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. The chimaeras were incubated up to stage 7−8 H & H and checked histologically with Feulgen staining for the presence of the heterochromatin marker in every detectable PGC. It was found that the PGCs are of epiblastic origin, contrary to previous data and speculations concerning avian PGCs.

The origin of primordial germ cells (PGCs) in animal embryos has been widely studied not only in its strict sense but also as a model of embryonic development. The predominant idea has been that already in the oocyte, even before fertilization, a cytoplasmic area exists whose fate will be to form the future germinal cells, and that on removal of this area sterile animals will develop. There is overwhelming evidence for this theory in invertebrates and in anuran amphibians. In spite of this there is evidence suggesting that PGCs of urodele amphibians may arise as a result of an inductive process (Kocher-Becker & Tiedeman, 1971; Boterenbrood & Nieuwkoop, 1973; Sutasurja & Nieuwkoop, 1974).

In birds the origin of PGCs is as yet an unsolved problem, the reason being that they can be identified with certainty using specific cytologic criteria only at about stage 4 H & H (Hamburger & Hamilton, 1951) which is relatively late in their development, after having populated the germinal crescent (Swift, 1914). The evidence of their location in earlier stages is indirect and based either on cutting and ablation experiments or on blocking of the movement and growth of embryonic layers (Dubois, 1969). It has been shown by several investigators that PGCs have been found in embryos developed from any fragment of a blastoderm, that is, posterior, anterior or lateral (Fargeix, 1967; 1969; Rogulska, 1968; Eyal-Giladi, Kochav & Menashi, 1976). To account for this, one of three conditions must prevail: (1) there is no specific germ plasm and the PGCs arise as a result of an inductive process; (2) there is a special germ plasm evenly distributed in the blastoderm; (3) there is a special germ plasm in a circular arrangement.

In order to accurately approach the problem of the PGCs’ early localization, a marker or a labelling method had to be found to permit tracing the fate of defined blastodermic areas, from early stages until the stage at which the PGCs may be recognized, without interrupting normal morphogenesis. We decided to recheck the possibility of using chick-quail chimaeras for this study despite data indicating that there is no recognizable heterochromatin marker in quail PGCs until about 6 days (stage 28 H & H) of incubation (Tachinante 1974). We now report that a heterochromatin marker can be recognized in quail PGCs of stage 6H & H. In head process embryos most quail PGCs already contain the marker and only in a few can it not be identified with certainty. Although the quail PGC marker is morphologically different, it is as reliable as the heterochromatin marker of quail somatic nuclei (Reynaud, 1969; Le Douarin & Barq, 1969; Le Douarin, 1973a, b). This finding reopened the possibility of using heteroplastic chick-quail transplantations at the earliest possible developmental stages and thereafter to grow the chimaeric blastoderms in vitro for the minimum time necessary for the PGCs to be cytologically identified as such. We used the marker in order to answer the first and basic question: do the avian PGCs originate from the epiblast or from the hypoblast of a stage XIII E.G & K (Eyal-Giladi & Kochav, 1976) blastoderm?

Detection of the heterochromatin marker

Stage-8 to -9 H & H embryos (4−8 somites) of both chick and quail were fixed in toto for 2 h in a mixture of formalin, ethanol and glacial acetic acid in the proportions of 2:1: 0-3. The embryos were then washed three times for 30 min in 70% ethanol and successively twice for 15 min in distilled water. Further staining was done according to Pearse (1968). Treatment time was prolonged because of the handling in toto. Attention was paid to heating the 1 N-HCI to 60 °C prior to the onset of the hydrolysis which then took about 15 min. Schiff’s reagent was prepared according to Barger and de Lamater (Pearse, 1968) and applied for h. After three 1 min washes with a freshly prepared bisulphite solution, a 5 − 10 min rinse in tap water and a brief rinse in distilled water, the material was transferred through 70% ethanol (5 min), twice through 95 % ethanol (5 min each) and into amylacetate. The material was stored in the latter or transferred after three 15 min changes of amylacetate and three 10 min changes of benzene into a 40 °C 1:1 mixture of benzeneparaffin for 30 min. It was then transferred into paraffin, blocks were prepared and serially sectioned at 8 μm.

For a light background staining that would not blur the Feulgen reaction, each of the four following stains proved adequate: 0·5% light green or 0·5% fast green for 10 sec; 0·1 % orange G for 1 min or picro-indigo carmine for 10 sec.

Chick-quail chimerae

Stage-XIII E.G & K (Eyal-Giladi & Kochav, 1976; Kochav, Ginsburg & Eyal-Giladi, 1980) chick and quail blastoderms were used.

(1) The hypoblast was removed from a blastoderm of one species and replaced by a similar hypoblast of the other species. The chimaeras were further incubated on a vitelline membrane, placed on solid albumin (New, 1966) until an embryo with 3−6 pairs of somites was observed. In some instances a blastoderm unlikely to survive was fixed at an earlier stage.

(2) Chimaeras were formed in the same way but in addition the area opaca and marginal zone were removed from the recipient epiblast, prior to further incubation in order to avoid regeneration of an inductive hypoblast from the epiblastic margin (Azar & Eyal-Giladi, 1979). At the end of the incubation period the chimaeras were fixed and further treated according to the procedure described above, but the serial sections were only 6 μm thick.

The heterochromatin marker of quail PGCs

By using our modification of the Feulgen-Rossenbeck procedure we have confirmed the observations of Reynaud (1969) and Le Dourin & Barq (1969) that in all somatic nuclei of the early quail blastoderms there is a characteristic heterochromatin marker. No similar marker has been observed in chick somatic nuclei. We nevertheless found that in quail blastoderms of stage 8 H & H, namely containing embryos with four somites, and even in slightly younger ones, the PGCs’ nuclei contain two or three typical heterochromatin granules (Figs. 6, 7). Such granules are absent from chick PGCs of the same stages (Fig. 1).

Fig. 1.

Stage-8 H & H chick embryo. No heterochromatin marker is visible after Feulgen staining either in somatic cells (cs) or in PGCs (eg). × 600. eg, chick PGC; eph, extraembryonic entoderm derived from chick primary hypoblast, cs, chick somatic cells, et, definitive embryonic entoderm, mr, mitotic figure, np, neural plate, nt, neural tube, qg, quail PGC. qph, extraembryonic entoderm derived from quail primary hypoblast, qs, quail somatic cells.

Fig. 1.

Stage-8 H & H chick embryo. No heterochromatin marker is visible after Feulgen staining either in somatic cells (cs) or in PGCs (eg). × 600. eg, chick PGC; eph, extraembryonic entoderm derived from chick primary hypoblast, cs, chick somatic cells, et, definitive embryonic entoderm, mr, mitotic figure, np, neural plate, nt, neural tube, qg, quail PGC. qph, extraembryonic entoderm derived from quail primary hypoblast, qs, quail somatic cells.

Fig. 2.

Feulgen-stained stage-6 H & H chimera chick epiblast, quail hypoblast. The embryo is formed of cells of chick origin. All the cells of hypoblastic origin with the heterochromatin marker (qph) have moved to the sides of the area pellucida and form a confluent sheet. The PGC in its vicinity is of chick origin (cg). All the dark spots in the chick cells are mitotic figures (mr). Fig. 2 × 200. Fig. 3 (left corner of Fig. 2) × 600.

Fig. 2.

Feulgen-stained stage-6 H & H chimera chick epiblast, quail hypoblast. The embryo is formed of cells of chick origin. All the cells of hypoblastic origin with the heterochromatin marker (qph) have moved to the sides of the area pellucida and form a confluent sheet. The PGC in its vicinity is of chick origin (cg). All the dark spots in the chick cells are mitotic figures (mr). Fig. 2 × 200. Fig. 3 (left corner of Fig. 2) × 600.

Fig. 3.

Feulgen-stained stage-6 H & H chimera chick epiblast, quail hypoblast. The embryo is formed of cells of chick origin. All the cells of hypoblastic origin with the heterochromatin marker (qph) have moved to the sides of the area pellucida and form a confluent sheet. The PGC in its vicinity is of chick origin (cg). All the dark spots in the chick cells are mitotic figures (mr). Fig. 2 × 200. Fig. 3 (left corner of Fig. 2) × 600.

Fig. 3.

Feulgen-stained stage-6 H & H chimera chick epiblast, quail hypoblast. The embryo is formed of cells of chick origin. All the cells of hypoblastic origin with the heterochromatin marker (qph) have moved to the sides of the area pellucida and form a confluent sheet. The PGC in its vicinity is of chick origin (cg). All the dark spots in the chick cells are mitotic figures (mr). Fig. 2 × 200. Fig. 3 (left corner of Fig. 2) × 600.

Fig. 4.

Feulgen staining of stage-8 H & H chimaera. Quail epiblast, chick hypoblast. All the cells from the chick hypoblast (cph) are confined in an extraembryonic area. The rest of the cells, embryonic as well as extraembryonic, show the heterochromatin marker. The PGC in the vicinity of the concentration of chick cells is of quail origin (qg). Fig. 4 × 200. Fig. 5 (left corner of Fig. 4) × 600.

Fig. 4.

Feulgen staining of stage-8 H & H chimaera. Quail epiblast, chick hypoblast. All the cells from the chick hypoblast (cph) are confined in an extraembryonic area. The rest of the cells, embryonic as well as extraembryonic, show the heterochromatin marker. The PGC in the vicinity of the concentration of chick cells is of quail origin (qg). Fig. 4 × 200. Fig. 5 (left corner of Fig. 4) × 600.

Fig. 5.

Feulgen staining of stage-8 H & H chimaera. Quail epiblast, chick hypoblast. All the cells from the chick hypoblast (cph) are confined in an extraembryonic area. The rest of the cells, embryonic as well as extraembryonic, show the heterochromatin marker. The PGC in the vicinity of the concentration of chick cells is of quail origin (qg). Fig. 4 × 200. Fig. 5 (left corner of Fig. 4) × 600.

Fig. 5.

Feulgen staining of stage-8 H & H chimaera. Quail epiblast, chick hypoblast. All the cells from the chick hypoblast (cph) are confined in an extraembryonic area. The rest of the cells, embryonic as well as extraembryonic, show the heterochromatin marker. The PGC in the vicinity of the concentration of chick cells is of quail origin (qg). Fig. 4 × 200. Fig. 5 (left corner of Fig. 4) × 600.

Fig. 6.

Stage-8 H & H quail blastoderm, Feulgen staining. All cells, somatic and PGCs are with marker. × 600.

Fig. 6.

Stage-8 H & H quail blastoderm, Feulgen staining. All cells, somatic and PGCs are with marker. × 600.

Fig. 7.

Stage-8 H & H quail blastoderm, Feulgen staining. All cells, somatic and PGCs are with marker. × 600.

Fig. 7.

Stage-8 H & H quail blastoderm, Feulgen staining. All cells, somatic and PGCs are with marker. × 600.

Chick-quail chimaeras

Twelve chimaeras developed into relatively normal embryos. Six had a chick epiblast and a quail hypoblast and the other six had a reciprocal combination. One blastoderm had to be fixed as early as stage 6 H & H, otherwise it would have been lost, and the others were fixed between stages 7 and 9 H & H.

The serial sections of the chimaeric blastoderms were checked for the presence of PGCs, the origin of which was assessed according to the presence or absence of a heterochromatin marker.

The total number of PGCs per blastoderm varied according to their developmental stage at the time of fixation from 17 at stage 6 H & H to 87 at stage 8 H & H and 169 at stage 9H&H.

The outcome of both variants of the experiment, namely chimaeras with or without marginal zone, seemed to be similar, and they are therefore all grouped into Table 1, but the blastoderms with intact marginal zone and area opaca are marked with an asterisk.

Table 1.

Origin of PGCs in chimaeric blastoderms

Origin of PGCs in chimaeric blastoderms
Origin of PGCs in chimaeric blastoderms

In the table the blastoderms were grouped according to the type of the chimaera.

The results of all chimaeras point in the same direction, and from the identifiable PGCs 95 % are of epiblastic origin and 5 % of hypoblastic origin.

The question of whether Feulgen staining can demonstrate the presence of a heterochromatin marker in quail PGCs has been dealt with previously.

Reynaud (1969) applied the Feulgen technique both to blastodermic layers and to whole quail embryos and concluded that with this method the existence of the nuclear marker is confirmed in somatic cells except blood cells. He also claimed that the marker has seldom been observed in primordial germ cells.

Tachinante (1974) observed more closely the appearance of the heterochromatin marker in quail PGCs as part of a study concerned with the influence of a female gonad on male PGCs. She concluded that a marker in the form of two to three large heterochromatin granules was visible in both male and female germ cells, but only after about 6 days of incubation, when the germ cells have already undergone morphological differentiation. In our study we have found that the characteristic two to three heterochromatin granules are present already at a much earlier stage, at about 24 h of incubation. We were therefore able to use chick-quail chimaeras at the earliest desired developmental stage so that after a short incubation period in vitro the outcome could be checked for the presence and origin of PGCs.

In their book Primordial Germ Cells in the ChordatesNieuwkoop & Sutasurya (1979) review all the available publications on the subject and conclude that: ‘avian PGCs seem to originate in the extraembryonic primary hypoblast during early development’. This statement, based among others on the conclusions of Dubois (1967) and Vakaet (1970) leads to the second step which is: ‘the early segregation of the avian PGCs from the primary hypoblast shows certain similarities to the history of the germ cells in the anuran amphibians’.

It is therefore clear that the clarification of which is the germ layer from which the PGCs arise is not only factually important but has an important theoretical implication.

Our findings show very clearly that at stage XIII E.G & K when the blastoderm is composed of two dististinct layers -the epiblast and the primary hypoblast -it is the epiblast which contributes the PGCs. The 5 % of PGCs of hypoblastic origin probably originated from epiblastic cells of the donor attached to the transplanted hypoblast. This might be due either to inaccurate treatment, in which a few epiblastic cells especially at the circumferential separation area have been carried along with the transplanted hypoblast, or to the fact that the first few future PGCs (not yet morphologically identifiable) already started to move out from the epiblast into the blastocoelic cavity, settling on top of the primary hypoblast with which they were later transplanted.

The demonstration of the epiblastic origin of the PGCs in birds is in agreement with several findings in mammals. Also in mammals the PGCs can be morphologically identified rather late in development. It was commonly believed that their first appearance was in the entoderm of the yolk sac (Brambell, 1956; Hamilton & Mossman, 1972). However Ożdżeński (1967) carefully checked the appearance of cells demonstrating the alkaline phosphatase reaction typical for mammalian PGCs. His results indicate that PGCs can first be identified in the embryonic rudiment of the allantois, which is an extension of the posterior part of the PS. This means that mouse PGCs are of epiblastic origin and only somewhat later in development do they migrate into the entoderm. This is also in agreement with the results of Gardner & Rossant (1976) and Gardner (1977) who have demonstrated, by injecting single ectodermal cells into a blastocyst, that they give rise to both somatic cells and PGCs. The calculations of Falconer & Avery (1978) on PGCs in chimaeric and mosaic mouse embryos, also point in the same direction.

By demonstrating the epiblastic origin of avian PGCs we have made only one step, for we do not yet have the answer as to how they arise from this layer. It will, however, be easier now to tackle the second problem, namely, is there a predetermined germinal region in the epiblast or do the PGCs arise from the epiblast by way of induction?

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