DrImpute: imputing dropout events in single cell RNA sequencing data.

BACKGROUND: The single cell RNA sequencing (scRNA-seq) technique begin a new era by allowing the observation of gene expression at the single cell level. However, there is also a large amount of technical and biological noise. Because of the low number of RNA transcriptomes and the stochastic nature of the gene expression pattern, there is a high chance of missing nonzero entries as zero, which are called dropout events. RESULTS: We develop DrImpute to impute dropout events in scRNA-seq data. We show that DrImpute has significantly better performance on the separation of the dropout zeros from true zeros than existing imputation algorithms. We also demonstrate that DrImpute can significantly improve the performance of existing tools for clustering, visualization and lineage reconstruction of nine published scRNA-seq datasets. CONCLUSIONS: DrImpute can serve as a very useful addition to the currently existing statistical tools for single cell RNA-seq analysis. DrImpute is implemented in R and is available at https://github.com/gongx030/DrImpute .

Feedback Mechanisms Regulate Ets Variant 2 (Etv2) Gene Expression and Hematoendothelial Lineages.

Etv2 is an essential transcriptional regulator of hematoendothelial lineages during embryogenesis. Although Etv2 downstream targets have been identified, little is known regarding the upstream transcriptional regulation of Etv2 gene expression. In this study, we established a novel methodology that utilizes the differentiating ES cell and embryoid body system to define the modules and enhancers embedded within the Etv2 promoter. Using this system, we defined an autoactivating role for Etv2 that is mediated by two adjacent Ets motifs in the proximal promoter. In addition, we defined the role of VEGF/Flk1-Calcineurin-NFAT signaling cascade in the transcriptional regulation of Etv2. Furthermore, we defined an Etv2-Flt1-Flk1 cascade that serves as a negative feedback mechanism to regulate Etv2 gene expression. To complement and extend these studies, we demonstrated that the Flt1 null embryonic phenotype was partially rescued in the Etv2 conditional knockout background. In summary, these studies define upstream and downstream networks that serve as a transcriptional rheostat to regulate Etv2 gene expression.

Kelch Repeat and BTB Domain Containing Protein 5 (Kbtbd5) Regulates Skeletal Muscle Myogenesis through the E2F1-DP1 Complex.

We have previously isolated a muscle-specific Kelch gene, Kelch repeat and BTB domain containing protein 5 (Kbtbd5)/Kelch-like protein 40 (Klhl40). In this report, we identified DP1 as a direct interacting factor for Kbtbd5 using a yeast two-hybrid screen and in vitro binding assays. Our studies demonstrate that Kbtbd5 interacts and regulates the cytoplasmic localization of DP1. GST pulldown assays demonstrate that the dimerization domain of DP1 interacts with all three of the Kbtbd5 domains. We further show that Kbtbd5 promotes the ubiquitination and degradation of DP1, thereby inhibiting E2F1-DP1 activity. To investigate the in vivo function of Kbtbd5, we used gene disruption technology and engineered Kbtbd5 null mice. Targeted deletion of Kbtbd5 resulted in postnatal lethality. Histological studies reveal that the Kbtbd5 null mice have smaller muscle fibers, a disorganized sarcomeric structure, increased extracellular matrix, and decreased numbers of mitochondria compared with wild-type controls. RNA sequencing and quantitative PCR analyses demonstrate the up-regulation of E2F1 target apoptotic genes (Bnip3 and p53inp1) in Kbtbd5 null skeletal muscle. Consistent with these observations, the cellular apoptosis in Kbtbd5 null mice was increased. Breeding of Kbtbd5 null mouse into the E2F1 null background rescues the lethal phenotype of the Kbtbd5 null mice but not the growth defect. The expression of Bnip3 and p53inp1 in Kbtbd5 mutant skeletal muscle are also restored to control levels in the E2F1 null background. In summary, our studies demonstrate that Kbtbd5 regulates skeletal muscle myogenesis through the regulation of E2F1-DP1 activity.

The transcription factor Mesp1 interacts with cAMP-responsive element binding protein 1 (Creb1) and coactivates Ets variant 2 (Etv2) gene expression.

Mesoderm posterior 1 (Mesp1) is well recognized for its role in cardiac development, although it is expressed broadly in mesodermal lineages. We have previously demonstrated important roles for Mesp1 and Ets variant 2 (Etv2) during lineage specification, but their relationship has not been defined. This study reveals that Mesp1 binds to the proximal promoter and transactivates Etv2 gene expression via the CRE motif. We also demonstrate the protein-protein interaction between Mesp1 and cAMP-responsive element binding protein 1 (Creb1) in vitro and in vivo. Utilizing transgenesis, lineage tracing, flow cytometry, and immunostaining technologies, we define the lineage relationship between Mesp1- and Etv2-expressing cell populations. We observe that the majority of Etv2-EYFP(+) cells are derived from Mesp1-Cre(+) cells in both the embryo and yolk sac. Furthermore, we observe that the conditional deletion of Etv2, using a Mesp1-Cre transgenic strategy, results in vascular and hematopoietic defects similar to those observed in the global deletion of Etv2 and that it has embryonic lethality by embryonic day 9.5. In summary, our study supports the hypothesis that Mesp1 is a direct upstream transactivator of Etv2 during embryogenesis and that Creb1 is an important cofactor of Mesp1 in the transcriptional regulation of Etv2 gene expression.

Inferring dynamic gene regulatory networks in cardiac differentiation through the integration of multi-dimensional data.

BACKGROUND: Decoding the temporal control of gene expression patterns is key to the understanding of the complex mechanisms that govern developmental decisions during heart development. High-throughput methods have been employed to systematically study the dynamic and coordinated nature of cardiac differentiation at the global level with multiple dimensions. Therefore, there is a pressing need to develop a systems approach to integrate these data from individual studies and infer the dynamic regulatory networks in an unbiased fashion. RESULTS: We developed a two-step strategy to integrate data from (1) temporal RNA-seq, (2) temporal histone modification ChIP-seq, (3) transcription factor (TF) ChIP-seq and (4) gene perturbation experiments to reconstruct the dynamic network during heart development. First, we trained a logistic regression model to predict the probability (LR score) of any base being bound by 543 TFs with known positional weight matrices. Second, four dimensions of data were combined using a time-varying dynamic Bayesian network model to infer the dynamic networks at four developmental stages in the mouse [mouse embryonic stem cells (ESCs), mesoderm (MES), cardiac progenitors (CP) and cardiomyocytes (CM)]. Our method not only infers the time-varying networks between different stages of heart development, but it also identifies the TF binding sites associated with promoter or enhancers of downstream genes. The LR scores of experimentally verified ESCs and heart enhancers were significantly higher than random regions (p <10(-100)), suggesting that a high LR score is a reliable indicator for functional TF binding sites. Our network inference model identified a region with an elevated LR score approximately -9400 bp upstream of the transcriptional start site of Nkx2-5, which overlapped with a previously reported enhancer region (-9435 to -8922 bp). TFs such as Tead1, Gata4, Msx2, and Tgif1 were predicted to bind to this region and participate in the regulation of Nkx2-5 gene expression. Our model also predicted the key regulatory networks for the ESC-MES, MES-CP and CP-CM transitions. CONCLUSION: We report a novel method to systematically integrate multi-dimensional -omics data and reconstruct the gene regulatory networks. This method will allow one to rapidly determine the cis-modules that regulate key genes during cardiac differentiation.

Cooperative interaction of Etv2 and Gata2 regulates the development of endothelial and hematopoietic lineages.

Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein-protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.

Cardiomyopathy in a dish: using human inducible pluripotent stem cells to model inherited cardiomyopathies.

Inherited cardiomyopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathies, arrythmogenic right ventricular cardiomyopathy, and other inherited forms of heart failure, represent a unique set of genetically defined cardiovascular disease processes. Unraveling the molecular mechanisms of these deadly forms of human heart disease has been challenging, but recent groundbreaking scientific advances in stem cell technology have allowed for the generation of patient-specific human inducible stem cell (hiPSC)-derived cardiomyocytes (CMs). hiPSC-derived CMs retain the genetic blueprint of the patient, they can be maintained in culture, and they recapitulate the phenotypic characteristics of the disease in vitro, thus serving as a disease in a dish. This review provides an overview of in vitro modeling of inherited cardiomyopathies with the use of patient-specific hiPSC-derived CMs.

ER71 directs mesodermal fate decisions during embryogenesis.

Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP(+) population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.

A critical role for endoglin in the emergence of blood during embryonic development.

Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here, we show that endoglin (Eng), a receptor for the TGFß superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng(+)Flk1(+) mesodermal precursor population at embryonic day 7.5 (E7.5), a cell fraction also endowed with endothelial potential. In Eng-knockout embryos, hematopoietic colony activity and numbers of CD71(+)Ter119(+) erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key bone morphogenic protein (BMP) target genes, including the hematopoietic regulators Scl, Gata1, Gata2, and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng(+)Flk-1(+) progenitors coexpressed TGFß and BMP receptors and target genes. Furthermore, Eng(+)Flk-1(+) cells presented high levels of phospho-SMAD1/5, indicating active TGFß and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of Eng-expressing cells was dependent on the TGFß superfamily ligands BMP4, BMP2, or TGF-ß1. These data demonstrate that the E(+)F(+) fraction at E7.5 represents mesodermal cells competent to respond to TGFß1, BMP4, or BMP2, shaping their hematopoietic development, and that Eng acts as a critical regulator in this process by modulating TGF/BMP signaling.

Expression levels of endoglin distinctively identify hematopoietic and endothelial progeny at different stages of yolk sac hematopoiesis.

Endoglin (Eng), an ancillary receptor of the transforming growth factor beta (TGFß) signaling pathway superfamily, has been well recognized for its important function in vascular development and angiogenesis since its discovery more than a decade ago. Recent studies show that this receptor is also critical for the emergence of blood during embryonic development, and that at E7.5, endoglin together with Flk-1 identifies early mesoderm progenitors that are endowed with hematopoietic and endothelial potential. These two lineages emerge in very close association during embryogenesis, and because they share the expression of the same surface markers, it has been difficult to distinguish the earliest hematopoietic from endothelial cells. Here, we evaluated the function of endoglin in hematopoiesis as development progresses past E7.5, and found that the hematopoietic and endothelial progenitors can be distinguished by the levels of endoglin in E9.5 yolk sacs. Whereas endothelial cells are Eng(bright), hematopoietic activity is primarily restricted to a subset of cells that display dim expression of endoglin (Eng(dim)). Molecular characterization of these subfractions showed that endoglin-mediated induction of hematopoiesis occurs in concert with BMP2/BMP4 signaling. This pathway is highly active in Eng(dim) cells but significantly downregulated in the Eng knockout. Taken together, our findings show an important function for endoglin in mediating BMP2/BMP4 signaling during yolk sac hematopoietic development and suggest that the levels of this receptor modulate TGFß versus bone morphogenetic protein (BMP) signaling.

Etv2 rescues Flk1 mutant embryoid bodies.

Independent mouse knockouts of Etv2 and Flk1 are embryonic lethal and lack hematopoietic and endothelial lineages. We previously reported that Flk1 activates Etv2 in the initiation of hematopoiesis and vasculogenesis. However, Flk1 and its ligand VEGF are expressed throughout development, from E7.0 to adulthood, whereas Etv2 is expressed only transiently during embryogenesis. These observations suggest a complex regulatory interaction between Flk1 and Etv2. To further examine the Flk1 and Etv2 regulatory interaction, we transduced Etv2 and Flk1 mutant ES cells with viral integrants that inducibly overexpress Flk1 or Etv2. We demonstrated that forced expression of Etv2 rescued the hematopoietic and endothelial potential of differentiating Flk1 and Etv2 mutant cells. We further discovered that forced expression of Flk1 can rescue that of the Flk1, but not Etv2 mutant cells. Therefore, we conclude that the requirement for Flk1 can be bypassed by expressing Etv2, supporting the notion that disruption of Etv2 expression is responsible for the early phenotypes of the Etv2 and Flk1 mutant embryos.

Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration.

Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals, possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.

Etv2 is expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene expression.

During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2(+), c-kit(+), and CD41(+) hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP(+) population. In the Etv2 mutant yolk sac, Tie2(+) cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2.

Nkx2-5 represses Gata1 gene expression and modulates the cellular fate of cardiac progenitors during embryogenesis.

BACKGROUND: Recent studies suggest that the hematopoietic and cardiac lineages have close ontogenic origins, and that an early mesodermal cell population has the potential to differentiate into both lineages. Studies also suggest that specification of these lineages is inversely regulated. However, the transcriptional networks that govern the cell fate specification of these progenitors are incompletely defined. METHODS AND RESULTS: Here, we show that Nkx2-5 regulates the hematopoietic/erythroid fate of the mesoderm precursors early during cardiac morphogenesis. Using transgenic technologies to isolate Nkx2-5 expressing cells, we observed an induction of the erythroid molecular program, including Gata1, in the Nkx2-5-null embryos. We further observed that overexpression of Nkx2-5 with an Nkx2-5-inducible embryonic stem cell system significantly repressed Gata1 gene expression and suppressed the hematopoietic/erythroid potential, but not the endothelial potential, of the embryonic stem cells. This suppression was cell-autonomous, and was partially rescued by overexpressing Gata1. In addition, we demonstrated that Nkx2-5 binds to the Gata1 gene enhancer and represses the transcriptional activity of the Gata1 gene. CONCLUSIONS: Our results demonstrate that the hematopoietic/erythroid cell fate is suppressed via Nkx2-5 during mesodermal fate determination, and that the Gata1 gene is one of the targets that are suppressed by Nkx2-5.

Etv2 regulates enhancer chromatin status to initiate Shh expression in the limb bud.

Sonic hedgehog (Shh) is essential for limb development, and the mechanisms that govern the propagation and maintenance of its expression has been well studied; however, the mechanisms that govern the initiation of Shh expression are incomplete. Here we report that ETV2 initiates Shh expression by changing the chromatin status of the developmental limb enhancer, ZRS. Etv2 expression precedes Shh in limb buds, and Etv2 inactivation prevents the opening of limb chromatin, including the ZRS, resulting in an absence of Shh expression. Etv2 overexpression in limb buds causes nucleosomal displacement at the ZRS, ectopic Shh expression, and polydactyly. Areas of nucleosome displacement coincide with ETS binding site clusters. ETV2 also functions as a transcriptional activator of ZRS and is antagonized by ETV4/5 repressors. Known human polydactyl mutations introduce novel ETV2 binding sites in the ZRS, suggesting that ETV2 dosage regulates ZRS activation. These studies identify ETV2 as a pioneer transcription factor (TF) regulating the onset of Shh expression, having both a chromatin regulatory role and a transcriptional activation role.

Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice.

The mammalian thalamus is located in the diencephalon and is composed of dozens of morphologically and functionally distinct nuclei. The majority of these nuclei project axons to the neocortex in unique patterns and play critical roles in sensory, motor, and cognitive functions. It has been assumed that the adult thalamus is derived from neural progenitor cells located within the alar plate of the caudal diencephalon. Nevertheless, how a distinct array of postmitotic thalamic nuclei emerge from this single developmental unit has remained largely unknown. Our recent studies found that these thalamic nuclei are in fact derived from molecularly heterogeneous populations of progenitor cells distributed within at least two distinct progenitor domains in the caudal diencephalon. In this study, we investigated how such molecular heterogeneity is established and maintained during early development of the thalamus and how early signaling mechanisms influence the formation of postmitotic thalamic nuclei. By using mouse genetics and in utero electroporation, we provide evidence that Sonic hedgehog (Shh), which is normally expressed in ventral and rostral borders of the embryonic thalamus, plays a crucial role in patterning progenitor domains throughout the thalamus. We also show that increasing or decreasing Shh activity causes dramatic reorganization of postmitotic thalamic nuclei through altering the positional identity of progenitor cells.

Feedback regulation of NEUROG2 activity by MTGR1 is required for progression of neurogenesis.

The sequential steps of neurogenesis are characterized by highly choreographed changes in transcription factor activity. In contrast to the well-studied mechanisms of transcription factor activation during neurogenesis, much less is understood regarding how such activity is terminated. We previously showed that MTGR1, a member of the MTG family of transcriptional repressors, is strongly induced by a proneural basic helix-loop-helix transcription factor, NEUROG2 in developing nervous system. In this study, we describe a novel feedback regulation of NEUROG2 activity by MTGR1. We show that MTGR1 physically interacts with NEUROG2 and represses transcriptional activity of NEUROG2. MTGR1 also prevents DNA binding of the NEUROG2/E47 complex. In addition, we provide evidence that proper termination of NEUROG2 activity by MTGR1 is necessary for normal progression of neurogenesis in the developing spinal cord. These results highlight the importance of feedback regulation of proneural gene activity in neurodevelopment.

Differential activity of Wnt/beta-catenin signaling in the embryonic mouse thalamus.

In neural development, several Wnt genes are expressed in the vertebrate diencephalon, including the thalamus. However, roles of Wnt signaling in the thalamus during neurogenesis are not well understood. We examined Wnt/beta-catenin activity in embryonic mouse thalamus and found that a Wnt target gene Axin2 and reporter activity of BAT-gal transgenic mice show similar, differential patterns within the thalamic ventricular zone, where ventral and rostral regions had lower activity than other regions. Expression of Wnt ligands and signaling components also showed complex, differential patterns. Finally, based on partially reciprocal patterns of Wnt and Shh signals in the thalamic ventricular zone, we tested if Shh signal is sufficient or necessary for the differential Axin2 expression. Analysis of mice with enhanced or reduced Shh signal showed that Axin2 expression is similar to controls. These results suggest that differential Wnt signaling may play a role in patterning the thalamus independent of Shh signaling.

Progenitor cell maturation in the developing vertebrate retina.

Progenitor cells in the developing retina initially divide so that each division produces two cells that divide again. Subsequently, progenitor cells change their mode of division so that one or both cells produced by a division can withdraw from the mitotic cycle and differentiate. We asked how these two progenitor cell stages differ molecularly and what controls the switch in the mode of division. We show that early preneurogenic progenitor cells express the transcription factor, Sox2, and the Notch ligand, Delta1. More mature neurogenic progenitor cells express Sox2 and the bHLH transcription factor, E2A, and not Delta1. Notch signaling maintains progenitor cells in the preneurogenic state. Sonic hedgehog expressed by newly differentiating cells initiates maturation of progenitor cells from preneurogenic to neurogenic at the neurogenic front, possibly by down-regulating Delta1 expression. Our results show that the preneurogenic-to-neurogenic transition is a highly organized unidirectional step made in unison by neighboring cells.

Generation of human endothelium in pig embryos deficient in ETV2.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney1,2. However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages3-7. ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.