epiblast

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visceral endoderm
epiblast
aka primitive endoderm
all of embryo
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external view
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allantois
amnion
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heart
heart
somites
node
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A short history of mammalian experimental
embryology and gene manipulation
Modern reproductive developmental biology was
born in the 17th century
Previously, theory of seeds, from the pluralistic
ideas of the Pythagorean school, led by
Anaxogoras of Clazomenae and Empedocles of
Acragas (5th century B.C.)
pluralism fetus results from mixing 2 seeds
Hippocrates (460-370 BC) seeds flow from all
parts of the body, each containing both the
masculine and feminine principle. Aristotle
(384-322 BC) only male seed contributes to
fetus, females only role in procreation is to
contribute menstrual blood.
Galen (130-201 AD) spiritual heir of
Hippocrates asserted seeds of both male and
female contribute to procreation, each providing
only one principle. inspired by the anatomist
Herophilus (340-300 BC), and led to the bold
notion that women have testes identical to mans,
but turned inwards
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Vesalius (1514-1564) father of modern anatomy
drew parallels between females uterine and
males semen-conveying tubes (function understood
only a century later - work of De Graaf
(considered the founder of modern reproductive
biology)
De Graaf (1641-1673) found the source of the
eggs, which were called testes, now (of course)
called ovaries.
Van Leeuwenhoek (1632-1723) observed
animalcules, or spermatic worms. Debates
ovists vs. animalculists as to which led to
animal and human offspring.
Until the 19th century, the century of the
Enlightenment saw preformationists favoring seed
theory, and epigenesists arguing that body parts
form gradually (e.g. Wolff 1733-1794).
1799-1825 fertilization documented in frogs, dogs
and rabbits 1827 precise microscopic description
of the ovum 1834 Adolph Bernhardt (student of Jan
Purkinje) observed in the ovum a germinal
vesicle (nucleus). Findings closely linked to
emergence of cell theory in 1828.
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1827 Karl Ernst von Baer noted resemblance
between dog and bird embryos Half-century later
(1833) early phases of egg and embryo development
described chromatin reduction in nematode
Ascaris (E. Van Beneden 1883) rabbit and bat,
formation of the 3 basic embryonic germ layers
(Van Beneden et al., 1875, 1880, 1884)
proclaimed that cleavage of zygote led to forming
a blastodermic vesicle, now called the
blastocyst
End 19th century emergence of new science
developmental mechanics (Wilhelm Roux)
chemistry physics to explain developmental
events, through experimentation Roux thus the
founder of experimental embryology, pulling in
Driesch who originated embryonic regulation
(cells talk to cells)
Brachet (1869-1930) strong proponent of new
science, called it embryologie causale, and
worried that mammalian eggs had eluded
experimentation. Up until 1940, descriptions of
early development of many mammals mole,
sheep pig, goat, hedgehog, tarsier, and
others. Late emergence of experimental
embryology in mammals was lack of interest, plus
very real technical difficulties (accessibility,
size, robustness after dissection, etc.)
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1890 first transfer of rabbit fertilized egg from
mother (Angora) to a Belgian line mother then
systematically applied to other species
1956, Whitten grew 8-cell mouse embryos to
blastocyst stage in defined medium. By
this time, intraspecific embryo transfer in
rabbit, goat, rat, mouse, cow, and pig achieved.
Then hamster, ferret, mink, horse, baboon, cat,
dog, and water buffalo. Attempts to transfer
between closely linked species also successful
in cattle and sheep, between donkey horse, or
zebra horse.
1956 maybe referred as the year when mammalian
eggs became manipulate-able.
Brachet (1912, 1913) had previously kept rabbit
blastocyst alive 48 hours in blood plasma
Similar studies on rabbit did not multiply until
late 1920s (W.H. Lewis, G. Pincus) 1929 Lewis
Gregory filmed rabbit egg to b/cyst, and later
macaque (2 to 8-cell stage) rat, mouse and
guinea pig eggs were refractory (only 1-2
cleavages)
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Others using rabbit eggs (including Pincus
mid-1930s) took steps towards defining chemical
requirements for suitable culture medium.
By taking into account metabolic requirements of
mouse eggs, Whitten (1957), and Ralph Brinster
(1963) improved culture conditions culturing
from 2-cell stage onward became reproducible a
major and long-awaited breakthrough
1960s (70 years after Rouxs new science)
mammalian embryology moved from descriptive to
experimental remember context 1978, first IVF
human baby born
1958 Anne McLarens group had mice born that had
been cultivated in vitro for a time as early
embryos
1959 rabbit made by in vitro fertilization,
uterine transfer and developed to term (Chang)
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Major question emerged and is still debated how
do various parts of mammalian embryos form
cells continually regulate each other, or
differential inheritance of substances localized
in the egg?
1942 Nicholas Hall showed that separated rat
2-cell embryos made complete embryos, and 2 eggs
stuck together could form a chimera embryos thus
regulative
also, killing 1 of 2 blastomeres normal embryo
still formed (Tarkowski 1959)
But the regulatory power of cells could be
controlled by inheritance of morphogenetic
substances. Some cells might be biased towards
trophoblast, others towards ICM. This major
concept is still under investigation (and
incredibly controversial even today are there
predispositions (and specific cleavage planes
with respect to orientation/placement of
different fates in developing zygotes?
Rearrangement experiments (1972, 1977) evidence
that blastomeres are totipotent until the time
they occupy an internal or external position.
Blastocyst reconstitution experiments (Gardner,
1970) demonstrated the committed state of ICM and
TE
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1986 role of cell polarization (apical-basal) in
8-cell stage mouse embryo (M. Johnson et al.)
Polarization gave credence to the inside-outside
theory
1960s gentle micromanipulation techniques were
largely developed. First mouse chimeras obtained
(Tarkowski, morula aggregation, 1959) and
(Gardner, ICM injection into Blastocysts,
1968) Also in 1960s, autoradiographic,
pharmacological and biochemical investigations
were beginning on general trends of gene
expression during early development
1970s many attempts made to parthenogenetically
activate and develop mammalian eggs never
worked
1984 (two groups McGrath Solter Barton,
Surani Norris) concluded maternal
and paternal genetic components are qualitatively
complementary - led to epigenetics of imprinting
and regulation through large-scale chromatin
modification.
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Non-egg models for mammalian developmental biology
Scarcity of embryos, resistance to hard
manipulation led to interests in other
more easily used models e.g., the malignant
teratocarcinoma, a variant of the more
benign, rare tumour, the teratoma (Pierce,
1967) 1954 first description in mouse (Stevens
Little) studies of a testicular teratoma that
was abnormally frequent in mouse line 129. Forms
many tissues corresponding to different stages
of differentiation. Derived from pluripotent
stem cells called embryonal carcinoma (EC) cells,
which derive from germline cells. EC
resemblance to pluripotent embryo cells
(morphology developmental potency) favored
new model for development.
Whole symposia devoted to EC cells (1975,
1982) Ovarian teratomas and EC cells could be
made experimentally, not just from 129/Sv
mice Stevens (1964, 1968, 1970) made some by
implanting mouse embryos into testes of isogenic
adult recipients.
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EC cell lines were also made by Martin and Evans
(1974) total in 1983 almost 100 lines.
Remained malignant, some nullipotential, others
differentiated into many cell types, in vitro,
and in vivo when injected into syngeneic adult
mice. But differentiation followed no order
under these conditions. Therefore, EC cells were
studied in association with normal embryonic
cells.
With blastocyst injection technique pioneered by
Gardner (1968), Brinster obtained first mouse
chimera from EC cells (1974). EC teratocarcinoma
cells had thus been normalized.
Experiments continued by K. Ilmensee in B.
Mintzs laboratory, Papaioannou McBurney in
Gardners lab (mid-to-late 1970s). Janet Rossant
also belonged to the latter small group.
Despite remarkable successes, with historic
papers, most EC cells were aneuploid. And had
lost pluripotential character.
Impossible to transfer a mutation through the
germline of any of the rare chimeras that were
obtained (Papaioannou, 1979). This method
therefore untenable for producing genetically
altered mouse lines.
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Road was opened developed methods applied to ES
cells, derived directly from blastocyst ICM
(Evans and Kaufman 1981 G. Martin 1981)
At around this time (1980, 1981, 1982), first
steps in transgenesis taken by injecting DNA
into the zygote.
1986 ES cells shown to allow derivation of
transgenic strains with specific genetic changes
(viral transfer E.J. Robertson et al.)
1987, site-directed mutagenesis of ES cells via
homologous recombination (2 groups Oliver
Smithies and Mario Capecchi) led to the era of
knockout mice. 1988, 2000-fold stimulation of HR
via negative selection.
1997 Cloning by nuclear transfer into oocytes
(Wilmut et al.)
Major advances in medicine and biomedical
researchdisease models, tools for drug
discovery, basic biology, cancer
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1975 Papaioannou, McBurney and Gardner Nature
258, 70-73
Teratocarcinoma cells convenient model for
mammalian development
Transplantable tumour cell types of germline or
embryonic origin, typically mixture of
differentiated tissues and undifferentiated stem
cells embryonic definition probably used here
Stem cells embryonal carcinoma, resemble cells
of early embryos propagatable in tissue
culture in hosts, make teratocarcinomas
differentiate in vitro similar to normal
embryonic development
Papaioannou et al. showed EC cells participating
in normal embryogenesis - validating their
utility as a model of normal embryonic
development
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Chimeras used Brinster had previously injected
cells from ascitic form of an in vivo
teratocarcinoma into blastocysts of different
genotype, and made a single overtly chimeric
mouse.
Papaioannou et al. injected cells from in vitro
cultures of EC lines - made good chimeras from 2
independent EC cell lines. A third line
colonized embryo but made large teratocarcinomas
postnatally.
Markers Gpi-1 isoenzymes used, plus albino or
pigmentation, and other coat colour markers.
Clumps of 20-40 EC cells injected into
blastocysts, analyzed initially at day 10 of
embryogenesis, or at term and several weeks
after. Organ analysis - wide chimerism.
Complementary normal embryonic cells converted
into teratocarcinomas by transplanting embryos
of various developmental stages into ectopic
sites in syngeneic adult hosts.
Their mice were being test bred at time of
publication germline transmission?
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Beddington and Robertson Development 105, 733-737
(1989)
ES cells into host blastocysts, as groups or
single cells
Analyze at mid-gestation for chimerism in the
embryo or extraembryonic tissue
chimera mythological fire breathing monster,
lions head, goats body, serpents tail or any
similar monster with disparate body
parts genetics organism with two or more
genetically distinct tissues
Chimerism tested by electrophoretic separation of
GPI isoenzymes
Major Findings trophectoderm colonized by ES
cells, but poorly High chimerism (ES
colonization) in embryo and extraembryonic
mesoderm
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Postnatal characterization in those papers
prevented determining contribution to
extraembryonic tissues, and thus an idea of which
cell in embryos ES cells most resemble. Paper
relevant to origin of ES cells making
tissues in which altered gene expression can be
studied in ES chimeras ES cells as in vitro
models for aspects of mouse development
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129/Sv/Ev inbred strain of mice ES cells were
derived, XY karyotype
Embryos MF1 outbred strain (homozygous for
Gpi-1b allele
ES cells into blastocoel cavity they fuse with
the ICM
Analyzed e10 embryos trophoblast giant cells,
placenta, ectoderm, parietal (extraembryonic)
endoderm, visceral yolk sac endoderm, VYS
mesoderm, amnion, fetus
Groups of cells most contribution to fetus
extraembryonic mesoderm-derived tissues, some
ES-derived isoenzymes in visceral and parietal
endoderm, and few trophoblast giant cells. Single
cells predominant pattern is chimaerism
restricted to the fetus, amnion and VYS mesoderm
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So what is an ES cell?
Late blastocyst three lineages of trophectoderm,
primitive endoderm, epiblast
Segregate sequentially early ICM cells
injected into blastocyst colonize mainly fetus
and EEM, but also produce TE and PE expanded
blastocyst ICM injected cells no longer give rise
to TE 5th day blastocyst, ICM comprises 2
distinct populations, PE and epiblast
Simplest conclusion by comparison is that ES
cells represent early ICM cells
Note that ES cells in this study derived from
implantationally delayed blastocysts and ICM
cells from delayed blastocysts do not make good
chimeras
ES cells derived from epiblast present in delayed
embryos, but growth in vitro reverts them to a
more primitive cell type. The restriction in
potential of epiblast cells may be lost in
clonal cell culture.
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Colonization patterns indicated utility in
analyzing certain genes in chimeras certain
genetic effects lethal if in all cell types,
rescued in chimeras via ES cells, can study
them in fetus and important extraembryonic
organs including placenta and visceral yolk
sac
ES cell rescue experiments - visceral endoderm
versus embryonic gene expression
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