Hefzibah Eyal-Giladi

From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology.

Abstract A manual method has been developed to extract eggs from the genital tract without harming the hen. Fourteen developmental stages preceding Hamburger and Hamiltons stage 2 have been studied from live material and photographed from both upper and lower surfaces. Three developmental periods have been distinguished: cleavage, formation of area pellucida, and hypoblast formation. The last two are morphogenetic periods which follow the determination of the embryonic axis. During cleavage the diameter of the germ becomes reduced and the cytoplasmic mass becomes thicker. Later there is a progressive increase of the germs area. The formation of the area pellucida is a result of an oriented massive loss of the yolk laden cells of the lower layers. The formation of the primary hypoblast is a result of posterio-anteriorly oriented polyinvagination and a later coalescence of the cell aggregates in the same direction. It is not yet possible to conclude whether there is also an anteriorly directed cell movement. The inconsistent usage of the terms blastodisc and blastoderm is discussed. The authors suggest avoiding the term blastodisc and using the general term germ for all the early stages. The term blastoderm may be applied from stage VI onward.

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.

Abstract 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.

Bilateral Symmetry in Chick Embryo Determination by Gravity

The determination of bilateral symmetry of chick blastodiscs occurring during the second half of the uterine period is only indirectly caused by the intrauterine rotation of the egg. As a result of this rotation the blastodisc is forced into an oblique position and the force of gravity affects the symmetrization process. The same result may be artificially achieved, without rotation, by forcing the blastodisc into an oblique position.

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 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.

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 chicks 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 embryo-forming potency of the posterior marginal zone in stages X through XII of the chick☆

At stage X a small posterior marginal zone (PM) fragment, when transplanted into similar-size hole in the lateral marginal zone, can initiate the development of an ectopic axis. The laterally transplanted PM, inhibits the regeneration of an axis at the original posterior side from the lateral section of the marginal zone (LM) inserted to replace it. At stage XI both the axis-forming and inhibitory capacities of the PM fragment become weaker and an axis-forming capacity starts to build up anterior to the PM, resulting in the formation of two primitive streaks at 90 degrees to each other. At stage XII the change of potencies exhibited at stage XI is more pronounced, the ability of the transplanted PM to promote axis formation at the new site is lost, and an axis is formed from the original posterior side of the blastoderm.

The inducing capacities of the primary hypoblast as revealed by transfilter induction studies

Summary1.The inducing powers of the primary hypoblast of the chick embryo were studied by the insertion of a TH millipore filter between the hypoblast and the reacting epiblast.2.Two successive inducing capacities were discovered in the hypoblast. The first to appear and disappear is the inductor of the primitive streak, while the second is a prosencephalic inductor.3.The formation of a mature primitive streak which contains a Hensens node is dependent on direct cellular contact between the epiblast and hypoblast, and most probably on the inclusion of hypoblastic cells in the forming node and notochord.4.The formation of spinal cord-like structures and undifferentiated neural plates can be related to the presence of nodeless primitive streaks in the blastoderms. These neural structures are probably induced by the mesoderm produced by the defective primitive streaks, which were unable to form notochord or somites.5.The primary hypoblast, by virtue of its prosencephalic inducing power, can be compared with the presumptive pharyngeal endoderm of the amphibian embryo.Zusammenfassung1.Die Induktionsfähigkeit des primären Hypoblasts des Hühnchenembryos wurde untersucht durch die Insertion eines TH-Millipore-Filters zwischen Hypoblast und reagierendem Epiblast.2.Zwei aufeinanderfolgende Induktionsfähigkeiten wurden im Hypoblast entdeckt. Die zuerst erscheinende und auch zuerst wieder verschwindende Fähigkeit ist die Induktion des Primitivstreifens; die zweite ist der prosencephale Induktor.3.Die Bildung eines reifen Primitivstreifens mit Hensenschem Knoten ist abhängig vom direkten cellulären Kontakt zwischen Epiblast und Hypoblast, und höchstwahrscheinlich abhängig von der Miteinschließung hypoblastischer Zellen in den sich bildenden Hensenschen Knoten und die Chorda.4.Die Bildung von undifferenzierten Neuralplatten und Strukturen, die dem Rückenmark ähneln, sind korreliert mit der Gegenwart von Primitivstreifen ohne Hensensche Knoten. Diese Neuralstrukturen werden wahrscheinlich durch das Mesoderm induziert, das durch die defekten Primitivstreifen hervorgebracht wird; diese defekten Primitivstreifen sind nicht in der Lage, Chorda oder Somiten zu bilden.5.Auf Grund seines prosencephalen Induktionsvermögens kann der primäre Hypoblast mit dem präsumtiven pharyngealen Endoderm des Amphibien-Embryos verglichen werden.

Axis determination in uterine chick blastodiscs under changing spatial positions during the sensitive period for polarity

Abstract The temporal limits of axis determination as well as the correlation between axis determination and the appearance of the area pellucida were investigated in 10-hr aborted uterine eggs. Between 14 and 16 hr of uterine age the axis of the blastodisc can be changed by altering its spatial position. Axis determination is a gradual process correlated with the morphogenetic process of the formation of the area pellucida. Changes in polarity are accompanied by the formation of a new area pellucida or the shifting of the center of the first area pellucida to one side.

The chicken homeo box genes CHox1 and CHox3: cloning, sequencing and expression during embryogenesis

Several Drosophila genes involved in the control of segmentation and segment identity share a 183-bp conserved sequence termed homeo box. Homeo box sequences have been detected and cloned from the genomes of insects like Drosophila to vertebrates such as mouse and man. Two chicken homeo box genes CHox1 and CHox3, are described. Cloning of the CHox1 and CHox3 homeo boxes was performed using Drosophila and murine homeo box sequences as probes under low-stringency conditions. Analysis of both chicken homeo box sequences revealed them to be homeo boxes that have diverged from the Antennapedia class with homologies to homeo boxes of other organisms in the range of 75-42% at the nucleotide level and 69-41% at the protein level. Analysis of CHox3 expression during early embryo development showed that the gene codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9 kb in size. Three of the transcripts (1.3, 1.9 and 5.6 kb) are also recognized by a flanking non-homeo box containing probe. The levels of the different transcripts changed during the first five days of development. The most abundant transcripts (1.3 and 1.9 kb) are already present at the time the egg is laid. Their transcription peaks at day 1 of incubation and then decreases. The CHox1 transcripts are present at very low levels between days 2.5 and 4 of development. These two chicken genes represent bona fide Hox genes in a branch of vertebrates that evolved parallel to mammals.