Chapter 3 (Lecture 3&4).pdf

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Cell and Systems Biology
Ashley Bruce

Chapter 3: Cell-cell Communication in Development  Development is more than just differentiation  Different cell types form organized structures – must be ordered to take different shapes and make different connections -> morphogenesis  Questions in morphogenesis: 1. How are separate tissues formed from populations of cells? 2. How are organs constructed from tissues? 3. How do organs form in particular locations, and how do migrating cells reach their destinations? 4. How do organs and their component cells grow, and how is their growth coordinated throughout development? 5. How do organs achieve polarity?  1850a, Robert Remak formulated the cell theory and showdd that all the different cells in the bosy are the mitotic products of a single cell, the fertilized egg  Mid 20 century, E.E. Just and Johannes Holtfreter predicted that embryonic cells could have differences in their cell membrane components that would enable the formation of organs o The molecules by which embryonic cells are able to adhere to, migrate over, and induce gene expression in neighbouring cells  The cells of an embryo are either epithelial or mesechymal o Epithelial cells adhere to one another and can form sheets and tubes o Mesenchymal cells often migrate individually and form extensive ECM that keep the individual cells separate  3 behaviours requiring cell-cell communication via the cell surface: cell adhesion, cell migration, and cell signaling Cell Adhesion Differential cell affinity  Morphogenesis focuses on the cell membrane and its interactions with the peripheral cytoplasm  Cell membrane differed among cell types  Townes and Holtfreter discovered that amphibian tissues become dissociated into single cells when placed in alkaline solutions o They prepared single cell suspensions from each of the 3 germ layers soon after the neural tube had formed o 2 or more of these suspensions could be combined in various ways:  When the pH of the soln was normalized, the cells adhered -> aggregates  By using embryos from species having cells of different sizes and colours, T&H were able to follow the behaviour of the recombined cells o Results:  Found that reaggregated cells become spatially segregated -> each type sorts into its own region  Thus, when epidermal and mesodermal cells are brought together in a mixed aggregate, the epidermal move to the periphery and meso move to the inside  Found that the final positions of the rearranged cells reflect their respective positions in the embryo  Reaggregated meso migrates centrally with respect to the epidermis, adhering to the inner epidermal surface  The meso also migrates centrally with respect to the gut or endoderm  When the 3 germ layers are mixed together, the endoderm separates from the ectoderm and meso and is then enveloped by them -> final configuration: ectoderm is on the periphery, endoderm is internal and the meso lies in the region between them  H interpreted this finding in terms of selective affinity: o Inner surface of ecto has a + affinity for meso and a – affinity for the endo o Meso has + affinities for both ecto and endo  Mimicry of normal embryonic structure by cell aggregates is also seen in the recombination of epidermis and neural plate cells  When axial meso (notochord) cells are added to a suspension of presumptive epi and presumptive neural cells, cell segregation results in an external epidermal layer, a centrally located neural tissue, and a layer of meso tissue between -> able to sort right  Selective affinities change during development  Expected because embryonic cells do not retain a single stable relationship with other cell types  For development to occur, cells must interact differently with other cell populations at specific times The thermodynamic model of cell interactions  Cells do not sort randomly but can actively move to create tissue organization – what directs this movement?  1964, Malcolm Steinberg proposed the differential adhesion hypothesis, a model that sought to explain patterns of cell sorting based on thermodynamic principles o Using cells derived from trypsinized embryonic tissues, S showed that certain cell types migrate centrally or peripherallywhen combined with a certain cell type o Figure: aggregates formed by mixing 7-day chick embryo neural retina (unpigmented retina cells) a. 5 hours – after single cell suspensions are mixed, aggregates of randomly distributed cells are seen b. 19 hours – pigmented retina cells are no longer in the periphery c. 2 days – geat majority of pigmented cells located in central internal mass, surrounded by neural retina cells  Such interactions form a hierarchy: o If the final position of A is internal to B, and the final of B is internal to C, then A will always be internal to C  This observation led S to propose that cells rearrange themselves into the most thermodynamically stable pattern o If the strength of AA connections is greater than that between AB and BB, sorting will occur with A cells becoming central o If AA is less than or equal to AB, aggregate will remain as a random mix o If AA is far greater than AB (almost no adhesivity), A and B will form separate aggregates  According to this hypothesis, the early embryo can be viewed as existing in an equilibrium state until some change in gene activity changes the cell surface molecules  New movements seek to restore a new equilibrium  For sorting to occur, cell types must differ in strengths of their adhesion  In several meticulous experiments using numerous tissue types, researchers showed that those cell types that had greater surface cohesion migrated centrally compared with cells that has less surface tension  In the simplest form of this model, all cells could have the same type of “glue” on the cell surface o The amount of “glue” could create a difference in the number of stable contacts made between cell types  In a more specific version, the thermodynamic differences could be caused by different types of adhesion molecules  Figure: hierarchy or cell sorting in order of decreased face tensions o The equilibrium config reflects the strength of cell cohesion, with the cell types having the greater cell cohesion segregating inside the cells with less cohesion Cadherins and cell adhesion  Recent evidence shows that boundaries between tissues can indeed be created by different cell types having both different types and different amounts of cell adhesion molecules  Major cell adhesion molecules: cadherins – Ca-dependent adhesion molecules o Critical for establishing and maintaining intercellular connections o Appear to be crucial to the spatial segregation of cell types and to the organization of animal forms o TM proteins that interact with other cadherins on adjacent cells o Anchored inside the cell by a complex of proteins called catenins  Cadherin-catenin complex forms the classic adherens junction that hold epithelial cells together o Bind to the actin (microfilament) cytoskeleton of the cell o Integrate the epithelial cells into a mechanical unit  Blocking cadherin function (Ab) or synthesis (antisense RNA) can prevent the formation of epithelial tissues -> disaggregate  Cadherins perform several related functions: o External domains serve to adhere cells together o Link to and help assemble the actin cytoskeleton -> mechanical forces for forming sheets and tubes o Serve as signaling molecules that change a cell’s gene expression  Vertebrate embryos, several major types: o E-cadherin: expressed on all early mammalian embryonic cells, even at the zygote stage  Later in development, restricted to epithelial tissues ofembryos and adults o P-cadherin: found predominantly on the placenta – helps the placenta stick to the uterus o N-cadherin: highly expressed on the cells of the developing central nervous system  May play roles in mediating neural signals o R-cadherin: critical in retina formation o Protocadherins – lack the attachment to the actin skeleton through catenins  Similar protocadherins: important means of keeping migrating epithelial cells together  Dissimilar protocadherins: important way of separating tissues  Differences in cell surface tension and the tendency of cells to bind together depend on the strength of cadherin interaction o Can be achieved quantitatively or qualitatively  Quantity and Cohesion o Steinberg and Takeichi collaborated on an experiment using 2 cell lines that were identical except that they synthesized different amounts of P-cadherin  When the 2 groups were mixed, the cells that expressed more P-cadherin had a higher surface cohesion and migrated internally to the lower-expressing group of cells o Foty and S demonstrated that this quantitative cadherin-dependent sorting directly correlated with surface tension o The surface tensions of these aggregates are linearly related to the amount of cadherin they express on the cell surface o The cell sorting hierarchy is strictly dependent on the cadherin interactions between the cells o The energetic value of cadherin-cadherin binding is remarkably strong  This free energy change associated with cadherin function could be dissipated by depolymerizing the actin skeleton  Underlying actin appears to be crucial in organizing the cadherins in a manner that allows them to form remarkably stable linkages between cells  Type, Timing, and Getting Along with Others o Duguay and colleagues showed that R-cad and B-cad do not bind well to each other o Expression of n-cad is important in separating the precursors of the neural cells from the precursors of the epidermal cells o All early embryonic cells originally contain e-cad but neural tube cells lose e-cad and gain n-cad o If epidermal cells are experimentally made to expressn-cad, the border between the skin and NS fails to form properly o The timing of particular developmental events can also depend on cadherin expression  N-cad appears in the mesenchymal cells of the developing chick leg just before these cells condense and form nodules of cartilage  N-cad is not seen prior to condensation nor afterward  If limbs areinjected just prior to condensation with Ab that block n-cad, the mesenchyme cells fail to condense and cartilage fails to form  Signal for cartilage formation: appearance of n-cad o During development, the many cadherins also work with other adhesion systems  If development is to continue after the embryo passes from the oviduct and enters the uterus, the embryo must adhere to and embed itself in the uterine wall  That is why the first differentiation event distinguishesthe trophoblast cells (outer cells that bind the uterine wall) from the inner cell mass (those will generate the embryo)  This occurs as the embryo travels from the upper regions of the oviduct on its way to the uterus  Trophoblast cells are endowed with several adhesion molecules that anchor the embryo to the uterine wall – contain both e and p-cad which recognize similar cadherins on the uterine wall Shape Change and Epithelial Morphogenesis: “The Force is Strong in You”  Epithelial cells’ ability to form sheets and tubes often depends on cell shape changes that usually involve cadherins and the actin cytoskeleton  The ECD of cadherins bind groups of cells together while the ICD alter the actin cytoskeleton  The proteins mediating this cadherin-depending remodeling of the cytoskeleton are usually: o Non-muscle myosin – provide the E for actin contraction o Rho family of GTPases – convert soluble actin into fibrous actin cables that anchor at the cadherins  Generally divided into 3 groups that have different but overlapping functions: 1. RhoA primarily organizes stress fibers 2. Rac1 is involved in producing lamellipodia 3. Cdc42 is used primarilyin constructing filopodia  2 examples of cadherin dependent remodeling of the cytoskeleton are: 1. The formation of the neural tube in vertebrates 2. Internalization of the mesoderm in Drosophila o In both cases, the cells are on the outside of the embryo and must migrate inside  Involution of the frog neural tube: o In the early frog embryo, each cell membrane can contain several types of cadherins o Each cell of the gastrula is covered with c-cad o But presumptive neural tube ectoderm also contain n-cad concentrated in their apical regions o Presumptive epidermal cells of the ectoderm express e-cad on their lateral and basal surfaces o The actin organized in the apical region of the neural cells causes them to change shape and enter the internal region of the embryo as a neural tube o The actin on the lateral sides enables the migratory movements of the epidermal cells over the surface of the embryo o If n-cad is experimentally removed, the cells still adhere (cause of c-cad) but the actin fail to assemble apically -> no neurulation (presumptive neural cells do not enter the embryo and no neural tube forms)  Drosophila mesoderm formation o In drosophila, the mesoderm is formed from epithelial cells on the ventral side of the embryo o These cells form a furrow and then migrate inside the embryo o Furrow is formed by cube-shaped cells becoming wedge-shaped, constricting the apical surfaces o This force is creased by the rearrangement of actin microfilaments and myosin II to the apical end of the cell - pushes the ventral cells inside the embryo o Actin microfilaments are part of the cytoskeleton and are often found on the periphery of the cell o Twist gene, expressed in the nuclei of the ventralmost cells, instruct for apical constriction  Twist protein activates other genes -> protein products cause the actin cytoskeleton to build up on the apical side –> accomplished by the binding of a Rho GTPase and β-catenin to e-cad on the apical portion in most ventral cells  Once stable, the actin-myosin complex constricts like a drawstring of a purse -> cells change shape, buckle inwards, and enter the embryo to form the mesoderm  External signals: insect trachea o Instructions for cell shape change can also arise outside the cell o The tracheal system in drosophila embryos develops from epithelial sacs o Approx.. 80 cells in each of these sacs become reorganized into primary, secondary, and tertiary branches without any cell division or death o Initiated when nearby cells secrete a protein called Branchless –> acts as a chemoattractant o Branchless binds to a receptor on the cell membranes of the epithelial cells o The cells with the most branchless protein lead the rest to form the tracheal tube o The lead cell will change its shape to migrate and to form a tube while keeping their integrity as an epithelium o Also, the dorsalmost secondary branches of the sacs move along a groove that forms between the developing muscles o These tertiary migrations cause the trachea to become segmented around the musculature Cell Migration  Common feature of both epithelial and mesenchymal cells o Eg. Gastrulation to form the 3 germ layers, neural tube folding in vertebrate embryo, mesoderm folding in fly embryo  A combination of motility and guidance  In epithelia, the motive force for migration is usually provided by the cells at the edge of the sheet, and the rest of the cells follow passively  In mesenchymal, individual cells become polarized and migrate through the extracellular milieu  In both cases, there is a broad reorganization of the actin cytoskeleton  First stage: polarization – a cell defines its front and its back o Polarization can be directed by diffusing signals or by signals from the ECM o These signals reorganize the cytoskeleton  Second stage: protrusion of the cell’s leading edge o Mechanical force: polymerization of the actin microfilaments at the cell membrane -> long parallel bundles (filopodia) or broad sheets (lamellipodia) o Membrane bound Rho GTPase activate the WASP-N proteins to nucleate actin and connect it to cadherins and the cell membrane  Third stage: adhesion of the cell to its extracellular substrate o Needs something to push off on by attaching to the surrounding matrix o Key molecules: integrins – span the cell membrane, connecting the ECM outside the cell to the actin cytoskeleton on the inside o These connections form focal adhesions on the cell membrane where the membrane contacts the ECM o Myosin and its regulators provide the motive force along these actin microfilaments and they are linked with the lamellipodial actin at the sites of adhesion  Fourth stage: release of adhesions in the rear, allowing the cell to migrate in the forward direction o Probable that stretch-sensitive Ca channels are opened and that the released Ca ions activate proteases that destroy the focal adhesion sites Cell Signaling Induction and competence  Throughout development, cell behaviours are regulated by signals from one cell being received by another cell  In the vertebrate eye, light is transmitted through the transparent corneal tissues and focused by the lens tissue, eventuallyimpinging on the tissue of the neural retina o Construction of organs is accomplished by one group of cells changing the behavior of an adjacent set of
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