From Hatching into Fetal Life in the Pig - Inicial — UFRGS Supl_s203-s221.pdf · 2013-05-24 · During the subsequent neurulation, which is a process overlapping with gastrulation

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

Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s203 - s221.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s203 - s221.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s203 - s221.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s203 - s221.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s203 - s221.

ISSN 1679-9216 (Online)

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

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Poul Hyttel, Kristian M. Kamstrup & Sara HyldigPoul Hyttel, Kristian M. Kamstrup & Sara HyldigPoul Hyttel, Kristian M. Kamstrup & Sara HyldigPoul Hyttel, Kristian M. Kamstrup & Sara HyldigPoul Hyttel, Kristian M. Kamstrup & Sara Hyldig

ABSTRACT

Background: Potential adverse effects of assisted reproductive technologies may have long term consequences on embryonicand fetal development. However, the complex developmental phases occurring after hatching from the zona pellucida are lessstudied than those occurring before hatching. The aim of the present review is to introduce the major post-hatchingdevelopmental 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 expressionof OCT4 and NANOG. In the pre-hatching porcine blastocyst, a different and yet unresolved mechanism is operating as OCT4is expressed in both the ICM and trophectoderm, and NANOG is not expressed at all. Around the time of hatching, OCT4becomes confined to the ICM. In parallel, the ICM is divided into a ventral cell layer, destined to form the hypoblast, and adorsal cell mass, destined to form the epiblast. The hypoblast gradually develops into a complete inner lining along theepiblast and the trophectoderm. Upon hatching (around Day 7-8 of gestation), the trophectoderm covering the developingepiblast (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 theembryo occurs, and by Day 13 it has formed a thin approximately one meter long filamentous structure. This elongation isparalleled with the initiation of placentation along with which, the embryonic disc undergoes gastrulation. The latter processis preceded by a thickening of the posterior region of the epiblast, putatively developing as a consequence of an absence ofinhibitory signals from a condensed portion of the hypoblast underlying the anterior epiblast. The thickened posterior epiblastexpresses the primitive streak marker BRACHYURY. Subsequently, the epiblast thickening extends in an anterior directionforming the primitive streak; also expressing BRACHYURY. Gastrulation is hereby initiated, and epiblast cells ingress throughthe primitive streak to form mesoderm and endoderm; the latter is inserted into the dorsal hypoblast whereas the mesodermforms a more loosely woven mesenchyme between the epiblast and the endoderm. The anterior mesoderm, ingressing throughthe anterior end of the primitive streak, referred to as the node, forms the rod-like notochord interposed between the epiblastand the endoderm. During the subsequent neurulation, which is a process overlapping with gastrulation in time, the notochordinduces the overlying epiblast to form neural ectoderm, which sequentially develops into the neural plate, neural groove, andneural tube, whereas the lateral epiblast develops into the surface ectoderm. In parallel with the development of the somaticgerm layers, ectoderm, mesoderm, and endoderm, the primordial germ cells, the predecessors of the germ line, develop in theposterior epiblast and initiates a migration finally bringing them to the genital ridges of the developing embryo. In parallel, theectoderm gives rise to the epidermis and neural tissue, the mesoderm develops into the cardiovascular system as well as theurogenital and musculoskeletal systems, whereas the endoderm forms the gastrointestinal system and related organs as theliver and pancreas.Conclusions: Porcine embryonic and fetal development is controlled by molecular mechanisms that to some degree differfrom those operating in the mouse. It is of importance to uncover the molecular control of development in ungulates as it hasgreat implications for assisted reproductive technologies as well as for biomedical model research.

Keywords: Biomedical models, embryology, blastulation, gastrulation, neurulation, embryonic staging.

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I. INTRODUCTIONII.BLASTULATION: DEVELOPMENT OF TRO-

PHECTODERM, ICM, EPIBLAST, HYPOBLAST,AND EMBRYONIC DISC

III. GASTRULATION: DEVELOPMENT OF MESO-DERM, ENDODERM, AND ECTODERM

3.1 The primitive streak3.2 Ingression of cells forming mesoderm and endodermIV. NEURULATION: DEVELOPMENT OF THE NEU-

RAL ECTODERM AND NEURAL CREST4.1 Neural ectoderm4.2 Neural crestV. DEVELOPMENT OF THE PRIMORDIAL GERM

CELLS (PGCS)VI. FURTHER DEVELOPMENT OF THE EMBRYO6.1 The ectoderm and its early derivatives6.2 The mesoderm and its early derivatives6.3 Paraxial mesoderm6.4 Paraxial mesoderm6.5 Lateral plate mesoderm and body folding6.6 Blood and blood vessel formation6.7 The endoderm and its early derivativesVII. PLACENTATION AND FORMATION OF EXTRA-

EMBRYONIC MEMBRANES AND CAVITIES7.1 Development of extra-embryonic membranes and

cavities7.2 PlacentationVIII. STAGING OF EMBRYONIC DEVELOPMENT

IX. CONCLUSIONS

I. INTRODUCTION

The wide-spread use of in vitro productionof, in particular, bovine embryos in animal husbandryhas paved the way for a detailed morphological andmolecular understanding of oocyte maturation,fertilization and initial embryonic development untilthe time of hatching. Hence, studies on these life pro-cesses have become facilitated by the easyaccessibility of oocytes, zygotes, and embryos.Cloning by somatic cell nuclear transfer (SCNT) isanother technology, which over the past decade hasresulted in alternative in vitro production ofconsiderable numbers of both bovine and porcineembryos adding to the accessibility of embryos forresearch. Development of the embryo to the blastocyststage includes several complex processes as e.g. theactivation of the embryonic genome (for review, seeOestrup et al. [31]). It is also clear, however, thatsuccess in developing into a blastocyst is not neces-

sarily a guarantee for further development into a fetusand newborn. Hence, it is well-documented that invitro embryo culture may impose long-term effects,which are revealed later during embryonic and fetaldevelopment [40]. This phenomenon becomes evenmore exaggerated when embryos are produced bySCNT, which imposes an even higher risk ofembryonic and fetal aberrations as well as neonatalloss [9,35].

In order to evaluate embryonic and fetaldevelopment resulting from assisted reproductivetechnologies more properly, increasing focus shouldbe put on the normality of some of the complex post-hatching processes, as e.g. gastrulation, neurulation,placentation and initial organogenesis, which areprerequisites for full term development. These pro-cesses are the focus of the present review. Over thepast decade the pig has attracted increasing attentionas a useful biomedical model, due to which thepresented data will mainly be derived in this species.Comparative notes will be made to cattle, wheneverthe variation between these two species are pro-nounced as e.g. at placentation. First, important deve-lopmental processes of the general embryology inclu-ding blastulation, gastrulation, neurulation, and deve-lopment 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 begiven.

II. BLASTULATION: DEVELOPMENT OFTROPHECTODERM, ICM, EPIBLAST, HYPOBLAST, AND

EMBRYONIC DISC

Blastulation (from the Greek term blastosmeaning sprout) is the process by which the embryodevelops into a fluid-filled structure in which the cellshave segregated into lines destined to produce theembryo proper (the ICM and epiblast) and suchdeveloping into the extra-embryonic membranes (thetrophectoderm and hypoblast).

In the pig, the blastocyst forms at around Day5 of gestation. The porcine embryo initiates com-paction as early as the 8-16-cell stage, when theembryo assumes a spherical appearance with asmoother surface where the protrusions of the indi-vidual blastomeres are no longer seen. The outer cells,allocated to the trophectoderm, become connectedby tight junctions and desmosomes sealing thedeveloping blastocyst cavity where the ICM forms

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as a cluster of lucent cells. Adjacent ICM cells com-municate through scarce gap junctions [12]. Thetrophectoderm is divided into a polar portion,covering the ICM, and a mural portion sealing theblastocyst cavity. In advance of hatching, the ICMdevelops into a distinct ventral cell layer, destined toform the hypoblast, and a dorsal mass of cells destinedto form the epiblast (Figure 1).

A dynamic change in gene expression is thedriving force for the first cell differentiation: i.e. thesegregation of the compacting blastomeres into theICM and trophectoderm. In the mouse, the ICM deve-lops a stable regulatory circuit, in which the tran-scription factors NANOG [5,25], OCT4 [27,36],SOX2 [1], and the more recently identified SAL4 [7],2006; [49]Zhang, et al., 2006) promote pluripotencyand suppress differentiation. In contrast, in the tro-phectoderm-destined cells, the transcription factorsCDX2 and EOMES are upregulated together withELF5 and TEAD4, which are transcription factors

that act upstream of CDX2 to mediate trophectodermdifferentiation [26,28,47]. On the other hand,expression of the trophectoderm-associated transcri-ption factors, CDX2, TEAD4, and ELF5, arerepressed in the ICM by the regulatory circuit ofNANOG, SOX2, and OCT4[34]. In the pig, theexpression of CDX2 during preimplantation deve-lopment appears conserved as compared with themouse [19]. OCT4 is, on the other hand, expressedin both the ICM and trophectoderm as opposed tothe mouse [17,18], and NANOG expression has notbeen observed in the porcine ICM [11]. Hence, thereare marked species differences with respect to themolecular background for ICM and trophectodermspecification.

The embryo expands in size and hatches fromthe zona pellucida by Days 7 to 8, and in parallel theOCT4 expression becomes confined to the ICM [43],whereas expression of NANOG is still lacking (Figu-re 2; [46]). At the time of hatching, the ICM is in the

Figure 1. Transmission electron micrograph of porcine Day 6 blastocystshowing the zona pellucida (ZP), polar trophectoderm (Te) and the innercell mass, which has already divided into ventral cells (VC), developinginto the hypoblast, and dorsal cells (DC), developing into the epiblast. BC:Blastocyst cavity. Insert: Light micrograph of the same blastocyst showingthe inner cell mass (ICM).

Figure 2. Confocal laser scanning micrographs of Day 8-9 hatched porcine blastocyst displaying OCT4 expression in the innercell mass, whereas NANOG expression is lacking. E-CADHERIN is used as an epithelial marker.

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process of separating into two distinct cellpopulations. Hence, the most “ventral” cell layertowards the blastocyst cavity flattens and, finally,delaminates forming the hypoblast. The “dorsal” cellpopulation establishes the epiblast. The hypoblastsubsequently extends along the inside of thetrophectoderm forming a complete inner epitheliallining of the embryo. The polar trophectodermcovering the epiblast (known as the Rauber´s layer)becomes very thin around Day 9 of gestation andgradually disintegrates exposing the epiblast to theuterine environment. Before the shedding of Rauber’slayer, tight junctions are formed between theoutermost epiblast cells to maintain the epithelialsealing the embryo despite the loss of the polartrophectoderm. Apparently, the porcine epiblast formsa small cavity, which finally opens dorsally followed

by an “unfolding” of the complete epiblast upon thedisintegration of Rauber’s layer (Figure 3; [12]). Afterthe loss of this component about Day 10 of gestation,the epiblast is discernable in the stereo microscopeas a circular lucent structure known as the embryonicdisc (Figure 4; [43]). Along with the formation of theembryonic disc, the blastocyst enlarges, and by Day10 it reaches a diameter of more than half acentimetre. In parallel, with the formation of theembryonic disc, the porcine epiblast starts to expressnot only OCT4, but also NANOG (Figure 5; [46]).

At this stage of development, the first sign ofanterior-posterior polarization develops in theembryonic disc: As mentioned earlier, the epiblast isunderlaid by the hypoblast, and an area of increaseddensity 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 ofthe trophectoderm (Te), covers the epiblast (Ep), in which a cavity (C) has developed. The epiblast is underlaid by the epiblast-related tallerhypoblast (Ep-Hy) and the trophectoderm by the trophectoderm-related lower hypoblast (Te-Hy). (B) Another section from the same epiblastshowing 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) Lightmicrograph of section of the embryonic disc showing the dome-shaped epiblast (Ep) underlaid by the hypoblast (Hy). The epiblast is continuouswith the trophectoderm (Te) indicated by the arrows.

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bryonic disc, but it is dislocated about one third of itsdiameter anteriorly as compared with the epiblast ofthe embryonic disc (Figure 6; [13,46]). It is likelythat this dense hypoblast region emits signals to theepiblast which suppress mesoderm-formation in theanterior epiblast regions [13]. In this sense, the hypo-blast may carry the blue-print for the specification ofthe epiblast.

During Days 11 to 12, the embryonic discdevelops into an oval shape, and a crescent-shapedaccumulation of cells are found in the posterior regionof the disc [43]. This crescent includes mesodermalprogenitors which express the mesodermal markers,T (BRACHYURY) and GOOSECOID [3,45], andapparently ingression of BRACHYURY-expressingextra-embryonic mesoderm is initiated from this

crescent even before the “true” gastrulation starts withthe appearance of the primitive streak (see later; [45]).A porcine embryo displaying BRACHYURY expres-sion in the posterior epiblast is displayed in Figure 6.

With the development of the embryonic disc,a very peculiar pattern of OCT4 and NANOG expres-sion develops in the porcine epiblast: The majority ofepiblast cells express OCT4, but small groups orislands of cells are OCT4 negative [46]. The lattercells, on the other hand, express NANOG resulting ina mutually exclusive expression pattern (Figure 7).Subsequently, NANOG expression is lost in almostthe entire epiblast, except for a few cell in the mostposterior region of the embryonic disc, in which OCT4is also expressed [46]. The latter cells are believed tobe the primordial germ cells (PGCs).

Figure 5. Confocal laser scanning micrographs of Day 9-10 hatched porcine blastocyst displaying OCT4 and NANOG expression in theepiblast.

Figure 6. Confocal laser scanning micrographs of Day 10-11 porcine blastocyst displaying expression of T (BRACHYURY) in the posteriorportion of the epiblast and of FOXA2 in the hypoblast. The open arrowheads in mark the periphery of the elongated embryonic disc. Theasterisks 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].

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III. GASTRULATION: DEVELOPMENT OF MESODERM,ENDODERM, AND ECTODERM

Gastrulation (from the Greek term gastrulameaning small stomach) is the process by which thethree somatic germ layers, ectoderm, mesoderm, andendoderm, as well as the PGCs (see later) are formed.

3.1 The primitive streakDuring Days 11-12 of gestation, the porcine

embryo initiates a dramatic elongation that over acouple of days results in the transformation of thespherical blastocyst to an approximately 1 m long,extremely thin filamentous structure. Gastrulation inthe porcine embryo is initiated around the time, whenelongation is in its initial progress [45]. Porcinegastrulation is not dependent on implantation, as it is

in man and mouse [3,10]. “True” gastrulation is de-fined by the presence of the primitive streak (Figure8). The porcine primitive streak apparently developsas an anterior extension of the BRACHYURY andGOOSECOID expressing crescent of epiblast cells[3,45]. The streak elongates in an anterior directionand forms a depression, termed the primitive groove,at the midline. The porcine primitive streak expres-ses BRACHYURY throughout its extension andGOOSECOID at least in the anterior portion [23,45].An example of a BRACHYURY expressing porcineprimitive streak is visualized in Figure 9. At ap-proximately Days 13-14 of gestation, the primitivestreak extends from the posterior pole of the epiblastand approximately two thirds of the length of theembryonic disc [42]. A key embryonic signalling cen-

Figure 7. Confocal laser scanning micrographs of Day 10 porcine blastocyst displaying mutually excluding epiblast cell populations expressingOCT4 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 thespace between the epiblast and the hypoblast (Hy/En), which is gradually exchanged by final endoderm. Loose mesoderm (Me) is also locatedin this area. A: Anterior; P: Posterior; D: Dorsal; V: Ventral.

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tre during gastrulation, found at the anterior end ofthe primitive streak, is the organizer region, termedthe node [37]. In the porcine embryo, the node ismorphologically evident as a thickening of cells inthe anterior part of the early primitive streak (Figure9); [42].3.2 Ingression of cells forming mesoderm andendoderm

Formation of the primitive streak involvesextensive movement of cells, where the epiblast cellsfirst 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 becomeeither mesoderm or definitive endoderm. When theprimitive streak has formed, epiblast cells continueto enter this structure, which, thus, contains a dynamicever changing cell population. The cells, which in-gress to the space between the epiblast and hypoblastform mesodermal and endodermal precursors. Untilrecently it was generally believed that the definitiveendoderm derived from the primitive streak replacedthe hypoblast cell layer. Recently however, it wasshown that in the gastrulating murine embryo the new-ly formed definitive endoderm cells insert themselvesinto the hypoblast epithelium in a dispersed manner

Figure 9. Confocal laser scanning micrographs of Day 12-13 porcine embryo. (A–C) Side-view of the embryonic disc. Note the expression ofT (BRACHYURY) in the primitive streak and the posterior epiblast, the intensive FOXA2 expression in the hypoblast, and the co-expressionof the two markers in the node (arrow and arrowhead in C). The periphery of embryonic disc marked with open arrowheads in A. Note theexpression 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 theprimitive 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 embryois not known. The mesodermal cells arrangethemselves as an intermediate cell layer between thetwo developing epithelial layers, i.e. the epiblast andendoderm.

The cells entering the primitive streak areexposed to distinct signaling factors at different con-centrations dependent on where in the primitive streakthe cells ingress through. Cell tracing studies hasshown that the fate of a given cell is related to the siteof ingression through the primitive streak: Cells in-gressing through anterior streak and node becomeprechordal plate mesoderm, notochord, and endo-derm, cells ingressing through “mid” streak becomeparaxial mesoderm, and cells migrating through theposterior streak become extra-embryonic and lateralplate mesoderm. These cell movements are, besidegeometrical differences, being conserved fromreptiles to mouse [24].

When the primitive streak has reached itsmaximum extension of about two thirds of the lengthof the embryonic disc, a subsequent posteriorretraction the streak occurs. Until recently, it wasthought that the primitive streak actually shortened

during this retraction. However, new investigationsin the mouse have shown that the node does notregress posteriorly, but that the streak becomesrelatively shorter due to the longitudinal growth ofthe embryonic disc (Yamanaka et al., 2007). Alongwith this process, cells ingressing through the nodeforms the notochord; a rod-shaped structureinterposed between the epiblast and the endodermextending from the rostral end of the embryonic discposteriorly to the node, from which it grows in a pos-terior direction (Figure 10). The notochord posteriorto the node is apparently formed by particular cellsmigrating posteriorly from the node [48]. Thenotochord expresses BRACHYURY [45].

With the formation of the three somatic germlayers; ectoderm, mesoderm, and endoderm and thePGCs (see later), the progenitors of all fetal tissuelineages are formed.

IV. NEURULATION: DEVELOPMENT OF THE NEURALECTODERM AND NEURAL CREST

Neurulation is the process leading to theformation of the neural tube, the precursor of the cen-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 anddeveloping 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) Opticalside view of the embryonic disc displaying the same features. A: Anterior; P: Posterior; D:Dorsal; V: Ventral. Modified from Wolf et al.(2011b)[45].

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cord. This organ system is the first to initiate itsdevelopment; functionally, however, it is overtakenby the later developing vascular system. Timewise,the process of neurulation overlaps with that ofgastrulation: Along with the posterior retraction ofthe primitive streak, neurulation progresses in an an-terior to posterior direction. Hence, over a certainperiod of time, the embryonic disc presents both theprimitive streak posteriorly and the developing neuralsystem anteriorly.

4.1 Neural ectodermThe epiblast cells anterior to the primitive node

are induced to differentiate at the second gestationalweek [44]. The notochord’s signalling molecules,including Sonic hedgehog, induce the overlying

epiblast to differentiate into neuroectoderm, whereasthe remaining more lateral portion of the epiblastdifferentiates into surface ectoderm. This notochord-induced neurulation is referred to as primary neu-rulation. The first morphological sign of primaryneurulation is a dorsal thickening in the anterior ec-toderm forming an elliptical region referred to as theneural plate. Subsequently, the neural plate undergoesa shaping which converts it into a more elongatedkey-hole shaped structure with broad anterior andnarrow posterior regions. Neural plate shaping isfollowed by the development of two lateral elevations,the neural folds, on either side of a depressedmidregion referred to as the neural groove. In pigsand cattle the neural folds become clear during thethird week of development (Figure 11).

Figure 11: Porcine Day 14-15 embryos. (A) Stereo micrograph of the embryonic disc showing the primitive streak (PS) posteriorly, delineatedby arrowheads, and the neural groove (NG) anteriorly, delineated by arrows. CAF: Chorioamniotic folding. (B) Section of embryonic disc alongthe broken line in A. Note the thick neural ectoderm (NE) continuous with the trophectoderm at the arrows. The mesoderm (Me) is seen betweenthe 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.

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The neural folds continue to elevate, apposein the midline, and, eventually, fuse to create the neu-ral tube which becomes covered by the surfaceectoderm. Primary neurulation creates the brain andmost of the spinal cord, whereas in the tail bud, theposterior neural tube is formed by secondary neu-rulation, where the spinal cord initially forms as asolid mass of epithelial cells, and a central lumendevelops secondarily by cavitation.

The primary neurulation is accompanied bya bending of the neural plate, which occurs at threeprincipal sites: the median hinge point (MHP), over-lying the notochord, and the paired dorsolateral hingepoints (DHLP) at the points of attachment of the sur-face ectoderm. The MHP is induced by signals fromthe notochord.

Gradually, the neural folds approach eachother in the midline, where they eventually fuse.Cellular protrusions extend from apical cells of theneural folds as they approach one another in thedorsal midline and interdigitate as the folds comeinto contact. This allows a first cell-cell recognitionand provides an initial adhesion pending laterestablishment of permanent cell contacts.

The subsequent fusion of the neural foldsbegins in the cervical region and proceeds in a zipper-like fashion anteriorly and posteriorly from there. Asa result of these processes, the neural tube is formedand separated from the overlying surface ectoderm.Until fusion is complete, the anterior and posteriorends of the neural tube communicate with theamniotic cavity via two openings, the anterior andposterior neuropores. Closure of the neuroporesoccurs at approximately Days 24 to 26 and Days 15to 16 in cattle and pig, respectively; the anteriorneuropore one to 2 days prior to the posterior.Neurulation is then complete. The central nervoussystem is represented at this time by a closed tubularstructure with a narrow posterior portion, the anlageof the spinal cord, and a much broader cephalicportion, the primordium of the encephalon. Duringneurulation, the neuroepithelium is entirely pro-liferative; cells do not begin to exit the cell cycle andstart neuronal differentiation until after the neural tubeclosure is complete.

During neurulation, cell proliferation is ac-companied by some degree of apoptosis in the neu-roepithelium. The rate of apoptosis appears to befinely 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 aspecial function. After opposing neural folds havemade contact and adhered to each other, midlineepithelial remodelling by apoptosis breaks thecontinuity between the neuroepithelium and surfaceectoderm.

4.2 Neural crestAlong with the elevation and fusion of the

neural folds, certain cells at the lateral border or crestof the neural folds become detached. This cellpopulation, known as the neural crest cells, will notparticipate in formation of the neural tube; insteadthey migrate widely and participate in the formationof many other tissues, such as the integument(melanocytes), other parts of the nervous system(including neurons for the central, sympathetic andenteric nervous system as well as glial and Swanncells), and large parts of the craniofacial mesenchymalderivatives [16].

The mechanism whereby the neural crest cellsdetach from the neural folds is comparable with thatoccurring during ingression of epiblast cells in theprimitive streak and node - a second example ofepithelio-mesenchymal transition. The term mesen-chyme refers to loosely organized embryonic tissueregardless of germ layer origin. Thus, both neu-roectoderm (through the neural crest cells) andmesoderm (at gastrulation) may give rise to me-senchyme.

The induction of neural crest cells is possiblymediated by a gradient of BMP4, BMP7, and WNTsecreted by the surface ectoderm. In the chick andpig, the neural crest cells express the transcriptionfactor PAX7 [2].

V. DEVELOPMENT OF THE PRIMORDIAL GERM CELLS(PGCS)

The development of the germ line involvesspecification of the cell linage and subsequentmigration of the individual cells through various em-bryonic tissues to the final destination in the genitalridges. After reaching the genital ridges, the cells ofthe germ line integrate and initiate the final steps ofdifferentiation towards mature germ cells; a processnot completed until adulthood.

At the stage where the embryo presents a clearprimitive streak the OCT4 expression gradually

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decreases in the epiblast. When that happens, theputative PGC precursors can be identified by theircontinuous expression of this marker (Figure 12;Hyldig, unpublished data). In addition, they alsoexpress NANOG another well known germ linemarker. These cells are seen dispersed within the cau-dal third of the embryonic disc [33]. From their po-

sition within the porcine embryonic disc, the putativePGC precursors move in caudal direction to the ex-tra-embryonic yolk sac wall. Initially, OCT4 positivecells are dispersed in both the embryonic and the ex-tra-embryonic part of the yolk sac wall. A small clustercan be identified in the junction between embryonicand extra-embryonic tissue (Figure 12). As the yolk

Figure 12. Schematic presentation of the position of the porcine germ line during early development. Sections of the porcineembryo are depicted below drawings of embryo proper. Broken lines across embryo proper indicate section sites. Red dotsrepresent 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 wallwhere 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.

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sac by Day 14-15 folds under the caudal area of theembryo to form the ventral wall of the hind gut, PGCsbecomes restricted to this. Subsequently, the PGCfollow the migration path from the ventral to dorsalside of the hind gut and further dorsolateral into thegenital ridges. Although a few PGCs are seen in thegenital ridge at Day 17, the vast majority is residedin the hindgut area at least until Day 18. By Day 20most PGCs are positioned in and around theattachment site of the elongated mesentery, howeverstill with a substantial part of the populationpositioned in the lower mesentery and hindgut area.The tubular mesonephric tissue forms voluminousbulges, forcing the genital ridges in towards themidline and effectively discontinuing the linear con-tact between them and the PGCs in the dorsalmesentery[14]. The final colonisation of the genitalridges occurs around E23-24 [15]. The integrationof the PGCs into the genital ridge tissue and thesubsequent differentiation of the germ line is to ourknowledge largely unexplored in the porcine species.The gonadal tissue begins organising by Day 42.Germ cell cords are present in both male and femalegonads, though larger and more regular in males.Male gonads are rounded with only a slim cellularconnection to the mesonephros [46].

In the newly formed PGCs, DNA is highlymethylated, as it is in their epiblast progenitors. Howe-ver, by the time the PGC have entered the genitalridge, DNA has become largely hypomethylated. Thedemethylation is well studied in the murine speciesand studies of various repeats and differentiallymethylated domain (DMD) sequences of imprintedgenes in porcine embryos show a comparabledemethylation. The process is completed by Day 28-31, where after remethylation is started [4,38].Immunostainings of genomewide CpG methylationhave indicated that the demethylation is initiatedaround Day 15 during PGC migration toward thegenital ridges [14]. During the subsequentgametogenesis, when oocytes and spermatozoa areformed from the PGC derivatives, de novomethylation of DNA occurs. Importantly, thisgenome-wide demethylation and remethylation alsoincludes the sex-specific DNA methylation of parti-cular 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 furtherdevelopment of organ systems collectively referredto as the area of special embryology (for review, seeHyttel et al. [16]).

6.1 The ectoderm and its early derivativesThe development of the neuroectoderm has

already been described in a former paragraph. Afterhaving allocated cells for endoderm, mesoderm, thegerm line, and neuroectoderm, most of the remainingmore laterally located epiblast will differentiate intosurface ectoderm. In parallel with the closure of theneuropores, two bilateral thickenings of the surfaceectoderm, the otic placode and the lens placode, areestablished in the embryonic cephalic ectoderm (Fi-gure 13A). The otic placode invaginates to form theotic vesicle, which will develop into the inner ear forhearing and balance, while the lens placodeinvaginates and forms the lens of the eye. Theremaining surface ectoderm gives rise to theepidermis and associated glands of the skin, as wellas the epithelium covering the oral and nasal cavitiesand the caudal portion of the anal canal. Theepithelium covering the oral cavity gives rise to theenamel of the teeth and also part of the pituitary gland,the adenohypophysis.

6.2 The mesoderm and its early derivativesFormation of the notochord provides an

embryonic midline axis as a template for the axialskeleton. Initially, cells of the mesoderm form a thinsheet of loosely woven mesenchyme on either sideof the notochord. Soon, however, the mesoderm clo-sest to the notochord (the paraxial mesoderm) pro-liferates and forms pairs of segmental thickened stru-ctures known as somites (Figure 13). This processstarts in the occipital region of the embryo, and inlarge animal species, somites are formed at a rate of,on average, about six pairs a day. The number of so-mites formed during this phase of developmenttherefore forms a basis for estimating embryonic age.

More laterally, the mesoderm remains thin andis therefore referred to as the lateral plate mesoderm.The lateral plate mesoderm is continuous with the

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extra-embryonic mesoderm. The extra-embryonicmesoderm is split into an outer component liningtrophectoderm and an inner component lining thehypoblast, and the cavity between these twocomponents is referred to as the extra-embryoniccoelom (Figure 11). With the continued developmentof the coelom, an intra-embryonic coelom similarlydivides the lateral plate mesoderm in such a way thatthe so-called somatic mesoderm associates with thesurface ectoderm to constitute the somatopleura whilethe so-called visceral mesoderm associates with theendoderm to form the splanchnopleura. Between theparaxial and lateral plate mesoderm, the intermediatemesoderm is established.6.3 Paraxial mesoderm

As a general rule, development proceeds inan anterior to posterior direction (one exception tothis was the development of the primitive streak). Ac-cordingly, formation of somites progresses from theoccipital region posteriorly. Each somite subsequentlydifferentiates into three components: The ventro-medial part of the somite associates with the notochordestablishing the sclerotome which patterns formationof the vertebral column. The dorso-lateral part of eachsomite forms regionalized precursors of both dermaland muscle tissue, the dermamyotome. From thisstructure, a dorso-medially located cell populationforms the myotome and a dorso-laterally locatedgroup becomes the dermatome. The myotome of eachsomite contributes to muscles of the back and limbs,while the dermatome disperses and forms the dermis

and subcutis of the skin. Later, each myotome anddermatome will receive its own segmental nervecomponent.6.4 Intermediate mesoderm

The intermediate mesoderm, which connectsparaxial and lateral plate mesoderm, differentiates intostructures of both the urinary system and the gonads,together referred to as the urogenital system. Theurinary system is first developed as an abortiveanteriorly located paired pronephros, succeeded bya very prominently developing paired mesonephros(Figure 13B). The mesonephros develops excretoryducts; the mesonephric ducts (Wollfian ducts). Finally,the even further posteriorly located paired metanephosdevelops and the persisting kidneys. The gonadsdevelop on the medial aspect of the mesonephros,initially as the genital ridges which receive the pri-mordial germ cells. In the male, the mesonephricducts develops into the epididymal ducts and theductus deferens. In the female, however, another duct,the paramesonephric duct (the Müllerian duct), formsparallel to the mesonephric duct and develops intothe oviduct, the uterus, and the cranial portion of thevagina.6.5 Lateral plate mesoderm and body folding

Through anterior-posterior and lateral fol-dings, the subdivision of the coelom into intra- andextra-embryonic cavities becomes progressivelybetter defined and the embryonic body gradually as-sumes the shape of a closed tube enclosing anothertube, the primitive gut. The somatopleura will form

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).

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the lateral and ventral body wall enclosing the intra-embryonic coelom of which the somatic mesodermwill provide the inner lining (the mesothelium of theperitoneum and pleura) and the ectoderm the outerlining (the epidermis). The splanchnopleura will formthe wall of the primitive gut and its derivatives inwhich the endoderm and the visceral mesoderm willprovide the inner lining (the lamina epithelialis ofthe tunica mucosa) and outer lining (the laminaepithelialis of the tunica serosa) respectively. Thevisceral mesoderm will also form the connectivetissue and muscular components of the gut and itsderivatives. Soon, the intra-embryonic coelom willbe divided into the peritoneal, pleural and pericardialcavities.

6.6 Blood and blood vessel formationBoth blood and blood vessels appear to arise

from common mesoderm precursor cells, thehemangioblasts. These differentiate into hema-topoietic stem cells (forming blood cells) and angio-blasts that form endothelial cells which coalesce to

form blood vessels. The first sign of blood and bloodvessel formation is seen in the visceral mesoderm ofthe splanchnopleura covering the yolk sac (see later).However, this appears to be only a transientphenomenon; later, hematopoiesis moves first to theliver and spleen and then to the bone marrow. Theheart is also of mesodermal origin; though with somecontribution of neural crest cells (Figure 13).

6.7 The endoderm and its early derivativesThe inner epithelial lining of the

gastrointestinal tract and its derivatives is the maincomponent derived from the endoderm. With theanterior-posterior and lateral foldings of the embryo,the endoderm-enclosed primitive gut becomesenclosed within the embryo, whereas the hypoblast-enclosed yolk sac becomes localized outside theembryo.

The primitive gut comprises cranial (foregut),middle (midgut) and caudal (hindgut) parts. Themidgut communicates with the yolk sac through thevitelline 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 diverticulumfrom the hindgut, is lined on the inside by endoderm covered by extra-embryonic mesoderm. The fusion between the twomembranes, the chorion and allantois, results in the chorioallantoic membrane, which forms foldings engaged inplacentation. The yolk sac (red), which is a diverticulum connected with the midgut through the vitelline duct, isrudimentary. The amnion (blue) surrounds the embryo and is fused with the chorion in the mesamnion (MA).

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but, as development proceeds, becomes long and nar-row and is eventually incorporated into the umbilicalcord. The endoderm forms the epithelium of thegastro-pulmonary system and the parenchyma of itsderivatives. Endoderm of the foregut gives rise tothe pharynx and its derivatives, including the middleear, the parenchyma of the thyroid gland, theparathyroid glands, the liver and the pancreas, andthe reticulated stroma of the tonsils and thymus, aswell as the oesophagus, stomach, liver, and pancreas.At its anterior end, the foregut is temporarily closedby an ectodermal-endodermal membrane, thebuccopharyngeal membrane. At a certain stage ofdevelopment, this membrane ruptures and opencommunication between the amniotic cavity and theprimitive gut is established. The midgut gives rise tomost of the small and the large intestine down to thetransverse colon whereas the hindgut gives rise tothe transverse and descending colon as well as therectum and part of the anal canal. At its caudal end,the hindgut temporarily dilates to form the cloaca, acavity transiently common to both the developinggastrointestinal and urogenital systems. The cloacais separated from the amniotic cavity by the cloacalmembrane, composed of closely apposed ectodermand endoderm, like the buccopharyngeal membrane.After separation of the gastrointestinal and urinarysystems, the cloacal membrane breaks down, openingthe two systems via the anus and urogenital sinus,respectively.

VII. PLACENTATION AND FORMATION OF EXTRA-EMBRYONIC MEMBRANES AND CAVITIES

7.1 Development of extra-embryonic membranes andcavities

During the early phases of gastrulation, thetrophectoderm becomes lined by a thin layer of ex-tra-embryonic mesoderm, the two layers togetherconstituting the outer extra-embryonic membrane, thechorion (Figure 14). During gastrulation, the chorionforms folds, the chorioamniotic folds, which surroundthe embryonic disc. Gradually, the folds extendupwards to meet and fuse above the embryonic discthereby enclosing the disc in a sealed amniotic cavity.The term amnion is generally used collectively forthe cavity and its wall. The inner epithelium of theamnion originates from the trophectoderm and so, atthe embryonic disc, it is continuous with the epiblast

and later the embryonic surface ectoderm. The outsidecovering of the amnion is composed of extra-embryonic mesoderm.

The site where the chorioamniotic folds meetand fuse is known as the mesamnion. In cattle andpig, the mesamnion persists; as a result, the amniongets torn during parturition and offspring are generallyborn without covering membranes.

With the body foldings and the formation ofthe endoderm-lined primitive gut, the hypoblast-linedyolk sac is transformed into an extra-embryonic cavitycommunicating with the primitive gut through thevitelline tube. The outside of the yolk sac is lined byvisceral mesoderm. In cattle and pig, the yolk sacserves a hematopoietic function for a short period oftime, but subsequently it regresses within one to twoweeks after its formation and never attains otherimportant functions.

During the second or third week ofdevelopment, depending on the species, the allantoisis formed as an outgrowth from the hindgut into theextra-embryonic coelom. In ruminants and the pig,the allantois assumes a T-shaped appearance with thetop bar of the T being located as a transverse cavityjust caudal to the embryo proper and the stem of theT connected with the hindgut. Like the vitelline duct,the allantoic duct, connecting the allantoic cavity andthe hindgut, becomes incorporated into the umbili-cal 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 ofendodermal origin and an outer layer derived fromthe visceral mesoderm. As the allantois enlarges, thevisceral mesodermal part of its wall fuses with thesomatic mesoderm of the chorion and, finally, moreor less covers the amnion. The fusion of the allantoicand chorionic walls forms the embryonic part of thechorioallantoic placenta found in the domesticanimals. The intra-embryonic proximal portion ofthe allantoic duct, extending from the hindgut to theumbilicus, is referred to as the urachus and gives riseto the urinary bladder. Throughout gestation, the al-lantoic cavity serves as a repository of the wastesexcreted through the embryo’s developing urinarysystem.

Prior to attachment the conceptus is solelynourished by uterine glandular secretions(histiotrophe), but with attachment of the chorio-

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allantoic placenta to the endometrial wall an exchangeof fetal/maternal blood-borne nutrients (hemotrophe)also contributes. Areolae, chorionic indentationsopposite the endometrial glands, are scattered in thediffuse porcine placenta and remain present duringgestation [21].7.2 Placentation

The placenta can be classified according tothe structure of the chorioallantoic surface and itsinteraction with the endometrium. Areas where thechorioallantois interacts with the endometrium andengages in placental formation are referred to aschorion frondosum, in contrast to the smooth chorionleave not included in the placenta. In the pig, chorionfrondosum is diffusely distributed over the entirechorioallantoic surface and so the placenta iscategorized as being diffuse. The porcine chorio-allantoic surface area is increased by foldings,revealed as primary plicae and secondary rugae, andis thus referred to as being folded. In cattle, the cho-rion frondosum is organized as arborizing chorionicvilli assembled into larger macroscopically visibletufts called cotyledons. Hence, the bovine placentais known as cotyledonary or multiplex and villous.The cotyledons combine with endometrialprominences known as caruncles, formingplacentomes in which the chorioallantoic villi of thecotyledon extend into crypts of the caruncle.

The placenta can also be classified based onthe number of tissue layers separating the fetal andmaternal circulations, thereby forming the placentalbarrier. There are always three fetal extra-embryoniclayers in the chorioallantoic placenta: the endotheliumlining the allantoic blood vessels; chorioallantoicmesenchyme, originating from the fused somatic(chorionic) and visceral (allantoic) mesoderm; andthe chorionic epithelium developed from thetrophectoderm and in the placenta referred to as thetrophoblast. However, the numbers of layers retainedin the maternal portion of the placenta varies withspecies. Before placentation, the endometrium pre-sents three layers that could contribute to the placental

barrier: the endometrial epithelium, connective tissue,and vascular endothelium.

In cattle and pig, the placenta is epithelio-chorial and the chorionic and endometrial epitheliaare apposed, and there is no loss of maternal tissue.The epitheliochorial placenta in ruminants is modifiedas particular trophoblast cells cross into, and fuse with,some of the endometrial epithelial cells. Hence, theplacenta is referred to as synepitheliochorial.

VIII. STAGING OF EMBRYONIC DEVELOPMENT

Simple measures have over the time been usedas a reference for embryonic development includinglength in mm [32], days of gestation [22], numbersof somites [15, 41], or external features [8]. Withinhuman embryology, a painstaking work has been putinto developing the Carnegie system; staging systemproviding a precise frame of reference of embryonicdevelopment [29,30]. This staging system utilizesmacroscopic as well as microscopic features in adeveloping embryo and fetus. The Carnegie systemhas been implemented in bats [6] and mouse [39].We are currently working on development of aCarnegie-based porcine staging system for thedomestic pig based on the examination of ap-proximately 600 specimens.

IX. CONCLUSIONS

An improved understanding of post-hatchingembryonic development holds an important key tonot only a more proper evaluation of the success orfailure of assisted reproductive technologies; it alsoforms an important basis for the understanding ofstem cell differentiation and cell replacement therapy.A wealth of contemporary data are published on themolecular regulation of the initial lineage segregationand cell differentiation taking place in the embryo,and it is a great challenge to align all the complexsets of information into integrated networks graduallyguiding the well-orchestrated embryonic and fetaldevelopment.

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