Establishment of the axis in chordates: facts and speculations.

A master plan for the early development of all chordates is proposed. The radial symmetry of the chordate ovum is changed at or after fertilization into a bilateral symmetry by an external signal. Until now two alternative triggers, sperm entry and gravity, have been demonstrated.

The spatial and temporal dynamics of Sax1 (CHox3) homeobox gene expression in the chick's spinal cord.

Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila NK1 and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain.

A direct approach to the study of the effect of gravity on axis formation in birds.

A system has been developed to enable the normal development of aborted very early uterine avian embryos, outside the female's uterus. The shell-less aborted egg was put into a foster shell of a sister egg, previously laid by the same female. The empty space between the shell and aborted egg was filled with artificial uterine fluid.

Changes in uterine fluid composition and acid-base status during shell formation in the chicken.

The aim of this study was to characterize the dynamic changes in uterine fluid composition and acid-base status during shell calcification in the chicken. Uterine eggs at timed intervals were manually aborted and the accompanying fluid collected and analyzed for composition of osmolytes, enzymes, and acid-base parameters. Blood samples were analyzed for comparison.

Immunohistochemical analysis of the segregation process of the quail germ cell lineage.

An antiserum against quail 7 day gonadal germ cells was found to react specifically with gonadal germ cells of both sexes. Transverse sections from a range of early quail developmental stages were submitted to the antibody PAP reaction. Blastodiscs from the earliest uterine stages (II to X E.

Primordial germ cell development in cultures of dispersed central disks of stage X chick blastoderms.

Central disk fragments cut from stage X chick blastoderms were dispersed and cultured on glass coverslips. After 48 hr of incubation the cultures showed various degrees of organization into three-layered aggregates in which no axis development was observed. Primordial germ cells (PGCs) were detected in all cultures.

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.

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.

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.

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).

Patterns of oxygen consumption during establishment of cephalocaudal polarity in the early chick embryo.

Oxygen uptake was studied during the establishment of cephalocaudal polarity in the very early chick embryo, i.e., 10 hr before (stage VI) and at laying (stage X).

Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo.

The migration of the PGCs from a stage XIII epiblast into the germinal crescent of a stage 10 chick blastoderm was experimentally investigated. Considerable numbers of PGCs start to come down from the epiblastic layer at stage XII-XIII and continue to do so in relatively smaller numbers until stage 6. The earliest PGCs land on the primary hypoblast and probably are carried by it into the GC.

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.

Developmental potencies of area opaca and marginal zone areas of early chick blastoderms.

The marginal zone, in pregastrulating chick blastoderms, has been defined as the intermediate ring between the epiblast proper and the most external region, the area opaca (Spratt & Haas, 1960). Azar & Eyal-Giladi (1979) have shown that the marginal zone of a stage XIII blastoderm has the capacity of regenerating an inductive layer which when in contact with a competent stage XIII epiblast can cause the formation of axial structures. The present work demonstrates that at stage X (Eyal-Giladi & Kochav, 1976) the marginal zone can both induce and respond to its own inductive stimulus by forming an embryonic axis.

Glycogen metabolism and the nuclear envelope-annulate lamella system in the early chick embryo.

The intracellular sites of glycogen degradation in the mid to late uterine chick embryo were determined by cytochemical localization of glucose-6-phosphatase at the ultrastructural level. Enzyme activity was found between the two membranes of the nuclear envelope, in the annulate lamellae and in specialized glycogen-containing membrane scrolls. Annulate lamellae and glycogen scrolls were most frequent during the stages of intensive glycogen degradation.

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.

The retention of primary hypoblastic cells underneath the developing primitive streak allows for their prolonged inductive influence.

An experimental study was made of the distribution of the primary hypoblastic cells in the lower layer of the avian blastoderm throughout primitive streak formation and until stage 10 (Hamburger & Hamilton, 1951). The primary hypoblast of stage XIII (Eyal-Giladi & Kochav, 1976) chick blastoderms was exchanged for either an [H3]thymidine-labelled similar chick hypoblast, or a quail primary hypoblast. During the entire period of primitive streak formation, the lower layer proved to be a mosaic of labelled hypoblastic and non-labelled entodermal cells (chick cells of epiblastic origin).

Nature of the hypoblastic influence on the chick embryo epiblast.

Stage XIII chick blastoderms deprived of the marginal zone, the area opaca and the posterior half of the hypoblast, when incubated further developed axes whose orientation in 50% of the cases was according to the original blastoderm's orientation, whilst in 50% of the cases they developed at 90 degrees from the posterior side. Those results illustrate the quantitative differences in inductivity between the anterior and the posterior hypoblastic halves. Normally the posterior region has the highest effect but other regions can also bring about the development of an embryonic axis if allowed to act upon the epiblast for a sufficiently long period of time.

The antigens of the chick blastula: a comparison between the epiblast, primary hypoblast, yolk entoderm, and extraembryonic yolk by immunoprecipitation in agar.

Antisera to chicken embryo epiblast, primary hypoblast, extraembryonic yolk, and yolk entoderm were elicited in rabbits. Extracts from the different sources were tested for their immunoprecipitation by these antisera in immunoelectrophoresis and double diffusion in agar (Ouchterlony) tests. The main findings were as follows: 1) Antigens recovered in the soluble fractions of the cells of the full hypoblast stage (Eyal-Giladi and Kochav, Stage XIII) are immunologically identical to antigens from the extraembryonic yolk, 2) Similar patterns of precipitation lines were formed between the soluble fractions of both the epiblast and primary hypoblast, and the different antisera.

Avian primordial germ cells are of epiblastic origin.

We have demonstrated that quail PGCs possess a characteristic heterochromatin nuclear marker demonstrable already at stage 6 H & H with Feulgen staining. Chimaeras of stage-XIII E. G & K chick epiblast and quail hypoblast and vice versa have been made.

A SEM study of cell shedding during the formation of the area pellucida in the chick embryo.

Intrauterine eggs at different stages of development were retrieved from hens and the blastodiscs were dissected out and prepared for scanning electron microscopy. Our observations trace the polarized shedding of cells from the central area of the lower layers of the blastodisc, which leads to the formation of the area pellucida. Prior to the shedding, single central blebs appear on the ventral side of the cells facing the sub-blastodermic cavity.

Hypoblastic cells can form a disk inducing an embryonic axis in chick epiblast.

The primitive streak of the chick embryo develops from one of the two layers of cells of the stage XIII blastoderm, the epiblast. The other layer of cells, the hypoblast, seems to be necessary for the induction of the primitive streak and also determines its orientation--rotation of the hypoblast by 90 degrees is followed by a similar rotation of the embryonic axis. After stage XIII, the hypoblast is replaced by the invaginating endoderm and plays no further part in the development of the embryo.

Interaction of epiblast and hypoblast in the formation of the primitive streak and the embryonic axis in chick, as revealed by hypoblast-rotation experiments.

Three types of experiments were performed to determine the interaction between the epiblast and hypoblast for primitive streak formation: (1) Hypoblasts of blastoderms from stages XIII E.G & K to 3 H & H were separated from the epiblasts and rotated by 90 degrees counterclockwise; (2) hypoblasts from stages XIII E.G & K to 3 H & H blastoderms were rotated by 180 degrees; (3) hypoblasts were exchanged between blastoderms of different developmental stages and placed at 90 degrees counterclockwise to the axis of the recipient epiblast.