Epithelial-to-mesenchymal transition (EMT) is fundamental to both embryogenesis and tumor metastasis. The Notch intercellular signaling pathway regulates cell fate determination throughout metazoan evolution, and overexpression of activating alleles is oncogenic in mammals. Here we demonstrate that Notch activity promotes EMT during both cardiac development and oncogenic transformation via transcriptional induction of the Snail repressor, a potent and evolutionarily conserved mediator of EMT in many tissues and tumor types. In the embryonic heart, Notch functions via lateral induction to promote a selective transforming growth factor-␤ (TGF␤)-mediated EMT that leads to cellularization of developing cardiac valvular primordia. Embryos that lack Notch signaling elements exhibit severely attenuated cardiac snail expression, abnormal maintenance of intercellular endocardial adhesion complexes, and abortive endocardial EMT in vivo and in vitro. Accordingly, transient ectopic expression of activated Notch1 (N1IC) in zebrafish embryos leads to hypercellular cardiac valves, whereas Notch inhibition prevents valve development. Overexpression of N1IC in immortalized endothelial cells in vitro induces EMT accompanied by oncogenic transformation, with corresponding induction of snail and repression of VE-cadherin expression. Notch is expressed in embryonic regions where EMT occurs, suggesting an intimate and fundamental role for Notch, which may be reactivated during tumor metastasis.[Keywords: Notch; endocardium; lateral induction; EMT; snail; TGF␤] Supplemental material is available at http://www.genesdev.org. Epithelial-to-mesenchymal transition (EMT) is fundamental to both normal development and the progression of malignant epithelial tumors (for review, see Thiery 2002). During EMT, epithelial cells undergo sweeping alterations in gene expression to lose apical/basolateral polarity, sever intercellular adhesive junctions, degrade basement membrane components, and become migratory. Several signaling pathways seem to be common to EMT regulation during both development and tumor progression, leading to the notion that developmentally regulated EMT and tumor metastasis are under the control of common molecular mechanisms (Thiery 2002), and raising the hypothesis that tumor metastasis could be regarded as a reactivation of at least some aspects of the embryonic program of EMT.The snail family of Zinc-finger-containing transcriptional repressors is believed to play a pivotal role in the process of EMT (Nieto 2002). Expression of various snail family members has been tightly associated with cells undergoing both metastatic and developmental EMT (Nieto et al. 1992;Romano and Runyan 2000). One important target of Snail repression is the E-cadherin gene, the primary cadherin that is responsible for homotypic adhesion between members of an epithelial sheet (Batlle et al. 2000;Cano et al. 2000).A classical example of developmentally regulated EMT occurs during the initial stages of cardiac morphogenesis. At embryonic day 8.5 (E8.5...

Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. We show that ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. We suggest that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease.

Notch is an evolutionarily conserved local cell signaling mechanism that participates in a variety of cellular processes: cell fate specification, differentiation, proliferation, apoptosis, adhesion, epithelial-mesenchymal transition, migration, and angiogenesis. These processes can be subverted in Notch-mediated pathological situations. In the first part of this review, we will discuss the role of Notch in vertebrate central nervous system development, somitogenesis, cardiovascular and endocrine development, with attention to the mechanisms by which Notch regulates cell fate specification and patterning in these tissues. In the second part, we will review the molecular aspects of Notch-mediated neoplasias, where Notch can act as an oncogene or as a tumor suppressor. From all these studies, it becomes evident that the outcome of Notch signaling is strictly context-dependent and differences in the strength, timing, cell type, and context of the signal may affect the final outcome. It is essential to understand how Notch integrates inputs from other signaling pathways and how specificity is achieved, because this knowledge may be relevant for future therapeutic applications.

Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-β2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3β, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification.

Venous malformations (VM) are vascular malformations characterized by enlarged and distorted blood vessel channels. VM grow over time and cause substantial morbidity because of disfigurement, bleeding, and pain, representing a clinical challenge in the absence of effective treatments (Nguyen et al., 2014; Uebelhoer et al., 2012). Somatic mutations may act as drivers of these lesions, as suggested by the identification of TEK mutations in a proportion of VM (Limaye et al., 2009). We report that activating PIK3CA mutations gives rise to sporadic VM in mice, which closely resemble the histology of the human disease. Furthermore, we identified mutations in PIK3CA and related genes of the PI3K (phosphatidylinositol 3-kinase)/AKT pathway in about 30% of human VM that lack TEK alterations. PIK3CA mutations promote downstream signaling and proliferation in endothelial cells and impair normal vasculogenesis in embryonic development. We successfully treated VM in mouse models using pharmacological inhibitors of PI3Kα administered either systemically or topically. This study elucidates the etiology of a proportion of VM and proposes a therapeutic approach for this disease.

Left-sided expression of Nodal in the lateral plate meso-derm is a conserved feature necessary for the establishment of normal left-right asymmetry during vertebrate embryogenesis. By using gain-and loss-of-function experiments in zebrafish and mouse, we show that the activity of the Notch pathway is necessary and sufficient for Nodal expression around the node, and for proper left-right determination. We identify Notch-responsive elements in the Nodal promoter, and unveil a direct relationship between Notch activity and Nodal expression around the node. Our findings provide evidence for a mechanism involving Notch activity that translates an initial symmetry-breaking event into asymmetric gene expression.

Signaling through Notch receptors, which regulate cell fate decisions and embryonic patterning, requires ligand-induced receptor cleavage to generate the signaling active Notch intracellular domain (NICD). Here, we show an analysis at specific developmental stages of the distribution of active mouse Notch1. We use an antibody that recognizes N1ICD, and a highly sensitive staining technique. The earliest N1ICD expression was observed in the mesoderm and developing heart, where we detected expression in nascent endocardium, presumptive cardiac valves, and ventricular and atrial endocardium. During segmentation, N1ICD was restricted to the presomitic mesoderm. N1ICD expression was also evident in arterial endothelium, and in kidney and endodermal derivatives such as pancreas and thymus. Ectodermal N1ICD expression was found in central nervous system and sensory placodes. We found that Notch1 transcription and activity was severely reduced in zebrafish and mouse Notch pathway mutants, suggesting that vertebrate Notch1 expression is regulated by a positive feedback loop. Developmental Dynamics 236: 2594 -2614, 2007.

EMT epithelial-mesenchyme transition NIC Notch intracellular domain TGFβ transforming growth factor beta ACKNOWLEDGEMENTS We would like to thank F.J. Díaz-Benjumea, A. García de Herreros, S. Mañes and J.M. Pérez-Pomares for critical reading of the manuscript and and C. Mark for editorial assistance. The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research (CSIC) and by Pfizer.