A novel role for the extraembryonic area opaca in positioning the primitive streak of the early chick embryo

Classical studies have established that the marginal zone, a ring of extraembryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extraembryonic region (area opaca) was only thought to have nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region: first, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extraembryonic tissues in early development.

An Introduction to Morphology of the Reproductive System and Anatomy of Hen’s Egg

The present study was designed to investigate the morphology of reproductive system and egg anatomy of the domestic hen (Gallus domesticus L.). The system consists of oviduct and ovary. The oviduct consists of infundibulum, magnum, isthmus, uterus and vagina which are sole distributor for making nutrition enriched egg. The anatomy of egg revealed that there are calcareous eggshell, shell membranes, egg white, vitelline membrane, egg yolk, and germinal disc. The fertilized egg showed a concentric circle around the nucleus known as blastoderm that contained area pellucida and area opaca whereas an unfertilized egg showed nucleus as white spot (blastodisc). Primordial germ cells (PGCs) are progenitor cells of ova and spermatozoa and they originate from the epiblast of the central part of the area pellucida. The microscopic structure of eggshell rendered leathery cuticle, fibrous matrix and shell membranes. The egg protects itself by its own mechanism from being injured and provides a complete diet for the developing embryo. DOI: http://dx.doi.org/10.3329/jles.v8i0.20133 J. Life Earth Sci., Vol. 8: 1-10, 2013

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.

Chordin regulates primitive streak development and the stability of induced neural cells, but is not sufficient for neural induction in the chick embryo

We have investigated the role of Bone Morphogenetic Protein 4 (BMP-4) and a BMP antagonist, chordin, in primitive streak formation and neural induction in amniote embryos. We show that both BMP-4 and chordin are expressed before primitive streak formation, and that BMP-4 expression is downregulated as the streak starts to form. When BMP-4 is misexpressed in the posterior area pellucida, primitive streak formation is inhibited. Misexpression of BMP-4 also arrests further development of Hensen's node and axial structures. In contrast, misexpression of chordin in the anterior area pellucida generates an ectopic primitive streak that expresses mesoderm and organizer markers. We also provide evidence that chordin is not sufficient to induce neural tissue in the chick. Misexpression of chordin in regions outside the future neural plate does not induce the early neural markers L5, Sox-3 or Sox-2. Furthermore, neither BMP-4 nor BMP-7 interfere with neural induction when misexpressed in the presumptive neural plate before or after primitive streak formation. However, chordin can stabilise the expression of early neural markers in cells that have already received neural inducing signals. These results suggest that the regulation of BMP signalling by chordin plays a role in primitive streak formation and that chordin is not sufficient to induce neural tissue.

Misexpression of chick Vg1 in the marginal zone induces primitive streak formation

In the chick embryo, the primitive streak is the first axial structure to develop. The initiation of primitive streak formation in the posterior area pellucida is influenced by the adjacent posterior marginal zone (PMZ). We show here that chick Vg1 (cVg1), a member of the TGFbeta family of signalling molecules whose homolog in Xenopus is implicated in mesoderm induction, is expressed in the PMZ of prestreak embryos. Ectopic expression of cVg1 protein in the marginal zone chick blastoderms directs the formation of a secondary primitive streak, which subsequently develops into an ectopic embryo. We have used cell marking techniques to show that cells that contribute to the ectopic primitive streak change fate, acquiring two distinct properties of primitive streak cells, defined by gene expression and cell movements. Furthermore, naive epiblast explants exposed to cVg1 protein in vitro acquire axial mesodermal properties. Together, these results show that cVg1 can mediate ectopic axis formation in the chick by inducing new cell fates and they permit the analysis of distinct events that occur during primitive streak formation.

The posterior section of the chick's area pellucida and its involvement in hypoblast and primitive streak formation

Posterior marginal zone sections with or without Koller's sickle were cut out of stage X, XI and XII E.G&K blastoderms, labelled with the fluorescent dye rhodamine-dextran-lysine (RDL) and returned to their original location. In control experiments, a similar lateral section of the marginal zone was identically treated. Different blastoderms were incubated at 37°C for different periods and were fixed after reaching stages from XII E.G&K to 4 H&H. The conclusions drawn from the analysis of the distribution pattern of the labelled cells in the serially sectioned blastoderms concern the cellular contributions to both the forming hypoblast and the forming primitive streak. Koller's sickle and the marginal zone behind it were found to contribute all the centrally located cells of the growing hypoblast. The lengthening pregastrulation PS (until stage 3+ H&H) was found to be entirely composed of epiblastic cells that at stage X were located in a narrow strip anterior to Koller's sickle. A model is proposed to integrate the results spatially and temporally.