Forming a Bilaminar Embryo PDF

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lecture notes on early embryogenesis: forming a bilaminar embryo...

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BS3570: Forming a Bilaminar Embryo from the Inner Cell Mass (08/10/2020)

RECAP:

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Trophoblast cells do not actually contribute to the embryo- they help to form the placenta (alongside maternal cells) Inner cell mass becomes epiblast and hypoblast Epiblast goes on to form the entire embryo

Inner cell mass- embryonic stem cells: -

ICM cells will happily proliferate as undifferentiated stem cells as long as there is Oct4, Nanog and Sox2 are expressed ICM forms all cell types of the embryo Embryonic stem cells are pluripotent Embryonic stem cells can be encouraged to form many different cell types within the dish Oct4, Nanog and Sx2- KEY

Changes in the embryo- day 7: -

ICM forms 2 types at this stage (after fertilisation) Epiblast and hypoblast- BILAMINAR MORPHOLOGY Hypoblast underlies the epiblast layer Hypoblast is single cell layer whereas epiblast is multiple

Molecular changes in the blastocyst, GATA6 and Nanog: -

THE DIFFERENTIATION OF THE ICM INTO EPIBLAST AND HYPOBLAST IS HIGHLY REGULATED The main thing is that the two cell types in epiblast and hypoblast cells express different transcription factors Epiblast contains nanog and hypoblast contains GATA6 Expression of these transcription factors causes the layers to arrange in this way

FGF signalling: -

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In early blastocyst, cells are more mixed upsome are expressing GATA6 and some are expressing nanog Both nanog and GATA6 are expressed at low levels at early stages- mutual repression When one cell begins to express more of one transcription factor, it represses the other More nanog represses Gata6 and more Gata6 represses nanog

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FGF signalling influences which one the cell expresses

Experiential evidence: -

Real time imaging- watch cell rearrangements Gene expression studies- single cell analysis

How system is regulated: -

Mouse embryonic DAY 3 (inner cell mass stage, not differentiated) ICM initiate both genetic transcription factors at low levels (nanog and Gata6) Stochastic effect or cell history(?) causes one to increase over the other FGF level expression could change before or after change in expression (unsure) FGF4 is secreted by one of the cells and starts binding to receptor on the other cell Increased Nanog, less GATA6 means FGF4 ligand secreted and FGFR2 is repressedepiblast fate Increased nanog represses Gata6 Gata6 inhibits FGF4 and activates FGFR2 Therefore, when Gata6 is repressed, FGF4 is expressed and FGFR2 is inhibited FGF- fibroblast growth factor molecule (whole family)

FGF signalling via receptor tyrosine kinase pathway: -

FGF ligand (signal) binds to FGFRs (FGF4 binds to FGFR2) Intracellular domain has tyrosine kinase enzyme activity When FGF binds, causes dimers to come together and catalyses itself Phosphorylation of Mek (kinase) This forms another kinase, Erk Erk enters via nuclear envelope and is phosphorylated into transcription factor

Experimental evidence: -

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Gene knockout studies- effect of removing function of a gene (can show you if a gene is important in a particular role) Overexpression studies- effect of increasing activity of a gene

Gene knock out study data: -

Embryonic day 3 in FGF4 mutant (no expression of FGF4)- starts out almost the same as ICM start the same- still express nanog Nothing can stimulate FGF receptor in adjacent cell, so they also retain nanog expression (communication between cells is lost) In the absence of FGF4, ICM cells cannot be instructed to become PrE (primitive endoderm) and by default mature to epiblast cells In the absence of nanog, there is no repression of Gata6, ICM cannot mature to epi fate Without nanog, cells do not secrete FGF4 to instruct other cells to become PrE ICM cells form intermediate PrE progenitors In the absence of Oct4. Oct4 is important in helping cells respond to FGFR So, if Oct4 gene is turned off, cells have undetermined identity ES cells can be used as tools to modify the genome to make things like gene knockouts Obtain ES cells, culture them in a dish, genetically modify in some way in a dish Then take these cells and add to another embryo where they integrate themselves as the ICM of the embryos

Forming the amniotic membrane- day 8: -

Some cells of the epiblast migrate to form a new layer called the amniotic membrane This is formed over the epiblasts Fluid filled cavity between amniotic membrane and epiblast at day 9

At 14 days, fully implanted blastocyst: -

Some epiblast cells have formed amniotic membrane Blastocyst is completely implanted into the uterine wall - At 14 days, the embryo has 2 layers (epiblast and hypoblast) - Informed by studies in mouse embryos - In human embryo- epiblast is disc-like structure sitting within the blastocyst - In a mouse, the disc is a different shape (like a cup?) - The epiblast is on the inside of the ‘cup’ in a mouse embryo and the hypoblast is on the outside of the ‘cup’ Monozygotic twinning:

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Occurs when an early embryo splits to form twins where they are both genetically identical Monozygotic- one zygote Depending on when the splitting happens, you can get different forms of twinning If early-stage splitting, the trophoblast and ICM form as completely separate entities in two different amniotic sacs- dichorionic If blastocyst forms and ICM splits on the inside, end up with monochorionic placenta (one) but separate amniotic sacs If splitting is later stage or incomplete, then you can end up with conjoined twins as embryos themselves do not split

Gene targeting: -

Introduce plasmid but this plasmid DNA undergoes homologous recombination with homologous regions on chromosomal DNA

Inner cell mass becomes embryonic stem cells under right conditions in a petri dish Depending on what conditions you put them in, they can develop into lots of different types of cells ES modifications are useful tools to modify genome to make gene knockouts: -

Obtain ICM from embryo and culture in a dish (ESC) Genetically modify them in the dish and then take those cells and add to another embryo where they will include themselves as ICM of that embryo Chimeric mice- mix f cells that contain the GMO cells and some do not (this is by chance)

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The chimeric mouse then breeds and some cells forming the gametes will be GMO so offspring can be identified if it has modified traits from GMO A lot of offspring will not carry modification by chance

Gene targeting scheme: -

Must be selection process for cells that have been genetically modified

Gene targeting: -

Plasmid introduced and plasmid DNA undergoes homologous recombination with chromosomal DNA Targeted region is inserted into chromosome Neomycin is selected resistance marker gene Presence of neo gene provides resistance to neomycin- allows for selection of cells Neo must be present- must culture cells with it Neo can be put into the middle of the gene to disrupt it- GENE KNOCKOUT as it disrupts function

Random integration: -

Possibility that plasmid sequence will insert itself elsewhere Could insert itself into damaged region, if DNA is damaged No control of this an this is bad

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Wherever it is inserted, if within another gene, it will disrupt the function of that gene These will survive from neomycin selection so cannot be stopped in this way Enzyme: thymidine kinase (tk) If there is random integration, tk will be included as opposed to correct integration where it will not be included Tk will enable cell to metabolise ganciclovir (reagent added to culture dish) -

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Gene targeting: -

Knockouts- targeted mice Delete or disrupt a functional gene

If this is metabolised, this forms lethal product that will kill the cells that have random integration This 2-step selection process means that the only cells that survive are the ones that have been correctly targeted

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Can be written as Tulp3 -/-: the minus shows that the gene function has been knocked out- null If writing paper, name it fully first but then you can just refer to it in the informal way Wildtype would be Tulp3 +/+ this does not mean you have additional function, this means normal function

Concept of conditional knockouts: -

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Gene is modified so it is flanked by loxP sites (floxed) By having loxP sites, no effect is had on gene, if inserted correctly Gene is only disrupted in presence of Cre enzyme Cre recognises the loxP sites and causes recombination between the 2 sites Effectively cuts out the sequence in the middle Conditional knockout where DNA mouse strain is modified in normal situation However, in some cells Cre is expressed and this deletes a gene Therefore, gene is only inactivated in cells expressing Cre

Cre-lox system: conditional knockouts: -

Tandem loxP sites results in deletion of the transgene Target gene of interest by floxing Then cross the floxed mouse line with mouse line carrying cre recombinase Cre gene driven by a promoter to give expression only in your tissue of interest This takes months or sometimes years Howveer, you always have 2 copies of the gene in the genome and cre recombinase may only affect 1

Inducible knockouts: -

Even if cre is expressed when promoter is active, cre may only be active if something else is expressed CreER protein binds to HSP90 and blocks activity of enzyme To allow Cre to be activated, must add tamoxifen- might inject into cell dish or into pregnant mother Tamoxifen allows the HSP90 to be displaced- does not bind to CreER protein and Cre recombinase activity is not inhibited Cre can now interact with loxP sites

Cre-LoxP system- genetic switch: -

Make a targeted gene but have a region flanked with LoxP sites (floxed) Inverted LoxP sites will lead to gene inversion, rather than deletion

Cre-expressing mice: -

Cre expression driven by promoter Create construct that has promoter region Add Cre-encoding gene into plasmid and inject plasmid into mouse DNA Additional sequence is called a transgene and the animal is called transgenic

Creating transgenic mouse line: -

Relatively quick method for over-expressing a gene Examples of use include: cDNA with CV promoter (expressed everywhere) Upstream region coupled to a reporter (for analysis of promoter function) Rescue of a mutant Tissue specific Cre expression...