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A family of proteins accumulating in ectoderm of sea urchin embryos specified by two related cDNA clones
DEVELOPMENTAL
BIOLOGY
91,31.7-324
(1982)
A Family of Proteins Accumulating in Ectoderm of Sea Urchin Embryos Specified by Two Related cDNA Clones ARTHUR M. BRUSKIN,*~’ *Program
in Molecular,
PIERRE-ANDRE BEDARD,t,’ ANGELA L. TYNER,* RICHARD M. SHOWMAN,* BRUCE P. Bk4mHowr,t AND WILLIAM H. KLEIN**’
Ce&lar, and Developmental Biology, Department and TDepartment of Biology, McGill University, Received
December
4, 1981; accepted
of Biology, Montreal
in revised
form
Indiana PQ, H3A
University, Bloomington,
February
4, 1982
lB1,
Indiana 47.405;
Canada
Two cDNA clones, pSpec 1 and pSpec 2, had been selected previously as corresponding to transcripts greatly enriched in ectoderm of pluteus-stage larvae of the sea urchin, Strmzglyocentrotus purpuratus. The two cDNA clones, corresponding to the 3’ ends of polysomal RNAs, have similar but distinct restriction maps. Messenger RNAs hybrid selected by these two cDNA clones were translated in a rabbit reticulocyte lysate cell-free system into the same set of 10 acidic proteins having mol.ecular weights of 14-17,000 daltons. These in vitro products have positions identical or similar on two-dimensional gels to a group of proteins whose synthesis in vivo is highly enriched in the ectoderm. The 1.5- and 2.2-kb transcripts corresponding to pSpec 1 and pSpec 2 increase in prevalence by at least loo-fold during embryonic development. The rates of synthesis of these ectoderm proteins also increase by over loo-fold, though the patterns of change are distinct. It is likely that pSpec 1 and pSpec 2 correspond to mRNAs which are part of a small family of genes coding for th’e group of similar ectoderm proteins. These proteins and their mRNAs are enriched in eetoderm by the early gastrula stage.
bryos. There is presently little information concerning the degree to which specific mRNAs are restricted to particular blastomeres either before or during the process of cellular differentiation occurring in later stages of embryogenesis. The recent development of methods for large-scale isolation of each of the primary germ layers of sea urchin embryos has provided a new approach to this problem (M&lay and Chambers, 1978; Harkey and Whitely, 1980). Recently, we have isolated clones representing mRNAs enriched in the ectoderm of sea urchin plutei (Bruskin et al., 1981). These mRNAs accumulate during embryogenesis, their mass per embryo increasing by more than loo-fold beginning just prior to or during gastrulation. The transcripts corresponding to these clones are found on polysomes, but the proteins they code for were not known. Using hybrid selected translation, we report here that two of these clones, which are similar but not identical in structure, code for a group of small, acidic polypeptides which are actively synthesized during the later stages of embryogenesis and are enriched in ectoderm. It is likely that the mRNAs coding for these proteins are derived from a small gene family.
INTRODUCTION
The timing and extent of change in mRNA populations during embryonic development have been the subject of many recent investigations. The population of rare mRNAs changes substantially during the development of sea urchin embryos, the general pattern being a gradual decline in sequence complexity (Galau et al., 1976; Hough-Evans et al., 1977). Populations of prevalent mRNAs also change during sea urchin embryogenesis (Shepherd and Nemer, 1980). Recently, the use of cloned DNA corresponding to several specific mRNAs has confirmed tbat the prevalence of both rare and abundant mRNA sequences can change substantially during development (Lev et al., 1980; Lasky et al., 1980; Bruskin et al., 1981; Xin et al., 1982). There are also many changes in the patterns of synthesis of hundreds of proteins which are detectible by two-dimensional electrophoresis (Brandhorst, 1976; Bedard and Brandhorst, 1982). By hybrid selection of mRNA using cloned DNA and cell-free translation, it is now possible to correlate specific mRNAs to specific proteins whose developmental patterns of synthesis have been determined. These investigations of mRNA populations and corresponding proteins have been applied to intact emi Contributed a To whom
MATERIALS
AND
Culturing and Fractionation Strongylocentrotus
equally to research reported. correspondence :should be addressed.
Pacific Biomarine 317
METHODS
of Embryos
purpuratus were purchased
(Venice, Calif.).
Embryos
from were cul-
0012-1606/82/060317-08$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
318
DEVELOPMENTALBIOLOGY
tured at 15°C and harvested at the appropriate times as described previously (Bruskin et al., 1981). In cases where egg or embryo proteins were to be radiolabeled, 10,000 eggs or embryos were suspended in 1 ml of artifical seawater and incubated for 2 hr with 50 &i of [35S]methionine (embryos) or for 4-6 hr with 250 &i of [35S]methionine (eggs). After labeling, the eggs or embryos were washed extensively in cold seawater. Pluteus endoderm/mesoderm and ectoderm fractions were isolated as described in Bruskin et al. (1981). Gastrula endoderm/mesoderm and ectoderm were isolated according to the procedure of Harkey and Whiteley (1980) with the following modification: glycine-dissociated embryos were brought to a final density of 1.058 (measured durectly using a hydrometer) with Percoll. Following centrifugation, the partially purified endoderm/mesoderm was further purified over sucrose step gradients. In a few cases, gastrula and pluteus endoderm/mesoderm was isolated by lysing the ectodermal cells in situ using Triton X-100. Gastrulae or plutei were suspended in 100 vol of cold 0.35 M KCl, 50 mM MgClz, 25 mM EGTA, 0.5% Triton X-100, 50 mM Pipes, pH 6.5. The embryos were incubated for a few seconds to lyse the ectodermal cells and then pelleted by gentle centrifugation. the resulting endoderm/mesoderm pellet was then washed extensively with cold seawater. Preparation and Electrophoresis Cellular RNA
of Total
RNA was isolated from embryos by the guanidinehydrochloride extraction procedure described in Bruskin et al. (1981). RNA was stored at 10 mg/ml in water at -70°C. RNA was electrophoresed on formaldehyde agarose gels, blotted onto nitrocellulose filters, and hybridized with nick-translated pSpec 1 or pSpec 2 [32P]DNA as detailed in Bruskin et al. (1981). Hybrid
Selection and in Vitro Translation
Fifteen micrograms of pSpec 1 or pSpec 2 DNA was heated at 100°C in 30 ~1 of 0.4 M NaOH in 0.8 M NaCl. The solutions were neutralized by adding an equal volume of 1 M Tris, pH 8, 3 M NaCl, and the DNA was spotted onto 0.5-cm2 pieces of nitrocellulose filter paper (Schleicher and Schuell, BA85). The filters were air dried, washed in 0.15 M sodium citrate, 1.5 M NaCl (pH 7.5) at room temperature for 30 min, and baked in a vacuum over at 80°C for 2 hr. The filters were then cut up into smaller pieces, placed in a 1.5-ml disposable Eppendorf tube, and prehybridized for l-2 h at 45°C in 100 ~1 of hybridization buffer (50% formamide, 100 mM Pipes, pH 6.5, 600 mM NaCl, 100 pg/ml poly rA). Following the prehybridization, fresh hybridization buffer
VOLUME91,1982
containing 100 pg of total cellular RNA was added. After hybridizing for 12-16 hr at 45”C, the filters were washed at 45°C five times each with 1.5 ml of 1 mM Pipes, pH 6.5, 6 mM NaCl, 0.5% SDS, and 1 mM Pipes, pH 6.5, 6 mM NaCl, respectively. After the final wash, 300 ~1 of water was added to elute the hybridized RNA. The filters were heated in a boiling water bath for 90 set and then immediately cooled in a dry ice-acetone bath. The eluted material was transferred to another 1.5-ml disposable Eppendorf tube and 10 pg of yeast tRNA, 30 ~1 of 2.5 M ammonium acetate, and 680 ~1 of ethanol were added. The RNA was precipitated overnight at -70°C. The RNA was pelleted by centrifugation and taken up in 10 ~1 of a supplemented rabbit reticulocyte lysate (Amersham) and 1~1 of [35S]methionine (1000 Ci/mmole; 10 Ci/ml). The reactions were incubated 90 min at 30°C. Two-Llimensional
Gel Electrophoresis
Eggs, embryos, embryonic tissues, or lyophilized rabbit reticulocyte lysates were suspended in lysis buffer (O’Farrell, 1975) containing 0.2% SDS. Two-dimensional electrophoresis on polyacrylamide gels was performed according to O’Farrell (1975). Silver staining of gels was according to Oakley et al. (1980). RESULTS
Sequence Homology
of pSpec 1 and pSpec 2
In a previous investigation we isolated two cDNA clones, pSpec 1 and pSpec 2, which were complementary to mRNAs enriched in the ectoderm of plutei (Bruskin et al., 1981). Two lines of evidence suggested that these DNAs have sequence homology. First, both clones hybridized to many fragments of the same size on blots of genomic DNA. Second, RNA gel blots showed that pSpec 1 hybridized strongly to a 1.5-kb RNA and weakly to a 2.2-kb RNA, while pSpec 2 hybridized strongly to a 2.2-kb RNA and weakly to a 1.5-kb RNA. Weak hybridization to other bands was also observed. To further examine the sequence homology, restriction maps were constructed for the two cDNA clones. Figure 1 shows that the two clones share four restriction sites for HueIII, KpnI, HpaII, and AU. Since the lengths of the inserted sea urchin sequences are only 0.5 to 0.6 kb, the coincidence of these restriction sites cannot be due to chance and provides strong evidence that the two clones are closely related. There are also differences in the maps: pSpec 2 contains HinfI and Hind11 sites not present in pSpec 1, and pSpec 1 contains an XbaI site not present in pSpec 2. In addition, the distances between the HaeIII-HpaII sites and HpaII-AZuI sites differ from the two clones by approximately 15 base pairs.
BRUSKIN
ET AL.
Embr-pnic
The significance of these small differences in length is as yet unclear. The clones pSpec 1 and pSpec 2 were constructed by cloning cDNA-RNA hybrids in pBR322 after reverse transcription of oligo(dT)-primed polyadenylated cytoplasmic RNA (Lasky et al., 1980). We have found that, as expected, these cloned DNAs are complementary to the 3’-terminal sequences of their respective RNAs. The evidence for this is based on the characterization of a recombinant X phage we have recently isolated which contains genomic DNA complementary to pSpec 1 (Bruskin and Klein, unpublished results). Trascription mapping of this X clone and orientation of pSpec 1 DNA with respect to transcription indicate that the sequence represented by pSpec :L must be at the 3’ end of the RNA. The direction of transcription of pSpec 1 is 3’ to 5’, left to right, on the restriction map shown in Fig. 1. We assume that pSpec 2 has a similar orientation. Thus, pSpec 1 and pSpec 2 apparently represent 0.5 to 0.6 kb at the 3’ ends of 1.5- and 2.2-kb RNAs, respectively. Because of the extensive sequence homology of the DNAs, both clones are capable of hybridizing to either transcript to sorne extent. Translation of RNA and pSpec 2
Hybrid
Selected by pSpec
1
We isolated pSpec 1 and pSpec 2 mRNAs by hybridizing total cellular RNA from gastrula-stage embryos with either pSpec 1 or pSpec 2 DNA immobilized on nitrocellulose filters. The hybridized RNA was eluted from the filters and translated in a rabbit reticulocyte lysate cell-free system supplemented with [35S]methionine. The r,adiolabeled proteins were analyzed by two-dimensio:nal electrophoresis, and results of a typical experiments are shown in Fig. 2. RNA selected by either cloned DNA coded for approximately 10 polypeptides which
Ectodermal
319
Proteins
wl-lwP,c 00 oaao IYIX
pSpec I
YI
I
y 1 a
(
,100bp,
FIG. 1. Restriction endonuclease maps of pSpec 1 and pSpec 2 DNAs. Maps were constructed by digesting the plasmid DNAs with one or two of the indicated enzymes and electrophoresing the digested material on 5% polyacrylamide gels. The direction of transcription of pSpec 1 is 3’ to 5’, left to right, on the map. This was determined by labeling pSpec 1 DNA at the single XbaI site, located within the insert sequence, with [3ZP]dCTP and reverse transcriptase. The labeled DNA was then digested with HhaI yielding two fragments labeled at the 3’ end of opposite strands. These fragments were isolated from 5% polyacrylamide gels and then used as probes in RNA gel blot hybridizations. The orientation was determined by the fragment that hybridized to the RNA.
Ectoderm-Enm’ched Proteins Synthesized in Vivo
Plutei were incubated for 2 hr with [35S]methionine and then separated into ectoderm and endoderm/mesoderm fractions. The newly synthesized proteins were analyzed by two-dimensional electrophoresis. Newly synthesized proteins enriched in the ectoderm fraction are pointed out by arrows in Fig. 3A. The corresponding positions of these spots in the endoderm/mesoderm fraction are indicated by circles in Fig. 3B. Some of the differences are very pronounced, others less obvious. Most of the very pronounced ectoderm-enriched proteins are small and acidic, having mobilities similar to the products of cell-free translation of pSpec 1 and pSpec 2 mRNAs. In Fig. 3B are shown the proteins which were being synthesized by endoderm/mesoderm of the pluteus-stage embryos. Arrows point to the few polypeptides enriched in the endoderm, in particular, two or three proteins having molecular weights of 24,000 and two having molecular weights of 37,000. The incorporation of radioactive methionine into proteins synthesized by endoderm/mesoderm of intact embryos indicates that methionine is accessible to internal tissues, validating analyses of protein synthesis on intact embryos. On the other hand, dissociation of embryos followed by incubation with [35S]methionine can lead to perturbations in protein synthesis (Bedard, unpublished observations). The results presented in Figs. 2 and 3 strongly suggest that some of the proteins whose synthesis is enriched in ectoderm in vivo are identical to the group of products of cell-free translation of hybrid-selected mRNAs
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corresponding to pSpec 1 and pSpec 2 DNAs. To confirm this the cell-free translation products of gastrula RNA hybrid selected with pSpec 1 and pSpec 2 DNAs were compared to proteins whose synthesis is enriched in ectoderm fractions of gastrulae. In this case the products were analyzed on the same series of two-dimensional gels, allowing for direct comparison of the spots
>Y
VOLUME
91, 1982
(data not shown). At least seven of the in vitro translation products comigrate with ectoderm-enriched proteins synthesized in vivo. These include the major 1’7K spots (marked 1 and 2 in Fig. 5B), as well as five other spots (labeled 3, 4, 6, 10, and 12 in Fig. 5B). Three or four other in vitro products did not comigrate but are very similar in size and isoelectric point to spots en-
B -100 -50
PH FIG. 2. Two-dimensional electrophoresis of products of translation of RNA selected by hybridization to pSpec 1 and pSpec 2 DNAs. One hundred micrograms of total cellular RNA from gastrula-stage embryos was hybridized to 15 pg of filter-bound pSpec 1 or pSpec 2 DNA. The hybridized RNA was eluted and translated in a rabbit reticulocyte lysate cell-free system supplemented with [35S]methionine. The radiolabeled proteins were electrophoresed in two dimensions. The second dimension uses a 10% polyacrylamide gel. The gels were prepared for fluorography with Enhance (New England Nuclear) and exposed to X-Omat RP X-ray film for 2 weeks at -70°C. (A) Translation of 10 pg of total cellular RNA from gastrula-stage embryos. (B) Translation with no exogenous RNA. (C) Translation with gastrula RNA hybrid selected by pSpec 1 DNA. (D) Translation with gastrula RNA hybrid selected by pSpec 2 DNA.
BRUSKIN
ET AL.
Embryonic
Ectodermal
Proteins
321
FIG. 3. Patterns of proteins synthesized in the ectoderm and endoderm/mesoderm fractions of plutei. Embryos were incubated with [?S]methionine and then separated into ectoderm and endoderm/mesoderm fractions (the latter prepared by lysis of ectoderm) according to procedures under Materials and Methods. Proteins were separated by two-dimensional electrophoresis and detected by autoradiography. Second-dimension gels consisted of an exponential gradient of 10-167~ polyacrylamide. Arrows indicate spots which are considerably more intensely labeled for that tissue fraction, while circles indicate that corresponding spot or area of the gel for the other tissue fraction. Shown are ectoderm (A) and endoderm/ectoderm (B).
riched in ectoderm in vivo, suggesting slight differences in their post-translational modifications or false initiation or termination in tlhe cell-free translation system. In any event, the fact that most of the in vitro products comigrated with proteins whose synthesis in vivo is enriched in ectoderm indicates that pSpec 1 and pSpec 2 code for mRNAs which direct the synthesis of a family of similar but distinct ectoderm proteins. Accumulation
of pSpec 1 and pSpec 2 mRNAs
Previously, we have shown that the two predominant transcript sizes corresponding to pSpec 1 and pSpec 2 accumulate during embryogenesis, their mass increasing by lOO- to ZOO-fold (Bruskin et al., 1981). In addition, our data indicated that the 1.5kb mRNA accumulated somewhat earlier than the 2.2-kb mRNA. To determine more precisely the kinetics of accumulation of these mRNAs, we isolated total cellular RNA from cultures of embryos 4 to 50 hr after fertilization (early cleavage stages). The RNA, resolved by through late gastrula electrophoresis on agarose gels, was blotted to nitrocellulose and hybridized with nick-translated 32P-pSpec
1 or 32P-pSpec 2 DNA. The resulting autoradiograms were scanned with a densitometer and the absorbances plotted as a fraction of the peak at 50 hr, as shown in Fig. 4A. These data demonstrate that the pSpec 1, 1.5kb transcripts begin accumulating about 10 hr earlier than the pSpec 2, 2.2-kb transcripts. We have previously shown that at the pluteus stage (96 hr) the levels of pSpec 1 and pSpec 2 mRNAs are about 30% of their maximum levels, which occur at the late gastrula-early prism stage (about 50 hr) (Bruskin et al., 1981). The pSpec 1 and pSpec 2 mRNAs might accumulate in all cells of the blastulaand early gastrula-stage embryos but be selectively degraded in the endoderm of plutei. To determine where in the embryo the pSpec 1 and pSpec 2 transcripts accumulate, early gastrula (40 hr)- and pluteus (96 hr)-stage embryos were fractionated into ectoderm and endoderm/mesoderm, and total cellular RNA was extracted. Nitrocellulose filters of RNA gel blots were hybridized to 32PpSpec 1 or 32P-pSpec 2 DNA. The autoradiograms of these filters are shown in Figs. 4B and C. The 1.5- and 2.2-kb transcripts from both the gastrula and pluteus stages are strongly enriched in the ectoderm fraction.
322
DEVELOPMENTALBIOLOGY
hatchinq
gastrulation
A
1.0 E 0.8 .-z $ 0.6 E % 0.4 .-E i 0.2 k IO
20 30 40 50 hrs after fertilization
60
FIG. 4. Accumulation of pSpec 1 and pSpec 2 mRNAs during embryogenesis. Ten micrograms of total cellular RNA from the indicated stage or cell type was electrophoresed on 1% agarose-formaldehyde gels and blotted to nitrocellulose filters. The filters were hybridized with 5 X lo7 cpm of nick-translated pSpec 1 and pSpec 2 [a’P]DNAs. (A) Accumulations of 1.5- and 2.2-kb transcripts based on densitometric scans of RNA gel blots probed with pSpec 1 or pSpec 2 DNA form 4 to 50 hr after fetilization (points earlier than 20 hr were at background levels). The peaks from each scan were normalized to the most intense peak which occurred at 50 hr. (B) pSpec 1 [a*P]DNA hybridized with RNA from pluteus ectoderm, lane 1; pluteus endoderm/mesoderm, lane 2; gastrula ectoderm, lane 3; gastrula endoderm/mesoderm, lane 4. (C) pSpec 2 [3zP]DNA hybridized with RNA from pluteus ectoderm, lane 1; pluteus endoderm/mesoderm, lane 2; gastrula ectoderm, lane 3; gastrula endoderm/mesoderm, lane 4.
These results, taken together with the data of Fig. 4A, show that the mRNA corresponding to pSpec 1 and pSpec 2 are not only enriched in the pluteus ectoderm but accumulate preferentially in the ectoderm during embryogenesis. Developmental Analysis of Ectoderm-Enriched Proteins
The proteins synthesized by intact eggs or embryos have been analyzed throughout embryonic development
VOLUME 91,1982
(Bedard and Brandhorst, 1982). In Figs. 5A and B, the low-molecular-weight acidic proteins are shown as synthesized in endoderm/mesoderm or ectoderm fractions of plutei. Newly synthesized proteins labeled 1-13 are enriched in ectoderm. Unfertilized eggs (Fig. 5C) have a distinctly different pattern of protein synthesis than plutei (Fig. 5D). In unfertilized eggs, synthesis of the ectoderm proteins relative to total synthesis is at a much lower rate or is not detectable. Proteins 1 and 2, the major ectoderm-enriched proteins synthesized in plutei, are synthesized at very reduced levels in the egg as shown in Fig. 5C, a gel exposed to a fivefold greater extent than that shown in Fig. 5D. Proteins 4 and 12, however, are synthesized in the egg at much higher levels. This suggests that the synthesis of the ectoderm proteins is noncoordinate. In another report (Bedard and Brandhorst, 1982), we show that some ectoderm proteins such as 1 and 2 appear during cleavage, while others such as 7,11, and 13 appear signficantly later in development, sometime during gastrulation. This group of small acidic ectoderm proteins is among the most actively synthesized in later embryonic stages and undergoes the most pronounced developmental changes observed during embryogenesis (Bedard and Brandhorst, 1982). The result of this synthesis is that they accumulate substantially in mass. This can be observed by comparing the silver-stained gel patterns of eggs (Fig. 5E) and plutei (Fig. 5F). Several proteins such as 1, 2, and 13 have accumulated considerably, while the mass of other proteins such as 3, 4, 5, and 6 have not changed much. DISCUSSION
It is clear that pSpec 1 and pSpec 2 correspond to mRNAs which are similar but not identical at their 3 ends. They have similar but distinct restriction maps, and the corresponding hybrid-selected mRNAs code for the same set of proteins. Most of these cell-free translation products comigrate with proteins whose synthesis is enriched in the ectoderm in vivo. The simplest and most likely interpretation of these results is that pSpec 1 and pSpec 2 cross-hybridize with the mRNAs of several members of a small gene family coding for the group of ectoderm-enriched proteins which are similar in size and isoelectric point. Other interpretations have not been excluded. The multiple forms of these ectoderm-enriched proteins could be derived from posttranslational modifications, some of which might occur in the rabbit reticulocyte lysate (e.g., Traugh and Sharp, 1977). When different batches of embryos are compared, there is some variation in the relative intensities of labeling of some members of this family of proteins
BRUSKIN
ET AL.
Ectodmal
Emtvymic
PH
517
Proteins
5.4
5.0
FIG. 5. Synthesis and accumulation of small, acidic ectodermal proteins during embryonic development. A-D are autoradiograms of twodimensional electrophoretic selparations of newly synthesized proteins. A and B are enlargements of corresponding autoradiograms shown in Fig. 3. Numbered spots are discussed in the text. Shown are newly synthesized proteins of pluteus endoderm/mesoderm (A), pluteus ectoderm (B), unfertilized eggs (C), and intact pluteus larvae (D). Silver-stained gels of eggs (E) and plutei (F) are also shown; these were derived from a different set of first-dimension gels, resulting in some differences in mobility compared to A-C. Proteins labeled 14 and 15 are shown for comparison. They are not enriched in pluteus endoderm/mesoderm or ectoderm but accumulate during embryonic development.
(Bedard, unpublished observations). This variation could be the result of variations in post-translational (or artifactual) modifications or genetic polymorphisms. Minor variations in peptide initiation or termination could generate some of the diversity observed. If there is indeed a family of distinct mRNAs they might be derived from a single allelic pair of genes by differential transcriptional initiation or terminal or differential splicing (Early et al., 1980; Young et al., 1981). The mRNAS corresponding to pSpec 1 and pSpec 2, having principle sizes of 1.5 and 2.2 kb, are large enough to code for several polypeptides of 14-1’7,000 daltons; thus they could be polycistronic mRNAs. Estimation of the number of related genes has been
complicated by the fact that pSpec 1 and pSpec 2 cDNA clones appear to contain a highly repeated element dispersed several hundred times throughout the genome. Most of these sites do not appear to code for ectodermal proteins. We have not as yet found other cDNA clones complementary to mRNAs in this family. The RNA gel blots (Figs. 4B, C) suggest that there are more than two transcipt sizes. We routinely observed faint bands at molecular weights higher than 2.2 kb. The 2.2-kb band itself is always broad and heterogeneous in intensity, suggesting the presence of several distinct transcripts. Thus far we have not been able to assign either cDNA clone (or its predominant mRNA) to specific ectoderm proteins. We have not found conditions which simplify
324
DEVELOPMENTAL
BIOLOGY
the patterns on RNA gel blots or hybrid-selected translations. For example, we have observed no changes in the relative intensities of hybridizing bands on RNA gel blots when the stringency of the hybridization conditions was increased several degrees. Comparison of the accumulation of specific transcripts as shown in Fig. 4 with the developmental changes in synthesis of ectoderm proteins has also not led to a definite assignment of a specific transcript size to a specific protein. The relative rates of synthesis of the 17K proteins 1 and 2 increase by over loo-fold during embryonic development (Bedard and Brandhorst, 1982), which is comparable to the increases in prevalence of both the 1.5- and 2.2-kb transcripts. The synthesis of the major ectoderm proteins such as 1 and 2 begins to increase well before hatching, being elevated by a factor of about 30 upon hatching (Bedard and Brandhorst, 1982). As shown by Bruskin et al. (1981), the 1.5-kb transcript also increases during cleavage, though apparently not as dramatically. After hatching, the 1.5-kb transcript increases by about loo-fold (see Fig. 4) while the rate of synthesis of the 1’7K ectoderm proteins 1 and 2 increases by about lo-fold. The 2.2-kb transcript begins to accumulate after the rise in synthesis of the 17K ectoderm proteins 1 and 2. This suggests that either the 2.2-kb transcripts code for the ectoderm proteins which appear later in development or that they are not translated. The latter alternative is possible since the shared 600 bases of the 3’ ends of the mRNAs corresponding to pSpec 1 and pSpec 2 may not include the coding sequences which might not be shared. The apparent quantitative discrepancy between the protein synthesis and transcript accumulation data might be explained if these mRNAs are more efficiently translated in earlier embryos. There is translatable mRNA coding for proteins 1 and 2 present in unfertilized eggs, albeit at low levels, relative to later stages (Bedard and Brandhorst, unpublished observations); and the 1.5-kb transcript corresponding to pSpec 1 can also be detected in low levels in eggs by RNA gel blotting (Bruskin et al., 1981). Perhaps these maternal transcripts are more efficiently translated than those synthesized subsequently. At this time we have no knowledge of the function of this family of ectoderm proteins. We plan further investigations to address this question and to establish the number, arrangement, and structures of the genes coding for them. This work the National
was supported by Grant PHS HD14182 (to W.H.K) from Institutes of Health and by a grant from the National
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91, 1982
Sciences and Engineering Research Council (to B.P.B). A.M.B. is supported by a predoctoral training grant from the National Institutes of Health (GM 7227). P.A.B. is supported by a scholarship from NSERC. Part of this work was performed at the Marine Biological Laboratories, Woods Hole, Massachusetts, and supported there by the National Institutes of Health Grant 232 HDO 7098 to the Embryology Course. REFERENCES BEDARD, A., and BRANDHORST, B. P. (1982). Manuscript in preparation. BRANDHORST, B. P. (1976). Two-dimensional gel patterns of protein synthesis before and after fertilization of sea urchin eggs. Develop. Biol. 52, 310-317. BRUSKIN, A. M., TYNER, A. L., WELLS, D. E., SHOWMAN, R. M., and KLEIN, W. H. (1981). Accumulation in embryogenesis of five mRNAs enriched in the ectoderm of the sea urchin pluteus. Develop. Biol. 87, 308-318. EARLY, P., ROGERS, J., DAVIS, M., COLANE, K., BOND, M., WALL, R., and HOOD, L. (1980). Two mRNAs can be produced from a single immunoglobin fi gene by alternative RNA processing pathways. Cell 20,313-319. GALAU, G. A., KLEIN, W. H., DAVIS, M. M., WOLD, B. J., BRITTEN, R. J., and DAVIDSON, E. H. (1976). Structural gene sets active in embryos and adult tissues of the sea urchin. Cell 7, 487-505. HARKEY, M. A., and WHITELEY, A. H. (1980). Isolation, culture and differentiation of echinoid primary mesenchyme cells. Wilhelm Roux’s Arch. Dev. Biol. 189, 111-122. HOUGH-EVANS, B. R., WOLD, B. J., ERNST, S. G., BRITTEN, R. J., and DAVIDSON, E. H. (1977). Appearance and persistence of maternal RNA sequences in sea urchin embryos. Develop. Biol. 60, 258-277. LASKY, L. A., LEV, Z., XIN, J.-H., BRITTEN, R. J., and DAVIDSON, E. H. (1980). Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs. Proc. Nat. Acad Sci. USA 77, 5317-5321. LEV, Z., THOMAS, T. L., LEE, A. S., ANGERER, R. C., BRITTEN, R. J., and DAVIDSON, E. H. (1980). Developmental expression of two cloned sequences coding for rare sea urchin embryo messages. Develop. Biol. 76, 322-340. MCCLAY, D. R., and CHAMBERS, A. F. (1978). Identification of four classes of cell surface antigens appearing at gastrulation in sea urchin embryos. Develop. Biol. 63, 179-186. O’FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem 250,4007-4021. OAKLEY, B. R., KERSH, D. R., and MORRIS, N. R. (1980). A simplified ultrasensitive silver strain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361-363. SHEPHERD, G. W., and NEMER, M. (1980). Development shifts in frequency distribution of polysomal mRNA and their posttranscriptional regulation in the sea urchin embryo. Proc. Nat. Acad Sci. USA 77, 4653-4656. TRAUGH, J. A., and SHARP, S. B. (1977). associated with protein-synthesizing locytes. J. Biol. Chem. 252, 3738-3744.
Protein modification enzymes complex from rabbit reticu-
XIN, J.-H., BRANDHORST, B. P., BRITTEN, R. J., and DAVIDSON, E. H. (1982). Cloned embryo mRNAs not detectably expressed in adult sea urchin coelomocytes. Develop. BioL 89, 527-531. YOUNG, R. A., HAGENBUCHLE, O., and SCHIBLER, V. (1981). mouse 01 amylase gene specifies two different tissue specific Cell 23, 451-458.
A single mRNAs.
BIOLOGY
91,31.7-324
(1982)
A Family of Proteins Accumulating in Ectoderm of Sea Urchin Embryos Specified by Two Related cDNA Clones ARTHUR M. BRUSKIN,*~’ *Program
in Molecular,
PIERRE-ANDRE BEDARD,t,’ ANGELA L. TYNER,* RICHARD M. SHOWMAN,* BRUCE P. Bk4mHowr,t AND WILLIAM H. KLEIN**’
Ce&lar, and Developmental Biology, Department and TDepartment of Biology, McGill University, Received
December
4, 1981; accepted
of Biology, Montreal
in revised
form
Indiana PQ, H3A
University, Bloomington,
February
4, 1982
lB1,
Indiana 47.405;
Canada
Two cDNA clones, pSpec 1 and pSpec 2, had been selected previously as corresponding to transcripts greatly enriched in ectoderm of pluteus-stage larvae of the sea urchin, Strmzglyocentrotus purpuratus. The two cDNA clones, corresponding to the 3’ ends of polysomal RNAs, have similar but distinct restriction maps. Messenger RNAs hybrid selected by these two cDNA clones were translated in a rabbit reticulocyte lysate cell-free system into the same set of 10 acidic proteins having mol.ecular weights of 14-17,000 daltons. These in vitro products have positions identical or similar on two-dimensional gels to a group of proteins whose synthesis in vivo is highly enriched in the ectoderm. The 1.5- and 2.2-kb transcripts corresponding to pSpec 1 and pSpec 2 increase in prevalence by at least loo-fold during embryonic development. The rates of synthesis of these ectoderm proteins also increase by over loo-fold, though the patterns of change are distinct. It is likely that pSpec 1 and pSpec 2 correspond to mRNAs which are part of a small family of genes coding for th’e group of similar ectoderm proteins. These proteins and their mRNAs are enriched in eetoderm by the early gastrula stage.
bryos. There is presently little information concerning the degree to which specific mRNAs are restricted to particular blastomeres either before or during the process of cellular differentiation occurring in later stages of embryogenesis. The recent development of methods for large-scale isolation of each of the primary germ layers of sea urchin embryos has provided a new approach to this problem (M&lay and Chambers, 1978; Harkey and Whitely, 1980). Recently, we have isolated clones representing mRNAs enriched in the ectoderm of sea urchin plutei (Bruskin et al., 1981). These mRNAs accumulate during embryogenesis, their mass per embryo increasing by more than loo-fold beginning just prior to or during gastrulation. The transcripts corresponding to these clones are found on polysomes, but the proteins they code for were not known. Using hybrid selected translation, we report here that two of these clones, which are similar but not identical in structure, code for a group of small, acidic polypeptides which are actively synthesized during the later stages of embryogenesis and are enriched in ectoderm. It is likely that the mRNAs coding for these proteins are derived from a small gene family.
INTRODUCTION
The timing and extent of change in mRNA populations during embryonic development have been the subject of many recent investigations. The population of rare mRNAs changes substantially during the development of sea urchin embryos, the general pattern being a gradual decline in sequence complexity (Galau et al., 1976; Hough-Evans et al., 1977). Populations of prevalent mRNAs also change during sea urchin embryogenesis (Shepherd and Nemer, 1980). Recently, the use of cloned DNA corresponding to several specific mRNAs has confirmed tbat the prevalence of both rare and abundant mRNA sequences can change substantially during development (Lev et al., 1980; Lasky et al., 1980; Bruskin et al., 1981; Xin et al., 1982). There are also many changes in the patterns of synthesis of hundreds of proteins which are detectible by two-dimensional electrophoresis (Brandhorst, 1976; Bedard and Brandhorst, 1982). By hybrid selection of mRNA using cloned DNA and cell-free translation, it is now possible to correlate specific mRNAs to specific proteins whose developmental patterns of synthesis have been determined. These investigations of mRNA populations and corresponding proteins have been applied to intact emi Contributed a To whom
MATERIALS
AND
Culturing and Fractionation Strongylocentrotus
equally to research reported. correspondence :should be addressed.
Pacific Biomarine 317
METHODS
of Embryos
purpuratus were purchased
(Venice, Calif.).
Embryos
from were cul-
0012-1606/82/060317-08$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
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tured at 15°C and harvested at the appropriate times as described previously (Bruskin et al., 1981). In cases where egg or embryo proteins were to be radiolabeled, 10,000 eggs or embryos were suspended in 1 ml of artifical seawater and incubated for 2 hr with 50 &i of [35S]methionine (embryos) or for 4-6 hr with 250 &i of [35S]methionine (eggs). After labeling, the eggs or embryos were washed extensively in cold seawater. Pluteus endoderm/mesoderm and ectoderm fractions were isolated as described in Bruskin et al. (1981). Gastrula endoderm/mesoderm and ectoderm were isolated according to the procedure of Harkey and Whiteley (1980) with the following modification: glycine-dissociated embryos were brought to a final density of 1.058 (measured durectly using a hydrometer) with Percoll. Following centrifugation, the partially purified endoderm/mesoderm was further purified over sucrose step gradients. In a few cases, gastrula and pluteus endoderm/mesoderm was isolated by lysing the ectodermal cells in situ using Triton X-100. Gastrulae or plutei were suspended in 100 vol of cold 0.35 M KCl, 50 mM MgClz, 25 mM EGTA, 0.5% Triton X-100, 50 mM Pipes, pH 6.5. The embryos were incubated for a few seconds to lyse the ectodermal cells and then pelleted by gentle centrifugation. the resulting endoderm/mesoderm pellet was then washed extensively with cold seawater. Preparation and Electrophoresis Cellular RNA
of Total
RNA was isolated from embryos by the guanidinehydrochloride extraction procedure described in Bruskin et al. (1981). RNA was stored at 10 mg/ml in water at -70°C. RNA was electrophoresed on formaldehyde agarose gels, blotted onto nitrocellulose filters, and hybridized with nick-translated pSpec 1 or pSpec 2 [32P]DNA as detailed in Bruskin et al. (1981). Hybrid
Selection and in Vitro Translation
Fifteen micrograms of pSpec 1 or pSpec 2 DNA was heated at 100°C in 30 ~1 of 0.4 M NaOH in 0.8 M NaCl. The solutions were neutralized by adding an equal volume of 1 M Tris, pH 8, 3 M NaCl, and the DNA was spotted onto 0.5-cm2 pieces of nitrocellulose filter paper (Schleicher and Schuell, BA85). The filters were air dried, washed in 0.15 M sodium citrate, 1.5 M NaCl (pH 7.5) at room temperature for 30 min, and baked in a vacuum over at 80°C for 2 hr. The filters were then cut up into smaller pieces, placed in a 1.5-ml disposable Eppendorf tube, and prehybridized for l-2 h at 45°C in 100 ~1 of hybridization buffer (50% formamide, 100 mM Pipes, pH 6.5, 600 mM NaCl, 100 pg/ml poly rA). Following the prehybridization, fresh hybridization buffer
VOLUME91,1982
containing 100 pg of total cellular RNA was added. After hybridizing for 12-16 hr at 45”C, the filters were washed at 45°C five times each with 1.5 ml of 1 mM Pipes, pH 6.5, 6 mM NaCl, 0.5% SDS, and 1 mM Pipes, pH 6.5, 6 mM NaCl, respectively. After the final wash, 300 ~1 of water was added to elute the hybridized RNA. The filters were heated in a boiling water bath for 90 set and then immediately cooled in a dry ice-acetone bath. The eluted material was transferred to another 1.5-ml disposable Eppendorf tube and 10 pg of yeast tRNA, 30 ~1 of 2.5 M ammonium acetate, and 680 ~1 of ethanol were added. The RNA was precipitated overnight at -70°C. The RNA was pelleted by centrifugation and taken up in 10 ~1 of a supplemented rabbit reticulocyte lysate (Amersham) and 1~1 of [35S]methionine (1000 Ci/mmole; 10 Ci/ml). The reactions were incubated 90 min at 30°C. Two-Llimensional
Gel Electrophoresis
Eggs, embryos, embryonic tissues, or lyophilized rabbit reticulocyte lysates were suspended in lysis buffer (O’Farrell, 1975) containing 0.2% SDS. Two-dimensional electrophoresis on polyacrylamide gels was performed according to O’Farrell (1975). Silver staining of gels was according to Oakley et al. (1980). RESULTS
Sequence Homology
of pSpec 1 and pSpec 2
In a previous investigation we isolated two cDNA clones, pSpec 1 and pSpec 2, which were complementary to mRNAs enriched in the ectoderm of plutei (Bruskin et al., 1981). Two lines of evidence suggested that these DNAs have sequence homology. First, both clones hybridized to many fragments of the same size on blots of genomic DNA. Second, RNA gel blots showed that pSpec 1 hybridized strongly to a 1.5-kb RNA and weakly to a 2.2-kb RNA, while pSpec 2 hybridized strongly to a 2.2-kb RNA and weakly to a 1.5-kb RNA. Weak hybridization to other bands was also observed. To further examine the sequence homology, restriction maps were constructed for the two cDNA clones. Figure 1 shows that the two clones share four restriction sites for HueIII, KpnI, HpaII, and AU. Since the lengths of the inserted sea urchin sequences are only 0.5 to 0.6 kb, the coincidence of these restriction sites cannot be due to chance and provides strong evidence that the two clones are closely related. There are also differences in the maps: pSpec 2 contains HinfI and Hind11 sites not present in pSpec 1, and pSpec 1 contains an XbaI site not present in pSpec 2. In addition, the distances between the HaeIII-HpaII sites and HpaII-AZuI sites differ from the two clones by approximately 15 base pairs.
BRUSKIN
ET AL.
Embr-pnic
The significance of these small differences in length is as yet unclear. The clones pSpec 1 and pSpec 2 were constructed by cloning cDNA-RNA hybrids in pBR322 after reverse transcription of oligo(dT)-primed polyadenylated cytoplasmic RNA (Lasky et al., 1980). We have found that, as expected, these cloned DNAs are complementary to the 3’-terminal sequences of their respective RNAs. The evidence for this is based on the characterization of a recombinant X phage we have recently isolated which contains genomic DNA complementary to pSpec 1 (Bruskin and Klein, unpublished results). Trascription mapping of this X clone and orientation of pSpec 1 DNA with respect to transcription indicate that the sequence represented by pSpec :L must be at the 3’ end of the RNA. The direction of transcription of pSpec 1 is 3’ to 5’, left to right, on the restriction map shown in Fig. 1. We assume that pSpec 2 has a similar orientation. Thus, pSpec 1 and pSpec 2 apparently represent 0.5 to 0.6 kb at the 3’ ends of 1.5- and 2.2-kb RNAs, respectively. Because of the extensive sequence homology of the DNAs, both clones are capable of hybridizing to either transcript to sorne extent. Translation of RNA and pSpec 2
Hybrid
Selected by pSpec
1
We isolated pSpec 1 and pSpec 2 mRNAs by hybridizing total cellular RNA from gastrula-stage embryos with either pSpec 1 or pSpec 2 DNA immobilized on nitrocellulose filters. The hybridized RNA was eluted from the filters and translated in a rabbit reticulocyte lysate cell-free system supplemented with [35S]methionine. The r,adiolabeled proteins were analyzed by two-dimensio:nal electrophoresis, and results of a typical experiments are shown in Fig. 2. RNA selected by either cloned DNA coded for approximately 10 polypeptides which
Ectodermal
319
Proteins
wl-lwP,c 00 oaao IYIX
pSpec I
YI
I
y 1 a
(
,100bp,
FIG. 1. Restriction endonuclease maps of pSpec 1 and pSpec 2 DNAs. Maps were constructed by digesting the plasmid DNAs with one or two of the indicated enzymes and electrophoresing the digested material on 5% polyacrylamide gels. The direction of transcription of pSpec 1 is 3’ to 5’, left to right, on the map. This was determined by labeling pSpec 1 DNA at the single XbaI site, located within the insert sequence, with [3ZP]dCTP and reverse transcriptase. The labeled DNA was then digested with HhaI yielding two fragments labeled at the 3’ end of opposite strands. These fragments were isolated from 5% polyacrylamide gels and then used as probes in RNA gel blot hybridizations. The orientation was determined by the fragment that hybridized to the RNA.
Ectoderm-Enm’ched Proteins Synthesized in Vivo
Plutei were incubated for 2 hr with [35S]methionine and then separated into ectoderm and endoderm/mesoderm fractions. The newly synthesized proteins were analyzed by two-dimensional electrophoresis. Newly synthesized proteins enriched in the ectoderm fraction are pointed out by arrows in Fig. 3A. The corresponding positions of these spots in the endoderm/mesoderm fraction are indicated by circles in Fig. 3B. Some of the differences are very pronounced, others less obvious. Most of the very pronounced ectoderm-enriched proteins are small and acidic, having mobilities similar to the products of cell-free translation of pSpec 1 and pSpec 2 mRNAs. In Fig. 3B are shown the proteins which were being synthesized by endoderm/mesoderm of the pluteus-stage embryos. Arrows point to the few polypeptides enriched in the endoderm, in particular, two or three proteins having molecular weights of 24,000 and two having molecular weights of 37,000. The incorporation of radioactive methionine into proteins synthesized by endoderm/mesoderm of intact embryos indicates that methionine is accessible to internal tissues, validating analyses of protein synthesis on intact embryos. On the other hand, dissociation of embryos followed by incubation with [35S]methionine can lead to perturbations in protein synthesis (Bedard, unpublished observations). The results presented in Figs. 2 and 3 strongly suggest that some of the proteins whose synthesis is enriched in ectoderm in vivo are identical to the group of products of cell-free translation of hybrid-selected mRNAs
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corresponding to pSpec 1 and pSpec 2 DNAs. To confirm this the cell-free translation products of gastrula RNA hybrid selected with pSpec 1 and pSpec 2 DNAs were compared to proteins whose synthesis is enriched in ectoderm fractions of gastrulae. In this case the products were analyzed on the same series of two-dimensional gels, allowing for direct comparison of the spots
>Y
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(data not shown). At least seven of the in vitro translation products comigrate with ectoderm-enriched proteins synthesized in vivo. These include the major 1’7K spots (marked 1 and 2 in Fig. 5B), as well as five other spots (labeled 3, 4, 6, 10, and 12 in Fig. 5B). Three or four other in vitro products did not comigrate but are very similar in size and isoelectric point to spots en-
B -100 -50
PH FIG. 2. Two-dimensional electrophoresis of products of translation of RNA selected by hybridization to pSpec 1 and pSpec 2 DNAs. One hundred micrograms of total cellular RNA from gastrula-stage embryos was hybridized to 15 pg of filter-bound pSpec 1 or pSpec 2 DNA. The hybridized RNA was eluted and translated in a rabbit reticulocyte lysate cell-free system supplemented with [35S]methionine. The radiolabeled proteins were electrophoresed in two dimensions. The second dimension uses a 10% polyacrylamide gel. The gels were prepared for fluorography with Enhance (New England Nuclear) and exposed to X-Omat RP X-ray film for 2 weeks at -70°C. (A) Translation of 10 pg of total cellular RNA from gastrula-stage embryos. (B) Translation with no exogenous RNA. (C) Translation with gastrula RNA hybrid selected by pSpec 1 DNA. (D) Translation with gastrula RNA hybrid selected by pSpec 2 DNA.
BRUSKIN
ET AL.
Embryonic
Ectodermal
Proteins
321
FIG. 3. Patterns of proteins synthesized in the ectoderm and endoderm/mesoderm fractions of plutei. Embryos were incubated with [?S]methionine and then separated into ectoderm and endoderm/mesoderm fractions (the latter prepared by lysis of ectoderm) according to procedures under Materials and Methods. Proteins were separated by two-dimensional electrophoresis and detected by autoradiography. Second-dimension gels consisted of an exponential gradient of 10-167~ polyacrylamide. Arrows indicate spots which are considerably more intensely labeled for that tissue fraction, while circles indicate that corresponding spot or area of the gel for the other tissue fraction. Shown are ectoderm (A) and endoderm/ectoderm (B).
riched in ectoderm in vivo, suggesting slight differences in their post-translational modifications or false initiation or termination in tlhe cell-free translation system. In any event, the fact that most of the in vitro products comigrated with proteins whose synthesis in vivo is enriched in ectoderm indicates that pSpec 1 and pSpec 2 code for mRNAs which direct the synthesis of a family of similar but distinct ectoderm proteins. Accumulation
of pSpec 1 and pSpec 2 mRNAs
Previously, we have shown that the two predominant transcript sizes corresponding to pSpec 1 and pSpec 2 accumulate during embryogenesis, their mass increasing by lOO- to ZOO-fold (Bruskin et al., 1981). In addition, our data indicated that the 1.5kb mRNA accumulated somewhat earlier than the 2.2-kb mRNA. To determine more precisely the kinetics of accumulation of these mRNAs, we isolated total cellular RNA from cultures of embryos 4 to 50 hr after fertilization (early cleavage stages). The RNA, resolved by through late gastrula electrophoresis on agarose gels, was blotted to nitrocellulose and hybridized with nick-translated 32P-pSpec
1 or 32P-pSpec 2 DNA. The resulting autoradiograms were scanned with a densitometer and the absorbances plotted as a fraction of the peak at 50 hr, as shown in Fig. 4A. These data demonstrate that the pSpec 1, 1.5kb transcripts begin accumulating about 10 hr earlier than the pSpec 2, 2.2-kb transcripts. We have previously shown that at the pluteus stage (96 hr) the levels of pSpec 1 and pSpec 2 mRNAs are about 30% of their maximum levels, which occur at the late gastrula-early prism stage (about 50 hr) (Bruskin et al., 1981). The pSpec 1 and pSpec 2 mRNAs might accumulate in all cells of the blastulaand early gastrula-stage embryos but be selectively degraded in the endoderm of plutei. To determine where in the embryo the pSpec 1 and pSpec 2 transcripts accumulate, early gastrula (40 hr)- and pluteus (96 hr)-stage embryos were fractionated into ectoderm and endoderm/mesoderm, and total cellular RNA was extracted. Nitrocellulose filters of RNA gel blots were hybridized to 32PpSpec 1 or 32P-pSpec 2 DNA. The autoradiograms of these filters are shown in Figs. 4B and C. The 1.5- and 2.2-kb transcripts from both the gastrula and pluteus stages are strongly enriched in the ectoderm fraction.
322
DEVELOPMENTALBIOLOGY
hatchinq
gastrulation
A
1.0 E 0.8 .-z $ 0.6 E % 0.4 .-E i 0.2 k IO
20 30 40 50 hrs after fertilization
60
FIG. 4. Accumulation of pSpec 1 and pSpec 2 mRNAs during embryogenesis. Ten micrograms of total cellular RNA from the indicated stage or cell type was electrophoresed on 1% agarose-formaldehyde gels and blotted to nitrocellulose filters. The filters were hybridized with 5 X lo7 cpm of nick-translated pSpec 1 and pSpec 2 [a’P]DNAs. (A) Accumulations of 1.5- and 2.2-kb transcripts based on densitometric scans of RNA gel blots probed with pSpec 1 or pSpec 2 DNA form 4 to 50 hr after fetilization (points earlier than 20 hr were at background levels). The peaks from each scan were normalized to the most intense peak which occurred at 50 hr. (B) pSpec 1 [a*P]DNA hybridized with RNA from pluteus ectoderm, lane 1; pluteus endoderm/mesoderm, lane 2; gastrula ectoderm, lane 3; gastrula endoderm/mesoderm, lane 4. (C) pSpec 2 [3zP]DNA hybridized with RNA from pluteus ectoderm, lane 1; pluteus endoderm/mesoderm, lane 2; gastrula ectoderm, lane 3; gastrula endoderm/mesoderm, lane 4.
These results, taken together with the data of Fig. 4A, show that the mRNA corresponding to pSpec 1 and pSpec 2 are not only enriched in the pluteus ectoderm but accumulate preferentially in the ectoderm during embryogenesis. Developmental Analysis of Ectoderm-Enriched Proteins
The proteins synthesized by intact eggs or embryos have been analyzed throughout embryonic development
VOLUME 91,1982
(Bedard and Brandhorst, 1982). In Figs. 5A and B, the low-molecular-weight acidic proteins are shown as synthesized in endoderm/mesoderm or ectoderm fractions of plutei. Newly synthesized proteins labeled 1-13 are enriched in ectoderm. Unfertilized eggs (Fig. 5C) have a distinctly different pattern of protein synthesis than plutei (Fig. 5D). In unfertilized eggs, synthesis of the ectoderm proteins relative to total synthesis is at a much lower rate or is not detectable. Proteins 1 and 2, the major ectoderm-enriched proteins synthesized in plutei, are synthesized at very reduced levels in the egg as shown in Fig. 5C, a gel exposed to a fivefold greater extent than that shown in Fig. 5D. Proteins 4 and 12, however, are synthesized in the egg at much higher levels. This suggests that the synthesis of the ectoderm proteins is noncoordinate. In another report (Bedard and Brandhorst, 1982), we show that some ectoderm proteins such as 1 and 2 appear during cleavage, while others such as 7,11, and 13 appear signficantly later in development, sometime during gastrulation. This group of small acidic ectoderm proteins is among the most actively synthesized in later embryonic stages and undergoes the most pronounced developmental changes observed during embryogenesis (Bedard and Brandhorst, 1982). The result of this synthesis is that they accumulate substantially in mass. This can be observed by comparing the silver-stained gel patterns of eggs (Fig. 5E) and plutei (Fig. 5F). Several proteins such as 1, 2, and 13 have accumulated considerably, while the mass of other proteins such as 3, 4, 5, and 6 have not changed much. DISCUSSION
It is clear that pSpec 1 and pSpec 2 correspond to mRNAs which are similar but not identical at their 3 ends. They have similar but distinct restriction maps, and the corresponding hybrid-selected mRNAs code for the same set of proteins. Most of these cell-free translation products comigrate with proteins whose synthesis is enriched in the ectoderm in vivo. The simplest and most likely interpretation of these results is that pSpec 1 and pSpec 2 cross-hybridize with the mRNAs of several members of a small gene family coding for the group of ectoderm-enriched proteins which are similar in size and isoelectric point. Other interpretations have not been excluded. The multiple forms of these ectoderm-enriched proteins could be derived from posttranslational modifications, some of which might occur in the rabbit reticulocyte lysate (e.g., Traugh and Sharp, 1977). When different batches of embryos are compared, there is some variation in the relative intensities of labeling of some members of this family of proteins
BRUSKIN
ET AL.
Ectodmal
Emtvymic
PH
517
Proteins
5.4
5.0
FIG. 5. Synthesis and accumulation of small, acidic ectodermal proteins during embryonic development. A-D are autoradiograms of twodimensional electrophoretic selparations of newly synthesized proteins. A and B are enlargements of corresponding autoradiograms shown in Fig. 3. Numbered spots are discussed in the text. Shown are newly synthesized proteins of pluteus endoderm/mesoderm (A), pluteus ectoderm (B), unfertilized eggs (C), and intact pluteus larvae (D). Silver-stained gels of eggs (E) and plutei (F) are also shown; these were derived from a different set of first-dimension gels, resulting in some differences in mobility compared to A-C. Proteins labeled 14 and 15 are shown for comparison. They are not enriched in pluteus endoderm/mesoderm or ectoderm but accumulate during embryonic development.
(Bedard, unpublished observations). This variation could be the result of variations in post-translational (or artifactual) modifications or genetic polymorphisms. Minor variations in peptide initiation or termination could generate some of the diversity observed. If there is indeed a family of distinct mRNAs they might be derived from a single allelic pair of genes by differential transcriptional initiation or terminal or differential splicing (Early et al., 1980; Young et al., 1981). The mRNAS corresponding to pSpec 1 and pSpec 2, having principle sizes of 1.5 and 2.2 kb, are large enough to code for several polypeptides of 14-1’7,000 daltons; thus they could be polycistronic mRNAs. Estimation of the number of related genes has been
complicated by the fact that pSpec 1 and pSpec 2 cDNA clones appear to contain a highly repeated element dispersed several hundred times throughout the genome. Most of these sites do not appear to code for ectodermal proteins. We have not as yet found other cDNA clones complementary to mRNAs in this family. The RNA gel blots (Figs. 4B, C) suggest that there are more than two transcipt sizes. We routinely observed faint bands at molecular weights higher than 2.2 kb. The 2.2-kb band itself is always broad and heterogeneous in intensity, suggesting the presence of several distinct transcripts. Thus far we have not been able to assign either cDNA clone (or its predominant mRNA) to specific ectoderm proteins. We have not found conditions which simplify
324
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BIOLOGY
the patterns on RNA gel blots or hybrid-selected translations. For example, we have observed no changes in the relative intensities of hybridizing bands on RNA gel blots when the stringency of the hybridization conditions was increased several degrees. Comparison of the accumulation of specific transcripts as shown in Fig. 4 with the developmental changes in synthesis of ectoderm proteins has also not led to a definite assignment of a specific transcript size to a specific protein. The relative rates of synthesis of the 17K proteins 1 and 2 increase by over loo-fold during embryonic development (Bedard and Brandhorst, 1982), which is comparable to the increases in prevalence of both the 1.5- and 2.2-kb transcripts. The synthesis of the major ectoderm proteins such as 1 and 2 begins to increase well before hatching, being elevated by a factor of about 30 upon hatching (Bedard and Brandhorst, 1982). As shown by Bruskin et al. (1981), the 1.5-kb transcript also increases during cleavage, though apparently not as dramatically. After hatching, the 1.5-kb transcript increases by about loo-fold (see Fig. 4) while the rate of synthesis of the 1’7K ectoderm proteins 1 and 2 increases by about lo-fold. The 2.2-kb transcript begins to accumulate after the rise in synthesis of the 17K ectoderm proteins 1 and 2. This suggests that either the 2.2-kb transcripts code for the ectoderm proteins which appear later in development or that they are not translated. The latter alternative is possible since the shared 600 bases of the 3’ ends of the mRNAs corresponding to pSpec 1 and pSpec 2 may not include the coding sequences which might not be shared. The apparent quantitative discrepancy between the protein synthesis and transcript accumulation data might be explained if these mRNAs are more efficiently translated in earlier embryos. There is translatable mRNA coding for proteins 1 and 2 present in unfertilized eggs, albeit at low levels, relative to later stages (Bedard and Brandhorst, unpublished observations); and the 1.5-kb transcript corresponding to pSpec 1 can also be detected in low levels in eggs by RNA gel blotting (Bruskin et al., 1981). Perhaps these maternal transcripts are more efficiently translated than those synthesized subsequently. At this time we have no knowledge of the function of this family of ectoderm proteins. We plan further investigations to address this question and to establish the number, arrangement, and structures of the genes coding for them. This work the National
was supported by Grant PHS HD14182 (to W.H.K) from Institutes of Health and by a grant from the National
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Sciences and Engineering Research Council (to B.P.B). A.M.B. is supported by a predoctoral training grant from the National Institutes of Health (GM 7227). P.A.B. is supported by a scholarship from NSERC. Part of this work was performed at the Marine Biological Laboratories, Woods Hole, Massachusetts, and supported there by the National Institutes of Health Grant 232 HDO 7098 to the Embryology Course. REFERENCES BEDARD, A., and BRANDHORST, B. P. (1982). Manuscript in preparation. BRANDHORST, B. P. (1976). Two-dimensional gel patterns of protein synthesis before and after fertilization of sea urchin eggs. Develop. Biol. 52, 310-317. BRUSKIN, A. M., TYNER, A. L., WELLS, D. E., SHOWMAN, R. M., and KLEIN, W. H. (1981). Accumulation in embryogenesis of five mRNAs enriched in the ectoderm of the sea urchin pluteus. Develop. Biol. 87, 308-318. EARLY, P., ROGERS, J., DAVIS, M., COLANE, K., BOND, M., WALL, R., and HOOD, L. (1980). Two mRNAs can be produced from a single immunoglobin fi gene by alternative RNA processing pathways. Cell 20,313-319. GALAU, G. A., KLEIN, W. H., DAVIS, M. M., WOLD, B. J., BRITTEN, R. J., and DAVIDSON, E. H. (1976). Structural gene sets active in embryos and adult tissues of the sea urchin. Cell 7, 487-505. HARKEY, M. A., and WHITELEY, A. H. (1980). Isolation, culture and differentiation of echinoid primary mesenchyme cells. Wilhelm Roux’s Arch. Dev. Biol. 189, 111-122. HOUGH-EVANS, B. R., WOLD, B. J., ERNST, S. G., BRITTEN, R. J., and DAVIDSON, E. H. (1977). Appearance and persistence of maternal RNA sequences in sea urchin embryos. Develop. Biol. 60, 258-277. LASKY, L. A., LEV, Z., XIN, J.-H., BRITTEN, R. J., and DAVIDSON, E. H. (1980). Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs. Proc. Nat. Acad Sci. USA 77, 5317-5321. LEV, Z., THOMAS, T. L., LEE, A. S., ANGERER, R. C., BRITTEN, R. J., and DAVIDSON, E. H. (1980). Developmental expression of two cloned sequences coding for rare sea urchin embryo messages. Develop. Biol. 76, 322-340. MCCLAY, D. R., and CHAMBERS, A. F. (1978). Identification of four classes of cell surface antigens appearing at gastrulation in sea urchin embryos. Develop. Biol. 63, 179-186. O’FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem 250,4007-4021. OAKLEY, B. R., KERSH, D. R., and MORRIS, N. R. (1980). A simplified ultrasensitive silver strain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361-363. SHEPHERD, G. W., and NEMER, M. (1980). Development shifts in frequency distribution of polysomal mRNA and their posttranscriptional regulation in the sea urchin embryo. Proc. Nat. Acad Sci. USA 77, 4653-4656. TRAUGH, J. A., and SHARP, S. B. (1977). associated with protein-synthesizing locytes. J. Biol. Chem. 252, 3738-3744.
Protein modification enzymes complex from rabbit reticu-
XIN, J.-H., BRANDHORST, B. P., BRITTEN, R. J., and DAVIDSON, E. H. (1982). Cloned embryo mRNAs not detectably expressed in adult sea urchin coelomocytes. Develop. BioL 89, 527-531. YOUNG, R. A., HAGENBUCHLE, O., and SCHIBLER, V. (1981). mouse 01 amylase gene specifies two different tissue specific Cell 23, 451-458.
A single mRNAs.