Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease (original) (raw)
The signaling pathways leading to the formation of the primitive streak. The term gastrulation comes from “gastrae,” as first used to describe gut formation in sponges (“gaster” means gut in Greek). Nowadays, “gastrulation” is conventionally used to describe the formation of the three embryonic germ layers, the ectoderm, mesoderm, and endoderm, from the epiblast, the initial epithelial embryonic layer. Ectoderm forms the skin and nervous system; mesoderm forms the skeletal and cardiac muscle, among other derivatives; and endoderm forms the gut. Gastrulation is observed in all metazoans (both diploblasts and triploblasts) and is accompanied by drastic changes that are necessary for remodeling a single epithelial layer such as the epiblast into a complex three-dimensional multilayered embryo. Essentially, this process serves to situate the mesoderm and endoderm inside the embryo. The internalization process can occur through two distinct mechanisms: invagination/involution or ingression. Invagination involves the infolding of an external layer into the interior of the embryo, whereas cell ingression refers to an EMT process whereby individual superficial cells detach from the external epithelial layer and are internalized. Here, we will only refer to ingression, as it is an EMT process and the mechanism used by avian and mammalian embryos.
In chick and mouse embryos, the cells that ingress at the primitive streak receive canonical Wnt signaling activated in the posterior region of the embryo (16). Wnt signaling seems to render these cells competent to respond to other extracellular signals that initiate EMT. Indeed, the primitive streak does not form in Wnt3-deficient mice, in which EMT does not occur (17). Conversely, mouse embryos overexpressing Wnt8c develop multiple primitive streaks (18). Downstream of Wnt signaling, proteins of the TGF-β superfamily, such as Nodal and Vg1, are key inducers of gastrulation in different species (19). Ectopic Vg1 expression alone is sufficient to induce the formation of an additional primitive streak in the chick blastula (20), and more than 50% of embryos mutant for the two Vg1 mouse homologs (GDF1 and GDF3) develop mesodermal defects (21).
Nodal signaling appears to induce EMT as cells ingress during mouse gastrulation, as highlighted by the failure of Nodal mutants to complete gastrulation. This phenotype can be rescued by transplantation of a few Nodal-expressing cells from wild-type embryos, suggesting that low levels of secreted Nodal are sufficient to drive ingression (22). The lower layer of the embryo (the hypoblast in the chick and the visceral endoderm in the mouse) secretes inhibitors of Nodal, which define the correct positioning of the streak and inhibit the formation of ectopic primitive streaks (23, 24). Thus, once Wnt signaling makes the epiblast competent to gastrulate, the cooperative activity of Vg1 and Nodal induces the formation of the primitive streak and mesoderm ingression (Figure 4). Signaling through FGF receptors (FGFRs) seems to be necessary to maintain the EMT regulatory network (25, 26). Indeed, in FGFR1-deficient mice, the primitive streak and mesodermal cells initially form, but their production is arrested (25).
Similar signaling pathways control the EMTs at gastrulation and neural crest delamination in the amniote embryo. Signaling molecules of the TFG-β superfamily (Nodal, Vg1, and BMPs), together with Wnt and FGF, initiate the formation of the primitive streak and operate at the neural folds to drive the ingression of the mesendoderm and the delamination of the neural crest, respectively. Some downstream targets are also conserved, such as the Snail genes. While Snail factors are key regulators of the EMT program during gastrulation, the coordinated induction of several transcription factors is required to control the robust program of neural crest delamination. EPB4L5, FERM and actin-binding domain–containing band 4.1 superfamily member; p38IP, p38-interacting protein; Rho, members of the Rho family of small GTPases.
Transcription factors downstream of signaling events in the primitive streak. Signaling events activate transcription factors that mediate EMT. In response to signals from Wnt, TGF-β, and FGF family members, cells activate transcription factors that in turn induce EMT (Figure 4). Members of the Snail family of zinc finger transcription factors are key molecules and are expressed in the primitive streak (27). Embryos lacking Snail activity fail to gastrulate, leading to an accumulation of epithelial cells still expressing E-cadherin that are unable to migrate (28, 29). Snail genes are induced in vitro and in vivo by cytokines of the TGF-β superfamily, and FGF-initiated signaling seems necessary to maintain Snail1 expression in the mouse primitive streak (25). Despite early studies in Drosophila leading to the proposal that snail is a mesodermal determinant (30, 31), there is no evidence that this gene family is involved in mesoderm specification. For example, mesoderm specification is not altered in Snail1-deficient mice; however, those mesodermal cells that form are unable to migrate as they fail to downregulate E-cadherin expression and thus retain cell-cell contacts. This is consistent with the existence of independent pathways governing cell fate specification and EMT during gastrulation (32). It is interesting to note that depending on the cellular context, the control of cell adhesion and migration by Snail may be expressed as full EMT, such as at the primitive streak in amniotes, or as collective cell migration, as occurs during axial mesendoderm migration in zebrafish (33) and during re-epithelialization of skin wounds in mice (34).
In the mouse, other transcription factors have recently been described as important elements in the control of EMT at gastrulation (Figure 4). Conditional inactivation of eomesodermin (Eomes) in the epiblast blocks EMT and mesoderm delamination, and although these mutant mice express Snail in the primitive streak, E-cadherin mRNA is only partially downregulated. It seems that eomesodermin increases the ability of Snail to repress E-cadherin through an unknown mechanism, perhaps involving induction of transcriptional partners of Snail or the epigenetic control of the availability of Snail-binding sites (35). Other recent studies have highlighted the role of mesoderm posterior (Mesp) transcription factors in inducing EMT in a Wnt-independent manner. Differentiated ES cells overexpressing either Mesp1 or Mesp2 undergo EMT through activation of the EMT inducers Snail and Twist (36), accounting for the block of mesodermal delamination from the primitive streak previously observed in mice lacking both Mesp1 and Mesp2 (37). Interestingly, as in Snail-deficient mice, mesoderm specification is not affected in the _Mesp1–/–Mesp2_–/– mice.
One consequence of the induction of EMT by Snail is the direct repression of E-cadherin expression (38–40), which, in turn, leads to the disruption of adherens junctions. However, it is worth noting that the Snail proteins are not only E-cadherin repressors but also regulators of the epithelial phenotype (4). Snail also represses expression of genes encoding tight junction components, such as claudins and occludins (41), as well as genes important for apico-basal polarity such as Crumbs3 and Discs large (5).
Nontranscriptional regulation of E-cadherin expression. Posttranscriptional events are also important for EMT. Gastrulation is a rapid process, and some of the proteins that must be downregulated for EMT to proceed (e.g., E-cadherin) have a long half-life. Thus, the regulation of gene transcription is not sufficient for gastrulation to proceed. Recent studies have revealed that posttranscriptional regulatory mechanisms are in play (Figure 4). An _N_-ethyl _N_-nitrosourea–induced (ENU-induced) mutagenesis screen identified two mouse mutants with impaired expression of an activator of p38 MAPK, p38-interacting protein (p38IP) (42). Although these mutant embryos can specify mesoderm, the cells do not undergo EMT and do not migrate normally due to impaired downregulation of E-cadherin protein. This phenotype is very similar to those observed in FGFR1-, FGF8-, and Snail1-deficient mice (29, 43, 44), although it is independent of the FGF pathway and Snail activation. Thus, it seems that p38 MAPK and p38IP may promote the active and rapid degradation of E-cadherin protein in cells undergoing EMT (42).
The mechanism by which p38 regulates E-cadherin protein turnover is still unknown, but it may be related to the control of E-cadherin trafficking, as occurs in mice lacking ADP-ribosylation factor–related protein 1 (ARFRP1) activity. These embryos fail to gastrulate after detachment of the epiblast and subsequently undergo apoptosis (45) due to the disruption of E-cadherin trafficking through the Golgi apparatus (46).
Another mutant mouse strain also obtained from an ENU-induced mutagenesis screen (lulu mutant mice) corresponds to a null allele of the FERM domain gene erythrocyte protein band 4.1-like 5 (Epb4.1l5). lulu mutants die early during embryonic development due to gross morphological defects resulting from a failure of EMT at gastrulation (47). E-cadherin transcription is inhibited in _Epb4.1l5_–/– mice, but the protein is not downregulated, further supporting the existence of posttranslational regulation of E-cadherin expression at gastrulation. Epb4.1l5 can bind to p120 and inhibit its association with E-cadherin at adherens junctions, contributing to the gastrulation phenotype in lulu mutants (48).
A key component of epithelial junctions is β-catenin, which is part of the protein complex that connects cadherins to the actin cytoskeleton at adherens junctions. In response to exogenous signals such as those provided by Wnts, β-catenin is translocated from the cell membrane to the cytoplasm, where it is either ubiquitinated and degraded or directed to the nucleus, where it can regulate gene expression and induce EMT. Thus, the relationship between the E-cadherin and β-catenin pools is crucial for the regulation of the epithelial phenotype. Interestingly, Snail interacts with β-catenin and stimulates its transcriptional activity (49), suggesting a new level of regulation of EMT.