element of the superoxide dismutase 3 (Sod3) gene to repress its expression. Increased levels of the extracellular SOD3 enzyme in Suv4-20h1 mutants increases hydrogen peroxide concentrations, causing vascular defects and impairing alveolarization. CONCLUSIONS: Our findings reveal a pivotal role of the histone modifier SUV4-20H1 in cardiopulmonary codevelopment and uncover the developmental origins of cardiopulmonary diseases. We assume that the study will facilitate the understanding of pathogenic events causing PH and COPD and aid the development of epigenetic drugs for the treatment of cardiopulmonary diseases.

multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets.

Targeting the coding genome to introduce nucleotide deletions/insertions via the CRISPR/Cas9 technology has become a standard procedure. It has quickly spawned a multitude of methods such as prime editing, APEX proximity labeling, or homology directed repair, for which supporting bioinformatics tools are, however, lagging behind. New CRISPR/Cas9 applications often require specific gRNA design functionality, and a generic tool is critically missing. Here, we introduce multicrispr, an R/bioconductor tool, intended to design individual gRNAs and complex gRNA libraries. The package is easy to use; detects, scores, and filters gRNAs on both efficiency and specificity; visualizes and aggregates results per target or CRISPR/Cas9 sequence; and finally returns both genomic ranges and sequences of gRNAs. To be generic, multicrispr defines and implements a genomic arithmetic framework as a basis for facile adaptation to techniques recently introduced such as prime editing or yet to arise. Its performance and design concepts such as target set-specific filtering render multicrispr a tool of choice when dealing with screening-like approaches.

ATAC-seq footprinting unravels kinetics of transcription factor binding during zygotic genome activation.

While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements.

Stimulation of glycolysis promotes cardiomyocyte proliferation after injury in adult zebrafish.

Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.

AP-1 Contributes to Chromatin Accessibility to Promote Sarcomere Disassembly and Cardiomyocyte Protrusion During Zebrafish Heart Regeneration.

RATIONALE: The adult human heart is an organ with low regenerative potential. Heart failure following acute myocardial infarction is a leading cause of death due to the inability of cardiomyocytes to proliferate and replenish lost cardiac muscle. While the zebrafish has emerged as a powerful model to study endogenous cardiac regeneration, the molecular mechanisms by which cardiomyocytes respond to damage by disassembling sarcomeres, proliferating, and repopulating the injured area remain unclear. Furthermore, we are far from understanding the regulation of the chromatin landscape and epigenetic barriers that must be overcome for cardiac regeneration to occur. OBJECTIVE: To identify transcription factor regulators of the chromatin landscape, which promote cardiomyocyte regeneration in zebrafish, and investigate their function. METHODS AND RESULTS: Using the Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq), we first find that the regenerating cardiomyocyte chromatin accessibility landscape undergoes extensive changes following cryoinjury, and that activator protein-1 (AP-1) binding sites are the most highly enriched motifs in regions that gain accessibility during cardiac regeneration. Furthermore, using bioinformatic and gene expression analyses, we find that the AP-1 response in regenerating adult zebrafish cardiomyocytes is largely different from the response in adult mammalian cardiomyocytes. Using a cardiomyocyte-specific dominant negative approach, we show that blocking AP-1 function leads to defects in cardiomyocyte proliferation as well as decreased chromatin accessibility at the fbxl22 and ilk loci, which regulate sarcomere disassembly and cardiomyocyte protrusion into the injured area, respectively. We further show that overexpression of the AP-1 family members Junb and Fosl1 can promote changes in mammalian cardiomyocyte behavior in vitro. CONCLUSIONS: AP-1 transcription factors play an essential role in the cardiomyocyte response to injury by regulating chromatin accessibility changes, thereby allowing the activation of gene expression programs that promote cardiomyocyte dedifferentiation, proliferation, and protrusion into the injured area.

TGF-ß Signaling Promotes Tissue Formation during Cardiac Valve Regeneration in Adult Zebrafish.

Cardiac valve disease can lead to severe cardiac dysfunction and is thus a frequent cause of morbidity and mortality. Its main treatment is valve replacement, which is currently greatly limited by the poor recellularization and tissue formation potential of the implanted valves. As we still lack suitable animal models to identify modulators of these processes, here we used adult zebrafish and found that, upon valve decellularization, they initiate a rapid regenerative program that leads to the formation of new functional valves. After injury, endothelial and kidney marrow-derived cells undergo cell cycle re-entry and differentiate into new extracellular matrix-secreting valve cells. The TGF-ß signaling pathway promotes the regenerative process by enhancing progenitor cell proliferation as well as valve cell differentiation. These findings reveal a key role for TGF-ß signaling in cardiac valve regeneration and establish the zebrafish as a model to identify and test factors promoting cardiac valve recellularization and growth.

Aging Triggers H3K27 Trimethylation Hoarding in the Chromatin of Nothobranchius furzeri Skeletal Muscle.

Aging associates with progressive loss of skeletal muscle function, sometimes leading to sarcopenia, a process characterized by impaired mobility and weakening of muscle strength. Since aging associates with profound epigenetic changes, epigenetic landscape alteration analysis in the skeletal muscle promises to highlight molecular mechanisms of age-associated alteration in skeletal muscle. This study was conducted exploiting the short-lived turquoise killifish Nothobranchius furzeri (Nfu), a relatively new model for aging studies. The epigenetic analysis suggested a less accessible and more condensed chromatin in old Nfu skeletal muscle. Specifically, an accumulation of heterochromatin regions was observed as a consequence of increased levels of H3K27me3, HP1α, polycomb complex subunits, and senescence-associated heterochromatic foci (SAHFs). Consistently, euchromatin histone marks, including H3K9ac, were significantly reduced. In this context, integrated bioinformatics analysis of RNASeq and ChIPSeq, related to skeletal muscle of Nfu at different ages, revealed a down-modulation of genes involved in cell cycle, differentiation, and DNA repair and an up-regulation of inflammation and senescence genes. Undoubtedly, more studies are needed to disclose the detailed mechanisms; however, our approach enlightened unprecedented features of Nfu skeletal muscle aging, potentially associated with swimming impairment and reduced mobility typical of old Nfu.

Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.

The phenotypes caused by morpholino-mediated interference of gene function in zebrafish are often not observed in the corresponding mutant(s). We took advantage of the availability of a relatively large collection of transcriptomic datasets to identify common signatures that characterize morpholino-injected animals (morphants). In addition to the previously reported activation of tp53 expression, we observed increased expression of the interferon-stimulated genes (ISGs), isg15 and isg20, the cell death pathway gene casp8, and other cellular stress response genes including phlda3, mdm2 and gadd45aa. Studies involving segmentation stage embryos were more likely to show upregulation of these genes. We also found that the expression of these genes could be upregulated by increasing doses of an egfl7 morpholino, or even high doses of the standard control morpholino. Thus, these data show that morpholinos can induce the expression of ISGs in zebrafish embryos and further our understanding of morpholino effects.

Genome-wide strategies reveal target genes of Npas4l associated with vascular development in zebrafish.

The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. Zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we have combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation. The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes that are crucial for hematoendothelial specification, such as etv2, tal1 and lmo2 We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.