CTO Lecture Notes - Semester 1 - PDF

lOMoARcPSD|35018169 CTO - Lecture notes for all CTO topics in Semester 1. Cells to Organisms 2 (The University of Edinburgh) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Multicellularity Lecture 2 an organism that consist of more than one cell type e.g algae volvox, colonial vs true multicellularity multicellularity “kingdoms”: plants, animals, fungi closest single cell relative to metazoa (animal): Choanoﬂagellates, bacteria eating eukaryotes they have an axis of symmetry, collar of actin rich ﬁlaments and a nucleus single cells can differentiate Choanoﬂagellates form multi-cellular colonies in particular environmental conditions Choanoﬂagellates: unicellular, porifera (sponges): colonial choanocytes: cell type in sponges that looks similar to choanoﬂagellates sponges have the same food source as choanoﬂagellates when comparing genome sequencing in choanoﬂagellates to sponges: differences in cell communication and cell adhesion Cell communication kinases: enzymes that add a phosphate: serine, threonine and tyrosine can be phosphorylated by kinases animals and choanoﬂagellates increased the number of receptor tyrosine kinases in their genomes before metazoan evolution: common ancestor had no or few receptor TKs phosphorylation of a`tyrosine residue can either activate or inhibit the function of an enzyme regulation of kinases increases in complexity 1 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Cell adhesion cadherins: adhesion molecules increase of cadherins in choanoﬂagellates; most of it is found in the collar on the microvilli can act as a bacterial receptor cadherins ﬁrst evolved to bind bacteria before being co-opted for cell adhesion Lecture 3 Body plan axis: at least one axis of symmetry; anterior-posterior, dorsal-ventral (bilateral animals), oral-aboral sponge larva and other radiata have a single anterior posterior axis, bilateral animals have 2 or 3 axes. other animal innovations: cell signalling and cell differentiation WNT pathway, etc. muscle, nerves WNT pathway WNT is a secreted ligand —> frizzled receptor —> beta-catenin —> response—> TCF active absence of WNT: off state frizzled is unbound GSK3ß (Glycogen-synthase-kinase-3,serine/ threonine protein kinase) phosphorylates ß- catenin ß-catenin is degraded TCF is inactive (T-cell factor, transcription factor that binds DNA in sequence speciﬁc manner) WNT responsive genes are off 2 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Binding of WNT ligand to a frizzled receptor causes stabilisation of the ß-catenin protein, which accumulates and enters the nucleus —> inside uncles it binds to TFC, activates the expression of a collection of WNT-responsive genes WNT can cause changes in gene expression and cell shape WNT pathway is only present in animals, no presence in choanoﬂagellates or sponges WNTs establish the primary axis of radiata (aboral-oral): one axis radial vs bilateral symmetry: aboral/oral anterior/posterior, dorsal/ventral WNTs establish the primary axis of all animals All multicellular animals express WNT at the tip of their major body axis, bilateral animals always express WNTs in the posterior end WNT inhibitor is found in the anterior Mouse embryo Wnt3a knockout: posterior defects Nerves nerves, muscles and guts not present in sponges, but present in ctenophores (next organism in line) Sensory-to-motor transformation drove the development of nerves Muscles and nerves work together to allow movement in response to a sensory input Ciliary locomotion: sensory-to-motor transformation in unicellular organisms in response to light stimulus Sensory input —> ciliary (cilia) locomotion (anicent) Sensory input —> muscle locomotion sponge larva: ﬂask cells for sensory input e.g. light —> transmit information to neighbouring ciliated cells —> guide swimming speed and direction ﬂask cells express many genes important for the function of synapses (structures responsible for communicating information between neurons) ﬂask cells may have evolved into neurons hypothesis: sensory ﬂask cells of the sponge evolved into neurons of the ctenophora 3 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Quiz Prior to their co-option as cell adhesion molecules, cadherins seem likely to have been sticky proteins to capture bacteria in unicellular organisms Although multicellularity has arisen independently many times during evolution, only one event led to metazoans. This explains why diverse animals share much of their molecular and cellular toolkit required for multicellularity e.g. WNT signalling. Choanoﬂagellates are not to be confused with choanocytes. Biﬂagellates and Paramecium are protozoans with ﬂagella and cilia but lack the key combination of features that make them good candidates for ancestry of metazoans. All these types of molecules are well represented in both multicellular and unicellular organisms (protozoa), although they may have been adapted for new functions. The exception is the class of receptor tyrosine kinases which are and innvovation of multicellular metazoans and choanoﬂagellates. 4 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Cell Adhesion Lecture 4 plants and animals are made entirely of cells cells are small and ﬂimsy but organisms can be large and strong but still ﬂexible multicellular organsims are built from organised collections of cells tissue can be deﬁned as a coopertaive assembly of cells and extracellular matrix woven together to form a multicellular fabric with a distinctive function multiple cell types in tissues: cells vary in appearance and function extracellular matrix gives strength to supportive tissues speciﬁc arrangement is essential for tissue function How do cells in multicellular organisms adhere? for cells to join together and become stable, they must have ways to transmit physical stress two strategies: extracellular matrix ECM cytoskeleton and cell adhesions that connect the cytoskeletons of neighbouring cells Plant tissues plant tissues are supported by cell walls —> supportive matrix can be thick and hard (wood) or ﬂexible and thin (leaf) plant cell control th production of their cell walls newly formed plant cells have a primary cell wall only —> thin and ﬂexible, can expand once cell growth stops, secondary cell wall —> thick, rigid, no need to expand, most common additional polymer is lignin (found in wood especially) cellulose microﬁbrils give cell wall strength —> most abundant macromolecule on earth long, unbranched chains of linked glucose subunits, hundreds of subunits in one cellulose molecule microﬁbril: approx. 16 cellulose molecules tensile strength comparable with steel cellulose predominates in secondary cell walls pectin provides resistance to compressive forces long, complex polysaccharides rich in galacturonic acid highly hydrated, binds cations, space-ﬁlling effect similar to glycosaminoglycans (GAGs) in animal ECM resists compression, crosslinks with cellulose to 5 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures form a matrix (when plants are squashed they can spring back to their original shape) abundant in primary cell walls Primary cell wall cellulose microﬁbrils —> tensile strength other polysaccharides crosslink the cellulose microﬁbrils pectin —> resistance to compression middle lamella (pectin rich) cements cell walls together Structure of wood cells have thick walls ,which remain when the cell dies, this forms long channels running vertically within the tree cell wall is composed of lignin lignin is a complex of polymer of cross-linked phenolic (phenol) compounds high wet strength (rigid when wet) because phenol has a hydrophobic property Plant cell wall composition main structural element are polysaccharides —> CO2 and H2O contains some proteins, for remodelling during growth animals use more proteins ﬁxed N2 is rate limiting for the growth of many plants —> reason why plants mainly use polysaccharides in cell walls Orientation of microﬁbrils turgor pressure in a plant cell = 5x car tyre—> cell wall helps sustain internal pressure microﬁbrils resist stretching the orientation inﬂuences the direction in which the cell elongates important during growth —> inﬂuences tissue shape Cellulose deposition beneath the plasma membrane, microtubules are aligned exactly with the microﬁbrils microtubules help direct the deposition of cellulose Cellulose synthesis glucose is supplied from cytosol cellulose synthase complex makes many cellulose molecules and assembles microﬁbril 6 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures cellulose microﬁbril is added to preexisting wall synthesis machinery is physically linked to underlying microtubules microtubules serve as tracks that guide enzyme complexes the cytoskeleton indirectly controls the architecture of plant tissue Animal tissues more diverse than plant tissus epithelial tissue, connective tissues, muscular tissue and nervous tissue specialised connective tissues (bone, tendon) have abundant ECM ECM directly bears mechanical stresses epithelial tissues have less ECM the cytoskeleton carries the mechanical load from cell to cell knee: bone connective tissues: hard and dense cartilage: absorbs shock, resilient tendons: tough and ﬂexible skin: soft and ﬂexible eye: vitreous humour: soft, transparent Connective tissue Bone bone-forming cells (osteoblasts) secrete collagen matrix calcium, magnesium and phosphate ions become incorporated into matrix, forming hydroxyapatite hard, ﬂexible but not brittle Osteons are composed of concentric rings of mineralised matrix deposited around central canal containing blood vessels and nerves bone is cell-sparse Cartilage present in mnay joints strong ﬂexible absorbs impact without breaking large amount of ECM abundant collagen and proteoglycan ( no mineralisation Vitreous humour between lens and retina clear, viscous gel, primarily water, collagen and hyaluronic acid, virtually acellular Collagen major protein of the ECM abundant in bone, tendon and skin (leather is pickled collagen) large family of ﬁbrous protein —> more than 40 collagen genes in mammals collagen makes up 25% of protein mass in mammals 7 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures produced by speciﬁc cell types within tissues e.g. ﬁbroblasts arrangement of collagen depends on the tissues function collagen ﬁbrils are organised in bundles: monomer—>trimer—>ﬁbril—>ﬁbre arrangement in skin: arranged in a plywood-like pattern allows skin to resist tensile stress in multiple directions arrangement in tendon: tendons attach muscle to bone ﬁbres align parallel along the axis of extension ﬁbroblasts inﬂuence the alignment by pulling and shaping the collagen they secrete Collagen precursors collagen molecules are secreted in a precursor form (procollagen) additional peptide extensions that obstruct premature ﬁbril assembly procollagen proteinase cleaves terminal procollagen extensions —> mature collagen molecule —> self- assembly into ﬁbril incorrect collagen assembly: can cause skin to be hyper- extensible, joint hyper-mobility, fragile skin (bruising) Lecture 5 How do cells attach to the ECM? ECM: ﬁbronectin, collagen ﬁbril, integrin dimer —> help cells attach to the ECM Integrins function as heterodimers, transmit tension across the plasma membrane —> anchored to the cytoskeleton internally and externally linked to the cells adaptor proteins in the cell link to the integrin dimer in the ECM 8 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Fibronectin Fibronectin serves as a molecular bridge between integrins and the ECM: extracellular matrix binding site (via collagen) and cell attachment site (via integrin) Integrin regulation consists of two different subunits (alpha and beta subunits): heterodimer regulated by signals from inside and outside the cell: bidirectional signalling subunits can switch between folded inactive form to an active conformation switches on when it binds molecules on either side of the plasma membrane on/off intern connections with the ECM enable cells to move through tissue reversible mechanical linkage between ECM and cytoskeleton Integrins coordinate cell movement new attachment points are established at the front of a moving cell old adhesions are released at the back cell enabled to move forward Resisting compression collagen provides tensile strength to resist stretching Glycosaminoglycans GAGs: repeating disaccharide subunits GAGs resist compression chains of GAGs are linked to a core protein to from proteoglycans negatively charged —> high cation conc. —> draws in water —> swelling pressure Proteoglycans and GAGs can form very large aggregates aggrecan: two types of GAGs combined, linked to core protein, linked to hyaluronan molecule through link protein tension in collagen ﬁbres balanced by GAG swelling pressure matrix is tough, resilient and resistant to compression (e.g. cartilage in the knee) 9 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Tissue speciﬁc ECM composition dense connective tissue (tendon, bone) has less GAG, more collagen vitreous humour, soft jelly, almost entirely GAG and little collagen GAGs are hydrophilic and adopt extended conformations relative to their mass space ﬁllers for connective tissue other proteoglycan functions include signalling e.g. binding growth factors and control migration of the cells through the ECM Epithelial tissue Epithelial sheets and cell junctions epithelia: sheet of cells joined together side by side barriers that maintain a different environment on either side of the epithelial layer very common structure in animals controls movement of molecules through that barrier; similar to a plasma membrane 4 forms of epithelia: columnar: lining of intestine, long column like epithelial cells side by side, secretion or absorption, great for absorbing nutrients cuboidal: lining of kidney tubules, secretion and absorption squamous: lining of lung, ﬁltration, stratiﬁed: epidermis of skin, protection all cells sit on basal lamina all epithelia have two things in common: side by side arrangement; touching adjacent cells polarity: apical and basal side 10 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Basal lamina thin, mat-like structure, tough sheet of ECM (type IV collagen supplied by stromal cells in the underlying connective tissue and laminin supplied by overlying epithelial cells) laminin: interacts with many proteins including integrins, provides adhesive sites for integrins —> similar linking role to that of ﬁbronectin in connective tissues separates the epithelial cells from the networks of collagen ﬁbres in the underlying connective tissues Epithelial polarity apical surface: free and exposed to air and bodily ﬂuids basal surface: attached to basal lamina external surface (skin): apical faces air internal surface (kidney duct): apical faces lumen polarity is essential for epithelial cell function the gut is lined by absorptive epithelial cells contains secretory goblet mucous epithelial cells: specialised cell type with a different function in the same epithelial sheet Tight junctions allow epithelial cells to function as barriers bind epithelial cells tightly to prevent leakage —> travel molecules are stopped by tight junctions in experiments occludin and claudin proteins seal interacting cells: proteins bind to the same protein on the opposing membrane —> claudin to claudin and occludin to occludin tight junctions segregate membrane proteins— > blocks diffusion of membrane proteins to keep apical and basal domains of the cell separate enables transport of glucose against its conc. gradient through a Na+ driven pump in from the gut lumen and down its conc. (passive transporter) into the bloodstream. Lecture 6 Cell-cell junctions tight junctions stitch cells together adherens junctions join actin ﬁlaments of neighbouring cells desmosomes join intermediate ﬁlaments of neighbouring cells mechanical attachments hemidesmosomes join intermediate ﬁlaments to the basal lamina gap junctions form tunnels of aqueous connectivity between cells 11 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Adherens junctions Cadherins are key components calcium dependent adherence proteins provide mechanical strength by linking cytoskeletons of adjoining cells homophilic binding of cadherins require extracellular calcium ions cadherin protein is linked to cytoskeleton by linker proteins cadherins relocate to positions of cell-cell contact leads to formation of adherens junctions Adhesion belts usually located near the apical cell surface, below tight junction bundle of actin ﬁlaments runs along the cytoplasmic surface of the membrane near the apex —> these bundles are linked to adjacent cells via adherens junctions adherens junctions form adhesion belts around many epithelial cells e.g. in the small intestine connections between adherens junctions and the cytoskeleton allow sheets of epithelial cells to change shape apical bundles of actin ﬁlaments can contract, causing epithelial cells to narrow at their apex —> epithelium rolls up into a tube or invaginate to form a vesicle —> forms neural tubes or retina of eye cup failure in epithelia tube closure causes disorders: spina biﬁda abnormalities of the spinal cord neurological deﬁcits motor sensory function affected 12 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Desmosomes connects keratin ﬁlaments in neighbouring epithelial cells keratin is a type of intermediate ﬁlament desmosomes contain cadherins —> different set of cadherins to those found in adherens junctions desmosomes are found mainly in touch exposed epithelia e.g. skin cadherins bind to plaque of intracellular linker proteins , keratin ﬁlaments are anchored to the plaque Hemidesmosomes epithelial cells must connect to a substratum —> basal lamina integrins in the epithelial cell membrane connect certain ﬁlaments in the cell to the basal lamina (integrins bind laminin in the basal lamina) Epidermolysis bullosa EB is a genetic skin disorder resulting in blisters and lesions gene therapy to correct defect in mutant gene e.g. encoding laminin —> skin grafting Gap junctions small regions of plasma membrane where two cells are very closely apposed in parallel allow small intercellular hate soluble molecules to pass from cell to cell, too small for proteins, sugars etc. connexon protein complexes (composed of six protein subunits) in the plasma membrane of each cell like up to form a water ﬁlled channel between the two adjacent cells physiology: molecular ﬂow creates an electrical and a mechanical coupling between the cells gap junctions between cardiac muscle cells provides electrical coupling allows waves of electrical stimulation to spread synchronously throughout the heart triggers coordinated contraction of cardiac cells that produces each heartbeat permeability: gap junctions are gated, permeability is regulated by extracellular signals 13 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures in retinal neurons, gap junctions close in response to dopamine increased light triggers dopamine release, which switches retina from using rod (good fro low light) to cone photoreceptors (good for bright light & color) Plasmodesmata plants lack tight junctions, adherens junctions, desmosomes and hemidesmosomes structural role carried out by the cell wall in plants, plasmodesmata carry out the function of gap junctions epithelial sheets: sparse ECM, cells rely on connections to one another, they carry mechanical load through a network of interlinked cytoskeletal ﬁlaments connective tissues: ECM is abundant and directly bears mechanical load: particularly collagen Lecture 7 Cell behaviour adherens junctions during compaction in embryo development compaction is a hallmark event of 8-cell stage mouse embryo development E-cadherins essential for the formation of adherens junctions for compaction during compaction, E-cadherin localisation becomes restricted to the basolateral cell-cell contact sites 14 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Types of cadherin expression pattern of some exemplar cadherins e-cadherin, mainly expressed on epithelial cells n-cadherin, expressed on nerve muscle and lens cells p-cadherins, expressed in placenta and epidermis cadherins mediate homophilic adhesion provides mechanism by which cells can recognise similar cells and stick to them thanks to the same cadherin binding site Cadherin-cadherin interaction individual interactions are relatively weak collectively many interactions are strong —> like velcro important because cell-cell interactions commonly break and re-from during embryonic development in adult tissues Selective Assembly of cells cells from different parts of an amphibian embryo will sort out according to cell type cells must be able to recognise other similar cells to sort themselves out suggests that tissue architecture is actively maintained by differential afﬁnity of cells for each other cells adhere to other cells expressing the same cadherin as distinct pools levels of cadherin expression affects afﬁnity of cell-cell interaction higher cadherin levels form a stronger interaction in the centre of the assembly 15 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Neural tube development ectoderm cells express e-cadherin when neural tube pinches off, cells turn of e-cadherin and turn on n-cadherin migrating neural crest turn off n-cadherin and turn on cadherin-7 over-expression of n-cadherin leads to cells failing to leave the neural tube Epithelial-to-mesenchymal transition EMT assembly of cells into an epithelium is reversible - EMT e.g. neural crest cell disposal EMT found in developing tissues EMT involved in cancel cell invasion key features epithelial: columnar morphology, high degree of cell-cell adhesion, cell junctions, specialist apical membrane (brush border), underlying base membrane, cells relatively static mesenchymal: irregular rounded or elongated morphology, loss of pico-basal polarity, front-back polarity, dynamic adhesions, lamellipodia and ﬁlopodia, cells highly motile EMT in cancer progression cancers that arise from epithelial cells —> carcinomas (accounts for approx. 80% of cancers) cancers develop gradually from increasingly aberrant cells many cancer are not diagnosed until a relatively late stage cancer metastasis accounts for 90% of cancer deaths neoplasia: the presence or formation of new abnormal tissue growth 16 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures selectins: specialised adhesion molecule cells surface proteins that bind to carbohydrates expressed in white blood cells and endothelial cells (blood vessel lining) linked to actin cytoskeleton via anchor proteins mediate transit cell adhesions in the bloodstream selectins in inﬂammation inﬂammation: bodys reaction to injury, tissue damaga or infection endothelial cells switch on expression of selectins, which recognise carbs on surface of leukocytes —> white blood cells loosely attach to blood vessel wall leukocytes activate integrins, which recognise endothelial membrane proteins integrins mediate stronger adhesion and tissue invasion (i-can ligand on endothelial cells) Defects in leukocyte adhesion leukocyte adhesion deﬁciency LAD disorders are caused by molecular defects in selectin ligands, integrin expression or integrin activation on leukocytes (rare disorder) n o r m a l l e u k o c y t e : p r e s e n t integrin proteins, present selectin ligands (carbohydrates) LAD-I: absent or decreased integrin expression, defective tight adhesion and invasion LAD-II: glycosylation (carbohydrate) defect, defective initial binding and rolling LAD-III: activation defect, defective tight adhesion and invasion symptoms: frequent bacterial and fungal infections because leukocytes don't ﬁght off infections 17 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Degradation of ECM Cells degrade ECM as well as make it remodelling of the ECM is important —> e.g. inﬁltration of tissue by white blood cells, ECM must be degraded to allow cels to pass between endothelial cells two main groups of ECM-degrading enzymes: Matrix metalloproteinases (MMPs) require bound Ca2+ or Zn2+ for activity Serine proteases have conserved serine residue in the active site some are highly substrate speciﬁc e.g. collagenase degrades only collagen localised degradation of ECM (e.g. collagen, laminin) maintains overall ECM structure but creates enough space for migrating cells to pass through some proteases are secreted in an inactive form —> localised activator converts them to active form —> e.g. tissue plasminogen activator activates plasminogen to dissolve blood clots some proteases are conﬁned by cell-surface receptors e..g found locally at growing tips of some of the migrating cells some proteases are inhibited by the actions of locally secreted inhibitors e.b. tissue inhibitors of metalloproteinases (TIMPs) ECM remodelling in cancer carcinoma in situ: basal lamina is restricting tumour cells from migrating —> basal lamina breakage —> tumour cells invade 18 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Quiz In plant cell walls, cellulose and lignin give strength and pectin resist compression. ECM is very important in plants, collagen is in animals. Microtubules are important for making and aligning the cellulose ﬁbrils, but do not provide mechanical strength themselves. Basal and apical surfaces are deﬁning characters of epithelia. Hemidesmosomes are connections between the cells basal surface and the basal lamina. E-cadherin is required for the early embryonic event of compaction, allowing cells to from a nice spherical ball together. Without E-cadherin there are no strong connections between the cells at this early stage and the cells lose their attachment with each other. This causes the embryo to fall apart and die. In connective tissues (such as bone), the ECM is a large component but they still contain cells. The ECM is the principle part bearing mechanical load. In epithelial tissues, ECM is still substantial and important but the mechanical load is largely carried through direct cell interactions e.g. through adherent junctions. 19 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Growth Lecture 8 Features of growth Anisotropy growth isn't the same in all directions Proportionality parts of our bodies are in proportion to each other Adaptability e.g. plants adapt their growth to the substrate and/or conditions in which they're growing, adapting to behavioural differences Discontinuous scaling cells and molecules aren’t scaled but tissues & organs are Limits to cellular growth transport - the bigger the cell, the more complicated it is to transport substances in and out of the cell communication/coordination - cells depend on the diffusion of chemical messages to coordinate their activities —> a limit halts their communication mRNA synthesis - there is a maximum rate at which mRNA can be made —> controlling restriction point Plant cells don't scale due to their cell walls holding vacuoles in place —> however less cytoplasm is present if the vacuole expands exclusive to plant cells Syncytia a cell with multiple nuclei Polytene chromosomes chromosomes with lots of DNA replication stacked up: extra copies of genes and lots of double stranded DNA parallel to one another Helper cells Granulosa cells have cytoplasmic connection with oocytes to run its metabolism Cell cycle cell division alternates with growth: alternation between growing and dividing this is normally described in a cell-cycle G1: growth 1 S: DNA synthesis G2: growth 2 M: mitotic phase cleavage division: no growth in cells, alternation between (S) synthesis and (M) mitotic division Cyclins are proteins that peak at 4 the different points of the cell-cycle cyclins work by controlling cyclin-dependent kinases (CDKs) 20 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures progression through the cell cycle works by each stage waiting for the last one to have occurred —> cyclins only activate once the one prior has deactivated —> right order transition between G2 and M is blocked if DNA is damaged transition between G1 and S is blocked unless the cell… has enough resources has enough room has external signals asking it to divide has no signals saying “do not divide” Lecture 9 Sources of anisotropic growth Environmental control of growth can directly drive morphogenesis Cells on the outer edge, with more surface are exposed, will be reached by more nutrients —> snowﬂake like model of growth branching out is reliant on agar stiffness Directional cell growth horizontal cell division axis: transverse, increases length veritcal cell division axis: anticlinal, increases circumference front-back cell-division axis: periclinal, increases cell radius Hertwig’s rule: Cells orientated their division planes in the direction that will reduce mechanical stress in tissues Crowding of cells can also orientate expansion uncrowded: random crowded, not signalled: orientated domains shown as an efﬁciency mechanism crowded, signalled: radial The gut and its shape the gut is connected to the rest oft the body via a mesentery removing the mesentery in vitro: curving of the gut fails —> becomes a long tube the gut proliferates much more than the mesentery —> the mesentery’s limited proliferation rate, compared to the gut, generates the guts particular shape due to the gut's need to ﬁt onto the mesentery ‘—> creates the loops Planar cell polarity on cell sheets —> see Olivia's notes Mitosis and astral microtubules Astral microtubules are no interloper, they stick out from the microtubule organising centre If there is no trapping, the cell can divide in any direction - if there is trapping proteins, when the astral proteins hit them, they bind and lock the direction of the division plane 21 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures the trapping proteins bind to both the north and south complexes: trapping leads to the mitosis direction working along a compass —> daughter cells come out on either side Lecture 10 What affects body shape and size? Environmental factors nutrition and freedom of disease e.g. foetal transfusion syndrome: two foetuses share a common placenta, the blood vessels can cross connect and feed only one foetus —> one baby is bigger than the other animals have a clear maximum size for a species, plants and fungi may not size is under genetic control: it can vary in different races of the same species e.g. in different dog breeds within a species, the two sexes can show marked differences in adult size How do fully grown organisms tend to respect a characteristic size? the vitruvian man: span of the out-stretched arms = height etc. —> human body is proportionate internally everything has to be proportionate too Outliers: 1. vitruvian proportions in different scales: larger/smaller than usual pituitary tumours are associated with gigantism —> growth hormone Pituitary gland —> growth hormone —> local tissues —> IGF1, IGF2 (growth factors - secondary signal) When this signalling pathway is compromised (cannot be made or there is no receptors for it), the person will be smaller than usual but still proportionate. growth hormone itself affects post-natal muscle growth directly, but other tissues only indirectly Rabbit leg experiment inhibit the growth of one leg of a young rabbit by restricting blood ﬂow to it unaffected leg grew normally —> lop-sided bunny once inhibition was released, the inhibited leg caught up —> faster growth than the other leg explanation: growth plates growth plate inside the bone is responsible for bone growth inside the growth plate: zone of proliferation —> cell enlargement —> cartilage mature (calciﬁes) —> cartilage cells die —> replacement with bone cells as the bone grows the growth rate decreases 22 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures small bone: distance between growth plate and the eye is small —> better diffusion of signals large bone: distance limits signalling —> in the rabbit, the bone remained small, therefore the bone was still capable of receiving signals quickly and easily The growth plate maintains itself using internal and external signals IGFI (for the zone of proliferation) and CNP (for the mature section) CNP signals to the proliferation cells to replace older cells of their own (signal to mature) PTHrP signal to zone of proliferation to increase stem cells production through an indirect pathway PTHrP —> mature cells —> Indian Hedgehog —> sheath of bone —> zone of proliferation 2. mutations that cause non-vitruvian phenotypes Achondroplasia: activating mutation in FGFR3 FGF signalling via FGFR3 usually inhibits both proliferation and differentiation of chondrocytes activating mutations in FGFR3 cause growth plates full of chondrocytes and premature closure of the growth plates In contrast, FGFR3-knockout in mice show overly long bones this mutation shows: some parts of the body keep growing anyway the amount of tissues (skin, tendon etc.) is still correct for a shortened limb, so tissues cannot be independent Lecture 11 Why is the amount of skin always correct for growth? hypothesis: applying a mechanical force (e.g. excessive stretch - longterm!) to human skin drives skin growth —> skin under tension will respond with mitosis to relieve tension 23 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Mechanically isolated organs: the growth of mechanically isolated organs cannot just be down to tension of skeleton mouse spleen experiment: total mass: all the spleens grew into the size of one normal spleen but if you do this with a thymus, each one grows normally hypothesis: tissues secrete tissue speciﬁc substances that inhibit the proliferation of immature cells Quorum sensing quorum: a critical number autocrine signalling: a cell contains the receptor for the factor which it secretes the signal goes through a second messenger pathway where it meets a threshold detector —> growth is either signalled to continue or stop Tropic theory Time course of neuronal development: at the start of embryonic stage: sufﬁcient increase in number of neurons for mitosis —> ﬁxed maximum at which it begins to fall off mitosis of the embryonic stage is independent of neurotrophins whereas survival depends on them target size increase = neurotrophins increase = more cells survive neurotrophins: NGF, BDNF, NT-3, GDNF, CNTF, HGF General trophic theory: any cell depends on survival signals from other cell types or it will die. “Cells have a death wish”. 24 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Cell Differentiation Lecture 12 Cell differentiation cells are different and there is specialised cell types cell differentiation is the process by which unspecialised cells become speciﬁc cell types this development happens in the embryo (development) continues throughout life (tissue homeostasis) Cell types there’s 200-600 different cell types epithelial cell fat cell nerve cell olfactory neuron red blood cell retinal rod How do cells differ? different macromolecules —> lipids, carbohydrates, proteins different metabolites different morphologies and behaviours It all depends on the presence of different sets of proteins speciﬁc to the cell type e.g. muscle: myosin ||, red blood cells: haemoglobin etc. enzymes e.g. the chromafﬁn cells of the adrenal gland contain enzymes needed to synthesise epinephrine (adrenaline) housekeeping and specialised proteins proteins found in most cell types for essential cell functions proteins speciﬁc to the particular cell type Cell differentiation in embryo development the egg divides to give many cells which are initially unspecialised —> they must differentiate into their various cell types this must be highly organised across the embryo —> cell signalling inﬂuences and coordinates cell function differentiation is progressive —> driven by interplay between cell history (lineage) and environment (cell interactions) intermediate states of differentiation progenitor cells or precursor cells ends in terminal differentiation —> ﬁnal cell form 25 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Germ layers formed in the earliest stages of embryonic development: gastrulation gastrulation: the formation of the germ layers by invagination germ layers are key intermediates: ectoderm, mesoderm, endoderm cells in each germ layer differentiate into tissues and embryonic organs mesoderm cells are more differentiated as a result of speciﬁc proteins (TWIST) characteristic cell behaviour (shape changes —> invagination —> gastrulation) restricted future differentiation potential (e.g. muscle cells) Haematopoiesis example of cell differentiation in tissue homeostasis cell fate determination cell lineage lineage restriction potency Gene constancy the nucleus of fertilised egg cells contains all genes for all possible proteins in all cell types that will be formed differentiated cells produce only a subset of proteins but the nucleus of a differentiated cell can still support development of a new organism BECAUSE all cells in a multicellular organism have a full set of genes —> gene constancy cells are therefore specialised because of differences in gene activity rather than gene content —> differential gene expression Gene expression gene expression is regulated —> switched on or off 26 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures different levels of activity in different cells much of the gene regulation in development is at the level of transcriotpin control of gene expression leads to gene expression patterns the cell-type speciﬁc distribution of proteins and their mRNAs are called gene expression patterns control of gene expression in cells determines: their protein content, which in turn determines: their morphology and function, their behaviour during development (e.g. cell division, adhesion, signalling etc.) gene activity —> cell behaviour —> cell specialisation & tissue formation —> tissue function Lecture 13 Summary regulation of gene expression is central to both aspects of cell differentiation cell specialisation (what makes cells different) cell behaviour during development (how cells become different) Regulation of initiation of transcription RNA polymerase is directed to gene transcription start sites by a series of helper proteins e.g. TATA box is a DNA signal sequence that binds TATA binding protein TFIID this is the same for all protein-coding genes this controls where transcription starts, not when the gene is transcribed there’s different signal sequences next to different genes (in a region called an enhancer) they are recognised and interpreted by proteins called transcription factors if the transcription factor is present in the cell, it binds its DNA signal this activates or represses RNA polymerase function —> the gene is switched on or off for transcription Transcription factors transcription factors may directly recruit RNA polymerase to the TATA box or they may indirectly recruit RNA polymerase by altering chromatin structure through either: Histone Acetyl Transferase (HAT): acetylation loosens histone interaction with DNA, making the gene more accessible 27 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Chromatin-remodelling complex in each case these chromatin modifying enzymes promote RNA polymerase binding and function a gene’s transcriptional activity in a cell depends on what binding sites are in its DNA enhancer sequences whether the appropriate transcription factor TFs are present in the cell a gene always has the binding site for a TF but the TF may not be present Muscle cell differentiation Myoblast: precursor of muscle cells MyoD (TF for muscle cell differentiation) is present in muscle cells during differentiation it activates expression of the gene for muscle myosin || MyoD switches on all the 100s of muscle-speciﬁc genes required for muscle cell differentiation e.g. muscle myosin ||, troponin, tropomyosin etc. —> target genes these genes all have MyoDs E-box recognition sequence mutated MyoD: undifferentiated myoblasts —> cell lineage progression is blocked MyoD can force cells in other lineages to become muscle cells Genetic analysis of transcription factor function TFs belong to a small number of protein families TFs usually work in combinations groups of transcription factor binding sites: enhancer or cis-regulatory element if a gene has binding sites for both TF2 and TF4 … 28 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures either TF2 or TF4 may be sufﬁcient to switch the gene on both TFs may be required together for the gene to switch on some TFs are repressors they may prevent binding of activators recruit proteins that tighten chromatin, making the gene less accessible GATA1 transcription factor zinc ﬁnger TF binds to A/T GATA A/G DNA sequence mutations of mouse Gata1 gene: anaemia due to death of erythroid precursor cells target genes include alpha and beta globin genes erythropoietin receptor haem biosynthesis enzymes spectrin GATA1 is expressed in several blood cell lineages it regulates different genes in each lineage works in combination with different TF in each lineage human mutations: anaemia and blood clotting disorders Lecture 14 What regulates the TFs? transcription factors are regulated through: controlling their activity e.g. protein modiﬁcatons controlling their gene expression (regulated by other TFs) they are regulated by environmental signals (e.g. signals from other cells, hormones, growth factors) developmental history: in cell lineages early transcription factors regulate the expression of later transcription factors complex regulatory interactions: gene regulatory networks GRNs Regulation by cell signalling the cascade reaction in cell signalling results in activation or inhibition of transcription factor —> change in gene expression Regulation by phosphorylation a common post-translational modiﬁcation the large negative phosphate group alters protein this may activate or inhibit the TF Growth factors signals that promote cell proliferation GFs regulate gene expression via phosphorylation of TFs they activate a protein cascade 29 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures mitogen activated protein kinase (MAP kinase) enters nucleus and phosphorylates transcription factors this activates them for target gene regulation Epidermal growth factor EGF is a mitogen and promotes cell proliferation Binding to EGF receptors leads to phosphorylation of MYC (a bHLH-LZ TF) MYC drives transcription of cyclin genes unphosphorylated form of MYC is unstable GATA1 in red blood cell differentiation in conditions of low O2 the hormone erythropoietin EPO is secreted from the kidney it stimulates progenitor proliferation and red blood cell differentiation (erythropoiesis) in the bone marrow binding to EPO receptors on precursor cells causes phosphorylation of GATA1, increasing its activity Developmental history - control of TFs gene expression TF genes are regulated by TFs important for orderly progression through to differentiation (cell lineages) cell fate determination decisions Plant leaf stomata stomata (stoma): for gas exchange opening controlled by osmosis in specialised guard cells three related bHLH TFs regulate sequential steps in guard cell differentiation speechless (SPCH) MUTE FAMA SPCH —> MUTE —> FAMA mutations in each gene —> stomata are missing 30 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Myogenesis - the muscle differentiation process paraxial mesoderm —> somite —> myotome —> progenitor etc —> muscle ﬁbre part of the process (myoblasts —> multinucleate myotube) is regulated by MyoD muscle differentiation pathway: closely related bHLH transcription factors: MyoD, Myf5, Mrf4, Myogenin Paired box homeodomain TF expressed in somite —> Pax3 regulator signals: growth factors maintain proliferation & WNT, SHH (sonig hedgehog), BMP signals from neighbouring tissues gene regulatory network (GRN) Activation of MyoD gene co-regulation of MyoD by a somite-speciﬁc TF (Pax3) and a signal-activated TF (Myf5) if both are present, MyoD expression proceeds MyoD activates its own expression (positive autoregulation) boosts the initial MyoD level given by Myf5 and Pax3 regulation MyoD expression becomes independent of Myf5 and Pax3 regulation Regulation of MyoD protein stability growth factors promote cell division (via cell cycle genes) cycling-dependent kinase phosphorylates MyoD and Myf5 proteolytic degradation muscle differentiation inhibited; myoblast proliferation enhanced Pax3 mRNA is degraded during differentiation Pax3 is required for early muscle development it must later be down-regulated for laters stages to proceed its mRNA is degraded speciﬁcally by RNA interference this is achieved by a speciﬁc regulatory RNA called microRNA miR-1 31 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Lecture 15 Cell fate determination in early embryogenesis Binary cell fate choices in the pancreas function: digestion (exocrine portion), maintaining blood sugar (endocrine portion Islets of Langerhans) cell types in the pancreas: acinar cells (exocrine) duct cells (NaHCO3) Islet of Langerhans (endocrine) α cells: Glucagon β cells: Insulin δ cells: Somatostatin PP cells: pancreatic polypeptide ε cells: Ghrelin Cell fate decisions Embryonic cells male fate decisions at successive steps of development determined by cell lineage and environment lineage (cell’s history) deﬁnes what choices are open to cell at that stage environment (signals & interactions) guides which choice is appropriate Developmental origin of the pancreas an outgrowth from early foregut (endoderm) cell lineage: Cdx2 expression gives gut cell the competence to become pre- pancreatic cells a signal from the notochord (Fgf2) in the mesoderm induces the pre-pancreatic fate in the correct part of the foregut environment: Cdx2 activates Pdx1 expression only if Fgf2 signal is received Muscle differentiation somite derives from mesoderm (lineage) it is competent to form muscle, dermis, bones surrounding tissues inﬂuence differentiation path (environment) myotome MY dermatome DM sclerotome SC The erythroid-myeloid fate choice in blood common myeloid progenitors transiently express both GATA1 and PU.1 gives them the potential to go down either cel lineage 32 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures antagonistic interactions between the TFs so that only one wins, perhaps decided stochastically PU.1 repression of GATA1 GATA1 bound to a target gene or GATA1 gene PU.1 binds to GATA1 knocks off a coactivator protein recruits chromatin methylation protein (tightens chromatin —> less accessible) Master regulators of organogenesis The Drosophila eye consists of highly ordered arrangement of many different cell types photoreceptors, lens cells, pigment cells, structural cells, glial cells etc. mutation of the PAX6 gene: eyeless whole eye is missing not just one cell type role of PAX6 gene: encodes Paired box + Homeodomain transcription factor expressed in early anterior ectoderm and before eye primordium, “master regulator" a single TF can determine the identity of a whole organ the eye comprises several cell types, all of which are lost in eyeless mutant all these cell types are present in well-formed extra eye when PAX6 is topically expressed (experiment where PAX6 gene is expressed on other body parts e.g. wings) loss of one PAX6 gene copy (heterozygote) in other animals —> genetic disease aniridia: loss of iris, defective retina, optic nerve cornea, cataracts 33 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Signalling Lecture 16 Signalling Highly coordinated heap of cells —> signalling allows the cells to coordinate within the organism Cells that are responding to a signal change their state in some way e.g. gene expression, cytoskeleton etc. Evolution of signalling Quantifying signalling capacity by counting ‘signalling genes’. The number of signalling proteins encoded by an organism’s genome gives a measure of its cell signalling capacity When you compare signalling capacity between multicellular and unicellular organisms, multicellular organisms devote 10x more genes to signalling Nature of signal Proteins (insulin, ﬁbroblast growth factors) Small hydrophobic molecules (animal steroid hormones) Small hydrophilic molecules (plant auxins) Gas (ethylene, nitric oxide) Electrical (nerve impulses) Signal ranges This is the distance between the signalling cell and the target cell. 34 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Long range = endocrine Intermediate range = paracrine Short range = contact dependent Sexual dimorphism Differences in appearance between the sexes is largely driven by long range signalling circuits using male and female hormones emanating from the gonads Flowering Long range signalling enables some plants to time their ﬂowering according to the day-length Allows plants of the same species to synchronise their ﬂowering, important for cross pollination A ﬂower that comes out by itself will not get pollinated by another ﬂower The sensor for sunlight is CO protein found in the leaf CO is a plant protein which regulates gene expression, and their levels in the leaf is sensitive to daylight CO accumulates when the days are long In plants, only the shoot, and not the leaf, is competent to respond to FT protein by ﬂowering Key concept in long range signalling = competence This describes the ability of a cell or tissue to respond to a signal Signal transmitted via blood or sap (plant ﬂuid) Many more cells are exposed to signal than respond to it —> only those carrying the receptor for the signal will respond to it 35 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Nervous system Nerve ﬁbres transmit signals within the brain and all over the body Neurons in the brain have elaborate shapes to connect to one another to make functional circuits Nerve signals move along axons and are transferred between cells at the synapse Electrical signal propagates from the cell body of the ‘pre-synaptic cell’ along its axon The two cells contact each other at the synapse where the axon of one cell touches the cell body of another Electrical signal then propagates along ‘post-synaptic cell’ The signalling between cells is over a very short range at the synapse Nerve impulses cause pre-synaptic cell to release neurotransmitter Neurotransmitter travels short distance across the synapse and binds to receptors on post-synaptic cell Triggers nerve impulse in post-synaptic cell Nerve cells also synapse with muscle cells Signal is transmitted over long distances along a nerve cell, the cell-cell signalling is over a short distance at the synapse During nervous system development nerve cells connect with each other via long axons to make functional circuits This involves nerve cells putting out axons that grow to their target Axon navigation is deﬁned by where the growth cone grows As the growth cone navigates through the brain it is guided by guidance molecules secreted by cells along its route Complex axon trajectories are a long journey, so they are broken into several steps In some cases, each step the growth cone is guided by a different ‘guidepost’: a cell that secretes a guidance cue Removal of a guidepost causes axon to become misrouted 36 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures The axon growth cone bears receptors that interact with axon guidance molecules (signals) via a paracrine or juxtacrine (contact-dependent) signalling mechanism 1. Paracrine short range (e.g., Slit/Robo + Netrin/DCC0) The growth cone reacts to these signals as it approaches the cell expressing them 2. Juxtacrine contact dependent (e.g., Eph/Ephrin = Semaphorin/plexin) The growth cone reacts to these signals when it comes into contact with the cell expressing them Lecture 17 Sexual dimorphism Sex determination produces two forms = female or male of a species Gonads = ovaries or testes Gametes = eggs or sperm Secondary sex characteristics = body mass, decoration etc. Mammals In mammals, sexual dimorphism is largely driven by long range signalling from the gonads to endocrine organs Sex of mammals is genetically determined at fertilisation 37 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Sex depends on sex chromosomes donated by sperm (X or Y) and egg (X). Once gonads are determined, they control development of secondary sex characteristics Sry (sex determining region Y chromosome) is a transcription factor only expressed from the Y chromosome (Sry is not a secreted protein signal) If the gonad cells are XY they will express Sry which regulates gene expression leading to testes differentiation During sex neutral developmental the mammalian embryo has both male and female structures. Male determination The Sry signal from the Y chromosome causes the gonads to become testes (not ovaries) and ‘male’ structures to persist and the ‘female’ structures to disappear XY testes secrete male hormones which act on distant target cells Müllerian-inhibiting substance inhibits Müllerian duct (ovaries). Testosterone signals to the Wolfﬁan duct to develop into the vas deferens which will carry sperm Testosterone signals secondary male sex character Testosterone: a hydrophobic steroid hormones that is released into the blood which can pass plasma membrane and bind to receptor proteins to regulate gene expression in target cells Female determination In absence of Sry signal from Y chromosome the gonads become ovaries and female structures persist, and male structures disappear No Mullerian inhibiting substance made so oviduct develops No testosterone made so vas deferens does not develop Ovaries secrete female hormones which signal to instruct female phenotype Key concept in this signal is default This is what happens unless a signal instructs an alternative fate In mammals the development of the female phenotype is the default Complete androgen insensitivity syndrome (CAIS) Some XY (genetically male) people lack functional receptors for androgens and have internal testes but develop as females at puberty This provides evidence that the response to androgen signals produced by the testis is essential for the development of male characteristics 38 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Summary Mammal sex determination is a series of signalling and differentiation events Males: Sry instructs gene expression for testes formation Testes secrete male hormones Male hormones act on target cells at long range Differentiation of secondary male characteristics Females: No signal to make testes develop Ovaries develop and produce female hormones Female hormones act on target cells at long range Differentiation of secondary female characteristics Birds Male and female chickens have distinct appearances Males have a wattle, gold plumage and spur and females have none of that Like mammals, they there are unequally sized sex chromosome W and Z ZZ male and ZW female embryos are initially sexually indifferent Rarely chickens have both male and female features —> they are called gynandromorphs This is a mixture of ZZ and ZW A mixture of genetic cells such as in the ZZ/ZW gynandromorph chicken could arise by two distinct mechanisms with similar outcomes Mosaic = genetic cell change in cell lineage derived from a single zygote Chimera = fusion of genetically distinct embryos The gynandromorph chicken shows that gonadal hormone secretions cannot be sufﬁcient to determine secondary sex characteristics in chickens The female and male sides of the same chicken are exposed to the same hormones secreted by the gonads into the bloodstream —> male (ZZ) and female (ZW) cells must have intrinsic sex identity Hormone signalling from the gonads are important but the interpretation of the signal by target cells is also critical This is an example of cell autonomous sex determination Arguably the primary component is NOT long range signalling Key concept in signalling: cell autonomy This describes whether the phenotype of a cell is determined by intrinsic or extrinsic factors In terms of cell signalling, ‘cell autonomous’ describes situation where external signals do not instruct phenotype The sex character of cells in birds is cell autonomous as the genotype (ZZ or ZW) of the cell determines whether it develops ‘male’ or female’ character 39 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures ‘Non-cell autonomous’ describes the opposite situation where external signals instruct phenotype. The sex character of mammal cells is non-cell autonomous and determined by hormones secreted by the gonads Lecture 18 Paracrine signalling Paracrine signalling operates over 1-100 cell diameters —> 1 cell diameter ≈10μm = 0.01 mm The development of the vertebrate ‘pentadactyl’ limb has provided a useful model system to study paracrine signalling during development The basic 5 digit structure is conserved between tetrapods (amphibians, reptiles, birds, and mammals) Evolution has developed many variations on a theme: Much of the work understanding the role of paracrine signalling in limb development was done in chick embryos The chick develops inside an egg so is accessible to surgical manipulation by removing the shell to access the embryo & then replace in incubator to continue growing 40 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Limb axis nomenclature The axes of the chick wing are the same as the human hand, the wing has 3 digits 2+3+4 [1 + 5 lost during wing evolution]. 41 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures How do cells adopt different fates along each of the three axes? Paracrine signalling plays a central role: 1. Patterning occurs when the limb is small 2. Subsequent growth to reach ﬁnal size 3. Mediated by cells signalling over 0-1mm range Apical ectodermal ridge The apical ectodermal ridge [AER] is the structure at the tip of the limb bud The position of the AER suggests the hypothesis that is important for limb formation along proximal-distal axis This hypothesis can be tested by manipulating the AER If limb formation is affected then this supports the hypothesis Conversely, if manipulating the AER has no impact on limb formation this falsiﬁes the hypothesis 42 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Manipulating the AER disrupts wing development along proximal-distal axis Supports hypothesis that AER important for proximodistal development —> The earlier the AER removed the less wing forms along the proximodistal axis Supports (additional) hypothesis that AER continues to be important over a developmental period (as opposed, for example, of just being important early on and then becoming dispensable) AER produces secreted signalling molecules which instructs limb formation along the proximo-distal axis Fibroblast growth factors (FGFs) are a family of secreted signalling proteins, FGF4 is expressed in the AER so is in the right place at the right time A source of FGF4 can rescue limb truncation caused by AER removal FGF4 soaked bud can replace function of surgically removed AER Morphogens A morphogen is a substance active in pattern formation whose spatial concentration varies and to which cells respond differently at different threshold concentrations For this to work there would need to be: A source of morphogen on one edge of the developing limb anterior-posterior axis Morphogen molecule(s) secreted on one side of limb-bud A resulting anterior-posterior concentration gradient Anterior (high) posterior (low) concentration gradient A speciﬁc range of morphogen concentration would specify each digit along the anterior posterior axis Digits instructed by different concentrations 43 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures The zone of polarising activity Zone of polarising activity (ZPA) of the chick limb: region at posterior edge of the limb bud Plausible hypothesis: the ZPA secretes morphogen molecules that specify digits along the anterior-posterior axis according to the ‘French ﬂag model’ What is the ZPA signal? Search for a secreted protein expressed in ZPA which can induce posterior digits ‘sonic hedgehog’ (SHH) is a secreted protein expressed in the ZPA and is a strong candidate for being the polarising activity 44 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Organiser concept An organiser is a signalling centre that directs the development of the whole embryo or part of the embryo The ZPA is an example of an organiser because it signals to the developing limb and directs differentiation between digits along the anterior-posterior axis Several mechanisms proposed for setting up morphogen gradients All produce a concentration gradient which allow the morphogen to give positional information about where a target cell is relative to the source by ‘reading’ morphogen levels Lecture 19 Spacing hairs on the plant surface Trichomes = ‘hairs’ on plant stems/leaves Evolution has adapted trichomes to many forms and functions: Stinging hairs on nettles Insect trapping hairs on sundew plant Seed dispersal structures on cotton plant Trichomes differentiate from an epidermal precursor cell: Initially the epidermal precursor cells are indistinguishable Then one cell (green) becomes selected as a trichome precursor This cell then differentiates into a mature trichome 45 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures For trichomes to function properly, two separate properties must both be regulated: Trichome spacing (patterning of the plant surface) Trichome anatomy (differentiation of individual trichomes) Trichomes are found at different densities on different plants, even closely related ones This observation indicates mechanism for regulating trichome spacing should be able to allow for variation in trichome spacing Plausible hypothesis 1: Each epidermal cell has a probability of developing into a trichome Whether one cell develops into a trichome or not is independent of the surrounding cells Cells do not need to signal to one another for this hypothesis This mechanism would give a random pattern of trichomes Hypothesis 1 would allow for trichome spacing to be varied Higher density: Increasing the probability of cells independently forming a trichome would increase the number of trichomes so decrease spacing Lower density: Decreasing the probability of cells independently forming a trichome would decrease the number of trichomes so increase spacing Plausible hypothesis 2: Each epidermal cell decides whether to develop into a trichome depending on signalling between it and other cells in the epidermis Whether one cell develops into a trichome or not is dependent on surrounding cells Cells need to signal to one another in this hypothesis This might not generate a random pattern Hypothesis 2 would allow for trichome spacing to be varied Higher or lower density trichome could be achieved by varying the signal Hypothesis 1 and hypothesis 2 could BOTH explain trichome spacing Hypothesis 1 predicts that trichome spacing is random 46 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Careful examination of trichomes on the leaf surface of Arabidopsis reveals they never normally touch ‘No touching rule’ shows trichomes not positioned by chance Argues against hypothesis 1 and suggests that cells do not independently decide whether to become trichomes Hypothesis 2 is consistent with the ‘no touching’ rule Spacing on the insect body surface ‘Hair’ on an insect are the sensory bristles, these allow the insect to sense its environment Each bristle is connected to a sensory neuron sending touch sensation to the brain Closer examination of bristle development shows that each bristle forms at the centre of a group of cells and bristles never form right next to one another Hypothesis: bristle precursor cells inhibit adjacent cells from adopting bristle cell fate Neighbouring cells are competent to develop into bristle but will only do so if inhibition from normal bristle cell is removed This mechanism ensures that only 1 bristle forms at this location Lateral inhibition allows one cell to inhibit touching cells from adopting the same fate 47 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Contact repulsion Organising cell structures by repulsive signalling at very close range Cell-cell contact in the gut Villi help digestion in the gut by increasing the surface area for absorption of digested food The gut is a hostile environment and villi are constantly being eroded and must be replaced Cells are constantly being shed from the tip of the villus Lost cells are replaced by division of stem cells in the crypt These new cells are displaced out of the crypt and replace the villus from the bottom as it is eroded from its tip It turns out that signalling by cell: cell contact helps in the development of the inwardly buckled structure of the crypt Speciﬁcally repulsive signalling between EphB and ephrinB proteins. EphB and EphrinB are transmembrane proteins Cells expressing EphB contacting a cell expressing EphrinB triggers a repulsive response EphB+ and EphrinB+ cells segregate because: an EphB+ cell moving into a region containing EphrinB+ cells will be repelled [and vice versa] 48 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Crypt formation by repulsive cell: cell contact signalling Proliferating stem cells in the crypt express EphB Adjacent cells express EphrinB As stem cells proliferate their numbers increase BUT they cannot escape from the crypt as this would involve mixing with EphinB expressing cells The pressure is resolved by the EphB+ cells buckling downwards to form the crypt Autocrine signalling 49 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Organogenesis Lecture 20 Making and shaping a kidney undifferentiated cells self assemble through a variety of processes into a mature organ mature organ: intricately organised collection of cells with specialised activities that function collectively to execute vital bodily functions building block of a kidney: nephron & collecting duct human kidney contains hundreds of thousands of nephrons, approx. 10k cells, 20 different cell types developmental origins: collecting duct (ureteric bud), nephron (metanephric mesenchyme) Origin of kidney progenitor cells regionalisation of the mesoderm paraxial mesoderm—> separated into blocks and makes somites —> skeletal muscle intermediate mesoderm generates the kidney and gonads lateral plate mesoderm generates circulatory system Kidney differentiation in the intermediate mesoderm early kidney tissue development is revealed by gene expression Lim-1 transcription factor Pax-2 and Pax-8 transcription factor —> these genes are speciﬁcally active in the intermediate mesoderm 50 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures the activation of these genes is the ﬁrst indication that these cells are different from surrounding cells mice with mutations in these genes do not develop kidney structures Lim-1 & Pax-2/8 —> downstream genes —> kidney tissues Kidney speciﬁcation a signal from the paraxial mesoderm t intermediate mesoderm (paracrine signal) induces kidney tissues when intermediate mesoderm develops far away from paraxial mesderm, Pax-2 doesn't develop a signal from the ;lateral side of the embryo (paracrine signal) induces kidney tissue bone morphogenetic protein 2 (BMP2) is a secreted morphogen that directs different cell fates according to concentration —> high conc. for lateral plate tissues, low conc. for paraxial tissues kidney tissue induced at medium level of BMP2 BMP2 & Paraxial mesoderm signal —> Lim-1 & Pax-2/8 —> downstream genes —> kidney tissues How cells self organise to make a kidney Within the intermediate mesoderm population of cells: 2 cell types emerge ureteric bud UB (collecting duct) & metanephric mesenchyme MM (nephrons) —> make up the entire kidney Reciprocal inductive interactions induction: one cell (inducer) regulates the behaviour of another cell (responder) the inducer send a signal that the responder must be competent to see and act on reciprocal induction: a two-way conversation between the two different populations of cells —> the behaviour of each cell is changed provides a mechanism by which different tissues that contribute to the same organ can grow and develop in a coordinated way —> proportionate growth and development this is an important feature of self organization of the kidney —> avoids mismatch in ratio or co- ordination 51 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures The transﬁlter induction assay 1. kidney rudiment dissected 2. MM separated from UB epithelium 3. cultured for a few days —> UB branching & nephron formation UB and MM are interacting with one another both tissues are altered in result of this dialogue growth and branching for the UB mesenchymal-to-epithelial transition MET in MM —> formation of the nephron Branching of the ureteric tree Signal: Glial cell line-derived neurotrophic factor (GDNF) —> active within the MM; Receptor: Ret —> expressed in the nephrite duct and tips of ureteric branches signalling induces the UB to grow into the MM signalling induces further branching to produce a ureteric tree dynamic regulation of Ret and GNDF expression Experiment 1: removing the function of the GDNF gene —> inhibits branching/ no outgrowth of the UB —> GDNF signalling is necessary for budding/branching Experiment 2: adding too much of the gene —> extra branching and outgrowth of the UB occurs next to the source of GDNF —> GDNF is sufﬁcient for budding/branching 52 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Observation of Ret/GDNF signalling with knockout Ret only Ret receptor positive cells contribute to branching, knockout cells don’t conclusion: branching behaviour requires Ret hypothesis: ret alters cells to induce branching behaviour Observation of Ret/GDNF signalling with elevated Ret activity relative levels of Ret activity determines the branching population cells with higher Ret activity exhibit stronger branching behaviour Cell behaviours of branching UB tip swells and bifurcate to form two new branches (occasionally form 3 branches) branch extends and bifurcates again branched organs often borsch with different patterns (e.g. kidney and lung branching) What cellular behaviours drive branching? Differential cell proliferation UB tip cells proliferate more than trunk —> suggests that differential cell proliferation contributes to UB growth elevated proliferation & branching at localised source of GDNF —> differential cell proliferation driven by Ret/GDNF signalling Cell rearrangement Ret signalling mediates rearrangement of epithelial cells before budding to forth UB tip domain Localised remodelling of the extracellular matrix 53 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Lecture 21 Overview of nephron development 1. metanephric mesenchymal cells condense around UB 2. Transform into small cysts with epithelial characteristics (called renal vesicles) —> Mesenchymal-to-epithelial transition MET 3. Differentiation & morphogenesis. Fuses with UB 4. Blood vessel progenitors invade and construct glomerular capillaries 5. Nephron differentiation e.g. specialised transporting segments of the nephron Making nephrons The transﬁlter induction assay 1. kidney rudiment dissected 2. MM separated from UB epithelium 3. cultured for a few days —> MM with UB —> MM - MET to form nephrons. —> Alternatively, if MM is isolated, it would die. —> Therefore, MM on its own isn't capable of producing a nephron. A signal from UB to MM induces MET. Experiment WNT signalling proteins could induce MET and nephron development in isolated MM Wnt9B is secreted from UB (paracrine signal) induces MET in MM 54 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Renal vesicle size renal vesicle size is approximately equal - how is it measured? Wnt9B also induces Wnt4 within MM Wnt4 acts in an autocrine manner (has an effect on the cell by which it is secreted) to propagate mesenchymal aggregation Wnt4 signalling allows renal vesicle to grow by recruiting more MM until Wnt4 concentration reaches a threshold (quorum sensing) —> RV then begins to differentiate this is a simple mechanism to ensure an appropriate size for RV MET - development of epithelial characteristics function of epithelial tubes: convey and modify ﬂuids development of epithelial structure: generate a tubular shape and form a lumen develop adhesion junctions e.g. cadherins (adherens junctions) establish apicobasal polarity development of occluding junctions (tight junctions) to ensure tubes are not leaky Pattern formation and regional differentiation in the nephron Nephron: approx. 10k cells Nephron cells differentiate into 20 distinct cell types with distinct functions Organised into segments along the Proximo-Distal (P-D) axis glomerulus - proximal tubules: recovery of salt, water, metabolites distal tubules: K+, Na+, Ca2+ regulation Stages of nephron formation: renal vesicle comma-shaped body s-shaped body mature nephron Bartter syndrome: Low potassium levels, increased blood pH, excessive loss of Na+, Cl-, K+ in urine caused by defect in the distal parts of the nephron and the loop of Henle (essential for producing hypertonic urine) Plumbing the kidney Development of glomerular capillaries migration of endothelial cells into developing glomerulus with podocyte precursors (s-shaped stage) —> Bowman’s capsule containing glomerular capillary and podocytes (mature glomerulus) 55 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Chemotaxis chemoattractant: cells move toward a signal source (up the conc. gradient) chemorepellent: cells move away from a signal source (down conc. gradient) Podocyte precursors release a chemoattractant which induces the migration of the endothelial cells into the developing glomerulus —> the cells move up the conc. gradient The chemoattractant signal is known as vascular endothelial growth factor (VEGF) endothelial cells are competent to respond to this signal because they express the VEGF receptor on their surface knockout VEGF: no migration of endothelial cells —> capillaries don't develop growth and development between tissues is proportionate within the organ —> Intimate relationship between podocytes and endothelial cells ensures that blood delivery (glomerular capillaries bring blood to kidney) and blood ﬂirtation (podocytes) is matched 56 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Shaping the tubules Polycystic kidney disease is one of the most common human genetic diseases (i) autosomal dominant polycystic kidney disease - ADPKD incidence of 1:1000 (ii) autosomal recessive polycystic kidney disease - ARPKD progressive formation of large ﬂuid ﬁlled cysts, increase in tubular diameter, nephron loss, kidney failure Two ways to shape an organ: (1) Oriented cell division in renal tubules cell proliferation tubules increase in length but not diameter suggests that orientation of cells is aligned long axis of tubule and not around circumferential axis while they separate example of anisotropic growth defects in the orientation of cell division result in abnormal tubule shape and the formation of cysts (2) Cell rearrangement in renal tubules renal tubules are sculpted by cell rearrangement renal tubule diameter reduces from 11 or 12 to 3 or 4 cells during embryonic development rearrangement is driven by cell intercalation highly ordered cell intercalation increases tubule length and decreases tubule circumference: convergent extension (converging in one axis and extending in the other axis) Renal tubule diameter: number of cells surround the lumen 57 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Tissue Homeostasis Lecture 22 What is a stem cell? At a tissue level it is a cell that renews, continuously turning over tissue Blood Skin Germ cells Cell that can renew a tissue undergoing regeneration after injury Muscle Pancreas Liver Cell biological deﬁnition: a cell that has a choice: to divide to give rise to: a) an exact copy of itself or b) a differentiated cell Tissue renewal could be considered a luxury in terms of energy use and evolutionary ﬁtness Tissues containing cells that meet continuous insults from the environment need to have expendable cells and continuous turnover Blood: Adult human red blood cells (2 x 1013 cells) last only 100 days To renew these cells we produce 2 million new red blood cells per second Gut: Lining of the gut (epithelium) is renewed every 5 days in mice 2 × 108 cells produced in mouse small intestine per day Back-of-envelope calculation mouse is about 1/3000 of human weight, so a human-sized mouse small intestine should produce 6 x 1011 cells/day Skin: Is completely replaced every 3-4 weeks in mice Why stem cells? Some differentiated cells are so highly specialised they lack a nucleus and so cannot replenish themselves Some cells (in the immune system) lose DNA sequences during differentiation So, these tissues need a reservoir of pristine cells Stem cell proliferation Stem cells divide very slowly and is immortal It generates very fats-dividing daughters (‘transit amplifying’ cells), which have a limited lifespan The ﬁnal differentiated cell may be completely non-proliferative, and also have a limited lifespan 58 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Symmetrical and asymmetric division Generation of asymmetry Effects of radiation Increasing radiation dose on bone marrow decreases survival Chromosomes were labelled by radiation itself: it induces (random) chromosomal breaks, which repair to give abnormal chromosomes that can be detected in nuclear preparations (‘chromosome spreads’) Total dose to the mouse’s own haematopoietic system is lethal, but dose to the transplanted bone marrow induces only limited chromosome damage It was found that all colonies were pure and not mixed Spleen colonies contain many differentiated cell types. So this was the ﬁrst demonstration that single bone marrow cells can differentiate to many cell types of the haematopoietic system 59 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Lecture 23 The Drosophila reproductive system: asymmetric division of a visible stem cell Genetics is much easier in Drosophila than in organisms that have long lifecycles and few offspring The function of many genes is known through mutations that disrupt them There are two clusters of germarium –-> this is where the eggs are laid Germarium manipulation: possible to introduce DNA or remove speciﬁc cells or organelles Stem cell and niche Niche: the environment of a stem cell that provides the factors needed for stem cell maintenance 60 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures The Drosophila female germline stem cells (GSC) 2-3 germline cells per germarium undergo asymmetric division to produce a cystoblast that divides 4x to produce the progenitors of 15 nurse cells and 1 oocyte Both male and female GSC are closely associated with somatic cells Features of the Drosophila germline Maintenance and number regulated by a niche (environment) Undergo asymmetric divisions segregating localised determinants (intracellular components) BAM BAM (bag-of-marbles) —> loss of function mutants have excessive numbers of germ cells BAM is not transcribed in the germline stem cell and is transcribed in the cytoplast The germline stem cells are the only cells attached to the cap cells The cap cells secrete molecules, these are the molecules that keep BAM off in germline stem cells 61 of 79 Downloaded by Maya Stables ([email protected]) lOMoARcPSD|35018169 CTO Lectures Spectrosome The spectrosome is a condensed organelle composed of spectrin, an actin-binding, contractile protein that normally mediates cell adhesion Ablating the spectrosome leads to random orientation of division planes, so it is responsible for anchoring the mitotic spindle The cell that inherits the spectrosome remains a GSC The GSC is anchored to the niche by the adherens junction, which is likely to be necessary for signalling to just the GSC Mammalian stem cells in the gut The epithelium of the small intestine consists of the villi (plural of villus), which absorb nutrients, and the crypts, which contain the stem cells for the gut Differentiated cell types in gut: There is a ﬁnal cell type: these cells expres

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