lecture 1 overview of early mammalian development tools for studying mammalian development

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Lecture 1 Overview of early mammalian development Tools for studying mammalian development Fertilisation and parthenogenesis Mosaic vs regulated development. You should understand Non-equivalence of maternal and paternal genomes Mammalian development is highly regulated. - PowerPoint PPT Presentation

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Lecture 1

Overview of early mammalian development Tools for studying mammalian development Fertilisation and parthenogenesis Mosaic vs regulated developmentYou should understand

Non-equivalence of maternal and paternal genomes Mammalian development is highly regulated Embryogenesis in mammals occurs in utero - difficult to observe. Important to study because of direct relevance for understanding and treating disease.Isolation of tissue culture models, e.g embryonic stem cells, is relatively easy. Also highly advantageous for genetic manipulation, knock-out, knock-in etc- Functional genomics studies- Disease models for basic science and pharmacology. Mouse is preferred model; Good genetics (inbred lines etc), short generation time.Mammalian DevelopmentWhere am I?Who am I?

Anterior (Head)Posterior (Tail)Ventral (Back)Dorsal (Front)LeftRightAn anthropomorphic view of development

In utero development in mouse occurs over 19-21 daysPreimplantation Development

TrophectodermPrimitive (primary) endodermInner cell massCleavage stagesZona pelucidaBlastocoel cavityActivation of embryonic genomeBlastomere01234days

Early Post-implantation Development

Gastrulation and Beyond

Extraembryonic tissuesExperimental Tools for studying mouse embryosEmbryological approaches;

Histological analysis and conventional microscopy Cell fate mapping (dyes and now tagged loci)

In vitro culture of preimplantation stages and in some cases postimplantation stages.In situ hybridization

Immunohistochemistry

Eed + NanogOct4 + EedSectionsWholemountEmbryological approaches; Gene expression profiling of embryos, dissected fragments, derivative tissue culture cell lines and single cells.

Chimera formation and embryo aggregation. Cell culture modelse.g. tetraploid chimeras for testing gene function in extraembryonic vs embryonic lineages.

Embryological approaches;

Embryonic stem (ES) cellsGenetic approaches;

Classical mouse mutantsBrachyury mouse with short tail is dominant mutation in gene fortranscription factor required for mesoderm formation. Genetic screens

Wild-type and Nodal (d/d) mutant embryos with staining for markers of primitive streak (brown) and ectoderm (dark blue).Chemical (ENU) mutagenesis requires lengthy genetic mapping and cloning to identify mutated locusInsertional or gene trap mutagenesis in ES cells can go directly to gene of interestSASDAntibiotic resistancemarkerReporter geneIRESPolyA signal Production of transgenic mice

- Gene construct injected into male pronucleus of 1-cell embryo

- DNA integrates randomly into the genome

- Usually at single site but in multiple copies

- Resulting mice can be bred and then maintainedby monitoring continued presence of the transgene using PCR etc.

Gene construct can be assembled in plasmid (up to 25kb) or bacterial artificial chromosome (BAC) vectors (100-200kb).Genetic approaches;

Transgene constructs;100kb- Intact gene in BAC complete with tissue specific regulatory sequencesenhancerpromoter- Engineered BAC with heterologous regulatory sequences, eg tetracycline inducible- Plasmid with tissue specific regulatory sequences and heterologous gene eg GFP reporter.Genetic approaches;Drawback; high copy number gives non-physiological expression levels Gene targeting in embryonic stem (ES) cellsGenetic approaches;

X

Homzygous mutants,double mutants etcHomzygous/double mutant ES cellsConventional gene knockout strategy (replacement vector)Potential drawbacks are redundancy and lethalityXXPositive selectableMarker geneNegative selectableMarker geneKnock-outXGFP OrfXKnock-inGenetic approaches;Conditional gene knockout strategy;Bacterial site specific recombinases (Cre-loxP or Flp-Frt)

Genetic approaches;Positive selectableMarker geneNegative selectableMarker geneXX+ site specific recombinase+Recombinase recognition sequenceConditional gene knockout strategy;Genetic approaches;

Homozygous conditional alleleTransgenic mouse expressing site specific recombinasein tissue specific patternXAnalyse phenotype in F1 embryos or adults

Examples of recombinase driver transgenics;- Cre recombinase driven by Nanog promoter- Estrogen receptor-Cre recombinase fusion driven by constitutive promoter. Addition of Tamoxifen to drinking water triggers nucleartranslocation of recombinase giving temporal control of gene deletion.Conditional gene knockout strategy;Genetic approaches;Fertilisation

Penetration of cumulus cellsAcrosomal reaction penetrates zona pellucida made up of glycoproteins Sperm and egg plasma membranes fuse and sperm nucleus enters egg.Fertilization triggers dramatic release of calcium in the egg, setting in train completion of female meiosis etc.Pronuclear Maturation1224ReplicationinitiationM-phasehr post fertilization0Second polar bodyZona pelucidaMaternal and paternal genome remain separate (pronuclei) unitil first metaphase. Male pronucleus. Female pronucleus. SyngamyParthenogenesis

Limited viability suggests either that sperm/fertilization confers essential properties for development or that maternal genome alone is incapable of supporting development Parthenogenetic activation

- Genetic background- In vitro manipulation- Pronase/hyalouronidase- Heat shock- Ethanol- Strontium chloride Oocytes can be activated in the absence of fertilization, leading to parthenogenetic development Parthenogenetic embryos have limited viability, contrasting with other model organisms

Non-equivalent contribution of maternal and paternal genomes?Recipient zygoteDonor zygoteBarton, Surani , Norris (1984)Nature 311, p374-6McGrath and Solter, (1984)Cell 37, p179-183 Gynogenetic embryos have retarded growth/development of extraembryonic tissues Androgenetic embryos have retarded growth/development of embryonic tissues

Epigenesis vs Preformation

Roux (1888) shows mosaic development of frog embryo following ablation of one cell in two-cell embryo formation of half embryo. Driesch (1895) finds opposite is true for sea urchin, normal albeit smaller embryo develops from one of two cells regulated development.Mosaic and Regulated developmentTarkowski, (1959)Nature 184, p1286-72-cellembryo

DonorRecipient

Regulated development in mouse embryosChimeras from aggregaton of 8-cell stage embryos

8-cell embryosRemove zona pellucidaAggregate in dishCulture in vitroChimeric blastocystTransfer to foster motherChimeric progenyTarkowski (1961) Nature 190, 857-860 Chimeras from transfer of ICM cells

Gardner later showed fate of TE and PE is determined by blastocyst stageGardner (1968), Nature 220, p596-7End lecture 1

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