AMERZOOL, 16:547-561 (1976).
Protein Synthesis and Differentiation During Pulmonate Development
JOHN B. MORRILL, ROBERT W. RUBIN, AND MITCHELL GRANDI
Division of Natural Sciences, New College-University of South Florida,
Sarasota, Florida 33580
SYNOPSIS An analysis of the changing patterns of protein synthesis during early development of Lymnaea palustns has been undertaken. An examination of the ingestion of capsule
fluid protein suggests that after gastrulation 30 to 65% of the total embryo protein is
undigested food protein. Starch gel electrophoresis reveals a sudden increase in the number
of hydrolases from four to twenty-six immediately following the trochophore stage with the
latter being present also in adult organs. Studies reported here and elsewhere demonstrate
rhythmic changes in uridine incorporation during early cleavage which peaks at the trochophore stage. Continuous treatment of embryos with 50 to 100 ^.g of actinomycin-D (AMD)
starting at the 2-cell stage slowed development through the trochophore stage but did not
prevent normal larval organ development. This AMD application reduced 3H uridine
incorporation more than 90% but did not appreciably alter the pattern of total or' 4C leucine
pulse labeled peptides on sodium dodecyl sulfate (SDS) ultrathin slab electrophoresis gels.
However, pronounced and numerous changes in the patterns of both labeled and unlabeled
peptides were observed during development through 4 days with the most notable alterations occurring at the 2.5 post-gastrula stage. This was true in normal and continuously
treated AMD embryos. The morphological and biochemical data suggest Lymnaea early
development is controlled by stable maternal messenger RNA.
biochemical development of the pulmonate
egg can offer interesting insights into the
The normal and experimental embryol- developmental processes of other spiralian
ogy of the freshwater pulmonate egg has eggs. In contrast to many other spiralian
been the subject of numerous studies (cf. eggs, the pulmonate egg exhibits a proHess, 1962, 1971; Raven, 1966, 1970). tected development (Cowden, 1972) with
However, present knowledge of the its major nutrient reserve being the extrachemistry of their development is in- embryonic albuminous capsule fluid and
adequate. The few data available are re- with nucleoli and RNA synthesis appearing
viewed by Horstmann, 1964; Norris and during the early cleavages (Van den
Morrill, 1964; Collier, 1965, 1966; Biggelaar, 1972).
Goudsmit, 1972; Raven, 1966, 1972; and
In this paper we will summarize a series
Brahmachary, 1973. In particular, our
of
continuing studies on the quantitative
knowledge of changing patterns of protein
and
qualitative changes in proteins and
synthesis in these spiralian eggs is limited
their
synthesis during the development of
and restricted to three species of Lymnaea.
one species of Lymnaea, L. palustris. We will
One reason for this is that there are apparent technical difficulties in obtaining review embryonic nutrition, electrophoretsufficient numbers of eggs to analyze ic analysis of hydrolases, synthesis of protein and RNA, and the effects of
biochemically.
actinomycin-D on morphogenesis and patNevertheless, the morphological and terns of protein synthesis.
INTRODUCTION
This work was supported in part by an NSF Grant
and restricted gifts to New College. The authors would
like to acknowledge the contributions of the following
students: Elaine Norris and Robert Bast, Wesleyan
Universityand Warren Rottmann, The College of William and Mary.
EMBRYONIC NUTRITION
The fertilized egg of pulmonates develops within a capsule containing a
perivitelline albuminous fluid (capsule
547
548
MORRILL ET AL.
The egg of L. palustris develops into a
fluid) that is the major nutrient reserve
until the larval snail hatches from the cap- definitive snail that hatches 8 days (25°C)
sule. Both Clement (1938) and Morrill after oviposition. During this period the
(1964) demonstrated nucleated egg frag- total embryo protein increases from 0.5 to
ments lacking the bulk of deutoplasmic 17.5 jug and the total capsule fluid protein
yolk reserves develop normally. However, decreases from 17 to 3 /xg. (Fig. 1) begindecapsulated eggs do not develop normally ning at the 2-day trochophore stage (Morbeyond the gastrula stage (Jockusch, 1968). rill, 1964). According to Raven (1946) the
The capsule fluid consists of a heterogene- yolk of the egg is consumed by the end of
ous array of polysaccharides and proteins gastrulation. Capsule fluid is ingested
that are species specific (Morrillet al., 1964). pinocytotically by various cells from the
In L. palustris there are 18 electrophoret- early cleavage stage until the trochophore
ically separable proteins (Fig. 8) and galac- when it is ingested orally and accumulates
in the vacuoles of enlarged cells of the larval
togen.
0
10
FIG. 1. Total protein, galactogen and glycogen of embryo and capsule fluid (C.F.) of Lymnaea palustris.
Units: x axis, days of development at 25°C; y axis,
micrograms per embryo or capsule fluid per capsule.
Vertical segments are standard error of the mean (SE).
Numbers above vertical segments are number of determinations. Dotted line, total C.F. protein; dashed
line, total embryo protein (From Morrill, 1964). Sym-
11
12
bols: solid circles, C.F. galactogen; solid squares, embryo galactogen; solid diamonds, embryo glycogen.
Galactogen and glycogen hydrolysates assayed with
galactostat and glucostat coupled enzyme systems
(Worthington Biochem. Corp.). Development references: Day 1, placode gastrula; Day 2, late
trochophore; Day 3, veliger; Day 4, "Hippo" stage with
beating heart; Day 8, larval snail hatches from capsule.
549
PROTEIN PATTERNS OF PULMONATE DEVELOPMENT
liver. Thus, changes in total embryo protein during the development are due in
part to varying amounts of ingested capsule
fluid.
In order to determine the percent of total
embryo protein composed of ingested capsule protein we first measured the amount
of galactogen and glycogen in the embryo
and capsule fluid at different stages of development with standard galactostat and
glucostat enzyme assay techniques. There
are about 24 fig of galactogen in the capsule
fluid of the oviposited egg (Fig. 1). At the
2.5 day stage the galactogen of the capsule
fluid begins to decrease measurably and the
galactogen of the embryo increases.
Glycogen-like polymers do not appear until
the fifth day. This decrease in capsule fluid
galactogen roughly parallels the decrease
in capsule fluid protein.
If one assumes that the galactogen content of the embryo reflects the amount of
undigested capsule fluid in the embryo, it is
possible to estimate the amount of capsule
fluid protein in the embryo at each stage
using the ratio of protein to galactogen in
the capsule fluid at oviposition (Table 1).
Following gastrulation (day 1) the percent
of total embryo protein that is capsule fluid
protein varies from a maximum of 64% on
the third day to a minimum of 29% on the
seventh day. Thus, following gastrulation,
between 30 to 65% of the total embryo protein is probably "food" protein. Although
the utilization of this nutrient reserve has
not been pursued, estimation of the
number of fig of actual embryonic protein
at different stages has been useful in adjusting the number of embryos per unit volume
in our biochemical analyses.
ELECTROPHORETIC PATTERNS OF HYDROLASES
Our first approach in following protein
differentiation involved a starch gel zy mogram analysis of soluble and electrophoretically mobile hydrolases using azo dye histochemical methods (Norris and Morrill,
1964). Hydrolases in particular may play
important roles in the mobilization of the
deutoplasm of the egg (Bluemink, 1967)
and in the utilization of the capsule fluid
(Goudsmit, 1962). In L. palustris the cleavage stage egg has only four electrophoretically resolvable hydrolase bands—two acid
phosphatases, one non-specific protease
and one galactosidase (Fig. 2). Presumably
these hydrolases are involved in mobilizing
nutrient reserves. Twenty-two additional
bands appear after the trochophore stage
during the period of growth and organ differentiation. Since the Lymnaea egg develops directly into a definitive snail, one
might expect that once new enzymic proteins appear during development they
would persist and increase in activity and/or
amount during development. Such is the
case for the hydrolases we have assayed.
Furthermore, all the enzymic bands in the
embryo extracts are detected in extracts of
one or more adult organs. In addition, we
have not detected any hydrolase bands
TABLE 1. Micrograms of galactogen and total protein per embryo and the calculated amount of total embryo protein less
ingested capsule fluid protein during L. palustris development.
Micrograms per embryo
Day of
development
Galactogen
Capsule
fluid
protein
0
1
2
3
4
5
6
7
8
0.0
0.3
0.6
1.6
1.6
3.3
6.1
6.9
8.5
0.0
0.2
0.4
1.1
1.1
2.2
4.3
4.9
6.0
Total
embryo
protein
0.5
0.6
0.8
1.8
3.4
7.1
9.8
16.5
17.5
Total protein
less capsule '
fluid protein
0.5
0.4
0.4
0.7
2.3
4.9
5.5
11.6
11.5
Percent of
total protein
as capsule
fluid protein
0
52
53
64
32
31
41
29
34
550
MORRILL ET AL.
Band
Enzyme
No.
Doys
2
of
Development
3
Esterose
Acid
phosphatose
Alkaline
phosphatose
L aucine
aminopeptidose
Alanine
ominopeptidose
0 -galactosidase
-glucosidose
c* -glucosidose
fi -glucuromdase
FIG. 2. AppearanceduringdevelopmentofZ../>a/itffris
of electrophoretically mobile enzymic bands. (From
Norris and Morrill, 1964).
peculiar to the early preorganogenesis
stages of development. Similar developmental patterns of electrophoretically
mobile alkaline and acid phosphatases
occur in Physa fontinalis and correlate with
changes in phosphatase activity in whole
embryo homogenates (Morrill, 1973).
While changes in zymogram patterns of
enzymic proteins during development
reflect changing states of differentiation,
they do not provide information on the
function and location of these enzymes in
the developing embryo. The localization of
these readily soluble enzymic proteins may
prove difficult because of their sensitivity to
cytochemical fixatives. For example, in P.
fontinalis 70 to 80% of the total phosphatase
activity in embryo homogenates consists of
readily soluble, electrophoretically mobile
forms sensitive to formaldehyde and
gluteraldehyde fixatives while 20 to 30% is
associated with insoluble or bound forms
that are relatively insensitive to these
cytochemical fixatives (Morrill, 1973).
TOTAL PROTEIN AND RNA SYNTHESIS
The electrophoretic patterns of enzymic
proteins in themselves do not necessarily
demonstrate changing patterns in protein
synthesis. Before analyzing the synthesis of
these and other specific proteins, we first
measured the incorporation of uridine-3H
into total embryo RNA and leucine-3H into
total embryo protein (Rottmann, 1967).
Preliminary experiments revealed that
when encapsulated embryos were pulsed
for 2 hours and homogenized in their capsules there were high counts of both labeled
precursors bound to the precipitate following perchloric acid (PCA) extraction. In
order to eliminate this non-specific binding, presumably by some component of the
capsule fluid or capsule membrane, it was
necessary to kill the embryos with 70%
ethanol following the 2-hour pulse and
then to decapsulate and wash the embryos
prior to extraction with ice cold 0.2 N PCA.
With this latter procedure the total
uridine and leucine incorporation in the
acid insoluble residue of whole embryos
was measured separately at 12 hour intervals during the first 4.5 days of development (Fig. 3). A low but measurable level of
incorporation of uridine-3H and leucine3
H occurred during the first 1.5 days of
development when the embryo underwent
cleavage and gastrulated. A sharp increase
in incorporation of both precursors relative
to changes in total embryo protein occurred at the early trochophore stage (2 day)
when the major larval organs have differentiated and several adult organ
primordia — foot, shell gland, and buccopharyngeal apparatus—are visible. A second increase in incorporation of precursors
at the 4.5 day stage is associated with the
differentiation and growth of the major
adult organ primordia and the appearance
of six hydrolase zymogram bands (Fig. 2).
Rhythmic RNA synthesis has been measured during the early cleavages beginning
during the maturation divisions of the uncleaved egg of an Indian species oiLymnaea
(Brahmachary, 1972). Using 14C uracil,
33
P uracil and methylmethionine- 14 C,
Brahmachary and his colleagues observed
that rhythmic synthesis during the early
PROTEIN PATTERNS OF PULMONATE DEVELOPMENT
150
FIG. 3. Incorporation of uridine 3H into RNA (solid
circles) andleucine 3 H into protein (solid squares) ofL.
palustns during development (24°C). Units: x axis, day
of development; y axis, dpm per microgram total embryo protein. At each stage of development 25 encapsulated embryos were incubated in either uridine-53
H (specific activity 4.4 curies/millimole, 5 /*c/ml) or
L-leucine-4,5-3H (specific activity; 5.0 curies/millimole, 5 fic/ml) for 2 hours at 24°C. Incorporation was
stopped by fixing in 70% ethanol for 10 minutes. Embryos were then rinsed in sterile pond water, transferred to 0.45 micron millipore filters, and washed 10
times (0.25 ml for 2 min per wash) with ice cold 0.2 N
PCA containing 1 mg/ml unlabeled precursor. The
embryos and filter were dissolved overnight in 0.3 ml
Nuclear Chicago Solubilizer. Radioactivity was measured with a Nuclear Chicago Model Series 720 liquid
scintillation counter, using a toluene base scintillation
fluid. Each point on the graph is the average of four
determinations. For each point the range was approximately 10% the average value (Rottmann, 1967).
cleavages is dominated by changes in incorporation in 4S, 18-28S and pre 28S
RNA. In autoradiographic analyses on L.
stagnalis, Van den Biggelaar (1971) detected the cytoplasmic localiztion of uridine
incorporation into RNA from the 8-cell
stage. RNA synthesis was localized in distinct nucleoli that reappeared at the 16-cell
stage. However, the cytochemically stainable RNA rich granules in the macromeres
of the 24-cell stage and later stages were not
labeled following incubation with uridine
3
H. In the Indian species as in L. palustris
total uridine incorporation increases dur-
551
ing development and peaks at the
trochophore stage, then declines through
the veliger stage. The major portion of
RNA synthesized at the trochophore stage
is a heterogeneous class of molecules between 4S and 16S which Brahmachary
(1972) refers to as 10S RNA.
Autoradiographic studies of leucine-3H
incorporation into L. stagnalis embryos revealed puromycin sensitive incorporation
beginning at the second maturation division and fluctuating during the cell cycle
during the early cleavages (Jockusch,
1968). Electron microscope auto radiography of the early cleavage eggs showed
that the labeled proteins were localized in
mitochondria, the nucleus, the surfaces of
intact yolk granules, the margins of vacuoles, and the diffuse matrix of disintegrating yolk granules (van der Wai, cited in
Raven, 1972). This latter localization
suggests that the newly synthesized proteins may represent enzymes such as hydrolases involved in the mobilization of the
yolk nutrients. In the Indian species of
Lymnaea incorporation of amino acid-14C
mixtures is low in the early stages but increases after the trochophore stage
(Brahmachary, 1972).
In summary, the several studies on Lymnaea suggest that the synthesis of RNA and
proteins begins during the maturation divisions, undergoes rhythmic changes during
the early cleavages, increases during development, and peaks at the trochophore
stage when larval organs have differentiated, adult primordia appear, the nutrient reserves of the capsule fluid begin to be
ingested orally, and overall growth of the
embryo increases measurably. Subsequent
to the trochophore stage RNA and protein
synthesis is associated with growth and differentiation of adult organ primordia, increasing rates of capsule fluid ingestion and
appearance of new species of hydrolytic enzymes. To what degree do these events depend on maternal mRN A mediated protein
synthesis and gene mediated protein synthesis? In pulmonates this question has
been pursued with the aid of agents that
suppress or block RNA synthesis.
A word of caution should be added here.
It is entirely inappropriate to correlate in-
552
MORRILL ET AL.
corporation of labeled precursors into proteins or nucleic acids during various stages
of development with RNA or protein or
even DNA synthesis. While rates of incorporation may reflect increases in synthesis it
is possible that they do not and that the
various changes observed represent
changes in transport of precursors into the
cells or differing rates of diffusion through
the extracellular matrices or other
phenomena that affect the rate of precursor entry into the cells of the embryo. We
have attempted to get around this problem
by determining the kinetics of pool size saturation for uridine and leucine during the
first three days of development. These
studies have been generally unsuccessful. It
appears that short pulses followed by washing whole embryos in pond water is an inefficient means of rapidly removing externally bound labeled precursors. The data
obtained from embryos washed with pond
water and then treated with cold 5% TCA
were variable and rarely showed a consistent
increase with increasing time of exposure
to the pulse. The reasons for this are not at
this time entirely clear, although they may
be related to the large amount of extracellular materials such as polysaccharides and
storage proteins which may bind the two
precursors and reduce the efficiency of
washing. Therefore the results described in
the literature and in this review must be
taken and interpreted in a cautious manner. Further studies dealing with the kinetics of the saturation of the soluble pool
of precursor molecules used to label these
embryos must be undertaken before an unequivocal interpretation of continuous or
pulse labeling experiments during development can be obtained.
MORPHOGENETIC EFFECTS OF ACTINOMYCIN
One of the agents most widely used to
block RNA synthesis and gene mediated
protein synthesis in developing embryos is
actinomycin-D (AMD). Its morphogenetic
and biochemical effects on molluscan eggs
have been reviewed by Collier (1966),
Brahmachary (1972), and Newrock and
Raff (1975). Treatment of L. stagnate eggs
with AMD at concentrations varying be-
tween 5 /xg and 200 fig/ml for 6 hrs after
first cleavage does not affect development
(Geilenkirchen, 1967). Treatment with
AMD in concentrations up to 100 /i.g/ml
does not suppress gastrulation (BoonNiermeijer, cited in Verdonk, 1973). However, in the Indian species oiLymnaea, eggs
treated with AMD (100 uglmX) for 1 hour
during the early cleavages develop until the
late veliger stage when abnormal growth
patterns of the shell gland appear. Treatment at the trochophore stage for 90 minutes blocks further development entirely
(Brahmachary and Banerjee, 1967).
In view of these differing results we
undertook an extensive series of experiments in which embryos of L. palustris were
treated at different stages of development
with concentrations of AMD ranging from
10 fjig to 200 /Ag/ml for various durations (2
hours to continuous treatment). We found
that eggs treated continuously from the
2-cell stage with 40 /u.g to 100 Mg/ml AMD
developed normally until gastrulation.
They gastrulated 6 to 12 hours later than
the controls and then developed into abnormal, arrested trochophores that lived
for several days before disintegrating. Typically these embryos attained the 2-day
trochophore stage 12 hours later than the
controls but had the normal compliment of
larval organs —head vesicle, ciliated velum,
paired protonephridia and larval liver.
As these arrested embryos aged they
tended to become hydropic; the larval liver
cells regressed and white crystalline deposits appeared in the cells and lumen of
the protonephridia (Fig. 4B). Since these
crystals were never observed in normal embryos, their presence in AMD treated embryos suggests an • alteration of nitrogen
catabolism. In addition to the larval organs
the shell gland and foot primordia were
usually present.
Although these arrested trochophores
were similar to normal trochophores, it appeared at low magnifications that the original blastopore had not closed and that the
stomodeum had not developed. Histological sections of AMD arrested trochophores
confirmed this (Fig. 5D). Furthermore, the
shell gland while present in AMD treated
embryos consisted of peculiar cells with
PROTEIN PATTERNS OF PULMONATE DEVELOPMENT
553
B
FIG. 4. Morphogenetic effects of actinomycin-D
' (AMD) on L. palustris development. A, normal 4 day
embryo; B, arrested, hydropic 2.5 day late
trochophore—early veliger treated continuously
from 2-cell stage with 50 /u.g/ml AMD. Note that the
larval kidney cells are filled with white crystalline de-
posits. C and D, differentiated snails with arrested
growth of shell gland 10 days following a 48-hour
treatment of 2-day embryos with 50 /u.g/m'- Symbols:
E, eye-tentacle of head; F, foot; K, larval kidney; M,
margin of shell gland or mantle fold. The bar is 200 p
long.
large nuclei (Fig. 5C,D,E). Embryos exposed
continuously with 50 /u.g/ml AMD for 4 days
were frequently small and compact with a
reduced blastocoel and a few scattered albumen droplets. Histological sections of
these embryos showed little tissue organization (Fig. 5F). Preliminary electron microscopic analysis indicates that the ultrastructure of the cells and organelles is normal
(Luchtel, unpublished).
Although the sectioned AMD arrested
trochophores had the normal compliment
of larval organs, none had a differentiated
stomodeum, radular sac, esophagus and
midgut that are normally present in 2.5 day
trochophore veligers. This suggests that
AMD arrested development at the
trochophore stage results partly from a
selective inhibition of the normal differentiation of those adult organs involved in
ingestion of capsule fluid nutrients. In
other words, arrested development at the
trochophore stage may result from starvation as well as direct inhibition of gene
transcription. A variety of agents [i.e.,
cobalt (Morrill, 1964); 5-fluorodeoxyuridine (Plotkin, unpublished); chloramphenicol (Sherbet & Lakashmi, 1964);
ethionine, 5-bromodeoxyuridine and d,Iparafluorophenylalanine (Morrill, unpub-
B
PROTEIN PATTERNS OF PULMONATE DEVELOPMENT
lished)] may arrest development at the
trochophore stage. A direct indication that
starvation is a key factor is seen in 2-day
trochophores isolated from their capsules.
Their development is arrested, their livers
regress and their protonephridia accumulate crystalline deposits within 36 hours.
Contrarily, 2-day trochophores isolated in
hanging drop cultures of albumen continue to develop normally (Morrill, unpublished).
That AMD appears to exert its first effects at gastrulation and causes arrested development at the trochophore stage
suggests that gene transcription is necessary for molecular events that accompany
gastrulation and that are necessary for the
differentiation of adult organs but not larval organs. Thus our data support the conclusions of Verdonk(1973) who found that
in progeny of irradiated snails gastrulation
was controlled by the genome of the embryo and that the stage at which most lethal
factors interfered with normal development was the early trochophore stage.
When L. palustris embryos were treated
with AMD (20-60 i*glm\) at the 2-day
trochophore stage, further development
and growth were retarded but adult organs
differentiated. Typically the treated embryos differentiated 1 to 3 days slower than
the controls but did not grow beyond the
size of a 4-day veliger-larva (Fig. 4A, C, D)
and did not hatch from their capsules. The
two-dimensional growth of the shell gland
was the most conspicuous morphogenetic
abnormality. While the shell gland secreted
a shell it did not grow over the visceral
hump as in the normal embryo but typically
remained at its origin on the left posterior
end of the embryo. Similar arrested development of the shell gland in pulmonates
is produced by other treatments [i.e.,
analogues of nucleotides and amino acids
(Dewan, 1968), sodium azide (Sherbet and
FIG. 5. Sections of resin embedded normal embryos
and embryos treated continuously from the 2-cell
stage with 50 /xg/ml AMD. A, normal 1.5 day post
gastrula; B, cross section of normal 2-day late
trochophore; C, 1.5 day AMD embryo; D, sagittal section of 2.5 day AMD embryo; E, sagittal section of
555
Lakshmi(1964)), and progeny of irradiated
adults (Verdonk, 1973)].
EFFECTS OF ACTINOMYCIN ON RNA AND PROTEIN
SYNTHESIS
When embryos treated continuously with
AMD (100 /xg/ml) from the 2-cell stage
were given 2 hour pulses of uridine-3H the
incorporation of uridine into RNA was
markedly reduced beginning at the 2-day
stage (Fig. 6). Brahmachary and Palchoudhury (1971) showed that in sucrose
FIG. 6. Effect of AMD on RNA synthesis during early
development of L. palustrts. Incorporation of uridine3
H into normal (solid circles) and AMD treated (open
circles) embryos. Units: x axis, day of development; y
axis, dpm per 25 embryos per 2 hours. All operations
were performed as described in Figure 3. AMD embryos were incubated continuously from the 2-cell
stage in sterile pond water containing 100/u.g/ml AMD.
3-day AMD embryo; F, sagittal section of 4-day AMD
embryo. Symbols: B,blastopore that has not closed; F,
foot primordium; K, larval kidney duct; L, larval liver
cells; Sg., shell gland primordium; S, presumptive
stomodeal area; V, velar cells. The bar is 50 /J. long.
556
MORRILL ET AL.
density profiles of newly synthesized RNA
from AMD arrested abnormal trochophoresthe4S, 10S, 18S and 23S peaks were
reduced; the 10S peak was also reduced in
the normal and AMD treated veligers. It
appears then that in Lymnaea the normal
differentiation and growth of adult organ
primordia begins at the 1.5 day post gastrula stage and requires RNA synthesis.
The retarded morphological development
in AMD (50-100 /xg/ml) embryos following
gastrulation is associated with a 12 hour
delay in the peak of leucine incorporation
in AMD embryos as compared to the peak
in the normal 2-day trochophore (Fig. 7).
Since there is over 90% reduction in
uridine incorporation at this stage, we conclude that protein synthesis leading to the
differentiation of larval organs requires
only the translation of maternal mRNAs as
does gastrulation. In Lymnaea as in sea ur-
chins (Gross et al., 1964) leucine incorporation during the cleavage period is greater in
AMD than normal embryos and is associated with the synthesis of unique proteins in AMD embryos (Fig. 13).
To further define the qualitative aspects
of the major classes of proteins during the
early development of L. palustris we
employed sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis followed by autoradiography. To determine
which proteins were synthesized on stable
maternal mRNA, replicate batches of embryos were pretreated continuously from
the 2-cell stage with AMD (50 Mg/ml). Figure 8 shows the Coomassie Blue stained
patterns of embryos and capsule fluid. With
this electrophoretic system it was possible to
determine which embryo bands were ingested capsule fluid proteins.
Comparison of stained gel patterns of
normal and AMD treated embryos (0.5 to 4
days after oviposition) showed that the
:
*
II
"a*
—
2
ft
M.w.
67,000
42.000
•31.000
15.100
MS «
1.5
2
2.5
_
3.0 C.F. Tryp DNA Act- Alb
ase in
FIG. 8. Qualitative peptide patterns from SDS slab gels
from 1.5, 2, 2.5 and 3 day L. palustris embryos and
capsule fluid (C.F.) and molecular weight markers
[bovine pancreas trypsin (Tryp.), bovine pancreas
DNAase, rabbit muscle actin and bovine serum albumin (Alb)]. Fifty embryos were removed from their
capsules, rinsed of capsule fluid, and lysed in a SDS
FIG. 7. Effect of actinomycin D or protein synthesis sample buffer. Samples were electrophoresed on 16
during the early development of L. palustris. Incorpo- cm x 12 cm x 0.08 cm polyacrylamide SDS slab gels,
ration of leucine-3H into protein in normal (solid cir- according to Laemmli (1970, Nature 227, 680, and
cles) and actinomycin-D (open circles). Units: x axis, O'Farrell, Gold, and Huang (1973J. Biol. Chem. 248,
day of development; y axis,dpm per 25 embryos per 2 5499); run at a constant current of 12.5 milliamps;
hours. All operations were performed as described in fixed and stained for 20 minutes in 25% TCA containFigure 3. Actinomycin D embryos were incubated con- ing 0.1% Coomassie Brilliant Blue; destained for 2-3
tinuously from the 2-cell stage in sterile pond water days in periodically changed 7% acetic acid; and dried
under vacuum and heat.
containing 100 A^g/ml AMD.
PROTEIN PATTERNS OF PULMONATE DEVELOPMENT
FIG. 9. SDS gels of normal (N) and AMD(A-D)
treated L. palustris embryos. Units: x axis, day of development. Proteins were isolated, electrophoresed
and stained as described in Figure 8. Actinomycin D
embryos were incubated continuously from the 2-cell
stage in 50 Mg/"1' AMD (Sigma Chem. Co.) in the dark.
major bands were present in both normal
and AMD embryos (Fig. 9). Similarly, densitometric profiles of autoradiographs of
the stained gels in Figure 9 showed that the
major bands in the autoradiographs of
normal embryos at each stage up to the
4-day stage were also present in the AMD
treated embryos (Figs. 10 and 11). However, there was an overall reduction in
leucine incorporation in the AMD embryos. Furthermore, the densitometric
profiles of the normal and AMD embryos
differed at each stage indicating both qualitative and quantitative changes in the synthesis of different groups of proteins during development. That protein synthesis
associated with major densitometric peaks
may be due to a time dependent rather than
a morphological stage dependent translation of maternal mRN As is best seen in the
densitometric profiles of 3 and 4 day old
normal and AMD embryos (Fig. 11) where
the latter were abnormal, arrested 2-day
trochophores.
To resolve further the significant qualitative features of the band patterns in the
stained gels and on the autoradiographs,
we measured the Rfs of individual bands
that were present only on the stained gels
(Fig. 12) and only on the autoradiographs
(Fig. 13) of normal and AMD embryos.
Both the stained gels and the autoradiographs of AMD embryos exhibited unique
bands at all stages. The number of unique
J
I ••> «»
FIG. 10. Densitometric profiles of 14C leucine labeled
peptides synthesized during the first 2 days of development of normal and AMD (A-D) treated embryos
of L. palustris. At 0.5, 1 and 2 day stages embryos were
pulsed for 2 hours with 14C reconstituted algal protein
hydrolysate (5 ^ic/ml), cooled to 4°C and washed before SDS electrophoresis as described in Figure 8.
Dried gels (Fig. 9) were autoradiographed on Kodak
medical X-ray film for 6 weeks. The emulsion was
removed from the unexposed side of the developed
film by treating that surface with 20% NaOH for 5
minutes, followed by a water rinse. The film was cut
into strips and individual auto radiograms were scanned at 700 nm with a Gilford 240 spectrophotometer.
This figure shows only the profiles of proteins > 40,000
daltons; auto radiograms of lower molecular weight proteins revealed no bands dense enough to register above
background.
AMD embryo bands increased in 2.5 day
old embryos (Table 2) coinciding with the
abrupt increase in total protein synthesis at
this time (Fig. 7).
558
MORRILL ET AL.
B
3 Day AD
4 Day lUrnal
« Day >D
FIG. 11. Densitometric profiles of 14C leucine labeled
peptides synthesized by 3 and 4 day normal (N) and
AMD (A-D) (50 /ig/ml continuously from 2-cell stage)
treated embryos (Fig. 9) of L. palustrts. Embryos were
pulse labeled, lysed in SDS-mercaptoethanol sample
buffer, electrophoresed and autoradiograms prepared as described in Figures 8 and 9. A, densitometric
profiles of synthesized polypeptides > 40,000 daltons;
B, < 40,000 daltons.
When the Rfs of bands on autoradiographs of consecutive stages (i.e., 0.5 day
versus 1 day) were compared additional
patterns of protein synthesis became apparent (Table 3). In both normal and AMD
embryos the majority of bands were present at both of two consecutive stages.
However, some bands were present at one
stage and not the other. This suggests that
the synthesis of some proteins is time
and/or stage specific. In normal embryos
the 2-day stage had 9 bands not present at
the 1.5 day stage and 6 bands not present at
the 2.5 day stage. Similarly, the 3 day stage
had 14 bands not present at the 2.5 day
stage. In AMD treated embryos the total
number of bands in the autoradiographs
increased from 44 at the 2-day stage to 68 at
the 2.5 day stage. Thirty-three of these
bands were detected at the 2-day stage but
not the 2.5 day stage and 57 at the 2.5 day
but not the 2-day stage. Interestingly there
559
PROTEIN PATTERNS OF PULMONATE DEVELOPMENT
TABLE 2. Total numbers of SDS electrophoretic bands stained with Coomassie Blue and/or on autoradiographs (Arg) of
normal and actinomycin D (AMD) treated embryos.
Day of development
Normal, stained
AMD, stained
Normal, arg.
AMD, arg.
Normal, stained & arg.
AMD, stained & arg.
Normal, stained only
AMD, stained only
Normal, arg. only
AMD, arg. only
0.5
1.0
1.5
2.0
2.5
3.0
28
37
34
36
27
27
1
10
7
9
30
41
35
39
29
35
1
6
6
4
33
40
36
41
32
37
1
3
4
4
46
44
45
44
41
40
5
4
4
46
60
39
68
38
39
8
21
1
29
55
62
53
69
50
41
5
21
3
28
4
were few differences in the band patterns of those newly synthesized as indicated by
of the AMD arrested 2.5 and 3-day em- incorporation of leucine during this period
of time. Early development in the pulmobryos.
nate embryo seems to follow the general
pattern seen in deuterostomes where
CONCLUSIONS
masked stable maternal messenger RNA
Our studies with L. palustris embryos provides the bulk of the synthetic template
demonstrate that during the first 4 days of during the early stages of development.
development morphological and func- Beginning at gastrulation (1-day) and intional differentiation is accompanied by creasing markedly at the 2-day trochoquantitative and qualitative changes in pro- phore stage selective gene mediated proteins and patterns of synthesis of proteins. tein synthesis becomes increasingly imporThe synthesis of some proteins appears to tant for normal development. AMD applibe time dependent rather than stage de- cation in concentrations sufficient enough
pendent. However, development to the to reduce3 below 90% the rate of incorporaearly trochophore (2-day) stage in part does tion of H uridine not only arrests denot require DNA dependent RNA synthe- velopment at the trochophore stage but
sis with the majority of proteins being syn- also produces embryos whose histomorthesized on long lived maternal mRNAs. phology and protein patterns differ from
The patterns of newly synthesized proteins normal embryos. Indeed many of the produring this period are with very few excep- teins synthesized in AMD embryos are not
tions identical in AMD treated and un- observed in normal embryos. This suggests
treated control embryos. This includes that AMD not only inhibits RNA synthesis
both the patterns observed for proteins not but either specifically or nonspecifically unbeing synthesized at any given period and couples the normal control of protein synTABLE 3. Numbers of bands on autoradiographs of SDS electrophorograms of normal and actinomycin D (AMD) treated
embryos at consecutive stages of development of L. palustris.
Days of development
Normal, both stages
Normal, 1st only
Normal, 2nd only
AMD, both stages
AMD, 1st only
AMD, 2nd only
0.5 vs. 1.0
1.0 vs. 1.5
1.5 vs. 2.0
2.0 vs. 2.5
2.5 vs. 3.0
31
4
4
29
7
10
29
6
7
35
4
6
31
5
14
38
3
6
32
13
7
11
33
57
35
4
18
67
1
2
560
150
MORRILL ET AL.
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140
130
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Deve I o o m e n t
D« v e I op men t
FIG. 12. Diagram of SDS electrophoretic patterns of
peptides stained with Coomassie Blue but not present
on autoradiograms of normal whole embryo lysates
(solid circles) and embryos treated continuously with
AMD prior to lysis in SDS sample buffer (solid
squares). Molecular weights were calculated by determining the relative mobility (Rf) of each band and
extrapolating from a linear, semi-logarithmic plot of
molecular weight versus Rf of four molecular weight
marker proteins run on each gel.
thesis and differentiation in this embryo.
Whether this apparent uncoupling is at the
transcriptional level of protein synthesis is
yet to be determined as are other details
such as changes in amino acid pool sizes and
cell permeability and selective uptake of
precursors and inhibitory agents.
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C labeled peptides that were synthesized at different
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