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DEVELOPMENTAL DYNAMICS 210:236–248 (1997)
Fates of the Blastomeres of the 32-Cell Stage
Pleurodeles waltl Embryo
MICHEL DELARUE,1* FRANCISCO JOSÉ SÁEZ,1 KURT E. JOHNSON,2 AND JEAN-CLAUDE BOUCAUT1
Moléculaire et Cellulaire du Développement, Groupe de Biologie Expérimentale, URA 1135 CNRS,
Université Pierre et Marie Curie, Paris, France
2Department of Anatomy and Cell Biology, George Washington University Medical Center, Washington, D.C.
1Biologie
ABSTRACT
Cell fate of the blastomeres at
the 32-cell stage in the Pleurodeles waltl embryo
was analyzed by injection of rhodamine or fluorescein lysinated-dextran (RLDx or FLDx). At the
tailbud stage, the progeny of each blastomere
contributed to more than one germ layer with
unequal distribution along the anteroposterior
and dorsoventral axis. Such a regionalized positioning of the descendants of the 32-cell blastomeres was found in the neuroectoderm, the epidermis, the notochord, the somites, the lateral
plate, and the endoderm, but not in the head
mesenchyme, the pronephros, or the blood islands. Results of double labeling of juxtaposed
blastomeres showed that cell mixing and rearrangement take place during organ formation.
Results are compared with those of the 32-cell
stage fate map in Xenopus and Rana and reveal
the more restricted fate of 32-cell stage blastomeres in Pleurodeles germ layers. Dev. Dyn.
1997;210:236–248. r 1997 Wiley-Liss, Inc.
Key words: Pleurodeles; embryo; cell lineage; regionalization
INTRODUCTION
In amphibians, since the first fate maps using vital
dyes at the early gastrula (Vogt, 1929; Pasteels, 1942;
Keller, 1975, 1976) or 32-cell stages (Nakamura and
Kishiyama, 1971; Nakamura et al., 1978), more accurate methods of cell lineage have been used involving
injection of individual cells with horseradish peroxidase (Jacobson and Hirose, 1978, 1981; Hirose and
Jacobson, 1979; Jacobson, 1983) or fluorescent-dextran
cell tracers (Gimlich and Gerhart, 1984; Gimlich and
Braun, 1985). New fate maps were thus constructed at
the eight-cell, 16-cell, and 32-cell stages of Xenopus
laevis, (Masho and Kubota, 1986; Moody, 1987a,b; Dale
and Slack, 1987) at the 32-cell stage of Rana pipiens
(Saint-Jeannet and Dawid, 1994), or at the early gastrula stage of Ambystoma mexicanum (Cleine and
Slack, 1985), Pleurodeles waltl (Delarue et al., 1992)
and Rana pipiens (Delarue et al., 1994).
Differences have been reported between anurans and
urodeles and between different species within the anuran group. First, in the urodeles Ambystoma mexicanum and Pleurodeles waltl, both deep and superficial
r 1997 WILEY-LISS, INC.
cells contribute to mesodermal derivatives (Smith and
Malacinski, 1983; Delarue et al., 1992), whereas, in
anurans, two modes have been reported: 1) Only deep
cells contribute to mesoderm, as in Xenopus (Keller,
1975; Smith and Malacinski, 1983); 2) deep and superficial cells contribute to axial and paraxial mesoderm, as
occurs in Ceratophrys, Rana, and Hymenochirus (Purcell and Keller, 1993; Delarue et al., 1994; Minsuk and
Keller, 1996). Second, in Xenopus, cell rearrangements
during the cleavage period lead to differences between
32-cell, blastula, or gastrula stage fate maps. For the
same relative spatial positioning, the fate is different
for each stage because the cells move from the original
founder cell location (Bauer et al. 1994).
Here, we study the anteroposterior and dorsoventral
distribution, mixing, and rearrangement of the descendants of the 32-cell blastomeres in the Pleurodeles waltl
embryo. Results are discussed in terms of the significance of cell lineages in setting up the body plan, and
we make comparisons with the fate map at the 32-cell
stage in Xenopus and Rana (Moody, 1987b; Dale and
Slack, 1987; Saint-Jeannet and Dawid, 1994).
RESULTS
Fate of the Blastomeres
The localization of the descendants of the labeled
blastomeres at the 32-cell stage was determined by the
method used for Xenopus and Rana (Bauer et al., 1994;
Saint-Jeannet and Dawid, 1994). A total of 217 rhodamine-lysine-dextran (RLDx) injections were made and
analyzed in tiers A (43), B (44), C (46), and D (84) (Table
1; Fig. 1). Each blastomere at the 32-cell stage invariably contributes to several germ layers. Blastomeres
from tier A contribute to ectoderm and mesoderm; tier B
to ectoderm, mesoderm, and in a few cases to endoderm;
tier C to ectoderm, mesoderm, and endoderm; and tier
D to mesoderm and endoderm. Tier A makes its major
Grant sponsor: CNRS; Grant number: URA 1135; Grant sponsor:
MEN; Grant sponsor: P. and Marie Curie University (France).
Francisco José Sáez is now at Universidad del Paı́s Vasco, Departamento de Biologı́a Celular y Ciencias Morfológicas, Facultad de
Medicina, 48940 Leioa (Vizcaya), Spain.
*Correspondence to: Michel Delarue, Biologie Moléculaire et Cellulaire du Développement, Groupe de Biologie Expérimentale, URA
1135 CNRS, Université Pierre et Marie Curie, 9 quai Saint-Bernard,
75005 Paris, France. E-mail: [email protected]
Received 12 December 1996; Accepted 22 July 1997
TABLE 1. Progeny of 32-Cell Stage Blastomeres, Peurodeles waltla
Ectoderm
ep
p
—
—
—
—
—
15
75
100
—
14
75
90
—
—
—
—
ol
—
—
18
77
—
—
—
—
—
—
—
—
—
—
—
—
ot
—
12
—
31
—
31
50
10
—
7
—
—
—
—
—
—
op
—
37
54
31
—
—
—
—
—
—
—
—
—
—
—
—
te
9
100
100
69
—
7
8
—
—
—
—
—
—
—
—
—
di
45
100
100
61
11
7
8
—
—
—
—
—
—
—
—
—
nt
my
100
87
73
—
78
54
41
10
—
—
—
—
—
—
—
—
sc
100
87
—
—
89
46
25
—
7
7
25
—
—
—
—
—
nc
—
62
73
31
—
—
17
—
—
—
—
—
—
—
—
—
hm
54
50
—
—
78
84
58
10
50
71
—
—
50
72
43
16
a
—
—
—
—
11
—
—
—
100
28
—
—
34
33
5
—
p
9
—
—
—
89
7
—
—
57
28
—
—
27
16
—
—
Mesoderm
so
a
p
pr
— 18 —
— 25 —
— — —
— — —
— 89 —
38 38
7
— 17
8
— — —
64 28 —
64 71 14
50 87 87
— 10 30
27 19
4
55 28 16
24 19 43
16 16 47
Endoderm
lp
—
—
—
—
—
15
—
—
—
28
62
100
4
—
10
10
he
—
—
—
—
—
—
33
—
—
35
12
—
8
44
28
31
bc
—
—
—
—
—
—
—
10
—
—
12
90
4
—
14
26
st
—
—
—
—
—
—
—
—
14
—
—
—
54
78
28
5
ph
—
—
—
—
—
—
—
—
100
85
12
—
81
100
62
21
br
—
—
—
—
—
7
—
—
14
—
—
—
46
50
14
—
li
—
—
—
—
—
—
—
—
71
71
—
—
84
27
5
—
a
—
—
—
—
—
—
—
—
57
35
12
—
100
100
81
68
gut
p
—
—
—
—
—
—
—
10
—
14
62
90
38
78
95
100
c
—
—
—
—
—
—
—
—
—
—
—
—
—
16
52
47
pt
—
—
—
—
—
—
—
—
—
—
12
40
—
—
19
10
aNumbers in parentheses indicate number of cases analyzed. Bold numbers are percentages of cases. %, percentage of cases; a, anterior; ag, anterior gut; ap, animal pole;
ar, archenteron; bc, blood cells; bl, blastocoel; bn, brain; bo, blastopore; bp, branchial pouches; br, branchial arches; c, caudal; CNS, central nervous system; d, dorsal; di,
diencephalon; dl, deep layer; en, endoderm; ep, epidermis; fb, forebrain; FLDx, fluorescein-lysine-dextran; hb, hindbrain; he, heart anlagen; hm, head mesenchyme; l,
lateral; li, liver anlagen; lp, lateral plate; m, middle; mb, midbrain; me, mesencephalon; mi, meninges; mt, midtrunk; my, myelecephalon; NAM, normal amphibian
medium; nb, number of cases; nc, neural crest cells; nt, notochord; ol, olfactory epithelium; op, optic vesicle; ot, otic vesicle; p, posterior; pg, posterior gut; ph, pharynx; pr,
pronephros; pt, proctodeum; RDLx, rhodamine-lysine-dextran; sc, spinal cord; sl, superficial layer; so, somites; st, stomodeum; t, trunk; te, telencephalon; v, ventral; vp,
vegetal pole.
CELL LINEAGE IN Pleurodeles EMBRYO
A1 (11)
A2 (8)
A3 (11)
A4 (13)
B1 (9)
B2 (13)
B3 (12)
B4 (10)
C1 (14)
C2 (14)
C3 (8)
C4 (10)
D1 (26)
D2 (18)
D3 (21)
D4 (19)
a
—
—
27
100
—
69
83
100
—
7
—
—
—
—
—
—
cns
me
73
100
82
38
11
—
—
—
—
—
—
—
—
—
—
—
237
238
DELARUE ET AL.
blastomere also makes a major contribution to the head
mesenchyme, the notochord, and the dorsal somites. A
few labeled cells occur in the fore- and midbrain.
The B2 blastomere makes its most significant contribution to the epidermis and a minor contribution to the
otic vesicle. Labeling is also widely scattered in the
head mesenchyme. There is major labeling in the
somites and minor labeling in the notochord, the pronephros, and the lateral plate. The B2 blastomere
contributes to the myelencephalon and the spinal cord
with a minor contribution to the forebrain.
The B3 blastomere makes its most significant contribution to the epidermis, with substantial labeling in the
otic vesicle. This blastomere also contributes to the
hindbrain, the spinal cord, the head mesenchyme, the
somites, the pronephros, and the heart. Minor labeling
occurs in the forebrain and the melanophores.
The B4 blastomere makes its most significant contribution to the epidermis from the stomodeum to the
tailbud regions and a less extensive contribution to the
otic vesicle, the head mesenchyme, the blood islands,
and the gut.
Fig. 1. Schematic drawing of the lateral view of the 32-cell stage
Pleurodeles waltl embryo. Nomenclature of blastomeres is that previously
defined for Xenopus laevis (Nakamura and Kishiyama, 1971).
contribution to the ectoderm, B and C to the mesoderm,
and D to the endoderm (Table 1).
Tier A
The A1 blastomere makes its most significant contribution to the hindbrain and the spinal cord. This
blastomere also makes a substantial contribution to all
the head mesenchyme and a minor contribution to the
forebrain, the somites, and the notochord.
The A2 blastomere makes its most significant contribution to the fore- and midbrain. Neural crest derivatives such as the meninges of the diencephalon, the
branchial arches, and the intersomitic ganglionic cells
were also labeled. A2 makes only a minor contribution
to the optic and otic vesicles, the hindbrain, and the
spinal cord. The A2 blastomere also makes a moderate
contribution to the head mesenchyme and the somites.
The A3 blastomere makes its most significant contribution to the forebrain. Labeling is extensive in the
neural crest derivatives such as the meninges of the
fore- and midbrain, the branchial arches, and the
melanophores. The A3 blastomere also contributes to
the epidermis and the olfactory epithelium but only
makes a minor contribution to the midbrain, the hindbrain, and the optic vesicle.
The A4 blastomere makes its most significant contribution to the head epidermis. Less labeling occurs in
the fore- and midbrain, as well as the optic and otic
vesicles. The A4 blastomere also makes a minor contribution to the meninges of the forebrain.
Tier B
The B1 blastomere makes its most significant contribution to the hindbrain and the spinal cord. The B1
Tier C
The C1 blastomere makes its most significant contribution to the pharynx, the head mesenchyme, the
notochord, and the somites. It makes a minor contribution to the gut, the liver, the branchial pouches, and the
stomodeum. Contribution to the neuroectoderm is restricted to the spinal cord.
The C2 blastomere makes its most significant contribution to the pharynx and the liver. This blastomere
also contributes to the head mesenchyme, the somites,
the notochord, the pronephros, the lateral plate, and
the heart. There is also a minor contribution to the gut,
the spinal cord, the epidermis, and the otic vesicle.
The C3 blastomere makes its most significant contribution to the pronephros, the lateral plate, and the
somites. This blastomere also contributes extensively
to the epidermis and slightly to the spinal cord. There
are also descendants in the gut and a minor contribution to the pharynx.
The C4 blastomere makes its most significant contribution to the epidermis, the lateral plate, the blood
islands, and the gut. A minor contribution occurs in the
somites and the pronephros.
Tier D
The D1 blastomere contributes most extensively to
the stomodeum and the branchial pouches. Substantial
labeling also occurs in the head mesenchyme, the
notochord, and the somites. A minor contribution occurs
in the lateral plate, the blood islands, and the heart.
The descendants of the D2 blastomere are concentrated in the pharynx, the gut, the head mesenchyme,
the somites, and the heart. A minor contribution occurs
in the branchial pouches, the liver, the notochord, and
the pronephros.
CELL LINEAGE IN Pleurodeles EMBRYO
The D3 blastomere makes its most significant contribution to the gut, the head mesenchyme, the somites,
the pronephros, and the heart. Only a few labeled cells
occur in the stomodeum, the branchial pouches, the
proctodeum, and the liver.
The D4 blastomere has most of its descendants in the
gut, somites, and the lateral plate. A few D4 progeny
are also found in the pharynx and the caudal endoderm.
239
Epidermis. Contributors to the anterior and posterior parts of the tailbud embryo come, respectively, from
tiers A,B and B,C of the 32-cell stage. For example, A4
derivatives are found in the head epidermis, and B4
derivatives extend posteriorly into the trunk (Fig. 2A).
Most of the dorsal and lateral contributors come from
columns 2 and 3, and most of the ventrolateral contributors come from column 4 (Tables 2, 3). For example,
the B3 lineage contributes to the dorsal epidermis, and
the B4 lineage contributes to the ventral epidermis
(Fig. 2B).
Neuroectoderm. Anteroposterior regionalization in
the CNS appears during neurulation. In vivo observations show a differential elongation of the A1, A2, A3,
and A4 clones along the anteroposterior axis (Fig.
3A–D). At the tailbud stage, major contributions of
these blastomeres are observed in the midbrain, the
hindbrain, and the spinal cord for A1, the entire brain,
and the spinal cord for A2, the forerain and the midbrain for A3, and the forebrain and the adjacent cephalic epidermis for A4 (Table 1; Figs. 2D, 3E,F). Tier B
contributes more prominently to the hindbrain and the
spinal cord.
The dorsoventral localization of progeny of A3, A2,
and A1 at the tailbud stage results from the mediolateral localization of progeny at the neurula stage. A1
forms the medial region, A2 the adjacent lateral region,
and A3 the more distal region of the neural plate. So, as
the neural plate curls, A1, A2, and A3 become, respectively, ventromedial, lateral, and laterodorsal (Table 2).
This is particularly clear after labeling A1 and A3,
respectively, with FLDx and RLDx. Progeny of A1 and
A3 are thus ventral and dorsal, respectively (Table 2;
Fig. 2E). Consequently, neural crest derivatives come
from dorsal and lateral blastomeres such as A2, A3, and
also B3.
Notochord. Anteroposterior regionalization in the
notochord is particularly clear with progeny distribution of C1 and B1 blastomeres (Table 1). C1 derivatives
contribute to most of the anterior notochord (Fig. 4A),
whereas B1 derivatives contribute to most of the remaining posterior notochord.
Somites. Contributors to the somites come from all
the tiers, and progeny are largely dispersed on the
anteroposterior and dorsoventral axis. However, a regionalization of some lineages is discernable in myotomes, particularly from blastomeres adjacent to the
dorsal midline. For example, the C1 lineage contributes
to the anterior somites (Table 1; Fig. 4B), from the
cephalic to the mid-trunk regions. Conversely, B1 derivatives populate the posterior somites (Table 1).
A dorsoventral regionalization is also apparent in the
somites. Some cases show a neat ventral (B3, D4) or
dorsal (A1, A2, B1) localization of the progeny (Tables 2,
3; Fig. 4C,D).
Both anteroposterior and dorsoventral regionalization is also observed in the somites. For example,
progeny of the C3 blastomere is more ventral in the
anterior region and both dorsal and ventral in the
posterior region (Table 3).
Pronephros. This derivative comes from blastomeres in the vegetal hemisphere and the marginal zone
such as B2, 3 and C2, 3, 4. It was not possible from a
32-cell stage fate map to find a regionalization of
lineages.
Fig. 2. (Overleaf) Anteroposterior and dorsoventral regionalization of
descendants of marked blastomeres. A: Anteroposterior regionalization
of A4 and B4 labeled cells in the epidermis at the tailbud stage in vivo.
Double-labeling experiments were performed with FLDx injected in A4
and RLDx injected in B4. Descendants of the green A4 blastomere are
essentially cephalic, anterior to the stomodeum (st). Descendants of the
red B4 blastomere are essentially localized in the trunk region (t). Notice
that both A4 and B4 lineages are mostly observable ventrally. my,
myelencephalon; ph, pharynx. B: Dorsoventral regionalization of descendants of B3 and B4 blastomeres in the epidermis at the tailbud stage.
Double labeling was performed with FLDx injected in B3 and RLDx
injected in B4. The red B3 descendants are essentially laterodorsal with a
slight contribution to the otic vesicle (ot). The green B4 descendants are
essentially lateroventral. Transverse section. C: Lateral view of a dissection of a tailbud stage. The epidermis was removed showing the lateral
plate. Double-labeled descendants of the red C2 blastomere and the
green C3 blastomere are distributed along the anteroposterior axis (a–p),
with red cells more anterior than green cells. D,E: Anteroposterior (D) and
dorsoventral regionalization (E) in the brain at tailbud stage on sagittal (D)
and transverse (E) sections. (D) Sagittal section in the anterior region of
the tailbud embryo. Anteroposterior distribution of descendants of A3
(FLDx) and A1 (RLDx) blastomeres is apparent. Green-labeled cells (A3)
distribute in the brain in more anterior region than red-labeled cells (A1).
(E) Detail in the ventral part of the myelencephalon (my) shows a
dorsoventral distribution of descendants of A1 and A3. Green descendants of A1 blastomeres are more ventral than red descendants of A3
blastomere. hb, hindbrain; mb, midbrain. F–H: Radial intercalation and
mixing between red B1 and green C1 descendants. F: At blastula stage,
green descendants of C1 blastomere and red descendants of
B1blastomere are arranged in two layers. At the frontier, slight mixing of
green and red cells is noticed. G: During gastrulation, green cells (C1)
involute, whereas red cells (B1) extend toward the blastopore. H: Higher
magnification of the frontier between B1 and C1 descendants, showing
mixing of cells. ar, archenteron; bl, blastocoel; bo, blatopore; dl, deep
layer; sl, superficial layer. I,J: Medial intercalation of descendants of C1
and contralateral C81 labeled with FLDx and RLDx, respectively. I:
Observation in vivo at the 64-cell stage. At this stage, two green cells (C1) and
two red cells (C81) are present in the dorsal region. Overlapping of the diffuse
green and red fluorescence makes a yellow light in the dorsal midline of the
embryo. J: Dissected embryo at the tailbud stage. At this stage, descendants of
C1 and C81 are mixed in the notochord. A medial intercalation is observed
laterally to the notochord and in somites. On both sides, the somites are
uniformly red and green labeled. nt, notochord; so, somites. Bars 5 500
µm in A,C,I, and J; 100 µm in B and G; 50 µm in D,E,F, and H.
Distribution of Labeled Cells Along the
Anteroposterior and Dorsoventral Axis
240
DELARUE ET AL.
Fig. 2.
241
CELL LINEAGE IN Pleurodeles EMBRYO
TABLE 2. Dorsoventral Regionalization of the Tier A and B Blastomeresa
ep
Blastomere
A1 (11)
A2 (8)
A3 (11)
A4 (13)
B1 (9)
B2 (13)
B3 (12)
B4 (10)
Region
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
a
—
—
—
—
—
—
—
—
27
23
61
100
—
—
—
69
—
—
50
33
—
—
70
100
p
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
15
7
—
25
50
—
—
70
100
te
—
—
9
25
100
37
45
91
9
38
38
38
—
—
—
—
—
7
8
—
—
—
—
—
cns
me
—
18
72
25
100
37
36
36
9
23
31
—
—
11
—
—
—
—
—
—
—
—
—
—
di
—
27
45
25
100
37
45
91
—
38
23
23
—
11
—
—
7
—
8
—
—
—
—
—
so
my
—
27
100
25
87
25
36
36
18
—
—
—
—
78
78
—
38
15
41
—
—
—
—
—
sc
—
18
100
—
87
25
—
—
—
—
—
—
—
—
89
23
7
15
25
—
—
—
—
—
a
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
15
23
23
—
—
—
—
—
—
p
18
—
—
25
—
—
—
—
—
—
—
—
89
—
—
7
31
31
—
—
17
—
—
—
aNumbers in parentheses indicate number of cases analyzed. Bold numbers are percentages of cases.
See abbreviations in Table 1 footnote.
TABLE 3. Dorsoventral Regionalization of the Tier C and D Blastomeresa
ep
Blastomere
C1 (14)
C2 (14)
C3 (8)
C4 (10)
D1 (26)
D2 (18)
D3 (21)
D4 (19)
Region
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
d
l
v
a
—
—
—
7
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
p
—
—
—
14
—
—
12
75
—
—
70
90
—
—
—
—
—
—
—
—
—
—
—
—
cns
sc
—
—
7
7
—
—
25
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
so
a
—
—
64
50
21
64
—
—
50
—
—
—
—
11
15
5
28
28
—
14
24
—
—
16
gut
p
7
7
28
50
14
64
50
—
87
—
—
10
—
8
15
5
16
11
—
9
19
—
—
16
ph
7
28
93
14
71
14
12
—
—
—
—
—
23
23
50
33
63
31
24
57
—
10
10
—
a
—
—
57
—
21
21
—
12
—
—
—
—
4
31
81
22
84
26
47
71
5
47
52
5
p
—
—
—
—
7
14
—
62
—
—
—
90
—
7
38
16
16
55
76
52
14
89
95
21
aNumbers in parentheses indicate number of cases analyzed. Bold numbers are percentages of cases.
See abbreviations in Table 1 footnote.
Lateral plate. An anteroposterior regionalization is
apparent in the lateral plate. For example, at the
tailbud stage, C2 progeny are distributed more anteriorly than C3 progeny (Fig. 2C).
Blood islands. This derivative comes from blastomeres in the vegetal hemisphere and the marginal zone
such as B4, C3, 4, and D1, 3, 4. It was not possible from a
32-cell stage fate map to find a regionalization of lineages.
242
DELARUE ET AL.
Fig. 3. Anteroposterior and dorsoventral regionalization of labeled
cells in the neuroectoderm. A–D: In vivo localization of the A1, A2, A3, and
A4 descendants in the neural plate at the early neurula stage (stage 14).
Notice the anteroposterior distribution of descendants of A1 (A), A2 (B),
A3 (C), and A4 (D). Descendants of marked A1 (A) are more posterior
than those of marked A4 (D). Descendants of A1 are proximal (A),
whereas descendants of A2 are lateral (B) and descendants of A3 are
distal (C). E,F: In vivo anteroposterior regionalization of labeled cells in
the central nervous system at the tailbud stage. E: The fore- and midbrain
come from the A3 blastomere. F: The hindbrain and spinal chord come
from the A2 blastomere. fb, forebrain; hb, hindbrain; mb, midbrain; op,
optic vesicle. Bars 5 1 mm in A–D, 500 µm in E and F.
Endoderm. Progeny of blastomeres C and D are
regionally distributed in the endodermal derivatives
(Table 1; Fig. 5). For example, the stomodeum is formed
by C1 and D1, 2, 3, 4 blastomeres, whereas the pharynx
is formed by tier C (C1, 2, 3). In the endodermal cell
mass of the trunk, D1 and D2 contribute mainly to the
anterior region; D3 and D4 contribute mainly to the
posterior region. Most of the proctodeal endoderm and
the caudal endoderm (post-proctodeal endoderm in the
growing tailbud) come from tier D (Table 1). A dorsoventral regionalization of the lineages may also be deduced. C1 and D1 progeny distribute in the ventral
regions of the pharynx and other anterior gut structures. The dorsoventral distribution also changes along
the anteroposterior axis. C2 and D2 progeny distribute
in the lateroventral regions of the pharynx and anterior
gut, but their distribution becomes more ventral posteriorly. Progeny of D3 show a lateral distribution in the
pharynx and the anterior gut and a more dorsal localization posteriorly. Conversely the lineage of D4 shows a
laterodorsal distribution in the anterior as well as
posterior gut (Table 3). The liver anlage, typically
ventral in the tailbud embryo, mainly comes from
laterodorsal blastomeres C1, 2 and D1, 2 (Table 3).
Cell mixing. Labeling of individual blastomeres at
the 32-cell stage resulted in a high rate of mixing
between labeled and unlabeled cells in the notochord
and somites (see Fig. 4A,B). Modalities of these rearrangements were investigated by double-labeling experiments. Adjacent dorsal B1 and C1 blastomeres were,
respectively, labeled with RLDx and FLDx, as were C1
and the adjacent contralateral C81 blastomeres.
During the cleavage period, neighboring clones
slightly overlap on their frontier (Fig. 2F). At the onset
of gastrulation (stage 8) progeny of C1 form the dorsal
lip of the blastopore. During gastrulation, progeny of
C1 involute into the embryo, whereas progeny of B1
extend toward the dorsal lip of the blastopore (Fig. 2G).
Examination of the B1–C1 frontier shows that the deep
and superficial layers are formed, respectively, with B1
and C1 clones which progressively intermix during
epiboly (Fig. 2H). This creates a progressive intermixing of cells between B1 and C1 clones during epiboly. At
the end of gastrulation, B1 cells also involute into the
embryo. Progeny of C1 and B1 form two consecutive
axial strips of cells, C1 anterior and B1 posterior largely
intermingled in the border area. This distribution leads
to the anteroposterior regionalization of labeled cells in
CELL LINEAGE IN Pleurodeles EMBRYO
243
Fig. 4. Anteroposterior and dorsoventral regionalization of marked
cells in the notochord and somites. A: Descendants of the C1 blastomere
contribute to the most anterior part of the notochord. Labeled cells are
mainly scattered in the middle (m) of the notochord. Sagittal section,
tailbud stage. B: Anterior somites contain descendants of the C2 blastomere at the level of the pharynx. Number of C2 descendants decreases
from trunk to caudal somites. Parasagittal section, tailbud stage. C,D:
Most of the descendants of the C1 blastomere localize ventrally in the
somites in the posterior half of the trunk (C). Most of the descendants of
the C2 blastomere localize dorsally in the somites in the anterior half of the
trunk (D). Parasagittal section, tailbud stage. a, anterior; en, endoderm;
m, middle; my, myelencephalon; nt, notochord; p, posterior; ph, pharynx;
sc, spinal cord; so, somites. Bars 5 100 µm in A and B, 25 µm in C and D.
the notochord at the tailbud stage so that the C1 clone
is more anterior and the B1 clone more posterior with a
large region of overlapping between them.
Another example illustrates cell mixing during the
formation of the notochord. Labeling of the C1 blastomere with RLDx and the contralateral blastomere C81
with FLDx (Fig. 2I) leads to the double labeling of a
large part of the notochord. Dissection of the dorsal
region at the tailbud stage shows that mediolateral
intercalation between the two clones results in a patchwork of red and green cells along the anteroposterior
axis (Fig. 2J).
At the tailbud stage, results show that descendants of
different blastomeres have characteristic patterns of
distribution along the anteroposterior and dorsoventral
axis in the CNS, the epidermis, the somites, and the
endoderm.
Dorsoventral regionalization in most of the territories of the 32-cell stage embryo is correlated to the
primitive dorsoventral axis. However, dorsoventral regionalization of the neuroectodermal fate map is inverse to the primitive dorsoventral axis. This becomes
clearly apparent when we compare the contribution to
epidermal and neuroectodermal derivatives (Fig. 6).
Also, examination of the endodermal fate map shows
that the anteroposterior localization of labeled cells
corresponds to the position of blastomeres along the
dorsoventral axis at the 32-cell stage. For example,
labeled cells in the rostral region of the pharynx come
from dorsal blastomeres and those in the posterior part
of the gut from ventral blastomeres (Fig. 7). In the
dorsal marginal zone of the Cynops pyrrhogaster em-
DISCUSSION
The results presented here show that in early
Pleurodeles waltl embryo 1) each blastomere contributes to more than one germ layer; 2) in certain tissues a
regionalized positioning of the descendants of the 32cell blastomeres is found; 3) during organ formation cell
mixing and rearrangements take place.
244
DELARUE ET AL.
Fig. 5. Anteroposterior and dorsoventral regionalization of labeled
cells in the endoderm. A: Descendants of the blastomere D1 contribute to
the ventral région of the embryo from the stomodeum to the midtrunk
region. Sagittal section, tailbud stage. B: Descendants of the blastomere
D2 contribute to the lateral region of the embryo from the branchial
pouches to the midtrunk region. Parasagittal section, tailbud stage. C:
Descendants of the blastomere D3 are visualized in the laterodorsal
region behind the pharynx. Parasagittal section, tailbud stage. D: Labeled
cells from D4 are present posteriorly from the midtrunk region to the
proctodeal region. Parasagittal section, tailbud stage. bp, branchial
pouches; mt, midtrunk region; pt, proctodeal region; st, stomodeum.
Bar 5 500 µm.
CELL LINEAGE IN Pleurodeles EMBRYO
245
Fig. 6. Comparison between epidermial and the neuroectodermal fate
maps at the 32-cell stage. The dorsoventral axis of the epidermal fate map
coincides with the dorsoventral axis of the cleaving embryo. Notice that
the dorsoventral axis of the neuroectodermal fate map is ordered
inversely to the dorsoventral axis of the cleaving embryo. Shading of the
blastomeres is proportional to the percentages of cases which contribute
to the epidermis and the neuroectoderm. AP, animal pole; Do, dorsal; Ve,
ventral; D, L, V, Dorsal, Lateral and Ventral fate of the blastomeres; VP,
vegetal pole.
Fig. 7. Schematic endodermal fate map at the 32-cell stage. Descendants of dorsal blastomeres contribute mainly to the anterior structures
(pharynx, anterior gut), and those of ventral blastomeres contribute
mainly to the posterior gut and caudal endoderm. Shading of the
blastomeres is proportional to the percentages of cases which contribute
to endodermal derivatives, respectively.
bryo, an anteroposterior regionalization was deduced
from vital staining as a projection of head-trunk-tail
resolution (Okada and Hana, 1945). Both in Pleurodeles
and Rana, the anteroposterior and dorsoventral specification of mesodermal territories in the marginal zone
was also observed (Delarue et al., 1994, 1995). Moreover, the superficial cell population of the early gastrula
makes a decreasing contribution to mesodermal derivatives from dorsal to ventral (Delarue et al., 1992).
In Xenopus and Rana, progeny of each blastomere of
the 32-cell stage are largely dispersed in the germ
layers (Moody 1987b; Dale and Slack, 1987; SaintJeannet and Dawid, 1994). In Xenopus, this large
dispersion is related to the pregastrula movements
reported in this species. Epiboly of the animal region
occurs by apical expansion of superficial cells from the
mid-blastula to the mid-gastrula stage, and the whole
DMZ has thinned before gastrulation occurs (Keller,
246
DELARUE ET AL.
1978, 1980). Epiboly moves material from the animal
half vegetally and forms dorsomarginal clones from
formerly animal cells. One of the consequences is that
progeny of B1 populate the dorsal blastoporal lip while
the descendants of the C1 blastomere populate the
sub-blastoporal layers (Bauer et al., 1994). In Pleurodeles embryos, we never observed such pregastrular
movements. Progeny at the blastula stage occupy the
same area as the founder cells so that dorsal lip is
formed mostly by C1 progeny.
We designed our study to be similar to those used in
lineage tracing in Xenopus (Moody, 1987b) and Rana
(Saint-Jeannet and Dawid, 1994). Comparison of the
32-cell stage fate maps in these three species reveals
significant differences (Table 4). For example, the major
contributing blastomere to the notochord is C1 in
Pleurodeles, whereas it is B1 in Xenopus and Rana. The
olfactory epithelium originates from A3, A4 in Pleurodeles but from A1, A2 in Xenopus and A2, A3 in Rana.
Similarly, the major contributor to the optic vesicle is
A3 in Pleurodeles but A1 in Xenopus (Huang and
Moody, 1993) and A1, A2, B1 in Rana. Another difference is that B4 descendants in Pleurodeles give epidermal cells; whereas, in Xenopus, they contribute mostly
to the posterior part of the somites. In Rana, B4
progeny localizes in epidermis and somites. The A1 and
A2 blastomeres make an important contribution to the
anterior epidermis in Xenopus and Rana. In Pleurodeles no descendant of A1 or A2 participates in epidermal
formation. Regarding endodermal derivatives, the contribution of blastomeres is more restricted in Pleurodeles than in Xenopus and Rana. In Pleurodeles, the
endoderm comes from tiers C and D (and for a minor
part from B2 and B4). In Xenopus and Rana all the tiers
contribute to the endodermal derivatives.
EXPERIMENTAL PROCEDURES
Embryos
Pleurodeles waltl embryos were obtained from natural matings in the laboratory. They were reared at 18°C
and staged according to the normal table of development (Gallien and Durocher, 1957; Shi and Boucaut,
1994). Jelly coats were manually removed with forceps.
Control and experimental embryos developed in 10%
normal amphibian medium (NAM) with 50 µg · ml21
gentamycin.
Labeling Procedures
In Pleurodeles, like in Xenopus, at the eight-cell
stage, the dorsoventral polarity of embryos displaying a
regular cleavage pattern was ascertained on the occurrence of a less pigmented dorsal side. Then, the presumptive territory of the dorsal lip was marked with
Nile blue sulfate applied directly through the vitelline
membrane.
At the 32-cell stage, one blastomere of each embryo
was injected with 1 nl of rhodamine-lysine-dextran
(RLDx) or fluorescein-lysine-dextran (FLDx) (Gimlich
and Gerhart, 1984) at 50 mg/ml in distilled water, as
previously described (Delarue et al., 1992). In order to
study cell movements, mixing, and the relative cell
distribution of neighboring blastomeres, we injected
two adjacent blastomeres, one with RLDx and the other
with FLDx. The nomenclature used for each blastomere
at this stage was that of Nakamura and Kishiyama
(1971) for Xenopus embryo: tiers A–D from animal to
vegetal pole and columns 1–4 from dorsal to ventral
side (Fig. 1). In some cases, adjacent lateral (A–D) and
contralateral (A8–D8) blastomeres were, respectively,
labeled with RLDx and FLDx.
We then allowed embryos to develop until the blastopore (stage 8a) became visible. Embryos in which the
vital dye was not coincident with the dorsal lip were
discarded.
Histological Procedures and Analysis
After selection, embryos were left in the dark (to
avoid bleaching of fluorochrome) to develop to the
desired stages. To study cell movements and mixing,
some embryos with two adjacent labeled blastomeres
were fixed during gastrulation (stages 8a–10). To study
the regionalization of progeny of each blastomere, most
embryos were fixed at tailbud stages (28–30) in 3.7%
formaldehyde in 10% NAM overnight at 4°C. These
stages were chosen because most of the organ rudiments are well developed and the tracer is still detectable in all the progeny. The embryos were then washed
in 10% NAM, dehydrated in an ethanol series, embedded in polyethylene glycol distearate 400 (Koch-Light),
and sectioned at 10 µm. After rehydration, sections
were mounted in Mowiol (Hoechst). Some doublelabeled embryos were dissected in 10% NAM to show
the extensive distribution and mixing between the
progeny of two adjacent labeled blastomeres.
The specimens were scored on a Leitz photomicroscope equipped with the appropriate filter sets for
rhodamine or fluorescein fluorescence. Injected embryos showing an abnormal morphology of their organs
and cell debris at the tailbud stage were disregarded.
According to Moody’s study (1987b), the scoring
method took into account all sections of embryos and
was based on the percentage of embryos that have any
descendants of a given blastomere in various tissues.
This allowed researchers to take into account even the
few labeled cells scattered in tissues in only some of the
sections (Saint-Jeannet and Dawid, 1994). The contribution of every blastomere to embryonic organs is shown
in Table 1.
ACKNOWLEDGMENTS
We are grateful to Dr. R.E. Keller for his suggestions
and comments, to M.M. Riou for assistance throughout
the preparation of the manuscript, and to Mr. J. Desrosiers for iconography. F.J.S. was the recipient of a
post-Doctoral Fellowship from the Dirección General de
Investigación Cientı́fica y Técnica (Spain) and a ShortTerm Fellowship from the European Science Foundation (EDB Program, 1994).
TABLE 4. Comparison Between the Contribution of the 32-Cell Stage Blastomeres in Pleurodeles waltl, Rana pipiens, and Xenopus laevisa
A1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
A3
1
1
1
1
1
1
1
A4
Pleurodeles waltl
B3 B4 C1 C2
1
1
1
B1
1
B2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ep
ol
ot
op
bn
sc
nc
hm
nt
so
pr
lp
he
bc
st
ph
br
li
ag
pg
pt
aSee
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
A1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
C3
1
C4
D1
D2
D3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
A2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
A3
1
A4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
B1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
A2
1
1
1
1
1
1
A3
1
1
1
A1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Rana pipiens (Saint-Jeannet and David, 1994)
A4 B1 B2 B3 B4 C1 C2 C3 C4 D1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Xenopus laevis (Moody, 1987b)
B2 B3 B4 C1 C2 C3
1
1
1
1
C4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
D1
D2
D3
D4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
D4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
D2
D3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
D4
1
1
1
1
1
CELL LINEAGE IN Pleurodeles EMBRYO
ep
ol
ot
op
bn
sc
nc
hm
nt
so
pr
lp
he
bc
st
ph
br
li
ag
pg
pt
A2
1
1
1
1
1
abbreviations in Table 1 footnote.
247
248
DELARUE ET AL.
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