Lecture 1 Overview of early mouse development and methodology nb reading list is at end of notes for this lecture Vertebrate development – classical models 1 cm 100 microns 1mm Phylotypic stage Similar Similar Vertebrates are triploblasts Three germ layers, Ectoderm, Mesoderm, and Endoderm (derm=layer), give rise to all cells and tissues of the developing embryo. Development = origami using layers/sheets of cells Why study the mouse? • The Victorian mouse fancy movement provided a ready made resource of inbred strains, variants and mutants • Fast generation time (21 day gestation) • Tissue culture models • Amenable to genetic manipulation Identity and location Cell Fate Positional information Anterior (Head) Right Dorsal (Back) Ventral (Front) Left Posterior (Tail) Mammals have extensive extraembryonic tissues In utero development in mouse occurs over 19-21 days • E (embryo stage) = dpc (days post coitum). Most commonly referred to from 0.5 onwards as mating takes place at night. • Preimplantation development occurs up to E3.5. All other development occurs postimplantation. Preimplantation Development 0 1 2 Cleavage stages 3 4 days Primitive (primary) endoderm Blastocoel cavity Zona pelucida Blastomere Activation of embryonic genome Trophectoderm Inner cell mass/ Primitive ectoderm Early Post-implantation Development Gastrulation and Beyond Confusing nomenclature! A ‘derm’ is a cell layer – not a cell type! Our understanding of the world can only be as good as the state of the art technology we use to measure it – knowledge is relative, not absolute. Experimental Tools for studying mouse embryos Embryological approaches; • Histological analysis and conventional microscopy • In vitro culture of preimplantation stages and in some cases postimplantation stages. • Cell fate mapping (dyes and now tagged loci) Embryological approaches; • Embryo manipulation/transplantation • Chimera formation and embryo aggregation. e.g. tetraploid chimeras for testing gene function in extraembryonic vs embryonic lineages. • Cell culture models Embryonic stem (ES) cells Molecular embryology; • Gene expression profiling of embryos, dissected fragments, derivative tissue culture cell lines and single cells. • In situ hybridization Sections • Wholemount Immunohistochemistry Oct4 + Eed Eed + Nanog Genetic approaches; • Classical mouse mutants Brachyury mouse with short tail is dominant mutation in gene for transcription factor required for mesoderm formation. • Genetic screens Chemical (ENU) mutagenesis – requires lengthy genetic mapping and cloning to identify mutated locus Insertional or ‘gene trap’ mutagenesis in ES cells – can go directly to gene of interest Antibiotic resistance marker IRES Reporter gene PolyA signal SA SD Wild-type and Nodal (d/d) mutant embryos with staining for markers of primitive streak (brown) and ectoderm (dark blue). Genetic manipulation in mouse; • Production of transgenic mice by pronuclear injection of DNA • Production of genetically modified mice by transferring ES cells to recipient embryo - Gene construct injected into male pronucleus of 1-cell embryo - Gene manipulation using homologous recombination in ES cells - DNA integrates randomly at single site, usually multicopy - Inject modified cells into Recipient embryo to produce chimeric animal that transmits donor genome through the germ-line. Genetic manipulation in mouse; • Gene targeting in embryonic stem (ES) cells Genetic manipulation in mouse; Conventional gene knockout strategy (replacement vector) Knock-out (or Knock-in) Positive selectable Marker gene X Negative selectable Marker gene X Genetic manipulation in mouse; Conditional gene knockout strategy; Recombinase recognition sequence Positive selectable Marker gene X Negative selectable Marker gene X + site specific recombinase (Cre or Flp) + Genetic manipulation in mouse; Conditional gene knockout strategy; Transgenic mouse expressing site specific recombinase in tissue specific pattern Homozygous conditional allele X Analyse 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 nuclear translocation of recombinase giving temporal control of gene deletion. Reading list Textbook; Principles of Development, Lewis Wolpert and Cheryl Tickle. Review papers; Lecture 1 -3 Alexandre (2001) International Journal of Developmental Biology 45, p457-467 Rossant (2001) Stem Cells 19, p477-82 Yamanaka et al, (2006). Developmental Dynamics 235, p2301-2314 Katsuyoshi and Hamada, (2012) Development 139, p3-14 Lecture 4 and 5 Arnold and Robertson (2009) Nature reviews Molecular cellular biology, 10, p91-103 Robb and Tam (2004) Seminars in Cell and Developmental biology 15, p43-54 Hayashi et al (2007) Science 316, p394-396. Hashimoto and Hamada (2010) , Curr Opin Genet Dev 20, p433-7 Hanna et al (2010) Cell 143, p508-525. Yamanaka and Blau (2010) Nature 465, p704-712 New innovations in ES cell manipulation (optional if time permits) Genetic manipulation in mouse; ZFN, TALEN and CrispR/cas systems; • Cys2-His2 zinc finger domain contacts 3bp of sequence in major groove with varying levels of selectivity. • TALE effector proteins secreted by Xanthomonas bacteria in order to activate host plant gene expression that aids infection. • Can use as modular component to get sequence specific targeting of Fokl restriction endonuclease monomer. Cleavage requires targeting second monomer to other strand to generate functional Fokl dimer. • Modular composition of sequence specific binding domains comprising 33-34 amino acids with positions 12 and 13 being highly variable. • Can be used to construct designer Transcription Activator Like Effector Nuclease (TALEN) to introduce DNA breaks at defined target sequence. • Provides substrate for error prone repair or HR using recombinant DNA template for custom modification. • Provides substrate for error prone repair or HR using recombinant DNA template for custom modification. Genetic manipulation in mouse; ZFN, TALEN and CrispR/cas systems; (Trans-encoded CRISPR RNA) PAM site • RNA mediated bacterial defense against viral or plasmid DNA. • Type II system adapted for genome engineering in many organisms. • Can use cas9 intrinsic nuclease to introduce ds break or ss nick. • Provides substrate for error prone repair or HR using recombinant DNA template for custom modification. • Can also mutate directly by injection into zygote. • Partially circumvents requirement for highly recombinogenic cell such as ES cell.