Download From Hatching into Fetal Life in the P e in the P e in the Pig

R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): s203 - s221.
ISSN 1679-9216 (Online)
Fr om Ha
etal Lif
e in the P
ig
Hatt ching in
intto FFetal
Life
Pig
Poul Hyttel, Kristian M. Kamstrup & Sara Hyldig
ABSTRACT
Background: Potential adverse effects of assisted reproductive technologies may have long term consequences on embryonic
and fetal development. However, the complex developmental phases occurring after hatching from the zona pellucida are less
studied than those occurring before hatching. The aim of the present review is to introduce the major post-hatching
developmental features bringing the embryo form the blastocyst into fetal life in the pig.
Review: In the pre-hatching mouse blastocyst, the pluripotency of the inner cell mass (ICM) is sustained through expression
of OCT4 and NANOG. In the pre-hatching porcine blastocyst, a different and yet unresolved mechanism is operating as OCT4
is expressed in both the ICM and trophectoderm, and NANOG is not expressed at all. Around the time of hatching, OCT4
becomes confined to the ICM. In parallel, the ICM is divided into a ventral cell layer, destined to form the hypoblast, and a
dorsal cell mass, destined to form the epiblast. The hypoblast gradually develops into a complete inner lining along the
epiblast and the trophectoderm. Upon hatching (around Day 7-8 of gestation), the trophectoderm covering the developing
epiblast (Rauber´s layer) is lost and the embryonic disc is formed by development of a cavity in the epiblast, which subsequently
“unfolds” resulting the establishment of the disc. In parallel, the epiblast initiates expression of NANOG in addition to OCT4.
The blastocyst enlarges to a sphere of almost 1 cm around Day 10 of gestation. Subsequently, a dramatic elongation of the
embryo occurs, and by Day 13 it has formed a thin approximately one meter long filamentous structure. This elongation is
paralleled with the initiation of placentation along with which, the embryonic disc undergoes gastrulation. The latter process
is preceded by a thickening of the posterior region of the epiblast, putatively developing as a consequence of an absence of
N
inhibitory signals from a condensed portion of the hypoblast underlying the anterior epiblast. The thickened posterior epiblast
expresses the primitive streak marker BRACHYURY. Subsequently, the epiblast thickening extends in an anterior direction
forming the primitive streak; also expressing BRACHYURY. Gastrulation is hereby initiated, and epiblast cells ingress through
the primitive streak to form mesoderm and endoderm; the latter is inserted into the dorsal hypoblast whereas the mesoderm
forms a more loosely woven mesenchyme between the epiblast and the endoderm. The anterior mesoderm, ingressing through
the anterior end of the primitive streak, referred to as the node, forms the rod-like notochord interposed between the epiblast
and the endoderm. During the subsequent neurulation, which is a process overlapping with gastrulation in time, the notochord
induces the overlying epiblast to form neural ectoderm, which sequentially develops into the neural plate, neural groove, and
neural tube, whereas the lateral epiblast develops into the surface ectoderm. In parallel with the development of the somatic
germ layers, ectoderm, mesoderm, and endoderm, the primordial germ cells, the predecessors of the germ line, develop in the
posterior epiblast and initiates a migration finally bringing them to the genital ridges of the developing embryo. In parallel, the
ectoderm gives rise to the epidermis and neural tissue, the mesoderm develops into the cardiovascular system as well as the
urogenital and musculoskeletal systems, whereas the endoderm forms the gastrointestinal system and related organs as the
liver and pancreas.
Conclusions: Porcine embryonic and fetal development is controlled by molecular mechanisms that to some degree differ
from those operating in the mouse. It is of importance to uncover the molecular control of development in ungulates as it has
great implications for assisted reproductive technologies as well as for biomedical model research.
Keywords: Biomedical models, embryology, blastulation, gastrulation, neurulation, embryonic staging.
CORRESPONDENCE: P. Hyttel [[email protected] – FAX: +45 353 32547]. Department of Basic Animal and Veterinary Sciences, Faculty of
Life Sciences, University of Copenhagen, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark.
s203
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
I. INTRODUCTION
II.BLASTULATION: DEVELOPMENT OF TROPHECTODERM, ICM, EPIBLAST, HYPOBLAST,
AND EMBRYONIC DISC
III. GASTRULATION: DEVELOPMENT OF MESODERM, ENDODERM, AND ECTODERM
3.1 The primitive streak
3.2 Ingression of cells forming mesoderm and endoderm
IV. NEURULATION: DEVELOPMENT OF THE NEURAL ECTODERM AND NEURAL CREST
4.1 Neural ectoderm
4.2 Neural crest
V. DEVELOPMENT OF THE PRIMORDIAL GERM
CELLS (PGCS)
VI. FURTHER DEVELOPMENT OF THE EMBRYO
6.1 The ectoderm and its early derivatives
6.2 The mesoderm and its early derivatives
6.3 Paraxial mesoderm
6.4 Paraxial mesoderm
6.5 Lateral plate mesoderm and body folding
6.6 Blood and blood vessel formation
6.7 The endoderm and its early derivatives
VII. PLACENTATION AND FORMATION OF EXTRAEMBRYONIC MEMBRANES AND CAVITIES
7.1 Development of extra-embryonic membranes and
cavities
7.2 Placentation
VIII. STAGING OF EMBRYONIC DEVELOPMENT
IX. CONCLUSIONS
sarily a guarantee for further development into a fetus
and newborn. Hence, it is well-documented that in
vitro embryo culture may impose long-term effects,
which are revealed later during embryonic and fetal
development [40]. This phenomenon becomes even
more exaggerated when embryos are produced by
SCNT, which imposes an even higher risk of
embryonic and fetal aberrations as well as neonatal
loss [9,35].
In order to evaluate embryonic and fetal
development resulting from assisted reproductive
technologies more properly, increasing focus should
be put on the normality of some of the complex posthatching processes, as e.g. gastrulation, neurulation,
placentation and initial organogenesis, which are
prerequisites for full term development. These processes are the focus of the present review. Over the
past decade the pig has attracted increasing attention
as a useful biomedical model, due to which the
presented data will mainly be derived in this species.
Comparative notes will be made to cattle, whenever
the variation between these two species are pronounced as e.g. at placentation. First, important developmental processes of the general embryology including blastulation, gastrulation, neurulation, and development of the germ line will be presented, and,
second, a short summary of the special embryology,
i.e. the development of the organ systems, will be
given.
I. INTRODUCTION
II. BLASTULATION: DEVELOPMENT OF
The wide-spread use of in vitro production
of, in particular, bovine embryos in animal husbandry
has paved the way for a detailed morphological and
molecular understanding of oocyte maturation,
fertilization and initial embryonic development until
the time of hatching. Hence, studies on these life processes have become facilitated by the easy
accessibility of oocytes, zygotes, and embryos.
Cloning by somatic cell nuclear transfer (SCNT) is
another technology, which over the past decade has
resulted in alternative in vitro production of
considerable numbers of both bovine and porcine
embryos adding to the accessibility of embryos for
research. Development of the embryo to the blastocyst
stage includes several complex processes as e.g. the
activation of the embryonic genome (for review, see
Oestrup et al. [31]). It is also clear, however, that
success in developing into a blastocyst is not neces-
TROPHECTODERM, ICM, EPIBLAST, HYPOBLAST, AND
EMBRYONIC DISC
Blastulation (from the Greek term blastos
meaning sprout) is the process by which the embryo
develops into a fluid-filled structure in which the cells
have segregated into lines destined to produce the
embryo proper (the ICM and epiblast) and such
developing into the extra-embryonic membranes (the
trophectoderm and hypoblast).
In the pig, the blastocyst forms at around Day
5 of gestation. The porcine embryo initiates compaction as early as the 8-16-cell stage, when the
embryo assumes a spherical appearance with a
smoother surface where the protrusions of the individual blastomeres are no longer seen. The outer cells,
allocated to the trophectoderm, become connected
by tight junctions and desmosomes sealing the
developing blastocyst cavity where the ICM forms
s204
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
as a cluster of lucent cells. Adjacent ICM cells communicate through scarce gap junctions [12]. The
trophectoderm is divided into a polar portion,
covering the ICM, and a mural portion sealing the
blastocyst cavity. In advance of hatching, the ICM
develops into a distinct ventral cell layer, destined to
form the hypoblast, and a dorsal mass of cells destined
to form the epiblast (Figure 1).
A dynamic change in gene expression is the
driving force for the first cell differentiation: i.e. the
segregation of the compacting blastomeres into the
ICM and trophectoderm. In the mouse, the ICM develops a stable regulatory circuit, in which the transcription factors NANOG [5,25], OCT4 [27,36],
SOX2 [1], and the more recently identified SAL4 [7],
2006; [49]Zhang, et al., 2006) promote pluripotency
and suppress differentiation. In contrast, in the trophectoderm-destined cells, the transcription factors
CDX2 and EOMES are upregulated together with
ELF5 and TEAD4, which are transcription factors
that act upstream of CDX2 to mediate trophectoderm
differentiation [26,28,47]. On the other hand,
expression of the trophectoderm-associated transcription factors, CDX2, TEAD4, and ELF5, are
repressed in the ICM by the regulatory circuit of
NANOG, SOX2, and OCT4[34]. In the pig, the
expression of CDX2 during preimplantation development appears conserved as compared with the
mouse [19]. OCT4 is, on the other hand, expressed
in both the ICM and trophectoderm as opposed to
the mouse [17,18], and NANOG expression has not
been observed in the porcine ICM [11]. Hence, there
are marked species differences with respect to the
molecular background for ICM and trophectoderm
specification.
The embryo expands in size and hatches from
the zona pellucida by Days 7 to 8, and in parallel the
OCT4 expression becomes confined to the ICM [43],
whereas expression of NANOG is still lacking (Figure 2; [46]). At the time of hatching, the ICM is in the
N
Figure 1. Transmission electron micrograph of porcine Day 6 blastocyst
showing the zona pellucida (ZP), polar trophectoderm (Te) and the inner
cell mass, which has already divided into ventral cells (VC), developing
into the hypoblast, and dorsal cells (DC), developing into the epiblast. BC:
Blastocyst cavity. Insert: Light micrograph of the same blastocyst showing
the inner cell mass (ICM).
Figure 2. Confocal laser scanning micrographs of Day 8-9 hatched porcine blastocyst displaying OCT4 expression in the inner
cell mass, whereas NANOG expression is lacking. E-CADHERIN is used as an epithelial marker.
s205
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
process of separating into two distinct cell
populations. Hence, the most “ventral” cell layer
towards the blastocyst cavity flattens and, finally,
delaminates forming the hypoblast. The “dorsal” cell
population establishes the epiblast. The hypoblast
subsequently extends along the inside of the
trophectoderm forming a complete inner epithelial
lining of the embryo. The polar trophectoderm
covering the epiblast (known as the Rauber´s layer)
becomes very thin around Day 9 of gestation and
gradually disintegrates exposing the epiblast to the
uterine environment. Before the shedding of Rauber’s
layer, tight junctions are formed between the
outermost epiblast cells to maintain the epithelial
sealing the embryo despite the loss of the polar
trophectoderm. Apparently, the porcine epiblast forms
a small cavity, which finally opens dorsally followed
by an “unfolding” of the complete epiblast upon the
disintegration of Rauber’s layer (Figure 3; [12]). After
the loss of this component about Day 10 of gestation,
the epiblast is discernable in the stereo microscope
as a circular lucent structure known as the embryonic
disc (Figure 4; [43]). Along with the formation of the
embryonic disc, the blastocyst enlarges, and by Day
10 it reaches a diameter of more than half a
centimetre. In parallel, with the formation of the
embryonic disc, the porcine epiblast starts to express
not only OCT4, but also NANOG (Figure 5; [46]).
At this stage of development, the first sign of
anterior-posterior polarization develops in the
embryonic disc: As mentioned earlier, the epiblast is
underlaid by the hypoblast, and an area of increased
density of closely apposed hypoblast cells develops.
This area is approximately the same size as the em-
Figure 3. Light micrograph of sections of the same porcine Day 9 blastocyst. (A) Rauber’ layer (RL), continuous with the remaining portion of
the trophectoderm (Te), covers the epiblast (Ep), in which a cavity (C) has developed. The epiblast is underlaid by the epiblast-related taller
hypoblast (Ep-Hy) and the trophectoderm by the trophectoderm-related lower hypoblast (Te-Hy). (B) Another section from the same epiblast
showing the opening of the cavity towards the external environment and the “unfolding” (arrows) of the epiblast to form the embryonic disc.
Figure 4. Porcine Day 10 blastocyst. (A) Stereo micrograph showing the blastocyst presenting the embryonic disc (arrow). (B) Light
micrograph of section of the embryonic disc showing the dome-shaped epiblast (Ep) underlaid by the hypoblast (Hy). The epiblast is continuous
with the trophectoderm (Te) indicated by the arrows.
s206
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
bryonic disc, but it is dislocated about one third of its
diameter anteriorly as compared with the epiblast of
the embryonic disc (Figure 6; [13,46]). It is likely
that this dense hypoblast region emits signals to the
epiblast which suppress mesoderm-formation in the
anterior epiblast regions [13]. In this sense, the hypoblast may carry the blue-print for the specification of
the epiblast.
During Days 11 to 12, the embryonic disc
develops into an oval shape, and a crescent-shaped
accumulation of cells are found in the posterior region
of the disc [43]. This crescent includes mesodermal
progenitors which express the mesodermal markers,
T (BRACHYURY) and GOOSECOID [3,45], and
apparently ingression of BRACHYURY-expressing
extra-embryonic mesoderm is initiated from this
crescent even before the “true” gastrulation starts with
the appearance of the primitive streak (see later; [45]).
A porcine embryo displaying BRACHYURY expression in the posterior epiblast is displayed in Figure 6.
With the development of the embryonic disc,
a very peculiar pattern of OCT4 and NANOG expression develops in the porcine epiblast: The majority of
epiblast cells express OCT4, but small groups or
islands of cells are OCT4 negative [46]. The latter
cells, on the other hand, express NANOG resulting in
a mutually exclusive expression pattern (Figure 7).
Subsequently, NANOG expression is lost in almost
the entire epiblast, except for a few cell in the most
posterior region of the embryonic disc, in which OCT4
is also expressed [46]. The latter cells are believed to
be the primordial germ cells (PGCs).
N
Figure 5. Confocal laser scanning micrographs of Day 9-10 hatched porcine blastocyst displaying OCT4 and NANOG expression in the
epiblast.
Figure 6. Confocal laser scanning micrographs of Day 10-11 porcine blastocyst displaying expression of T (BRACHYURY) in the posterior
portion of the epiblast and of FOXA2 in the hypoblast. The open arrowheads in mark the periphery of the elongated embryonic disc. The
asterisks mark the hypoblast area with higher cell density, which is about one third dislocated anteriorly as compared with the embryonic disc.
A: Anterior; P: Posterior. Modified from Wolf et al. (2011b)[45].
s207
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
III. GASTRULATION: DEVELOPMENT OF MESODERM,
ENDODERM, AND ECTODERM
Gastrulation (from the Greek term gastrula
meaning small stomach) is the process by which the
three somatic germ layers, ectoderm, mesoderm, and
endoderm, as well as the PGCs (see later) are formed.
3.1 The primitive streak
During Days 11-12 of gestation, the porcine
embryo initiates a dramatic elongation that over a
couple of days results in the transformation of the
spherical blastocyst to an approximately 1 m long,
extremely thin filamentous structure. Gastrulation in
the porcine embryo is initiated around the time, when
elongation is in its initial progress [45]. Porcine
gastrulation is not dependent on implantation, as it is
in man and mouse [3,10]. “True” gastrulation is defined by the presence of the primitive streak (Figure
8). The porcine primitive streak apparently develops
as an anterior extension of the BRACHYURY and
GOOSECOID expressing crescent of epiblast cells
[3,45]. The streak elongates in an anterior direction
and forms a depression, termed the primitive groove,
at the midline. The porcine primitive streak expresses BRACHYURY throughout its extension and
GOOSECOID at least in the anterior portion [23,45].
An example of a BRACHYURY expressing porcine
primitive streak is visualized in Figure 9. At approximately Days 13-14 of gestation, the primitive
streak extends from the posterior pole of the epiblast
and approximately two thirds of the length of the
embryonic disc [42]. A key embryonic signalling cen-
Figure 7. Confocal laser scanning micrographs of Day 10 porcine blastocyst displaying mutually excluding epiblast cell populations expressing
OCT4 and NANOG. Modified from Wolf et al. (2011a)[46].
Figure 8. Median section though embryonic disc from Day 12-13 porcine embryo. The epiblast (Ep) is continuous with the trophectoderm (Te),
indicated by the arrows. In the posterior two third of the epiblast, more loosely arranged cells ingress through the primitive streak (PS) to the
space between the epiblast and the hypoblast (Hy/En), which is gradually exchanged by final endoderm. Loose mesoderm (Me) is also located
in this area. A: Anterior; P: Posterior; D: Dorsal; V: Ventral.
s208
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
tre during gastrulation, found at the anterior end of
the primitive streak, is the organizer region, termed
the node [37]. In the porcine embryo, the node is
morphologically evident as a thickening of cells in
the anterior part of the early primitive streak (Figure
9); [42].
3.2 Ingression of cells forming mesoderm and
endoderm
Formation of the primitive streak involves
extensive movement of cells, where the epiblast cells
first gather at the posterior end of the embryonic disc,
then rearrange to extend anteriorly in the streak itself,
and, finally, undergo an epithelial-mesenchymal
transition through the primitive streak to become
either mesoderm or definitive endoderm. When the
primitive streak has formed, epiblast cells continue
to enter this structure, which, thus, contains a dynamic
ever changing cell population. The cells, which ingress to the space between the epiblast and hypoblast
form mesodermal and endodermal precursors. Until
recently it was generally believed that the definitive
endoderm derived from the primitive streak replaced
the hypoblast cell layer. Recently however, it was
shown that in the gastrulating murine embryo the newly formed definitive endoderm cells insert themselves
into the hypoblast epithelium in a dispersed manner
N
Figure 9. Confocal laser scanning micrographs of Day 12-13 porcine embryo. (A–C) Side-view of the embryonic disc. Note the expression of
T (BRACHYURY) in the primitive streak and the posterior epiblast, the intensive FOXA2 expression in the hypoblast, and the co-expression
of the two markers in the node (arrow and arrowhead in C). The periphery of embryonic disc marked with open arrowheads in A. Note the
expression of T in the primitive streak as well as in intra- and extra embryonic mesoderm (EEM) underlying the epiblast and trophectoderm,
respectively. (D-F) Optical transversal sections of the embryonic disc corresponding to the broken line in A. Note the expression of T in the
primitive streak as well as in intra- and extra embryonic mesoderm underlying the epiblast and trophectoderm, respectively. A: Anterior; P:
Posterior.
s209
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
[20]. Whether this is the case in the porcine embryo
is not known. The mesodermal cells arrange
themselves as an intermediate cell layer between the
two developing epithelial layers, i.e. the epiblast and
endoderm.
The cells entering the primitive streak are
exposed to distinct signaling factors at different concentrations dependent on where in the primitive streak
the cells ingress through. Cell tracing studies has
shown that the fate of a given cell is related to the site
of ingression through the primitive streak: Cells ingressing through anterior streak and node become
prechordal plate mesoderm, notochord, and endoderm, cells ingressing through “mid” streak become
paraxial mesoderm, and cells migrating through the
posterior streak become extra-embryonic and lateral
plate mesoderm. These cell movements are, beside
geometrical differences, being conserved from
reptiles to mouse [24].
When the primitive streak has reached its
maximum extension of about two thirds of the length
of the embryonic disc, a subsequent posterior
retraction the streak occurs. Until recently, it was
thought that the primitive streak actually shortened
during this retraction. However, new investigations
in the mouse have shown that the node does not
regress posteriorly, but that the streak becomes
relatively shorter due to the longitudinal growth of
the embryonic disc (Yamanaka et al., 2007). Along
with this process, cells ingressing through the node
forms the notochord; a rod-shaped structure
interposed between the epiblast and the endoderm
extending from the rostral end of the embryonic disc
posteriorly to the node, from which it grows in a posterior direction (Figure 10). The notochord posterior
to the node is apparently formed by particular cells
migrating posteriorly from the node [48]. The
notochord expresses BRACHYURY [45].
With the formation of the three somatic germ
layers; ectoderm, mesoderm, and endoderm and the
PGCs (see later), the progenitors of all fetal tissue
lineages are formed.
IV. NEURULATION: DEVELOPMENT OF THE NEURAL
ECTODERM AND NEURAL CREST
Neurulation is the process leading to the
formation of the neural tube, the precursor of the central nervous system including the brain and spinal
Figure 10. Confocal laser scanning micrographs of Day 14 porcine embryo. (A) Dorsal view of the embryonic disc showing the epiblast and
developing ectoderm, identified from persisting OCT4 expression, and T (BRACHYURY) expression in the primitive streak (PS) posteriorly,
in the node (N), and in the notochord (No) anteriorly. Note the cluster of OCT4 expressing primordial germ cells posteriorly (arrow). (B) Optical
side view of the embryonic disc displaying the same features. A: Anterior; P: Posterior; D:Dorsal; V: Ventral. Modified from Wolf et al.
(2011b)[45].
s210
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
cord. This organ system is the first to initiate its
development; functionally, however, it is overtaken
by the later developing vascular system. Timewise,
the process of neurulation overlaps with that of
gastrulation: Along with the posterior retraction of
the primitive streak, neurulation progresses in an anterior to posterior direction. Hence, over a certain
period of time, the embryonic disc presents both the
primitive streak posteriorly and the developing neural
system anteriorly.
4.1 Neural ectoderm
The epiblast cells anterior to the primitive node
are induced to differentiate at the second gestational
week [44]. The notochord’s signalling molecules,
including Sonic hedgehog, induce the overlying
epiblast to differentiate into neuroectoderm, whereas
the remaining more lateral portion of the epiblast
differentiates into surface ectoderm. This notochordinduced neurulation is referred to as primary neurulation. The first morphological sign of primary
neurulation is a dorsal thickening in the anterior ectoderm forming an elliptical region referred to as the
neural plate. Subsequently, the neural plate undergoes
a shaping which converts it into a more elongated
key-hole shaped structure with broad anterior and
narrow posterior regions. Neural plate shaping is
followed by the development of two lateral elevations,
the neural folds, on either side of a depressed
midregion referred to as the neural groove. In pigs
and cattle the neural folds become clear during the
third week of development (Figure 11).
N
Figure 11: Porcine Day 14-15 embryos. (A) Stereo micrograph of the embryonic disc showing the primitive streak (PS) posteriorly, delineated
by arrowheads, and the neural groove (NG) anteriorly, delineated by arrows. CAF: Chorioamniotic folding. (B) Section of embryonic disc along
the broken line in A. Note the thick neural ectoderm (NE) continuous with the trophectoderm at the arrows. The mesoderm (Me) is seen between
the neural ectoderm and the endoderm (En). The mesoderm also forms extra-embryonic portions lining both the trophectoderm and the yolk sac
(YS) with the extra-embryonic coelom (EC) between the layers. The latter opens into the primitive gut forming the hindgut (Hg) and the foregut
(Fg). CAF: Chorioamniotic folding. (C) Section through the dorsal portion of the neural tube (NT) showing the neural ectoderm (NE)
overlaid by the surface ectoderm (SE) characterized by expression of Pankeratin. Note the PAX7 expressing neural crest cells.
A: Anterior; P: Posterior; D:Dorsal; V: Ventral.
s211
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
The neural folds continue to elevate, appose
in the midline, and, eventually, fuse to create the neural tube which becomes covered by the surface
ectoderm. Primary neurulation creates the brain and
most of the spinal cord, whereas in the tail bud, the
posterior neural tube is formed by secondary neurulation, where the spinal cord initially forms as a
solid mass of epithelial cells, and a central lumen
develops secondarily by cavitation.
The primary neurulation is accompanied by
a bending of the neural plate, which occurs at three
principal sites: the median hinge point (MHP), overlying the notochord, and the paired dorsolateral hinge
points (DHLP) at the points of attachment of the surface ectoderm. The MHP is induced by signals from
the notochord.
Gradually, the neural folds approach each
other in the midline, where they eventually fuse.
Cellular protrusions extend from apical cells of the
neural folds as they approach one another in the
dorsal midline and interdigitate as the folds come
into contact. This allows a first cell-cell recognition
and provides an initial adhesion pending later
establishment of permanent cell contacts.
The subsequent fusion of the neural folds
begins in the cervical region and proceeds in a zipperlike fashion anteriorly and posteriorly from there. As
a result of these processes, the neural tube is formed
and separated from the overlying surface ectoderm.
Until fusion is complete, the anterior and posterior
ends of the neural tube communicate with the
amniotic cavity via two openings, the anterior and
posterior neuropores. Closure of the neuropores
occurs at approximately Days 24 to 26 and Days 15
to 16 in cattle and pig, respectively; the anterior
neuropore one to 2 days prior to the posterior.
Neurulation is then complete. The central nervous
system is represented at this time by a closed tubular
structure with a narrow posterior portion, the anlage
of the spinal cord, and a much broader cephalic
portion, the primordium of the encephalon. During
neurulation, the neuroepithelium is entirely proliferative; cells do not begin to exit the cell cycle and
start neuronal differentiation until after the neural tube
closure is complete.
During neurulation, cell proliferation is accompanied by some degree of apoptosis in the neuroepithelium. The rate of apoptosis appears to be
finely tuned and it seems to be equally detrimental if
the intensity of apoptosis is increased or decreased.
Apoptosis at the tips of the neural folds may serve a
special function. After opposing neural folds have
made contact and adhered to each other, midline
epithelial remodelling by apoptosis breaks the
continuity between the neuroepithelium and surface
ectoderm.
4.2 Neural crest
Along with the elevation and fusion of the
neural folds, certain cells at the lateral border or crest
of the neural folds become detached. This cell
population, known as the neural crest cells, will not
participate in formation of the neural tube; instead
they migrate widely and participate in the formation
of many other tissues, such as the integument
(melanocytes), other parts of the nervous system
(including neurons for the central, sympathetic and
enteric nervous system as well as glial and Swann
cells), and large parts of the craniofacial mesenchymal
derivatives [16].
The mechanism whereby the neural crest cells
detach from the neural folds is comparable with that
occurring during ingression of epiblast cells in the
primitive streak and node - a second example of
epithelio-mesenchymal transition. The term mesenchyme refers to loosely organized embryonic tissue
regardless of germ layer origin. Thus, both neuroectoderm (through the neural crest cells) and
mesoderm (at gastrulation) may give rise to mesenchyme.
The induction of neural crest cells is possibly
mediated by a gradient of BMP4, BMP7, and WNT
secreted by the surface ectoderm. In the chick and
pig, the neural crest cells express the transcription
factor PAX7 [2].
V. DEVELOPMENT OF THE PRIMORDIAL GERM CELLS
(PGCS)
The development of the germ line involves
specification of the cell linage and subsequent
migration of the individual cells through various embryonic tissues to the final destination in the genital
ridges. After reaching the genital ridges, the cells of
the germ line integrate and initiate the final steps of
differentiation towards mature germ cells; a process
not completed until adulthood.
At the stage where the embryo presents a clear
primitive streak the OCT4 expression gradually
s212
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
decreases in the epiblast. When that happens, the
putative PGC precursors can be identified by their
continuous expression of this marker (Figure 12;
Hyldig, unpublished data). In addition, they also
express NANOG another well known germ line
marker. These cells are seen dispersed within the caudal third of the embryonic disc [33]. From their po-
sition within the porcine embryonic disc, the putative
PGC precursors move in caudal direction to the extra-embryonic yolk sac wall. Initially, OCT4 positive
cells are dispersed in both the embryonic and the extra-embryonic part of the yolk sac wall. A small cluster
can be identified in the junction between embryonic
and extra-embryonic tissue (Figure 12). As the yolk
N
Figure 12. Schematic presentation of the position of the porcine germ line during early development. Sections of the porcine
embryo are depicted below drawings of embryo proper. Broken lines across embryo proper indicate section sites. Red dots
represent PGCs. At embryonic Day 12, the putative germ line precursors are positioned in the caudal third of the embryo proper,
scattered around the primitive streak. At Day 13 the distribution is similar, but with some PGCs in the extra-embryonic yolk sac wall
where a specific cluster of PGCs is formed. At Day 15 the PGCs are seen in the ventral wall of the hind gut in all its length.
Subsequently, the population moves in dorsal direction towards the genital ridges so that by Day 20, most PGCs reside in this tissue.
The Day 28 gonads are beginning to form and PGCs are restricted to these organs. PS: Primitive streak; NG: Neural groove; Mn:
Mesonephros; Me: Mesenterium; Li: Liver.
s213
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
sac by Day 14-15 folds under the caudal area of the
embryo to form the ventral wall of the hind gut, PGCs
becomes restricted to this. Subsequently, the PGC
follow the migration path from the ventral to dorsal
side of the hind gut and further dorsolateral into the
genital ridges. Although a few PGCs are seen in the
genital ridge at Day 17, the vast majority is resided
in the hindgut area at least until Day 18. By Day 20
most PGCs are positioned in and around the
attachment site of the elongated mesentery, however
still with a substantial part of the population
positioned in the lower mesentery and hindgut area.
The tubular mesonephric tissue forms voluminous
bulges, forcing the genital ridges in towards the
midline and effectively discontinuing the linear contact between them and the PGCs in the dorsal
mesentery[14]. The final colonisation of the genital
ridges occurs around E23-24 [15]. The integration
of the PGCs into the genital ridge tissue and the
subsequent differentiation of the germ line is to our
knowledge largely unexplored in the porcine species.
The gonadal tissue begins organising by Day 42.
Germ cell cords are present in both male and female
gonads, though larger and more regular in males.
Male gonads are rounded with only a slim cellular
connection to the mesonephros [46].
In the newly formed PGCs, DNA is highly
methylated, as it is in their epiblast progenitors. However, by the time the PGC have entered the genital
ridge, DNA has become largely hypomethylated. The
demethylation is well studied in the murine species
and studies of various repeats and differentially
methylated domain (DMD) sequences of imprinted
genes in porcine embryos show a comparable
demethylation. The process is completed by Day 2831, where after remethylation is started [4,38].
Immunostainings of genomewide CpG methylation
have indicated that the demethylation is initiated
around Day 15 during PGC migration toward the
genital ridges [14]. During the subsequent
gametogenesis, when oocytes and spermatozoa are
formed from the PGC derivatives, de novo
methylation of DNA occurs. Importantly, this
genome-wide demethylation and remethylation also
includes the sex-specific DNA methylation of particular loci, forming the basis of genomic imprinting.
VI. FURTHER DEVELOPMENT OF THE EMBRYO
The three germ layers, ectoderm, mesoderm,
and endoderm, form the basis for the further
development of organ systems collectively referred
to as the area of special embryology (for review, see
Hyttel et al. [16]).
6.1 The ectoderm and its early derivatives
The development of the neuroectoderm has
already been described in a former paragraph. After
having allocated cells for endoderm, mesoderm, the
germ line, and neuroectoderm, most of the remaining
more laterally located epiblast will differentiate into
surface ectoderm. In parallel with the closure of the
neuropores, two bilateral thickenings of the surface
ectoderm, the otic placode and the lens placode, are
established in the embryonic cephalic ectoderm (Figure 13A). The otic placode invaginates to form the
otic vesicle, which will develop into the inner ear for
hearing and balance, while the lens placode
invaginates and forms the lens of the eye. The
remaining surface ectoderm gives rise to the
epidermis and associated glands of the skin, as well
as the epithelium covering the oral and nasal cavities
and the caudal portion of the anal canal. The
epithelium covering the oral cavity gives rise to the
enamel of the teeth and also part of the pituitary gland,
the adenohypophysis.
6.2 The mesoderm and its early derivatives
Formation of the notochord provides an
embryonic midline axis as a template for the axial
skeleton. Initially, cells of the mesoderm form a thin
sheet of loosely woven mesenchyme on either side
of the notochord. Soon, however, the mesoderm closest to the notochord (the paraxial mesoderm) proliferates and forms pairs of segmental thickened structures known as somites (Figure 13). This process
starts in the occipital region of the embryo, and in
large animal species, somites are formed at a rate of,
on average, about six pairs a day. The number of somites formed during this phase of development
therefore forms a basis for estimating embryonic age.
More laterally, the mesoderm remains thin and
is therefore referred to as the lateral plate mesoderm.
The lateral plate mesoderm is continuous with the
s214
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
Figure 13. Stereo micrographs of porcine embryos. (A) Day 15-16 embryo showing lens placode (LP), otic placode (OP), somites (S),
developing heart (H), yolk sac (YS), and allantois (Al). (B) Day 18-19 embryo showing pharyngeal arches (PA), developing heart with atrial
(At) and ventricular (Ve) compartments, forelimb bud (FB), hind limb bud (HB), and prominent mesonephros (Mn).
extra-embryonic mesoderm. The extra-embryonic
mesoderm is split into an outer component lining
trophectoderm and an inner component lining the
hypoblast, and the cavity between these two
components is referred to as the extra-embryonic
coelom (Figure 11). With the continued development
of the coelom, an intra-embryonic coelom similarly
divides the lateral plate mesoderm in such a way that
the so-called somatic mesoderm associates with the
surface ectoderm to constitute the somatopleura while
the so-called visceral mesoderm associates with the
endoderm to form the splanchnopleura. Between the
paraxial and lateral plate mesoderm, the intermediate
mesoderm is established.
6.3 Paraxial mesoderm
As a general rule, development proceeds in
an anterior to posterior direction (one exception to
this was the development of the primitive streak). Accordingly, formation of somites progresses from the
occipital region posteriorly. Each somite subsequently
differentiates into three components: The ventromedial part of the somite associates with the notochord
establishing the sclerotome which patterns formation
of the vertebral column. The dorso-lateral part of each
somite forms regionalized precursors of both dermal
and muscle tissue, the dermamyotome. From this
structure, a dorso-medially located cell population
forms the myotome and a dorso-laterally located
group becomes the dermatome. The myotome of each
somite contributes to muscles of the back and limbs,
while the dermatome disperses and forms the dermis
and subcutis of the skin. Later, each myotome and
dermatome will receive its own segmental nerve
component.
6.4 Intermediate mesoderm
The intermediate mesoderm, which connects
paraxial and lateral plate mesoderm, differentiates into
structures of both the urinary system and the gonads,
together referred to as the urogenital system. The
N
urinary system is first developed as an abortive
anteriorly located paired pronephros, succeeded by
a very prominently developing paired mesonephros
(Figure 13B). The mesonephros develops excretory
ducts; the mesonephric ducts (Wollfian ducts). Finally,
the even further posteriorly located paired metanephos
develops and the persisting kidneys. The gonads
develop on the medial aspect of the mesonephros,
initially as the genital ridges which receive the primordial germ cells. In the male, the mesonephric
ducts develops into the epididymal ducts and the
ductus deferens. In the female, however, another duct,
the paramesonephric duct (the Müllerian duct), forms
parallel to the mesonephric duct and develops into
the oviduct, the uterus, and the cranial portion of the
vagina.
6.5 Lateral plate mesoderm and body folding
Through anterior-posterior and lateral foldings, the subdivision of the coelom into intra- and
extra-embryonic cavities becomes progressively
better defined and the embryonic body gradually assumes the shape of a closed tube enclosing another
tube, the primitive gut. The somatopleura will form
s215
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
the lateral and ventral body wall enclosing the intraembryonic coelom of which the somatic mesoderm
will provide the inner lining (the mesothelium of the
peritoneum and pleura) and the ectoderm the outer
lining (the epidermis). The splanchnopleura will form
the wall of the primitive gut and its derivatives in
which the endoderm and the visceral mesoderm will
provide the inner lining (the lamina epithelialis of
the tunica mucosa) and outer lining (the lamina
epithelialis of the tunica serosa) respectively. The
visceral mesoderm will also form the connective
tissue and muscular components of the gut and its
derivatives. Soon, the intra-embryonic coelom will
be divided into the peritoneal, pleural and pericardial
cavities.
6.6 Blood and blood vessel formation
Both blood and blood vessels appear to arise
from common mesoderm precursor cells, the
hemangioblasts. These differentiate into hematopoietic stem cells (forming blood cells) and angioblasts that form endothelial cells which coalesce to
form blood vessels. The first sign of blood and blood
vessel formation is seen in the visceral mesoderm of
the splanchnopleura covering the yolk sac (see later).
However, this appears to be only a transient
phenomenon; later, hematopoiesis moves first to the
liver and spleen and then to the bone marrow. The
heart is also of mesodermal origin; though with some
contribution of neural crest cells (Figure 13).
6.7 The endoderm and its early derivatives
The inner epithelial lining of the
gastrointestinal tract and its derivatives is the main
component derived from the endoderm. With the
anterior-posterior and lateral foldings of the embryo,
the endoderm-enclosed primitive gut becomes
enclosed within the embryo, whereas the hypoblastenclosed yolk sac becomes localized outside the
embryo.
The primitive gut comprises cranial (foregut),
middle (midgut) and caudal (hindgut) parts. The
midgut communicates with the yolk sac through the
vitelline duct (Figure 14). This duct is wide initially
Figure 14. Schematic drawing of the extra-embryonic membranes and cavities in the pig. The outer membrane, chorion,
is formed by trophectoderm underlaid by extra-embryonic mesoderm. The allantois (green), which is a diverticulum
from the hindgut, is lined on the inside by endoderm covered by extra-embryonic mesoderm. The fusion between the two
membranes, the chorion and allantois, results in the chorioallantoic membrane, which forms foldings engaged in
placentation. The yolk sac (red), which is a diverticulum connected with the midgut through the vitelline duct, is
rudimentary. The amnion (blue) surrounds the embryo and is fused with the chorion in the mesamnion (MA).
s216
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
but, as development proceeds, becomes long and narrow and is eventually incorporated into the umbilical
cord. The endoderm forms the epithelium of the
gastro-pulmonary system and the parenchyma of its
derivatives. Endoderm of the foregut gives rise to
the pharynx and its derivatives, including the middle
ear, the parenchyma of the thyroid gland, the
parathyroid glands, the liver and the pancreas, and
the reticulated stroma of the tonsils and thymus, as
well as the oesophagus, stomach, liver, and pancreas.
At its anterior end, the foregut is temporarily closed
by an ectodermal-endodermal membrane, the
buccopharyngeal membrane. At a certain stage of
development, this membrane ruptures and open
communication between the amniotic cavity and the
primitive gut is established. The midgut gives rise to
most of the small and the large intestine down to the
transverse colon whereas the hindgut gives rise to
the transverse and descending colon as well as the
rectum and part of the anal canal. At its caudal end,
the hindgut temporarily dilates to form the cloaca, a
cavity transiently common to both the developing
gastrointestinal and urogenital systems. The cloaca
is separated from the amniotic cavity by the cloacal
membrane, composed of closely apposed ectoderm
and endoderm, like the buccopharyngeal membrane.
After separation of the gastrointestinal and urinary
systems, the cloacal membrane breaks down, opening
the two systems via the anus and urogenital sinus,
respectively.
VII. PLACENTATION AND FORMATION OF EXTRAEMBRYONIC MEMBRANES AND CAVITIES
7.1 Development of extra-embryonic membranes and
cavities
During the early phases of gastrulation, the
trophectoderm becomes lined by a thin layer of extra-embryonic mesoderm, the two layers together
constituting the outer extra-embryonic membrane, the
chorion (Figure 14). During gastrulation, the chorion
forms folds, the chorioamniotic folds, which surround
the embryonic disc. Gradually, the folds extend
upwards to meet and fuse above the embryonic disc
thereby enclosing the disc in a sealed amniotic cavity.
The term amnion is generally used collectively for
the cavity and its wall. The inner epithelium of the
amnion originates from the trophectoderm and so, at
the embryonic disc, it is continuous with the epiblast
and later the embryonic surface ectoderm. The outside
covering of the amnion is composed of extraembryonic mesoderm.
The site where the chorioamniotic folds meet
and fuse is known as the mesamnion. In cattle and
pig, the mesamnion persists; as a result, the amnion
gets torn during parturition and offspring are generally
born without covering membranes.
With the body foldings and the formation of
the endoderm-lined primitive gut, the hypoblast-lined
yolk sac is transformed into an extra-embryonic cavity
communicating with the primitive gut through the
vitelline tube. The outside of the yolk sac is lined by
visceral mesoderm. In cattle and pig, the yolk sac
serves a hematopoietic function for a short period of
time, but subsequently it regresses within one to two
weeks after its formation and never attains other
important functions.
During the second or third week of
development, depending on the species, the allantois
is formed as an outgrowth from the hindgut into the
extra-embryonic coelom. In ruminants and the pig,
the allantois assumes a T-shaped appearance with the
top bar of the T being located as a transverse cavity
just caudal to the embryo proper and the stem of the
N
T connected with the hindgut. Like the vitelline duct,
the allantoic duct, connecting the allantoic cavity and
the hindgut, becomes incorporated into the umbilical cord as a consequence of embryonic foldings.
Since the allantois is a diverticulum of the hindgut,
its wall is composed of an inner epithelial lining of
endodermal origin and an outer layer derived from
the visceral mesoderm. As the allantois enlarges, the
visceral mesodermal part of its wall fuses with the
somatic mesoderm of the chorion and, finally, more
or less covers the amnion. The fusion of the allantoic
and chorionic walls forms the embryonic part of the
chorioallantoic placenta found in the domestic
animals. The intra-embryonic proximal portion of
the allantoic duct, extending from the hindgut to the
umbilicus, is referred to as the urachus and gives rise
to the urinary bladder. Throughout gestation, the allantoic cavity serves as a repository of the wastes
excreted through the embryo’s developing urinary
system.
Prior to attachment the conceptus is solely
nourished by uterine glandular secretions
(histiotrophe), but with attachment of the chorio-
s217
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
allantoic placenta to the endometrial wall an exchange
of fetal/maternal blood-borne nutrients (hemotrophe)
also contributes. Areolae, chorionic indentations
opposite the endometrial glands, are scattered in the
diffuse porcine placenta and remain present during
gestation [21].
7.2 Placentation
The placenta can be classified according to
the structure of the chorioallantoic surface and its
interaction with the endometrium. Areas where the
chorioallantois interacts with the endometrium and
engages in placental formation are referred to as
chorion frondosum, in contrast to the smooth chorion
leave not included in the placenta. In the pig, chorion
frondosum is diffusely distributed over the entire
chorioallantoic surface and so the placenta is
categorized as being diffuse. The porcine chorioallantoic surface area is increased by foldings,
revealed as primary plicae and secondary rugae, and
is thus referred to as being folded. In cattle, the chorion frondosum is organized as arborizing chorionic
villi assembled into larger macroscopically visible
tufts called cotyledons. Hence, the bovine placenta
is known as cotyledonary or multiplex and villous.
The cotyledons combine with endometrial
prominences known as caruncles, forming
placentomes in which the chorioallantoic villi of the
cotyledon extend into crypts of the caruncle.
The placenta can also be classified based on
the number of tissue layers separating the fetal and
maternal circulations, thereby forming the placental
barrier. There are always three fetal extra-embryonic
layers in the chorioallantoic placenta: the endothelium
lining the allantoic blood vessels; chorioallantoic
mesenchyme, originating from the fused somatic
(chorionic) and visceral (allantoic) mesoderm; and
the chorionic epithelium developed from the
trophectoderm and in the placenta referred to as the
trophoblast. However, the numbers of layers retained
in the maternal portion of the placenta varies with
species. Before placentation, the endometrium presents three layers that could contribute to the placental
barrier: the endometrial epithelium, connective tissue,
and vascular endothelium.
In cattle and pig, the placenta is epitheliochorial and the chorionic and endometrial epithelia
are apposed, and there is no loss of maternal tissue.
The epitheliochorial placenta in ruminants is modified
as particular trophoblast cells cross into, and fuse with,
some of the endometrial epithelial cells. Hence, the
placenta is referred to as synepitheliochorial.
VIII. STAGING OF EMBRYONIC DEVELOPMENT
Simple measures have over the time been used
as a reference for embryonic development including
length in mm [32], days of gestation [22], numbers
of somites [15, 41], or external features [8]. Within
human embryology, a painstaking work has been put
into developing the Carnegie system; staging system
providing a precise frame of reference of embryonic
development [29,30]. This staging system utilizes
macroscopic as well as microscopic features in a
developing embryo and fetus. The Carnegie system
has been implemented in bats [6] and mouse [39].
We are currently working on development of a
Carnegie-based porcine staging system for the
domestic pig based on the examination of approximately 600 specimens.
IX. CONCLUSIONS
An improved understanding of post-hatching
embryonic development holds an important key to
not only a more proper evaluation of the success or
failure of assisted reproductive technologies; it also
forms an important basis for the understanding of
stem cell differentiation and cell replacement therapy.
A wealth of contemporary data are published on the
molecular regulation of the initial lineage segregation
and cell differentiation taking place in the embryo,
and it is a great challenge to align all the complex
sets of information into integrated networks gradually
guiding the well-orchestrated embryonic and fetal
development.
REFERENCES
1 Avilion A.A., Nicolis S.K., Pevny L.H., Perez L., Vivian N. & Lovell-Badge R. 2003. Multipotent cell lineages in early
mouse development depend on SOX2 function. Genes & Development. 17: 126-140.
s218
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
2 Basch M.L., Bronner-Fraser M. & Garcia-Castro M.I. 2006. Specification of the neural crest occurs during gastrulation
and requires Pax7. Nature. 441: 218-222.
3 Blomberg L.A., Garrett W.M., Guillomot M., Miles J.R., Sonstegard T.S., Van Tassell C.P. & Zuelke K.A. 2006.
Transcriptome profiling of the tubular porcine conceptus identifies the differential regulation of growth and developmentally
associated genes. Molecular Reproduction and Development. 73: 1491-1502.
4 Byskov A.G., Hoyer P.E., Bjorkman N., Mork A.B., Olsen B. & Grinsted J. 1986. Ultrastructure of germ cells and adjacent
somatic cells correlated to initiation of meiosis in the fetal pig. Anatomy and Embryology. 175: 57-67.
5 Chambers I., Colby D., Robertson M., Nichols J., Lee S., Tweedie S. & Smith A. 2003. Functional expression cloning of
Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 113: 643-655.
6 Cretekos C.J., Weatherbee S.D., Chen C.H., Badwaik N.K., Niswander L., Behringer R.R. & Rasweiler J.J.T. 2005.
Embryonic staging system for the short-tailed fruit bat, Carollia perspicillata, a model organism for the mammalian order
Chiroptera, based upon timed pregnancies in captive-bred animals. Developmental Dynamics. 233: 721-738.
7 Elling U., Klasen C., Eisenberger T., Anlag K. & Treier M. 2006. Murine inner cell mass-derived lineages depend on SaII4
function. Proceedings of the National Academy of Sciences of the United States of America. 103: 16319-16324.
8 Evans H.E. & Sack W.O. 1973. Prenatal development of domestic and laboratory mammals: growth curves, external
features and selected references. Zentralbl Veterinarmed C. 2: 11-45.
9 Everts R.E., Chavatte-Palmer P., Razzak A., Hue I., Green C.A., Oliveira R., Vignon X., Rodriguez-Zas S.L., Tian X.C.,
Yang X., Renard J.P. & Lewin H.A. 2008. Aberrant gene expression patterns in placentomes are associated with
phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer. Physiology and Genomics. 33: 65-77.
10 Flechon J.E., Degrouard J. & Flechon B. 2004. Gastrulation events in the prestreak pig embryo: Ultrastructure and cell
markers. Genesis. 38: 13-25.
11 Hall V.J., Christensen J., Gao Y., Schmidt M.H. & Hyttel P. 2009. Porcine pluripotency cell signaling develops from the
inner cell mass to the epiblast during early development. Developmental Dynamics. 238: 2014-2024.
12 Hall V.J., Jacobsen J.V., Rasmussen M.A. & Hyttel P. 2010. Ultrastructural and molecular distinctions between the porcine
inner cell mass and epiblast reveal unique pluripotent cell states. Developmental Dynamics. 239: 2911-2920.
13 Hassoun R., Schwartz P., Feistel K., Blum M. & Viebahn C. 2009. Axial differentiation and early gastrulation stages of the
N
pig embryo. Differentiation. 78: 301-311.
14 Hyldig S.M., Croxall N., Contreras D.A., Thomsen P.D. & Alberio R. 2011. Epigenetic reprogramming in the porcine
germ line. BMC Developmental and Biolology. 11: 11.
15 Hyldig S.M., Ostrup O., Vejlsted M., Thomsen P.D. 2011. Changes of DNA methylation level and spatial arrangement of
primordial germ cells in embryonic Day 15 to embryonic Day 28 pig embryos. Biology of Reproduction. 84(6): 10871093.
16 Hyttel P. 2009. Essentials of domestic animal embryology. Edinburgh ; New York: Saunders/Elsevier.
17 Keefer C.L., Pant D., Blomberg L. & Talbot N.C. 2007. Challenges and prospects for the establishment of embryonic stem
cell lines of domesticated ungulates. Animal Reproduction Science. 98: 147-168.
18 Kuijk E.W., Du Puy L., Van Tol H.T., Oei C.H., Haagsman H.P., Colenbrander B. & Roelen B.A. 2008. Differences in
early lineage segregation between mammals. Developmental Dynamics. 237: 918-927.
19 Kuijk E.W., du Puy L., van Tol H.T.A., Haagsman H.P., Colenbrander B. & Roelen B.A.J. 2007. Validation of reference
genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Developmental Biology. 31:
57-58.
20 Kwon G.S., Viotti M. & Hadjantonakis A.K. 2008. The endoderm of the mouse embryo arises by dynamic widespread
intercalation of embryonic and extraembryonic lineages. Developmental Cell. 15: 509-520.
21 Leiser R. & Dantzer V. 1994. Initial vascularisation in the pig placenta: II. Demonstration of gland and areola-gland
subunits by histology and corrosion casts. The Anatomical Record. 238: 326-334.
22 Marrable A.W. 1971. The embryonic pig: a chronological account. London: Pitman Medical.
23 Meijer H.A., Van de Pavert S.A., Stroband H.W.J. & Boerjan M.L. 2000. Expression of the organizer specific homeobox
gene Goosecoid (gsc) in porcine embryos. Molecular Reproduction and Development. 55: 1-7.
24 Mikawa T., Poh A.M., Kelly K.A., Ishii Y. & Reese D.E. 2004. Induction and patterning of the primitive streak, an
organizing center of gastrulation in the amniote. Developmental Dynamics. 229: 422-432.
s219
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
25 Mitsui K., Tokuzawa Y., Itoh H., Segawa K., Murakami M., Takahashi K., Maruyama M., Maeda M. & Yamanaka S.
2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 113:
631-642.
26 Ng R.K., Dean W., Dawson C., Lucifero D., Madeja Z., Reik W. & Hemberger M. 2008. Epigenetic restriction of
embryonic cell lineage fate by methylation of Elf5. Nature Cell Biology. 10: 1280-1290.
27 Nichols J., Zevnik B., Anastassiadis K., Niwa H., Klewe-Nebenius D., Chambers I., Scholer H. & Smith A. 1998.
Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 95: 379391.
28 Nishioka N., Yamamoto S., Kiyonari H., Sato H., Sawada A., Ota M., Nakao K. & Sasaki H. 2008. Tead4 is required for
specification of trophectoderm in pre-implantation mouse embryos. Mechanisms of Development. 125: 270-283.
29 O’Rahilly R. & Muller F. 2010. Developmental Stages in Human Embryos: Revised and New Measurements. Cells Tissues
Organs. 192: 73-84.
30 O’Rahilly R., Müller F. & Streeter G.L. 1987. Developmental stages in human embryos : including a revision of Streeter’s
“Horizons” and a survey of the Carnegie collection. Washington, D.C.: Carnegie Institution of Washington.
31 Oestrup O., Hall V., Petkov S.G., Wolf X., Hyldig S. & Hyttel P. 2009. From zygote to implantation: morphological and
molecular dynamics during embryo development in the pig. Reproduction in Domestic Animals. 44: 39-49.
32 Patten B.M. 1948. Embryology of the pig. Philadelphia: Blakiston Co.
33 Petkov S.G., Reh W.A. & Anderson G.B. 2008. Methylation changes in porcine primordial germ cells. Molecular
Reproduction and Development. 76(1): 22-30.
34 Ralston A. & Rossant J. 2005. Genetic regulation of stem cell origins in the mouse embryo. Clinical Genetics. 68: 106-112.
35 Schmidt M., Kragh P.M., Li J., Du Y., Lin L., Liu Y., Bogh I.B., Winther K.D., Vajta G. & Callesen H. 2010. Pregnancies
and piglets from large white sow recipients after two transfer methods of cloned and transgenic embryos of different pig
breeds. Theriogenology. 74:1233-1240.
36 Scholer H.R., Ruppert S., Suzuki N., Chowdhury K. & Gruss P. 1990. New Type of Pou Domain in Germ Line-Specific
Protein Oct-4. Nature. 344:435-439.
37 Solnica-Krezel L. 2005. Conserved patterns of cell movements during vertebrate gastrulation. Current Biology. 15: 213228.
38 Takagi Y., Talbot N.C., Rexroad C.E.Jr. & Pursel V.G. 1997. Identification of pig primordial germ cells by
immunocytochemistry and lectin binding. Molecular Reproduction and Development. 46: 567-580.
39 Theiler K. 1989. The House Mouse - Atlas of Embryonic Development. Springer Verlag.
40 Thompson J.G., Mitchell M. & Kind K.L. 2007. Embryo culture and long-term consequences. Reproduction Fertility and
Development. 19: 43-52.
41 van Straaten H.W., Peeters M.C., Hekking J.W. & van der Lende T. 2000. Neurulation in the pig embryo. Anatomy and
Embryology. 202: 75-84.
42 Vejlsted M., Du Y.T., Vajta G. & Maddox-Hyttel P. 2006. Post-hatching development of the porcine and bovine embryodefining criteria for expected development in vivo and in vitro. Theriogenology. 65: 153-165.
43 Vejlsted M., Offenberg H., Thorup F. & Maddox-Hyttel P. 2006. Confinement and clearance of OCT4 in the porcine
embryo at stereomicroscopically defined stages around gastrulation. Molecular Reproduction and Development. 73: 709718.
44 Wilson S.I. & Edlund T. 2001. Neural induction: toward a unifying mechanism. Nature Neuroscience. 4: 1161-1168.
45 Wolf X.A., Serup P. & Hyttel P. 2011. Three-Dimensional Immunohistochemical Characterization of Lineage Commitment
by Localization of T and FOXA2 in Porcine Peri-implantation Embryos. Developmental Dynamics. 240: 890-897.
46 Wolf X.A., Serup P. & Hyttel P. 2011. Three-dimensional localisation of NANOG, OCT4, and E-CADHERIN in porcine
pre- and peri-implantation embryos. Developmental Dynamics. 240: 204-210.
47 Yagi R., Kohn M.J., Karavanova I., Kaneko K.J., Vullhorst D., DePamphilis M.L. & Buonanno A. 2007. Transcription
factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development. 134:
3827-3836.
48 Yamanaka Y., Tamplin O.J., Beckers A., Gossler A. & Rossant J. 2007. Live imaging and genetic analysis of mouse
notochord formation reveals regional morphogenetic mechanisms. Developmental Cell. 13: 884-896.
49 Zhang J., Tam W.L., Tong G.Q., Wu Q., Chan H.Y., Soh B.S., Lou Y., Yang J., Ma Y., Chai L., Ng H.H., Lufkin T., Robson
s220
R.C. C
heb
el. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in
Cheb
hebel.
Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.
Dairy Cattle.
p. & Lim B. 2006. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the
transcriptional regulation of Pou5f1. Nature Cell Biology. 8: 1114-1123.
N
39(Suppl 1)
www.ufrgs.br/actavet
s221