/. Embryol. exp. Morph. Vol. 52, pp. 79-88, 1979 79 Printed in Great Britain © Company of Biologists Limited 1979 Marginal zone cells-the primitive streak-inducing component of the primary hypoblast in the chick By YEHUDIT AZAR1 AND HEFZIBAH EYAL-GILADI1 From the Department of Zoology, The Hebrew University of Jerusalem SUMMARY (1) Removal of both the area opaca and the marginal zone of the area pellucida from a blastoderm stripped of its hypoblast (type-lV operation) prevents the regeneration of a normally functioning primary hypoblast. (2) Stage-XIIl E.G & K blastoderms (prior to the appearance of PS) after a type-lV operation do not form a primitive streak. (3) In older type-lV operated blastoderms in which the primitive streak has already begun to appear, the regeneration of a non-functional hypoblast did not support the normal matura- tion of the primitive streak, and in many cases the already existing rudimentary streak was absorbed. (4) Type-IV operated blastoderms from stage 3+ H & H and on developed normally. (5) It is concluded that the cellular contribution of the marginal zone to the primary hypoblast is instrumental in the latter's capacity to induce a PS. INTRODUCTION The chick blastoderm at about 10 h of incubation consists of two germ layers: an upper layer, the epiblast, and a lower layer, the primary hypoblast. Some- what later the primitive streak (PS) appears as the first axial tissue and cells from the epiblast invaginate through it, either into the forming mesoblast, or deeper down into the primary hypoblast, to form the definitive entoblast (secondary hypoblast). The new entodermal cells occupy at stage 30 H & H (Hamburger & Hamilton, 1951) a limited central position in the lower layer (Vakaet, 1962, 1970), but as they increase in number, they spread centrifugally and push the primary hypoblast to the periphery. The cells of the primary hypoblast later form only extra-embryonic structures such as the yolk-sac entoderm (Rosenquist, 1972; Wolk & Eyal-Giladi, 1977). Despite the fact that its cells do not participate in the formation of embryonic organs (Vakaet, 1962; Rosenquist, 1966; Modak, 1966; Nicolet, 1970), it was proposed (Eyal-Giladi & Wolk, 1970; Eyal-Giladi, 1970) that the young primary hypoblast has a major role in inducing the primitive streak and in stabilizing the embryonic axis. 1 Authors' address: Department of Zoology, The Hebrew University of Jerusalem, Jerusalem, Israel. 82 Y. AZAR AND H. EYAL-GILADI (a) Fig. 1. (a-c) Transverse sections of blastoderms subjected to type-lV operation at stages: 3~H & H (a); 2+H & H (b) and X1IL E.G & K (c) respectively. BI, Blood islands; Ch, notocord; PL, lateral plates; NT, neural tube; PS, primitive streak; Som., somites, (d) Control embryo, (e) Curved embryo after type-Ill operation at stage XIII E.G & K. (/) Blastoderm without axis; the thickness is caused by the massive development of mesenchyme and blood islands. Marginal zone cells in chick 83 Table 1. Scheme of operations and results of experiments with stage-XIIIE.G & K blastoderms No. of Stage Mil Typeof Fragments No. of h.Ci&K. Ar...i"itirm Steps of operation Fragments discarded blasto- blastoderms blastoderm opudtI°' cultured derms witli axial development 15 12 18 15 19 IV 37 Area opaca t( ^Marginal zone ffiffl Ilypoblast f.:[) Hpiblast ----- Incision Table 2. Results of type-IV operations carried out at four consecutive develop- mental stages Group (a) (6) (c) (d) Stage of operation XIII E.G &K 2-2+ H & H 3"-3 H & II 3+ H & 11 No. of blastoderms 37 19 19 8 Fragment cultured delineated by broken line No. of blastoderms 2 with embryo 7 i. 8 No. of blastoderms 2 with P.S. 1 3 - No. of blastoderms in which P.S. - 10 5 - disappeared 84 Y. AZAR AND H. EYAL-GILADI tion was made between blastoderms which developed an embryo and others in which only a primitive streak developed. Subgroup (a) consisted of 37 stage-XIII E.G & K blastoderms, only two of which developed an embryo and one a primitive streak. Thirty-five blastoderms developed into relatively compact fragments with mesenchyme and blood islands (Fig. lc,f). Subgroup (b) consisted of 19 stage-2 to 2+ H & H blastoderms, of which seven developed an embryo. Two blastoderms succeeded in completing the rudi- mentary PS present at the beginning of the experiment (Fig. \b), as compared to ten of the blastoderms in which the existing small PS disappeared at the end of incubation. Subgroup (c) included 19 stage-3~ to 3 H & H blastoderms in 11 of which embryonic development took place (Fig. la). Additional three completed the formation of the PS and in five the PS present at the time of operation dis- appeared at the end of the experiment. Subgroup (d) included eight stage-3+ H & H blastoderms with a 100% embryonic development. The results were statistically analysed for x2 distribution. The difference between group a and each of the other groups was highly significant (0-003 or less) while between b and c and c and d the significance was 0-05. DISCUSSION The aim of the present study was to follow the dynamics of the inductive influence of the primary hypoblast on the epiblast by removing the entire hypoblast from blastoderms of stages XIII E.G & K to 3 H & H, which covers the period starting just prior to the formation of the PS until about two-thirds PS. Waddington (1932, 1933) and Vakaet (1967) showed, by horizontally turning the hypoblast relative to the epiblast in blastoderms with PS of various lengths, that the orientation of the developing embryo was influenced by the polarity of the rotated hypoblast. Waddington suspected that an induction might be involved. However, he made no distinction between the primary and secondary hypoblasts. Eyal-Giladi & Wolk (1970), using pre-streak developmental stages, showed by making trans-millipore filter combinations of epiblasts and primary hypo- blasts that the primary hypoblast is indeed the inductor of the PS. Two processes are involved in the formation of a primary hypoblast as well as in the regeneration of a removed primary hypoblast. The process of poly- invagination, which proceeds simultaneously at many isolated spots, and an anteriorly directed growth of marginal zone cells from Roller's sickle (Vakaet, 1962, 1967, 1970; Spratt & Haas, 1965; Eyal-Giladi & Kochav, 1976). At stages older than stage XIII E.G & K the regeneration of a lower layer is quite different, the cells being derived mostly from the PS, from which they Marginal zone cells in chick 85 spread centrifugally (Vakaet, 1962, 1967, 1970; Modak, 1966; Nicolet, 1970, 1971; Rosenquist, 1972). At the same time the contribution to such a regenerat- ing hypoblast by the processes of polyinvagination and posterio-anterior growth from the marginal zone is gradually suppressed. Thus, the two different modes of the regeneration of the lower layer in stage XIII E.G & K and at the PS stages is obviously based on the normal development of the same stages (Modak, 1966; Vakaet, 1967) where the cells of the definitive endoblast or the secondary hypoblast which will form the intestinal tract of the chick invaginate from the streak, invade the primary hypoblast and gradually push its cells to the periphery of the lower layer. With the above information in mind we tried to remove the inductive lower layer at different developmental stages and also to interfere with its normal regeneration which would inevitably result, after a certain lag, in the develop- ment of a normal embryonic axis (Modak, 1966; Wolk, 1968; and results of type-I experimental group). Of the two processes involved in the formation of the inductive-primary hypoblast, the process of polyinvagination taking place at stages X-XIII E.G & K seemed difficult if not impossible to tackle. However, the centripetal growth from the marginal zone, predominantly expressed along the posterio-anterior axis, seemed a suitable candidate for blocking. Spratt & Haas (1960) prevented growth from the marginal zone mechanically, by putting large clumps of carmine powder on the front of the anteriorly moving cells. Eyal-Giladi & Wolk (1970) prevented the formation of normal primary hypoblasts capable of PS induction both in unincubated stage-X E.G & K blastoderms and in stage-XIII blastoderms stripped of their already existing primary hypoblast, by applying a millipore filter to their lower surface. However, from other experiments of the same series, it appeared that the filter might have interfered mechanically with the normal differentiation of the mesodermal tissues in experiments in which primitive streaks were induced. In the present study we therefore decided to discard the marginal contribution to the primary hypoblast by excising the entire marginal zone from the rest of the area pellucida, which also required the removal of the area opaca external to it. Bellairs, Bromham & Wylie (1967) studied the effect of the removal of the area opaca on the differentiation of blastoderms of stage 4 H & H and concluded that after 24 h of incubation these embryos were poorly differentiated and smaller as compared to their controls. We checked the development of blastoderms with their area opaca removed either at stage XIII E.G & K or at stages 2-3+ H & H grown for 48 h in culture, and found that the blastoderms were small, and the embryos short and curved, probably due to the lack of the normal tension exerted on the area pellucida by the centrifugal growth of the area opaca (Bellairs, Boyde & Heaysman, 1969). However, more than 70 % of axial developments in these blastoderms justified the application of the above experi- mental procedure. 86 Y. AZAR AND H. EYAL-GILADJ The experimental results were compared along two different parameters.