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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. 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