Download Avian primordial germ cells are of epiblastic origin

J. Embryol. exp. Morph. Vol. 65, pp. 139-147, 1981
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Avian primordial germ cells are of epiblastic origin
By HEFZIBAH EYAL-GILADI, 1 MALKA GINSBURG
AND AHARON FARBAROV
From the Department of Zoology, Hebrew University oj Jerusalem
SUMMARY
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-Xl] I
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 histoiogically 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.
INTRODUCTION
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 (KocherBecker & 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
1
Author's address: Department of Zoology, Hebrew University of Jerusalem, 91904
Jerusalem, Israel.
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H. EYAL-GILADI, M. GINSBURG AND A. FARBAROV
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 6 H & 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, 1973/7, 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?
MATERIALS AND METHODS
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 - H C I to 60 °C prior to the onset of the hydrolysis which then took about
15 min. Scruff's reagent was prepared according to Barger and de Lamater
(Pearse, 1968) and applied for 1^-2 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 /on.
For a light background staining that would not blur the Feulgen reaction,
Avion primordial germ cells are of epiblastic origin
141
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/*m thick.
RESULTS
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).
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 9 H & H.
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H. EYAL-GILADI, M. GINSBURG AND A. FARBAROV
1
Avian primordial germ cells are of epiblastic origin
143
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.
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.
DISCUSSION
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 Chordates Nieuwkoop & 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
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), x 600. eg, chick PGC;
cph, 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.
Figs. 2 and 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 (eg). All the dark spots in the chick cells are mitoticfigures(mr). Fig. 2 x 200.
Fig. 3 (left corner of Fig. 2) x 600.
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H. EYAL-GILADI, M. GINSBURG AND A. FARBAROV
Avian primordial germ cells are of epiblastic origin
145
Table 1 Origin of PGCs in chimaeric blastoderms
Un-
Blastoderm
M44
M48
M45
M50
M47
*M55
M49
M43
M46
*M51
*M56
*M52
Epiblast/
hypoblast
q/c
q/c
q/c
q/c
q/c
q/c
c/q
c/q
c/q
c/q
c/q
c/a
Stage of
fixation
7H&H
7H&H
7H&H
7H&H
8H&H
9H&H
6H&H
7H&H
7H&H
7H&H
7H&H
7-8 H & H
Total no.
of PGCs
Chick
PGCs
Quail
PGCs
identified
PGCs
25
34
43
47
87
169
17
36
48
57
64
70
1
3
3
2
5
9
14
32
39
53
61
67
23
30
38
42
82
160
2
2
4
1
1
3
1
1
2
3
—
—
1
2
5
3
—
(The blastoderms marked with an asterisk are with area opaca and marginal
zone. From all other blastoderms the area opaca and marginal zone were removed
at the time of hypoblast exchange.)
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,
Figs. 4 and 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 x 200. Fig. 5 (left corner of Fig. 4) x 600.
Figs. 6 and 7. Stage-8 H & H quail blastoderm, Feulgen staining. All cells, somatic
and PGCs are with marker, x 600.
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H. EYAL-GILADI, M. GINSBURG AND A. FARBAROV
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 Ozdzeriski (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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