/ . Embryol. exp. Morph., Vol. 17, 2, pp. 341-348, April 1967
Printed in Great Britain
341
Inducing activity in extracts from Xenopus
embryos and from isolated dorsal lips
By E. M. DEUCHAR 1
From the Department of Anatomy, University College London
Tiedemann and his collaborators (Tiedemann, Becker & Tiedemann, 1961) have
demonstrated both neural- and mesodermal-inducing activity in extracts from
whole gastrulae and neurulae and from ventral gastrula ectoderm of Triturus
alpestris. They have also obtained a wide range of inductions from subcellular
fractions of 9-day chick embryos (Tiedemann, Kesslring, Becker & Tiedemann,
1962). Microsomal and ribosomal fractions were particularly active. A further
finding was that the inducing activity of gastrula ectoderm could be enhanced
by phenol treatment (see Tiedemann, 1963, for review). Latterly most of the
work has been pursued with chick extracts, and no systematic study of the
inducing ability of extracts from different embryonic stages of an amphibian has
so far been reported.
In the present work, Xenopus embryos were used as source material because
of their ready availability in large numbers at all times of year. Since microsomal
and mitochondrial fractions from amphibian embryos have previously been
found to be active in neural and mesodermal induction (Yamada, 1961; KocherBecker & Tiedemann, 1966), a simple method was used to obtain mitochondriaand microsome-rich fractions from blastulae, gastrulae, tailbud stages and
dorsal lips of Xenopus laevis. These extracts, after drying to pellet form, were
tested for inducing ability by implantation of pieces into the blastocoel of early
gastrulae. It was of some interest to see if there would be maximal inducing
activity at the early gastrula stage, falling off later, as the classical grafting
experiments (see Spemann, 1938) have always indicated for induction by living
mesoderm. If the extracts used here have any relation to the normal inducing
agent (a matter which is often in doubt, particularly when foreign tissues are
used as sources of extracts) it should also be expected that extracts from isolated
dorsal lips would show particularly high inducing activity. Some extracts from
samples of dorsal lips were therefore prepared. The effects of phenol on the
inducing activity of all extracts was also tested, to see if Tiedemann's findings
were borne out in the case of Xenopus embryo extracts.
1
Author's address: Dept. of Anatomy, University College, Gower Street, London,
W.C.I, U.K.
342
E. M. DEUCHAR
MATERIALS AND METHODS
Embryos of the South African clawed toad, Xenopus laevis, were demembranated with watchmaker's forceps in Holtfreter saline, and dorsal lips (in series 4)
were dissected off with mounted steel needles. Samples of 100 whole embryos,
or 200 dorsal lips, were transferred to chilled, buffered sucrose-saline which
had the following composition: 0-25 M sucrose; 005 M tris buffer, pH 8-0;
0-002 M MgCl2; 0-025 M KC1. After two washes in this saline the embryos were
taken up in a minimal volume of it (c. 0-2 ml) and sucked up and down 3-5
times into a chilled syringe through a wide-bore needle (gauge 20,0-90 mm bore).
This procedure broke the cells of early stage embryos without damaging the
nuclei or yolk-platelets; with tailbud-stage embryos, however, a light homogenization in a Potter-Evejehm homogenizer was necessary first. In preliminary
trials the homogenate was examined under a phase-contrast microscope to
check that cell disruption to the right degree had occurred.
The homogenate was then pipetted gently on to the surface of 1 ml of sucrosesaline (similar to the above except that it contained 0-35 M sucrose) in a narrow
2 ml centrifuge tube. After centrifugation in the cold at 3000 rev/min for 10 min
phase-contrast examination of the layers showed that large yolk platelets, nuclei
and most of the pigment fell to the bottom of the tube, smaller yolk platelets,
some pigment and some mitochondria were at the interface between the two
sucrose solutions, and the upper sucrose layer contained mainly mitochondria,
with some small yolk platelets too. There was a lipid cap on the upper surface.
The upper sucrose layer was saved and recentrifuged to throw down most of the
yolk. The supernatant was retained as the extract for induction tests.
Normal extracts. The supernatant was dialysed for 24 h in the cold against
double-distilled water (two changes of 5 1. each) to remove any toxic components
of the buffered sucrose. Two volumes of absolute ethanol were then added and
after 30 min in the cold a white precipitate formed which was centrifuged at
3000 rev/min for 10 min. The precipitate, after washing and recentrifuging once
in 96 % ethanol and once in 66 % ethanol, was dried in a vacuum desiccator for
2 h. It was stored at 2 °C until used for implanting into gastrulae.
Phenol-treated extracts. The sucrose supernatant was not dialysed, but instead
was mixed immediately with an equal volume of 80 % (v/v) phenol (previously
purified by redistilling three times). The mixture was stirred on a water bath at
60 °C for 5 min. After chilling on ice it was centrifuged at 3000 rev/min for
15 min. The upper (aqueous) layer was discarded and the lower (phenol) layer
was extracted three times with an equal volume of 0-05 M NaCl to remove
sucrose and other contaminants, recentrifuging at 3000 rev/min for 15 min
each time. Four volumes of methanol were then added, and after 10 min in the
deep freeze a flocculent precipitate formed which was centrifuged for 10 min at
3000 rev/min. The precipitate was washed once with 96 % ethanol, three times
with 1:1 ethanol: ether (peroxide-free), a further four times with 96 % ethanol
Inducing activity in Xenopus extracts
343
and then twice with 66 % echanol (c. 5 ml for each wash). It was then dried in a
vacuum desiccator for 2 h and stored at 2 °C before use. The numerous
washes were necessary in order to ensure removal of the phenol, which would
be toxic in implants.
Implantation. The dried extract pellets were put into Holtfreter saline for \ h
to soak them and to get rid of air bubbles, then pieces were cut off with a needle
and inserted into the blastocoels of early gastrulae of Xenopus. They were left
1-1 •£• h to heal (no longer, or exogastrulation was liable to occur), then transferred
to agar-covered dishes of one-tenth Holtfreter saline and kept till hatching stages.
They were finally fixed in Bouin's fluid, dehydrated, and cleared in methyl
benzoate. They were embedded in wax and sectioned at 10-13 fi, then stained
with Weigert's haematoxylin and eosin and mounted in DPX mounting medium.
EXPERIMENTAL RESULTS
(1) Extracts from blastulae (stage 8 of Nieuwkoop & Faber, 1956)
(a) Normal extracts. Eighty-seven embryos with implants of blastula-stage
extracts survived. Thirty-one of them (= 35-6%) developed induced secondary
tissue. This was in no way connected with the host's axis: usually it lay ventrally
but in a few cases it was lateral. In seventeen embryos there was apparently a
complete secondary tail with neural tube and, in sixteen of them, chorda and
somites too. The results are set out fully in Table 1 (top row), and the percentages
of each type of induction calculated in Table 2. The induction classed as
' neuroid' was a large, solid mass of neural tissue which might have represented
incipient brain. In the two cases described as ' undifferentiated mesoderm' a
round, knob-like process was present, covered with epidermis and having a
solid core of apparently undifferentiated mesoderm cells. In contrast the two
cases headed 'mesenchyme and blood' showed loosely arranged, rounded cells
within the epithelial protrusion. The ' epithelial thickenings' of the right-hand
column of the Table appeared to be due to epidermal hyperplasia and were not
clearly neural or neuroid in character.
(b) Phenol-treated extracts. Fifty-one of these embryos survived. There were
only 11 with inductions (21-6%) (see Tables 1 and 2, second row) and only 4 of
these included neural tissue; the others were of mesodermal tissue excepting
for one in which there was apparent thickening of the epidermis.
(2) Extracts from early gastrulae (Nieuwkoop & Faber's stage 10-10%).
(a) Normal extracts. There were 58 survivors of these implants. The yield of
inductions (Tables 1 and 2, third row) was again not high, only 18 (= 31 %) in
all. Eight had recognizable neural tube, 1 was classed as 'neuroid', and 6
showed epidermal thickenings, usually with finger-like outgrowths resembling
an extra fin-tip. The rest of the inductions were mesodermal.
(b) Phenol-treated extracts. Out of 30 successful implants, 7 (= 23-3%)
22
J E K M 17
344
E. M. DEUCHAR
showed inductions. None of these was neural: three were mesenchyme and
epithelial processes: the other 4 were epithelium alone (Tables 1 and 2, fourth
row).
Table 1. Numbers of inductions of various types, in each
experimental series
Total
embryos
Total
inductions
(a)* 87
(b)* 51
31
11
MesenNeural
Undiff. chyme and Epithelial
tube Neuroid Chorda Somites meso.
blood
thickenings
17
4
Series 1: blastula
1
23
23
—
2
5
2
5 4
3
2
3
6
7
1
(a) 58
(b) 30
18
7
6
Series 2: gastrula
—
10
9
(a) 105
(6)41
43
26
15
9
Series 3: tailbud
1
16
15
1
9
9
12
13
1
16
11
(a) 38
(b) 32
27
22
6
3
Series 4: dorsal lip
3
5
4
3
5
1
7
10
—
13
12
* (a) Normal extract; (b) phenol-treated extract.
Table 2. Percentages of inductions of various types in each
experimental series
Embryos
All
types of
MesenUndiff. chyme and Epithelial
induc- Neural
tion
tube Neuroid Chorda Somites meso.
blood
thickenings
100
(b)* 100
35-6
21-6
19-6
7-8
Series 1: blastula
1-2
26-4
26-49-8
7-8
—
2-3
9-8
2-3
—
—
20
(a) 100
(b) 100
310
23-3
10-3
—
Series 2: gastrula
—
17-2
15-5
—
—
—
5-2
—
3-4
100
10-3
23-3
400
63-4
14-3
21-9
Series 3: tailbud
15-2
14-3
0-9
2-4
21-9
21-9
11-4
31-7
0-9
—
15-2
26-8
15-8
9-4
Series <X: dorsal lip
7-9
13-2
10-5
9-4
15-6
31
18-4
31-2
—
—
34-2
37-5
(a) 100
(b) 100
(a) 100
(b) 100
711
68-8
!
(a) Normal extract; (b) phenol-treated extract.
Inducing activity in Xenopus extracts
345
(3) Extracts from tailbud stage embryos (Nieuwkoop & Faber's stage 24-25)
(a) Normal extracts. There were a total of 105 survivors, and 41 of these
( = 4 0 %) showed induced secondary tissue, i.e. a very slightly higher percentage
than with other extracts (compare the percentage figures in Table 2). Fifteen
of the inductions appeared to be secondary tails, with neural tube, notochord
and somites all showing. One further neural induction was of the 'neuroid' type.
In addition there were 16 inductions of epithelial thickenings, and 12 inductions
of knobs of undifferentiated mesoderm. The data are given fully in Tables 1 and 2.
(b) Phenol-treated extracts. There were only 41 survivors, but a higher
proportion than usual (26, i.e. 63-4%, see Table 2) showed inductions. The
distribution as between neural and mesodermal inductions was much the same
as with the normal extracts (cf. rows 5 and 6 in Tables 1 and 2).
(4) Extracts of early gastrula dorsal lip tissue
(a) Normal extracts. Out of 38 implanted embryos, 27 (= 71%) showed
inductions, a considerably higher percentage than with the other extracts
(see Table 2). Though only 6 ( = 15-8 %) of these showed neural tube, an additional 3 ( = 7-9 %) had neuroid tissue. Four of the inductions were of complete
secondary tails and therefore had chorda and somites as well as neural tube. But
by far the most frequent types of induction were epithelial thickenings and
outgrowths (see Tables 1 and 2, row 7).
(b) Phenol-treated extracts. Of the 32 survivors, again a high proportion
(22; 68-8%) had inductions. The majority of these were epithelial processes or
thickenings. Only 3 (= 9-4%) showed neural tube, though an additional 3 had
neuroid tissue. On the whole, there was a slight increase in the percentages of
mesoderm inductions as compared with group (a) (cf. rows 7 and 8, Table 2),
but the numbers are too small for the difference to be statistically significant.
DISCUSSION
Judging from the percentages of embryos in each series that showed any
induced tissue at all, it would appear that the tailbud-stage extracts were a little
more active as inductors than were the extracts from blastulae and gastrulae.
None of the totals were very high, however. This may have been partly due to
the fact that some of the implants were extruded a few hours after the operation.
They were found lying in the agar dishes, but as it could not be determined with
certainty which embryos had extruded them, it was not possible to correlate
extrusion of the implant with failure of induction.
Surprisingly, the early gastrula extracts did not give very high percentages
of inductions. Dorsal-lip extracts, however, were apparently more active than
those from whole embryos. This encourages one to believe that the inducing
factors operating here may have some relation to those normally active in the
living embryo, which should be most highly concentrated in the dorsal lip of
346
E. M. DEUCHAR
the early gastrula. It was unfortunate that the small bulk of extract obtainable
from these small pieces of tissue made it possible to do only small numbers of
implants in this series, but the numbers used are sufficient to point up the
difference for whole embryo extracts. It was clear too that there is both mesodermal and neural activity present. A further point of interest was the higher
percentage of neuroid (perhaps incipient brain) inductions in this group than
in the other series (where they were very rare).
The effects of phenol treatment were not at all clear. The number of neural
(and of total) inductions was apparently reduced by phenol treatment of
gastrula extracts, and there was no proportionate increase in mesoderm inductions. Only with the tailbud extracts was there a higher proportion of total
inductions after phenol treatment, and the percentages of mesodermal inductions were increased very slightly more (taking all kinds together, including
'undifferentiated' mesoderm) than were neural inductions. In dorsal-lip
extracts after phenol treatment, there was also an increase in the percentage of
undifferentiated mesoderm inductions, but on the other hand a decrease in the
somite inductions.
As pointed out under Methods, the supernatant fraction used for extracts
contained, besides mitochondria, the bulk of the ribosomes. That it should
show considerable neural and mesodermal inducing capacity is not surprising.
This agrees with Yamada's (1961) findings on extracts from Urodele embryos,
and with Tiedemann et al. (1962) who reported finding spinocaudal inducing
activity in 'microsomal' fractions from 9-day chick embryos. They also noted
hindbrain induction by the chick embryo material, however, which could not be
identified in the present Xenopus series, though some of the few 'neuroid' cases
may really have been underdeveloped brain tissue. Holtfreter (personal communication, 1958) has, however, observed that in operations on Xenopus, unlike
other amphibians, it is very rare to obtain inductions of anything other than
trunk and tail.
We do not know of any special role that mitochondria may have as inductive
agents. It may be remarked that their DNA, the presence of which has recently
been confirmed by Dawid (1966), should have been retained in the phenol
treatment as well as in the dialysis, so that the inductions produced by both
types of extract could be at least partly due to activity of their DNA or its
products. Until purer fractions are obtained of separated mitochondria and
ribosomes the inducing properties of the mitochondria themselves cannot, of
course, be assessed. In other tissues, somes are active inductors.
It may be concluded from these experiments that active inducers of neural
tube and mesoderm can readily be extracted from the small-particle supernatants
of blastula, gastrula and tailbud stages of Xenopus laevis, and that a more active
fraction is obtainable from the dorsal lip alone. TJie inducers remain active
after phenol treatment, but there is no evidence so far to suggest that phenoltreatment of the extracts enhances either neural or mesoderm induction.
Inducing activity in Xenopus extracts
341
SUMMARY
Supernatant fractions, containing mitochondria and ribosomes, were obtained
from homogenates of early gastrulae, neurulae, tailbud stages, or dorsal lips of
Xenopus laevis. These supernatants were either dialysed and then precipitated
with ethanol (normal extracts), or treated with hot phenol, then precipitated
after removal of the phenol. Both types of extracts were tested for inducing
activity by implanting pieces of the dried pellets into the blastocoels of early
gastrulae of Xenopus. All extracts gave both mesodermal and neural tube
inductions but none gave brain, though there were a few neuroid masses that
may have represented poorly formed brain.
The extracts of dorsal-lip tissue gave much higher proportions of inductions
(c. 70 %) than those from whole embryos (30-40 %).
There was no significant difference in inducing activity between the extracts
of whole embryos at different stages.
Phenol treatment did not appear to enhance the inducing activity of extracts,
but had varying effects in the different experimental series.
RESUME
Activite inductrice dans des extraits d'embryons de Xenopus et de
levres dorsales isolees
Des fractions de surnageant, contenant mitochondries et ribosomes, furent
obtenues a partir d'homogenats de jeunes gastrulas, de neurulas, et de stades du
bourgeon caudal, ou de levres dorsales de Xenopus laevis. Ces surnageants
furent dialyses et ensuite precipites par l'ethanol (extraits normaux), ou bien
traites par le phenol chaud, puis precipites apres elimination du phenol. Les
deux types d'extraits furent eprouves pour l'activite inductrice en implantant
des morceaux de comprinees seches dans les blastoceles de jeunes gastrulas de
Xenopus. Tous les extraits donnerent a la fois des inductions mesodermiques et
de tube neural, mais aucun ne donna du cerveau, bien qu'il y eut quelques
masses neuroides pouvant representer un cerveau mediocrement differencie.
Les extraits de levre dorsale donnerent des proportions bien plus fortes
d'inductions (70%) que ceux d'embryons entiers (30-40%).
II n'y avait pas de difference significative dans l'activite inductrice entre les
extraits d'embryons entiers des differents stades.
Le traitement par le phenol n'a pas semble renforcer l'activite inductrice des
extraits, mais il a eu des effets varies dans les differentes series experimental.
I should like to thank Professor Tiedemann and his colleagues for the facilities given me
at the Max-Planck Institut, Wilhelmshaven, where this work was carried out. It is a pleasure
to thank Miss Gill Weedon and Miss Susan Bevan for help with the histological work and Miss
C. Martin for typing the manuscript.
348
E. M. DEUCHAR
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(Manuscript received 3 October 1966)