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/. Embryol. exp. Morph. Vol. 21, 3, pp. 383-90, June 1969
383
Printed in Great Britain
The influence of
the nucleus on sequential determination in frog
ectoderm following induction by lithium ion
ByKRYSTYNA D. ANSEVIN 1
From the Department of Biology, Rice University, Houston, Texas
It appears certain that the process of cellular differentiation is an outcome of
interactions between the cell nucleus and cell cytoplasm. Differentiation of
embryonic amphibian ectoderm involves two fairly distinct phases: during the
first short period an inductor (or some intrinsic factor, if an inductor is absent)
determines the course of future differentiation in a multipotential cell; during
the second, longer interval of time presumably a complex sequence of reactions
leads to physiological and morphological differentiation. Little is known about
the nature of reactions which take place during the first phase when the cells
become developmentally determined by the inductor. It appears that the first
step in translation of the inductive instruction in the competent cell is accomplished during the first 2 or 3 h following the treatment with the inductor
(Ansevin, 1966). The step is not actinomycin-sensitive (Ansevin, 1965), as was
shown by cells that completely recovered after actinomycin treatment (in
conditions when it was unlikely that they could have failed to take up some
inhibitor). This suggests that transcription of RNA does not have to occur
during cell determination, but it does not exclude the possibility that induction
removes biological repressors from specific genes which thus become available
to direct cellular differentiation along a specific line. Barth (1965) has drawn
attention to a striking similarity between the power of several inorganic ions to
bind to DNA and the capacity of the same ions to act as embryonic inductors.
He suggested that ions might compete with histones for active sites on DNA
molecules, and by binding on these sites protect the molecules from the inactivating action of histones.
With the present lack of definite evidence for or against the involvement of
genes in cell determination, no hypothesis concerning the nature of induction
can be more than a speculation, and two possibilities have to be explored:
(a) that cell determination involves nuclear activity; (b) that the phenomenon is
restricted to cytoplasmic reactions. The following report presents an attempt to
analyse further the role of cell nucleus and cytoplasm in determination of frog
1
Author's address: Department of Biology, Rice University, Houston, Texas 77001, U.S.A.
25
J E E M 21
384
K. D. ANSEVIN
ectoderm in which the genetic and nucleo-cytoplasmic relations were experimentally modified in various ways. In this study induction and differentiation
were tested on cells in vitro, since Barth & Barth recently found (1966) that cells
of arrested hybrid embryos do not necessarily lose capacity for differentiation:
if isolated and cultured, in some cases they respond to induction and differentiate into a variety of tissues. Lithium ion was employed as the inductor,
since in isolated ectoderm of Rana pipiens gastrula it induces a time-dependent
sequence of cell types (Barth & Barth, 1963):
radial
spreading
epithelium ->• nerve -> nerve ->- melanaphore -> astrocyte ->• neuroglia
MATERIALS AND METHODS
Ovulation was induced in females of Rana pipiens, R. catesbeiana and
R. palustris by pituitary injections. R. sylvatica females had been caught while
in amplexus and contained ripe oocytes in their uteri. The eggs were fertilized
with sperm of either the same or different species.
Table 1. Genetic composition of tested ectoderm and number of cultures that
were prepared from tissues of each nucleo-cytoplasmic combination
Species of cross
R. pipiens diploid
7?. pipiens haploid
7?. palustris diploid
7?. sylvatica diploid
7?. catesbeiana diploid
7?. pipiens 2x7?. sylvatica <5
R. sylvatica 2x7?. pipiens S
R. pipiens 2x7?. catesbeiana 3
R. palustris 2x7?. sylvatica <$
No. of
cultures*
37
72
15
22
31
40
17
18
65
* The average number of aggregates for culture was 10.
Haploid pipiens embryos were obtained by fertilizing pipiens eggs with u.v.inactivated sperm of R. pipiens, or, in other cases, of R. catesbeiana. For irradiation, sperm suspension was pipetted into Syracuse dishes, using only as many
drops per dish as were necessary to cover the bottom. The dishes were placed
under a GE-G8T5 germicidal u.v. lamp and irradiated for 5-10 min. with a
dose of 39-5 erg/mm2/sec. Haploid condition of the embryos from eggs fertilized
with treated sperm was assumed if the haploid syndrome (Porter, 1939) later
occurred in nearly 100 % of the unused embryos from a given batch of eggs.
Table 1 lists the nucleo-cytoplasmic combinations which were experimentally
produced in the course of this study and which characterized the ectoderm
Nucleus in cell determination
385
tested for its ability to respond to induction by lithium chloride at a concentration of 3 mg/ml in the culture medium.
Altogether eggs from ovulations of 8 R. catesbeiana, 10 R. pipiens, 4 R. palustris and 4 R. sylvatica were used in these experiments. Ectoderm was dissected
from stage 11 gastrulae, or, in the case of the hybrids, from an equivalent stage.
Handling of the tissue and preparation of the cultures were similar to those
Table 2. Sequential induction at 25-27 °C in diploid, haploid and
hybrid ectoderm of Rana pipiens
( + ) one or a few cells of this type were found in an occasional culture; + small
numbers of cells of this type were usually present in a culture; + + most or all of
the cells in the culture were of this type.
Outcome
Time of
Species
R. pipiens diploid
to LiCl
(h)
Epithelium
0
++
1
R. pipiens haploid
H
0
*
R. pipiens ? x R. sylvatica 3
H
o
i
1
R. pipiens ? x R. catesbeiana 3
Nerve
Melanophores
-
++
++*
++
++
++
l
+
H
+
-
-
+
(+)
(+)
—
(+)
++
+
+
++
++
All cells died in 1-2 days of cultivation
* Epithelium of this species formed swirling ciliated aggregates instead of tissue sheets
attached to the glass.
employed by the author in previous work (1965, 1966), according to the method
outlined by Barth & Barth (1962). On the average, 12 embryos of the same
nucleo-cytoplasmic type were used for a single experiment. About ten cell
aggregates were included in each culture dish. The cultures were maintained
for 2 or 3 weeks without changing the medium and examined under the microscope every second day. At the end of the culture period they were fixed and
stained with hemalum and acid fuchsin and mounted in toto.
RESULTS
It is evident from Tables 2 and 3 that different rates of response to the
inductor and different patterns of differentiation characterized tissues with
different genetic composition. Pipiens and palustris diploid ectoderm showed a
25-2
386
K. D. ANSEVIN
higher incidence of neural cells as a result of induction by the lithium ion than
catesbeiana or sylvatica ectoderm after similar treatment; however, ectoderm
of the latter two species had lower thresholds for induction to the melanophore
level. Hybrid pipiens $ x sylvatica $ or palustris $ x sylvatica $ ectoderm had a
lower threshold for melanophore induction than either parental species involved
Table 3. Sequential induction at 25-27 °C in the ectoderm of three other
species of Rana and of their hybrids
( + ) one or a few of cells of this type were found in an occasional culture; + small
number of cells of this type were usually present in a culture; + + most or all of the
cells in the culture were of this type.
Time of
Species
R. palustris diploid
Outcome
to LiCl
(h)
Epithelium
0
++
Nerve
Melanophores
1
H
R. palustris ?xi?. sylvatica <?
0
l
i
H
R. sylvatica diploid
0
l
H
R. sylvatica $ x R. pipiens c?t
+
+
+*
+*
+*
+*
0
\
H
R. catesbeiana diploid
0
l
i
H
2
+*
i
*
+*
+*
+*
* Epithelium of this species formed swirling ciliated aggregates instead of tissue sheets
attached to the glass.
f The number of surviving cells of this cross was very small.
in a particular hybrid. Whether the same was true for the cells of the reciprocal
cross R. sylvatica ? x R. pipiens $ could not be stated with certainty, because of
poor viability of these cultures; however, in the surviving cultures nerve cells
were more frequent than might have been expected, if quantitative relations
between cell types had been the same as in the two former crosses, or if sylvatica
cytoplasm had been responsible for the outcome. Thus, both the nucleus and
Nucleus in cell determination
387
the cytoplasm, rather than the nucleus alone, seemed to control the course of cell
determination. It should be added that the modified patterns of cell determinations did not merely result from the presence of only one set of pipiens
chromosomes, since haploid condition alone did not seem to modify the diploid
pipiens pattern of response to lithium induction.
Since for the type of culture which was employed there is no satisfactory
method for estimating the numbers of cells of different kinds, and the observer
has to rely on visual impressions, great caution was exercised in evaluating the
differences between patterns of differentiation in the cultures. For this reason the
real differences between cultures of cells of different genetic compositions are
underestimated here rather than overestimated, and it is possible that existing
less conspicuous differences have been altogether excluded. Unfortunately, cells
of the hybrid pipiens $ x catesbeiana S died too soon for differentiation to take
place, and thus to contribute any information.
The presence of a hybrid nucleus in the cell manifested itself in some other
ways. Pipiens $ x sylvatica $ and palustris $ x sylvatica £ gastrula cells, when
spread on the glass, showed distinctly larger diameters than those of pipiens or
palustris diploid cells at that stage; however, differentiated hybrid cells were of
the normal size. Cells of these two hybrids also showed a considerably greater
migratory activity and, apparently, an increased adhesiveness to the glass
substrate. In the author's experience almost no culture of homospecific ectodermal cells had developed such spectacular extensive sheets of cells spreading
out of the explants as did the hybrid tissue cultures. Although viability of the
cells of the sylvatica $ xpipiens $ was poor, in several cultures some aggregates
or single cells survived and differentiated. There was a conspicuous difference in
the length of survival and degree of differentiation of these cells depending on
the pair of animals used for this type of hybridization: cells of embryos derived
from one pair did fairly well; those of embryos of another pair gradually died off
so that, actually, only a single nerve cell survived until full differentiation, and
all ectoderm that had come from a third pair died very soon after explantation.
Uninduced by lithium, catesbeiana and sylvatica diploid ectoderm behaved
differently in culture from ectoderm of the other two species. Only those cells
adhered to the glass substrate which later differentiated as melanophores. The
majority formed aggregates which never adhered to the glass substrate; however,
cells of these aggregates seemed to undergo differentiation since they digested
yolk, developed and then lost cilia, and eventually formed some type of epithelial tissue.
DISCUSSION
The purpose of the investigation was to learn whether the cell nucleus
participates in cellular reactions associated with determination in ectoderm. This
was done by comparing patterns of response to time-graded induction by the
lithium ion in ectoderm of several species of Rana and of their hybrids. As the
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K. D. ANSEVIN
results showed, there were differences in the level of induction after the same
treatment between ectoderm of different species; also, hybrid ectoderm appeared
to differ with respect to its inducibility from ectoderm of either of its parental
species. The differences in the hybrid pipiens ectoderm did not depend on the
presence of only one set of pipiens chromosomes, since haploid pipiens cells did
not show these differences. Unfortunately, the complementary test with androgenetic hybrid haploid cells did not seem feasible in view of the limited viability
of the hybrid (R. pipiens) x R. sylvatica, and, therefore, was not attempted.
These results are considered as evidence for the involvement of the nucleus in
cell determination: in ectoderm of hybrid embryos the presence of a paternal
genome of a different species was found to modify the response to induction
which would have been characteristic for the maternal species. Furthermore, the
way in which the response was modified suggested that the nucleus influences the
process of determination itself regardless of whether or not it also earlier
influences the establishment of competence, or later participates in postinductive
reactions of differentiation.
The last statement requires some comment concerning what types of nuclear
influence should be considered to demonstrate involvement of the nucleus in a
particular stage of differentiation. It is unknown when competence is determined by the genes. If it were determined in oogenesis, hybridization should not
affect competence; if it were established after fertilization, and if, in a hybrid,
genes were incompatible for this purpose, one might expect that: (1) competence would not develop at all, and thus that the hybrid cells could not be
induced; (2) competence would be decreased in which case fewer cells would
respond to induction, or induction would take longer in time to produce the
same result; or (3) competence would be increased and a larger number of cells
would become induced or induction would be faster. If, however, genetic incompatibility between maternal and paternal factors affected some postinductive stage of differentiation, one might expect total or partial arrest of
differentiation. None of these predictions corresponded to the actual results.
Hybrid cells responded to induction which excluded the first possibility and, if
anything, induction occurred faster which speaks against the second. Since even
very short treatments with lithium ions produced only very few neural cells in
hybrid ectoderm, the third possibility seems unlikely. In viable hybrid cells
differentiation was never arrested, either totally or at some morphological stage
of the process, and thus the postulate that the observed effect was due to an
interference with a postinductive reaction becomes untenable. This leaves the
hypothesis that the nucleus influences the process of cell determination per se.
However, it still remains to be answered whether this nuclear 'control' of cell
determination is exerted more or less directly on some specific genes, or whether
it affects determination only in a very indirect way. For instance, it is conceivable that the hybrid nucleus might affect the properties of the cell membrane,
thus modifying its permeability to ions. The results of some experiments of
Nucleus in cell determination
389
Barth & Barth (1966) suggest that, indeed, permeability of hybrid cells was not
the same as that of the normal cell surface, and, in the present experiments,
increased adhesiveness and migratory activity of hybrid cells also pointed to
changed properties of their membranes. However, modified permeability could
probably account for speeding or slowing induction, but not for the qualitative
differences in its pattern that have been actually observed. The qualitative aspect
suggests a more direct role of the nucleus in the process of cell determination.
Some differences in results of the present experiments and of earlier investigations might probably be explained by different techniques (Moore, 1947;
Moore & Moore, 1953), or by different temperatures at which induction and
cultivation of cells was carried on (Barth & Barth, 1966) or in the case of lower
viability of R. sylvatica $xi?. pipiens 6* cells in Barth & Barth's experiments
(1968), by genetic variability of different pairs of parent animals.
SUMMARY
1. It is known that gastrula ectoderm of R. pipiens, when treated in vitro,
displays a time-or-concentration dependent, sequentially appearing spectrum of
cell types following induction with lithium ion.
2. The pattern of response to lithium ion induction of haploid pipiens
ectoderm does not differ from that of diploid pipiens tissue.
3. Ectoderm of two other species of the genus, R. sylvatica and R. catesbeiana,
appears to have somewhat different patterns of response to induction with
lithium chloride, while the pattern of ectodermal response of R. palustris is very
similar to that of pipiens.
4. In hybrid cells of R. pipiens $ x R. sylvatica S, R. palustris $ x R. sylvatica S and probably also of R. sylvatica $ x R. pipiens 6* the maternal species
response to lithium ion inductor is modified by the presence of a foreign set of
chromosomes.
5. These results are discussed as evidence for participation of ectodermal cell
nuclei in the process of cell determination.
RESUME
Vinfluence du noyau sur la determination sequentielle de Vectoderme de
grenouille a la suite d'une induction par les ions lithium
1. On sait que l'ectoderme gastruleen de Rana pipiens, traite in vitro, presente
une gamme de types cellulaires apparaissant successivement, et dependant de la
duree du traitement ou de la concentration utilisee, a la suite d'une induction
par les ions lithium.
2. Le type de reaction de l'ectoderme haploide pipiens, a l'egard de l'induction
par le lithium, n'est pas different de celui d'un tissu diploide pipiens.
3. L'ectoderme de deux autres especes du genre, Rana sylvatica et R. cates-
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K. D. ANSEVIN
beiana, a reagi de maniere quelque peu differente a l'induction par le lithium,
tandis que le type de reaction ectodermique de Rana palustris etait tres semblable
a celui de R. pipiens.
4. Dans des cellules hybrides de R. pipiens ? x i ? . sylvatica <$, R. palustris $ x
R. sylvatica <$, et probablement aussi de R. sylvatica $ x R. pipiens <$, la
reaction de l'espece maternelle a l'induction par le lithium a ete modifiee par la
presence d'un lot etranger de chromosomes.
5. On discute ces resultats comme demontrant la participation des noyaux des
cellules ectodermiques dans le processus de la determination cellulaire.
The author is grateful to Dr L. G. Barth, Dr L. J. Barth and to Dr S. Subtelny for valuable
comments concerning this manuscript. She thanks also Mrs E. M. Lewis for technical
assistance.
REFERENCES
ANSEVIN, K. D. (1965). The effects of RNA synthesis inhibition and of protein synthesis
inhibition on embryonic induction and cell differentiation in Rana pipiens. J. Morph. Ill,
171-83.
ANSEVIN, K. D. (1966). Reversal of primary embryonic induction by cold, and the 'fixation'
of induction in cells activated by lithium chloride. /. Morph. 118, 1-9.
BARTH, L. G. (1965). The nature of the action of ions as inductors. Biol. Bull. mar. biol. Lab.,
Woods Hole 129, 471-81.
BARTH, L. G. & BARTH, L. J. (1962). Further investigation of the differentiation in vitro of
presumptive epidermis of the Rana pipiens gastrula. /. Morph. 110, 347-74.
BARTH, L. G. & BARTH, L. J. (1963). The relation between intensity of inductor and type of
cellular differentiation of Rana pipiens presumptive epidermis. Biol. Bull. mar. biol. Lab.,
Woods Hole 124, 125-40.
BARTH, L. J. & BARTH, L. G. (1966). Differentiation and competence of cells from hybrid
embryos. Devi Biol. 13, 95-111.
BARTH, L. J. & BARTH, L. G. (1968). Further studies of differentiation of cells from hybrid
gastrulae. Devi Biol. 17, 272-92.
MOORE, J. A. (1947). Studies in the development of frog hybrids. II. Competence of gastrula
ectoderm of Rana pipiens x Rana sylvatica hybrids. /. exp. Zool. 105, 349-70.
MOORE, J. A. & MOORE, B. C. (1953). Studies on the development of frog hybrids. IV. Competence of gastrula ectoderm in androgenetic hybrids. Biol. Bull. mar. biol. Lab., Woods
Hole 104, 68-74.
PORTER, K. R. (1939). Androgenetic development of the egg of Rana pipiens. Biol. Bull. mar.
biol, Lab., Woods Hole 11, 233-57.
(Manuscript received 29 July 1968)