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Acta Scientiae Veterinariae ISSN: 1678-0345 [email protected] Universidade Federal do Rio Grande do Sul Brasil

Hyttel, Poul; Kamstrup, Kristian M.; Hyldig, Sara From Hatching into Fetal Life in the Pig Acta Scientiae Veterinariae, vol. 39, núm. 1, 2011, pp. s203-s221 Universidade Federal do Rio Grande do Sul Porto Alegre, Brasil

Available in: http://www.redalyc.org/articulo.oa?id=289060016027

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. Acta Scientiae Veterinariae, 2011. 39(Suppl 1): s203 - s221.

ISSN 1679-9216 (Online)

From Hatching into Fetal Life in the 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 form the into fetal life in the pig. Review: In the pre-hatching mouse blastocyst, the pluripotency of the inner 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 , and a dorsal cell mass, destined to form the . 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 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 . The latter process is preceded by a thickening of the posterior region of the epiblast, putatively developing as a consequence of an absence of inhibitory signals from a condensed portion of the hypoblast underlying the anterior epiblast. The thickened posterior epiblastN expresses the marker . 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 and ; 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 interposed between the epiblast and the endoderm. During the subsequent , which is a process overlapping with gastrulation in time, the notochord induces the overlying epiblast to form neural , which sequentially develops into the , , and , whereas the lateral epiblast develops into the . 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 , 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, , , gastrulation, neurulation, embryonic staging.

CORRESPONDENCE: P. Hyttel [[email protected] – FAX: +45 353 32547]. Department of Basic and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark.

s203 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.

I. INTRODUCTION sarily a guarantee for further development into a II.BLASTULATION: DEVELOPMENT OF TRO- and newborn. Hence, it is well-documented that in PHECTODERM, ICM, EPIBLAST, HYPOBLAST, vitro embryo culture may impose long-term effects, AND EMBRYONIC DISC which are revealed later during embryonic and fetal III. GASTRULATION: DEVELOPMENT OF MESO- development [40]. This phenomenon becomes even DERM, ENDODERM, AND ECTODERM more exaggerated when are produced by 3.1 The primitive streak SCNT, which imposes an even higher risk of 3.2 Ingression of cells forming mesoderm and endoderm embryonic and fetal aberrations as well as neonatal IV. NEURULATION: DEVELOPMENT OF THE NEU- loss [9,35]. RAL ECTODERM AND In order to evaluate embryonic and fetal 4.1 Neural ectoderm development resulting from assisted reproductive 4.2 Neural crest technologies more properly, increasing focus should V. DEVELOPMENT OF THE PRIMORDIAL GERM be put on the normality of some of the complex post- CELLS (PGCS) hatching processes, as e.g. gastrulation, neurulation, VI. FURTHER DEVELOPMENT OF THE EMBRYO 6.1 The ectoderm and its early derivatives placentation and initial , which are 6.2 The mesoderm and its early derivatives prerequisites for full term development. These pro- 6.3 cesses are the focus of the present review. Over the 6.4 Paraxial mesoderm past decade the pig has attracted increasing attention 6.5 and body folding as a useful biomedical model, due to which the 6.6 Blood and blood vessel formation presented data will mainly be derived in this species. 6.7 The endoderm and its early derivatives Comparative notes will be made to cattle, whenever VII. PLACENTATION AND FORMATION OF EXTRA- the variation between these two species are pro- EMBRYONIC MEMBRANES AND CAVITIES nounced as e.g. at placentation. First, important deve- 7.1 Development of extra-embryonic membranes and lopmental processes of the general embryology inclu- cavities ding blastulation, gastrulation, neurulation, and deve- 7.2 Placentation lopment of the germ line will be presented, and, VIII. STAGING OF second, a short summary of the special embryology, i.e. the development of the organ systems, will be IX. CONCLUSIONS given.

I. INTRODUCTION II. BLASTULATION: DEVELOPMENT OF The wide-spread use of in vitro production TROPHECTODERM, ICM, EPIBLAST, HYPOBLAST, AND EMBRYONIC DISC of, in particular, bovine embryos in animal husbandry has paved the way for a detailed morphological and Blastulation (from the Greek term blastos molecular understanding of oocyte maturation, meaning sprout) is the process by which the embryo fertilization and initial embryonic development until develops into a fluid-filled structure in which the cells the time of hatching. Hence, studies on these life pro- have segregated into lines destined to produce the cesses have become facilitated by the easy embryo proper (the ICM and epiblast) and such accessibility of oocytes, , and embryos. developing into the extra-embryonic membranes (the Cloning by somatic cell nuclear transfer (SCNT) is trophectoderm and hypoblast). another technology, which over the past decade has In the pig, the blastocyst forms at around Day resulted in alternative in vitro production of 5 of gestation. The porcine embryo initiates com- considerable numbers of both bovine and porcine paction as early as the 8-16-cell stage, when the embryos adding to the accessibility of embryos for embryo assumes a spherical appearance with a research. Development of the embryo to the blastocyst smoother surface where the protrusions of the indi- stage includes several complex processes as e.g. the vidual are no longer seen. The outer cells, activation of the embryonic genome (for review, see allocated to the trophectoderm, become connected Oestrup et al. [31]). It is also clear, however, that by tight junctions and desmosomes sealing the success in developing into a blastocyst is not neces- developing blastocyst cavity where the ICM forms

s204 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. as a cluster of lucent cells. Adjacent ICM cells com- that act upstream of CDX2 to mediate trophectoderm municate through scarce gap junctions [12]. The differentiation [26,28,47]. On the other hand, trophectoderm is divided into a polar portion, expression of the trophectoderm-associated transcri- covering the ICM, and a mural portion sealing the ption factors, CDX2, TEAD4, and ELF5, are blastocyst cavity. In advance of hatching, the ICM repressed in the ICM by the regulatory circuit of develops into a distinct ventral cell layer, destined to NANOG, SOX2, and OCT4[34]. In the pig, the form the hypoblast, and a dorsal mass of cells destined expression of CDX2 during preimplantation deve- to form the epiblast (Figure 1). lopment appears conserved as compared with the A dynamic change in is the mouse [19]. OCT4 is, on the other hand, expressed driving force for the first cell differentiation: i.e. the in both the ICM and trophectoderm as opposed to segregation of the compacting blastomeres into the the mouse [17,18], and NANOG expression has not ICM and trophectoderm. In the mouse, the ICM deve- been observed in the porcine ICM [11]. Hence, there lops a stable regulatory circuit, in which the tran- are marked species differences with respect to the scription factors NANOG [5,25], OCT4 [27,36], molecular background for ICM and trophectoderm SOX2 [1], and the more recently identified SAL4 [7], specification. 2006; [49]Zhang, et al., 2006) promote pluripotency The embryo expands in size and hatches from and suppress differentiation. In contrast, in the tro- the zona pellucida by Days 7 to 8, and in parallel the phectoderm-destined cells, the factors OCT4 expression becomes confined to the ICM [43], CDX2 and EOMES are upregulated together with whereas expression of NANOG is still lacking (Figu- ELF5 and TEAD4, which are transcription factors re 2; [46]). At the time of hatching, the ICM is in the

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Figure 1. Transmission electron micrograph of porcine Day 6 blastocyst showing the zona pellucida (ZP), polar trophectoderm (Te) and the , 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- is used as an epithelial marker. s205 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. process of separating into two distinct cell by an “unfolding” of the complete epiblast upon the populations. Hence, the most “ventral” cell layer disintegration of Rauber’s layer (Figure 3; [12]). After towards the blastocyst cavity flattens and, finally, the loss of this component about Day 10 of gestation, delaminates forming the hypoblast. The “dorsal” cell the epiblast is discernable in the stereo microscope population establishes the epiblast. The hypoblast as a circular lucent structure known as the embryonic subsequently extends along the inside of the disc (Figure 4; [43]). Along with the formation of the trophectoderm forming a complete inner epithelial embryonic disc, the blastocyst enlarges, and by Day lining of the embryo. The polar trophectoderm 10 it reaches a diameter of more than half a covering the epiblast (known as the Rauber´s layer) centimetre. In parallel, with the formation of the becomes very thin around Day 9 of gestation and embryonic disc, the porcine epiblast starts to express gradually disintegrates exposing the epiblast to the not only OCT4, but also NANOG (Figure 5; [46]). uterine environment. Before the shedding of Rauber’s At this stage of development, the first sign of layer, tight junctions are formed between the anterior-posterior polarization develops in the outermost epiblast cells to maintain the epithelial embryonic disc: As mentioned earlier, the epiblast is sealing the embryo despite the loss of the polar underlaid by the hypoblast, and an area of increased trophectoderm. Apparently, the porcine epiblast forms density of closely apposed hypoblast cells develops. a small cavity, which finally opens dorsally followed 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. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. bryonic disc, but it is dislocated about one third of its crescent even before the “true” gastrulation starts with diameter anteriorly as compared with the epiblast of the appearance of the primitive streak (see later; [45]). the embryonic disc (Figure 6; [13,46]). It is likely A porcine embryo displaying BRACHYURY expres- that this dense hypoblast region emits signals to the sion in the posterior epiblast is displayed in Figure 6. epiblast which suppress mesoderm-formation in the With the development of the embryonic disc, anterior epiblast regions [13]. In this sense, the hypo- a very peculiar pattern of OCT4 and NANOG expres- blast may carry the blue-print for the specification of sion develops in the porcine epiblast: The majority of the epiblast. epiblast cells express OCT4, but small groups or During Days 11 to 12, the embryonic disc islands of cells are OCT4 negative [46]. The latter develops into an oval shape, and a crescent-shaped cells, on the other hand, express NANOG resulting in accumulation of cells are found in the posterior region a mutually exclusive expression pattern (Figure 7). of the disc [43]. This crescent includes mesodermal Subsequently, NANOG expression is lost in almost progenitors which express the mesodermal markers, the entire epiblast, except for a few cell in the most T (BRACHYURY) and GOOSECOID [3,45], and posterior region of the embryonic disc, in which OCT4 apparently ingression of BRACHYURY-expressing is also expressed [46]. The latter cells are believed to extra-embryonic mesoderm is initiated from this be the primordial germ cells (PGCs).

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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. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.

III. GASTRULATION: DEVELOPMENT OF MESODERM, in man and mouse [3,10]. “True” gastrulation is de- ENDODERM, AND ECTODERM fined by the presence of the primitive streak (Figure Gastrulation (from the Greek term gastrula 8). The porcine primitive streak apparently develops meaning small stomach) is the process by which the as an anterior extension of the BRACHYURY and three somatic germ layers, ectoderm, mesoderm, and GOOSECOID expressing crescent of epiblast cells endoderm, as well as the PGCs (see later) are formed. [3,45]. The streak elongates in an anterior direction and forms a depression, termed the , 3.1 The primitive streak at the midline. The porcine primitive streak expres- During Days 11-12 of gestation, the porcine ses BRACHYURY throughout its extension and embryo initiates a dramatic elongation that over a GOOSECOID at least in the anterior portion [23,45]. couple of days results in the transformation of the An example of a BRACHYURY expressing porcine spherical blastocyst to an approximately 1 m long, primitive streak is visualized in Figure 9. At ap- extremely thin filamentous structure. Gastrulation in proximately Days 13-14 of gestation, the primitive the porcine embryo is initiated around the time, when streak extends from the posterior pole of the epiblast elongation is in its initial progress [45]. Porcine and approximately two thirds of the length of the gastrulation is not dependent on implantation, as it is 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. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. tre during gastrulation, found at the anterior end of transition through the primitive streak to become the primitive streak, is the organizer region, termed either mesoderm or definitive endoderm. When the the node [37]. In the porcine embryo, the node is primitive streak has formed, epiblast cells continue morphologically evident as a thickening of cells in to enter this structure, which, thus, contains a dynamic the anterior part of the early primitive streak (Figure ever changing cell population. The cells, which in- 9); [42]. gress to the space between the epiblast and hypoblast 3.2 Ingression of cells forming mesoderm and form mesodermal and endodermal precursors. Until endoderm recently it was generally believed that the definitive Formation of the primitive streak involves endoderm derived from the primitive streak replaced extensive movement of cells, where the epiblast cells the hypoblast cell layer. Recently however, it was first gather at the posterior end of the embryonic disc, shown that in the gastrulating murine embryo the new- then rearrange to extend anteriorly in the streak itself, ly formed definitive endoderm cells insert themselves and, finally, undergo an epithelial-mesenchymal into the hypoblast in a dispersed manner

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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.

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[20]. Whether this is the case in the porcine embryo during this retraction. However, new investigations is not known. The mesodermal cells arrange in the mouse have shown that the node does not themselves as an intermediate cell layer between the regress posteriorly, but that the streak becomes two developing epithelial layers, i.e. the epiblast and relatively shorter due to the longitudinal growth of endoderm. the embryonic disc (Yamanaka et al., 2007). Along The cells entering the primitive streak are with this process, cells ingressing through the node exposed to distinct signaling factors at different con- forms the notochord; a rod-shaped structure centrations dependent on where in the primitive streak interposed between the epiblast and the endoderm the cells ingress through. Cell tracing studies has extending from the rostral end of the embryonic disc shown that the fate of a given cell is related to the site posteriorly to the node, from which it grows in a pos- of ingression through the primitive streak: Cells in- terior direction (Figure 10). The notochord posterior gressing through anterior streak and node become to the node is apparently formed by particular cells prechordal plate mesoderm, notochord, and endo- migrating posteriorly from the node [48]. The derm, cells ingressing through “mid” streak become notochord expresses BRACHYURY [45]. paraxial mesoderm, and cells migrating through the With the formation of the three somatic germ posterior streak become extra-embryonic and lateral layers; ectoderm, mesoderm, and endoderm and the plate mesoderm. These cell movements are, beside PGCs (see later), the progenitors of all fetal tissue geometrical differences, being conserved from lineages are formed. reptiles to mouse [24]. IV. NEURULATION: DEVELOPMENT OF THE NEURAL When the primitive streak has reached its ECTODERM AND NEURAL CREST maximum extension of about two thirds of the length of the embryonic disc, a subsequent posterior Neurulation is the process leading to the retraction the streak occurs. Until recently, it was formation of the neural tube, the precursor of the cen- thought that the primitive streak actually shortened tral 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. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. cord. This organ system is the first to initiate its epiblast to differentiate into , whereas development; functionally, however, it is overtaken the remaining more lateral portion of the epiblast by the later developing vascular system. Timewise, differentiates into surface ectoderm. This notochord- the process of neurulation overlaps with that of induced neurulation is referred to as primary neu- gastrulation: Along with the posterior retraction of rulation. The first morphological sign of primary the primitive streak, neurulation progresses in an an- neurulation is a dorsal thickening in the anterior ec- terior to posterior direction. Hence, over a certain toderm forming an elliptical region referred to as the period of time, the embryonic disc presents both the neural plate. Subsequently, the neural plate undergoes primitive streak posteriorly and the developing neural a shaping which converts it into a more elongated system anteriorly. key-hole shaped structure with broad anterior and narrow posterior regions. Neural plate shaping is 4.1 Neural ectoderm followed by the development of two lateral elevations, The epiblast cells anterior to the the neural folds, on either side of a depressed are induced to differentiate at the second gestational midregion referred to as the neural groove. In pigs week [44]. The notochord’s signalling molecules, and cattle the neural folds become clear during the including , induce the overlying third week of development (Figure 11).

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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 (YS) with the extra-embryonic (EC) between the layers. The latter opens into the primitive gut forming the (Hg) and the (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.

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The neural folds continue to elevate, appose the intensity of apoptosis is increased or decreased. in the midline, and, eventually, fuse to create the neu- Apoptosis at the tips of the neural folds may serve a ral tube which becomes covered by the surface special function. After opposing neural folds have ectoderm. Primary neurulation creates the brain and made contact and adhered to each other, midline most of the spinal cord, whereas in the tail bud, the epithelial remodelling by apoptosis breaks the posterior neural tube is formed by secondary neu- continuity between the neuroepithelium and surface rulation, where the spinal cord initially forms as a ectoderm. solid mass of epithelial cells, and a central lumen 4.2 Neural crest develops secondarily by . The primary neurulation is accompanied by Along with the elevation and fusion of the a bending of the neural plate, which occurs at three neural folds, certain cells at the lateral border or crest principal sites: the median hinge point (MHP), over- of the neural folds become detached. This cell lying the notochord, and the paired dorsolateral hinge population, known as the neural crest cells, will not points (DHLP) at the points of attachment of the sur- participate in formation of the neural tube; instead face ectoderm. The MHP is induced by signals from they migrate widely and participate in the formation the notochord. of many other tissues, such as the integument Gradually, the neural folds approach each (melanocytes), other parts of the nervous system other in the midline, where they eventually fuse. (including neurons for the central, sympathetic and Cellular protrusions extend from apical cells of the enteric nervous system as well as glial and Swann neural folds as they approach one another in the cells), and large parts of the craniofacial mesenchymal dorsal midline and interdigitate as the folds come derivatives [16]. into contact. This allows a first cell-cell recognition The mechanism whereby the neural crest cells and provides an initial adhesion pending later detach from the neural folds is comparable with that establishment of permanent cell contacts. occurring during ingression of epiblast cells in the The subsequent fusion of the neural folds primitive streak and node - a second example of begins in the cervical region and proceeds in a zipper- epithelio-mesenchymal transition. The term mesen- like fashion anteriorly and posteriorly from there. As chyme refers to loosely organized embryonic tissue a result of these processes, the neural tube is formed regardless of origin. Thus, both neu- and separated from the overlying surface ectoderm. roectoderm (through the neural crest cells) and Until fusion is complete, the anterior and posterior mesoderm (at gastrulation) may give rise to me- ends of the neural tube communicate with the senchyme. amniotic cavity via two openings, the anterior and The induction of neural crest cells is possibly posterior neuropores. Closure of the neuropores mediated by a gradient of BMP4, BMP7, and WNT occurs at approximately Days 24 to 26 and Days 15 secreted by the surface ectoderm. In the chick and to 16 in cattle and pig, respectively; the anterior pig, the neural crest cells express the transcription neuropore one to 2 days prior to the posterior. factor PAX7 [2]. Neurulation is then complete. The central nervous V. DEVELOPMENT OF THE PRIMORDIAL GERM CELLS system is represented at this time by a closed tubular (PGCS) structure with a narrow posterior portion, the anlage of the spinal cord, and a much broader cephalic The development of the germ line involves portion, the primordium of the encephalon. During specification of the cell linage and subsequent neurulation, the neuroepithelium is entirely pro- migration of the individual cells through various em- liferative; cells do not begin to exit the and bryonic tissues to the final destination in the genital start neuronal differentiation until after the neural tube ridges. After reaching the genital ridges, the cells of closure is complete. the germ line integrate and initiate the final steps of During neurulation, cell proliferation is ac- differentiation towards mature germ cells; a process companied by some degree of apoptosis in the neu- not completed until adulthood. roepithelium. The rate of apoptosis appears to be At the stage where the embryo presents a clear finely tuned and it seems to be equally detrimental if primitive streak the OCT4 expression gradually

s212 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. decreases in the epiblast. When that happens, the sition within the porcine embryonic disc, the putative putative PGC precursors can be identified by their PGC precursors move in caudal direction to the ex- continuous expression of this marker (Figure 12; tra-embryonic yolk sac wall. Initially, OCT4 positive Hyldig, unpublished data). In addition, they also cells are dispersed in both the embryonic and the ex- express NANOG another well known germ line tra-embryonic part of the yolk sac wall. A small cluster marker. These cells are seen dispersed within the cau- can be identified in the junction between embryonic dal third of the embryonic disc [33]. From their po- and extra-embryonic tissue (Figure 12). As the yolk

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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: ; Me: Mesenterium; Li: Liver.

s213 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. sac by Day 14-15 folds under the caudal area of the VI. FURTHER DEVELOPMENT OF THE EMBRYO embryo to form the ventral wall of the hind gut, PGCs The three germ layers, ectoderm, mesoderm, becomes restricted to this. Subsequently, the PGC and endoderm, form the basis for the further follow the migration path from the ventral to dorsal development of organ systems collectively referred side of the hind gut and further dorsolateral into the to as the area of special embryology (for review, see genital ridges. Although a few PGCs are seen in the Hyttel et al. [16]). genital ridge at Day 17, the vast majority is resided in the hindgut area at least until Day 18. By Day 20 6.1 The ectoderm and its early derivatives most PGCs are positioned in and around the The development of the neuroectoderm has attachment site of the elongated mesentery, however already been described in a former paragraph. After still with a substantial part of the population having allocated cells for endoderm, mesoderm, the positioned in the lower mesentery and hindgut area. germ line, and neuroectoderm, most of the remaining The tubular mesonephric tissue forms voluminous more laterally located epiblast will differentiate into bulges, forcing the genital ridges in towards the surface ectoderm. In parallel with the closure of the midline and effectively discontinuing the linear con- neuropores, two bilateral thickenings of the surface tact between them and the PGCs in the dorsal ectoderm, the and the placode, are mesentery[14]. The final colonisation of the genital established in the embryonic cephalic ectoderm (Fi- ridges occurs around E23-24 [15]. The integration gure 13A). The otic placode invaginates to form the of the PGCs into the genital ridge tissue and the , which will develop into the inner ear for subsequent differentiation of the germ line is to our hearing and balance, while the knowledge largely unexplored in the porcine species. invaginates and forms the lens of the eye. The The gonadal tissue begins organising by Day 42. remaining surface ectoderm gives rise to the Germ cell cords are present in both male and female epidermis and associated glands of the skin, as well gonads, though larger and more regular in males. as the epithelium covering the oral and nasal cavities Male gonads are rounded with only a slim cellular and the caudal portion of the anal canal. The connection to the mesonephros [46]. epithelium covering the oral cavity gives rise to the In the newly formed PGCs, DNA is highly enamel of the teeth and also part of the pituitary gland, methylated, as it is in their epiblast progenitors. Howe- the adenohypophysis. ver, by the time the PGC have entered the genital ridge, DNA has become largely hypomethylated. The 6.2 The mesoderm and its early derivatives demethylation is well studied in the murine species Formation of the notochord provides an and studies of various repeats and differentially embryonic midline axis as a template for the axial methylated domain (DMD) sequences of imprinted skeleton. Initially, cells of the mesoderm form a thin genes in porcine embryos show a comparable sheet of loosely woven mesenchyme on either side demethylation. The process is completed by Day 28- of the notochord. Soon, however, the mesoderm clo- 31, where after remethylation is started [4,38]. sest to the notochord (the paraxial mesoderm) pro- Immunostainings of genomewide CpG methylation liferates and forms pairs of segmental thickened stru- have indicated that the demethylation is initiated ctures known as (Figure 13). This process around Day 15 during PGC migration toward the starts in the occipital region of the embryo, and in genital ridges [14]. During the subsequent large animal species, somites are formed at a rate of, gametogenesis, when oocytes and spermatozoa are on average, about six pairs a day. The number of so- formed from the PGC derivatives, de novo mites formed during this phase of development methylation of DNA occurs. Importantly, this therefore forms a basis for estimating embryonic age. genome-wide demethylation and remethylation also More laterally, the mesoderm remains thin and includes the sex-specific DNA methylation of parti- is therefore referred to as the lateral plate mesoderm. cular loci, forming the basis of genomic imprinting. The lateral plate mesoderm is continuous with the

s214 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.

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 (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 and subcutis of the skin. Later, each myotome and mesoderm is split into an outer component lining dermatome will receive its own segmental nerve trophectoderm and an inner component lining the component. hypoblast, and the cavity between these two 6.4 components is referred to as the extra-embryonic The intermediate mesoderm, which connects coelom (Figure 11). With the continued development paraxial and lateral plate mesoderm, differentiates into of the coelom, an intra-embryonic coelom similarly structures of both the urinary system and the gonads, divides the lateral plate mesoderm in such a way that together referred to as the urogenital system. TheN the so-called somatic mesoderm associates with the urinary system is first developed as an abortive surface ectoderm to constitute the somatopleura while anteriorly located paired , succeeded by the so-called visceral mesoderm associates with the a very prominently developing paired mesonephros endoderm to form the splanchnopleura. Between the (Figure 13B). The mesonephros develops excretory paraxial and lateral plate mesoderm, the intermediate ducts; the mesonephric ducts (Wollfian ducts). Finally, mesoderm is established. the even further posteriorly located paired metanephos 6.3 Paraxial mesoderm develops and the persisting kidneys. The gonads As a general rule, development proceeds in develop on the medial aspect of the mesonephros, an anterior to posterior direction (one exception to initially as the genital ridges which receive the pri- this was the development of the primitive streak). Ac- mordial germ cells. In the male, the mesonephric cordingly, formation of somites progresses from the ducts develops into the epididymal ducts and the occipital region posteriorly. Each subsequently ductus deferens. In the female, however, another duct, differentiates into three components: The ventro- the paramesonephric duct (the Müllerian duct), forms medial part of the somite associates with the notochord parallel to the and develops into establishing the sclerotome which patterns formation the oviduct, the uterus, and the cranial portion of the of the vertebral column. The dorso-lateral part of each vagina. somite forms regionalized precursors of both dermal 6.5 Lateral plate mesoderm and body folding and muscle tissue, the dermamyotome. From this Through anterior-posterior and lateral fol- structure, a dorso-medially located cell population dings, the subdivision of the coelom into intra- and forms the myotome and a dorso-laterally located extra-embryonic cavities becomes progressively group becomes the dermatome. The myotome of each better defined and the embryonic body gradually as- somite contributes to muscles of the back and limbs, sumes the shape of a closed tube enclosing another while the dermatome disperses and forms the dermis tube, the primitive gut. The somatopleura will form

s215 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. the lateral and ventral body wall enclosing the intra- form blood vessels. The first sign of blood and blood embryonic coelom of which the somatic mesoderm vessel formation is seen in the visceral mesoderm of will provide the inner lining (the mesothelium of the the splanchnopleura covering the yolk sac (see later). and pleura) and the ectoderm the outer However, this appears to be only a transient lining (the epidermis). The splanchnopleura will form phenomenon; later, hematopoiesis moves first to the the wall of the primitive gut and its derivatives in liver and spleen and then to the bone marrow. The which the endoderm and the visceral mesoderm will heart is also of mesodermal origin; though with some provide the inner lining (the lamina epithelialis of contribution of neural crest cells (Figure 13). the tunica mucosa) and outer lining (the lamina 6.7 The endoderm and its early derivatives epithelialis of the tunica serosa) respectively. The The inner epithelial lining of the visceral mesoderm will also form the connective and its derivatives is the main tissue and muscular components of the gut and its component derived from the endoderm. With the derivatives. Soon, the intra-embryonic coelom will anterior-posterior and lateral foldings of the embryo, be divided into the peritoneal, pleural and pericardial the endoderm-enclosed primitive gut becomes cavities. enclosed within the embryo, whereas the hypoblast- 6.6 Blood and blood vessel formation enclosed yolk sac becomes localized outside the Both blood and blood vessels appear to arise embryo. from common mesoderm precursor cells, the The primitive gut comprises cranial (foregut), hemangioblasts. These differentiate into hema- middle () and caudal (hindgut) parts. The topoietic stem cells (forming blood cells) and angio- midgut communicates with the yolk sac through the blasts that form endothelial cells which coalesce to (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 (blue) surrounds the embryo and is fused with the chorion in the mesamnion (MA).

s216 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. but, as development proceeds, becomes long and nar- and later the embryonic surface ectoderm. The outside row and is eventually incorporated into the umbilical covering of the amnion is composed of extra- cord. The endoderm forms the epithelium of the embryonic mesoderm. gastro-pulmonary system and the parenchyma of its The site where the chorioamniotic folds meet derivatives. Endoderm of the foregut gives rise to and fuse is known as the mesamnion. In cattle and the and its derivatives, including the middle pig, the mesamnion persists; as a result, the amnion ear, the parenchyma of the thyroid gland, the gets torn during parturition and offspring are generally parathyroid glands, the liver and the pancreas, and born without covering membranes. the reticulated stroma of the tonsils and thymus, as With the body foldings and the formation of well as the oesophagus, stomach, liver, and pancreas. the endoderm-lined primitive gut, the hypoblast-lined At its anterior end, the foregut is temporarily closed yolk sac is transformed into an extra-embryonic cavity by an ectodermal-endodermal membrane, the communicating with the primitive gut through the buccopharyngeal membrane. At a certain stage of vitelline tube. The outside of the yolk sac is lined by development, this membrane ruptures and open visceral mesoderm. In cattle and pig, the yolk sac communication between the amniotic cavity and the serves a hematopoietic function for a short period of primitive gut is established. The midgut gives rise to time, but subsequently it regresses within one to two most of the small and the large intestine down to the weeks after its formation and never attains other transverse colon whereas the hindgut gives rise to important functions. the transverse and descending colon as well as the During the second or third week of rectum and part of the anal canal. At its caudal end, development, depending on the species, the allantois the hindgut temporarily dilates to form the , a is formed as an outgrowth from the hindgut into the cavity transiently common to both the developing extra-embryonic coelom. In ruminants and the pig, gastrointestinal and urogenital systems. The cloaca the allantois assumes a T-shaped appearance with the is separated from the amniotic cavity by the cloacal top bar of the T being located as a transverse cavity membrane, composed of closely apposed ectoderm just caudal to the embryo proper and the stem of the and endoderm, like the buccopharyngeal membrane. T connected with the hindgut. Like the vitelline duct,N After separation of the gastrointestinal and urinary the allantoic duct, connecting the allantoic cavity and systems, the breaks down, opening the hindgut, becomes incorporated into the umbili- the two systems via the anus and , cal cord as a consequence of embryonic foldings. respectively. Since the allantois is a diverticulum of the hindgut, its wall is composed of an inner epithelial lining of VII. PLACENTATION AND FORMATION OF EXTRA- EMBRYONIC MEMBRANES AND CAVITIES endodermal origin and an outer layer derived from the visceral mesoderm. As the allantois enlarges, the 7.1 Development of extra-embryonic membranes and visceral mesodermal part of its wall fuses with the cavities somatic mesoderm of the chorion and, finally, more During the early phases of gastrulation, the or less covers the amnion. The fusion of the allantoic trophectoderm becomes lined by a thin layer of ex- and chorionic walls forms the embryonic part of the tra-embryonic mesoderm, the two layers together chorioallantoic found in the domestic constituting the outer extra-embryonic membrane, the . The intra-embryonic proximal portion of chorion (Figure 14). During gastrulation, the chorion the allantoic duct, extending from the hindgut to the forms folds, the chorioamniotic folds, which surround umbilicus, is referred to as the urachus and gives rise the embryonic disc. Gradually, the folds extend to the . Throughout gestation, the al- upwards to meet and fuse above the embryonic disc lantoic cavity serves as a repository of the wastes thereby enclosing the disc in a sealed amniotic cavity. excreted through the embryo’s developing urinary The term amnion is generally used collectively for system. the cavity and its wall. The inner epithelium of the Prior to attachment the conceptus is solely amnion originates from the trophectoderm and so, at nourished by uterine glandular secretions the embryonic disc, it is continuous with the epiblast (histiotrophe), but with attachment of the chorio-

s217 R.C. Chebel. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221. allantoic placenta to the endometrial wall an exchange barrier: the endometrial epithelium, connective tissue, of fetal/maternal blood-borne nutrients (hemotrophe) and vascular endothelium. also contributes. Areolae, chorionic indentations In cattle and pig, the placenta is epithelio- opposite the endometrial glands, are scattered in the chorial and the chorionic and endometrial epithelia diffuse porcine placenta and remain present during are apposed, and there is no loss of maternal tissue. gestation [21]. The epitheliochorial placenta in ruminants is modified 7.2 Placentation as particular cells cross into, and fuse with, The placenta can be classified according to some of the endometrial epithelial cells. Hence, the the structure of the chorioallantoic surface and its placenta is referred to as synepitheliochorial. interaction with the endometrium. Areas where the chorioallantois interacts with the endometrium and VIII. STAGING OF EMBRYONIC DEVELOPMENT engages in placental formation are referred to as Simple measures have over the time been used chorion frondosum, in contrast to the smooth chorion as a reference for embryonic development including leave not included in the placenta. In the pig, chorion length in mm [32], days of gestation [22], numbers frondosum is diffusely distributed over the entire of somites [15, 41], or external features [8]. Within chorioallantoic surface and so the placenta is human embryology, a painstaking work has been put categorized as being diffuse. The porcine chorio- into developing the Carnegie system; staging system allantoic surface area is increased by foldings, providing a precise frame of reference of embryonic revealed as primary plicae and secondary rugae, and development [29,30]. This staging system utilizes is thus referred to as being folded. In cattle, the cho- macroscopic as well as microscopic features in a rion frondosum is organized as arborizing chorionic developing embryo and fetus. The Carnegie system villi assembled into larger macroscopically visible has been implemented in bats [6] and mouse [39]. tufts called cotyledons. Hence, the bovine placenta We are currently working on development of a is known as cotyledonary or multiplex and villous. Carnegie-based porcine staging system for the The cotyledons combine with endometrial domestic pig based on the examination of ap- prominences known as caruncles, forming proximately 600 specimens. placentomes in which the chorioallantoic villi of the cotyledon extend into crypts of the caruncle. IX. CONCLUSIONS The placenta can also be classified based on the number of tissue layers separating the fetal and An improved understanding of post-hatching maternal circulations, thereby forming the placental embryonic development holds an important key to barrier. There are always three fetal extra-embryonic not only a more proper evaluation of the success or layers in the chorioallantoic placenta: the endothelium failure of assisted reproductive technologies; it also lining the allantoic blood vessels; chorioallantoic forms an important basis for the understanding of mesenchyme, originating from the fused somatic stem cell differentiation and cell replacement therapy. (chorionic) and visceral (allantoic) mesoderm; and A wealth of contemporary data are published on the the chorionic epithelium developed from the molecular regulation of the initial lineage segregation trophectoderm and in the placenta referred to as the and cell differentiation taking place in the embryo, trophoblast. However, the numbers of layers retained and it is a great challenge to align all the complex in the maternal portion of the placenta varies with sets of information into integrated networks gradually species. Before placentation, the endometrium pre- guiding the well-orchestrated embryonic and fetal sents three layers that could contribute to the placental development.

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