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.