The chick's marginal zone and primitive streak formation. II. Quantification of the marginal zone's potencies--temporal and spatial aspects

When a posterior fragment of the chick's marginal zone (PM) was exchanged with equal sized lateral marginal zone fragment (LM), of the same blastoderm, its capacity to initiate an ectopic primitive streak (PS) was found to be both size and stage dependent. Good correlation was demonstrated between the areas of PM fragments and the number of cells they contained. In stage X blastoderms, PM fragments containing less than 1200 cells were incapable of initiating an ectopic PS. Transplanted PMs containing between 1200 and 1500 cells initiated a lateral ectopic PS in 50% of the cases, while in the other 50% a posterior PS developed from the original posterior side. PMs containing 1500 cells or more in all cases initiating an ectopic PS and inhibited the formation of a posterior PS. At stage XI, laterally transplanted PMs containing less than 1800 cells were not effective. Stage XI PMs containing 1800-2300 cells in some cases succeeded in initiating a lateral ectopic PS, in addition to the posterior one. Stage XI PMs containing 2300 cells or more invariably promoted the development of an ectopic PS, but were unable to suppress the formation of a posterior PS, so that two PSs developed in the same blastoderm, one posterior and one ectopic. When a stage XI PM fragment was laterally transplanted into a younger, stage X blastoderm, the minimal effective cell number needed to initiate an ectopic PS increased to at least 3000 cells, again without inhibiting the formation of a posterior PS. The inductive potential of a stage X PM is therefore at least twice that of a stage XI PM. The marginal zone belt of stage X blastoderms was checked for the decrease in its developmental potential from the posterior to the lateral side by evaluating its effect on the developmental expression of two competing stage X PMs, one located posteriorly and the other inserted laterally. The developmental expression of the laterally inserted PM was consistently inferior to that of the posterior PM. The developmental expression of each PM was not related to absolute size, but depended on the size ratio of lateral PM/posterior PM. When the ratio was 1.2 or less, only posterior PSs developed. When the ratio was 1.3-1.4, three different results were encountered: (1) only a posterior PS, (2) posterior plus lateral, and (3) only lateral PS. When the ratio was 1.5 or more, only a lateral PS developed, which suppressed the posterior PS.     

The dynamics of antigenic changes in the epiblast and hypoblast of the chick during the processes of hypoblast, primitive streak and head process formation, as revealed by immunofluorescence.

Antisera were prepared to epiblast, primary hypoblast, yolk, yolk entoderm, and extraembryonic yolk sac ectoderm and were submitted to various absorption procedures. The absorbed antisera were used in the indirect immunofluorescent method to stain microscopic sections of developing chick blastoderms at different developmental stages. The antigens revealed by the staining at the periods studied were divided into groups of persistent, nonspecific, and specific antigens. The epiblast does not appear to form or include specific antigens until stage XIII (full hypoblast). The primary hypoblast is the layer which during its formation acquires specificity by the inclusion of antigenic components through a cytoplasmic segregation and probably by one or two waves of appearance of primary hypoblast specific antigens. The inductive role of the hypoblast is discussed in relation to the above antigenic manifestations. The anti-hypoblast and anti-epiblast sera after absorption with yolk were found to be suitable reagents for the detection of morphogenetic movements.     

5-Bromodeoxyuridine inhibition of the epiblast competence for primitive streak formation in the young chick blastoderm

The competence of stage XIII chick epiblast which under the influence of an inductive hypoblast is directed to form a normal primitive streak, is affected by 5-bromodeoxyuridine (BUdR). The BUdR-treated epiblast forms an atypical primitive streak and no axial mesoderm. However, a nonorganized mesenchymal layer is formed between the epiblast and the hypoblast, and atypical neural tissue in the epiblast. BUdR interferes neither with hypoblast formation nor with its inductivity even when blastoderms are treated with BUdR as early as uterine stage VIII and later.     

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.     

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.     

Differentiation of dissociated-reconstituted epiblasts of the chick under the influence of a normal hypoblast

Abstract. A cell suspension of chick epiblast cells cultured under defined conditions to form a flat disk, can differentiate and generate axial embryonic structures when covered with a primary hypoblast. Macroscopically identifiable axes developed in 26 out of 33 cases. In all cases axes developed in a direction consistent with the posteroanterior polarity of the normal hypoblast. Almost invariably the epiblast cells differentiated into ectoderm, neural plates or tubes, and endoderm. In some cases typical primitive streaks were found, sometimes accompanied by signs of axial mesoderm, whereas in other cases the primitive streaks seemed to regress. In the absence of a hypoblast no differentiation of neural tissue or any signs of axial development were observed.     

The chick's marginal zone and primitive streak formation. I. Coordinative effect of induction and inhibition

A variety of transplantation experiments of posterior and lateral marginal zone fragments at stages X, XI, and XII have been carried out in order to test their relevance to the development of a primitive streak (PS). At the stages studied the marginal zone (MZ) was shown to behave as a ring-like gradient field, the maximal value of which was at the posterior end (PM). The PM was found to be capable at the same time of promoting the development of a PS and of suppressing the inductive potential of other regions of the MZ. By systematically evaluating inductive and suppressive capacities of PMs, at different developmental stages, it was found that both features are maximal at stage X. During stages XI and XII, both properties gradually decrease in the MZ and build up in the forming hypoblast.     

The sorting-out of thymidine-labelled chick hypoblast cells in mixed epiblast--hypoblast aggregates.

Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa. The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.