Cellular dynamics of EMT: lessons from live in vivo imaging of embryonic development

Abstract

Epithelial-mesenchymal transition (EMT) refers to a process in which epithelial cells lose apical-basal polarity and loosen cell-cell junctions to take on mesenchymal cell morphologies and invasive properties that facilitate migration through extracellular matrix. EMT-and the reverse mesenchymal-epithelial transition (MET)-are evolutionarily conserved processes that are used throughout embryonic development to drive tissue morphogenesis. During adult life, EMT is activated to close wounds after injury, but also can be used by cancers to promote metastasis. EMT is controlled by several mechanisms that depend on context. In response to cell-cell signaling and/or interactions with the local environment, cells undergoing EMT make rapid changes in kinase and adaptor proteins, adhesion and extracellular matrix molecules, and gene expression. Many of these changes modulate localization, activity, or expression of cytoskeletal proteins that mediate cell shape changes and cell motility. Since cellular changes during EMT are highly dynamic and context-dependent, it is ideal to analyze this process in situ in living organisms. Embryonic development of model organisms is amenable to live time-lapse microscopy, which provides an opportunity to watch EMT as it happens. Here, with a focus on functions of the actin cytoskeleton, I review recent examples of how live in vivo imaging of embryonic development has led to new insights into mechanisms of EMT. At the same time, I highlight specific developmental processes in model embryos-gastrulation in fly and mouse embryos, and neural crest cell development in zebrafish and frog embryos-that provide in vivo platforms for visualizing cellular dynamics during EMT. In addition, I introduce Kupffer's vesicle in the zebrafish embryo as a new model system to investigate EMT and MET. I discuss how these systems have provided insights into the dynamics of adherens junction remodeling, planar cell polarity signaling, cadherin functions, and cytoskeletal organization during EMT, which are not only important for understanding development, but also cancer progression. These findings shed light on mechanisms of actin cytoskeletal dynamics during EMT, and feature live in vivo imaging strategies that can be exploited in future work to identify new mechanisms of EMT and MET. Video Abstract.

Keywords: Actin cytoskeleton; Cancer metastasis; Cell migration; Embryonic development; Epithelial-mesenchymal transition (EMT); Gastrulation; In vivo live imaging; Kupffer’s vesicle; Mesenchymal-epithelial transition (MET); Neural crest cell development.

Fig. 1

Overview of EMT and MET transitions. Epithelial-mesenchymal transition (EMT) is a dynamic process in which cells turn on EMT-promoting transcription factors (EMT-TFs), disassemble cell–cell junctions, lose apical-basal polarity, and upregulate new cadherins. Cells also undergo extensive rearrangements of actin cytoskeleton that mediate shape changes, front-rear polarity, invasive behavior, and migration. In many cases in vivo, EMT is partial and cells have both epithelial and mesenchymal properties. A mesenchymal-epithelial transition (MET) is the reverse process

Fig. 2

Actomyosin contractility and FGF signaling regulate adherens junction dynamics during Drosophila gastrulation. A During apical constriction of ventral mesoderm cells, increased contraction of the actin-myosin cytoskeleton mediates remodeling of adherens junction (AJ) complexes from sub-apical to apical positions. The polarity protein Par3 localizes to remodeled AJs, and Myosin II-generated tension protects AJs from Snail-mediated disassembly [32]. FGF receptor (Heartless) localizes with apical AJs, and FGF signaling modulates AJ number and cell division (not shown) during gastrulation and EMT [42]. B As Myosin II levels decrease, Snail activity leads to Par3 downregulation and AJ disassembly to promote EMT [32, 37]

Fig. 3

Planar cell polarity protein Prickle 1 mediates the transition of neural crest cells to a mesenchymal morphology. A Wild-type pre-migratory neural crest cells (NCCs) in the zebrafish neuroepithelium undergo EMT behaviors that include detachment from the apical surface, cell rounding and membrane blebbing at the basal surface, and a transition to protrusive mesenchymal morphology for lateral migration. B In embryos with a mutation in the core PCP Prickle 1 genes pk1a or pk1b, NCCs cluster and undergo abnormal anterior migration along the neural tube [55]. These mutant NCCs bleb for an extended period and largely fail to transition to mesenchymal morphology. Some NCCs that do become mesenchymal and migratory fail to separate from adjacent NCCs. Knockdown of Pk1b results in an increase of E-cadherin and a decrease of N-cadherin in migratory NCCs as compared to wild-type

Fig. 4

Development of the zebrafish Kupffer’s vesicle as a model to investigate mechanisms that control EMT and MET. A Schematic of Kupffer’s vesicle development in zebrafish. Boxes on embryo diagrams on the left indicate location of cells depicted on the right. At 6 h post-fertilization (hpf), precursor cells called dorsal forerunner cells (DFCs) are specified at the dorsal margin. Mesenchymal DFCs migrate and then form a rosette structure at 10 hpf. DFCs undergo MET to form the epithelial Kupffer’s vesicle (KV). A cross section through the KV depicts epithelial cells lining a fluid-filled lumen at 12 hpf. After KV functions to establish the left–right body axis, KV collapses at 18 hpf and the epithelial cells undergo EMT and migrate away. B Confocal microscopy images of live transgenic Tg(sox17:GFP-CAAX) embryos that express membrane-targeted GFP in DFC and KV cells at developmental stages corresponding to diagrams in A. The planar cell polarity (PCP) proteins Prickle 1a (Pk1a) and Wnt11 regulate cell–cell adhesion between DFCs during MET and rosette formation [67]. Mechanisms that control KV breakdown and EMT of KV cells remain unknown

Fig. 5

Cadherin 6 regulates active Rho GTPase distribution, F-actin accumulation, and apical detachment in zebrafish neural crest cells during EMT. A Wild-type zebrafish pre-migratory neural crest cells (NCCs) show an apical accumulation of active Rho GTPases and filamentous actin (F-actin), which are necessary for actomyosin-mediated apical detachment and subsequent lateral migration during EMT. B In most Cadherin 6 knockdown NCCs, F-actin fails to accumulate in the apical tail, active Rho is more widely distributed, the apical tail does not detach, and the cells do not undergo EMT or initiate migration [85]

Fig. 6

Crumbs2 mediates adherens junction disassembly and apical detachment of epiblast cells as they undergo EMT during gastrulation in the mouse embryo. A During gastrulation, wild-type epiblast cells near the primitive streak undergo EMT that involves basement membrane breakdown, apical constriction and basal positioning of the nucleus, and loss of E-cadherin containing adherens junctions (AJs). EMT correlates with downregulation of the transcription factor (TF) Sox2 and upregulation of the EMT-TF Snail1. Cells that successfully undergo EMT detach from the epithelium and migrate into the primitive streak in a process called ingression. B In Crumbs2 mutant (Crb2-/-) embryos, mosiacally labeled epiblast cells undergo apical constriction and basal nuclear displacement, but fail to detach from the epithelium [91]. Crb2-/- cells develop an elongated morphology and remain attached to the epithelium via E-cadherin. Apical Myosin II accumulation is reduced in Crb2-/- epiblast relative to wild-type (not shown), suggesting a link between Crb2 and actomyosin activity during EMT [91]