/ . Embryol. exp. Morph. Vol. 31, 1, pp. 75-87, 1974
Printed in Great Britain
75
Germ cell migration in toad (Bufo bufo): effect of
ventral grafting of embryonic dorsal regions
ByPIERO P. GIORGI1
From the Institute of Zoology, University of Bologna, Italy
SUMMARY
Bufo bufo embryos were used at the tail-bud stage for the grafting of two different dorsal
regions (cephalic and caudal tracts) into the ventral side of the embryo (cf. Fig. 1). Germ cell
localization was studied at the beginning of larval life. The results seem to confirm the original
finding of Gipouloux (1970) who suggested that in anurans germ cells migrate under the
attraction of a substance produced by the dorsal mesodermal tissues.
The attractive action of dorsal tissue was confined to the caudal region of the embryo. In
operated specimens the migration of germ cells was drastically altered. The genital ridges of
host embryos were almost sterile, while numerous germ cells appeared associated with caudal
grafts. A considerably smaller number of germ cells was associated with cephalic grafts.
About 80 % of germ cells associated with caudal grafts were present at the same levels
where a well-developed dorsal mesentery was also present. It is suggested that the formation
of the dorsal mesentery plays a morphogenetic role in segregating primordial germ cells from
other endodermal cells and contributes to their final localization in the genital ridges.
INTRODUCTION
In anuran amphibians the early localization of primordial germ cells and their
migration into the genital ridges have been described in detail (Bounoure,
1939; Bladder, 1958) and confirmed by experimental studies (Bladder & Fischberg, 1961; Padoa, 1963; Smith, 1966). For a review see Bladder (1970).
During cleavage the 'germinal cytoplasm' (Bounoure, 1939; Smith, 1966;
Czolowska, 1969; Mahowald & Hennen, 1971) becomes localized in a number
of endodermal cells, which will be situated deep within the endodermal material
later at the neurula stage. During the following developmental stages these
primordial germ cells migrate towards the dorsal side of the embryo and form a
medial ridge under the dorsal mesodermal tissue. As the formation of the
dorsal mesentery takes place, the primordial germ cells leave the endodermal
ridge and reach their final localization at each side of the dorsal root of the
mesentery. This last stage of migration has been studied and discussed previously (Vannini & Giorgi, 1969).
1
Author's address: Station de Zoologie Experimental, Universite de Geneve, 154 Route
de Malagnou, 1224 Geneve, Switzerland.
76
P. P. G I O R G I
Fig. 1. Grafting of dorsal regions into the ventral side of Bufo bufo embryos at
tail-bud stage, d, donor embryo; h, host embryo; gr, graft.
The embryonic dorsal tissue situated between the primordium of the pronephros
and the base of the tail-bud was divided into its caudal half (A) and cephalic half
(B) for two different experimental groups.
The aim of this work was to study the mechanism of germ cell migration
throughout the endodermal tissue. Gipouloux (1970) first worked on this problem in a number of anurans, providing evidence that primordial germ cells
actively migrate under the attractive influence of the dorsal mesodermal tissue.
After extirpation and grafting experiments, Gipouloux (1970) showed that
notochord, Wolffian duct and somites are co-operatively responsible for the
attraction of germ cells. The present experimental study aimed to confirm the
hypothesis formulated by Gipouloux and to provide further information on this
problem. In particular this study was designed to find out if the attractive effect
is limited to a certain region of the dorsal embryonic tissue. Particular emphasis
was given to the establishment of an experimental situation in which the extra
dorsal region grafted ventrally would be sufficiently complete and natural to
oppose that of the host embryo acting in the opposite direction.
MATERIAL AND METHODS
Bufo bufo embryos were obtained in the laboratory from adult couples captured in nature during the mating season. The operation (Fig. 1) was performed
at the tail-bud stage (stage III-l of Cambar & Gipouloux, 1956) with micropins and hair loops in commercial spring water (Cerelia, Bologna) without the
use of antibiotics. The dorsal region of the donor embryos was taken between
the primordium of the pronephros and the base of the tail-bud. The anterior
half of this region was used as a cephalic graft (Fig. 1B), the posterior as a
caudal graft (Fig. 1 A). A small cut was made in the ventral region of the host
embryo and the graft was gently placed inside in such a way that it was held by
the host body wall until healing. Particular care was taken not to introduce
11
Germ cell migration in toad
Table 1. Cephalic grafts
Germ cell counts and graft length. The protocol numbers correspond to those used
in Fig. 2. The number of germ cells present in the genital ridges of control specimens was
about 80.
Protocol
number of
specimens
Length
of the
graft Om)
Germ cells
Germ cells
associated with in the host's
genital ridges
the graft
52 a
52b
52 d
52e
52 f
52 h
52 i
521
52m
52 n
53 c
55a
55 b
55c
55d
55e
55g
55 h
55i
551
55 m
55 n
56i
650
650
870
1000
1000
650
1050
450
1070
900
620
650
500
420
800
800
670
650
900
770
620
650
1350
2
—
—
9
—
2
1
2
6
1
7
—
1
3
—
—
1
—
—
—
6
—
—
8
4
6
3
21
13
—
4
3
6
21
17
7
10
—
4
4
2
1
13
6
6
16
Average
769
2
8
endodermal cells from the donor embryo into the host embryo and not to lose
those of the latter.
After healing of the wound, the embryos developed in tap water until stage
IV1 (the beginning of larval life) when they were fixed in Bouin's liquid,
embedded in wax, cut in transverse serial sections of 10 /an and stained with
Mayer's haemalum-eosin. The location of germ cells was analysed in each section
and recorded in diagrammatic form, as shown in Figs. 2 and 3. Control specimens were taken from the same spawn and fixed at the same developmental
stage.
RESULTS
Among 43 embryos operated, 5 extruded the graft, while the other 38 reached
stage IV1 with a well-grown graft.
78
P. P. GIORGI
Table 2. Caudal grafts
Germ cell counts, graft length and relative position of germ cells and dorsal mesentery.
The protocol numbers correspond to those used in Fig. 3. The number of germ cells present
in the genital ridges of control specimens was about 80.
Germ cells associated with the graft
c
Protocol
number of
specimens
Length of
the graft
Om)
Total
At the same
levels where a
dorsal mesentery
was present
54 a
54 b
54 c
54 d
54e
54 f
54 g
56a
56b
56c
56d
56e
56 h
561
56 m
1750
1000
1000
1870
1250
820
650
970
1250
1370
1070
450
850
1470
1450
7
54
10
12
16
29
9
8
6
10
30
1
4
25
55
7
43
6
12
16
29
9
8
4
—
20
—.
.—.
23
52
—•—
7
5
—10
2
7
9
5
13
1148
18
15
5
Average
Germ cells in
the host's
genital ridges
—
•
8
4
5
General features of the graft
Only occasionally did the organs of the graft appear deformed and incomplete,
especially at its terminal sides, but generally the neural tube, the notochord,
dorsal muscles and mesenchyme were present and their aspect was similar to
that of the host (Figs. 4, 5, 6,7). The renal system presented a different situation.
The pronephros often developed within the cranial grafts (Fig. 6). In this type
of graft the Wolffian duct was large and hydropic, because of the absence of a
cloacal drain. No renal organ was present in the caudal grafts. At the time of
the operation in fact, the growing Wolffian duct had not yet reached the caudal
region. Without the stimulus of this organ, the competent mesoderm does not
differentiate into mesonephric tubules, as already shown in Bufo bufo by
Vannini & Giorgi (1969).
The presence of an extra dorsal region in the ventral side of the embryo
affected the normal development of the intestinal coils. The dorsal mesentery
was not always present under the graft.
It is worth noting (Tables 1 & 2) that the average length of the caudal grafts
(1150/tm) was found to be noticeably higher than that of the cranial grafts
(770 /OTI), although the length of the two types of graft was the same at the time
Germ cell migration in toad
52 e
55 c
52 f
79
55 a
Fig. 2. Cephalic grafts. Some examples of germ-cell localization in control specimens
(C) and experimental embryos. The latter are indicated by their protocol numbers
(at the top) which correspond to those used in Table 1.
For each experimental embryo the localization of germ cells in both genital ridge
(H) and graft (G) are illustrated. The genital ridges of control specimens (C) and host
embryos (H) were screened from the level of the cloaca (bottom of the diagram) until
the cephalic end of the organ (top of the diagram). The beginning of the genital ridge
(somatic elements) at caudal levels is represented by a baseline block which continues throughout the diagram; the presence of germ cells is represented by parallel
blocks, their height being proportional to the number of germ cells. The aim of the
diagrams is to provide a visual pattern of germ-cell localization. A precise quantitation of these elements is given in Table 1. The grafts (G) were screened throughout
their length. The relative position between the graft and the host's genital ridge in
the embryo is maintained in this representation. The diagrams of grafts (G) contain
blocks for germ cells, but no baseline block for somatic elements, which were
missing.
80
P. P. G I O R G I
54 b
54 f
56 m
561
H
G
Fig. 3. Caudal grafts. Some examples of germ-cell localization in control specimens
(C) and experimental embryos. The latter are indicated by their protocol number
(at the top) which corresponds to those used in Table 2. For each experimental
embryo the localization of germ cells in both genital ridge (H) and graft (G) are
illustrated. For further explanations cf. Fig. 2. The vertical dashed lines alongside
the diagrams of grafts (G) indicate the presence of a dorsal mesentery associated
with the graft.
81
Germ cell migration in toad
FIGURES
4-7
Transverse sections of experimental embryos bearing different types of grafts:
Dorsal tissue of the host embryo: H. Grafted dorsal tissue: G. Germ cells: gc.
H
0-5 mm
Fig. 4. Caudal graft. Two groups of germ cells are localized underneath the grafted
tissue on each side of a dorsal mesentery.
of the operation (Fig. 1). Probably this is due to the fact that the process of
axial elongation takes place mainly in the posterior (caudal) half of the embryo.
Effect of the graft upon the gonad of the host
The number of germ cells present in the genital ridges of the control specimens was about 80, which is normal at stage IV1. The higher concentration
of germ cells in the cephalic region of the gonad (Figs. 2 and 3, control specimens) is typical of Bufo and represents the first stage of differentiation of the
Bidder's organ, an abortive ovary present in both sexes (Witschi, 1933).
Tables 1 and 2 show a striking decrease in the number of germ cells present
in the genital ridges of the host embryos, without any significant difference seen
(l
EM B 3 1
82
P. P. G I O R G I
Fip. 5. Caudal graft. A group of germ cells associated with the graft. Note the
presence of a dorsal mesentery.
between individuals bearing a cephalic graft and those with a caudal one. No
specific localization of these germ cells was detected (Figs. 2 and 3).
The genital ridges of six experimental specimens were completely devoid of
germ cells. In view of the problem of the relative role played by germinal and
somatic cells in the differentiation of the gonad (Burns, 1955) attention has been
given to the cephalo-caudal distribution of the somatic cells of these sterile
gonads. It was observed that the Bidderian (cephalic) region of the sterile
gonads was always formed by a number of somatic cells larger than that of the
true (caudal) genital region (Figs. 8C, D). In control specimens (Figs. 8A, B)
the Bidderian region of the gonad was also formed by a larger number of both
somatic and germinal cells. The same phenomenon was already reported by Di
Grande (1968) in a different experimental situation, and it is in line with the
view that primary gonad differentiation is expressed in the somatic cells (Vannini
& Sabbadin, 1954; Witschi, 1956).
83
Germ cell migration in toad
Fig. 6. Cephalic graft. The grafted tissue included part of the primordium of
pronephros and of a limb. No germ cells.
Attraction of germ cells by the graft
The number of cells associated with cephalic grafts (Table 1) was considerably lower than that of cells associated with caudal grafts (Table 2). As pointed
out in the previous paragraph, at the stage when experimental animals were
studied caudal grafts were longer than the cephalic ones. Thus it could be
possible that the number of germ cells attracted by the grafts was related to their
length. But an analysis of individual cases (cf. Tables 1 and 2) does not favour
this hypothesis, showing no correlation between these two parameters.
The dorsal mesentery seems to be of some importance in the mechanism of
germ-cell migration of anurans (Bounoure, 1939; Sabbadin, 1959). Therefore
the relationship between the presence of a dorsal mesentery in the graft and the
presence of germ cells was considered. This study was carried out only in the
group bearing caudal grafts, because the small number of germ cells associated
with cranial grafts did not allow a significant analysis. As shown in Table 2 and
6-2
84
P. P. G I O R G I
Fig. 7. Cephalic graft. Well-developed dorsal tissue in the graft. No germ cells.
Fig. 3, a very large percentage (83 %) of germ cells associated with the grafts
were localized at those levels where a dorsal mesentery was present.
DISCUSSION
Gipouloux (1970) has suggested that in anurans the migration of primordial
germ cells takes place under the attraction of the dorsal mesodermic tissue.
This hypothesis seems confirmed by the present experimental work, where a
complete dorsal region was used to attract primordial germ cells from the
endoderm.
Grafts obtained from the caudal half of the embryonic dorsal region were
able to attract a number of germ cells considerably higher than that attracted
by the cephalic half (Tables 1 and 2). It can be suggested, therefore, that the
attractive ability of dorsal mesodermal tissue is localized in the caudal region of
the embryo. This would be in agreement with the fact that in normal develop-
Germ cell migration in toad
85
D
Fig. 8. Comparison between the genital ridges of a control specimen (A and B) and
an experimental specimen (C and D). The latter was sterile as a consequence of the
operation. The Bidderian (cephalic) tract of the control gonad (A) contains more
germinal and somatic elements than the true gonadic (caudal) tract (B). Also cf.
diagrams of control specimens in Figs. 2 and 3. In the host's genital ridges of sterile
experimental embryos a similar relationship can be seen between the somatic
elements of the Bidderian tract (C) and those of the true gonadic tract (D), which
are less numerous.
ment the genital ridges are indeed formed at the medio-caudal levels of the
embryo (cf. control specimens Figs. 2 and 3). It should be noted, however, that
the growth of an extra dorsal region must also cause an unspeciflc disturbance in
the normal migration of germ cells to the host's genital ridges. Probably this
effect has no relationship with the attractive capacity of the graft. In fact the
degree of sterility of recipient embryos was very similar with both types of
grafts (Tables 1 and 2 and Figs. 2 and 3). The total number of germ cells
recovered in each experimental animal (present in the genital ridge, plus those
associated with the graft) never reaches the normal set of about 80, suggesting
that a number of primordial germ cells have remained in the endoderm without
being finally attracted by either dorsal region.
The early development of the gonad in bufonids is characterized by a precocious differentiation of its cephalic region into Bidder's organ (Ponse, 1927;
Witschi, 1933). Soon after the migration of germ cells into the genital ridges,
the region corresponding to Bidder's organ typically contains more germ cells
than the posterior region, the future true gonad (control specimens Figs. 2 and
86
P. P. GIORGI
3, Figs. 8 A-B). One of the purposes of the present investigation was to check
the possibility that this differential localization of germ cells in bufonids could
be due to a different attractive capacity of the cephalic and caudal tract of their
dorsal embryonic region. The present results show that this is not the case,
and other mechanisms must underlie the development of Bidder's organ.
The attractive action of dorsal mesodermic tissue does not seem, however,
to be the only mechanism leading to the localization of germ cells in the genital
ridges. A clear relationship between germ cells associated with grafted tissue
and the presence of a well-formed dorsal mesentery at the same levels is shown
in Fig. 3 and Table 2. It is likely that the opposition of the lateral coelomic
folds to form the dorsal mesentery causes the segregation of germ cells from the
mass of other endodermal cells. Thus, the final stages of germ cell localization
in the genital ridges could be simply due to the movements of the surrounding
tissue, as already suggested by Vannini & Giorgi (1969). To summarize, three
major events seem involved: (a) active attraction of primordial germ cells
towards the posterior (caudal) region of dorsal mesodermic tissue; (b) passive
segregation of the dorsal ridge of germ cells from other endodermal tissue by
the fusion of lateral mesodermal plates to form the dorsal mesentery; (c)
passive displacement of germ cells at both sides of the root of dorsal mesentery
by the fusion of the medial postcardinal veins to form the posterior vena cava
(Vannini & Giorgi, 1969).
This investigation was supported by a grant from the Consiglio Nazionale delle Ricerche
(Rome). The author is grateful to Professor E. Vannini for his interest and advice throughout
this work.
REFERENCES
A. W. (1958). Contributions to the study of germ cells in the Anura. /. Embryol.
exp. Morph. 6, 491-503.
BLACKLER, A. W. (1970). The integrity of the reproductive cell line in the Amphibia. Curr.
Top. Devi Biol. 5, 71-87.
BLACKLER, A. W. & FISCHBERG, M. (1961). Transfer of primordial germ cells in Xenopus
laevis. J. Embryol. exp. Morph. 9, 634-641.
BOUNOURE, L. (1939). UOrigine des Cellules Reproductrices et le Probleme de la Ligne'e
Germinale. Paris: Gauthier-Villars.
BURNS, R. K. (1955). The urogenital system. In Analysis of Development (ed. by B. H. Willier
& P. A. Weiss), p. 462. Philadelphia: W. B. Saunders Co.
CAMBAR, R. & GIPOULOUX, J. D. (1956). Table chronologique du developpement embryonnaire et larvaire du Crapaud commun: Bufo bufo L. Bull. biol. Fr. Belg. 90, 198-217.
CZOLOWSKA, R. (1969). Observations on the origin of the 'germinal cytoplasm' in Xenopus
laevis. J. Embryol. exp. Morph. 22, 229-251.
Di GRANDE, F. (1968). Sviluppo postmetamorfico delle gonadi e degli organi di Bidder in
esemplari di Bufo bufo precocemente panirradiati can raggi X. Rend. Accad. Naz. Lincei
ser. vnr, 45, 437-442.
GIPOULOUX, J. D. (1970). Recherches experimentales sur l'origine, la migration des cellules
germinales, et l'edification des cretes genitales chez les Amphibiens Anoures. Bull. biol. Fr.
Belg. 104, 22-93.
1
MAHOWALD, A. P. & HENNEN, S. (1971). Ultrastructure of the 'germ plasm in eggs and
embryos of Rana pipiens. Devi Biol. 24, 37-53.
BLACKLER,
Germ cell migration in toad
87
PADOA, E. (1963). Le gonadi di girini di Rana esculenta da uova irradiate con ultravioletto.
Monit. Zool. Ital. 71, 238-249.
PONSE, K. (1927). L'evolution de l'organe de Bidder et la sexualite chez le Crapaud. Revue
suisseZool. 34, 217-220.
SABBADIN, A. (1959). Alcune osservazioni sullo sviluppo de l'apparato renale, dell'interrenale
e delle creste genitali di Discoglossus pictus in esperimenti di trapianto autoplastico del
mesoderma intermedio. Archo. ital. Anat. Embriol. 64, 87-104.
SMITH, L. D. (1966). The role of a 'germinal plasm' in the formation of primordial germ cells
in Rana pipiens. Devi Biol. 14, 330-347.
VANNiNr, E. & GIORGI, P. P. (1969). Organogenesi dell-apparato urogenitale degli Anfibi:
agenesi ed interruzione del dotto di Wolff in embrioni di Bufo bufo. Archo ital. Anat.
Embriol. 74, 111-143.
VANNiNr, E. & SABBADIN, A. (1954). The relation of the interrenal blastema to the origin of
the somatic tissues of the gonad in frog tadpoles. J. Embryol. exp. Morph. 2, 275-289.
WITSCHI, E. (1933). Studies on sex differentiation and sex determination in amphibians. VI.
The nature of Bidder's organ in the toad. Amer. J. Anat. 52, 461-515.
WiTSCHr, E. (1956). Development of Vertebrates. Philadelphia: W. B. Saunders Co.
{Received 2 April 1973, revised 3 July 1973)
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