Tomáš Bárta tbarta@med.muni.cz The role of physical phenomena on development 1 Contents * Introduction Mechanosensing: Cadherins, Integrins, Hippo Shear forces and heart development Mechanical forces and morphogenesis Bonus: Optogenetics Bonus: Organoids Conclusion 2 Mechanosensing in embryogenesis •Mechanical force is an invisible but ubiquitous part of biological systems. Forces such as gravity and osmotic pressure set physical limits for the body's plane. At the same time, the cells of the embryo use these forces to create the complex shapes that we find in the animal kingdom. The mechanical forces generated by living cells at the molecular level have a great impact on embryogenesis. The direct result of the action of force is the movement that occurs during the separation of chromosomes, the migration of cells or the folding of tissues. A less direct, but equally important effect of force is the activation of mechanosensitive signaling, which allows cells to explore their mechanical surroundings and communicate with each other over short and long distances = > mechanical forces are a way of communicating 3 Mechanosensing in embryogenesis * The ability of cells to "perceive" mechanical signals and convert them into biochemical signals. • •And how/what does it "perceive"? Ion channels Primary cilium Integrins Cadherins Actin/Myosin cytoskeleton Notch Growth factor receptors 4 5 Mechanosensing in embryogenesis Mechano reception in embryogenesis https://doi.org/10.1016/j.ceb.2020.08.007 6 •Many mechanical forces act on the cell: • •Intrinsic: osmotic pressure, contractility of actin and myosin External: shear stress, gravity, stretching • •Forces are perceived and interpreted by mechanosensors (adhesive molecules, ion channels), which then lead to a change in gene expression. This leads to the cell reaction -> a change in cell/tissue morphology 7 Mechanosensing in embryogenesis – Cadherin/β-catenin – gastrulation and induction of mesoderm https://doi.org/10.1016/j.ceb.2020.08.007 •During the gastrulation, there is a movement of cells = > the action of mechanical forces. These forces activate the β-catenin pathway, which contributes to the mesoderm specification by activating the expression of genes that specify the mesoderm. 8 Link to previous lecture Mechanosensing in embryogenesis – Cadherin/β-catenin – gastrulation and induction of mesoderm •β-Catenin is a primary molecule in mechanoreception • in the nucleus as a transcription factor (regulates gene expression) but also structural role in adherens junctions (link between E-cadherin and cytoskeleton) Cadherin-catenin-actin "axis" is under constant mechanical pressure not only due to the internal contractility of actin. An external mechanical stimulus acts on the intercellular contacts and increases this pressure, resulting in a sense of mechanical pressure (1 nm). 9 Mechanosensing in embryogenesis – Cadherin/β-catenin – gastrulation and induction of mesoderm 10 Mechanosensing in embryogenesis – Cadherin/β-catenin – gastrulation and induction of mesoderm •Blockage of gastrulation movements (genetic, pharmacol.) leads to inhibition of mesodermal genes – repeated action of mechanical forces saves the phenotype and expression of mesoderm. Genes Feedback for signal pathways. Blebbistatin = specific non-muscle myo-II inhibitor 11 Mechanosensing in embryogenesis – Cadherin/β-catenin – gastrulation and induction of mesoderm 12 Mechanosensing in embryogenesis – Cadherin/β-catenin – gastrulation and induction of mesoderm 13 Intercellular mechanosensing and tissue growth - Hippo Hippo not only in size control 14 Intercellular tension and tissue growth - Hippo •ECM stiffness, cell density, cytoskeleton pressure affect YAP/Yki localization Molecular mechanism is unclear https://doi.org/10.1016/j.ceb.2020.08.007 15 Obsah obrázku barevné, zelená, různé, barvy Popis byl vytvořen automaticky NOTCH 16 NOTCH 17 •After fertilization, the mammalian zygote produces about 100 cells within about 4 days. • Embryoblast (ICM): Oct4, Nanog, Sox2 Trophectoderm (TE): CDX2 •These factors determine individual cell lines, but how did the individual lines were generated? The position of blastomeres in the embryo. Different polarity and adhesion of cells. NOTCH 18 Integrins 19 Integrins 20 21 Shear forces 22 Shear forces and heart development •The heart begins its development as a tube and gradually develops into a multi-chamber apparatus. But in the course of development, it constantly draws blood. Blood pressure exerts shear forces on endothelial cells. Shear forces are perceived by endothelial cells = > affects their organization and physiology. 23 Shear forces and heart development 24 Shear forces and heart development 25 Shear forces and heart development The development of heart valves therefore depends on the perception of shear forces by mechanosensitive channels in endothelial cells, thus determining the right place for valve development and activation of the corresponding genes. Disruption of shear forces (genetically, surgically, change in viscosity) leads to disorders in valve development 26 Shear forces and heart development 27 Shear forces and heart development 28 Food for thought 29 30 31 Mechanical forces and morphogenesis •During the development of mechanical forces cause changes in the shape, size, number and position of cells, which is accompanied by a change in gene expression => impact on morphogenesis. All of these cellular processes that lead to a change in tissue shape are a form of force between individual cells, normally mediated by intercellular adhesion. • This force is generated through: •Actin Polymerization of micro-domes Osmotic pressure Molecular motors - Myosin 32 Mechanosensing in embryogenesis – gastrulation and induction of mesoderm 33 Mechanical forces and morphogenesis •Changes in the cytoskeleton are transmitted to neighboring cells and the ECM through the interconnection of the cytoskeleton to adhesive molecules that provide cell-to-cell, cell-ECM interaction (cadherins, integrins). Actin-myosin contraction and interaction mediated by cadherins are basic and evolutionarily conserved mechanisms that generate and transmit forces for the formation of morphogenesis (see differential adhesion hypothesis/equlibrium state in ECM and cell adhesion – all forces in equilibrium, the state of lowest energy). 34 Mechanical forces and morphogenesis 35 Mechanical forces and morphogenesis 36 Mechanical forces – cell differentiation and proliferation * The ability of cells to perceive external mechanical forces affects tissue size and architecture not only by changing their adhesive and cytoskeletal organization, but also by influencing their differentiation. 37 BONUS: Vibrational cues alter developmental timing Agalychnis callidryas •It lays eggs on the leaves, which are located above the water Normal development lasts 7 days. In the case of a predator attack, larvae hatch (within seconds!) if the eggs have about 5 days of development. Signaling an attack of eggs by a predator is through vibration This leads to the production of enzymes that disrupt the shell 38 39 BONUS: optogenetics 40 BONUS: optogenetics 41 42 BONUS: optogenetics 43 BONUS: Optogenetics in Developmental Biology 44 •Early activation -> shortening of the body and cyclops Late activation -> symmetry of the heart BONUS: optogenetics 45 BONUS: organoids for studying the influence of physical phenomena on development and physiology 46 •Organoids are three-dimensional miniatures of organs that have a similar structure and function to the organ BONUS: organoids for the study of physical phenomena on development and physiology 47 BONUS: 48 49 50 Obsah obrázku text, papoušek, různé, pták Popis byl vytvořen automaticky BONUS: 51 BONUS: 52 Conclusion and questions 53 •How does the cell perceive mechanical forces? The role of Cadherins and Integrins in mechno reception? And how does it work? Hippo signal pathway and perception of mechanical force. How do shear forces affect the development of the heart? The importance of mechanoreception during gastrulation Thank you for your attention 54 Tomáš Bárta tbarta@med.muni.cz