Lecture 1 Overview of early mouse development and methodology nb reading list is at end of notes for this lecture

Vertebrate development – classical models 1mm 1 cm 100 microns Phylotypic stage Similar

Development = origami using layers/sheets of cells Three germ layers, Ectoderm, Mesoderm, and Endoderm (derm=layer), give rise to all cells and tissues of the developing embryo. Vertebrates are triploblasts

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) Amenable to genetic manipulation Tissue culture models

Positional informationCell Fate Anterior (Head) Posterior (Tail) Dorsal (Back) Ventral (Front) Left Right Identity and location

Mammals have extensive extraembryonic tissues

In utero development in mouse occurs over 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 Trophectoderm Primitive (primary) endoderm Inner cell mass/ Primitive ectoderm Cleavage stages Zona pelucida Blastocoel cavity Activation of embryonic genome Blastomere days

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 Cell fate mapping (dyes and now tagged loci) In vitro culture of preimplantation stages and in some cases postimplantation stages.

Chimera formation and embryo aggregation. Cell culture models e.g. tetraploid chimeras for testing gene function in extraembryonic vs embryonic lineages. Embryological approaches; Embryonic stem (ES) cells Embryo manipulation/transplantation

In situ hybridization Immunohistochemistry Eed + Nanog Oct4 + Eed SectionsWholemount Molecular embryology; Gene expression profiling of embryos, dissected fragments, derivative tissue culture cell lines and single cells.

Genetic approaches; Classical mouse mutants Brachyury mouse with short tail is dominant mutation in gene for transcription 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 locus Insertional or ‘gene trap’ mutagenesis in ES cells – can go directly to gene of interest SA SD Antibiotic resistance marker Reporter gene IRES PolyA signal

Production of transgenic mice by pronuclear injection of DNA - Gene construct injected into male pronucleus of 1-cell embryo - DNA integrates randomly at single site, usually multicopy Genetic manipulation in mouse; Production of genetically modified mice by transferring ES cells to recipient embryo - Gene manipulation using homologous recombination in ES cells - Inject modified cells into Recipient embryo to produce chimeric animal that transmits donor genome through the germ-line.

Gene targeting in embryonic stem (ES) cells Genetic manipulation in mouse;

Conventional gene knockout strategy (replacement vector) X X Positive selectable Marker gene Negative selectable Marker gene Knock-out (or Knock-in) Genetic manipulation in mouse;

Positive selectable Marker gene Negative selectable Marker gene X X + site specific recombinase (Cre or Flp) + Recombinase recognition sequence Conditional gene knockout strategy; Genetic manipulation in mouse;

Homozygous conditional allele Transgenic mouse expressing site specific recombinase in tissue specific pattern 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. Conditional gene knockout strategy; Genetic manipulation in mouse;

Textbook; Principles of Development, Lewis Wolpert and Cheryl Tickle. Review papers; Lecture 1 -3 Alexandre (2001) International Journal of Developmental Biology 45, p Rossant (2001) Stem Cells 19, p Yamanaka et al, (2006). Developmental Dynamics 235, p Katsuyoshi and Hamada, (2012) Development 139, p3-14 Lecture 4 and 5 Arnold and Robertson (2009) Nature reviews Molecular cellular biology, 10, p Robb and Tam (2004) Seminars in Cell and Developmental biology 15, p43-54 Hayashi et al (2007) Science 316, p Hashimoto and Hamada (2010), Curr Opin Genet Dev 20, p433-7 Hanna et al (2010) Cell 143, p Yamanaka and Blau (2010) Nature 465, p Reading list

New innovations in ES cell manipulation (optional if time permits)

ZFN, TALEN and CrispR/cas systems; Genetic manipulation in mouse; Cys 2 -His 2 zinc finger domain contacts 3bp of sequence in major groove with varying levels of selectivity. 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. Provides substrate for error prone repair or HR using recombinant DNA template for custom modification. TALE effector proteins secreted by Xanthomonas bacteria in order to activate host plant gene expression that aids infection. Modular composition of sequence specific binding domains comprising 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.

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. ZFN, TALEN and CrispR/cas systems; Genetic manipulation in mouse; PAM site (Trans-encoded CRISPR RNA)