Tara L. Rasmussen

BOP, a regulator of right ventricular heart development, is a direct transcriptional target of MEF2C in the developing heart.

The vertebrate heart is assembled during embryogenesis in a modular manner from different populations of precursor cells. The right ventricular chamber and outflow tract are derived primarily from a population of progenitors known as the anterior heart field. These regions of the heart are severely hypoplastic in mutant mice lacking the myocyte enhancer factor 2C (MEF2C) and BOP transcription factors, suggesting that these cardiogenic regulatory factors may act in a common pathway for development of the anterior heart field and its derivatives. We show that Bop expression in the developing heart depends on the direct binding of MEF2C to a MEF2-response element in the Bop promoter that is necessary and sufficient to recapitulate endogenous Bop expression in the anterior heart field and its cardiac derivatives during mouse development. The Bop promoter also directs transcription in the skeletal muscle lineage, but only cardiac expression is dependent on MEF2. These findings identify Bop as an essential downstream effector gene of MEF2C in the developing heart, and reveal a transcriptional cascade involved in development of the anterior heart field and its derivatives.

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.

Getting to the Heart of Myocardial Stem Cells and Cell Therapy

Heart disease is both common and deadly. Cardiovascular disease is a global epidemic, because it is the number 1 cause of death worldwide, and it is estimated that 1 in 3 adults in the United States have cardiovascular disease.1 Although a number of pioneering initiatives have transformed our treatment of cardiovascular disease, new therapies are required to further address the growing incidence of this deadly disease. Intense interest has focused on regenerative medicine as an emerging strategy for chronic diseases such as cardiovascular disease. A number of human tissues, including skin,2 gut, liver,3,–,6 and skeletal muscle3,7 have a tremendous regenerative capacity. For example, skeletal muscle is able to completely restore its cellular architecture and function after an injury that destroys >80% of the muscle.7,8 This regenerative response lacks a fibroproliferative response (ie, formation of scar) and is associated with restoration of the vasculature, myofibers, and extracellular matrix. Compared with skeletal muscle, the regenerative capacity of the adult heart is more limited. Recent studies suggest that the adult heart is capable of cellular turnover and limited regeneration after injury, although the networks that govern this process are ill defined. The use of genetic mouse models and molecular biological techniques is unveiling cell populations, pathways, and extracellular cues that may direct cardiac regeneration and provide a platform for further investigation. The goal of the present review is to examine the endogenous regenerative capacity of the adult heart and highlight new experimental regenerative therapies aimed at restoring myocardial architecture and function. Previous studies have demonstrated that metazoans such as the newt and zebrafish are capable of cardiac regeneration in response to a significant injury.9,–,12 This myocardial regenerative response is complex, and occurs over a 2-month …

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. STEM CELLS2012;30:1611–1623

VEGF/Flk1 signaling cascade transactivates Etv2 gene expression.

Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.

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

Background: Mesp1 and Etv2 are essential transcription factors in the regulation of mesodermal lineage development, but their relationship is unclear. Results: Mesp1 interacts physically with Creb1 and transcriptionally regulates Etv2 gene expression. Conclusion: Etv2 is a direct downstream target gene of Mesp1. Significance: This is the first report to identify Creb1 as a coactivator of Mesp1 to regulate 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.

Dpath software reveals hierarchical haemato-endothelial lineages of Etv2 progenitors based on single-cell transcriptome analysis

Developmental, stem cell and cancer biologists are interested in the molecular definition of cellular differentiation. Although single-cell RNA sequencing represents a transformational advance for global gene analyses, novel obstacles have emerged, including the computational management of dropout events, the reconstruction of biological pathways and the isolation of target cell populations. We develop an algorithm named dpath that applies the concept of metagene entropy and allows the ranking of cells based on their differentiation potential. We also develop self-organizing map (SOM) and random walk with restart (RWR) algorithms to separate the progenitors from the differentiated cells and reconstruct the lineage hierarchies in an unbiased manner. We test these algorithms using single cells from Etv2-EYFP transgenic mouse embryos and reveal specific molecular pathways that direct differentiation programmes involving the haemato-endothelial lineages. This software program quantitatively assesses the progenitor and committed states in single-cell RNA-seq data sets in a non-biased manner.

Defective myogenesis in the absence of the muscle-specific lysine methyltransferase SMYD1.

The SMYD (SET and MYND domain) family of lysine methyltransferases harbor a unique structure in which the methyltransferase (SET) domain is intervened by a zinc finger protein-protein interaction MYND domain. SMYD proteins methylate both histone and non-histone substrates and participate in diverse biological processes including transcriptional regulation, DNA repair, proliferation and apoptosis. Smyd1 is unique among the five family members in that it is specifically expressed in striated muscles. Smyd1 is critical for development of the right ventricle in mice. In zebrafish, Smyd1 is necessary for sarcomerogenesis in fast-twitch muscles. Smyd1 is expressed in the skeletal muscle lineage throughout myogenesis and in mature myofibers, shuttling from nucleus to cytosol during myoblast differentiation. Because of this expression pattern, we hypothesized that Smyd1 plays multiple roles at different stages of myogenesis. To determine the role of Smyd1 in mammalian myogenesis, we conditionally eliminated Smyd1 from the skeletal muscle lineage at the myoblast stage using Myf5(cre). Deletion of Smyd1 impaired myoblast differentiation, resulted in fewer myofibers and decreased expression of muscle-specific genes. Muscular defects were temporally restricted to the second wave of myogenesis. Thus, in addition to the previously described functions for Smyd1 in heart development and skeletal muscle sarcomerogenesis, these results point to a novel role for Smyd1 in myoblast differentiation.

Smyd1 facilitates heart development by antagonizing oxidative and ER stress responses

Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense—a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.

The Etv2-miR-130a Network Regulates Mesodermal Specification

MicroRNAs (miRNAs) are known to regulate critical developmental stages during embryogenesis. Here, we defined an Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors toward the endothelial lineage. Gain-of-function experiments demonstrated that miR-130a promoted the endothelial program at the expense of the cardiac program without impacting the hematopoietic lineages. In contrast, CRISPR/Cas9-mediated knockout of miR-130a demonstrated a reduction of the endothelial program without affecting hematopoiesis. Mechanistically, miR-130a directly suppressed Pdgfra expression and promoted the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a overexpression and knockout phenotypes, respectively. In summary, we report the function of a miRNA that specifically promotes the divergence of the hemato-endothelial progenitor to the endothelial lineage.