/ . Embryol. exp. Morph. Vol. 29, 1, pp. 15-25, 1973
\ 5
Printed in Great Britain
Autoradiographic patterns of
[ H]uridine incorporation during the development
of the mollusc, Acmaea scutum
3
By GERALD C. KARP 1
From the Department of Zoology and Friday Harbor Marine Laboratories,
University of Washington, Seattle
SUMMARY
Autoradiographic experiments on eggs and embryos of the gastropod mollusc, Acmaea
scutum, have provided information on the time of initiation of [3H]uridine incorporation
into RNA, the relative degree to which different embryonic regions are participating, and
the relative rates of incorporation at different times of development. The unfertilized egg
does not incorporate exogenous [3H]uridine. After fertilization the first indication of
incorporation in the stages examined was at the beginning of the sixth cleavage. There is a
marked increase in the level of incorporation during the sixth cleavage which marks the
beginning of gastrulation in these embryos. After this stage there is a gradual increase in
incorporation per embryo, throughout development to the mid-veliger. Very little indication
of significant differences in the level of incorporation among the cells of any embryo was
found. The most pronounced exception was the lower activity of the anterior ectodermal
cells of the trochophore larva. At later stages the derivatives of these cells were as active as
the cells of other regions.
INTRODUCTION
A large number of investigations have been made into various aspects of
RNA metabolism in a wide variety of embryos. These studies provide information on the time of initiation of detectable RNA synthesis, the relative degree
to which different embryonic regions are participating, and the relative rate of
RNA synthesis at different times of development. Not unexpectedly, few
generalizations concerning these aspects of gene activity can be made to cover
the entire range of embryos studied. In many of the animals studied the first
detectable expression of the embryonic genome occurs during cleavage. This is
true for the sea urchin (Wilt, 1964; Rinaldi & Monroy, 1969), Xenopus (Brown
& Littna, 1964), mouse (Mintz, 1964; Ellem & Gwatkin, 1968; Woodland &
Graham, 1969; Piko, 1970), rabbit (Manes, 1969; Karp, Manes & Hahn, in preparation) and several species of molluscs (Dauwalder, 1963; Davidson etal. 1965;
Brahmachary, Banerjee & Basu, 1968). In Ascaris a relatively high level of
[3H]uridine incorporation has been demonstrated very soon after fertilization
(Kaulenas & Fairbairn, 1968) while in Ascidia incorporation cannot be detected
1
Author's address: Department of Zoology, University of Florida, Gainesville, Florida
32601, U.S.A.
16
G. C. KARP
until after the formation of the tadpole larva (Lambert, 1971). Presumably the
stage at which RNA synthesis is initiated is dependent upon the nature of the
RNAs produced during oogenesis and the requirements for newly synthesized
RNA during the development of each of the individual species. A detailed
compilation of studies of RNA metabolism during embryogenesis can be found
in Davidson (1968).
Few investigations have been made of the topographical distribution of sites
of RNA synthesis throughout the various embryonic regions. Where this has
been studied, marked differences in the level of RNA synthesis exist among the
different embryonic cells at specific times of development. Czihak, Wittman &
Hindennach (1967) have shown that in the 16-cell stage of the sea urchin only
the micromeres are actively synthesizing RNA. In later stages regional
differences are less apparent (Markman, 1961). In Xenopus the presumptive
endodermal and mesodermal cells synthesize much greater quantities of RNA
at the beginning of gastrulation than do the cells of the presumptive ectoderm
(Bachvarova & Davidson, 1966). In the amphibian these differences can be
correlated with the onset of determination of each of these presumptive germ
layers.
The present autoradiographic study of RNA metabolism during the development of the gastropod mollusc, Acmaea scutum, was undertaken to complement
the results of DNA-RNA hybridization studies on these embryos (Karp &
Whiteley, 1971) and to provide a further comparative study to those on other
species.
MATERIALS AND METHODS
Handling of gametes
Gametes used in this study were collected during the winter months, a period
during which adult Acmaea will not spawn naturally in the laboratory. To
obtain unfertilized eggs, oocytes are stripped mechanically from the ovary,
large-sized oocytes selected by use of nylon bolting silk, and these oocytes
treated with sea water adjusted to pH 9-5-9-9 for 1-3 h until germinal vesicles
are observed to have broken down. These eggs are then washed several times
and inseminated. Millipore-filtered sea water containing 100 /^g/ml streptomycin
sulfate and 50 /^g/ml penicillin G potassium was used for all of the above
procedures as well as in culturing the embryos. Sperm was collected by
dissecting small pieces of testis into a test-tube with ice-cold sea water at pH 9-5
containing 10~ 3 M EDTA (ethylenedinitrilotetraacetic acid) and 5 x 10~5 M
ammonium hydroxide. The tissue was macerated in this solution and drops of
the sperm suspension were quickly added to the eggs. Fertilized eggs were
washed and prepared for culturing to the desired stage. The use of stripped
gametes causes a delay in the rate of cleavage and a considerable degree of
asynchrony of development in contrast to naturally spawned gametes, but does
not visibly alter development in other ways.
[*H]uridine in mollusc development
17
Autoradiography of eggs and embryos
Eggs and embryos were incubated in [3H]uridine (lOO^Ci/ml, 21 Ci/mM,
New England Nuclear) for varying times, washed with Millipore-filtered sea
water, and fixed by the addition of 9 5 % ethanol: glacial acetic acid (3:1).
In later swimming stages the embryos were first relaxed by the addition of
chloretone. Embryos were fixed on ice for 45 min, the fixative removed, and
cold 70 % ethanol added for a minimum of 90 min. The embryos were stained
with light green in 70 % ethanol, dehydrated, cleared in toluene, embedded in
paraffin and sectioned at 5 /.im. Sections were deparaffinized, hydrated, treated
with ice-cold 5 % TCA (trichloroacetic acid), washed in several changes of
70 % ethancl, hydrated, and dipped in liquid Kodak NTB-3 or Ilford K-5
nuclear track emulsion. Slides were exposed at 0 °C in the dark over Drierite
for varying lengths of time and developed in the 1/3 strength Dektol. Slides
were stained either with Harris's haematoxylin and eosin or alternatively with
nuclear fast red and indigo carmine (Mortreuil-Langlois, 1962). Certain slides
were incubated with 0-2 mg/ml RNase (boiled for 10 min to inactivate DNase)
for 2 h at 37 °C, treated with ice-cold 5 % TCA, washed, and dipped into the
emulsion.
RESULTS
Autoradiography of pre-blastula stages
Unfertilized eggs incubated in [3H]uridine (100 fiCxfmX) for 1 h showed no
evidence of incorporation of uridine in autoradiographs exposed up to 5 months
prior to photographic development. Fertilization caused no observable increase
in the capacity of these eggs to incorporate RNA precursors. Eggs incubated
continually in [3H]uridine (100/tCi/ml) from 45 min to 5 h 15 min after
insemination, at which time they were at the 2-4 cell stage, also showed no
evidence of uridine incorporation. Fig. 1 is an autoradiograph of such an
embryo showing two of the nuclei and the surrounding basophilic region. A
few scattered grains are present over the cytoplasm representing the background
formed during the long exposure time. Nuclei in this section are completely
devoid of silver grains.
Autoradiography of post-cleavage embryos
In the following series of autoradiographs the embryos were incubated in
[3H]uridine (100 /^Ci/ml) for 90 min prior to fixation and the autoradiographs
were exposed for 5 months prior to photographic development. Figs. 2A-E
are autoradiographs of 16-5 h embryos arranged on the basis of an ascending
number of cells as determined by nuclear counts of serial sections. All of these
embryos were present on the same slide and thus received identical treatment. It
is clear that these embryos are now capable of incorporating exogenous
2
E M B 29
18
G. C. KARP
2A
2C
2D
2E
[3H]uridine in mollusc development
3
19
[ H]uridine and that the level of this incorporation increases markedly as the
embryo progresses from 34 cells in Fig. 2 A to 62 cells in Fig. 2E. There is very
little evidence of regional differences in incorporation in any of the embryos
examined at this time of development. The same approximate grain density is
found over all of the nuclei throughout all of the serial sections of each embryo.
Nucleoli are present in all of the nuclei of embryos by the start of the sixth
cleavage, yet there is no preferential accumulation of grains over this organelle.
Fig. 3 shows one nucleus with grains distributed in as high a density over nonnucleolar regions as over nucleolar ones.
Fig. 4 is an autoradiograph of a 28 h trochophore larva. Some indication of
regional variation of [3H]uridine incorporation can be seen at this stage as is
demonstrated by the lower grain density over the nuclei of the anterior ectodermal cells. These cells appear to have a lower incorporation rate than cells at
an earlier stage from which they were derived. In the 28 h embryo a greater
level of incorporation is found in the posterior ectodermal and interior endodermal and mesodermal cells (not shown in Fig. 4). In the 28 h embryo, as
in the 16-5 h stage, there is no evidence of predominating nucleolar activity.
Fig. 5 is an autoradiograph of a 40 h embryo showing the general increase in
[3H]uridine incorporation per embryo as compared to earlier stages. This
section passes through the large velar cells, derivatives of the anterior ectodermal
cells of the 28 h stage, which are now as active as the posterior ectodermal
cells and the two endodermal cells shown in this section. Very little regional
variation was found in cells of the 40 h embryo. Fig. 6 is a section of a 51 h
embryo that was treated with ribonuclease prior to being dipped into the
liquid emulsion. A low percentage of residual radioactivity remains after
enzyme treatment, suggesting that either a small percentage of the newly
synthesized RNA is unavailable for enzyme digestion or that the label is
contained in a molecule other than RNA, presumably DNA. In either case the
great bulk of the silver grains represent incorporation into RNase-sensitive
material.
Beyond the 40 h stage much shorter exposure times are suitable to reveal the
location of sites of [3H]uridine incorporation. Figs. 7 A-E are autoradiographs of
Fig. 1. Autoradiograph (exposed 5 months) of a 4-cell stage incubated in
[3H]uridine (100/tCi/ml) from 45 min post-insemination until 5 h 15 min, at which
time the embryos were fixed. No evidence of uridine incorporation is found at this
stage, n, Nucleus, x 640.
Fig. 2. Autoradiographs (exposed 5 months) of embryos at various stages of the
sixth cleavage (16-5 h post-fertilization), incubated for 90 min in [3H]uridine
(lOO^Ci/ml) prior to fixation. Incorporation is shown to increase many fold during
this cleavage period. Cell number was determined by nuclear counts of serial
sections, x 500. (A) Embryo containing 34 cells; arrows denote cell nuclei.
(B) Embryo containing 42 cells; arrows denote cell nuclei. (C) Embryo containing
50 cells; arrows denote cell nuclei. (D) Embryo containing 60 cells. (E) Embryo
containing 62 cells.
20
G. C. K A R P
Fig. 3. Autoradiograph showing one nucleus of a 16-5 h embryo. Conditions are
identical to those of Fig. 2. The dotted line illustrates the outline of the nucleolus
within this nucleus, x 1900.
Fig. 4. Autoradiograph (exposed 5 months) of a 28 h trochophore larva incubated
for 90 min in [3H]uridine (100 /iCi/ml) prior to fixation. The anterior ectodermal
nuclei synthesize RNA at a lower rate than those of other embryonic regions.
Posterior ectodermal, endodermal, and mesodermal cells have increased their rate
of incorporation as compared to earlier stages, op, Apical plate; e, endodermal;
pr, prototroch. x 450.
Fig. 5. Autoradiograph (exposed 5 months) of a 40 h embryo incubated for 90 min
in [3H]uridine (100/iCi/ml) prior to fixation. At this stage there is once again a
uniform level of RNA synthesis throughout the embryo, de, Dorsal ectoderm;
e, endodermal nucleus; v, velar cell nucleus; ve, ventral ectoderm, x 500.
Fig. 6. Autoradiograph (exposed 5 months) of a 51 h embryo incubated for 90 min
in [3H]uridine (100/*Ci/ml) prior to fixation. This section was incubated in
ribonuclease (0-2 mg/ml for 2 h at 37 °C) prior to being dipped into the emulsion.
The enzyme removes nearly all of the radioactivity in comparison to untreated
sections, bp, Blastopore;^, foregut; hg, hindgut. x 600.
\?H]uridine in mollusc development
7A
7B
7D
7E
Fig. 7. Autoradiographs (exposed 3 days) of embryos at various stages of
development that had been incubated for 90 min in [3H]uridine (100 /iCi/ml) prior
to fixation. Grain densities are low enough at this exposure time to allow comparisons of the rate of [3H]uridine incorporation throughout development.
(A) Embryo at 28 h of development; x 500. (B) Embryo at 40 h of development;
x 550. (C) Embryo at 51 h of development; x 600. (D) Embryo at 87 h of development; x 600. (E) Embryo at 109 h of development, x 450.
21
22
G. C. KARP
embryos of 28, 40, 51, 87, and 109 h of development that were exposed for
3 days prior to photographic development. All of these embryos were handled
identically after incubation in [3H]uridine and can be directly compared. This
series of micrographs illustrates the general increase in incorporation that
occurs through development to the mid-veliger. Very little indication of regional
differences in nuclear activity is found in these later stages, though there are
marked differences in cell density among the various embryonic tissues.
DISCUSSION
The results of studies on the unfertilized Acmaea egg show no evidence of the
ability to incorporate [3H]uridine at this stage. Results of the following experiment suggest that this lack of observed incorporation is not the result of the
impermeability of the egg to precursor. Fully grown oocytes of Acmaea are
freely permeable to uridine as demonstrated by its incorporation within the
germinal vesicle. The nucleotide pool of these oocytes was labeled by incubation
in [3H]uridine and the oocytes allowed to mature to the unfertilized egg stage by
treatment with basic sea water. These eggs remained in a fertile state for at
least 1 h before fixation, yet autoradiographs of these eggs gave no evidence of
localized incorporation that might indicate the presence of active chromatin.
Experiments with other animals having eggs that are fertilized at a stage
following germinal vesicle breakdown have also revealed little or no RNA
synthesis in the unfertilized egg (Wilt, 1964; Brown & Littna, 1964; Siekevitz,
Maggio & Catalano, 1966). In contrast, the unfertilized Urechis egg still
contains a large germinal vesicle and is actively synthesizing RNA, the rate of
which drops to an undetectable level following fertilization and germinal
vesicle breakdown (Gould, 1969). Fertilization causes no immediate detectable
increase in the ability of the Acmaea egg to incorporate exogenous uridine,
though it cannot be proven that this is not due to the impermeability of the egg
to uridine at this stage. If the fertilized egg is impermeable to uridine, then the
transformation from the permeable condition of the oocyte must have occurred
rapidly during the times these oocytes were maturing in vitro under the influence
of basic sea water.
In Acmaea, incorporation of exogenous [3H]uridine begins at approximately
the end of the fifth cleavage. Embryos at the end of the fifth cleavage (Fig. 2 A)
show a low but significant level of incorporation. The demonstration of such
synthesis requires high concentrations of isotope and long periods of exposure
of the autoradiographs, suggesting that this is very near the time at which RNA
synthesis is initiated.
Since gastrulation, as defined by the enclosure of the presumptive endodermal
and mesodermal cells by presumptive ectodermal cells, occurs at a stage having
a much smaller cell number in gastropod embryos than in those of echinoderms
and vertebrates, comparisons of transcriptional events must consider more
[*H]uridine in mollusc development
23
than simply the number of cleavages that have occurred before gene expression
begins. In Acmaea, during the sixth cleavage, the cells of the vegetal pole
become elongated anteriorly, causing them to project into the blastocoel. This
stage marks the beginning of gastrulation as described in the detailed study of
the development of a related species by Patten (1886). It is during the sixth
cleavage in Acmaea that a marked increase in the rate of uridine incorporation
by all cells of the embryo occurs. The activation of incorporation in Acmaea
occurs at the same stage (gastrulation) as that in which a similar abrupt increase
occurs in Xenopus (Bachvarova & Davidson, 1966) and Arbacia (Karasaki,
1968), though a much greater number of cells is present at this time in the
amphibian and sea urchin.
Not only is the sixth cleavage the time when gastrular processes are initiated
during the embryogenesis of Acmaea, it is also the cleavage when the 3D cell
divides into the 4D and 4d blastomeres, the latter cell being the one from which
the mesodermal tissues will eventually derive. Though the division of the 3D
cell is of great importance in the future organogenesis of the molluscan embryo,
there is no a priori reason to expect that this event would be accompanied by
the activation of transcription either in that cell or throughout the embryo. The
significance, therefore, of the general nuclear activation during the sixth
cleavage is unclear.
Figs. 7 A-E illustrate the relative levels of [3H]uridine incorporation occurring
at several stages of development. These autoradiographs suggest that there is a
general increase in the amount of RNA synthesized per embryo as development
proceeds. This is the typical pattern found in incorporation studies of other
embryos. Whether there is a continual increase in the incorporation per cell is a
difficult question to answer using autoradiography of a tritiated precursor and
thick sections. As the embryonic cell number increases, the nuclear volumes
greatly decrease, resulting in a higher silver grain density per unit of RNA
synthesized. From this study there is no reason to believe that development
results in the continual increase in the rate of RNA synthesis per unit of
embryonic DNA.
The process of embryonic differentiation is characterized by the regional
appearance of specific properties in the various parts of the developing embryo.
Together with the many localized morphological changes that occur during
development there is evidence that many biochemical processes, including
transcription, maintain regional differences at even the earliest stages (Markman,
1961; Flickinger, Greene, Kohl & Miyagi, 1966; Czihak et ah 1967; Woodland
& Gurdon, 1968; Bachvarova & Davidson, 1966; Flickinger, 1969). In Acmaea
the initiation of incorporation of [3H]uridine after the fifth cleavage as well as
the marked increase during the sixth cleavage occurs in all cells to approximately the same extent. Autoradiographs of embryos were made at approximately 12 h intervals throughout the first 6 days of development, and it was
generally found that all cells of the embryo showed similar levels of RNA
24
G. C. KARP
synthesis. The strongest exception occurred in trochophore larvae of 28 h of
development. In these sections there is a much lower level of RNA synthesis in
the anterior ectodermal cells, including the prototroch, than in other cell types.
Indications from studies on members of diverse phyla are that the appearance
of nucleoli is closely correlated with the synthesis of ribosomal RNA. If this is
true for molluscan development, initiation of ribosomal RNA synthesis would
vary considerably within the group, since nucleoli have been reported to appear
from the 2-cell stage to the blastula (reviewed in Raven, 1966). In Acmaea,
nucleoli are present at the beginning of the sixth cleavage and silver grains are
found over them but at no greater density than over the nuclear sap. It appears
likely, therefore, that the synthesis of ribosomal RNA has begun by the 32-cell
stage. The presence of high levels of nucleoplasmic label suggests that these
early embryos are synthesizing species of RNA other than ribosomal. The
alternative explanation, that nucleoplasmic label is totally a result of transport
from the nucleolus during the 1 h pulse with [3H]uridine, cannot be eliminated.
Results from DNA-RNA hybridization experiments (Karp & Whiteley,
1971) indicate that Acmaea development is characterized by the lack of
appearance of new species of RNA through to the mid-veliger stage. Since it is
clear that the synthesis of RNA occurs relatively early in development this
suggests that these embryos are synthesizing the same species of RNA that were
already present at the time of fertilization. This conclusion is supported by
indications from the DNA-RNA hybridization data that there is an increase in
the concentration of these RNAs as development proceeds. The alternative
explanation that at least a percentage of the newly synthesized RNA molecules
detected in the autoradiographs represent species not being detected in the
hybridization studies cannot be ruled out.
I wish to thank Dr Arthur H. Whiteley for his encouragement and guidance during the
progress of this work and Dr R. L. Fernald for making available the facilities at the Friday
Harbor Laboratories.
The author was supported by an NSF Predoctoral Fellowship during the course of this
work.
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{Manuscript received 25 April 1972, revised 26 June 1972)