EMT and embryonic development

EMT and embryonic development EMT: Definition? Examples during development: Gastrulation Neural crest cell migration Cadherin switch? What is required to make epithelial cells migratory? Regulation of adhesion? Example of EpCAM

Classical view of EMT (cancer cell lines)

Classical view of EMT: genetic program

Classical view of EMT: cellular changes

Apical constriction Classical view of EMT: cellular changes What about natural developmental EMT-related processes? Cell elongation E-cadherin downregulation Apical junction disassembly Cadherin switch (E- to N-??) Lower cell-cell adhesion Basal lamina degradation Protrusions Integrin activation Cell migration

Embryonic development for dummies:

1) Gastrulation: Ectoderm outside, mesoderm and endoderm inside Ectoderm Migration, including EMT Germ layers Mesoderm Endoderm Ectoderm Mesoderm Endoderm epithelial mesenchymal epithelial

2) Patterning of the germ layers Ectoderm Neuroderm Mesoderm Notochord Endoderm

2) Secondary epithelization Mesoderm Notochord Splanchopleura Mesoderm Somatopleura Mesoderm Somites

3) Neurulation Ectoderm Neural tube

4) Formation of extensions (diverticula) Endoderm Liver Pancreas

5) Secondary EMT

Figure 1. Successive EMT during Embryonic Development (A) Primary EMT occurs early during embryonic development, even before implantation such as during the formation of the parietal endoderm in mice. The first EMT after implantation is that undergone by the mesendodermal progenitors during gastrulation, whereas the delamination of neural crest cells from the dorsal neural tube is a later event. (B) Early mesodermal cells are subdivided into axial, paraxial, intermediate, and lateral plate mesodermal cells that will condense into transient epithelial structures: the notochord, the somites, and the somatopleure and splanchnopleure, respectively. These transient structures will undergo secondary EMT, leading to the generation of mesenchymal cells that differentiate into specific cell types. Endodermal tissues, including the pancreas bud and the liver diverticulum, exhibit morphological changes reminiscent of a secondary EMT to induce the dissociation of endocrine cells and hepatoblasts from their respective epithelial primordia. (C) An example of tertiary EMT arises during the formation of the cushion mesenchyme in the heart from the atrioventricular canal (AV) or the outflow tract (OT). The cushion mesenchyme is the precursor of the cardiac valves. Successive EMT during Embryonic Development

Gastrulation Many different types Various versions of cell migration ~ +/- EMT Conserved genes, conserved modules

Genetic program for EMT during Gastrulation in Amniotes Genetic pathways controlling gastrulation in amniotes. Convergence of signaling pathways at the posterior part of the embryo leads to primitive streak formation and initiation of the EMT as well as the mesodermal fate program. Snail genes are key regulators of the EMT program during gastrulation in amniotes as they control cell-cell adhesion, cell shape, and motility. Additional mechanisms such as endocytosis, lysosomal targeting, and degradation of the E-cadherin protein together with the control of basement membrane integrity explain the rapid and drastic changes occurring in ingressing cells during gastrulation. The induction of endodermal and mesodermal fates is mainly governed by the FGF and Nodal pathways through specific regulators and the contribution of some of the genes involved in the EMT program. EPB4L5, FERM and actin-binding domains-containing band 4.1 superfamily member; FLRT3, Fibronectin-leucine-rich-transmembrane protein-3; Net-1, neuroepithelial transforming factor 1; MMP, metalloproteinases; p38ik, p38 interacting kinase.

Various modes of gastrulation

Drosophila gastrulation

Sea urchin gastrulation

Various modes of gastrulation

Various modes of gastrulation

Gastrulation in the chick embryo

Other gastrulation modes Ingression (birds, mammals)

Conserved regulators of EMT in development Transcription factors: General for EMT: Snail/Slug Twist Typical for mesoderm: T/Brachyury Goosecoid

Snail sequence alignment

Snail expression in cnidarians and Drosophila

Dynamic pattern of Lvsnail mrna expression during sea urchin development. Shu-Yu Wu, and David R. McClay Development 2007;134:1061-1070 2007.

Snail expression in Xenopus Early gastrula Early neurula

Snail and T/Brachyury expression in the chicken gastrula Primitive streak

Brachyury expression in sea urchin and Xenopus Blastopore lip, prospective mesoderm

Key features of the EMT of sea urchin primary mesenchyme cells basal lamina junctional complex microvilli hyaline layer Mechanisms of Development 120 (2003) 1351 1383

Gastrulation can use partial aspects of EMT

Invagination in cnidarians: apical constriction, elongation, basal expansion Developmental Biology 305 (2007) 483 497 Gastrulation in the cnidarian Nematostella vectensis occurs via invagination not ingression Craig R. Magie a, Marymegan Daly b, Mark Q. Martindale

Invagination in cnidarians: apical constriction, elongation, basal expansion

Gastrulation in Xenopus (and fish): Collective migration Dorsal section of early gastrula ectoderm inner layer outer layer leading mesendoderm endoderm (not shown) involuted mesoderm non-involuted mesoderm bottle cells blastopore lip

E-cadherin downregulation is not required for gastrulation Zebrafish: Only E-cadherin Xenopus: Only C-cadherin (~E-cadherin) Drosophila: Switch E- to N-cadherin, but..

Mis-expression of E-cadherin in the embryonic mesoderm of Drosophila Cadherin switching during the formation and differentiation of the Drosophila mesoderm implications for epithelial-tomesenchymal transitions Gritt Scha fer1,*, Maithreyi Narasimha2,*, Elisabeth Vogelsang1 and Maria Leptin1 Journal of Cell Science (2014) 127, 1511 1522

F-actin/C-cadherin Gastrulation in Xenopus: Ectoderm cells not polarized Changes in adhesion and migration without changes in adhesion molecules Dorsal section of early gastrula ectoderm inner layer outer layer leading mesendoderm Ectoderm bleb Mesoderm endoderm (not shown) involuted mesoderm non-involuted mesoderm lamellipodium bottle cells blastopore lip cell-cell contact cell-cell contact lamellipodium lamellipodia blebs

GFP-membrane marker Gastrulation in Xenopus: Ectoderm cells not polarized Changes in adhesion and migration without changes in adhesion molecules Ectoderm cells Mesoderm cells

Regulation of adhesion via cell cortex contractility cadherin levels -> No need to control cadherin expression to modulate adhesion!! Example of EpCAM = tumor-associated protein, marker of carcinomas

EpCAM = tumor-associated protein, major marker of carcinomas Comparison between immunohistochemical epithelial cell adhesion molecule (EpCAM) expression in primary breast cancer and metastases. Invasive ductal carcinoma Brain metastases from the same patient Invasive lobular carcinoma What is the cellular/molecular function of this molecule? Peritoneal metastases normal breast ducts Gilbert Spizzo et al. J Clin Pathol 2011;64:415-420 Copyright by the BMJ Publishing Group Ltd & Association of Clinical Pathologists. All rights reserved.

EpCAM controls cadherin levels Whole embryo (mosaic)

EpCAM controls tissue integrity C COMO EpCAM MO EpCAM MO + EpCAM mrna D ec F ec ne som v ne no C-cad mgfp ar COMO C-cad mgfp EpCAM MO

EpCAM controls cadherin levels EpCAM controls tissue integrity? Molecular mechanism? 1) Need for an assay

A simple assay to study the cellular/molecular control of tissue integrity A A B COMO Ectoderm explant EpCAM C-Cadh - 45kDa - 120kDa Outer layer Inner cells A B EpCAM MO α-tubulin COMO EpCAM MO - 50kDa Overnight culture

EpCAM depletion causes cadherin endocytosis

EpCAM controls cadherin levels through regulation of myosin Blebbistatin = myosin inhibitor pmlc = phosphorylated myosin light chain Rock inhibitor

EpCAM directly binds and inhibits PKCs immunoprecipitation Pulldown of endogenous PKCs Surface plasmon resonance affinity measurement Pulldown of purified PKCs In vitro kinase assay: EpCAM tail inhibits phosphorylation of MBP, a PKC susbtrate

EpCAM cytoplasmic tail contains a pseudosubstrate sequence for PKC Principle of PKC regulation Consensus substrate sequence: cluster of positive amino acids (KR)

EpCAM cytoplasmic tail contains a pseudosubstrate sequence for PKC A EpCAM Extracellular TM TAIL Position -7-6 -5-4 -3-2 -1 0 +1 +2 +3 +4 +5 GST-xEpCAM tail TM F T R R K R G K Y Q K A E GST-hEpCAM2 tail TM T N R R K S G K Y K K V E GST-zEpCAM2 tail TM L A R R Q K A H Y S K A Q PKCδ consensus subst. A R R K R K G S F F Y G G PKCη consensus subst. A R R R R R R S F R R X R PKCδ pseudosubstrate P T M N R R G A I K Q A K PKCη pseudosubstrate F T R K R Q R A M R R R V

EpCAM cytoplasmic tail contains a pseudosubstrate sequence for PKC A EpCAM Extracellular TM TAIL Position -7-6 -5-4 -3-2 -1 0 +1 +2 +3 +4 +5 GST-xEpCAM tail TM F T R R K R G K Y Q K A E GST-hEpCAM2 tail TM T N R R K S G K Y K K V E GST-zEpCAM2 tail TM L A R R Q K A H Y S K A Q PKCδ consensus subst. A R R K R K G S F F Y G G PKCη consensus subst. A R R R R R R S F R R X R PKCδ pseudosubstrate P T M N R R G A I K Q A K PKCη pseudosubstrate F T R K R Q R A M R R R V

Protein transmembrane domains are clamped by charged amino acids - - - + ++ + ++ + + + ++

Selected transmembrane proteins have evolved a pseudosubstrate sequence for PKC B Position -7-6 -5-4 -3-2 -1 0 +1 +2 +3 +4 +5 +6 Main resid cons δ/η + + + + + X S F xepcam TM F T R R K R G K Y Q K A E M hepcam2 TM T N R R K S G K Y K K V E I EphA4 TM I S R R R S - K Y S K A K Q NrCAM TM I R R N K G G K Y P V K E K ICAM TM N R Q R K I K K Y R L Q E A C Input D ctrl EphA4 mrna PKCδ PKCη EphA4 ppkd 1 * GST loading 0 ctrl EphA4 mrna

EpCAM directly inhibits PKCs cytoplasm ps PKC (active form) Catalytic site + EpCAM A PKC Erk myosin ATP P+1 P-3 C2 C1A C1B cell membrane P-4 P-5 Hydrophobic pocket EpCAM

EpCAM stabilizes cadherin levels AND stimulates cell migration!!

EpCAM is overexpressed in primary and secondary tumors but not necessarily in invasive cells!! Comparison between immunohistochemical epithelial cell adhesion molecule (EpCAM) expression in primary breast cancer and metastases. Invasive ductal carcinoma Brain metastases from the same patient Invasive lobular carcinoma? Peritoneal metastases normal breast ducts Gilbert Spizzo et al. J Clin Pathol 2011;64:415-420 Copyright by the BMJ Publishing Group Ltd & Association of Clinical Pathologists. All rights reserved.

Different EpCAM levels -> different cell behavior?

Neural crest cells Roberto Mayor, and Eric Theveneau Development 2013;140:2247-2251

Roberto Mayor, and Eric Theveneau Development 2013;140:2247-2251 2013. Published by The Company of Biologists Ltd

Neural crest cells

Jaws = modification of anterior pharyngeal arches Origin of neural crest cells

Neural crest cells

Evolution of vertebrates as viewed from the crest Stephen A. Green1, Marcos Simoes-Costa1 & Marianne E. Bronner Neural crest cells

EMT in neural crest cells: Differential temporal sequences Eric Theveneau, Roberto Mayor Neural crest delamination and migration: From epithelium-to-mesenchyme transition to collective cell migration Developmental Biology, Volume 366, Issue 1, 2012, 34 54

Contact inhibition of locomotion in neural crest cells Dom neg Dsh PCP inhibition

Role of E- to N-cadherin switch in neural crest cells Pre-migratory Migratory

Contact Inhibition of Locomotion (CIL) Is a Developmentally Regulated Property of NC Cells Scarpa et al., 2015, Developmental Cell 34, 421 434

Different Dynamics of Junction Disassembly in Migratory and Premigratory NC Cells Scarpa et al., 2015, Developmental Cell 34, 421 434

E-Cadherin Suppresses CIL In Vivo Scarpa et al., 2015, Developmental Cell 34, 421 434

E-Cadherin Suppresses CIL In Vitro Scarpa et al., 2015, Developmental Cell 34, 421 434

E-Cadherin Suppresses CIL In Vitro Scarpa et al., 2015, Developmental Cell 34, 421 434

Scarpa et al., 2015, Developmental Cell 34, 421 434 E-Cadherin Suppresses CIL In Vitro

Collective cell migration of neural crest cells

Collective cell migration of neural crest cells

Collective cell migration of neural crest cells

Collective cell migration of neural crest cells

Directional collective migration: Chemotaxis and CIL ( Chase and run )