Ptk7 Marks the First Human Developmental EMT In Vitro

Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development, but these events are essentially impossible to study at the molecular level in vivo in humans. The first EMT event that has been described morphologically in human development occurs just prior to generation of the primitive streak. Because human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are thought to most closely resemble cells found in epiblast-stage embryos prior to formation of the primitive streak, we sought to determine whether this first human EMT could be modeled in vitro with pluripotent stem cells. The data presented here suggest that generating embryoid bodies from hESCs or hiPSCs drives a procession of EMT events that can be observed within 24–48 hours after EB generation. These structures possess the typical hallmarks of developmental EMTs, and portions also display evidence of primitive streak and mesendoderm. We identify PTK7 as a novel marker of this EMT population, which can also be used to purify these cells for subsequent analyses and identification of novel markers of human development. Gene expression analysis indicated an upregulation of EMT markers and ECM proteins in the PTK7+ population. We also find that cells that undergo this developmental EMT retain developmental plasticity as sorting, dissociation and re-plating reestablishes an epithelial phenotype.     

Organoids and the genetically encoded self‐assembly of embryonic stem cells

Understanding the mechanisms of early embryonic patterning and the timely allocation of specific cells to embryonic regions and fates as well as their development into tissues and organs, is a fundamental problem in Developmental Biology. The classical explanation for this process had been built around the notion of positional information. Accordingly the programmed appearance of sources of Morphogens at localized positions within a field of cells directs their differentiation. Recently, the development of organs and tissues from unpatterned and initially identical stem cells (adult and embryonic) has challenged the need for positional information and even the integrity of the embryo, for pattern formation. Here we review the emerging area of organoid biology from the perspective of Developmental Biology. We argue that the events underlying the development of these systems are not purely linked to “self‐organization,” as often suggested, but rather to a process of genetically encoded self‐assembly where genetic programs encode and control the emergence of biological structures.     

Epithelial-Mesenchymal Transition in Cancer: A Historical Overview

Epithelial-mesenchymal transitions (EMTs), the acquisition of mesenchymal features from epithelial cells, occur during some biological processes and are classified into three types: the first type occurs during embryonic development, the second type is associated with adult tissue regeneration, and the third type occurs in cancer progression. EMT occurring during embryonic development in gastrulation, renal development, and the origin and fate of the neural crest is a highly regulated process, while EMT occurring during tumor progression is highly deregulated. EMT allows the solid tumors to become more malignant, increasing their invasiveness and metastatic activity. Secondary tumors frequently maintain the typical histologic characteristics of the primary tumor. These histologic features connecting the secondary metastatic tumors to the primary is due to a process called mesenchymal-epithelial transition (MET). MET has been demonstrated in different mesenchymal tumors and is the expression of the reversibility of EMT. EMT modulation could constitute an approach to avoid metastasis. Some of the targeted small molecules utilized as antiproliferative agents have revealed to inhibit EMT initiation or maintenance because EMT is regulated through signaling pathways for which these molecules have been designed.     

Ingression of primary mesenchyme cells of the sea urchin embryo: a precisely timed epithelial mesenchymal transition

Epithelial-mesenchyme transitions (EMTs) are familiar to all scholars of development. Each animal system utilizes an EMT to produce mesenchyme cells. In vertebrates, for example, there are a number of EMTs that shape the embryo. Early, entry of epiblast cells into the primitive streak is followed by the emergence of mesoderm via an EMT process. The departure of neural crest cells from the margin of the neural folds is an EMT process, and the delamination of cells from the endomesoderm to form the supporting mesenchyme of the lung, liver, and pancreas are EMTs. EMTs are observed in Drosophila following invagination of the ventral furrow, and even in Cnidarians, which have only two germ layers, yet mesoglial and stem cells delaminate from the epithelia and occupy the matrix between the ectoderm and endoderm. This review will focus on a classic example of an EMT, which occurs in the sea urchin embryo. The primary mesenchyme cells (PMCs) ingress from the vegetal plate of this embryo precociously and in advance of archenteron invagination. Because ingression is precisely timed, the PMC lineage precisely known, and the embryo easily observed and manipulated, much has been learned about how the ingression of PMCs works in the sea urchin. Though the focus of this review is the sea urchin PMCs, there is evidence that all EMTs share many common features at both cellular and molecular levels, and many of these mechanisms are also shown to be involved in tumor progression, especially metastasizing carcinomas.     

Degradation of type IV collagen by matrix metalloproteinases is an important step in the epithelial-mesenchymal transformation of the endocardial cushions

Morphogenesis of some tissues and organs in the developing embryo requires the transformation of epithelial cells into mesenchyme followed by cell motility and invasion of surrounding connective tissues. Details of the mechanisms involved in this important process are beginning to be elucidated. The epithelial-mesenchymal transformation (EMT) process involves many steps, one of which is the upregulation and activation of specific extracellular proteinases including members of the matrix metalloproteinase (MMP) family. Here we analyze the role of MMPs in the initiation of the mesenchymal cell phenotype in the developing heart, and find that they are necessary for the invasion of mesenchymal cells into the extracellular matrix of the endocardial cushion tissues. An important requirement in the formation of this mesenchyme is the turnover of type IV collagen along the basal surface of endocardial cells. In vitro experiments suggest that type IV collagen does not provide a suitable migratory substrate for endocardial cushion cells unless MMP-2 and MT-MMP are active. Relevant MMPs were found to be upregulated by factors known to be involved in the induction of the EMT such as TGFbeta3. These results provide evidence of an important role for MMPs during a specific stage of the epithelial mesenchymal transformation in the embryonic heart, and suggest that specific cell-matrix interactions which facilitate cell migration only occur when the composition of the surrounding extracellular matrix is proteolytically altered.     

The role of EMT in renal fibrosis

It is clear that the well-described phenomenon of epithelial–mesenchymal transition (EMT) plays a pivotal role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer progression. EMTs have been classified into three subtypes based on the functional consequences and biomarker context in which they are encountered. This review will highlight findings on type II EMT as a direct contributor to the kidney myofibroblast population in the development of renal fibrosis, specifically in diabetic nephropathy, the signalling molecules and the pathways involved in type II EMT and changes in the expression of specific miRNA with the EMT process. These findings have provided new insights into the activation and development of EMT during disease processes and may lead to possible therapeutic interventions to suppress EMTs and potentially reverse organ fibrosis.     

Transforming growth factor beta 1 is an epithelial-derived signal peptide that influences otic capsule formation.

Interactions between epithelial and mesenchymal tissues in the developing inner ear direct the formation of its cartilaginous capsule. Recent work indicates that many growth factors are distributed in the early embryo in vivo in a temporal-spatial pattern that correlates with sites of ongoing morphogenetic events. We report here that the localization of transforming growth factor beta 1 (TGF-beta 1) in both epithelial and mesenchymal tissues of the mouse inner ear between 10 and 16 days of embryonic development (E10-E16). In addition, utilizing a high-density culture system as an in vitro model of otic capsule chondrogenesis, we show that modulation of chondrogenesis by TGF-beta 1 in cultured mouse periotic mesenchyme mimics the in vitro effects of otic epithelium on the expression of chondrogenic potential. We provide evidence of a causal relationship of this growth factor to otic capsule formation in situ by demonstrating that the actual sequence of chondrogenic events that occur in the developing embryo is reproduced in culture by the addition of exogenous TGF-beta 1 peptide. Furthermore, in cultures of mesenchyme containing otic epithelium, we demonstrate the localization of endogenous TGF-beta 1, first within the epithelial tissue and later within both the epithelium and its surrounding periotic mesenchyme, contrasted to an absence of endogenous TGF-beta 1 in cultures of mesenchyme alone. Our results suggest that TGF-beta 1 is one of the signal molecules that mediate the effects of otic epithelium in influencing the formation of the cartilaginous otic capsule.     

Transitions between epithelial and mesenchymal states in development and disease

The ancestors of modern Metazoa were constructed in large part by the foldings and distortions of two-dimensional sheets of epithelial cells. This changed approximately 600 million years ago with the evolution of mesenchymal cells. These cells arise as the result of epithelial cell delamination through a reprogramming process called an epithelial to mesenchymal transition (EMT) [Shook D, Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev 2003;120:1351-83; Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131-42]. Because mesenchymal cells are free to migrate through the body cavity, the evolution of the mesenchyme opened up new avenues for morphological plasticity, as cells evolved the ability to take up new positions within the embryo and to participate in novel cell-cell interactions; forming new types of internal tissues and organs such as muscle and bone [Thiery JP, Sleeman, JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131-42; Hay ED, Zuk A. Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J Kidney Dis 1995;26:678-90]. After migrating to a suitable site, mesenchymal cells coalesce and re-polarize to form secondary epithelia, in a so-called mesenchymal-epithelial transition (MET). Such switches between mesenchymal and epithelial states are a frequent feature of Metazoan gastrulation [Hay ED, Zuk A. Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J Kidney Dis 1995;26:678-90] and the neural crest lineage [Duband JL, Monier F, Delannet M, Newgreen D. Epitheliu-mmesenchyme transition during neural crest development. Acta Anat 1995;154:63-78]. Significantly, however, when hijacked during the development of cancer, the ability of cells to undergo EMT, to leave the primary tumor and to undergo MET at secondary sites can have devastating consequences on the organism, allowing tumor cells derived from epithelia to invade surrounding tissues and spread through the host [Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131-42; Hay ED, Zuk A. Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J Kidney Dis 1995;26:678-90]. Thus, the molecular and cellular mechanisms underpinning EMT are both an essential feature of Metazoan development and an important area of biomedical research. In this review, we discuss the common molecular and cellular mechanisms involved in EMT in both cases.     

Src activation is not necessary for transforming growth factor (TGF)-beta-mediated epithelial to mesenchymal transitions (EMT) in mammary epithelial cells. PP1 directly inhibits TGF-beta receptors I and II

Epithelial to mesenchymal transitions (EMTs) are key events during embryonic development and cancer progression. It has been proposed that Src plays a major role in some EMT models, as shown by the overexpression of viral Src (v-Src) in epithelial cells. It is clear that Src family kinases can regulate the integrity of both adherens junctions and focal adhesions; however, their significance in EMT, especially in the physiological context, remains to be elucidated. Here we showed that Src is activated in transforming growth factor-beta1 (TGF-beta1)-mediated EMT in mammary epithelial cells and that the Src family kinase inhibitor, PP1, prevents EMT. However, neither a more specific Src family kinase inhibitor, SU6656, nor a dominant-negative Src inhibited TGF-beta1-mediated EMT, leading us to speculate that Src activation is not an essential component of TGF-beta1-mediated EMT. Unexpectedly, PP1 prevented Smad2/3 activation by TGF-beta1, whereas SU6656 did not. Most interestingly, an in vitro kinase assay showed that PP1 strongly inhibited the TGF-beta receptor type I, and to a lesser extent, the TGF-beta receptor type II. Taken together, our data indicated that PP1 interferes with TGF-beta1-mediated EMT not by inhibiting Src family kinases but by inhibiting the Smad pathway via a direct inhibition of TGF-beta receptor kinase activity.     

Development of the vertebrate small intestine and mechanisms of cell differentiation

The intestinal epithelium represents an attractive biological model of differentiation from stem cells to highly differentiated epithelial cells, not only during particular developmental events depending upon the vertebrate species considered but also throughout adult life. The ontogenic maturation of the intestinal epithelium arises from both a programmed expression of specific genes and epigenetic influences mainly due to epithelial and mesenchymal interactions and hormonal participation. In the present paper we review the structural and functional changes that occur in the amphibian, avian and mammalian intestine during embryonic and/or post-embryonic development. Furthermore, we review the data concerning the mechanisms which control the cytodifferentiation of the intestinal epithelium.     

The existence of multipotent stem cells with epithelial-mesenchymal transition features in the human liver bud

During early stage of embryonic development, the liver bud, arising from the foregut endoderm, is the beginning for the formation of future liver three-dimensional structure. While the gene expression profiles associated with this developmental stage have been well explored, the detailed cellular events are not as clear. Epithelial-mesenchymal transition (EMT) was thought to be essential for cell migration in the early vertebrate embryo but seldom demonstrated in human liver development. In this study, we tried to identify the cell populations with both stem cell and EMT features in the human liver bud. Our in situ studies show that the phenotype of EMT occurs at initiation of human liver development, accompanied by up-regulation of EMT associated genes. A human liver bud derived stem cell line (hLBSC) was established, which expressed not only genes specific to both mesenchymal cells and hepatic cells, but also octamer-binding protein 4 (OCT4) and nanog. Placed in appropriate media, hLBSC differentiated into hepatocytes, adipocytes, osteoblast-like cells and neuron-like cells in vitro. When transplanted into severe combined immunodeficiency mice pre-treated by carbon tetrachloride, hLBSC engrafted into the liver parenchyma and proliferated. These data suggests that there are cell populations with stem cell and EMT-like properties in the human liver bud, which may play an important role in the beginning of the spatial structure construction of the liver.     

Transitions between epithelial and mesenchymal states and the morphogenesis of the early mouse embryo

Multicellular organisms arise from the generation of different cell types and the organization of cells into tissues and organs. Cells of metazoa display two main phenotypes, the ancestral epithelial state and the recent mesenchymal derivative. Epithelial cells are usually stationary and reside in twodimensional sheets. By contrast mesenchymal cells are loosely packed and can move to new positions, thereby providing a vehicle for cell rearrangement, dispersal and novel cell-cell interactions. Transitions between epithelial and mesenchymal states drive key morphogenetic events in the early vertebrate embryo, including gastrulation, germ layer formation and somitogenesis. The cell behaviors and molecular mechanisms promoting transitions between these two states in the early mouse embryo are discussed in this review.Key words: mouse embryo, EMT, MET, morphogenesis, gastrulation, somitogenesis, epiblast, mesoderm, endoderm, primitive streak, paraxial mesoderm     

Cadherin-6B stimulates an epithelial mesenchymal transition and the delamination of cells from the neural ectoderm via LIMK/cofilin mediated non-canonical BMP receptor signaling

We previously provided evidence that cadherin-6B induces de-epithelialization of the neural crest prior to delamination and is required for the overall epithelial mesenchymal transition (EMT). Furthermore, de-epithelialization induced by cadherin-6B was found to be mediated by BMP receptor signaling independent of BMP. We now find that de-epithelialization is mediated by non-canonical BMP signaling through the BMP type II receptor (BMPRII) and not by canonical Smad dependent signaling through BMP Type I receptor. The LIM kinase/cofilin pathway mediates non-canonical BMPRII induced de-epithelialization, in response to either cadherin-6B or BMP. LIMK1 induces de-epithelialization in the neural tube and dominant negative LIMK1 decreases de-epithelialization induced by either cadherin-6B or BMP. Cofilin is the major known LIMK1 target and a S3A phosphorylation deficient mutated cofilin inhibits de-epithelialization induced by cadherin-6B as well as LIMK1. Importantly, LIMK1 as well as cadherin-6B can trigger ectopic delamination when co-expressed with the competence factor SOX9, showing that this cadherin-6B stimulated signaling pathway can mediate the full EMT in the appropriate context. These findings suggest that the de-epithelialization step of the neural crest EMT by cadherin-6B/BMPRII involves regulation of actin dynamics via LIMK/cofilin.