From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick: II. Microscopic anatomy and cell population dynamics

The microscopic anatomy of uterine and freshly laid unincubated and briefly incubated chick germs is described. Special attention is paid to the difference between the three developmental periods involved: cleavage, area pellucida formation, and primary hypoblast formation. During cleavage the cytoplasm of the germinal disc divides into blastomeres, which become constantly smaller, and the subgerminal cavity is formed. The germ is accumulating extensive glycogen reserves for utilization during the next period. The most fascinating period is the formation of the area pellucida, which arises as a result of a polarized cell-shedding process. During this process all the subepithelial cells round up and fall into the subblastodermic cavity, where they assemble beneath the future anterior side of the blastoderm. The cell-shedding process is presumably energy consuming and the glycogen reserves are utilized as cell shedding progresses, starting at the posterior and terminating at the anterior side of the germ. The germ loses about one-fifth of its initial cytoplasmic mass during this process. The formation of the primary hypoblast is again polarized, posterioanteriorly. The onset of the process of polyinvagination takes place concomitantly with the shedding of the last subepithelial cells.     

Post-nodal mesoblast caudalizes the host axis and inhibits cell population growth, and induces new primitive streaks in chick embryos

One to eight post-nodal fragments (PN) or Hensen's nodes (HN) from full primitive streak stage chick embryos were transplanted onto the area pellucida or area opaca of stage 4 embryos and cultured for 20 h. Thirteen morphological and numerical parameters were affected in the host embryos and analyzed by multiple logistic regression for parametric hierarchy. In the area pellucida, both PN and HN transplants inhibited cell population growth while only PN caudalized the host axis and induced supernumerary primitive streaks expressing the mesoderm-specific gene Brachyury. In the area opaca, neither grafts influenced host axis morphogenesis, but PN inhibited the cell population growth significantly. Tracking [(3)H]TdR labeled grafts showed that PN cells migrated towards the host axis and participated in the formation of supernumerary somites and hearts. When placed near the host axis, PN caudalized it and inhibited cell population growth.     

Nucleolar ontogenesis in the uterine chick germ correlated with morphogenetic events

Nucleolar development in the cleaving chick germ up to the formation of the primary hypoblast was followed through a series of well-defined uterine and early incubated stages both by light and electron microscopy. Well-established criteria of nucleolar morphology were used for determining the developmental stage of onset of rRNA synthesis. By these criteria rRNA synthesis was first observed at midcleavage in uterine stage VII [1] germs. This could be correlated with the first morphogenetic event—the posterio-anteriorly orientated formation of the area pellucida which results in a bilaterally symmetrical blastoderm.     

Effect of spatial position of uterine quail blastoderms cultured in vitro on bilateral symmetry formation

Summary A method of in vitro culture for uterine quail blastoderms has been developed, which allows them to develop from cleavage throughout gastrulation and further: stages 4–10 of Hamburger and Hamilton (1951). The method consists of cultivating the blastoderms on egg albumen in a vertical position; this permits about 50% of the blastoderms explanted before area pellucida formation to develop bilateral symmetry and to form normal primitive streak, somites and head structures. Development of the blastoderms explanted after their area pellucida was already formed, occurred normally and was not influenced by their spatial position in the culture.This work was performed as part of project no. 09.7.1.5.2 of the Polish Academy of Sciences     

Primordial germ cells of the young chick blastoderm originate from the central zone of the area pellucida irrespective of the embryo-forming process

Early chick blastoderms (stages X-XII) were divided by a circular cut into two fragments. In one experimental group, the area opaca was separated from the marginal zone and the central disc of the area pellucida, while in another group the area opaca plus marginal zone were separated from the central disc. Other blastoderms of equivalent stages were each cut into three strips of equal size (either perpendicular or parallel to the axis of symmetry). The fragments were isolated and incubated for 43-48 h after which they were PAS-stained, whole-mounted and checked for the presence of primordial germ cells (PGCs). The results showed that most of the PGCs originated from the central disc and not from the periphery of the area pellucida and that they segregated from this zone even if no embryonic axis developed in the explant. In such cases, the PGCs were found to be dispersed throughout the entire explant, usually in association with forming blood islands. When an axis did develop in the explant, the PGCs were found to be concentrated around its anterior end, in a pattern resembling the germinal crescent. No indication of a quantitative regulation of PGCs was found in the explants and the sum of PGCs, calculated for the complementary fragments of a blastoderm, matched the range of numbers in control blastoderms. Our results suggest that PGCs may already be determined as early as stage X and that their further differentiation is independent of the embryo-forming process.     

Turkey embryo staging from cleavage through hypoblast formation

The progressive development of the turkey embryo from first cleavage through hypoblast formation was examined in order to determine the applicability of a chicken embryo staging procedure. It was concluded that the temporal and spatial events associated with the development of the early turkey embryo are sufficiently different from those of the chicken embryo to warrant a separate staging procedure. Cleavage is asynchronous and often results in asymmetrical segmentation. Unlike the chicken embryo, which at oviposition has already formed the area pellucida and area opaca and is classified as a Stage X embryo, the turkey embryo at oviposition is only at the beginning of area pellucida formation and is classified as a Stage VII embryo. After about 3 hr of incubation and prior to completion of the area pellucida, hypoblast formation begins at the posterior end, thereby establishing the bilaterally symmetrical pattern of the embryo. When viewed from the dorsal surface, an opaque region is observed at the center of the area pellucida. This opacity is unique to the turkey embryo and is referred to as the area alba. When viewed from the ventral surface, the area alba appears to be composed of large whitish cells. To conclude, the rate of turkey embryo development through the completion of hypoblast formation, which consists of 11 stages, lags behind that of the chicken. Furthermore, the organization as well as origin of the area pellucida and hypoblast observed in the turkey embryo differ from that of the chicken embryo. © 1993 Wiley-Liss, Inc.     

Axis formation in half blastoderms of the chick: Stage at separation and the relative positions of fused halves influence axis development

The blastoderm of the avian embryo acts during the early stages of development as an integrative system programmed to form a single embryonic axis. Isolated parts of the blastoderm are known to each form an axis, owing to the system's properties. In the work reported here, the regulative capability of the right and left halves of chick blastoderms to form an embryonic axis was examined systematically at different stages. This revealed a progressive change in the developing blastoderm. After early separation, the axis in each half will form at some distance from the blastoderm's original midline, while with late separation the axis will form next to the original midline and may even lack one row of somites at the medial rim. Since development stops in culture after about 2 days, axis development after early separation ceases before somites are formed, whereas after late separation somites and brain vesicles can develop. In addition, an attempt was made to learn whether the two halves of blastoderm, when shifted along the midline and then reunited in staggered fashion, act as a single or two separate embryonic fields. When reunion of the right and left halves was achieved so that the posterior end of one half was adjoining the posterior area pellucida region of the other half, a single embryonic axis developed. When, on the other hand, the shift was larger so that the posterior end was fused to the central area pellucida of the other half, two separated embryonic axes developed.     

Early embryo development in the Siberian hamster (Phodopus sungorus)

Development of preimplantation embryos of the Siberian hamster (Phodopus sungorus) in vivo and in vitro was examined. The timing of early development in vivo was found to be slower than that reported for the golden hamster. Progression through the cleavage stages, cavitation, and hatching from the zona pellucida occurred later, with blastocyst formation beginning on the afternoon of day 4 and uterine attachment occurring early on day 5. In vitro, morulae, and early blastocysts collected on day 4 and cultured in serum-containing medium formed expanded blastocysts and some began to hatch from the zona pellucida. With extended culture, blastocysts attached and formed trophoblast outgrowths. Outgrowth was characterized by an initial migration of small cells from the blastocyst, followed by formation of a sheet of trophoblast giant cells. Differences in the morphology of outgrowth between the hamster and mouse suggest that further comparative studies with the Siberian hamster may be useful.     

Reproductive failure due to the entrapment of oocytes in luteinized follicles of the little bulldog bat (Noctilio albiventris)

The reproductive tracts of 112 female little bulldog bats collected around the onset of a breeding season in the Cauca Valley of Colombia were examined histologically. Of the 88 females with luteinizing/luteinized follicles or new CL, 72 carried tubal ova or uterine blastocysts and 16 were non-pregnant. In 14 of the latter follicle rupture had apparently failed to occur, and the oocyte or a collapsed zona pellucida was found within a luteinizing or luteinized follicle. These structures appeared to be functional because most of the affected bats demonstrated preferential stimulation of the ipsilateral oviduct and/or uterine horn. Two of these animals also had a second, older luteinized follicle which contained the remnants of an oocyte. None of these 14 bats exhibited the uterine modifications thought to be associated with a previous pregnancy or had prominent mammary glands. Such reproductive features were, in contrast, frequently demonstrated by other females in the population. These observations suggest that the luteinization of unruptured follicles may have occurred in prepubertal members of the population and reflect immaturity of the hypothalamo-pituitary -ovarian axis in these individuals.     

Experimentally induced variations in vitelline artery development and circulatory pattern in the early chick embryo

White Leghorn eggs were incubated to desired prevasular stages. Each embryo, upon its intact yolk and surrounded by albumen, was rolled from the shell into a sterile 50 ml beaker. In an uppermost position, the blastoderm was lightly stained with neutral red. Three types of localized cuts were made as indicated below, and the beaker-embryo unit placed in a sterile humidified chamber for further incubation. Results: (1) Unilateral cuts adjacent to the body and parallel with its axis blocked or suppressed formation of the vitelline artery on the cut side, even though healing occurred; (2) A specific site was found in the area pellucida opposite the sinus rhomboidalis which, when lightly cut perpendicular to the body axis, resulted in blockage or shifting of the final junction between aorta and vitelline artery as far cephalad as the thirteenth somite level. Formation of a dual final junction with the aorta also occurred. (3)Transverse cuts through the body axis and into the area pellucida bilaterally, frequently resulted in bilateral blockage or bilateral shifting. In still other cases, growth of a functional loop around the cut to connect the aortae in anterior and posterior segments with the vitelline artery were observed. Other unique circulatory patterns are described. Apparently, slight interference with the capillary plexus which precedes the vitelline artery causes anomalous development. Circulation is considered a major factor in arterial differentiation. Cutting probably alters the plexus and relation of its components to the onset of blood flow.     

Interactions between Wnt and Vg1 signalling pathways initiate primitive streak formation in the chick embryo

The posterior marginal zone (PMZ) of the chick embryo has Nieuwkoop centre-like properties: when transplanted to another part of the marginal zone, it induces a complete embryonic axis, without making a cellular contribution to the induced structures. However, when the PMZ is removed, the embryo can initiate axis formation from another part of the remaining marginal zone. Chick Vg1 can mimic the axis-inducing ability of the PMZ, but only when misexpressed somewhere within the marginal zone. We have investigated the properties that define the marginal zone as a distinct region. We show that the competence of the marginal zone to initiate ectopic primitive streak formation in response to cVg1 is dependent on Wnt activity. First, within the Wnt family, only Wnt8C is expressed in the marginal zone, in a gradient decreasing from posterior to anterior. Second, misexpression of Wnt1 in the area pellucida enables this region to form a primitive streak in response to cVg1. Third, the Wnt antagonists Crescent and Dkk-1 block the primitive streak-inducing ability of cVg1 in the marginal zone. These findings suggest that Wnt activity defines the marginal zone and allows cVg1 to induce an axis. We also present data suggesting some additional complexity: first, the Vg1 and Wnt pathways appear to regulate the expression of downstream components of each other's pathway; and second, misexpression of different Wnt antagonists suggests that different classes of Wnts may cooperate with each other to regulate axis formation in the normal embryo.     

Multi-level and multi-scale integrative approach to the understanding of human blastocyst implantation

Implantation is a complex process which results in fixation of zona pellucida free blastocyst to the maternal uterine endometrium. In the human, it involves progesterone mediated preparation of endometrium, age- and stage-matched development of pre-implantation embryo, and interaction between embryo and endometrium. In the present essay, we present the case to explain why there is a necessity of undertaking multi-level, multi-scale integrative approach to deconstruct the succession process of endometrial development to the climax of implantation.     

Nodal signal is required for morphogenetic movements of epiblast layer in the pre-streak chick blastoderm

During axis formation in amniotes, posterior and lateral epiblast cells in the area pellucida undergo a counter-rotating movement along the midline to form primitive streak (Polonaise movements). Using chick blastoderms, we investigated the signaling involved in this cellular movement in epithelial-epiblast. In cultured posterior blastoderm explants from stage X to XI embryos, either Lefty1 or Cerberus-S inhibited initial migration of the explants on chamber slides. In vivo analysis showed that inhibition of Nodal signaling by Lefty1 affected the movement of DiI-marked epiblast cells prior to the formation of primitive streak. In Lefty1-treated embryos without a primitive streak, Brachyury expression showed a patchy distribution. However, SU5402 did not affect the movement of DiI-marked epiblast cells. Multi-cellular rosette, which is thought to be involved in epithelial morphogenesis, was found predominantly in the posterior half of the epiblast, and Lefty1 inhibited the formation of rosettes. Three-dimensional reconstruction showed two types of rosette, one with a protruding cell, the other with a ventral hollow. Our results suggest that Nodal signaling may have a pivotal role in the morphogenetic movements of epithelial epiblast including Polonaise movements and formation of multi-cellular rosette.     

Glycogen metabolism in the prelaid chick embryo.

Glycogen metabolism has been studied during the development of the early chick embryo, at the cytochemical and ultrastructural levels. Two waves of glycogen synthesis and breakdown have been found. In the first, free clusters of glycogen particles are synthesized at late oogenesis. These clusters are found later in invaginations of the membrane of vesicles containing a floccular material (FLOV). The glycogen clusters are degraded there during ovulation and the first hours in the oviduct. The second wave of glycogen synthesis begins before cleavage, reaching a maximum at mid-uterine age. This second wave occurs in another type of vesicle (GLYV), which eventually disintegrates releasing free clusters of glycogen granules. This glycogen is degraded in membranous structures containing a floccular material, as in the first wave of degradation. The degradation ends at the late uterine stages, and at the same time numerous ribosomes are formed. This period corresponds to area pellucida formation, which probably depends on the energy liberated during the second wave of glycogen degradation.     

The neural inductive response of competent chick ectoblast decreases away from the host axis and correlates with an increased proliferative activity.

We have assessed the quality and quantity of the neural inductive response of the chick gastrula ectoblast located at increasing distancefrom the host axis. In a stage 4 gastrula, entire ectoblast exhibits neural competence. The quality of induced neural tissue shifts from deuterencephalic type in the area pellucida to archencephalic type in the area opaca and primitive medullary or palisade type atthe margin of overgrowth with a concomitant reduction in the number of induced neural cells. In contrast, the mitotic and 3H-TdR labelling frequencies in the competent ectoblast increase with increasing distance from the host axis and in a proportion inverse to the amount of induced neural tissue. It is suggested that the strong neural inductive response is correlated with low proliferative activity, or longer cell cycle time, of the competent ectoblast.     

Blastocyst implantation in the Chinese hamster (Cricetulus griseus)

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete area. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.     

Sites of zona pellucida shedding by mouse embryo other than muran trophectoderm

The sites at which a mouse embryo sheds the zona pellucida in preparation for its hatching were investigated in vitro. Of all cases examined, 24% shedding occurred at the mural site, 24% at the inner cell mass site, 17% at the equatorial site, and 35% at other sites. It thus appears reasonable to conclude that there is no particular site destined for shedding and the site at which it does take place in vitro is irrelevant to that at which the uterus later becomes attached. Expressing its frequency in terms of a unit of area indicated shedding to occur markedly at the axis penetrating the inner cell mass and mural trophectoderm. The ratio of hatching frequency along this axis to that at other areas was determined as 7:1.     

Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos.

Studies in Xenopus laevis and zebrafish suggest a key role for beta-catenin in the specification of the axis of bilateral symmetry. In these organisms, nuclear beta-catenin demarcates the dorsalizing centers. We have asked whether beta-catenin plays a comparable role in the chick embryo and how it is adapted to the particular developmental constraints of chick development. The first nuclear localization of beta-catenin is observed in late intrauterine stages of development in the periphery of the blastoderm, the developing area opaca and marginal zone. Obviously, this early, radially symmetric domain does not predict the future organizing center of the embryo. During further development, cells containing nuclear beta-catenin spread under the epiblast and form the secondary hypoblast. The onset of hypoblast formation thus demarcates the first bilateral symmetry in nuclear beta-catenin distribution. Lithium chloride exposure also causes ectopic nuclear localization of beta-catenin in cells of the epiblast in the area pellucida. Embryos treated before primitive streak formation become completely radialized, as shown by the expression of molecular markers, CMIX and GSC. Lithium treatments performed during early or medium streak stages cause excessive development of the anterior primitive streak, node and notochord, and lead to a degeneration of prospective ventral and posterior structures, as shown by the expression of the molecular markers GSC, CNOT1, BMP2 and Ch-Tbx6L. In summary, we found that in spite of remarkable spatiotemporal differences, beta-catenin acts in the chick in a manner similar to that in fish and amphibia.     

The gradual establishment of cell commitments during the early stages of chick development

The early development of the chick (Stages I-XIII E.G&K) can be regarded as an ideal model for epigenetic development and for the study of the forces and factors involved in the establishment of cellular heterogeneity and the imprinting of polarity. It seems that a physical vectorial force is utilized to imprint upon the cleaving radial-symmetric multilayered blastodisc, a postero-anterior metabolic gradient, which is translated into the first morphogenetic phenomenon - a polarized cell shedding, causing the formation of the area pellucida. In a freshly laid egg, the Stage X blastoderm despite its radial appearance, has a concealed bilateral symmetry which is not susceptible any longer to spatial changes, but nevertheless is still labile and can be changed by several other drastic experimental procedures. This means that the individual cells are still pluripotential and not yet specifically committed. During the first 10 h of incubation a second polar morphogenetic process, the formation of Hyp, occurs following the same orientation of the previous cell shedding and thus stressing and enforcing the postero-anterior axis of bilateral symmetry. In the resulting double layered blastula, each layer separately expresses its own polar characteristics along the mutual axis of symmetry. Even at Stage XIII the Ep still remains a totipotential system which can regenerate a normal Hyp, and its polarity is probably in the form of a labile gradient field of competence for PS formation. The Hyp, on the other hand, has sorted out from the single layered Stage X blastoderm and is more specialized, as demonstrated by several developmental, metabolic, and immunologic criteria. Its only developmental potential is the polar ability to induce a PS in a competent Ep. The Hyp was found to contain two different cell populations, of which only one, of marginal zone origin, has the PS-inducing capacity. Even after the PS has started to form, the induction is still uncompleted and has to go on until the PS is more than half its full length. In order to render possible normal PS formation, at least one of the two layers has to retain its polarity. In this respect the polarity of the competent Ep seems to be more crucial than that of the inductive Hyp. Polarity and inductivity of the Hyp can be dissociated from each other, and a disorganized Hyp can still induce a normal PS as long as the Ep retains its polarity.     

Ultrastructural assessment of a blastocyst recovered from a cow with subclinical purulent endometritis

A blastocyst was recovered from a cow with subclinical purulent endometritis from which Actinomyces pyogenes was isolated. Ultrastructurally the blastocyst appeared viable, although the zona pellucida and pervitelline cell debris were undergoing phagocytosis by neutrophils. An increase in lysosomal activity was observed in the inner cell mass, which may represent sublethal injury due to inflammatory alteration of the uterine milieu. It was concluded that the embryo was resistant to the bystander effect of the suppurative inflammation but that the altered uterine microenvironment may have caused sublethal blastocyst injury.