Epithelial-to-mesenchymal transition in development Tomáš Bárta tbarta@med.muni.cz Duševní vlastnictví a poskytované studijní materiály Copyright notice ̶ Tato prezentace je autorským dílem vytvořeným zaměstnanci Masarykovy univerzity. ̶ Studenti kurzu/předmětu mají právo pořídit si kopii prezentace pro potřeby vlastního studia. ̶ Jakékoliv další šíření prezentace nebo její části bez svolení Masarykovy univerzity je v rozporu se zákonem. ̶ The presentation is copyrighted work created by employees of Masaryk university. ̶ Students are allowed to make copies for learning purposes only ̶ Any unauthorised reproduction or distribution of the presentation or individual slides is against the law. 2 Outline * Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) * Mechanis * EMT and MET in development * BONUS: The role of EMT in cancer metastasis Link to the previous lecture Are all cells in differentiated status? NO! Terminally differentiated epithelial cells are capable to change their phenotype using an activation of EMT „programme“ that allows to switch epithelial cell into a mesenchymal cell during development and adulthood. The previous concept that terminally differentiated cells just execute their function(s) and they are more or less „static“ is not valid anymore. •Series of events leading to transformation of mesenchymal cells into epithelial cells. Epithelial-to-mesenchymal transition Mesenchymal-to-epithelial transition EMT EMT vs MET MET •A polarized, stationary epithelial cell, which interacts with a basal membrane, becomes a mesenchymal cell with an increased migration potential that is capable to invade tissues. •During embryogenesis this transformation is critical for organ formation. Obsah obrázku jídlo, závěs, štětec Popis byl vytvořen automaticky Basal membrane Apical Basal •Series of events that lead to transformation of epihelial cells into mesenchymal cells Obsah obrázku jídlo, závěs, štětec Popis byl vytvořen automaticky EMT MET •Polygonal/column cell shape •Apico-basal polarisation •Strong cell-to-cell interction •Migration potential is limited •Markers (expressed genes): •E-cadherin, Cytokeratins, Occludin, Claudin •Spindle-shaped cell morphology •Anterior-posterior polarization •Focal interaction between cells •Strong migration potential •Markers (expressed genes): •N-cadherin, Vimentin, Fibronectin Typically, epithelial cells execute some function(s) in a tissue, while mesenchymal cells have rather supportive function. EMT These are mouse embryonic stem cells expressing a fluorescent reporter of Wnt/Beta-Catenin activity. As they are differentiated towards mesoderm, there is a huge increase in reporter activity and cells begin the process of an epithelial to mesenchymal transition (EMT). MET EMT vs MET – sum up •You must be able to discrimine epithelial vs mesenchymal cells •to describe EMT and MET and know the difference between them EMT vs MET – questions? EMT - mechanism There is no „master regulator“ of EMT or MET! EMT - mechanism •Downregulation of Cadherins expression •Complete rebuilding/reorganization of cytoskeletal actin •Production of enzymes that are capable to degrade the basal membrane •Cell proliferation EMT - mechanism Remember 5 major pathways EMT - mechanism Where/When the EMT take place: EMT in •Embryonic development •Cancer (metastasis) •Inflamation and fibrosis • EMT – in embryonic development •Embryo implantation •Embryogenesis •Organ development •Regeneration and homeostasis maintenance •During the development some epithelial cells are more „plastic“ – are capable of transition epithel <–> mesenchym using EMT and/or MET processes. EMT – in development Key role of EMT in development – it would not be possible without it •Embryo implantation •Gastrulation and mesoderm generation •Neural crest formation •Formation of vertebrae We will explain using 4 examples: EMT – in development Key role of EMT in development – it would not be possible without it •Embryo implantation •Gastrulation and mesoderm generation •Neural crest formation •Formation of vertebrae We will explain using 4 examples: EMT – implantation of embryo EMT – in development Key role of EMT in development – it would not be possible without it •Embryo implantation •Gastrulation and mesoderm generation •Neural crest formation •Formation of vertebrae We will explain using 4 examples: EMT – during gastrulation Gastrulation: •Process of transition from blastula/blastocyst to gastrula •Before the gastrulation an embryo is just a layer of epithelial cells •Individual layers of the gastrula are transformed into germ layers ecto-, endo-, and mesoderm -> therefore EMT is critical here •Before the gastrulation an embryo fully rely on maternal mRNA, only after the gastrulation is capable of synthetize own mRNA Obsah obrázku semafor Popis byl vytvořen automaticky EMT – during gastrulation Obsah obrázku text, interiér Popis byl vytvořen automaticky There are 5 major kinds of the gastrulation process: Invagination – invagination of a group of cells Involution – involution of the outer cell layer, so it covers cell layer beneath it. Ingression – migration of cells into the inner part Delamination – division of a cell layer into two paraler cell layers Epiboly – the outer epithelial layer overgrow the prospective endoderm EMT – during gastrulation EMT – during gastrulation EMT – during gastrulation Skeletogenic mesenchyme cells breaking through extracellular matrix. The matrix laminin is stained pink, the mesenchyme cells are green and cell nuclei are blue. (B) Laminin matrix is uniformly spread throughout the lining of the blastocoel. (C) Hole is made in blastocoel laminin above the vegetal cells, and the mesenchyme begins to pass through it into the blastocoel. (D) Within an hour, cells are in the blastocoel. (E) Scanning electron micrograph of skeletogenic mesenchyme cells enmeshed in the extracellular matrix of an early Strongylocentrotus gastrula. (F) Gastrula-stage mesenchyme cell migration. The extracellular matrix fibrils of the blastocoel lie parallel to the animal-vegetal axis and are intimately associated with the skeletogenic mesenchyme cells. EMT – during gastrulation Regulated by canonical Wnt – Wnt3, TGF-β (Nodal, Vg1) a FGF Momose, Development EMT – during gastrulation Rescue of the phenotype by injecting Wnt3-mRNA EMT – during gastrulation FGF Ciruna, 2001 Mesoderm is not formed – failure of EMT (accumulation of E-cad positive cells) EMT – in development Key role of EMT in development – it would not be possible without it •Embryo implantation •Gastrulation and mesoderm generation •Neural crest formation •Formation of vertebrae We will explain using 4 examples: •EMT – Neural crest formation •„Fourth germ layer“ - “the only interesting thing about vertebrates is the neural crest” (Thorogood 1989) •Ectodermal origin •Transient – is absent after embryo development •Is generated from the neural tube by EMT. Cells are migrating alongside the anterior-posterior axis and are differentiating (changes of the cell environment leads to the generation of different cell types). What is the neural crest? •EMT – Neural crest formation •EMT – Neural crest formation •EMT – Neural crest formation Prospective neural crest cells are loosing adhesive junctions and are released from epithel – this process is called delamination •EMT – Neural crest formation Neural crest delamination and migration by contact inhibition. The process of neural crest delamination is shown here at the time when the neural and surface ectoderms have separated and are both in the process of fusing at the midline into the neural tube and epidermis respectively. BMP and Wnt signals specify the three major regions of the neuroepithelium, which are distinguished by their expression of unique adhesion proteins: surface ectoderm (E-cadherin), neural tube (N-cadherin), and the premigratory neural crest (cadherin-6B). In the premigratory domain, BMP levels are the highest, with Wnt at intermediate amounts; this situation supports the upregulation of Snail-2 (and Zeb-2) in these cells. Snail-2 proteins repress N-cadherin and E-cadherin in this domain. Cadherin-6B is upregulated only in the apical half of premigratory neural crest cells, and functions to activate RhoA and actomyosin contractile fibers for apical constriction and the initiation of delamination. Noncanonical Wnt signaling (not shown) establishes the polar activity of RhoA (red) and Rac1 (yellow) along the migratory axis of migrating neural crest cells. When neural crest cells contact one another, they experience contact inhibition, during which they will stop, turn, and migrate away in the opposite direction. •EMT – Neural crest formation •EMT – Neural crest formation EMT – in development Key role of EMT in development – it would not be possible without it •Embryo implantation •Gastrulation and mesoderm generation •Neural crest formation •Formation of vertebrae We will explain using 4 examples: EMT – formation of vertebrae from somites Somites are epthelial blocks (clusters) of cells that are localized in a close proximity of the neural tube •Body segmentation - vertebrae Somites and body segmentation „How can a tissue be developmentally cut up into precisely sized segments? How can snakes have some 300 segments while humans have only about 35?“ – Gilbert and Barresi EMT – formation of vertebrae from somites Three neural tubes: loss of the Tbx6 gene transforms the paraxial mesoderm into neural tubes. In situ hybridization for the mRNA expression (blue) of the neural specification markers Sox2 and Pax6 in wildtype mice (A) and Tbx6 knockout mice (B). Sox2 is ectopically expressed throughout the presumptive paraxial mesoderm in the Tbx6–/– embryo, which has also taken on a neural tube-like morphology, even displaying a central lumen (arrows). Similarly, the dorsal neural tube marker Pax6 shows regional cell specification within these ectopic neural tubes (arrowheads). (From Takemoto et al. 2011.) MET MET – during development – formation of somites •Archtecture of somites is formed by epithelial blocks, but presomitic (paraxial) mesoderm is formed by mesenchymal cells. •Therefore, mesenchymal cells must be transformed into epithelial cells => MET Mesp (Mesodermal posterior) MET – during development – formation of somites Eph-Ephrin signaling regulates epithelialization during somite boundary formation. Expression of (A) Mesodermal posterior-a (Mesp-a; dark purple) and (B) Eph-A4 (black arrows) and ephrin-B2 (red arrows) in the paraxial mesoderm of zebrafish embryos (dorsal views). (C) Model of Mesp-a and Eph-Ephrin signaling fostering mesenchymal-to-epithelial transitions that define the apposing cells of a somite boundary. Mesp-a becomes restricted to the anterior half of the S-I somitomere, which upregulates Eph-A4 within this domain. In turn, Eph-A4 upregulates its binding partner ephrin-B2 in the cells of the presumptive posterior S-0 somitomere, which triggers epithelialization and formation of a boundary. Fissure formation is facilitated by repression of Cdc42 and activation of integrin α5-fibronectin interactions downstream of ephrin-B2. MET – during development – formation of somites Link to the previous lecture: Do you rember blastoids? Liu, 2021, Nature There are gastruloids as well! Van der Brink, 2021 Stainings and time-lapse imaging of somite formation in gastruloids Van der Brink, Nature 2020 Another example of MET – Development of nephrons •Cells of mesenchymal origin are able to differentiate into progenitor cells of a nefron. •They are responsive to Wnt9b and Wnt6 that is produced from uretetic bud •Wnt9b and 6 are crucial for the transformation of metanefric mesenchyme into tubular epithel •Mesenchyme has receptors for these Wnt molecules, leading to production of Wnt4 that finishes the transformation. •In the absence of Wnt5 the mesenchyme is condensed, but epithelium is not formed. Questions? EMT – inflamation and fibrosis BONUS: EMT - cancer EMT - rakovina BONUS: Embryonic origin of pituitary gland (hypophysis) BONUS: Embryonic origin of pituitary gland (hypophysis) Thank you for your attention Tomáš Bárta tbarta@med.muni.cz