DEVELOPMENTAL
BIOLOGY
106, 236-242 (1984)
Developmental Time, Cell Lineage, and Environment Regulate the
Newly Synthesized Proteins in Sea Urchin Embryos
DEBRA PITTMAN AND SUSAN G. ERNST’
Department
of Biology,
Received November
Tufts University,
Meow-d,
Massachusetts
15, 1983; accepted in revised form
02155
June 25, 1984
Strwngylocentrotus
purpuratus
embryos were fractionated
into two cell populations of defined lineages at times
corresponding to two critical developmental events: determination
(16-cell stage) and early differentiation
(mesenchyme
blastula). The 16-cell stage blastomeres, labeled with [35S]methionine, exhibited identical protein synthesis patterns
by fluorography,
and this pattern was not significantly
altered by cell separation. In comparing the proteins of the
mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography
of newly
synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary
mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm
cells. Additionally,
in contrast to the
earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially
altered by cell
separation. The ability to alter protein synthesis in response to environmental
factors may be a further demonstration
of the differentiation
of these cells, (c IW Academic PWS, ant
INTRODUCTION
In sea urchin embryos, the patterns of newly synthesized proteins remain relatively similar from before
fertilization
until hatching when significant
changes
occur. Bedard and Brandhorst
(1983) have recently
carried out an extensive two-dimensional
gel electrophoretic analysis of newly synthesized proteins and
report that 60% of the changes in protein synthesis
occurring from the time of fertilization
to the pluteus
stage, take place in the relatively
short time period
from hatching to early gastrulation.
This period corresponds to about 20% of the developmental
time
studied and encompasses the mesenchyme blastula
stage. The time from hatching to early gastrulation
is
characterized by the first morphological changes in sea
urchin embryogenesis
and also represents the time
when the three germ layers start to differentiate.
Cells of the sea urchin are fated by the 16-cell stage,
which occurs about 5 hr postfertilization
in Strongylocentrotus purpuratus.
The four small micromeres at
the vegetal pole are the progenitors
of the primary
mesenchyme cells in the mesenchyme blastula, which
then secrete the spicules of the larval skeletal system.
The eight animal pole mesomeres give rise to the
majority of the ectoderm. The remainder of the ectodermal cells are derived from descendants of the four
large macromeres at the 16-cell stage, whose descendants also make up the endoderm and secondary mesenchyme (reviewed, Okazaki, 1975). Both one- and two’ To whom all correspondence
should be addressed.
0012-1606/84 $3.00
Copyright
All rights
0 1984 by Academic Press, Inc.
of reproduction
in any form reserved.
236
dimensional polyacrylamide gel electrophoretic analysis
of newly synthesized proteins in the isolated cells of
the 16-cell stage embryo, have demonstrated that the
patterns of protein synthesis in micromeres, mesomeres, and macromeres are essentially identical (Senger
and Gross, 1978; Tufaro and Brandhorst, 1979; Harkey
and Whiteley, 1983).
In sea urchin embryos, the first morphological
signs
of cell specialization
and the most significant change
in the pattern of protein synthesis are apparent at the
mesenchyme blastula stage. Our interest is in the cellspecific regulation of expression of genes in the strictly
determined micromere-primary
mesenchyme lineage.
Therefore, the experiments in the present study were
designed to determine if any of the changes in the
pattern of protein synthesis seen at the mesenchyme
blastula stage are specific to or enhanced in either the
micromere-primary
mesenchyme lineage or the combined lineages of the mesomeres and macromeres to
presumptive endoderm and ectoderm.
MATERIALS
AND
METHODS
Sea urchin embryos. S. purpuratus
were obtained
from Pacific Biomarine (Venice, Calif.) and from Patrick
Leahy (Corona de1 Mar, Calif.). Animals were maintained in Instant Ocean tanks at 11-13°C in a mix of
natural and artificial seawaters. Spawning, fertilization,
and growth of the embryos were as previously described
(Ernst et ah, 1980).
Cell separations. Mesenchyme blastula embryos were
separated into two cell fractions: primary mesenchyme
PITTMAN
AND ERNST
Cell-Specijc
and a population of presumptive endoderm and ectoderm (endoderm/ectoderm)
as described by Harkey
and Whiteley (1982) with minor modification
of S. G.
Ernst, F. R. Bushman, and W. R. Crain, Jr. (manuscript
submitted).
Labeling of cells, “Mini-separations”
prepared by a
modification
of the standard large scale procedure,
were used for mesenchyme blastula embryos labeled
before the cell separation. At the mesenchyme blastula
stage 1.2 X lo5 embryos were pelleted at 1200 rpm for
3 min. Embryos were suspended in 12 ml of MPFSW
with Pen-Strep (20 units/ml penicillin, 20 pg/ml streptomycin) and a total of 840 &i of [35S]methionine.
Embryos were labeled at 12-14°C for 2 hr with constant
stirring. If appropriate, a 2-ml aliquot was removed at
this time for a sample of mesenchyme blastula labeled
before the cell separation.
Embryos were pelleted,
washed twice in 15 ml of ice-cold CMFSW (calciummagnesium-free
seawater) with 100 PM EDTA (ethylenediaminetetraacetic
acid), and washed once in 12 ml
of 1 Mglycine (pH 8.0). When embryos were dissociated,
they were pelleted at 1200 rpm for 3 min and the pellet
was resuspended in 1.5 ml 1 M dextrose:CMFSW
with
100 PM EDTA (40:60) and Percoll was added to 17.5%.
Samples for total mesenchyme blastula embryos labeled
after the cell separation were taken at this point, if
appropriate. Dissociated embryos, suspended in Percoll,
were centrifuged
at 1750 rpm for 15 min and the
primary
mesenchyme
and endoderm/ectoderm
cell
fractions were collected and washed as described for
the larger preparations. Isolated primary mesenchyme
cells and endoderm/ectoderm
cells labeled after the
cell separation was suspended in 1 ml of MPFSW with
Pen-Strep and incubated at 12-14°C for 30 min, at
which time 250 &I of [35S]methionine were added and
the cells labeled for 2 hr with constant stirring.
Labeling was stopped by pelleting the cells in an Eppendorf centrifuge and washing twice in MPFSW.
Sample preparations,
quantitations,
and electrophv
resis. Samples of total embryos or isolated cell populations were suspended and dispersed in lysis buffer
(O’Farrell, 1975) containing 9.5 M urea, 2% NonidetP40, and 5% 2-mercaptoethanol.
Incorporation
of
[35S]methionine was determined by trichloroacetic
acid
precipitation
of sample aliquots onto glass fiber filters,
followed by scintillation
counting in Aquasol (New
England Nuclear). The amount of protein in each
sample was determined by Bio-Rad protein assay according to the manufacturer’s
directions. Samples were
frozen and stored at -70°C. Protein samples for electrophoresis were thawed and aliquots precipitated with
8-10 vol of 80% acetone at -20°C. Pellets were collected
by centrifugation,
dried, and resuspended in 0.1 M Tris
(pH 7.6), 5 mM MgC&, 10 mM dithiothreitol,
and 1
Protein
237
Synthesis
mM PMSF (phenylmethylsulfonylfluoride)
with 50 pg/
ml of deoxyribonuclease
(Worthington,
2005 D) and 50
pg/ml of ribonuclease A (Sigma). Samples were incubated for 20 min on ice, followed by precipitation
with
8-10 vol of 80% acetone at -20°C as above. The dried
samples were then resuspended in the lysis buffer plus
2% ampholytes (80% pH 5-7 (LKB), 20% pH 3-10 (BioRad)). Approximately
equal amounts of protein of
similar specific activities were loaded onto gels. Generally 2.5-5 X lo5 cpm, representing approximately
lo20 pg of protein, were loaded per gel. Electrophoresis
was as described by O’Farrell (1975). One gel of each
focus was run in parallel to determine the pH gradient.
On the second dimension, molecular weight markers
were run in a well at the end of each gel. Gels were
treated with Enhance (New England Nuclear), dried,
and exposed to preflashed Kodak XAR-5 film at -70°C
for a length of time proportional
to the counts loaded
on the gel (Laskey and Mills, 1975). Newly synthesized
proteins with isoelectric points between 4.5 and 7, in
the molecular weight range of lO,OOO-150,000 were
compared.
RESULTS
Changes in protein synthesis between the l&cell stage
and mesenchyme blastula embryos. Embryos were labeled in the presence of [35S]methionine for 2 hr, from
the late 4-cell stage through the 16-cell stage, and
others were labeled for 2 hr at the mesenchyme blastula
stage. Total proteins were isolated and separated on
two-dimensional
gels, followed by fluorography.
Although there is substantial
overlap in the proteins
synthesized at these two stages, there are a number of
proteins that appear to be exclusively,
or predominantly, synthesized at one stage compared to the other.
Of approximately
400 reproducibly
resolvable proteins
in these gels, the synthesis of 3.8% (15) of these was
enriched (at least threefold-determined
by visual inspection) in the 16-cell stage and 10.8% (43) was
enriched at the mesenchyme blastula stage. Similar
observations were reported by Bedard and Brandhorst
(1983).
Dramatic changes in the pattern of protein synthesis
in, cells labeled after the mesenchyme blastula cell separation procedures. To determine the extent of differentiation at the level of protein synthesis exhibited by
mesenchyme blastula cells, the embryos were dissociated and a sample taken of the total embryos and
the rest were fractionated
into primary mesenchyme
and endoderm/ectoderm.
These three cell populations
were incubated in the presence of [35S]methionine for
2 hr. Proteins were isolated from these cell populations
and analyzed by two-dimensional
gel fluorography.
238
DEVELOPMENTALBIOLOGY
Before a detailed comparison of the newly synthesized
proteins was made, it was necessary to determined if
the cell separation procedure caused any changes in
the pattern of protein synthesis. Therefore mesenchyme
blastula
embryos
were labeled
for 2 hr with
[35S]methionine and the unincorporated
label washed
out. A sample was taken for protein from unfractionated embryos and the rest of the embryos were fractionated as described for “mini-separations.”
Proteins
from these three samples were isolated and analyzed
VOLUME106.1984
on two-dimensional
gels, followed by fluorography.
Comparison of the proteins synthesized by cells labeled
before and after the cell separation, produced some
surprising and unexpected results. Figure 1 represents
fluorographs of newly synthesized proteins from primary mesenchyme cells labeled before (A) and after
(C) the cell separation and from endoderm/ectoderm
cells labeled before (B) and after (D) the cell separation.
Total embryos labeled before and after cell dissociation
gave essentially
the same results as observed for
1 If5
92.5
($53
x
3
H
2 M -
FIG. 1. Comparison of newly synthesized proteins at the mesenchyme blastula stage, labeling before and after the cell separation. Primary
mesenchyme cells were labeled before (A) and after (C) the cell separation and endoderm/ectoderm
cells were also labeled before (B) and
after (D) the separation. 0, Increases in synthesis as a result of cell separation; 0, decreases resulting from cell separation. Each of the
increases are also identified by a number for analysis in Table 1. A, actin; T, tubulin.
PITTMAN AND ERNST
Cell-Speci&
endoderm/ectoderm
(data not shown). The synthesis
of some proteins was induced as a result of the cell
separation while the synthesis of others was greatly
decreased. Most changes in either direction were common to both cell types, however, there were a few cellspecific changes. Between the two cell populations
a
total of 37 proteins, marked by squares, showed significant decrease in synthesis (threefold or greater). Of
the 37 proteins, 28 were common to both primary
mesenchyme cells and the endoderm/ectoderm
population. Two proteins, designated a and b, showed a
decrease in primary mesenchyme, but no corresponding
reduction of synthesis in endoderm/ectoderm.
The synthesis of 5 proteins was decreased in endoderm/ectoderm with no corresponding
decrease in the primary
mesenchyme cells. A total of 20 proteins, marked by
circles, were induced as a result of the cell separation
procedure. Table 1 presents an analysis of the 20
TABLE
SILVER
BLASTULA
1
STAIN AND FLITOROGRAPHIC
ANALYSIS OF MESENCHYME
PROTEINS INDUCED BY THE CELL SEPARATION PROCEDURE
Silver staind
“S fluorography’
Protein”
1
P
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
P. M.
before
E./E.
before
P. M.
after
E./E.
after
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
-
-
-
+
+
-
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
16-Cell
Mesenchyme
blastula
+
++
+
?
++
+
?
+
++
++
-
+
+
+
?
+
+
+
+
+
+
-
+
+
+
+
+
++
‘Twenty
proteins, identified by numeral on Fig. 1, were induced
in one or both of the cell fractions as a result of the cell separation
procedures.
‘2 represents a cluster of four proteins.
’ Fluorographs
were compared of proteins labeled in primary
mesenchyme cells before (1A) and after (1C) the cell separation and
in endoderm/ectoderm
before (1B) and after (1D) cell separation.
Undetectable
or barely detectable
proteins
are scored ‘I-” and
proteins whose synthesis is significantly
induced are marked “+“.
“Each of the proteins was identified by silver staining. Proteins
clearly identified by silver staining are marked by + and those that
are distinctly
more abundant in one stage over the other are marked
++. A 1 indicates that the resolution
in this area of the gel made
definite identification
impossible.
Protein
Synthesis
239
proteins which showed increased synthesis as a result
of cell separation. The protein numbers correspond to
markings on the fluorographs,
with number 2 representing a group of 4 proteins enclosed in parenthesis.
The induced synthesis of ten of the proteins was
common to both cell populations, while the synthesis
of each of the remaining 10 was induced in only one
cell population. There is one protein, 12, which was
actually turned on in primary mesenchyme cells and
off in endoderm/ectoderm.
Of approximately
450 proteins, the synthesis of 57,
or 12.7% of them, is significantly
and reproducibly
affected by the cell separation procedure at the mesenchyme blastula stage. We used silver-stained
gels of
proteins at the 16-cell and mesenchyme blastula stages
to determine whether the induced proteins were normal
cellular proteins or totally new proteins, present as a
result of the stress induced by the cell separation
procedure. Table 1 shows that 15 of the 20 proteins
are clearly normal cellular proteins, based on silverstain analysis (data not shown). Three additional proteins (12-14) are normally synthesized in endoderm/
ectoderm and induced in primary mesenchyme. The
resolution of the gels in the area of protein 4 made it
impossible to be sure if 4 is faintly detectable on silver
stains. Protein 11 appears to be the only induced
protein that is not normally synthesized or detectably
present at either the 16-cell or mesenchyme blastula
stages.
Synthesis
of cell-specijk w enriched proteins at the
mesenchyme
blast&a stage. The experiment in Fig. 1
demonstrated that a comparison of the normal pattern
of newly synthesized proteins in primary mesenchyme
cells to the normal pattern in endoderm/ectoderm
cells
has to be made with embryos that were labeled before
the cell separation. Figure 2 presents the same fluorographs of newly synthesized proteins in primary mesenchyme (A) and endoderm/ectoderm
cells (B) as shown
in Fig. 1A and B. These embryos were labeled with
[35S]methionine
for 2 hr, the unincorporated
label
washed out, and the embryos harvested and fractionated according to the “mini-separation”
procedure.
Differences marked between these two cell populations
represent reproducible protein differences between the
two cell populations as detected on 12 different presumptive endoderm/ectoderm
gels from 8 different cell
separations and 8 primary mesenchyme gels from 4
different cell separations. Of approximately 450 proteins
the synthesis of 13 of these (2.9%), represented by
circles, is specific to or enriched (threefold or greater)
in primary
mesenchyme cells. The synthesis of 37
additional proteins (8.2%), marked by squares, is enriched or confined to the endoderm/ectoderm
cell population. No attempts were made to distinguish between
DEVELOPMENTALBIOLOGY
240
VOLUME106, 1984
B
200
-
116
-
92.568
m
0
vx
z
B
m
0
40
x
b
E
FIG. 2. Comparison of the newly synthesized proteins in the mesenchyme blastula cell populations. Primary mesenchyme (A) and
endoderm/ectoderm cells (B) were labeled before the cell separation. 0, primary mesenchyme-enriched proteins; 0, endoderm/ectodermenriched proteins, A, actin; T, tubulin.
qualitative and quantitative
synthesis differences since
it has been demonstrated that the distinction
between
them is not absolute (Bedard and Bandhorst, 1983).
Since we compared the results of numerous gels from
several cell separations to determine reproducibility,
there are some protein difference detectable between
Figs. 2A and B that are not scored as cell specific.
DISCUSSION
Analysis of newly synthesized proteins in primary
mesenchyme and endoderm/ectoderm
cells has demonstrated that, in contrast to the 16-cell stage embryo,
the mesenchyme blastula synthesizes cell-specific and
enriched proteins. In the course of this investigation
it was also discovered that cells labeled after the cell
separation procedure had a substantially
altered pattern of protein synthesis compared to cells labeled
before cell separation. Mesenchyme blastula cells were
labeled with [35S]methionine before and after cell separation and analyzed by two-dimensional
gel electrophoresis.
Micromeres of the 16-cell stage sea urchin embryo
are morphologically
distinct and strictly determined.
However, within the limits of detection, newly synthesized proteins are shared between the micromeres and
the mesomeres/macromeres
which comprise the rest
of the 16-cell stage embryo (Chamberlain,
1977; Tufaro
and Brandhorst,
1979; Ernst and Pittman, 1982). We
have previously demonstrated that the total polysomal
RNA populations in micromeres and mesomeres/mac-
romeres, including both abundant and rare sequences,
were between 66-100% overlapping (Ernst et cd., 1980).
In comparing the 16-cell stage to the mesenchyme
blastula stage, synthesis of 3.8% of the proteins was
enriched in, or confined to, the 16-cell stage while
10.8% of the newly synthesized proteins were enriched
or confined to the mesenchyme blastula. The finding
that the synthesis of more proteins is enriched in the
more advanced stage of development is consistent with
the recent report of Bedard and Brandhorst (1983) that
there is a greater number of increases compared to
decreases in protein synthesis as development proceeds.
Labeling cells before and after embryo fractionation
at the two stages produced surprising and important
results. Previous reports (Tufaro and Brandhorst, 1979;
Harkey and Whiteley,
1983) and our present work
(data not shown) demonstrated
that at the 16-cell
stage, labeling before or after the cell separation
appeared to have little effect on the pattern of newly
synthesized proteins. However, a substantial difference
was seen at the mesenchyme blastula stage. Possible
factors responsible for the differences in labeling patterns seen with cells labeled before or after the cell
separation fall into two separate, but related, categories.
The alteration
in the protein synthesis pattern of
separated cells may be a response to loss of normal
cell-cell or cell-substrate
contacts. Alternatively,
this
may be a more general “stress response” induced by
the cell separation procedure. The stress response may
include, but would not be limited to lack of normal cell
contacts.
PITTMAN AND ERNST
Cell-Specijc
The effect of dissociation on protein synthesis observed here may be similar to reports on tissue culture
cells in which it has been demonstrated that disruption
of normal cell-cell contacts can lead to considerable
quantitative
variation in the synthesis of normal cellular proteins (Spiegelman and Farmer, 1982; Farmer
et al, 1983). During dissociation
of the mesenchyme
blastula, not only are cell-cell contacts broken, but cell
contacts with the hyaline layer and basement membrane substrates are also disrupted. The blastomeres
of the 16-cell stage embryos have smooth membrane
surfaces where the cells come in contact with each
other. It is late in cleavage that adhesion stabilities
among cells increase (Watanabe et al., 1982) and normal
cell membrane attachments, such as desmosomes, are
formed (Okazaki, 1975). It is also toward the end of
cleavage when the basement membrane starts to form.
If the dramatic change in the protein synthesis pattern
seen in mesenchyme blastula cells following dissociation
is due to the disruption
of cell-cell and cell-surface
contacts, it is consistent that we do not observe these
protein changes at the 16-cell stage, before the cellcell and cell-surface
contacts have been established.
In a recent study Harkey and Whiteley (1983) report
that isolated micromeres grown in culture for 90 hr
and primary mesenchyme cells grown in culture for 50
hr, synthesized similar proteins when they both reached
98 hr of development. During these extended culture
conditions, the cells are able to reestablish their normal
cell-cell and, at least in the case of those isolated at
the mesenchyme blastula stage, cell-substrate
contacts
(Okazaki, 1975; Harkey and Whiteley, 1983).
We demonstrate that the pattern of protein synthesis
is significantly
altered in mesenchyme blastula cells
due to the cell separation. Determination
of whether
or not this is a general “stress response” would require
additional information.
The lack of response at the 16cell stage to dissociation,
compared to the strong
response in mesenchyme blastula embryos, is similar
to the situation observed when sea urchins were “heat
shocked.” Exposing embryos to elevated temperatures
at any developmental stage decreased the overall rate
of protein synthesis, but it was not until after hatching
that specific heat-shock
proteins
were synthesized
(Roccheri et al., 1981). As shown in Table 1, at least 15
of the 20 proteins induced by the cell separation
procedure are proteins that are present in sufficient
abundance to be detected by silver-stain
analysis. Interestingly,
half of these silver-stained
proteins, including the cluster of 4 proteins labeled 2 (Fig. 2), are
significantly
more abundant in 16-cell stage embryos
when compared to mesenchyme blastula. Cell separation inducing the synthesis of proteins at the mesenthyme blastula stage that normally are synthesized at
Protein
Synthesis
241
other developmental stages is particularly
interesting
in light of the results of two recent studies. In Drosophila, it has been shown that proteins very similar
to heat shock proteins are synthesized in non-heat
shocked adult tissues (Craig et ah, 1983). In the mouse,
two of the first normal major zygotic proteins of the
embryo are identical
to mouse heat-shock proteins
HSP68 and ‘70 (Bensaude et al., 1983).
The significant alteration
in the pattern of protein
synthesis, including both inhibition
and induction of
proteins, in mesenchyme blastula cells labeled before
and after the cell separation, was about equal to the
number of changes in protein synthesis in going from
16-cell stage to mesenchyme blastula. Bruskin et al.
(1982) also report observing alterations in the pattern
of newly synthesized proteins in sea urchin cells following a similar dissociation, but do not characterize
these alterations. Harkey and Whiteley (1982) reported
no difference between the pattern of proteins synthesized in mesenchyme blastula cells labeled before or
after the cell separation procedure. The conflicting
reports on the effect of the cell separation on the
pattern of protein synthesis cannot be explained by
the cell separation procedure itself, since both studies
use essentially
the same method. However, several
possibilities for this difference do exist. The experiment
we report in Fig. 1 and Table 1 was designed to detect
any changes caused by the cell separation procedure
used by Harkey and Whiteley (1982) and ourselves,
and our results are representative
of several gels of
total embryo, primary mesenchyme, and endoderm/
ectoderm proteins. Since they report considerable difficulty
labeling
primary
mesenchyme
cells with
[3H]valine
before the cell separation,
Harkey and
Whitely (1982) compared primary mesenchyme cells
labeled after the cell separation to endoderm/ectoderm
cells labeled before the cell separation. In their study,
in the one experiment in which primary mesenchyme
cells were labeled before the cell separation, at least
60% of the cells were endoderm/ectoderm
and the cell
separation and gel system they used were different
from those employed in the rest of the study. Therefore,
the only comparison made of primary
mesenchyme
cells labeled before and after the cell separation was
between cells isolated by different
procedures and
proteins separated on a different gel system.
In addition to possessing an ability to alter protein
synthesis in response to environmental
factors, the
cells of the mesenchyme blastula embryo are different
from 16-cell stage blastomeres in that the cells of
distinct lineages have nonidentical
patterns of protein
synthesis. Synthesis of 2.9% of the proteins at this
stage is primary mesenchyme enriched or specific, and
8.2% is endoderm/ectoderm
enriched or specific. The
242
DEVELOPMENTAL BIOLOGY
higher percentage of endoderm/ectoderm
enriched
proteins, relative to primary mesenchyme, probably
foretells the immediate future of these cells. Within a
few hours after the mesenchyme blastula stage, the
cells of the endoderm/ectoderm
will separate into
three distinct cell populations, recognizable by morphology and position: endoderm, ectoderm, and secondary mesenchyme. Mesenchyme blastula embryos show
changes, including increases and decreases, of 14.6% of
the newly synthesized proteins compared to the 16-cell
stage. Bedard and Brandhorst (1983), analyzing about
twice as many proteins as in the present study, report
a similar change in protein synthesis, 11.2%, during
the same developmental
time. It has been reported
(Harkey and Whiteley, 1982) that 28% of the newly
synthesized proteins are cell enriched or specific at the
mesenchyme blastula stage. This is twice that demonstrated for the percentage of change between the 16cell stage, when there is no blastomere-enriched
synthesis, and the mesenchyme blastula stage. Because of
the significant alterations resulting from the cell separation procedure, and since Harkey and Whiteley
(1982) compared endoderm/ectoderm
labeled before
the cell separation procedure to primary mesenchyme
cells labeled after the cell separation, it is possible
that their study represents an overestimate
of cell
enriched/specific
protein synthesis at the mesenchyme
blastula stage. We report here that the synthesis of
11% of the 450 most abundantly synthesized proteins
at the mesenchyme blastula stage is cell specific or
enriched.
We thank Andre Bedard in Dr. Bruce Brandhorst’s
laboratory for
initial instructions
in running two-dimensional
gels. We also thank
Dr. W. R. Crain and Dr. S. Slapikoff for critically
reading this
manuscript. The work was supported by an NIH grant and a grant
from Tufts University
Biomedical research funds to S.G.E.
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