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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.

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Presentation Transcript
slide1

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
slide2

Mammalian Development

  • Embryogenesis in mammals occurs in utero - difficult to observe.
  • Important to study because of direct relevance for understanding and treating disease.
  • Mouse is preferred model;

Good genetics (inbred lines etc), short generation time.

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.

slide3

An anthropomorphic view of development

Who am I?

Where am I?

Anterior (Head)

Right

Ventral (Back)

Dorsal (Front)

Left

Posterior (Tail)

slide5

0

1

2

3

4

Preimplantation Development

days

Cleavage stages

Primitive (primary) endoderm

Blastocoel cavity

Blastomere

Inner cell mass

Zona pelucida

Trophectoderm

Activation of embryonic genome

slide9

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)
slide10

Embryological approaches;

  • Gene expression profiling of embryos, dissected fragments, derivative

tissue culture cell lines and single cells.

  • In situ hybridization

Sections

Wholemount

  • Immunohistochemistry

Eed +Nanog

Oct4 +Eed

slide11

Embryological approaches;

  • 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

slide12

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

Reporter gene

PolyA signal

IRES

SA

Wild-type and Nodal (d/d) mutant embryos with staining for markers of primitive streak (brown) and ectoderm (dark blue).

SD

slide13

Genetic approaches;

  • 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 maintained
  • by 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).
slide14

100kb

enhancer

promoter

Genetic approaches;

Transgene constructs;

- Intact gene in BAC complete with tissue specific regulatory sequences

- Engineered BAC with heterologous regulatory sequences, eg tetracycline inducible

- Plasmid with tissue specific regulatory sequences and heterologous gene eg GFP reporter.

Drawback; high copy number gives non-physiological expression levels

slide15

Homzygous/double

mutant ES cells

X

Homzygous mutants,

double mutants etc

Genetic approaches;

  • Gene targeting in embryonic stem (ES) cells
slide16

Knock-out

Negative selectable

Marker gene

Positive selectable

Marker gene

X

X

Knock-in

GFP

Orf

X

X

Genetic approaches;

Conventional gene knockout strategy (replacement vector)

Potential drawbacks are redundancy and lethality

slide17

Genetic approaches;

Conditional gene knockout strategy;

Bacterial site specific recombinases (Cre-loxP or Flp-Frt)

slide18

X

X

+ site specific recombinase

+

Genetic approaches;

Conditional gene knockout strategy;

Negative selectable

Marker gene

Recombinase recognition sequence

Positive selectable

Marker gene

slide19

Genetic approaches;

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.

slide20

Fertilisation

  • Penetration of cumulus cells
  • Acrosomal 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.
slide21

Pronuclear Maturation

Second polar body

Zona pelucida

Syngamy

Male pronucleus.

Female pronucleus.

12

24

0

hr post fertilization

Replication

initiation

M-phase

  • Maternal and paternal genome remain separate (pronuclei) unitil first metaphase.
slide22

Parthenogenesis

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
  • Limited viability suggests either that sperm/fertilization confers essential properties for development or
  • that maternal genome alone is incapable of supporting development
slide23

Recipient zygote

Donor zygote

Non-equivalent contribution of maternal and paternal genomes

?

Barton, Surani , Norris (1984)

Nature 311, p374-6

McGrath 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
slide25

Mosaic and Regulated development

  • 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’.
slide26

Regulated development in mouse embryos

Donor

Recipient

2-cell

embryo

Tarkowski, (1959)

Nature 184, p1286-7

slide27

8-cell embryos

Remove zona pellucida

Aggregate in dish

Culture in vitro

Transfer to foster mother

Chimeric blastocyst

Chimeric progeny

Chimeras from aggregaton of 8-cell stage embryos

Tarkowski (1961) Nature 190, 857-860

slide28

Chimeras from transfer of ICM cells

Gardner (1968), Nature 220, p596-7

  • Gardner later showed fate of TE and PE is determined by blastocyst stage