Mouse Embryonic Stem Cells Research Articles

POGZ suppresses 2C transcriptional program and retrotransposable elements

The POGZ gene has been found frequently mutated in neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) and intellectual disability (ID). We have recently shown that POGZ maintains mouse embryonic stem cells (ESCs). However, the exact mechanisms remain unclear. Here, we show that POGZ plays an important role in the maintenance of ESCs by silencing Dux and endogenous retroviruses (ERVs). POGZ maintains a silent chromatin state at Dux and ERVs by associating with and recruiting TRIM28 and SETDB1, and its loss leads to decreased levels of H3K9me3/H4K20me3, resulting in up-regulation of 2C transcripts and ESC transition to a 2C-like state. POGZ suppresses different classes of ERVs through direct (IAPEy, the intracisternal A-type particle elements) and indirect regulation (MERVL). Activation of POGZ-bound ERVs is associated with up-regulation of nearby neural disease genes such as Serpina3m. Our findings provide important insights into understanding the disease mechanism caused by POGZ dysfunction.

Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells.

The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.

A genome-wide screen reveals new regulators of the 2-cell-like cell state.

In mammals, only the zygote and blastomeres of the early embryo are totipotent. This totipotency is mirrored in vitro by mouse '2-cell-like cells' (2CLCs), which appear at low frequency in cultures of embryonic stem cells (ESCs). Because totipotency is not completely understood, we carried out a genome-wide CRISPR knockout screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a gene expressed in 2CLCs. Here we report the identification of four mutants that reactivate Dazl and a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc-Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. All four factors function upstream of DPPA2 and DUX, but not via p53. In addition, SPOP binds DPPA2, and KDM5C interacts with ncPRC1.6 and inhibits 2CLC gene expression in a catalytic-independent manner. These results extend our knowledge of totipotency, a key phase of organismal life.

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops.

Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci-at different length scales-can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.

FOXO1 promotes endothelial cell elongation and angiogenesis by up-regulating the phosphorylation of myosin light chain 2.

The forkhead box O1 (FOXO1) is an important transcription factor related to proliferation, metabolism, and homeostasis, while the major phenotype of FOXO1-null mice is abnormal vascular morphology, such as vessel enlargement and dilation. In in vitro mouse embryonic stem cell (ESC)-differentiation system, Foxo1-/- vascular endothelial cells (ECs) fail to elongate, and mimic the abnormalities of FOXO1-deficiency in vivo. Here, we identified the PPP1R14C gene as the FOXO1 target genes responsible for elongating using transcriptome analyses in ESC-derived ECs (ESC-ECs), and found that the FOXO1-PPP1R14C-myosin light chain 2 (MLC2) axis is required for EC elongation during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP). PPP1R14C is an inhibitor of PP1, the catalytic subunit of MLCP. The abnormal morphology of Foxo1-/- ESC-ECs was associated with low level of PPP1R14C and loss of MLC2 phosphorylation, which were reversed by PPP1R14C-introduction. Knockdown of either FOXO1 or PPP1R14C suppressed vascular cord formation and reduced MLC2 phosphorylation in human ECs (HUVECs). The mouse and human PPP1R14C locus possesses an enhancer element containing conserved FOXO1-binding motifs. In vivo chemical inhibition of MLC2 phosphorylation caused dilated vascular structures in mouse embryos. Furthermore, foxo1 or ppp1r14c-knockdown zebrafish exhibited vascular malformations, which were also restored by PPP1R14C-introduction. Mechanistically, FOXO1 suppressed MLCP activity by up-regulating PPP1R14C expression, thereby promoting MLC2 phosphorylation and EC elongation, which are necessary for vascular development. Given the importance of MLC2 phosphorylation in cell morphogenesis, this study may provide novel insights into the role of FOXO1 in control of angiogenesis.

Quantitative Analysis of Differentiation Activity for Mouse Embryonic Stem Cells by Deep Learning for Cell Center Detection using Three-Dimensional Confocal Fluorescence Microscopy Images.

Accurate single cell segmentation provides means to monitor the behavior of single cell within a population of cells. Time-lapse fluorescence images are used to reveal heterogeneous nature of single mouse embryonic stem cell (ESC) colony and monitor fluctuations of the cell states. Automatic quantification of speed and status shifts of the ESCs depends on accurate single cell segmentation that is used to calculate the 3D center of every cell and track this cell for the quantification. This study proposes a new 3D U-net to accurately detect center of each single cell in 3D confocal images. The dimension of input 3D images to the U-net is flexible so that multiple center detections from different image directions can be implemented simultaneously to improve the center detection accuracy. This study showed that our method can improve accuracy for cell center detection and thus the quantification for ESC speeds and status shifts.

Stem Cell Therapy in Stargardt Disease: A Systematic Review.

This article aimed to review current literature on the safety and efficacy of stem cell therapy in Stargardt disease. A comprehensive literature search was performed, and two animal and eleven human clinical trials were retrieved. These studies utilized different kinds of stem cells, including human or mouse embryonic stem cells, mesenchymal stem cells, bone marrow mononuclear fraction, and autologous bone marrow-derived stem cells. In addition, different injection techniques including subretinal, intravitreal, and suprachoroidal space injections have been evaluated. Although stem cell therapy holds promise in improving visual function in patients with Stargardt disease, further investigation is needed to determine the long-term benefits, safety, and efficacy in determining the best delivery method and selecting the most appropriate stem cell type.

Histone proteomics implicates H3K36me2 and its regulators in mouse embryonic stem cell pluripotency exit and lineage choice

Abstract Objectives Gene expression changes during embryonic stem cell (ESC) differentiation is regulated by epigenetic mechanisms. Understanding these can help uncover how cell fate decisions are made during early embryonic development. Comparison of chromatin of ESCs with lineage-committed cells can implicate chromatin factors functional in exit from pluripotency and the choice of proper lineages. Therefore, we quantitatively analyzed histone modifications in mouse ESC differentiation towards neuroectoderm and endoderm. Methods We cultured mouse ESCs (mESCs) and differentiated them towards neuroectoderm or endoderm lineages and performed mass spectrometry on total histones. Subsequent Western blots verified significantly altered H3K36me2. RT-qPCR analyses showed changes in H3K36-specific methyltransferases, demethylases and readers at mESC stage or during neuroectoderm/endoderm commitment. Results We presented quantitative histone modification levels in mESCs and lineage-committed cells. H3K36me2 increased specifically in neuroectoderm compared to mESCs or endoderm-committed cells. Regulation of H3K36 methylation might have a role in pluripotency exit and/or differentiation. Nsd2, Dnmt3b and Zmynd11 increased during differentiation regardless of lineage. Conversely, mESCs had higher Kdm4c and Msh6 expression than differentiated cells. Comparing neuroectoderm and endoderm-committed cells, we revealed Nsd1, Setd5 and Dnmt3a had lineage specific expression pattern. Conclusions Our results show quantitative changes in histone modifications during mESC lineage commitment and implicate H3K36me2 regulation for not only pluripotency exit but also lineage choice. Its regulatory proteins show stage (mESC vs. committed) or lineage (neuroectoderm vs. endoderm) dependent expression changes. Further work will be needed to discover their possible involvement in cell fate decisions and target genes.

SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells.

Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs. SAFB predominantly associated with introns of protein-coding genes through purine-rich motifs. The transcript most enriched in SAFB association was the lncRNA Malat1, which also contains a purine-rich region in its 5' end. Knockout of SAFB/SAFB2 led to differential expression of approximately 1000 genes associated with multiple biological processes, including apoptosis, cell division, and cell migration. Knockout of SAFB/SAFB2 also led to splicing changes in a set of genes that were largely distinct from those that exhibited changes in expression level. The spliced and nascent transcripts of many genes whose expression levels were positively regulated by SAFB also associated with high levels of SAFB, implying that SAFB binding promotes their expression. Reintroduction of SAFB into double-knockout cells restored gene expression toward wild-type levels, an effect again observable at the level of spliced and nascent transcripts. Proteomics analysis revealed a significant enrichment of nuclear speckle-associated and RS domain-containing proteins among SAFB interactors. Neither Xist nor Polycomb functions were dramatically altered in SAFB/2 knockout ESCs. Our findings suggest that among other potential functions in ESCs, SAFB promotes the expression of certain genes through its ability to bind nascent RNA.

Long-read sequence analysis of MMEJ-mediated CRISPR genome editing reveals complex on-target vector insertions that may escape standard PCR-based quality control

CRISPR genome editing is a powerful tool for elucidating biological functions. To modify the genome as intended, it is essential to understand the various modes of recombination that can occur. In this study, we report complex vector insertions that were identified during the generation of conditional alleles by CRISPR editing using microhomology-mediated end joining (MMEJ). The targeting vector contained two loxP sequences and flanking 40-bp microhomologies. The genomic regions corresponding to the loxP sequences were cleaved with Cas9 in mouse embryonic stem cells. PCR screening for targeted recombination revealed a high frequency of bands of a larger size than expected. Nanopore sequencing of these bands revealed complex vector insertions mediated not only by MMEJ but also by non-homologous end joining and homologous recombination in at least 17% of the clones. A new band appeared upon improving the PCR conditions, suggesting the presence of unintentionally modified alleles that escape standard PCR screening. This prompted us to characterize the recombination of each allele of the genome-edited clones using heterozygous single nucleotide polymorphisms, leading to confirmation of the presence of homozygous alleles. Our study indicates that careful quality control of genome-edited clones is needed to exclude complex, unintended, on-target vector insertion.

DPPA5A suppresses the mutagenic TLS and MMEJ pathways by modulating the cryptic splicing of Rev1 and Polq in mouse embryonic stem cells

Genetic alterations are often acquired during prolonged propagation of pluripotent stem cells (PSCs). This ruins the stem cell quality and hampers their full applications. Understanding how PSCs maintain genomic integrity would provide the clues to overcome the hurdle. It has been known that embryonic stem cells (ESCs) utilize high-fidelity pathways to ensure genomic stability, but the underlying mechanisms remain largely elusive. Here, we show that many DNA damage response and repair genes display differential alternative splicing in mouse ESCs compared to differentiated cells. Particularly, Rev1 and Polq, two key genes for mutagenic translesion DNA synthesis (TLS) and microhomology-mediated end joining (MMEJ) repair pathways, respectively, display a significantly higher rate of cryptic exon (CE) inclusion in ESCs. The frequent CE inclusion disrupts the normal protein expressions of REV1 and POLθ, thereby suppressing the mutagenic TLS and MMEJ. Further, we identify an ESC-specific RNA binding protein DPPA5A which stimulates the CE inclusion in Rev1 and Polq. Depletion of DPPA5A in mouse ESCs decreased the CE inclusion of Rev1 and Polq, induced the protein expression, and stimulated the TLS and MMEJ activity. Enforced expression of DPPA5A in NIH3T3 cells displayed reverse effects. Mechanistically, we found that DPPA5A directly regulated CE splicing of Rev1. DPPA5A associates with U2 small nuclear ribonucleoprotein of the spliceosome and binds to the GA-rich motif in the CE of Rev1 to promote CE inclusion. Thus, our study uncovers a mechanism to suppress mutagenic TLS and MMEJ pathways in ESCs.

OBOX regulates mouse zygotic genome activation and early development.

Zygotic genome activation (ZGA) activates the quiescent genome to enable thematernal-to-zygotic transition1,2. However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcriptionfactor family (OBOX1-OBOX8)3-5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a two-cellto four-cellarrest, accompanied by impaired ZGA. TheObox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the'preconfiguration'of RNA polymerase II,as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.

Geometric Confinement-Mediated Mechanical Tension Directs Patterned Differentiation of Mouse ESCs into Organized Germ Layers.

The self-organization of embryonic stem cells (ESCs) into organized tissues with three distinct germ layers is critical to morphogenesis and early development. While the regulation of this self-organization by soluble signals is well established, the involvement of mechanical force gradients in this process remains unclear due to the lack of a suitable platform to study this process. In this study, we developed a 3D microenvironment to examine the influence of mechanical tension gradients on ESC-patterned differentiation during morphogenesis by controlling the geometrical signals (shape and size) of ESC colonies. We found that changes in colony geometry impacted the germ layer pattern, with Cdx2-positive cells being more abundant at edges and in areas with high curvatures. The differentiation patterns were determined by geometry-mediated cell tension gradients, with an extraembryonic mesoderm-like layer forming in high-tension regions and ectodermal-like lineages at low-tension regions in the center. Suppression of cytoskeletal tension hindered ESC self-organization. These results indicate that geometric confinement-mediated mechanical tension plays a crucial role in linking multicellular organization to cell differentiation and impacting tissue patterning.

Identification and Functional Evaluation of Alternative Splice Variants of Dax1 in Mouse Embryonic Stem Cells.

Dax1 (Nr0b1; Dosage-sensitive sex reversal-adrenal hypoplasia congenital on the X-chromosome gene-1) is an important component of the transcription factor network that governs pluripotency in mouse embryonic stem cells (ESCs). Functional evaluation of alternative splice variants of pluripotent transcription factors has shed additional insight on the maintenance of ESC pluripotency and self-renewal. Dax1 splice variants have not been identified and characterized in mouse ESCs. We identified 18 new transcripts of Dax1 with putative protein-coding properties and compared their protein structures with known Dax1 protein (Dax1-472). The expression pattern analysis showed that the novel isoforms were cotranscribed with Dax1-472 in mouse ESCs, but they had transcriptional heterogeneity among single cells and the subcellular localization of the encoded proteins differed. Cell function experiments indicated that Dax1-404 repressed Gata6 transcription and functionally replaced Dax1-472, while Dax1-38 and Dax1-225 partially antagonized Dax1-472 transcriptional repression. This study provided a comprehensive characterization of the Dax1 splice variants in mouse ESCs and suggested complex effects of Dax1 variants in a self-renewal regulatory network.

Cooperative regulation of Zhx1 and hnRNPA1 drives the cardiac progenitor-specific transcriptional activation during cardiomyocyte differentiation

The zinc finger proteins (ZNFs) mediated transcriptional regulation is critical for cell fate transition. However, it is still unclear how the ZNFs realize their specific regulatory roles in the stage-specific determination of cardiomyocyte differentiation. Here, we reported that the zinc fingers and homeoboxes 1 (Zhx1) protein, transiently expressed during the cell fate transition from mesoderm to cardiac progenitors, was indispensable for the proper cardiomyocyte differentiation of mouse and human embryonic stem cells. Moreover, Zhx1 majorly promoted the specification of cardiac progenitors via interacting with hnRNPA1 and co-activated the transcription of a wide range of genes. In-depth mechanistic studies showed that Zhx1 was bound with hnRNPA1 by the amino acid residues (Thr111–His120) of the second Znf domain, thus participating in the formation of cardiac progenitors. Together, our study highlights the unrevealed interaction of Zhx1/hnRNPA1 for activating gene transcription during cardiac progenitor specification and also provides new evidence for the specificity of cell fate determination in cardiomyocyte differentiation.

The cytoplasmic fraction of the histone lysine methyltransferase Setdb1 is essential for embryonic stem cells

The major lysine methyltransferase (KMT) Setdb1 is essential for self-renewal and viability of mouse embryonic stem cells (mESCs). Setdb1 was primarily known to methylate the lysine 9 of histone 3 (H3K9) in the nucleus, where it regulates chromatin functions. However, Setdb1 is also massively localized in the cytoplasm, including in mESCs, where its role remains elusive. Here, we show that the cytoplasmic Setdb1 (cSetdb1) is essential for the survival of mESCs. Yeast two-hybrid analysis revealed that cSetdb1 interacts with several regulators of mRNA stability and protein translation machinery, such as the ESCs-specific E3 ubiquitin ligase and mRNA silencer Trim71/Lin41. We found that cSetdb1 is required for the integrity of Trim71 complex(es) involved in mRNA metabolism and translation. cSetdb1 modulates the abundance of mRNAs and the rate of newly synthesized proteins. Altogether, our data uncovered the cytoplasmic post-transcriptional regulation of gene expression mediated by a key epigenetic regulator.

Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells

Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

Hyperosmotic stress induces 2-cell-like cells through ROS and ATR signaling.

Mouse embryonic stem cells (ESCs) display pluripotency features characteristic of the inner cell mass of the blastocyst. Mouse embryonic stem cell cultures are highly heterogeneous and include a rare population of cells, which recapitulate characteristics of the 2-cell embryo, referred to as 2-cell-like cells (2CLCs). Whether and how ESC and 2CLC respond to environmental cues has not been fully elucidated. Here, we investigate the impact of mechanical stress on the reprogramming of ESC to 2CLC. We show that hyperosmotic stress induces 2CLC and that this induction can occur even after a recovery time from hyperosmotic stress, suggesting a memory response. Hyperosmotic stress in ESCs leads to accumulation of reactive-oxygen species (ROS) and ATR checkpoint activation. Importantly, preventing either elevated ROS levels or ATR activation impairs hyperosmotic-mediated 2CLC induction. We further show that ROS generation and the ATR checkpoint act within the same molecular pathway in response to hyperosmotic stress to induce 2CLCs. Altogether, these results shed light on the response of ESC to mechanical stress and on our understanding of 2CLC reprogramming.

The Development of Methods for the Production of New Molecular Vaccines and Appropriate RNA Fragments to Counteract Unwanted Genes: A Pilot Study.

The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations.

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia

STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. In mouse embryonic stem cells (ESCs) we manage to determine the specific subset of hypoxia-induced genes that need STAT3 to be properly transcribed and, among them, fundamental genes like Vegfa, Hk1, Hk2, Pfkp and Hilpda are worth mentioning. Unexpectedly, we also demonstrated that the absence of STAT3 does not affect the expression of Hif1α mRNA nor the stabilization of HIF1α protein, but the STAT3-driven regulation of the hypoxia-dependent subset of gene could rely on the physical interaction between STAT3 and HIF1α. To further elucidate the physiological roles of this STAT3 non-canonical nuclear activity, we used a CRISPR/Cas9 zebrafish stat3 knock-out line. Notably, hypoxia-related fluorescence of the hypoxia zebrafish reporter line (HRE:mCherry) cannot be induced when Stat3 is not active and, while Stat3 Y705 phosphorylation seems to have a pivotal role in this process, S727 does not affect the Stat3-dependent hypoxia response. Hypoxia is fundamental for vascularization, angiogenesis and immune cells mobilization; all processes that, surprisingly, cannot be induced by low oxygen levels when Stat3 is genetically ablated. All in all, here we report the specific STAT3/HIF1α-dependent subset of genes in vitro and, for the first time with an in vivo model, we determined some of the physiological roles of STAT3-hypoxia crosstalk.