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In Vivo and in Vitro Studies on the Hypoblast and Definitive Endoblast of Avian Embryos

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In Vivo and in Vitro Studies on the Hypoblast and Definitive Endoblast of Avian Embryos /. Embryol. exp. Morph. Vol. 46, pp. 187-205, 1978 Printed in Great Britain © Company of Biologists Limited 1978 In vivo and in vitro studies on the hypoblast and definitive endoblast of avian embryos ByE. J. SANDERS,1 RUTH BELLAIRS2 AND P. A. PORTCH2 From the Department of Anatomy and Embryology, University College, London SUMMARY An unusual example of the invasion of one tissue by another occurs during gastrulation in the chick embryo when the definitive endoblast becomes inserted into the hypoblast. The two tissues were examined morphologically by SEM and TEM. They resemble each other in being of an epithelial type, though neither possesses a basal lamina. The definitive endoblast cells are flatter than the hypoblast cells and more closely attached to one another. When they were explanted in hanging drop cultures, the two tissues were found to exhibit differences in their behaviour. In comparison with the definitive endoblast, the hypoblast cells attached more readily to the glass, produced larger ruffle membranes, moved more rapidly, showed poorer contact-inhibition of locomotion and showed a greater tendency to break away from the main explant. When a hypoblast explant was confronted with a definitive endoblast explant, the hypo- blast cells became displaced by the definitive endoblast. The hypoblast explant tended to fragment into smaller groups of cells, many of which migrated around the definitive endo- blast, thus mimicking the situation in vivo. Control experiments comprised confronting hypoblast with hypoblast, hypoblast with somites, definitive endoblast with definitive endoblast, and definitive endoblast with somites. The hypoblast explants behaved in a consistent manner, always fragmenting when coming into contact with cells from a con- fronting explant. The definitive endoblast explants showed more contact inhibition of locomotion when confronted with definitive endoblast or with somites than when con- fronted with hypoblast. It is suggested therefore that the ability of the hypoblast cells to separate from one another may play an important role in the penetration of the hypoblast by the definitive endoblast both in vitro and in vivo. INTRODUCTION Until recently, it was believed that the endoderm of birds arose in a totally different way from that of amphibians. In amphibians it is invaginated through the blastopore at the same time as the mesoderm. In birds it was considered to arise as a distinct lower layer prior to the imagination of the mesoderm. There was some controversy as to the precise source of this lower layer (see discussion 1 Author's address: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada. 2 Authors' address: Department of Anatomy and Embryology, University College London, Gower Street, London WC1E 6BT, U.K. 188 E. J. SANDERS, R. BELLAIRS AND P. A. PORTCH XI XII 2 Hypoblast Definitive endoblast Junctional endoblast Fig. 1. Diagram to illustrate the relations of the hypoblast and the definitive endo- blast. Initially, the area pellucida is underlain by hypoblast alone, though subse- quently this tissue becomes replaced by definitive endoblast medially and junctional endoblast posteriorly. The definitive endoblast cells invaginate through the primi- tive streak whilst the junctional endoblast cells migrate from the posterior germ wall. XI, XII = Eyal-Giladi & Kochar stages. 2-5 = Hamburger-Hamilton stages. (Composite diagram, partially after Vakaet, 1970; Rosenquist, 1971; and Fontaine & Le Douarin, 1977.) by Bellairs, 1971) but it was generally accepted that it appeared first of all at the posterior end of the area pellucida, and it was shown by marking experi- ments that the cells then migrated anteriorly until they formed a continuous sheet, the lower layer of the area pellucida (Spratt & Haas, 1960; Vakaet, 1970). It is now known that this lower layer, the hypoblast (the 'sickle endoblast' of Vakaet, 1970), does not contribute to the embryonic endoderm, but that instead it spreads out to the periphery of the area pellucida and subsequently forms extra-embryonic endoderm (Vakaet, 1962, 1970; Rosenquist, 1971, 1972; Fontaine & Le Douarin, 1977). The hypoblast also contains among its cells the primordial germ cells (the 'endophyll' cells of Vakaet, 1970) which subsequently collect in the anterior germ wall (area opaca) prior to their trans- port to the gonad at a later stage of development (Dubois, 1969; Vakaet & Hertoghs-De Maere, 1973). The embryonic endoderm which we call the definitive endoblast (following Vakaet, 1970) is located around the anterior end of the primitive streak at a later stage (Bellairs, 1953a, b) and like the mesoderm is derived from cells of the epiblast or upper layer, which invaginate through the primitive streak (Modak, 1965, 1966; Nicolet, 1965, 1967, 1970; Hypoblast and definitive endoblast of avian embryos 189 Rosenquist, 1966, 1972; Vakaet, 1970; Fontaine & Le Douarin, 1977). The definitive endoblast inserts itself into the hypoblast and spreads out so that it is like a halo in the central part of the lower layer, whilst the hypoblast moves distally (Fig. 1). This means that the lower layer in the anterior part of the area pellucida consists of two types of cell, and these not only have a different origin but they also have a different fate. This paper is concerned with the relationship between these two types of cell, the hypoblast and the definitive endoblast, which we have examined by transmission and scanning electron microscopy, as well as by time-lapse cinematography. The endoderm at the posterior end of the area pellucida is called the junctional endoblast and is thought to be derived by ingrowth from the germ wall (Vakaet, 1970; Modak, 1966) but we shall not be considering this region in this paper. MATERIALS AND METHODS In order to obtain pure hypoblast, the dissection was carried out before the stage at which endoblast inserts. To obtain pure definitive endoblast, it was necessary to dissect the tissue after it had already inserted. The hypoblast was obtained by dissecting the lower layer from chick or quail embryos which had been incubated for about 4-10 h and were therefore at stages X-XIV of Eyal-Giladi & Kochav (1976). The endoblast was obtained by dissecting the lower layer from beneath the region around the anterior part of the primitive streak of embryos which had been incubated for about 24 h and were at stages 3 + to 5 of Hamburger & Hamilton (1951). Roman numerals are used to indicate Eyal-Giladi & Kochav stages, whilst arabic ones are used for Hamburger & Hamilton ones. Somites for use in control experiments were dissected from embryos of stage 12 (Hamburger & Hamilton, 1951) which were first treated with 0-1 % trypsin in Ca2+ and Mg2+-free saline. Twenty-five specimens were prepared for transmission electron microscopy (TEM) and 16 for scanning electron microscopy (SEM). Specimens for TEM were fixed in 2-5% glutaraldehyde in 0-1 M sodium cacodylate for 1-4 h, and then washed three times in 0-1 M sodium cacodylate which contained 0-333 g CaCl2, for a total of \\ h. They were treated with 1 % osmium tetroxide in phosphate buffer for 1 h at 4 °C, then rinsed in phosphate buffer. After dehy- dration in graded ethanols followed by two changes of propylene oxide, they were embedded in araldite. Sections were stained with 2% uranyl acetate at 38 °C for 20 min, then counterstained in lead citrate. Thick sections for light microscopy were stained in toluidine blue. Specimens for SEM were fixed for periods of 4-24 h in 3 % glutaraldehyde, made up in 0-15 M cacodylate buffer, or in half strength Karnovsky's fixa- tive (Karnovsky, 1965). The pH of the fixative was 7-2. After washing in the cacodylate buffer, the specimens were immersed in 1 % osmium tetroxide for 30 min, and then washed again in cacodylate buffer. They were dehydrated in 13 EMB 46 190 E. J. SANDERS, R. BELLAIRS AND P. A. PORTCH graded ethanols and dried in a Polaron critical point drying apparatus from liquid CO2. They were mounted on stubs with Uhu glue (Fishmar Ltd., Waterford, Eire) and coated with gold. A total of 278 hanging drop cultures was prepared, and these were of three types. The first consisted of solitary explants of hypoblast or of definitive endo- blast or of somites. The second was composed of confronted cultures in which two homologous tissues were grown in close proximity; these were hypoblast with hypoblast, definitive endoblast with definitive endoblast, or somites with somites. The third type consisted of confronted cultures in which two hetero- logous tissues were grown in close proximity; the combinations were hypo- blast with definitive endoblast, hypoblast with somites, or definitive endoblast with somites. In the homologous confronted cultures it was difficult to dis- tinguish between the two explants but the problem was overcome by con- fronting chick with quail tissues, this technique (Le Douarin, 1969) enabling us to identify the two types of cell correctly on the basis of their nucleolar morphology. In the heterologous confronted cultures the hypoblast, definitive endoblast and somite cells could be distinguished from one another with confidence because of their morphological differences. The culture medium was 9 ml Earle's 199, 1 ml foetal calf serum, 0-5 ml penicillin and streptomycin (5000/*g/ml). Cultures were maintained at 37 °C for periods ranging from 1^ to 3 days. Fixation was generally in formal saline for 24 h. The explants were stained with Harris' haematoxylin, unless the tissues were quail-chick combinations, in which case, Feulgen's stain was used. Time-lapse filming studies were made using a Bolex camera with Wild Variotimer controls. Most of the films were taken at an interval of 12 sec using a x 16 phase contrast objective, giving a film magnification of x 38. Other films were taken at an interval of 2 min, using a x 2-5 objective with dark ground illumination, and a film magnification of x 8.
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