/. Embryol. exp. Morph. Vol. 33, 4, pp. 969-978, 1975
Printed in Great Britain
969
Xenopus laevis cement gland as an experimental
model for embryonic differentiation
II. The competence of embryonic cells
By J. J. PICARD 1
From the Laboratoire d'Embryologie, Louvain-la-Neuve
SUMMARY
Explants of the blastocoelic roof of Xenopus laevis embryos were incubated for 6 h in
Holtfreter solution containing 10 mM ammonium chloride and 10 mM sodium bicarbonate.
After this incubation they were transferred for 5 days to Barth's physiological salt solution.
Under these conditions the explants underwent differentiation into cement gland tissue. The
ability to produce cement gland was highest when explants were dissected from young
gastrulae (stage 10). Explants from younger and older embryos displayed much smaller
cement glands.
There is no difference in the response of ventral or dorsal portions of the blastocoelic roof
of stage-10 embryos under these in vitro conditions involving ammonium chloride stimulation.
Although the treatment changed the fate of some ectoblastic tissues, it was unable to force
endodermal or mesodermal tissues of the blastoporal lip to differentiate into cement gland.
The amount of cement gland in the ectoblastic explants varied according to the egg-batch
and on the average accounted for 55 % of the total tissue. The remaining tissue was undifferentiated. Most of this undifferentiated tissue originates from the adjacent layer of the
ectoblast. Therefore it may be estimated that the cement gland accounts for 80 to 100 % of
the volume of the competent superficial layer.
INTRODUCTION
Ectoblastic cells of young Xenopus laevis gastrulae explanted in standard salt
solutions for 5 days produce atypical epidermis. However, when these explants
are first incubated for 6 h in Holtfreter solution containing 10 mM ammonium
chloride at pH 7-5 and are then transferred for 5 days to a standard salt solution
they undergo differentiation into typical cement gland (Picard, 1975). Under
these in vitro conditions about 80 to 90 % of the explanted tissue is cement
gland and the additional 20 to 10 % remains undifferentiated.
The aim of the present article is to specify more precisely which cells of the
embryo are able to differentiate into cement gland after stimulation with 10 mM
ammonium chloride. This problem deals with the position of the cells in the
embryo and the time they become competent to respond to the stimulation.
We also wanted to know whether the fate of gastrula cells which differentiate
1
Author's address: Laboratoire d'Embryologie, Sciences 12, 1348 Louvain-la-Neuve,
Belgium.
970
J. J. PICARD
in vivo and in vitro into ectodermal or mesodermal tissues may be changed by
ammonium chloride stimulation and forced to differentiate into cement gland.
Finally, the variability of the results according to the egg-batch will be estimated.
MATERIAL AND METHODS
The animals (Xenopus laevis, Daudin), the methods and the experimental
conditions were the same as previously described (Picard, 1975). The volumes
of individual tissues were estimated by morphometry of histological sections
(Hennig & Meyer-Arendi, 1963).
RESULTS
(1) The temporal competence of ectoblastic cells
Explants were prepared from embryos ranging from stage 8 to stage \\\
(Nieuwkoop & Faber, 1967). The explanted tissue was a cup of the blastocoelic
roof extending from about 20° below the animal pole dorsally to about 70°
below the animal pole ventrally.
The explants were first incubated for 6 h in a modified Holtfreter solution
containing 10 mM ammonium chloride and 10 mM sodium bicarbonate at
pH 7-6. After this stimulation they were cultured for 5 days in a standard salt
solution (Barth & Barth, 1959), processed for histological examination and the
individual volumes estimated.
The mean total volume of individual explants is given in Fig. 1 A. This volume
decreased progressively to reach at stage lOJr a value of 59 % of the volume
observed when the explants were prepared from embryos at stage 8. The total
volume of explants from embryos at stages 11 and 1 1 | was slightly higher than
the volume observed at stage 10^.
The mean volume of cement gland per explant is given for each stage in
Fig. 1B. The response of the ectoderm to incubation in ammonium chloride
was weak at stages 8 and 9. The best differentiation occurred in stage 10 explants.
Explants from older gastrulae formed smaller cement glands under the same
conditions.
The percentage of the total explanted tissue occupied by the cement gland was
low at stages 8 and 9, reached a maximum (62 %) at stages 10 and 10|, and finally
decreased to a value of about 2 % at stage 11^ (Fig. 1C).
The cement gland was the only differentiated tissue to be observed in the
explants. The remaining tissue was atypical epidermis. Therefore, in each series
the total explanted tissue is the sum of the undifferentiated tissue and the
cement gland tissue. This undifferentiated tissue was predominant in stage-8
explants, decreased at stage 10^ to about 24 % of the stage-8 value and was
again predominant at stage 11^ (Fig. ID).
In vitro studies on Xenopus cement gland. II
60
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Fig. 1. Influence of a 6 h incubation in 10 HIM ammonium chloride on explants of
the blastocoelic roof at different stages. The volumes were measured after 5 days of
culture in Barth's solution. The ordinate indicates the average volume of several
explants whose number is given. Vertical bars are the confidence limits at 95 %.
For each explant the total volume is the sum of the volume of the cement gland
and of the undifferentiated tissue. A, Total volume of the explanted tissue; B, volume
of the cement gland; C, relative volume of the cement gland; D, volume of the
undifferentiated tissue.
(2) The regional competence of ectoblastic cells
Two series of circular pieces of the ectoderm were dissected from stage-10
embryos. The centre of the explants was situated in the sagittal plane. The
explants extended from 5° to 50° from the animal pole, ventrally in the first series
and dorsally in the second one (Fig. 2).
The explants were stimulated for 6 h in 10 mM ammonium chloride as described previously. The volumes of the tissues were measured after 5 days of
culture in Barth's solution.
Table 1 shows that the ventral and dorsal portions of the ectoblast have about
the same ability to respond to the stimulation by producing cement gland tissue.
The mean cement gland volumes per explant are not statistically different. The
972
J. J. PICARD
Blp.
P. I V.
Fig. 2. Diagram of a median section through a stage-10 gastrula indicating the
position of explanted portions, v and d, ventral and dorsal portions respectively
explanted in the experiment described in section 2. Expl, upper blastoporal lip
explanted in the experiment described in section 3. P.A., Animal pole; P.V.,
vegetal pole; Blp., blastopore; Blc, blastocoelic cavity.
percentages of the total explanted tissue occupied by the cement gland are very
similar in both the ventral and dorsal portions of the ectoblast. No other differentiated tissue was observed in these explants. The mean total explanted
tissue was significantly lower in the series of dorsal ectoderm than in the series
of ventral ectoderm. This may be explained either by a more important tissue
loss in the dorsal explants during the incubation or more probably by a smaller
initial volume due to restraint during the dissection of dorsal explants to avoid
reaching mesoblastic tissues.
(3) Influence of ammonium chloride on the differentiation of the
upper blastoporal lip
The upper blastoporal lip of stage-10 embryos was dissected. The lower limit
of the explants was the rim of the lip and the upper limit was about 5° above the
equatorial plane (Fig. 2). Laterally, the explants extended to about 15° from the
plane of symmetry.
The explanted lips were divided into two series. The control series was first
cultured for 6 h in Holtfreter solution and then transferred for 5 days in Barth's
solution. The experimental series was incubated for 6 h in Holtfreter solution
containing 10 mM ammonium chloride and 10 mM sodium bicarbonate. After
this incubation the explants were cultured for 5 days in Barth's solution.
In vitro studies on Xenopus cement gland. II
973
Table 1. Influence ofa 6 h stimulation with 10 mM ammonium chloride on ventral
and dorsal portions of the ectoblast of stage-10 embryos
The volumes measured after 5 days of culture in Barth's solution are given with
their standard deviation, S.D. The number of explants, n, is given in the first column.
The total tissue is the sum of cement gland and of undifferentiated tissue.
Volume, 106/tm3±s.D.
f
Ectoblast
Ventral
in = 13)
Dorsal
(n = 12)
/ test
Undifferentiated
tissue
38-9
± 19 2
260
±15-2
1-79
Not significant
Cement
gland
23-1
±17-6
171
±7-5
110
Not significant
Total
tissue
620
±16-3
431
± 13-5
309
P< 0005
Percentage
cement
gland
37-5
39-6
The volumes of the tissues are given in Table 2. Cement gland, neural tissue
and notochord were measured separately. The volumes of the remaining tissues,
containing muscle, kidney tubules and endodermal structures, were measured
together. The mean total volume of the explants remaining after 5 days of culture
was markedly lower in the experimental series than in the controls. The volume
of the former was about 59 % that of the latter. The difference is highly significant. The observation of living explants had shown that they were losing cells
during the first 3 days of culture mostly from the underlying layer.
All the tissues which were measured had a volume significantly smaller in
the experimental series than in the control series. The notochord was the most
affected and accounted for only 28-6 % of the volume observed in the controls.
This proportion was respectively 47-7 % and 60-4 % for cement gland and
neural tissue.
The percentage of the total explanted tissue occupied by cement gland and
neural tissue was approximately equal in the two culture series. On the other
hand this percentage was only 7-7 % for notochordal tissue in the experimental
series compared with 16-0 % in the control series.
(4) The response to ammonium chloride stimulation in individual egg-batches
In order to estimate the reproducibility of the results, ectodermal explants
from stage-10 embryos belonging to twelve different egg-batches were stimulated for 6 h by 10 mM ammonium chloride and subsequently cultured for 5 days
in Barth's solution (Table 3).
The mean volume of the cement gland per explant varied from 10-3 x 106 /tma
to 36-0 x 106 jLtm3. The weighted mean was 24-9 x 106 /*m3. The percentage of the
total explanted tissue occupied by the cement gland varied from 26-1 % to 80-4 %
and the percentage corresponding to the weighted means was 55-0 %.
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J. J. PICARD
Table 2. Influence of a 6 h stimulation with 10 mM ammonium chloride on
dorsal blastoporal lip
The control lips were incubated for 6 h in Holtfreter solution. Both series were
cultured for 5 days in Barth's solution. For each tissue measured, the volumes
are given in /tm 3 (xl0~ 6 ) with their standard deviation and the corresponding
percentage of total tissue. The volume of each tissue in the experimental series is
compared with the corresponding tissue in the control series (treatment x 100/
control). The column on the right gives the statistical analysis of the results.
Tissues
Volume
Percentage total
tissue
Treatment x 100
control
Control
(n = 20)
NH4C1, 10
(/i = 20)
Cement gland
11-4
5-5
±7-3
±4-8
test
306
P < 0005
71
8-8
47-7
Neural
Volume
Percentage total
tissue
Treatment x 100
control
28-2
±8-0
28-9
36-7
±6-5
28-3
6-32
P < 0001
60-4
Notochord
Volume
Percentage total
tissue
Treatment x 100
control
5-9
20-8
±5-9
160
±40
9-72
P < 0001
7-7
28-6
Others
Volume
Percentage total
tissue
61 1
±22-9
470
43-3
± 15-8
2-86
P < 001
56-3
Treatment x 100
control
70-9
Total tissue
Volume
Treatment x 100
control
1300
±34-2
76-9
±23-1
591
5-76
P < 0001
In vitro studies on Xenopus cement gland. II
975
The weighted means of the total and of the undifferentiated tissue per
explant were 45-1 x 106/*m3 and 20-3 x 106/*m3, respectively.
In order to check the homogeneity of the variances Bartlett's test was applied
to these data (Snedecor, 1959). Calculations indicated a x2 of 62-8 (P <| 0-01)
Table 3. Influence of the layings on the response of the ectoblastic explants to a
6 h stimulation with 10 mm ammonium chloride
The volumes were measured after 5 days of culture in Barth's solution. Standard
deviations are given with the volumes. The weighted means of the three tissues
were calculated from the data of the twelve experimental series.
Volume, 10 6 /*m 3 ±s.D.
Date
n
9. xi. 72
15
21. xi. 72
39
22. xi. 72
13
11. i. 73
15
24. i. 73
18
21. ii. 73
16
28. ii. 73
15
21. iii. 73
9
30. iii. 73
14
11. iv. 73
18
18. iv. 73
17
19. iv. 73
18
Weighted mean
Undifferentiated
tissue
Cement
gland
Total
tissue
15-9
±9-5
12-2
±5 4
7-6
±80
20-6
±11-4
14-7
±83
32-4
±14-2
15-7
± 10 9
32-3
±25-8
32-4
330
±11-4
360
±12-3
28 1
± 13-1
190
±10-1
33-2
±11-7
11-4
±90
17-5
±7-2
191
±8-7
20-8
48-9
±14-4
48-2
±110
35-7
± 17-4
596
±90
47-9
±13-8
43-8
±140
33-2
±9-6
±8-8
25-7
±7-1
260
±13-8
23-6
± 10-7
20-3
10-3
±7-1
22-7
± 11-8
28-4
±9-2
24-9
Percentage
cement
gland
67-5
74-7
80-4
47-9
69-3
261
52-7
±7-9
51-4
±16-8
53-2
±12-6
360
37-2
391
28-6
±6-2
48-7
± 15 6
520
±120
45 1
46-7
54-6
550
for undifferentiated tissue, of 14-0 {P < 0-3) for cement gland tissue and of 26-7
(P < 0-01) for total explanted tissue. The variances are therefore only homogeneous for the volumes of the cement gland. The heterogeneity of variances of
the total explanted tissue appears to be essentially the result of the heterogeneity
observed in the undifferentiated tissue.
976
J. J. PICARD
Analysis of variance was applied to the data corresponding to the volumes of
the cement gland and the variance ratio was calculated to be F = 14-0
(P <^ 0-01). Hence the amount of cement gland appearing in the explants as
a result of a 6 h ammonium chloride stimulation varies significantly according
to different egg-batches.
DISCUSSION
The response of ectoblastic cells to a 6 h stimulation with ammonium
chloride varies considerably according to the stage of the embryos. The beginning of gastrulation (stage 10) is the time when the explants produce the greatest
amount of cement gland tissue under these conditions (Fig. 1B). This stage
may be accepted as the time of optimum competence and will be routinely used
in further studies on this experimental mode. The mean volume of the total
tissue per explant measured after 5 days culture in Barth's solution also varied
according to the stage of the embryos (Fig. 1 A). The lowest value was observed
at stage 10^. This may probably be explained by the epibolic movements
occurring during gastrulation. Indeed, as a result of these cellular movements
the blastocoelic roof becomes thinner and hence the same explanted area will
have a smaller starting volume as gastrulation proceeds. A slight increase of
the mean volume of the total tissue per explant occurs at stages 11 and 11|.
This probably results from a better resistance of older ectoblastic cells to
incubation in ammonium chloride. Indeed, we observed that explants from
stage 11 and 11£ lose fewer dissociated cells than younger explants.
In these experiments a large portion of the blastocoelic roof was explanted.
If smaller explants had been used, the results could have been very different
according to the presence or absence of the presumptive cement gland area in
the explanted tissue. It could indeed be possible that only this presumptive
area is able to respond to ammonium chloride stimulation and that the
remaining ectoblast would only produce atypical epidermis. The presumptive
area of the cement gland on amphibian gastrulae is located either ventrally
(Discoglossus) or dorsally (Bombinator) as regards the animal pole of the
embryo (Vogt, 1929). The fate map of Xenopus laevis gastrula is not known.
However, the fate maps of the two above-mentioned anuran species indicate
that the presumptive areas of their cement glands do not exceed 10° in the
sagittal plane. This most probably applies to the presumptive area in Xenopus
laevis too. Therefore, in the experiment described in section 2 the presumptive
area of the cement gland will not be situated in both explanted portions. The
volume of the cement gland was not significantly different in these two portions
(Table 1). It is therefore safe to conclude that other parts of the ectoblast and
possibly the whole ectoblast is able to differentiate into cement gland in response to ammonium chloride stimulation. Hence the fate of certain parts of
the ectoblast can be changed by this treatment. Moreover, if the presumptive
In vitro studies on Xenopus cement gland. II
977
area was situated in one of the two explanted portions it would appear that the
ability of that area to produce cement gland tissue in response to ammonium
chloride stimulation is not greater than that of the remaining ectoblast.
It is well known that blastoporal lips of young amphibian gastrulae explanted
in a physiological salt solution will differentiate mainly into cephalic endodermal and mesodermal structures (Holtfreter, 1938). When Xenopus laevis
blastoporal lips were incubated for the first 6 h in 10 mM ammonium
chloride the treatment was unable to change massively the fate of this tissue
(Table 2). Under these conditions the volume of the total tissue of explanted
blastoporal lips remaining after 5 days culture was 41 % lower than in the
controls. All the tissues suffered from the loss of material and the most affected
one was the notochord. The volume of the cement gland was also smaller in
the experimental series. This is probably not due to a direct inhibition of the
differentiation into cement gland. Indeed, the explants contain the natural
inductors of the cement gland, i.e. endoderm and/or brain (Yamada, 1933,
1938). As the latter tissues are affected by the treatment, it seems more likely
that the smaller volume of the cement gland is due to an indirect influence.
The response of the explants to incubation in ammonium chloride displays
some quantitative variations according to egg-batches. The 12 identical experiments performed over a five-month period are insufficient to disclose a relationship with seasonal changes. The most important variability was observed in the
undifferentiated tissue. The volume of the cement gland appearing in the
explants also differs significantly according to the egg-batches. This biological
variability is not surprising and may have numerous causes which are impossible to analyse.
In these series the weighted mean of the volume of the cement gland per
explant was 24-9 x 106 /«n3. This accounts for more than twice the volume of an
individual cement gland differentiated in vivo (unpublished results). Cells of
cement glands differentiated in vivo and in vitro will probably display approximately the same volume. Therefore these results represent an independent confirmation that the ability of the ectoblast to differentiate into cement gland in
response of ammonium chloride stimulation is found in a broader region than
the presumptive area.
On average the cement gland tissue accounted for 55 % of the total tissue
measured after 5 days of culture. The cement gland originates exclusively from
the superficial layer of the ectoderm (Lieberkind, 1937). After 5 days of culture
it is not possible to detect by histological means what part of the undifferentiated tissue originates either from the superficial or from the underlying layer
of the ectoderm. However, results to be published elsewhere have demonstrated
that the volume of the underlying layer accounts for about 62 % of the ectodermal portion at the time of explantation and that the greater part of this
tissue remains in the explants during cultivation. Therefore most of the
undifferentiated tissue (45 % of total tissue) originates from the underlying
978
J. J. PICARD
ectodermal layer. A rough estimation indicates that after a 5-day culture the
cement gland accounts for 80-100 % of the volume of the superficial layer.
This conclusion is interesting in view of the possibility of isolating cells from
the latter layer by density gradient centrifugation (Morrill & Kostellow, 1965;
MacMurdo & Zalik, 1970, 1971). This improvement would reduce further the
volume of contaminating undifferentiated tissue.
Finally, the absence of any significant amount of any other differentiated
tissue in the stimulated explants is important because the homogeneity of the
material will facilitate interpretation of biochemical studies.
REFERENCES
L. G. & BARTH, L. J. (1959). Differentiation of cells of the Rana pipiens gastrula in
unconditioned medium. /. Embryol. exp. Morph. 7, 210-222.
HENNIG, A. & MEYER-ARENDI, J. R. (1963). Microscopic volume determination and probability. Lab. Invest. 12, 460-464.
HOLTFRETER, J. (1938). Differenzierungspotenzen isolierter Teile der Anurengastrula. Wilhelm
Roux Arch. EntwMech. Org. 138, 657-738.
LIEBERKIND, I. (1937). Vergleichende Studien iiber die Morphologie und Histogenese der larvalen Haftorgane bei den Amphibien. Kobenhavn: C. A. Reitzels Forlag.
MACMURDO, H. L. & ZALIK, S. E. (1970). Embryonic cell surface: electrophoretic mobilities
of blastula cells. Experientia 26, 406-408.
MACMURDO, H. L. & ZALIK, S. E. (1971). Microelectrophoresis of early amphibian embryonic cells. Devi Biol. 24, 335-347.
MORRILL, G. A. & KOSTELLOW, A. B. (1965). Phospholipid and nucleic acid gradients in the
developing amphibian embryo. J. Cell Biol. 25, 21-29.
NIEUWKOOP, P. D. & FABER, J. (1967). Normal table o/Xenopus laevis (Daudin), 2nd ed.
Amsterdam: North-Holland Publ. Co.
PICARD, J. J. (1975). Xenopus laevis cement gland as an experimental model for embryonic
differentiation. I. Stimulation of differentiation by ammonium chloride. /. Embryol. exp.
Morph. 33, 957-967.
SNEDECOR, G. W. (1959). Statistical methods, 5th ed. Ames, Iowa: Iowa State College Press.
VOGT, W. (1929). Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung. 2.
Teil: Gastrulation und Mesodermbildung bei Urodelen und Anuren. Wilhelm Roux Arch.
EntwMech. Org. 120, 384-706.
YAMADA, T. (1933). Uber die Determination der Haftdriisen bei Rana nigromaculata. J. Fac.
Sci. Tokyo Univ. Sect. IV 3, 239-254.
YAMADA, T. (1938). Weitere Analyse der Determination der Haftdriise bei Rana nigromaculata, mit einigen Bemerkungen iiber die Induktion anderer Kopforgane. /. Fac. Sci. Tokyo
Univ. Sect. IV 5, 133-163.
BARTH,
(Received 19 November 1974, revised 9 January 1975)