mES Cells Research Articles

Nuclear Magnetic Resonance Detects Phosphoinositide 3-Kinase/Akt-Independent Traits Common to Pluripotent Murine Embryonic Stem Cells and Their Malignant Counterparts

Pluripotent embryonic stem (ES) cells, a potential source of somatic precursors for cell therapies, cause tumors after transplantation. Studies of mammalian carcinogenesis using nuclear magnetic resonance (NMR) spectroscopy have revealed changes in the choline region, particularly increased phosphocholine (PCho) content. High PCho levels in murine ES (mES) cells have recently been attributed to cell pluripotency. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated in tumor-like properties of mES cells. This study aimed to examine a potential link between the metabolic profile associated with choline metabolism of pluripotent mES cells and PI3K/Akt signaling. We used mES (ES-D3) and murine embryonal carcinoma cells (EC-F9) and compared the metabolic profiles of 1) pluripotent mES (ESD0), 2) differentiated mES (ESD14), and 3) pluripotent F9 cells. Involvement of the PI3K/Akt pathway was assessed using LY294002, a selective PI3K inhibitor. Metabolic profiles were characterized in the extracted polar fraction by (1)H NMR spectroscopy. Similarities were found between the levels of choline phospholipid metabolites (PCho/total choline and PCho/glycerophosphocholine [GPCho]) in ESD0 and F9 cell spectra and a greater-than five-fold decrease of the PCho/GPCho ratio associated with mES cell differentiation. LY294002 caused no significant change in relative PCho levels but led to a greater-than two-fold increase in PCho/GPCho ratios. These results suggest that the PCho/GPCho ratio is a metabolic trait shared by pluripotent and malignant cells and that PI3K does not underlie its development. It is likely that the signature identified here in a mouse model may be relevant for safe therapeutic applications of human ES cells.

Noninvasive Monitoring of Embryonic Stem CellsIn Vivowith MRI Transgene Reporter

Reporter gene-based magnetic resonance imaging (MRI) offers unique insights into behavior of cells after transplantation, which could significantly benefit stem cell research and translation. Several candidate MRI reporter genes, including one that encodes for iron storage protein ferritin, have been reported, and their potential applications in embryonic stem (ES) cell research have yet to be explored. We have established transgenic mouse ES (mES) cell lines carrying human ferritin heavy chain (FTH) as a reporter gene and succeeded in monitoring the cell grafts in vivo using T(2)-weighted MRI sequences. FTH generated MRI contrast through compensatory upregulation of transferrin receptor (Tfrc) that led to increased cellular iron stored in ferritin-bound form. At a level sufficient for MRI contrast, expression of FTH posed no toxicity to mES cells and did not interfere with stem cell pluripotency as observed in neural differentiation and teratoma formation. The compatibility and functionality of ferritin as a reporter in mES cells opens up the possibility of using MRI for longitudinal noninvasive monitoring of ES cell-derived cell grafts at both molecular and cellular levels.

Effects of Ethanol on Mouse Embryonic Stem Cells

Fetal alcohol syndrome (FAS) reflects a constellation of congenital abnormalities caused by excess maternal consumption of alcohol. It is likely that interference with embryonic development plays a role in the pathogenesis of the disorder. Ethanol-induced apoptosis has been suggested as a causal factor in the genesis of FAS. Mouse embryonic stem (mES) cells are pluripotent cells that differentiate in vitro to cell aggregates termed embryoid bodies (EBs), wherein differentiation capacity and gene expression profile are similar to those of the early embryo. To investigate the effects of ethanol during differentiation, mES cells were cultured on a gelatin surface in the presence of leukemia inhibitory factor which maintains adherent undifferentiated cells or in suspension to promote formation of EBs. All cells were treated (1-6 days) with 80 mM ethanol. The pluripotency and differentiation of mES cells were evaluated by western blotting of stage-specific embryonic antigen (SSEA-1), transcription factors Oct-3/4, Sox-2, and Nanog, using alkaline phosphatase staining. Apoptosis (early to late stages) was assessed by fluorescence-activated cell sorting using TdT-mediated biotin-dUTP nick-end labelling assay and fluorescein isothiocyanate-Annexin V/propidium iodide staining. Ethanol increased apoptosis during in vitro differentiation of mES cells to EBs, whereas undifferentiated cells were not affected. Ethanol exposure also interfered with pluripotency marker patterns causing an upregulation of SSEA-1 under self-renewal conditions. In EBs, ethanol delayed the downregulation of SSEA-1 and affected the regulation of transcription factors during differentiation. Our findings suggest that ethanol may contribute to the pathogenesis of FAS by triggering apoptotic pathways during differentiation of embryonic stem cells and deregulating early stages of embryogenesis.

Gene Regulation and Epigenetic Remodeling in Murine Embryonic Stem Cells by c-Myc

BackgroundThe Myc oncoprotein, a transcriptional regulator involved in the etiology of many different tumor types, has been demonstrated to play an important role in the functions of embryonic stem (ES) cells. Nonetheless, it is still unclear as to whether Myc has unique target and functions in ES cells.Methodology/Principal FindingsTo elucidate the role of c-Myc in murine ES cells, we mapped its genomic binding sites by chromatin-immunoprecipitation combined with DNA microarrays (ChIP-chip). In addition to previously identified targets we identified genes involved in pluripotency, early development, and chromatin modification/structure that are bound and regulated by c-Myc in murine ES cells. Myc also binds and regulates loci previously identified as Polycomb (PcG) targets, including genes that contain bivalent chromatin domains. To determine whether c-Myc influences the epigenetic state of Myc-bound genes, we assessed the patterns of trimethylation of histone H3-K4 and H3-K27 in mES cells containing normal, increased, and reduced levels of c-Myc. Our analysis reveals widespread and surprisingly diverse changes in repressive and activating histone methylation marks both proximal and distal to Myc binding sites. Furthermore, analysis of bulk chromatin from phenotypically normal c-myc null E7 embryos demonstrates a 70–80% decrease in H3-K4me3, with little change in H3-K27me3, compared to wild-type embryos indicating that Myc is required to maintain normal levels of histone methylation.Conclusions/SignificanceWe show that Myc induces widespread and diverse changes in histone methylation in ES cells. We postulate that these changes are indirect effects of Myc mediated by its regulation of target genes involved in chromatin remodeling. We further show that a subset of PcG-bound genes with bivalent histone methylation patterns are bound and regulated in response to altered c-Myc levels. Our data indicate that in mES cells c-Myc binds, regulates, and influences the histone modification patterns of genes involved in chromatin remodeling, pluripotency, and differentiation.

Abstract 5440: Forward Programming of Pluripotent Stem Cells Towards Distinct Cardiovascular Cell Types

Aims: The proliferative potential of pluripotent stem cell derived cardiomyocytes is limited and reasonable yields for novel therapeutic options have yet to be achieved. In addition various clinical applications will require the generation of specific cardiac cell types. Whereas early cardiovascular precursors appear to be important for novel approaches such as reseeding decellularized hearts, direct cell transplantation may require ventricular cells. Our recent work demonstrated that MesP1 represents a master regulator sufficient to induce cardiovasculogenesis in pluripotent cells. This led to our hypothesis that “forward programming” towards specific subtypes may be feasible via overexpression of distinct early cardiovascular transcription factors. Methods and results: Here we demonstrate that forced expression of Nkx2.5 similar to MesP1 is sufficient to enhance cardiogenesis in murine embryonic stem cells. In comparison to control transfected mES cells a 5 fold increased appearance of beating foci was observed as well as upregulated mRNA and protein expression levels. In contrast to MesP1 no increase of the endothelial lineage within the cardiovasculogenic mesoderm was observed. Likewise Flk-1, the earliest known cardiovascular surface marker was not induced via Nkx2.5 as opposed to MesP1. Detailed patch clamping analyses showed electrophysiological characteristics corresponding to all subtypes of cardiac ES cell differentiation in Nkx2.5 as well as MesP1 programmed embryoid bodies (EBs) but fractions of cardiomyocytes had distinct characteristics: MesP1 forced the appearance of early/intermediate type cardiomyocytes in comparison to control transfected ES cells whereas Nkx2.5 led to preferentially differentiated ventricular cells. Conclusion: Our findings show proof of principle for cardiovascular subtype specific programming of pluripotent stem cells and confirm the molecular hierarchy for cardiovascular specification initiated via MesP1 with differentiation factors such as Nkx2.5 further downstream.

Cripto-1 Is Required for Hypoxia to Induce Cardiac Differentiation of Mouse Embryonic Stem Cells

Cripto-1 is a membrane-bound protein that is highly expressed in embryonic stem cells and in human tumors. In the present study, we investigated the effect of low levels of oxygen, which occurs naturally in rapidly growing tissues, on Cripto-1 expression in mouse embryonic stem (mES) cells and in human embryonal carcinoma cells. During hypoxia, Cripto-1 expression levels were significantly elevated in mES cells and in Ntera-2 or NCCIT human embryonal carcinoma cells, as compared with cells growing with normal oxygen levels. The transcription factor hypoxia-inducible factor-1alpha directly regulated Cripto-1 expression by binding to hypoxia-responsive elements within the promoter of mouse and human Cripto-1 genes in mES and NCCIT cells, respectively. Furthermore, hypoxia modulated differentiation of mES cells by enhancing formation of beating cardiomyocytes as compared with mES cells that were differentiated under normoxia. However, hypoxia failed to induce differentiation of mES cells into cardiomyocytes in the absence of Cripto-1 expression, demonstrating that Cripto-1 is required for hypoxia to fully differentiate mES cells into cardiomyocytes. Finally, cardiac tissue samples derived from patients who had suffered ischemic heart disease showed a dramatic increase in Cripto-1 expression as compared with nonischemic heart tissue samples, suggesting that hypoxia may also regulate Cripto-1 in vivo.

Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells

Growing evidence suggests that physical microenvironments and mechanical stresses, besides soluble factors, help direct mesenchymal stem cell fate. However, biological responses to a local force in embryonic stem (ES) cells remain elusive. Here we show that a local cyclic stress via focal adhesions induced spreading in mouse ES (mES) cells but not in mES cell-differentiated (ESD) cells that were 10-fold stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src, or Cdc42 were essential in the spreading response. The applied stress led to Oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might play far more important roles in early developments of soft embryos than previously appreciated.

Dimeric integrin α5β1 ligands confer morphological and differentiation responses to murine embryonic stem cells

We present the first report utilizing, and showing the functional relevance of, self-assembling polyvalent ligands specific for integrin α5β1 in murine embryonic stem (mES) cell adhesion. Di, tri and tetrameric 9th–10th type III fibronectin domains (FIII9′10) were used to generate clustered integrin α5β1 ligand surfaces for mES cell culture. Compared to gelatin, FIII9′10 (monomer), FIII9′10-trimer and -tetramer, the FIII9′10-dimer supported the highest number of mES cell colonies. No evidence of domain unfolding upon surface adsorption was found. Colonies appeared disperse with a spread cell morphology unless subdued back to a tight morphology with increasing concentrations of leukemia inhibitory factor (LIF). In the presence of LIF, mES cells adherent to the FIII9′10-dimer showed transient upregulation of Oct-4, the mesodermal transcription factor, Brachyury, and the ectodermal marker, Nestin. However, dual upregulation of Nanog maintained the mES cells in a pluripotent state, confirmed by alkaline phosphatase staining. Therefore, the behavior of mES cells adherent to dimeric integrin α5β1 ligands is a largely morphological phenomenon conferring pro-differentiation signals towards mesodermal and ectodermal lineages. This work will be of interest to cell and tissue engineering groups aiming to control ES cell behavior through integrin ligand presentation and synthetic substrates.

Effects of Reconstituted Collagen Matrix on Fates of Mouse Embryonic Stem Cells Before and After Induction for Chondrogenic Differentiation

Embryonic stem (ES) cells are pluripotent cells with great potential in regenerative medicine. However, controlling their differentiation toward homogeneous lineages is challenging. In this study, we aim to investigate the effects of reconstituted 3D collagen matrix on the fates of mouse ES (mES) cells before and after induction for chondrogenic differentiation. Specifically, mES cells were encapsulated and cultured in 3D collagen microspheres and exposed to induction signals at different time points. Growth characteristics and differentiation status of mES cells were then evaluated. Collagen microspheres provided a suitable microenvironment supporting mES cell growth and maintaining their undifferentiated status for certain period of time. At later time points, the proportion of undifferentiated mES cells gradually decreased, accompanied by increasing proportions of mesenchymal progenitor cells. This suggests the inductive role of collagen matrix in differentiating mES cells toward mesenchymal lineages. Moreover, a lower initial collagen monomer concentration facilitated the differentiation of mES cells into chondrogenic lineages, while induction at a later time point associated with a more advanced stage of chondrogenic differentiation. This indicates that both the initial collagen concentration and the time to induce differentiation significantly affected the fates of mES cells. This study contributes to future development of ES cell-based therapies.

Dazl Promotes Germ Cell Differentiation from Embryonic Stem Cells

It has been demonstrated that through the formation of embryoid bodies (EBs) germ cells can be derived from embryonic stem (ES) cells. Here, we describe a transgene expression approach to derive germ cells directly from ES cells in vitro without EB formation. Through the ectopic expression of Deleted in Azoospermia-Like (Dazl), a germ cell-specific RNA-binding protein, both motile tailed-sperm and oocytes were induced from mouse ES (mES) cells in culture. Furthermore, transient overexpression of Dazl led to suppression of Nanog but induced germ cell nuclear antigen in mES cells. Dazl knockdown resulted in reduction in the expression of germ cell markers including Stella, MVH and Prdm1. Our study indicates that Dazl is a master gene controlling germ cell differentiation and that ectopic expression of Dazl promotes the dynamic differentiation of mouse ES cells into gametes in vitro.

Microarrays of over 2000 hydrogels – Identification of substrates for cellular trapping and thermally triggered release

In this paper we describe an approach whereby over 2000 individual polymers were synthesized, in situ, on a microscope slide using inkjet printing. Subsequent biological analysis of the entire library allowed the rapid identification of specific polymers with the desired properties. Herein we demonstrate how this array of new materials could be used for the identification of polymers that allow cellular adherence, proliferation and then mild thermal release, for multiple cell lines, including mouse embryonic stem (mES) cells. The optimal, identified hydrogels were successfully scaled-up and demonstrated excellent cell viability after thermal detachment for all cell lines tested. We believe that this approach offers an avenue to the discovery of a specific thermal release polymer for every cell line.

SysPTM: A Systematic Resource for Proteomic Research on Post-translational Modifications

With the rapid expansion of protein post-translational modification (PTM) research based on large-scale proteomic work, there is an increasing demand for a suitable repository to analyze PTM data. Here we present a curated, web-accessible PTM data base, SysPTM. SysPTM provides a systematic and sophisticated platform for proteomic PTM research equipped not only with a knowledge base of manually curated multi-type modification data but also with four fully developed, in-depth data mining tools. Currently, SysPTM contains data detailing 117,349 experimentally determined PTM sites on 33,421 proteins involving nearly 50 PTM types, curated from public resources including five data bases and four web servers and more than one hundred peer-reviewed mass spectrometry papers. Protein annotations including Pfam domains, KEGG pathways, GO functional classification, and ortholog groups are integrated into the data base. Four online tools have been developed and incorporated, including PTMBlast, to compare a user's PTM dataset with PTM data in SysPTM; PTMPathway, to map PTM proteins to KEGG pathways; PTMPhylog, to discover potentially conserved PTM sites; and PTMCluster, to find clusters of multi-site modifications. The workflow of SysPTM was demonstrated by analyzing an in-house phosphorylation dataset identified by MS/MS. It is shown that in SysPTM, the role of single-type and multi-type modifications can be systematically investigated in a full biological context. SysPTM could be an important contribution to modificomics research. SysPTM is freely available online at www.sysbio.ac.cn/SysPTM.

Use of the Sleeping Beauty Transposon System for Stable Gene Expression in Mouse Embryonic Stem Cells

INTRODUCTIONSleeping Beauty (SB) transposon-based transfection is a two-component system consisting of a transposase and a transposon containing inverted repeat/direct repeat (IR/DR) sequences that result in precise integration into a TA dinucleotide. The transposon is designed with an expression cassette of interest flanked by IR/DRs, and SB transposase mediates stable integration and reliable long-term expression of the gene of interest. It has recently been demonstrated that SB efficiently mediates gene transfer and stable gene expression in human embryonic stem (ES) cells. Here, we describe a method for transfecting and establishing stable cell lines in mouse embryonic stem (mES) cells with the SB system.

MES cell growth medium

mES cell growth medium

An effort to test the embryotoxicity of benzene, toluene, xylene, and formaldehyde to murine embryonic stem cells using airborne exposure technique

Benzene, toluene, xylene, and formaldehyde are well-known indoor air pollutants, especially after house decoration. They are also common pollutants in the working places of the plastic industry, chemical industry, and leather industry. It has been reported that these pollutants cause people to be irritated, sick, experience a headache, and be dizzy. They also have the potential to induce asthma, aplastic anemia, and leukemia, even cause abortion or fetus malformation in humans. In this study, the airborne toxicity of benzene, toluene, xylene, and formaldehyde to murine embryonic stem cells (mES cells) were tested using airborne exposure technique to evaluate the mES cell airborne exposure model on embryotoxicity prediction. Briefly, mES cells were cultured on Transwell inserts and were exposed to an airborne surrounding of test chemicals in a chamber for 1 h at 37°C. Cytotoxicity was determined using the MTT assay after further culture for 18 h at 37°C in normal medium. The airborne IC50 (50% inhibition concentration) of benzene, toluene, xylene, and formaldehyde derived from the fitted dose-response curves were 17,400 ± 1290, 16,000 ± 250, 4680 ± 500, and 620 ± 310 ppm, respectively. Formaldehyde was found to be the compound most toxic to mES cells compared to benzene homologues. The toxicity data had good correlation with the in vivo data. The results showed that the mES airborne exposure model may be used to predict embryotoxicity of volatile organic compounds.

Delivery of Polyethylenimine/DNA Complexes Assembled in a Microfluidics Device

Polyethylenimine (PEI) and plasmid DNA (pDNA) complexes (PEI/pDNA) are nonviral vectors for gene delivery. The conventional method for producing these complexes involves bulk mixing (BM) of PEI and DNA followed by vortexing which at low N/P ratios results in large particle size distribution, low cytotoxicity, and poor gene transfection, while at high N/P ratios it results in small particle size and better gene transfection but high cytotoxicity. To improve size control, gene transfection efficiency, and cytotoxicity, in this study, we used a microfluidic hydrodynamic focusing (MF) device to prepare PEI/pDNA complexes at N/P = 3.3 and 6.7. We used bulk mixing as control, mouse NIH 3T3 fibroblast cells and mouse embryonic stem (mES) cells as model cell lines, plasmid encoding green fluorescent protein (pGFP) and secreted alkaline phosphatase (pSEAP) as the reporter gene, and commercially available Lipofectamine 2,000 as a positive control. The complexes were characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), and zeta potential (zeta) measurement. Confocal laser scanning microscopy (CLSM) and fluorescent labeling techniques were used to visualize the complex size distribution, complexation uniformity, and cellular distribution. The results showed that MF produced complexes were smaller and more uniformly complexed and had higher cell viability and improved exogenous gene expression.

Deciphering Induction of Pluripotency in a Bovine Model.

Strategies utilized in deriving embryonic stem (ES) cells from mouse and primate embryos have not been sufficient for maintaining self renewal and inhibiting spontaneous differentiation in ungulate embryo explant cultures, implying that ungulate ESC may require a unique signature of signal transduction pathways, transcription factor hierarchies, and chromatin modifications. To determine how key transcription factors OCT4 (POU5F1) and SOX2 induce changes in developmental potential within differentiated bovine cells, we constructed inducible bOCT4 and bSOX2 expression constructs and a live cell, pluripotency-specific transcriptional marker using the bovine NANOG promoter. The open reading frames of transcription factors bOCT4 and bSOX2 were obtained via RT-PCR from bovine CT-1 and bovine fetal fibroblast (BFF) cell lines, respectively. The bovine NANOG promoter was obtained by PCR from genomic DNA. The bOCT4 ORF was joined to a hemagglutinin (HA) epitope tag sequence and cloned into the tetracycline-inducible pTHE vector, which also contains a bicistronic tetracycline repressor (TetR)-IRES-neomycin resistance cassette. The bSOX2 ORF was joined to a MYC epitope tag sequence and cloned into a modified pTHE vector containing a bicistronic TetR-IRES-nuclear localized dsRED2 cassette. Each construct was transiently transfected into HEK293 cells and then doxycycline induced for initial protein expression analysis. Recombinant protein levels were analyzed by western blot. Both constructs were shown to be responsive to doxycycline in a dose dependent manner (0 ng/ml - 10 ng/ml). Immunocytochemistry of induced, transfected HEK293 cells demonstrated that these recombinant transcription factors localized to nuclei, indicating that the HA and MYC epitopes did not obstruct proper protein transport and localization. The transcriptional marker construct, bNANOG promoter linked to nuclear localized green fluorescent protein (bNANOGp-nGFP) was also created using a portion of the bNANOG promoter 440 base pairs upstream of the translational start site. Stable expression of nGFP driven by the bNANOG promoter was obtained in both NANOG expressing R1 mouse ESC and human NTERA cell lines. Expression of nGFP was down-regulated in differentiating R1 mES cells and was not observed in BFF. These studies demonstrated that the bNANOG promoter functioned as expected and that when linked to a marker gene can be used to study induction, maintenance, and loss of pluripotency. These results validate the constructs for appropriate use in studies exploring induction and loss of pluripotency, thus allowing us to follow changes in the transcriptome of selected BFF cells throughout the initial stage of reprogramming and uncover signature markers indicating increased developmental potential. This research was supported by CSREES USDA NRI grant 200405447 and Maryland Agricultural Experimental Station grants. (poster)

Forward programming of pluripotent stem cells towards distinct cardiovascular cell types

The proliferative potential of pluripotent stem cell-derived cardiomyocytes is limited, and reasonable yields for novel therapeutic options have yet to be achieved. In addition, various clinical applications will require the generation of specific cardiac cell types. Whereas early cardiovascular precursors appear to be important for novel approaches such as reseeding decellularized hearts, direct cell transplantation may require ventricular cells. Our recent work demonstrated that MesP1 represents a master regulator sufficient to induce cardiovasculogenesis in pluripotent cells. This led to our hypothesis that 'forward programming' towards specific subtypes may be feasible via overexpression of distinct early cardiovascular transcription factors. Here we demonstrate that forced expression of Nkx2.5 similar to MesP1 is sufficient to enhance cardiogenesis in murine embryonic stem cells (mES). In comparison to control transfected mES cells, a five-fold increased appearance of beating foci was observed as well as upregulated mRNA and protein expression levels. In contrast to MesP1, no increase of the endothelial lineage within the cardiovasculogenic mesoderm was observed. Likewise, Flk-1, the earliest known cardiovascular surface marker, was not induced via Nkx2.5 as opposed to MesP1. Detailed patch clamping analyses showed electrophysiological characteristics corresponding to all subtypes of cardiac ES cell differentiation in Nkx2.5 as well as MesP1 programmed embryoid bodies, but fractions of cardiomyocytes had distinct characteristics: MesP1 forced the appearance of early/intermediate type cardiomyocytes in comparison to control transfected ES cells whereas Nkx2.5 led to preferentially differentiated ventricular cells. Our findings show proof of principle for cardiovascular subtype-specific programming of pluripotent stem cells and confirm the molecular hierarchy for cardiovascular specification initiated via MesP1 with differentiation factors such as Nkx2.5 further downstream.

Stochastic response of mES cells toward BMP signaling is improved under dynamic microfluidic conditions

Stochastic response of mES cells toward BMP signaling is improved under dynamic microfluidic conditions

Somitovasculin, a Novel Endothelial-Specific Transcript Involved in the Vasculature Development

We recently isolated and characterized endothelial-like CD31(+) cells derived from mouse embryonic stem (mES) cells and identified their transcriptome. The main objective of this study was to determine the functional relevance of the transcripts of unknown function (TUF) for vasculature development. We selected 2 TUFs of more than 27 to study their role for blood vessel development in zebrafish. Morpholino (MO) knockdown of the zebrafish orthologs of the first TUF (TUF1, mouse cDNA BC022623) showed disruption of the intersegmental vessels (ISV) at 2 days postfertilization as observed by live imaging of fli:EGFP-transgenic embryos. The morphants showed abnormal blood circulation, but no effect on hematopoiesis was observed as demonstrated by gata-1 in situ hybridizations. Because knockdown of TUF1 resulted in disruption of the ISV patterning we named the TUF1 somitovasculin. TUF2 has been identified as cDNA clone BC020535. The MO knockdown of TUF2 resulted in a phenotype with an enlarged heart and the embryos lacked circulation completely. We have shown the participation of a novel transcript (named somitovasculin) in circulatory vessel development. The combination of expression profiling in differentiating mES cells and the zebrafish model has the potential for rapid identification and functional characterization of TUFs.