Overview of Cell Signalling and Development

LECTURE 7 Cell signalling is the cascade of processes by which an extracellular stimulus (typically a neurotransmitter or hormone) effects a change in cell function Fight or flight response is mediated by adrenaline whereas the nerve to nerve signlling is mediated by glutamate Signalling through ion channels or receptors Ion channels are proteins in membrane and when ions flow thru these channels this initiates the signalling cascade , Ion channels are often selective for a particular ion Receptor binds an extracellular ligand triggering a cascade of events which leads to a change ION CHANNELS Ca2+ is a ubiquitous signalling ion, calcium spikes within egg cells when the sperm fertilises the egg , so calcium is needed to activate the egg and begin cell division These processes are dependent on calcium signals Huge time range over which calcium can regulate processes, calcium takes hours in fertilisation but nerve to nerve transmission takes microseconds Charge prevents free diffusion of ions so require channel and carrier proteins Channel proteins are water-filled pores, they allow charged substances ions) to diffuse through the cell membrane, The diffusion of these ions does not occur freely, most channel proteins are ‘gated’, meaning that part of the channel protein on the inside surface of the membrane can move in order to close or open the pore Channel p[roteins normally remain similar in shape but carrier proteins undergo a conformational change allowing the ion to be moved down conc gradient The binding site of the carrier protein is open to one side of the membrane first, and then open to the other side of the membrane when the carrier protein switches shape There are also ion gradients within organelles ER has higher conc inside than out Low pH is high proton conc so higher in lysosome than cytosol Subtler grad in mito , more protons cytosol than mito When move down conc gradient passive , when move against active and need energy from atp Movement is determined not only by conc grad but also electrical grad , alw move from positive to negative membrane potential so if conc gradient is opposite to direction of electrical gradient like in the third example then movement can be inhibited Ion gradients are generated by active transport, it requires atp hydrolysis Ejects sodium out of cell and potassium into cell , despite the actual concs of sodium being greater outside and potassium being greater inside . remember the table Atpase pump transports 3 sodium ions out for 2 potassium ions in Look at arrows , an inward sodium electrochem grad and outward potassium gradient , how do we activ transport them opp direction? P TYPE stands for phosphorylation as we n=know phosphate involved in cycle steps : na can bind to binding site on open side of protein , then atp hydrolysed and pi bind to protein causing conformational change such that na can no longer bind and is released to other side of membrane , conformai=tional change also causes K to be able to bind to its binding site , then dephosphorylation occurs so another shape change K is released to other side as no longer fits in binding site and resting protein shape is restored Pmca is a calcium carrier protein dont need to know structure but know its very complex got transmembrane structures plus loads of domains PM plasma membrane Calcium Atpase Ejects calcium outside cell , from low inside conc to high outside against conc grad SER sarcoplasmic(muscle|) reticulum/ ER endoplasmic reticulum, Calcium Atpase Transports from cytoplasm into lumen of ser Remember conc in er is higher inside than out so this is against conc gradient from out to in If cotransported ion same direction its symport if not then antiport N(sodium)CalciumXchanger We transport 3 sodium in for one calcium out , na drives movement against the ca gradient to move it out despite ca being higher out than in This is antiport as na and ca move opp Cotransport is secondary active transport Vg are opened by changes in voltage Ligand gated opened by attachment of small intra or extracellular molecules Mechanically gated are opened by stretch/bend/ mechanical force Learn this structure 6 transmembrane domains , first 4 make up v sensing domain which responds to the changed in v , last 2 make up pore domain in which there is hole thru which ions can flow Evolution can put these building blocks tg in dif ways to perform dif functions 4 of the building block subunits can make a vg K channel , whereas by joining each building block into a single protein we form vg naca channel He first crystallized/ found structure of K channels How do K channels only allow potassium to pass thru/ why r they so selective? Well if pores were just holes and we know na is smaller than k we would think na would also pass thru but 3d structure is more complicated the 4 middle amino acids are known as the selectivity filter and they have 4 o atoms which bind to a k ion stabilising it , whereas only 2 o bind to na which is less energetically favourable Each k ion channel was found to have positively charged amino acid residues : arginine and or lysine , because they are + charged they can detect changes in voltage which control opening and closing of the channel , these residues are in the fourth domain of the vg channel S4 regions point down but when voltage change (depolarisation) s4 moves up and it is this that yanks on pore opening and allowing k to move thru LECTURE 8 Membrane potential is the difference between the voltage inside and outside the cell , typically -70mv at rest Cells are generally electrically neutral; the 5 negative charges neutralise the 5 potassium charges ,positive charges are generally fixed by matched anions if cell membrane permeable, shown by dashed line, the potassium will move down its conc gradient outside of the cell creating voltage difference so +2 charges outside and uncovered 2 negative charges inside , difference between the charges is 4 as 2- -2 V = -4 because cell is now negative relative to the outside However due to electrical difference the positive charges are gonna wanna move back into cell from + to - so potassium moves in via electrical gradient , so now weve got a V = -2 , these forces eventually balance out outward movement by chemical and inward by electrical gradient until reach an equilibrium , T is time btw Final potential is eq potential Co/ci is gradient Z is valence/ charge of ion Rest are physical constants Temp is calculated in kelvin and done under physiological conditions so around 37 This is the electrical potential/difference that would arise if membrane was permeable to k Hence -70 is close to -90 Due to the presence of leak channels more permeable to k as k can go thru these k leak channels The new term P refers to permeability Eq takes into acc the permeability of each ion relative to each other Permeability of k is much higher than cl and na at rest P of K is close to 1 and the other close to 0 so we cna cancel out these terms and we are left back with nernst The longest dendrite referred to as axon Nerve cells connected to other nerve cells Transient change in membrane potential known as action potential allowing neurons to communicate at junction called synapse During transmission vg channel involved Action potentials arise due to the concerted action of voltage gated Na+ and K+ channels During stim u get small depolarisation so membrane become less negative, this opens vg sodium channels allowing sodium into cell further depolarising membrane Now because these vg sodium ions are open the p value for na becomes close to 1 and the rest of the terms close to 0 so cancel and the equation becomes the equilibrium potential of sodium , so basc the resting membrane potential is the eq potential of k and the depolarised potential is eq potential of sodium Voltage–gated Na+ channels depolarize the membrane from the equilibrium potential for K+ (-90 mV) to Na+ (60 mV) Ap is transient so not long lasting so the depolarisation is followed by repolarisation The na channels become inactivated , only open for short period , stops na coming in , the vg k channels then open , they are slower to open than vg na channels , opening vg channel for k takes membrane potential to the eq potential of k , as the k ions move OUT of cell The delay causes the shape of the hill , up and then down Depolarisation of the membrane triggers opening of vg ca channels , ca ions flow in down conc gradient causing neurot vesicles to fuse with presynaptic membrane and releases nt into synaptic cleft Ligand gated ion channels are present on postsynaptic cells , they respond to the nt release at synaptic cleft Voltage-Gated Channels 🔌 Location: Mainly on the presynaptic membrane * These open in response to a change in membrane voltage. * Example: When an action potential arrives at the presynaptic terminal, voltage-gated calcium channels open → Ca²⁺ enters → triggers neurotransmitter release. Ligand-Gated Channels 🧬 Location: Mainly on the postsynaptic membrane * These open when a specific neurotransmitter (ligand) binds to them. * Example: Acetylcholine binds to receptors on the postsynaptic side → Na⁺ channels open → depolarization of the postsynaptic neuron. Epilepsy occurs due to messing with balance of inhibition and excitation Glutamate is the key nt that activates these channels 4 subunits come together to form ion channel , tetramers just like vg channels Unlike vg channel where s5 and s6 form pore u can see in this lg channel the transmembrane structure domains within the box form the pore which is also inverted hence the inverted pore topology , tm2, transmembrane 2 forma the invented pore Allow flow of na and ca into cell down conc gradient and due to increasing membrane potential inside cell , they are excitatory Gaba is key nt that activates these receptors 4 transmembrane regions , but 5 subunits come tg to form channel unlike glutamate receptors Also referred to as cys loop, they are part of cys loop family , bc we find disulfide bond in extracellular loop, they allow flow of chloride ions into cell and because are neg , influx of neg into cell so they are inhibitory Respond to ach Pentameric so 5 subunits come tg to form channel , belong to same family as gaba , so cys loop family Respond to sodium allow na to go in , so excitatory They can also be found on postsnyaptic muscle cells at the nmjunction nACh receptors, like GABA, are part of the Cys-loop receptor family, with pentameric structure and 4 TM domains per subunit. Glutamate receptors are tetrameric, with an inverted topology and a unique pore-forming TM2 loop. Voltage-gated channels differ structurally with 6 TM segments and a voltage-sensing S4 domain. Not alw nerve to nerve communication but also nerve to muscle The ach in cleft opens up nicotinic ach receptors and influx of na gets activation of muscle cells which opens up vg na channels and signalling continues in muscle cells , causing contraction Depolarisation of muscle cell causes opening of ca vg channels causing influx of ca from INTRACELLULAR stores, this causing contraction , whole process called excitation contraction coupling, excitation is na depolarisation and contraction is ca part Lecture 9 Receptor proteins are found typically at cell surface but also sometimes found inside cell so the ligand must be smth that can cross the membrane Addition of Phosphate turns protein on and when remove turns off Phosphate comes from atp , depicted as appp so adenosine and 3 phosphates and the terminal phosphate is transferred onto signalling protein to turn on Second switch is gtp , gtp shown as gppp and terminal phosphate transferred to turn on protein When gtp is bound to signalling protein then protein is turned on , when bound to gdp then off state molecular switches in cell signalling: (A) proteins activated by phosphorylation (adding a phosphate group) and turned off by dephosphorylation, and (B) GTP-binding proteins switched on by binding GTP and off when GTP is hydrolyzed to GDP. Second messengers are small molecules which act as relays in intracellular signalling cascade Dont need to know structures G protein GPCRs are activated by a variety of ligandsHormones (e.g. adrenaline) Neurotransmitters (e.g. glutamate) Odorants But many are “orphans” Heptahelical receptors Heterotrimeric cus 3 parts alpha beta gamma In unactivated state alpha bound to gdp In activated alpha bound to gtp , alpha dissociates from beta and gamma , Odorants work via g protein , things that smell , don't know what activates many types of gpcr Beta and gamma can also activate signalling cascades These are made/mobilised during activation Different g proteins activate different cascades , alpha s subunit activate adenylyl fyi Regulation of messenger synthesis by G-protein α subunits αs subunits activate adenylyl cyclase αi subunits inhibit adenylyl cyclase Adenylyl cyclase is reciprocally regulated by distinct α subunits Catalytic subunits act on target by phosphorylation Structure of PKA: * PKA is inactive when its two catalytic (C) subunits are bound to two regulatory (R) subunits — this forms a tetramer (R₂C₂). * Each R subunit has two cAMP binding sites. 🔹 Activation by cAMP: * When cAMP levels rise 1. cAMP binds to the regulatory (R) subunits. 2. This causes a conformational change that releases the catalytic (C) subunits. 3. The freed catalytic subunits are now active enzymes. These catalytic subunits phosphorylate target proteins at serine or threonine residues using ATP. Phosphodiesterase + H₂O: This enzyme breaks the cyclic bond in cAMP using water (hydrolysis).converted to 5′-AMP (right structure): This cannot activate PKA, so the signal is terminated. Phospholipase c enzyme converts pip2 into dag and ip3 Acts at endoplasmic reticulum Ca bp = calcium binding protein like calmodulin Ip3r is receptor Signalling thru ion channels and receptors 1. Gqα activates PLCβ, which cleaves PIP₂ into: * IP₃ (inositol triphosphate) * DAG (diacylglycerol) 2. IP₃ binds IP₃R (receptor) on the endoplasmic reticulum (ER) → opens Ca²⁺ channels → Ca²⁺ released into cytosol. 3. DAG stays in membrane and, with Ca²⁺, activates PKCγ. 4. Released Ca²⁺ binds calcium-binding proteins (CaBPs) like calmodulin (CaM), activating downstream targets. Can bind 4 ions of calcium Ca²⁺/calmodulin-dependent protein kinase II (CaMKII Inactive CaMKII: The catalytic domain is blocked by an inhibitory domain. Ca²⁺ binds to calmodulin, and the complex binds CaMKII → partial activation. Autophosphorylation: CaMKII autophosphorylates itself using ATP → fully active. Memory effect: Even after calmodulin detaches, the kinase remains active (Ca²⁺-independent) due to autophosphorylation. Cant metabolise calcium so shuttles move calcium away from target The pumps and exchanger are all activated and turn off the reaction Dag works with ip3 to activate 1. IP₃ releases Ca²⁺ from the ER. 2. DAG and Ca²⁺ together activate PKC, which moves to the membrane. 3. Activated PKC phosphorylates target proteins to produce cellular effects. Enzyme linked receptors dont have set structure , such as rtks U get activation very quickly by growth hormones like epidermal growth factor , fgf, etc, unlike gpcr which have to go thru many steps Dimer is 2 subunits coming together 1. Activation by Growth Hormones * RTKs are membrane-bound receptors activated by growth factors like: * EGF (Epidermal Growth Factor) * FGF (Fibroblast Growth Factor) * PDGF (Platelet-Derived Growth Factor) * Compared to GPCRs, RTKs initiate faster signaling due to direct activation of internal enzymes., rtk dont need second messenger 🔹 2. Dimerization and Autophosphorylation * Ligand binding (e.g., FGF, PDGF) causes dimerization (two RTK subunits come together). * The dimerized receptors autophosphorylate (add phosphate groups to each other’s tyrosine residues using ATP). phosphate tagging activates the intracellular signaling domains. 🔹 3. Signal Transduction * Phosphorylated RTKs act like docking stations for downstream signaling proteins- phosphotyrosines (p-Tyr) now recruit specific intracellular proteins which either get activated themselves (e.g., enzymes like PI3K), or link the receptor to other signaling pathways p switching , activation rtk by autophos, then recruiting 3 proteins needed in transduction, one called grb2 activator protein as grabs next protein in cascade known as sos which activates ras , if ras mutates gain of function and can cause cancer , ras is a g protein Ras In presence of sos bound to gtp and not when not activated by sos This diagram explains part of the MAPK (Mitogen-Activated Protein Kinase) signaling pathway, a key cell signaling cascade that controls growth, division, and survival: 1. Stimulus (e.g., growth factor) binds to a receptor tyrosine kinase (RTK). 2. RTK autophosphorylates, creating docking sites for proteins. 3. Grb-2 (an adaptor protein) binds the phosphorylated RTK. 4. Grb-2 recruits Sos (a guanine nucleotide exchange factor). 5. Sos activates Ras (a monomeric G-protein) by exchanging GDP for GTP. 6. Active Ras triggers a kinase cascade: MAPKKK → MAPKK → MAPK, leading to cellular responses. Note: Mutated Ras (always active) can lead to cancer by promoting uncontrolled cell division. What is a mitogen, chemical that bind to trigger mitosis Map kinase phos in order to be activated , what phosphorylates upstream it is called map kinase kinase and even more upstream protein map kinase kinase kinase which activates downstream This diagram shows the MAPK signaling cascade: 1. Stimulus activates receptors. 2. Transducers (e.g., Grb-2, Sos, Ras) pass the signal. 3. A phosphorylation cascade occurs: * MAPKKK (e.g., Raf) activates * MAPKK (e.g., MEK) which activates * MAPK (e.g., ERK) Each step phosphorylates the next, ultimately leading to changes in gene expression or cell behavior. Fgf singalling thru map kinase pathway in a process mesoderm induction for instance, Grb-2 and Sos are not G-proteins—they’re adaptor and exchange factor proteins, respectively. G protein is ras Ras is a small (monomeric) GTPase, meaning it binds GDP (inactive) or GTP (active). Rewatch edit notes to the above lecture ^ Lecture 10 - developmental bio 1 Developmental biology How do we go from an undifferentiated egg to diversity of differentiated cell types Model organisms: simple systems to understand more complicated ones There is Conservation of processes and principles across model systems so we can study human embryo by studying zebrafish etc cus same mechanisms embryo micromanipulation refers to precise techniques used to physically handle or alter embryos using fine instruments under a microscope A genetic knockout is when a specific gene is completely disabled or deleted in an organism, so it no longer produces its protein product.Purpose: To study what that gene normally does by seeing what happens when it's missing. Mammalian , vertebrate so similar to humans Not as high reproductive rate as other organisms Knocked out genes in mice to learn abt development won nobel Expensive lots of cages Long life cycle so takes several months for a mouse to develop Large embryos so good micromanipulation Hard to make transgenic chickens Big tanks of circulating water so expensive 2-3 months from embryo to adult so not as long as mice Can make transgenic fish , gfp mutants etc ________________ Been worked on for a long time , old model system , 1995 knocking out genes to understand development Free soil living worm found in compost heaps , 1mm long, this is a grant proposal for working on nematodes showing advantages of model systems Few cells fixed cells can be an advantage First animal to have its connectome fully transcribed so how each neuron connects to each neuron Human would be similar size to mouse embryo Chicken egg derives all its nutrition from the egg so big but we derive from placenta so egg can be smaller CHICKEN EGG Maternal FACTORS(INSTEAD OF GOODIES) GIANT NUCLEUS , lots of mito cus need energy to move Acrosome full of enzymes that can break zona pellucida Ca wave in egg ends meiosis 2 , triggers enzymes Contractile ring forms around embryo to separate the 2 cells Both centrioles come from father in c elegans If egg with all proteins needed small, if placental development then less yolk but other protein involved in processes still there but if not provided anything egg big lot of yolk No growth ! half in size when double Discoidal is when restricted to disk of cells that sit on the yolk Superficial is where division occurs at edge Usually synchronous so divide at same time in waves , Often synchronous initially—especially in early embryonic stages like in frogs and mammals, where all cells (blastomeres) divide at the same time. But it becomes asynchronous later as development progresses and cells start dividing at different rates based on their position and function. Xenopus develops externally not placental so large eggs * Meroblastic cleavage: Only part of the egg divides (not the whole thing), because the yolk is too dense to cleave. * Discoidal: Division happens only in a small disc-shaped area at the top of the egg (the animal pole, where there's little yolk). * Early cleavages (first 3 divisions): * Are meridional (top to bottom) * Only cleave the yolk-free cytoplasm * 4th cleavage: * Is equatorial (side to side) * Produces two layers of 8 cells sitting like a mound on top of the yolk (which remains uncleaved below) Summary: In zebrafish, only the upper disc of cytoplasm divides, while the large yolk stays intact—this is called discoidal meroblastic cleavage. Firstly embryo is bag of proteins, The dots are all nuclei there is no pm as no cytokinesis occurs until stage 10, they then go to periphery of egg and division occurs at the edge of the membrane 1. First 8 divisions (images 1–8): * The fertilized egg begins dividing its nucleus, but no cell membranes form yet. * These nuclear divisions happen in the center of the egg (no cytokinesis = no individual cells yet). * The embryo is called a syncytium (a single cell with many nuclei). 2. Nuclei migrate outward (images 8–10): * Most of these nuclei move to the outer edge (periplasm) of the egg. * This creates the syncytial blastoderm—a single cell with many nuclei just under the surface. 3. 4 more divisions at the periphery (images 10–12): * The nuclei continue dividing near the outer surface. * Still, no individual cells—just lots of nuclei in one shared cytoplasm. 4. Cell membranes form (images 13–14): * The plasma membrane folds inward around each nucleus. * This forms individual cells for the first time, creating the cellular blastoderm. Holoblasic cleavage occus in cells w little yolk ________________ Human is similar to mouse In order to be this rapid the early cleavage divisions have modification of the cell cycle Its usually just S mitosis S mitosis Only nuclear divisions no pm division S phase can happen quicker bc mum alw provides many of the proteins required in dna replication in the egg In droso ONLY no cytokinesis but it is there in other organisms Asynchronous means different cells divide at different times based on what they are gonna become , mostly early cleavage stage divisions are synchronous and modified cell cycle This transition from syn to async is mid blastula transition, happens at mid blastula stage , mother loads proteins into egg cus zygotic transcription hasn't started yet , so dna of embryo is not being transcribed or translated into proteins in these early stages , this also occurs at the mbt Rna translated to make proteins is loaded into egg by mum , at mbt zygotic genes are now turned on so can make own proteins , this is true of most model orgs but not nematode where zygotic transcription starts at 4 cell stage Developmental lecture 2 Ingression is individual cells delaminating from surface moving interiorly Imvagination is not individual cell movement but coordinated movement of a sheet of cells moving inwards creating an invagination Involution is cells migrating across the under surface of the outer layer , similar to invagination Each embryo has vegetal pole and animal pole In animal development, the animal pole is the region of the egg with less yolk and more cytoplasm, while the vegetal pole is the opposite end with more yolk and less cytoplasm, influencing early embryonic development. Common in chick, mammals, sea urchin Sea urchins, droso, nematode also display invagination Seems as though some external force pushes and forces cells inwards but force generated by apical constriction by the actin myosin skeleton - this is only for the invagination In order to generate enough force to push the mesenchymal cells all the way to the animal pole use another mechanism called convergent extension gut=endoderm Lemellipodia all begin to face same directions then carry out convergent extension Cells migrate around to the dorsal side of the embryo , which is thickest part and forms most of the tissues * Ectoderm (blue) stays on the outside. * Mesoderm (red) and endoderm (yellow) move inward through the blastopore via a process called involution. * These internalized cells migrate along the inner surface, forming the archenteron (the future gut). * Movement is directed toward the dorsal side (top), which becomes the main body axis and forms most tissues. Convergent extension towards the dorsal side aswell Blastoderm sits on yolk: * The early embryo (blastoderm) sits atop the large yolk. * It consists of two layers: a deep layer and an outer enveloping layer. Spreading of blastoderm by epiboly: * The blastoderm spreads to cover the yolk via a movement called epiboly, where cells thin out and expand over the yolk. Gastrulation begins with involution: * Cells at the margin of the blastoderm move inward (involution) at the dorsal side, initiating gastrulation, which establishes the body plan. Convergence and extension: * Cells move medially and elongate the embryonic axis through convergent extension, especially toward the dorsal side, helping to form the notochord and other midline structures. Via compaction end up with blastocoel Inner cell mass form all of the tissues Sync starts to form lacunae for transport of nutrients become blood vessels Epiblast cells ingress at the primitive streak Mesodermal cells spread out laterally Myotome forms skeletal muscle Somites form every 30 minutes in zebrafish, every 90 minutes in chick and every 5 hours in humans Line of cells neural plate cells, thicken and then apical constriction causes neural tube closure They are between roof of neural tube and external ectoderm despite dif sizes of eggs , at end of gastrulation all vertebrate embryos have remarkable similarities , see same shape and structure , but end up looking super different at end Phylotypic stage at end of gastrulation where all look very similar and are molecularly similar This is in situ hybridisation , method of revealing where certain mrna are , brain of mouse chick frog and fish can see 2 expressions of this gene , shared molecularity , very similar at phylotypic stage Developmental lecture 3 - i didnt rewatch but u could if there's stuff missing Fluorescent labelling shows expression of dif genes Green is dna , red is differentiation drivers Transcription factors are being turned on specifically in these germ layers , t.f binds to a promoter upstream of dna to turn off n on genes Each germ layer dif t.f , turn on n off dif sets of genes to acquire dif characteristics to adopt their dif fates Determination: commitment to a particular fate that cannot be changed Specification: commitment to a particular fate that can be changed if cells are moved to a new environment Once differentiated can't go back , irreversible Mechanisms of commitment: • Localised determinants • Embryonic inductions • Morphogens Fertilised egg loaded with proteins, proteins induce ecto/meso fate , proteins qre localised to one part of the egg, so part of the cell becomes ecto other becomes meso , cell gets partitioned Edwin first discovered localised determinants Called it yellow crescent in cytoplasm, was localized in early embryo and only ends up in particular cells, give rise to muscle, cell gets partitioned into those who have and those who don't , yellow contained muscle determinant We learnt it is a transcription factor, not just protein localised in yellow crescent but also rna of this gene is localised in only one cell , can only make protein in one cell, If inject into other cells when not present can get ectopic expression of these genes What is Macho-1? * Macho-1 is a transcription factor. * Its mRNA is localized in the yellow cytoplasm of only one blastomere during early cleavage. * It is responsible for activating muscle-specific genes. ________________ 🧬 How Macho-1 Works: * The diagram shows Macho-1 binding to DNA (muscle gene promoter) to activate transcription (red arrow) of muscle-specific genes, such as muscle actin. ________________ 🧪 Experimental Results: * Control (n): Normal embryo — Macho-1 is present → muscle-specific actin mRNA is expressed in the expected cells. * Depleted (o): Macho-1 mRNA removed → no muscle gene expression (no purple stain). * Injected (q): Macho-1 mRNA artificially added to other cells → ectopic expression (purple stain in abnormal locations), showing Macho-1 is sufficient to induce muscle fate in other cells. worm has invariant lineage showing that every worm all develop the exact same way , depends on localized determinants displays asymmetric segregation (asymmetric cell division where mother cell gives rise to 2 daughter cells with 2 dif fates)when protein ends up in one part of the embryo , polarise the protein, Asymmetric segregation depends on PAR proteins: partition protein , asymmetrically localised in embryo Invariant Lineage * C. elegans has a completely invariant cell lineage. * This means every individual worm develops the same way, with the same cell divisions and fates. * Scientists can trace each adult cell back to its origin with precision. 2. Localized Determinants Drive Cell Fate * Early development relies on localized cytoplasmic determinants (molecules like mRNAs or proteins placed in specific parts of the egg or embryo). * These determinants influence what each daughter cell becomes. 3. Asymmetric Segregation * During asymmetric cell division, the mother cell produces two daughters with different fates. * This happens because the cytoplasmic determinants (proteins/mRNA) are not evenly distributed. * Example: One daughter inherits a determinant → becomes a neuron; the other doesn't → becomes skin. 4. PAR Proteins Are Crucial * PAR (Partitioning-defective) proteins are responsible for this asymmetric localization. * They polarize the embryo — meaning they mark "front" vs. "back" or "top" vs. "bottom." * This polarity guides the uneven distribution of fate determinants. * If PAR proteins are mutated, the embryo can't polarize properly → symmetric division → wrong cell fates. CELL SIGNALLING , embryonic induction, inductive events: All of cells have ability to give rise to all cell types , but signals restrict that potential, refer to this as regulative development , means development cant only be about localised determinants but ALSO signals Competence is ability of cell to respond to the signal , do u have receptor to signal or not None of embryo makes mesoderm when you cut it out and put it separate , mesoderm must be INDUCED if put yellow and blue together u get induction of pink mesoderm , endoderm and and ectoderm send signals to pattern mesoderm The Experiment: 1. The animal cap (blue ectodermal cells) is isolated from the embryo. 2. It's tested with and without MIF. Results: * Without MIF (-MIF): * The animal cap stays a round ball. * It forms only ectoderm-derived tissue → epidermis. * With MIF (+MIF): * The cap elongates due to convergent extension. * It now forms mesodermal tissues like notochord and muscle (in red), and neural ectoderm (neural tube, in purple) Animal cap assay, mesoderm cells can undergo conversion extension Nodal is part of tgfb fam If in contact, signal can induce fate Does it get different descendants, that's evidence of inductive signal , if produces what expected it is intrinsic determinate Chatgpt this As ab starts to form which is meant to be at front of embryo push with glass needle to back to be in contact to p2 , then produces a abp cell , u get a viable worm but swap fates fo 2 blastomeres, says signal is from p2 , cells have equal potential , equipotent , both competent , final fate is determined by signal , this is binary decision either be aba or abp depending on whether u get signal Can generate multiple fates , dif types of neurons , even tho same germ layer and same cell type , signal can diffuse away from the source, establish gradient of the signal cus more down than up, If have row of cells , signalling source and field of cells, highest in conc left and as diffuses less and less , what if cells can read out the CONC of signal, based on this dif cell types develop Notochord and floor plate is inducer, can regulate neural fate , if take notochord tissue and put it elsewhere then it induces production of floorplate , shh is signal from notochord and acts as a morphogen Developmental lecture 4 Specification: commitment to a particular fate that can be changed if cells are moved to a new environment (determinants and induction) Many major cell types can be divided into different subtypes Can see diversity within tissue :sensory/motor/interneurons Genes sit on chromo waves of gene expression and end genes have to show end cell type These are the batteries of genes needed to be turned on to make a skeletal muscle Nebullin n titin unexpected List is dif for neuron bc need genes fo chemical electrical signaling etc Mutually exclusive set of genes for neuron and muscle Turn on dif parts of genome for dif tissues There are certain things some neurons develop more and some less , have unique expression of other genes like neuro receptors/ion channels There `are dif subtypes of neurons , all neurons express the neuronal gene battery but some express additional proteins Neuron a is expressed in all these genes , neuron b expressed in 2 etc Thomas also suggests that “initial differences in the [cytoplasmic] regions may be supposed to affect the activity of genes. The genes will then in turn affect the cytoplasm, which will start a new series of reciprocal interactions. In this way we can picture the gradual elaboration and differentiation of the various regions of the embryo.” Why are dif genes expressed in dif cells: 1 idea: muscle cells lose all cells normally expressed in neuron or 2 idea the genes required are amplified - this is true in some but this is not the general mechanism Therefore concluded it must be gene expression that is controlled: turning particular genes on and off Proposed there is a cascade of t.f which in turn turn on a cascade of genes, progressive restriction for genes u can turn on and off so depending on what is expressed upstream u have a diff selection of genes u can turn on downstream T.f bind to enhancers to turn genes on n off If certain activating t.f bind upstream to the enhancers , this elicits other t.f to turn gene on/off, end result is to turn on battery of genes for the tissue type If dna becomes more tightly coiled , t.f cannot access the dna so not expressed, it could be that for expression of a neuronal cell the muscle genes are tightly wound we can see that the transcribed genes are loosely wound and t.f can access these genes for transcription Also when genes are meant to be transcribed , chromatid modifying enzymes recruited by transcription factors to keep gene open for further transcription Initial inputs like asymmetric segregation factors result in dif differentiation driver which is t.f result in dif gene battery , so it is technically a cascade Invertebrates , myod =tf regulates gene battery for muscle , it is differentiation drive/master regulator/selector gene , we often find one tf can regulate many genes in one go This terminal selector regulate general neuronal gene and extra subtype specific factors Terminal selector genes encode transcription factors that activate and maintain the full battery of cell-specific effector genes (e.g. ion channels, neurotransmitter enzymes) required for a neuron’s final differentiated identity. Myod only expressed in the somites Fruit fly undergoes metamorphosis wherein the eye/leg/wing discs enlarge and form the organ Most of the time the disk was the same as original but occasionally transdetermination despite determination being very stable A coordinated, hormone-driven developmental program in which a mature larva or juvenile remodels its tissues and organs to become the sexually mature adult. Transdetermination The reversal or switch of a cell that’s already “determined” (committed) to one fate, so that it adopts a different developmental pathway. * Classic model: Drosophila imaginal discs in culture can change from leg to wing precursors when exposed to different signals * Key features: plasticity of lineage-restricted cells, often revealed under experimental manipulation or injury We can reprogram cells to revert to original stem cell, we can reprogram cells and force to switch cell fate , and we know cells can switch fate even during determination Take nucleus out of muscle cell from a frog injected into a non dna filled egg sometime su get normal frog sometimes u can graft bits of cells into part of fog The later u take the cells the less likely u are to get a viable embryo Think about genome of a particular differentiated cell which only expressed a specific gene battery , but moving this into an early egg must somehow get rid of all those genetic marks . open up dna again so can produce whole organism Look at mouse embryos Therefore we can take differentiated adult cells , give them these t.f and revert back to pluripotent stem cells , or transplant into ealy egg and form pluripotency Sir John Gurdon and Shinya Yamanaka received the 2012 Nobel Prize for Physiology or Medicine “for the discovery that mature cells can be reprogrammed to become pluripotent” Instead of going back to pluripotency , can we switch from differentiated to different type of differentiated , well yes if we give enough of that maser regulator such as myod In worms which are male glial cell switches fate to neuron , this is natural transdifferentiation , this neuron can help with mating behaviours etc Natural transdifferentiation is the direct conversion of one fully differentiated cell type into another in vivo, without reverting through a pluripotent or progenitor state Virology lecture 1 Viruses are inert, have no intrinsic ,metabolic characteristics , most have rna genomes Viruses jump from species to species called zoonosis U may be wondering well can you define them as alive, well it doesnt fucking matter its just a word 8mm is a grain of rice Fibroblast is 30microns Rbc is 8micron , bactria is 3micron 12 nano is antibody , 20-300 is virusessss Circles are glycoproteins which have role of identifying host cell and getting the virus inside host Phages are viruses that infect bacteria Complex-pox, corona Helical-tmv Icosahedral- polio They look the same but very little structural similarity Each hiv is closer to its primate progenitor than the other hivs Foldseek AI used to find virus sequences within global metagenome, These are phylogenetic trees of viruses , can see how many unknown families there are

LECTURE 7 
Cell signalling is the cascade of processes by which an extracellular stimulus (typically a neurotransmitter or hormone) effects a change in cell function

Fight or flight response is mediated by adrenaline whereas the nerve to nerve signlling is mediated by glutamate

Signalling through ion channels or receptors

Ion channels are proteins in membrane and when ions flow thru these channels this initiates the signalling cascade , Ion channels are often selective for a particular ion

Receptor binds an extracellular ligand triggering a cascade of events which leads to a change

ION CHANNELS
Ca2+ is a ubiquitous signalling ion, calcium spikes within egg cells when the sperm fertilises the egg , so calcium is needed to activate the egg and begin cell division

These processes are dependent on calcium signals 
Huge time range over which calcium can regulate processes, calcium takes hours in fertilisation but nerve to nerve transmission takes microseconds

Charge prevents free diffusion of ions so require channel and carrier proteins

Channel proteins are water-filled pores, they allow charged substances ions) to diffuse through the cell membrane, 
The diffusion of these ions does not occur freely, most channel proteins are ‘gated’, meaning that part of the channel protein on the inside surface of the membrane can move in order to close or open the pore
Channel p[roteins normally remain similar in shape but carrier proteins undergo a conformational change allowing the ion to be moved down conc gradient  
The binding site of the carrier protein is open to one side of the membrane first, and then open to the other side of the membrane when the carrier protein switches shape

There are also ion gradients within organelles

ER has higher conc inside than out 
Low pH is high proton conc so higher in lysosome than cytosol 
Subtler grad in mito , more protons cytosol than mito

When move down conc gradient passive , when move against active and need energy from atp

Movement is determined not only by conc grad but also electrical grad , alw move from positive to negative membrane potential so if conc gradient is opposite to direction of electrical gradient like in the third example then movement can be inhibited

Ion gradients are generated by active transport, it requires atp hydrolysis

Ejects sodium out of cell and potassium into cell , despite the actual concs of sodium being greater outside and potassium being greater inside . remember the table  
Atpase pump transports 3 sodium ions out for 2 potassium ions in 
Look at arrows , an inward sodium electrochem grad and outward potassium gradient , how do we activ transport them opp direction?

P TYPE stands for phosphorylation as we n=know phosphate involved in cycle 
steps : na can bind to binding site on open side of protein , then atp hydrolysed and pi bind to protein causing conformational change such that na can no longer bind and is released to other side of membrane , conformai=tional change also causes K to be able to bind to its binding site , then dephosphorylation occurs so another shape change K is released to other side as no longer fits in binding site and resting protein shape is restored 
   
Pmca is a calcium carrier protein dont need to know structure but know its very complex got transmembrane structures plus loads of domains 
PM plasma membrane Calcium Atpase 
Ejects calcium outside cell , from low inside conc to high outside against conc grad

SER sarcoplasmic(muscle|) reticulum/ ER endoplasmic reticulum, Calcium Atpase
Transports from cytoplasm into lumen of ser 
Remember conc in er is higher inside than out so this is against conc gradient from out to in

If cotransported ion same direction its symport if not then antiport

N(sodium)CalciumXchanger
We transport 3 sodium in for one calcium out , na drives movement against the ca gradient to move it out despite ca being higher out than in 
This is antiport as na and ca move opp 
Cotransport is secondary active transport

Vg are opened by changes in voltage 
Ligand gated opened by attachment of small intra or extracellular molecules 
Mechanically gated are opened by stretch/bend/ mechanical force

Learn this structure 
6 transmembrane domains , first 4 make up v sensing domain which responds to the changed in v , last 2 make up pore domain in which there is hole thru which ions can flow 
Evolution can put these building blocks tg in dif ways to perform dif functions

4 of the building block subunits can make a vg K channel , whereas by joining each building block into a single protein we form vg naca channel

He first crystallized/ found structure of K channels

How do K channels only allow potassium to pass thru/ why r they so selective?
Well if pores were just holes and we know na is smaller than k we would think na would also pass thru but 3d structure is more complicated
  
 the 4 middle amino acids are known as the selectivity filter and they have 4 o atoms which bind to a k ion stabilising it , whereas only 2 o bind to na which is less energetically favourable

Each k ion channel was found to have positively charged amino acid residues : arginine and or lysine , because they are + charged they can detect changes in voltage which control opening and closing of the channel , these residues are in the fourth domain of the vg channel

S4 regions point down but when voltage change (depolarisation) s4 moves up and it is this that yanks on pore opening and allowing k to move thru

LECTURE 8 
Membrane potential is the difference between the voltage inside and outside the cell , typically -70mv at rest

Cells are generally electrically neutral; the 5 negative charges neutralise the 5 potassium charges ,positive charges are generally fixed by matched anions
 if cell membrane permeable, shown by dashed line,  the potassium will move down its conc gradient outside of the cell creating voltage difference so +2 charges outside and uncovered 2 negative charges inside , difference between the charges is 4 as 2- -2
V = -4 because cell is now negative relative to the outside 
However due to electrical difference the positive charges are gonna wanna move back into cell from + to - so potassium moves in via electrical gradient , so now weve got a V = -2 , these forces eventually balance out outward movement by chemical and inward by electrical gradient until reach an equilibrium , T is time btw 
Final potential is eq potential

Co/ci is gradient 
Z is valence/ charge of ion 
Rest are physical constants 
Temp is calculated in kelvin and done under physiological conditions so around 37

This is the electrical potential/difference  that would arise if membrane was permeable to k

Hence -70 is close to -90
Due to the presence of leak channels more permeable to k as k can go thru these k leak channels

The new term P refers to permeability

Eq takes into acc the permeability of each ion relative to each other 
Permeability of k is much higher than cl and na at rest 
P of K is close to 1 and the other close to 0 so we cna cancel out these terms and we are left back with nernst

The longest dendrite referred to as axon

Nerve cells connected to other nerve cells 
Transient change in membrane potential known as action potential allowing neurons to communicate at junction called synapse

During transmission vg channel involved 
Action potentials arise due to the concerted action of voltage gated Na+ and K+ channels

During stim u get small depolarisation so membrane become less negative, this opens vg sodium channels allowing sodium into cell further depolarising membrane

Now because these vg sodium ions are open the p value for na becomes close to 1 and the rest of the terms close to 0 so cancel and the equation becomes the equilibrium potential of sodium , so basc the resting membrane potential is the eq potential of k and the depolarised potential is eq potential of sodium 
Voltage–gated Na+ channels depolarize the membrane from the equilibrium potential for K+ (-90 mV) to Na+ (60 mV)
Ap is transient so not long lasting so the depolarisation is followed by repolarisation

The na channels become inactivated , only open for short period , stops na coming in , the vg k channels then open , they are slower to open than vg na channels  , opening vg  channel for k takes membrane potential to the eq potential of k , as the k ions move OUT of cell 
The delay causes the shape of the hill , up and then down

Depolarisation of the membrane triggers opening of vg ca channels , ca ions flow in down conc gradient causing neurot vesicles to fuse with presynaptic membrane and releases nt into synaptic cleft

Ligand gated ion channels are present on postsynaptic cells , they respond to the nt release at synaptic cleft 
Voltage-Gated Channels
🔌 Location: Mainly on the presynaptic membrane
* These open in response to a change in membrane voltage.

* Example: When an action potential arrives at the presynaptic terminal, voltage-gated calcium channels open → Ca²⁺ enters → triggers neurotransmitter release.

Ligand-Gated Channels
🧬 Location: Mainly on the postsynaptic membrane
   * These open when a specific neurotransmitter (ligand) binds to them.

* Example: Acetylcholine binds to receptors on the postsynaptic side → Na⁺ channels open → depolarization of the postsynaptic neuron.

Epilepsy occurs due to messing with balance of inhibition and excitation

Glutamate is the key nt that activates these channels 
4 subunits come together to form ion channel , tetramers just like vg channels 
Unlike vg channel where s5 and s6 form pore u can see in this lg channel the transmembrane structure domains within the box form the pore which is also inverted hence the inverted pore topology , tm2, transmembrane 2 forma the invented pore
  
 
Allow flow of na and ca into cell down conc gradient and due to increasing membrane potential inside cell , they are excitatory

Gaba is key nt that activates these receptors 
4 transmembrane regions , but 5 subunits come tg to form channel unlike glutamate receptors 
Also referred to as cys loop, they are part of cys loop family  , bc we find disulfide bond in extracellular loop, they allow flow of chloride ions into cell and because are neg , influx of neg into cell so they are inhibitory

Respond to ach 
Pentameric so 5 subunits come tg to form channel , belong to same family as gaba , so cys loop family 
Respond to sodium allow na to go in , so excitatory 
They can also be found on postsnyaptic muscle cells at the nmjunction

nACh receptors, like GABA, are part of the Cys-loop receptor family, with pentameric structure and 4 TM domains per subunit.

Glutamate receptors are tetrameric, with an inverted topology and a unique pore-forming TM2 loop.

Voltage-gated channels differ structurally with 6 TM segments and a voltage-sensing S4 domain.

Not alw nerve to nerve communication but also nerve to muscle

The ach in cleft opens up nicotinic ach receptors and influx of na gets activation of muscle cells which opens up vg na channels and signalling continues in muscle cells , causing contraction
Depolarisation of muscle cell causes opening of ca vg channels causing influx of ca from INTRACELLULAR stores, this causing contraction , whole process called excitation contraction coupling, excitation is na depolarisation and contraction is ca part

Lecture 9

Receptor proteins are found typically at cell surface but also sometimes found inside cell so the ligand must be smth that can cross the membrane

Addition of Phosphate turns protein on and when remove turns off 
Phosphate comes from atp , depicted as appp so adenosine and 3 phosphates and the terminal phosphate is transferred onto signalling protein to turn on 
Second switch is gtp , gtp shown as gppp and terminal phosphate transferred to turn on protein
When gtp is bound to signalling protein then protein is turned on , when bound to gdp then off state 
molecular switches in cell signalling: (A) proteins activated by phosphorylation (adding a phosphate group) and turned off by dephosphorylation, and (B) GTP-binding proteins switched on by binding GTP and off when GTP is hydrolyzed to GDP.

Second messengers are small molecules which act as relays in intracellular signalling cascade

Dont need to know structures 
G protein

GPCRs are activated by a variety of ligandsHormones (e.g. adrenaline)
Neurotransmitters (e.g. glutamate)
Odorants
But many are “orphans”

Heptahelical receptors

Heterotrimeric cus 3 parts alpha beta gamma
In unactivated state alpha bound to gdp 
In activated alpha bound to gtp , alpha dissociates from beta and gamma ,

Odorants work via g protein , things that smell , don't know what activates many types of gpcr

Beta and gamma can also activate signalling cascades

These are made/mobilised during activation 
Different g proteins activate different cascades , alpha s subunit activate adenylyl fyi

Regulation of messenger synthesis by G-protein α subunits 
αs subunits activate adenylyl cyclase 
αi subunits inhibit adenylyl cyclase Adenylyl cyclase is reciprocally regulated by distinct α subunits

Catalytic subunits act on target by phosphorylation 
Structure of PKA:
      * PKA is inactive when its two catalytic (C) subunits are bound to two regulatory (R) subunits — this forms a tetramer (R₂C₂).

* Each R subunit has two cAMP binding sites.

🔹 Activation by cAMP:
         * When cAMP levels rise
         1. cAMP binds to the regulatory (R) subunits.

2. This causes a conformational change that releases the catalytic (C) subunits.

3. The freed catalytic subunits are now active enzymes. These catalytic subunits phosphorylate target proteins at serine or threonine residues using ATP.

Phosphodiesterase + H₂O: This enzyme breaks the cyclic bond in cAMP using water (hydrolysis).converted to  5′-AMP (right structure): This cannot activate PKA, so the signal is terminated.

Phospholipase c enzyme converts pip2 into dag and ip3

Acts at endoplasmic reticulum 
Ca bp = calcium binding protein like calmodulin 
Ip3r is receptor 
Signalling thru ion channels and receptors

1. Gqα activates PLCβ, which cleaves PIP₂ into:

* IP₃ (inositol triphosphate)

* DAG (diacylglycerol)

2. IP₃ binds IP₃R (receptor) on the endoplasmic reticulum (ER) → opens Ca²⁺ channels → Ca²⁺ released into cytosol.

3. DAG stays in membrane and, with Ca²⁺, activates PKCγ.

4. Released Ca²⁺ binds calcium-binding proteins (CaBPs) like calmodulin (CaM), activating downstream targets.

Can bind 4 ions of calcium

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII
Inactive CaMKII: The catalytic domain is blocked by an inhibitory domain.
Ca²⁺ binds to calmodulin, and the complex binds CaMKII → partial activation.
Autophosphorylation: CaMKII autophosphorylates itself using ATP → fully active.
Memory effect: Even after calmodulin detaches, the kinase remains active (Ca²⁺-independent) due to autophosphorylation.

Cant metabolise calcium so shuttles move calcium away from target 
  
 
The pumps and exchanger are all activated and turn off the reaction

Dag works with ip3 to activate
                     1. IP₃ releases Ca²⁺ from the ER.

2. DAG and Ca²⁺ together activate PKC, which moves to the membrane.

3. Activated PKC phosphorylates target proteins to produce cellular effects.

Enzyme linked receptors dont have set structure , such as rtks

U get activation very quickly by growth hormones like epidermal growth factor , fgf, etc,  unlike gpcr which have to go thru many steps

Dimer is 2 subunits coming together

1. Activation by Growth Hormones
                        * RTKs are membrane-bound receptors activated by growth factors like:

* EGF (Epidermal Growth Factor)

* FGF (Fibroblast Growth Factor)

* PDGF (Platelet-Derived Growth Factor)

* Compared to GPCRs, RTKs initiate faster signaling due to direct activation of internal enzymes., rtk dont need second messenger

🔹 2. Dimerization and Autophosphorylation
                                 * Ligand binding (e.g., FGF, PDGF) causes dimerization (two RTK subunits come together).

* The dimerized receptors autophosphorylate (add phosphate groups to each other’s tyrosine residues using ATP). phosphate tagging activates the intracellular signaling domains.
🔹 3. Signal Transduction
                                    * Phosphorylated RTKs act like docking stations for downstream signaling proteins- phosphotyrosines (p-Tyr) now recruit specific intracellular proteins which either get activated themselves (e.g., enzymes like PI3K), or link the receptor to other signaling pathways

p switching , activation rtk by autophos, then recruiting 3 proteins needed in transduction, one called grb2 activator protein as grabs next protein in cascade known as sos which activates ras , if ras mutates gain of function and can cause cancer , ras is a g protein 
Ras In presence of sos bound to gtp and not when not activated by sos

This diagram explains part of the MAPK (Mitogen-Activated Protein Kinase) signaling pathway, a key cell signaling cascade that controls growth, division, and survival:
                                    1. Stimulus (e.g., growth factor) binds to a receptor tyrosine kinase (RTK).

2. RTK autophosphorylates, creating docking sites for proteins.

3. Grb-2 (an adaptor protein) binds the phosphorylated RTK.

4. Grb-2 recruits Sos (a guanine nucleotide exchange factor).

5. Sos activates Ras (a monomeric G-protein) by exchanging GDP for GTP.

6. Active Ras triggers a kinase cascade: MAPKKK → MAPKK → MAPK, leading to cellular responses.

Note: Mutated Ras (always active) can lead to cancer by promoting uncontrolled cell division.
What is a mitogen, chemical that bind to trigger mitosis

Map kinase phos in order to be activated , what phosphorylates upstream it is called map kinase kinase and even more upstream protein map kinase kinase kinase which activates downstream
This diagram shows the MAPK signaling cascade:
                                       1. Stimulus activates receptors.

2. Transducers (e.g., Grb-2, Sos, Ras) pass the signal.

3. A phosphorylation cascade occurs:

* MAPKKK (e.g., Raf) activates

* MAPKK (e.g., MEK) which activates

* MAPK (e.g., ERK)

Each step phosphorylates the next, ultimately leading to changes in gene expression or cell behavior.

Fgf singalling thru map kinase pathway in a process mesoderm induction for instance,

Grb-2 and Sos are not G-proteins—they’re adaptor and exchange factor proteins, respectively.
G protein is ras Ras is a small (monomeric) GTPase, meaning it binds GDP (inactive) or GTP (active).

Rewatch edit notes to the above lecture ^

Lecture 10 - developmental bio 1 
Developmental biology 
How do we go from an undifferentiated egg to diversity of differentiated cell types 
Model organisms: simple systems to understand more complicated ones
There is Conservation of processes and principles across model systems so we can study human embryo by studying zebrafish etc cus same mechanisms

embryo micromanipulation refers to precise techniques used to physically handle or alter embryos using fine instruments under a microscope
A genetic knockout is when a specific gene is completely disabled or deleted in an organism, so it no longer produces its protein product.Purpose: To study what that gene normally does by seeing what happens when it's missing.

Mammalian , vertebrate so similar to humans 
Not as high reproductive rate as other organisms 
Knocked out genes in mice to learn abt development won nobel 
Expensive lots of cages 
Long life cycle so takes several months for a mouse to develop

Large embryos so good micromanipulation 
Hard to make transgenic chickens

Big tanks of  circulating water so expensive 
2-3 months from embryo to adult so not as long as mice 
Can make transgenic fish , gfp mutants etc   
________________
Been worked on for a long time , old model system , 1995 knocking out genes to understand development

Free soil living worm found in compost heaps , 1mm long, this is a grant proposal for working on nematodes showing advantages of model systems 
Few cells  fixed cells can be an advantage

First animal to have its connectome fully transcribed so how each neuron connects to each neuron

Human would be similar size to mouse embryo 
Chicken egg derives all its nutrition from the egg so big but we derive from placenta so egg can be smaller 
CHICKEN EGG

Maternal FACTORS(INSTEAD OF GOODIES)

GIANT NUCLEUS , lots of mito cus need energy to move 
Acrosome full of enzymes that can break zona pellucida

Ca wave in egg ends meiosis 2 , triggers enzymes

Contractile ring forms around embryo to separate the 2 cells 
Both centrioles come from father in c elegans

If egg with all proteins needed small, if placental development then less yolk but other protein involved in processes still there but if not provided anything egg big lot of yolk

No growth ! half in size when double 
Discoidal is when restricted to disk of cells that sit on the yolk 
Superficial is where division occurs at edge

Usually synchronous so divide at same time in waves , Often synchronous initially—especially in early embryonic stages like in frogs and mammals, where all cells (blastomeres) divide at the same time.
But it becomes asynchronous later as development progresses and cells start dividing at different rates based on their position and function.

Xenopus develops externally not placental so large eggs

* Meroblastic cleavage: Only part of the egg divides (not the whole thing), because the yolk is too dense to cleave.

* Discoidal: Division happens only in a small disc-shaped area at the top of the egg (the animal pole, where there's little yolk).

* Early cleavages (first 3 divisions):

* Are meridional (top to bottom)

* Only cleave the yolk-free cytoplasm

* 4th cleavage:

* Is equatorial (side to side)

* Produces two layers of 8 cells sitting like a mound on top of the yolk (which remains uncleaved below)

Summary:
In zebrafish, only the upper disc of cytoplasm divides, while the large yolk stays intact—this is called discoidal meroblastic cleavage.

Firstly embryo is bag of proteins, The dots are all nuclei there is no pm as no cytokinesis occurs until stage 10, they then go to periphery of egg and division occurs at the edge of the membrane 
                                                         1. First 8 divisions (images 1–8):

* The fertilized egg begins dividing its nucleus, but no cell membranes form yet.

* These nuclear divisions happen in the center of the egg (no cytokinesis = no individual cells yet).

* The embryo is called a syncytium (a single cell with many nuclei).

2. Nuclei migrate outward (images 8–10):

* Most of these nuclei move to the outer edge (periplasm) of the egg.

* This creates the syncytial blastoderm—a single cell with many nuclei just under the surface.

3. 4 more divisions at the periphery (images 10–12):

* The nuclei continue dividing near the outer surface.

* Still, no individual cells—just lots of nuclei in one shared cytoplasm.

4. Cell membranes form (images 13–14):

* The plasma membrane folds inward around each nucleus.

* This forms individual cells for the first time, creating the cellular blastoderm.

Holoblasic cleavage occus in cells w little yolk
________________

Human is similar to mouse 
In order to be this rapid the early cleavage divisions have modification of the cell cycle

Its usually just S mitosis S mitosis 
Only nuclear divisions no pm division 
S phase can happen quicker bc mum alw provides many of the proteins required in dna replication in the egg 
In droso ONLY  no cytokinesis but it is there in other organisms

Asynchronous means different cells divide at different times based on what they are gonna become , mostly early cleavage stage divisions are synchronous and modified cell cycle 
This transition from syn to async is mid blastula transition, happens at mid blastula stage , mother loads proteins into egg cus zygotic transcription hasn't started yet , so dna of embryo is not being transcribed or translated into proteins in these early stages , this also occurs at the mbt

Rna translated to make proteins is loaded into egg by mum , at mbt zygotic genes are now turned on so can make own proteins , this is true of most model orgs but not nematode where zygotic transcription starts at 4 cell stage

Developmental lecture 2

Ingression is individual cells delaminating from surface moving interiorly 
Imvagination is not individual cell movement but coordinated movement of a sheet of cells moving inwards creating an invagination 
Involution is cells migrating across the under surface of the outer layer , similar to invagination

Each embryo has vegetal pole and animal pole 
In animal development, the animal pole is the region of the egg with less yolk and more cytoplasm, while the vegetal pole is the opposite end with more yolk and less cytoplasm, influencing early embryonic development. 
Common in chick, mammals, sea urchin

Sea urchins, droso, nematode also display invagination 
Seems as though some external force pushes and forces cells inwards but force generated by apical constriction by the actin myosin skeleton - this is only for the invagination 
In order to generate enough force to push the mesenchymal cells all the way to the animal pole use another mechanism called convergent extension 
gut=endoderm

Lemellipodia all begin to face same directions then carry out convergent extension

Cells migrate around to the dorsal side of the embryo , which is thickest part and forms most of the tissues 
                                                                                 * Ectoderm (blue) stays on the outside.

* Mesoderm (red) and endoderm (yellow) move inward through the blastopore via a process called involution.

* These internalized cells migrate along the inner surface, forming the archenteron (the future gut).

* Movement is directed toward the dorsal side (top), which becomes the main body axis and forms most tissues.

Convergent extension towards the dorsal side aswell 
Blastoderm sits on yolk:

* The early embryo (blastoderm) sits atop the large yolk.

* It consists of two layers: a deep layer and an outer enveloping layer.

Spreading of blastoderm by epiboly:

* The blastoderm spreads to cover the yolk via a movement called epiboly, where cells thin out and expand over the yolk.

Gastrulation begins with involution:

* Cells at the margin of the blastoderm move inward (involution) at the dorsal side, initiating gastrulation, which establishes the body plan.

Convergence and extension:

* Cells move medially and elongate the embryonic axis through convergent extension, especially toward the dorsal side, helping to form the notochord and other midline structures.

Via compaction end up with blastocoel
Inner cell mass form all of the tissues

Sync starts to form lacunae for transport of nutrients become blood vessels

Epiblast cells ingress at the primitive streak

Mesodermal cells spread out laterally

Myotome forms skeletal muscle 
Somites form every 30 minutes in zebrafish, every 90 minutes in chick and every 5 hours in humans

Line of cells neural plate cells, thicken and then apical constriction causes neural tube closure

They are between roof of neural tube and external ectoderm

despite dif sizes of eggs , at end of gastrulation all vertebrate embryos have remarkable similarities , see same shape and structure , but end up looking super different at end 
Phylotypic stage at end of gastrulation where all look very similar and are molecularly similar

This is in situ hybridisation , method of revealing where certain mrna are , brain of mouse chick frog and fish can see 2 expressions of this gene , shared molecularity , very similar at phylotypic stage

Developmental lecture 3 - i didnt rewatch but u could if there's stuff missing

Fluorescent labelling shows expression of dif genes 
Green is dna , red is differentiation drivers 
Transcription factors are being turned on specifically in these germ layers , t.f binds to a promoter upstream of dna to turn off n on genes 
Each germ layer dif t.f , turn on n off dif sets of genes to acquire dif characteristics to adopt their dif fates

Determination: commitment to a particular fate that cannot be changed
Specification: commitment to a particular fate that can be changed if cells are moved to a new environment
Once differentiated can't go back , irreversible

Mechanisms of commitment:  • Localised determinants • Embryonic inductions • Morphogens

Fertilised egg loaded with proteins, proteins induce ecto/meso fate , proteins qre localised to one part of the egg, so part of the cell becomes ecto other becomes meso , cell gets partitioned

Edwin first discovered localised determinants

Called it yellow crescent in cytoplasm, was localized in early embryo and only ends up in particular cells, give rise to muscle, cell gets partitioned into those who have and those who don't , yellow contained  muscle determinant

We learnt it is a transcription factor, not just protein localised in yellow crescent but also rna of this gene is localised in only one cell , can only make protein in one cell, 
If inject into other cells when not present can get ectopic expression of these genes

What is Macho-1?
                                                                                             * Macho-1 is a transcription factor.

* Its mRNA is localized in the yellow cytoplasm of only one blastomere during early cleavage.

* It is responsible for activating muscle-specific genes.

________________

🧬 How Macho-1 Works:
                                                                                                * The diagram shows Macho-1 binding to DNA (muscle gene promoter) to activate transcription (red arrow) of muscle-specific genes, such as muscle actin.

________________

🧪 Experimental Results:
                                                                                                   * Control (n): Normal embryo — Macho-1 is present → muscle-specific actin mRNA is expressed in the expected cells.

* Depleted (o): Macho-1 mRNA removed → no muscle gene expression (no purple stain).

* Injected (q): Macho-1 mRNA artificially added to other cells → ectopic expression (purple stain in abnormal locations), showing Macho-1 is sufficient to induce muscle fate in other cells.

worm has invariant lineage showing that every worm all develop the exact same way , depends on localized determinants 
 displays asymmetric segregation (asymmetric cell division where mother cell gives rise to 2 daughter cells with 2 dif fates)when protein ends up in one part of the embryo , polarise the protein, Asymmetric segregation depends on PAR proteins: partition protein , asymmetrically localised in embryo

Invariant Lineage
                                                                                                      * C. elegans has a completely invariant cell lineage.

* This means every individual worm develops the same way, with the same cell divisions and fates.

* Scientists can trace each adult cell back to its origin with precision.

2. Localized Determinants Drive Cell Fate
                                                                                                            * Early development relies on localized cytoplasmic determinants (molecules like mRNAs or proteins placed in specific parts of the egg or embryo).

* These determinants influence what each daughter cell becomes.

3. Asymmetric Segregation
                                                                                                               * During asymmetric cell division, the mother cell produces two daughters with different fates.

* This happens because the cytoplasmic determinants (proteins/mRNA) are not evenly distributed.

* Example: One daughter inherits a determinant → becomes a neuron; the other doesn't → becomes skin.

4. PAR Proteins Are Crucial
                                                                                                                     * PAR (Partitioning-defective) proteins are responsible for this asymmetric localization.

* They polarize the embryo — meaning they mark "front" vs. "back" or "top" vs. "bottom."

* This polarity guides the uneven distribution of fate determinants.

* If PAR proteins are mutated, the embryo can't polarize properly → symmetric division → wrong cell fates.

CELL SIGNALLING , embryonic induction, inductive events:

All of cells have ability to give rise to all cell types , but signals restrict that potential, refer to this as regulative development , means development cant only be about localised determinants but ALSO signals

Competence is ability of cell to respond to the signal , do u have receptor to signal or not 
None of embryo makes mesoderm when you cut it out and put it separate , mesoderm must be INDUCED if put yellow and blue together u get induction of pink mesoderm , endoderm and and ectoderm send signals to pattern mesoderm

The Experiment:
                                                                                                                              1. The animal cap (blue ectodermal cells) is isolated from the embryo.

2. It's tested with and without MIF.

Results:
                                                                                                                                 * Without MIF (-MIF):

* The animal cap stays a round ball.

* It forms only ectoderm-derived tissue → epidermis.

* With MIF (+MIF):

* The cap elongates due to convergent extension.

* It now forms mesodermal tissues like notochord and muscle (in red), and neural ectoderm (neural tube, in purple)

Animal cap assay, mesoderm cells can undergo conversion extension

Nodal is part of tgfb fam

If in contact, signal can induce fate 
Does it get different descendants, that's evidence of inductive signal , if produces what expected it is intrinsic determinate 
Chatgpt this

As ab starts to form which is meant to be at front of embryo push with glass needle to back to be in contact to p2 , then produces a abp cell , u get a viable worm but swap fates fo 2 blastomeres, says signal is from p2 , cells have equal potential , equipotent , both competent , final fate is determined by signal , this is binary decision either be aba or abp depending on whether u get signal

Can generate multiple fates , dif types of neurons , even tho same germ layer and same cell type , signal can diffuse away from the source, establish gradient of the signal cus more down than up,

If have row of cells , signalling source and field of cells, highest in conc left and as diffuses less and less , what if cells can read out the CONC of signal, based on this dif cell types develop

Notochord and floor plate is inducer, can regulate neural fate , if take notochord tissue and put it elsewhere then it induces production of floorplate , shh is signal from notochord and acts as a morphogen

Developmental lecture 4

Specification: commitment to a particular fate that can be changed if cells are moved to a new environment (determinants and induction)

Many major cell types can be divided into different subtypes
Can see diversity within tissue :sensory/motor/interneurons

Genes sit on chromo
waves of gene expression and end genes have to show end cell type

These are the batteries of genes needed to be turned on to make a skeletal muscle 
Nebullin n titin unexpected

List is dif for neuron bc need genes fo chemical electrical signaling etc 
Mutually exclusive set of genes for neuron and muscle 
Turn on dif parts of genome for dif tissues 
There are certain things some neurons develop more and some less , have unique expression of other genes like neuro receptors/ion channels

There `are dif subtypes of neurons , all neurons express the neuronal gene battery but some express additional proteins 
Neuron a is expressed in all these genes ,  neuron b expressed in 2 etc

Thomas also suggests that “initial differences in the [cytoplasmic] regions may be supposed to affect the activity of genes. The genes will then in turn affect the cytoplasm, which will start a new series of reciprocal interactions. In this way we can picture the gradual elaboration and differentiation of the various regions of the embryo.”

Why are dif genes expressed in dif cells: 1 idea: muscle cells lose all cells normally expressed in neuron or 2 idea the genes required are amplified - this is true in some but this is not the general mechanism

Therefore concluded it must be gene expression that is controlled: turning particular genes on and off

Proposed there is a cascade of t.f which in turn turn on a cascade of genes, progressive restriction for genes u can turn on and off so depending on what is expressed upstream u have a diff selection of genes u can turn on downstream 
T.f bind to enhancers to turn genes on n off

If certain activating t.f bind upstream to the enhancers , this elicits other t.f to turn gene on/off, end result is to turn on battery of genes for the tissue type

If dna becomes more tightly coiled , t.f cannot access the dna so not expressed, it could be that for expression of a neuronal cell the muscle genes are tightly wound

we can see that the transcribed genes are loosely wound and t.f can access these genes for transcription 
Also when genes are meant to be transcribed , chromatid modifying enzymes recruited by transcription factors to keep gene open for further transcription

Initial inputs like asymmetric segregation factors result in dif differentiation driver which is t.f result in dif gene battery , so it is technically a cascade

Invertebrates , myod =tf regulates gene battery for muscle , it is differentiation drive/master regulator/selector gene , we often find one tf can regulate many genes in one go

This terminal selector regulate general neuronal gene and extra subtype specific factors 
Terminal selector genes encode transcription factors that activate and maintain the full battery of cell-specific effector genes (e.g. ion channels, neurotransmitter enzymes) required for a neuron’s final differentiated identity.

Myod only expressed in the somites

Fruit fly undergoes metamorphosis wherein the eye/leg/wing discs enlarge and form the organ 
  
Most of the time the disk was the same as original but occasionally transdetermination despite determination being very stable 
A coordinated, hormone-driven developmental program in which a mature larva or juvenile remodels its tissues and organs to become the sexually mature adult.
Transdetermination
 The reversal or switch of a cell that’s already “determined” (committed) to one fate, so that it adopts a different developmental pathway.
                                                                                                                                             * Classic model: Drosophila imaginal discs in culture can change from leg to wing precursors when exposed to different signals

* Key features: plasticity of lineage-restricted cells, often revealed under experimental manipulation or injury

We can reprogram cells to revert to original stem cell, we can reprogram cells and force to switch cell fate , and we know cells can switch fate even during determination

Take nucleus out of muscle cell from a frog injected into a non dna filled egg sometime su get normal frog sometimes u can graft bits of cells into part of fog 
The later u take the cells the less likely u are to get a viable embryo 
Think about genome of a particular differentiated cell which only expressed a specific gene battery , but moving this into an early egg must somehow get rid of all those genetic marks . open up dna again so can produce whole organism

Look at mouse embryos 
  
Therefore we can take differentiated adult cells , give them these t.f and revert back to pluripotent stem cells , or transplant into ealy egg and form pluripotency

Sir John Gurdon and Shinya Yamanaka received the 2012 Nobel Prize for Physiology or Medicine “for the discovery that mature cells can be reprogrammed to become pluripotent”

Instead of going back to pluripotency , can we switch from differentiated to different type of differentiated , well yes if we give enough of that maser regulator such as myod 
  
In worms which are male glial cell switches fate to neuron , this is natural transdifferentiation , this neuron can help with mating behaviours etc

Natural transdifferentiation is the direct conversion of one fully differentiated cell type into another in vivo, without reverting through a pluripotent or progenitor state

Virology lecture 1 
Viruses are inert, have no intrinsic ,metabolic characteristics , most have rna genomes 
Viruses jump from species to species called zoonosis

U  may be wondering well can you define them as alive, well it doesnt fucking matter its just a word

8mm is a grain of rice 
Fibroblast is 30microns
Rbc is 8micron , bactria is 3micron
12 nano is antibody , 20-300 is virusessss

Circles are glycoproteins which have role of identifying host cell and getting the virus inside host

Phages are viruses that infect bacteria

Complex-pox, corona 
Helical-tmv
Icosahedral- polio

They look the same but very little structural similarity

Each hiv is closer to its primate progenitor than the other hivs

Foldseek AI used to find virus sequences within global metagenome,
These are phylogenetic trees of viruses , can see how many unknown families there are

Transcript for:Overview of Cell Signalling and Development

Transcript for:
Overview of Cell Signalling and Development