Developmental patterns of cytoplasmic transcript prevalence in sea urchin embryos

DEVELOPMENTAL

BIOLOGY

91,

2735 (1982)

Developmental

Patterns

of Cytoplasmic Transcript Urchin Embryos

Prevalence

in Sea

CONSTANTIN N. ~FLYTZANIS, BRUCE P. BRANDHORST,’ ROY J. BRITTEN, AND ERIC H. DAVIDSON The Division

of Biology,

California

Institute

of Technology, Pasadena, California

Received September 8, 1981; accepted in revised form

91125

December 4, 1981

Several hundred two-cell stage, gastrula stage, and pluteus stage sea urchin embryo cDNA clones were screened with [32P]cDNA transcribed from a developmental series of cytoplasmic and polysomal poly(A) RNAs. Transcript prevalence was estimated by reference to the reaction of a series of standard clones complementary to RNAs of known abundance. We describe the dominant pattern of prevalence change during development for abundant sequences, i.e., those present in the range 5 X 104-lo6 molecules/embryo. These transcripts are most often about equally abundant in egg and pluteus stage embryos, and are severalfold less prevalent at gastrula stage. Less than 10% of the sequences displaying this pattern undergo >lO-fold changes in prevalence during development. A search for later embryo sequences not represented detectably in egg or cleavage stage embryo poly(A) RNA yielded a number of examples. These were confirmed by the RNA gel blot method. Sequences regulated in this manner all belonged to lower abundance classes, and are present at about 40 copies/cell or less at the gastrula stage.

species clearly display sharp developmental changes in their rate of synthesis, particularly in the blastula-gastrula period (Brandhorst, 1976; Bedard and Brandhorst, unpublished results). Lasky et al. (1980) argued that mRNA sequences whose translation products are detectable in two-dimensional gel separations of newly synthesized proteins are sufficiently prevalent so that they should also be detected by their colony screening procedures. Therefore, poly(A) RNA sequences whose prevalence varies impressively during development should be clearly evident in colony screening experiments, though no examples were found in this initial study. Lasky et al. (1980) suggested that highly regulated sequences are likely to be found mainly in less abundant classes of transcripts, and mentioned a few possible instances of such sequences. We report here developmental prevalence measurements on about 200 cDNA clones represented by abundant embryo poly(A) RNAs. These clones were selected after preliminary screening with mitochondrial DNA and are all believed to represent nuclear genes. We confirm the overall conclusion of Lasky et al. (1980) with respect to the dominant patterns of change in sequence prevalence within the abundant transcript classes. In addition we describe a set of clones discovered in additional screening experiments whose transcripts are absent or very infrequent in egg poly(A) RNA, but become relatively prominent at the gastrula or pluteus stage. Even when significantly expressed, however, these sequences do not approach the prevalence of highabundance embryo transcripts.

INTRODUCTION

Initial explorations of transcript prevalence during development by the cDNA clone colony screening method showed small changes for most highly abundant cloned sequences. Lasky et al. (1980) investigated a sample of 40 of the most prevalent sequences represented in a 500-clone gastrula library and a 1400-clone pluteus library. When these 40 clones were reacted with cDNAs transcribed from egg, gastrula, and pluteus stage poly(A) RNAs, complementary transcripts were found in almost every case at all three stages, though many sequences were more prevalent in egg and pluteus poly(A) RNAs than in gastrula poly(A) RNA. It was concluded that prevalent late embryo sequences are almost always already abundant in the maternal RNA of the sea urchin egg, and that major changes in the level of representation omfsuch sequences during embryonic development occur very infrequently. This result was in accord with prior measurements indicating that almost all of the rare polysomal mRNA sequences of gastrula and pluteus stage embryos are also represented (at similar levels) in egg RNA (Galau et al., 1976; Hough-Evans et al, 1977). Brandhorst (1976) had shown, furthermore, that most prominent species of protein are synthesized in sea urchin embryos throughout development, and are coded initially by abundant maternal mRNAs. On the other hand, a minority of protein ’ Present address: Department treal, Quebec, Canada.

of Biology, McGill

University,

Mon-

27 0012-1606/82/050027-09$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

28

DEVELOPMENTAL BIOLOGY

VOLUME 91, 1982

were screened with nick translated mitochondrial DNA prepared by the method of Devlin (1976). All clones Growth of sea urchin embryos. Eggs of Strongylocentracer were removed trotus purpuratus were collected, fertilized, and cul- reacting with the mitochondrial from this study and are not considered further except tured at 15°C as described (Smith et al., 1974; Houghwhere noted. About 50% of the abundant sequence subEvans et ah, 1977). Pluteus and gastrula stage embryos libraries (and thus about 20% of the overall unselected were harvested at 36 and 90 hr after fertilization, recDNA libraries) consisted of mitochondrial transcripts. spectively. Some of the remaining clones in each of the abundant Preparation of RNAs. Egg RNA was isolated by hosequence sublibraries failed to grow or transferred mogenizing eggs in urea-SDS lysis buffer followed by poorly in particular screening reactions. Clones which phenol extraction steps as described in detail by Houghdid not provide acceptable data for all three embryonic Evans et al. (1977). Embryo cytoplasmic RNAs were also stages tested were dropped from the sample that was prepared as previously described (Galau et al, 1976; Lev analyzed. This left 85 clones in the pluteus sublibrary et aZ., 1980). Total embryo or adult intestine RNAs were and 116 clones in the two-cell sublibrary. isolated by homogenization in guanidinium thiocyanate The approximate prevalence of transcripts complebuffer as described by Chirgwin et al. (1979). The RNA mentary to the selected clone sets was estimated by the was pelleted through cesium chloride gradients (Glisin method of Lasky et al. (1980). In this procedure (see also et al., 1974). Embryo polysomal RNAs were isolated as Xin et al., 1982) the clone matrix is transferred to nidescribed by Galau et al. (1974). The poly(A) RNA was trocellulose filter paper by a modification of the colony isolated by affinity chromatography on oligo(dT)-celscreening method originally described by Grunstein lulose as described by Aviv and Leder (1972). and Hogness (1975), and is hybridized with [32P]cDNA. Colony hybridization. The desired sets of cDNA clones The amount of [32P]cDNA bound to each clone is deterwere replicated from microtiter plates onto nitrocelmined by cutting out the appropriate area of the filter lulose filters, and after lysis the filters were hybridized paper and measuring its radioactivity in a scintillation with [32P]cDNAs synthesized from poly(A) RNA at the counter. The relation between the amount of [32P]cDNA indicated stages. The procedure used was similar to that reacting with the standard clones and the known numreported by Lasky et al. (1980) with the modifications ber of transcripts in the embryo complementary to described by Xin et al. (1982). these clones provides a means of estimating the repRNA gel blot hybridizations. The RNA gel blots were resentation of unknown clones present in the same carried out basically according to B. Seed and D. Goldmicrotiter matrix. Lasky et al. (1980) showed a linear berg (unpublished results) as described in detail by relationship on a logarithmic plot, as illustrated in Fig. Scheller et al. (1981). 1 for the standard clones used in the present study. The three curves shown in Fig. 1 were obtained with cDNA transcribed from egg poly(A) RNA, and from the cyRESULTS toplasmic poly(A) RNAs of gastrula and pluteus stage Prevalence Changes for Abundant Sequences during embryos. These curves are almost identical throughout Development the abundance range determined by the standards. The use of standard curves in screening experiments parIn preliminary experiments about 190 clones were tially normalizes for differences in cDNA preparations selected from a two-cell stage cDNA library kindly proand other experimental variables (Lasky et ah, 1980), vided by Dr. William R. Crain, and a similar number and in addition, the congruity of the standard curves of clones were selected from the pluteus stage cDNA shown in Fig. 1 increases confidence in the developlibrary described earlier by Lasky et al. (1980). The two cDNA libraries had been constructed according to al- mental comparisons which they control. An overall summary of the results obtained with the most identical protocols (Zain et aZ., 1979; Lasky et aC, sequences is shown in Fig. 2. 1980). The selected clones were chosen on the basis of selected high-abundance The individual values for each clone were calculated at a relatively strong reaction with [32P]cDNAs traneach stage as the average of two or three independent scribed from egg or embryo poly(A) RNAs, and were regarded as a representative sample of sequences be- observations. Though separate measurements on the same clone were generally in agreement within a factor longing to the abundant embryo transcript classes of 2, a single measurement for any given clone at any (Lasky et al., 1980). These clones were replated in four given stage could be in error by as much as a factor of 96-well microtiter matrices along with sets of “stan3-5. However, this degree of accuracy is acceptable for dard” clones complementary to transcripts of known the purpose of obtaining a statistical description of the prevalence. The experiments described in this section overall patterns of representation for the large number were carried out with the two abundant sequence subof abundant sequences in our sample. libraries carried in these plates. These sublibraries MATERIALS

AND METHODS

FLYTZANISETAL.

Sea Urchin Embryo

IO’L------a Id

IO” Transcripts

IO8 /embryo

FIG. 1. “Standard curves” for colony hybridization, obtained with clones complementary to transcripts of known prevalence. The cDNA clones used as standards in order of increasing prevalence (at all stages) were SpG6, SpG30, and SpP389. SpP389 and SpG30 are mitochondrial in origin, while SpG6 is genomic. Mitochondrial and genomic clones are equally applicable as prevalence standards since the cytoplasmic RNA was prepared with detergents that quantitatively lyse mitochondria. The number of cytoplasmic poly(A) RNA transcripts per embryo reacting with these clones were measured in prior studies in titration reactions with excess strand separated cloned tracer (Lev et aL, 1980; Lasky et aL, 1980; Xin et al., 1982). Countsper-minute hybridized (0rdina.t.e) have been corrected for background, measured as the average cpm hybridized to pBR322 and to the 20 clones hybridizing the least cDNA on each filter. These values were usually slightly below the hybridization observed with colonies containing only pBR322. Backgrounds in this study were about 30 cpm (above counter background). ‘Thus 60 cpm net hybridized is approximately 2X background for samples reacted with cDNA transcribed from egg poly(A) RNA and represents about 5 X lOa copies/embryo for the embryo cDNAs. The lowest possible limit of sensitivity in these experiments was judged. to be around 2 X lo3 copies. Curves are shown for cDNAs transcribed from egg poly(A) RNA and from gastrula and pluteus cytoplasmie poly(A) RNAs. The data were fit by least squares to the expression: log NsTo = k log Csrn + log Nisrn, where Nsm is cpm hybridized to the standard clones, k is the value of the slope in the log-log plot, CSTDis the number of complementary cytoplasmic transcripts per embryo, established independently as indicated above, and N1srn is lthe number of cpm that would be hybridized if there were one complementary transcript per embryo. The values of k for the egg, gastmla, and pluteus curves, respectively, are 0.736,0.726, and 0.635. It was observed by Lasky et aL (1980) that the slope in any given experiment depends largely on characteristics of the cDNA and in this case the three cDNA preparations were closely comparable. The approximate number of transcripts (C,) complementary to an unknown clone on the same filter matrix as the standards was calculated from the amount of [32P]cDNA hybridized to that clone (N,), as follows: C, = 10 exp[(Uog N, - log NISTD)/k], where the values of NSTD and k were determined from the standard curve for that stage as above. (0 0) Egg cDNA standard curve; (0 - - - 0) gastrula cDNA standard curve; and (A . . . A) pluteus cDNA standard curve.

Cytoplasmic

Transcript

Prevalence

29

Lasky et al. (1980) pointed out that a single sequence could account for the highest prevalence class in pluteus poly(A) RNA. The most highly represented standard clone, SpP389, is in fact that sequence, which is now known to be of mitochondrial origin (unpublished data). The developmental behavior of the SpP389 sequence is indicated by the dashed line in Fig. 2b. All highly represented sequences will of course be included more times in a cDNA library than are less highly represented ones, With few exceptions the genomic clones in these sublibraries are represented by between 2 X lo4 and 3 X lo5 transcripts per egg. The calculation given by Lasky et al. (1980) indicates that the set of sequences present at 2105 copies per egg in Fig. 2 probably represent between 10 and 40 different clones. The diversity of the less abundant transcripts of Fig. 2 is of course greater. Figure 2 shows that at gastrula stage, in the typical case, there are severalfold less copies in the cytoplasm per embryo than there were originally in the egg. By pluteus stage, however, the number of transcripts has recovered to a level close to or slightly greater than that in the maternal RNA. This is clearly the dominant pattern of developmental change for the abundant cytoplasmic poly(A) RNA sequences. In the two-cell sublibraries about 95% (111/116) of the selected sequences follow this pattern (Fig. 2a) and in the pluteus sublibrary all do likewise. For those clones that follow the typical pattern of expression, the average value of the ratio [(transcripts/egg)/(transcripts/gastrula)] is 3.4 for the pluteus sublibrary (65 X 103/19 X 103), and 3.4 for the two-cell sublibrary (96 X 103/28 X 103). The average value of the ratio [(transcripts/pluteus)/(transcripts/gastrula)] is 4.2 (80 X 103/19 X 103) for the pluteus sublibrary and 4.0 (112 X 103/28 X 103) for the twocell sublibrary. Figure 2 indicates that a few of the sequences indeed describe other courses, but when considered in view of the three- to fivefold uncertainty estimate we prefer for this type of measurement, these examples are not necessarily signigicant. However, about 8% of the clones shown in Fig. 2 demonstrate greater than lo-fold stage-related changes that probably are significant. These 16 clones and the prevalence values we estimate for their complementary transcripts are listed in Table 1. It is striking that in almost every case shown in Table 1 the developmental pattern of abundance change is the same as the typical pattern, except that it is quantitatively more extreme.

Cloned Sequences That Are Rare in Maternal RNA and Moderately Prevalent in Embryos A search was carried out for sequences which differ from those described earlier in that they are rare or not represented in maternal RNA and become signifi-

DEVELOPMENTAL BIOLOGY

VOLUME 91, 1982 IO'

_,’

(a)

__.’

_’

-“i

._,’

,-_

b)

106

_,,/’

lo3

Egg

1,’ Gastrula

1 Pluteus

Egg

Gastrula

Pluteus

FIG. 2. Patterns of prevalence change for abundant cytoplasmic transcripts. The approximate transcript prevalence per egg or embryo at three developmental stages is shown for all the clones in the high-abundance sublibraries for which complete data were available, except those with homology to mitochondrial transcripts, as described in the text. Data are expressed as poly(A) RNA transcripts per egg, per gastrula (cytoplasmic compartment), or per pluteus (cytoplasmic compartment). Prevalence values were calculated from several observations on each clone at each stage, as described in the legend to Fig. 1. Light solid lines represent those clones displaying the “typical” developmental pattern, i.e., more complementary transcripts in egg and pluteus than in gastrula, and the heavier lines represent those few clones which display other patterns. Prevalence patterns shown with heavy lines were those in which either the gastrula value was r3X the egg value, or the pluteus value was 5 one third of the gastrula value. Since only three developmental points were investigated the lines shown are not expected to represent the actual kinetics of abundance change, but are used only to connect the values obtained at the measured stages. (a) 116 clones from the two-cell cDNA clone sublibrary; (b) 85 clones from the pluteus cDNA clone sublibrary. The dashed line represents the behavior of the superprevalent mitochondrial sequence carried in the clone SpP389 (data from Fig. 1).

cantly more abundant during development. About 450 unselected pluteus cDNA clones and 450 gastrula cDNA clones were screened with [32P]cDNAs transcribed from egg poly(A) RNA, from 16-cell embryo, blastula, and gastrula polysomal poly(A) RNAs, and from total

poly(A) RNA of pluteus. This experiment (not shown) yielded 36 clones that appeared to have the desired characteristics. These clones reacted very little with cDNA representing egg or 16-cell polysomal RNAs, but reacted significantly with some or all of the later em-

FLYTZANIS ET AL.

Sea Urchin Embryo

Cytoplasmic

bryo preparations. These 36 clones were transferred to a separate microtiter plate along with the cloned standards described above. Figure 3 shows an experiment in which this matrix of selected clones was screened with [32P]cDNAs representing early and late embryo polysomal RNAs, and also with [32P]cDNA transcribed from intestine poly(A) RNA. At least 28 of the 36 clones screened (not including the standards) display dramatic increases in the intensity of hybridization with the gastrula and pluteus polysomal cDNAs, relative to their hybridization with 16cell polysomal RNA. These differences could not be quantitated because polysomal poly(A) RNA prevalence standards were not ava.ilable for all of the various stages tested. However, inspection of the strong reactions of some of the clones shown in Fig. 3 with the [32P]cDNA transcribed from 16-cell polysomal RNA shows that the absence of hybridization of most of the 36 selected clones with this [32P]cDNA cannot be explained as an artifact of a nonreactive cDNA preparation. Two of the standard clones included in Fig. 3 are mitochondrial in origin, as indicated in the legend.

A

TABLE 1 CLONES COMPLEMENTARY TO ABUNDANT EGG OR PLTJTE~S TRANSCRIPTS FOR WHICH THE LEVEL OF REPRESENTATION CHANGES >lOFOLD DURING DEVELOPMENT

D

Transcripts/egg cDNA Clone”

E&x?

or embryo (X10m3)*

Gastrula

Pluteus

SpP4-F2 SpP5-BlO SpP5-DlO SpP5-H6 SpP5-H7

100 50 140 16 20

10 3 25 0.5 1

100 20 300 40 100

SpP8-B2 SpPlZ-E4 SpP7-F4 SpP7-F12 SpP7-Gl

100 250 100 130 150

10 25 13 15 17

80 350 130 190 250

SpZCl-A9 SpZCl-All SpZCl-B8 SpZCl-Gl SpZCl-G2 SpZCZ-Bl

50 50 80 90 40

12 12 17 9 30

300 190 250 90 300

50

3

50

a The nomenclature for cDNA clones in use in this laboratory indicates the location of the specific clone on the permanent microtiter matrices in which the clones are stored. Thus, for example, SpP3-G6 indicates an S. purpuratus plasmid clone located in position G6 of plate 3 of the pluteus library. Sp2Cl denotes plate 1 of the two-cell stage library, etc. *The values shown are all supported by multiple, independent observations each displaying stage-to-stage ratios rlO-fold. Averages of available measurements are listed. Poly(A) RNA transcripts are shown per egg, and per gastrula or pluteus cytoplasm.

1

Transcript 2

3

4

31

Prevalence 5

6

7

0

9 "!

a

lo

11

12

*

b C d

0 a b C d

a b C d

a b C d

FIG. 3. Colony screen hybridization demonstrating embryonic expression of specific sequences. Selected cDNA clones that had been identified in prior comparative screens (see text) were replated in the matrix shown, together with several standards. This matrix was screened with [32P]cDNA transcribed from (A) 16-cell polysomal poly(A) RNA; (B) gastrula polysomal poly(A) RNA; (C) polysomal poly(A) RNA from pluteus; and (D) total poly(A) RNA from adult intestine. Row d of the matrix contained the following standards and blank controls: dl-d7, blank; d8, SpGZ, an actin cDNA clone (Scheller et al., 1981); d9, SpP389; d10, SpG30; dll, SpG6; d12, pBR322. The experiment indicates that the representation of the actin clone rises sharply between early cleavage and gastrula stage, as shown by other means elsewhere (Scheller et al., 1981; Crain et al., 1981). The clones in positions a2, a5, a10, all, a12, b5, b6 and c4 either were not detectably expressed during embryogenesis or were ubiquitously expressed at a low level. However, the remaining 28 clones all appear qualitatively to be represented by far more prevalent transcripts in the gastrula polysomes than in the 16-cell polysomes. A number of these clones, e.g., those at positions b7, b9, ~2, ~5, c9, ~10, and cl2 are undetectable or barely detectable in intestine poly(A) RNA, relative to their level of expression in the embryo poly(A) RNAs.

Their reaction with the polysomal poly(A) RNAs probably indicates contamination of these preparations with mitochondrial transcripts that are present in the embryo cytoplasm. Figure 3 also shows that those clones

32

DEVELOPMENTAL BIOLOGY

TABLE 2 PREVALENCE ESTIMATES BY COLONY SCREENING FOR CLONES WHOSE TRANSCRIPTS ARE RARE IN EGG PoLY(A) RNA AND RISE IN ABIJNDANCE DURING EMBRYOGENESIS Poly(A) RNA transcripts/egg or embryo (X10-‘)

Location

Clone matrix position SpGl-A8 SpGl-GlZ spa-G5 SpGZ-D12 SpG2-F4 SpG3-B3 SpG3-F2 SpG4-B9 SpGI-HlO SpG6-B9 SpG6-H2 SpPlP-BlO SpPlO-H? SpP14-H3 SpP14-H6 Average prevalence

In Fig. 3

In Fig. 4

J&g

a8 a7 a6 a3 al bll b10 b9 b7 bl cl2 c6 c8 c2

(a) -

10 < < * < 3 < < 5 < < 2.7 < 18

Cl

-

(b) (cl (4 (0

Gastrula cytoplasm 41 5 < 2.1 < 2.5 5 2 14 < 5 35 < 42

Pluteus tot*1

<

<

22 37 22 68 21 10 10 < < 15 < 14 10 85 41

53.8

a10.9

524.5

Note. Values are listed for 15 of the clones shown in Fig. 3, including all but one of the clones used for the experiment of Fig. 4. The clones included in this screen were replated in a new matrix for the purpose of these measurements. Data for egg poly(A) RNA and gastrula cytoplasmic poly(A) RNA derive from a single experiment, while those for pluteus total poly(A) RNA are from a different experiment. All the estimates shown were based on comparisons to standard clone hybridizations (not shown) exactly as described in the legend to Fig. 1. In many cases (denoted <) the signal obtained was less than twice background, and in one case no detectable signal was observed (‘). The estimates shown are probably accurate only within a factor of 3-5, as discussed above. The data are not expected to be exactly comparable to those in Fig. 3 since the latter experiment was carried out with polysomal RNAs. However, in moat cases there is acceptable qualitative agreement. Data for three clones initially included in this experiment have been excluded, because the number of counts per minute bound was extremely high, indicating some form of contamination or washing failure in that area of the filter. Averages are calculated on the assumption that samples giving a small signal (<2x background) are represented by the maximum number of transcripts consistent with this result. i.e., by 2 X l@ transcripts per egg or embryo and where no signal is obtained, by 0 transcripts.

not represented in 16-cell polysomal poly(A) RNA but clearly represented in gastrula polysomal RNA are also easily detected in pluteus polysomal RNA. In at least 7 such cases homologous transcripts are not evident in adult intestine poly(A) RNA. The clones in Fig. 3 that are represented at low levels in the polysomal RNA of early embryos, and at much higher levels in later embryos, constitute about 3% of the total number of clones screened in this series of observations. Many others following the same pattern of expression could of course exist among the 40-50% of cloned sequences whose complementary transcripts are too rare to be easily detected by colony screening (Lasky et al., 1980). However, had 3% of the clones in the abundant sequence sublibraries described in the preceding section behaved in this manner, they would surely have been detected. It follows that sequences displaying the pattern of regulation observed in Fig. 3 must generally belong to a lower prevalence transcript class than those represented in the high abundance sublibraries. Cursory observation of Fig. 3 in fact indicates that in later embryos most of the clones are represented

VOLUME 91, 1982

by transcripts of lower prevalence than even the SpG6 standard (position dll). This clone is complementary to transcripts present at about 18 copies/cell at the gastrula stage, or about lo4 copies/embryo (Lev et ab, 1980). To provide data directly comparable to those in Fig. 2, further screening experiments on the selected clones were carried out using [32P]cDNA transcribed from cytoplasmic rather than polysomal poly(A) RNA. Table 2 lists some prevalence estimates obtained from these screens. As Fig. 3 suggests, most of the clones turn out to be represented in egg poly(A) RNA at levels too low for reliable measurement by the screening technique (i.e., Q2 X lo3 copies/egg). However, even at gastrula stage the level of representation is usually
with Clones Expressed

DNA from six of the clones included in Fig. 3 and Table 2 were nick translated and used for RNA gel blot hybridizations. The purpose of these experiments was to provide independent confirmation of the pattern of expression implied for these clones by the colony hybridization measurements, using a method that is considerably more sensitive than the latter. Thomas et al. (1981) and unpublished results have shown that transcripts present at less than lo3 copies per sea urchin egg can easily be detected in RNA gel blot hybridizations. Autoradiographs of some of the gel blot experiments are reproduced in Fig. 4. All of the six sequences investigated are indeed dramatically regulated during development. The amount of transcript present in gastrula polysomal poly(A) RNA is in each case many times greater than in egg poly(A) RNA. The sequences in lanes a, c, and e are represented in intestine poly(A) RNA by the same transcripts as in gastrula polysomal poly(A) RNA, while that in lane d is represented to a small extent in intestine poly(A) RNA, but by a larger transcript. The sequences in lanes b and f do not appear at all in the intestine poly(A) RNA, as also shown in Fig. 3 for the same clones. The observations shown in Fig. 4 confirm the qualitative results of the colony screening hybridizations presented in Fig. 3. More importantly, they demonstrate independently the existence of moderately prominent embryo sequences that are not significantly represented in the maternal poly(A) RNA of the egg.

FLYTZANIS

E

G

(a)

I

El

G

(b)

ET AL.

Sea Urchin Embryo

I

E

G

(c)

I

Cgtvplosmic

E

G

(d)

Transcript

I

33

Prevalence

E

G

(e)

I

E

G

I

if)

FIG. 4. RNA gel blot hybridizations with selected cDNA clones. Each trio of lanes contain in order (left to right) 1 Gg of poly(A) RNA from eggs (E), gastrula polysomes (G), and intestine (I). The six probes used, and the location of these clones in the experiment shown in Fig. 3, were as follows: (a) SpG2-D12, position a3 in Fig. 3; (b) SPG4-B9, position b9 in Fig. 3; (c) SpG4-HIO, position b? in Fig. 3; (d) SpP6-H2, position cl2 in Fig. 3; (e) SpPL2-G8, position c3 in Fig. 3; (f) SpP14-H3, position c2 in Fig. 3. The autoradiographs shown in lanes (b)-(e) were exposed 5 times longer than ,those in lanes (a) and (f).

synthesized transcripts can account for the total quantity of transcripts in the embryo differs for each seIn sea urchin embryo;s the prevalence of specific tran- quence studied. For some abundant sequences this point scripts is determined by four parameters. These are the occurs in the blastula stage, for most by the gastrula amount of the transcript in the maternal RNA, the stage, but for others not until well after gastrulation decay rate of the maternal transcript, the rate of flow (Cabrera et al, 1982). The shape of the dominant repof newly synthesized transcripts of that species into the resentation patterns shown in Fig. 2 thus reflect (a) the cytoplasm, and their decay rate. The dominant devel- initial prevalence of the maternal transcripts; (b) a deopmental prevalence pattern shown in this paper for velopmental decline in prevalence that is apparently the abundant transcripts can be interpreted in terms due to turnover of the maternal transcript, since much of current knowledge of these parameters. Cabrera et of this decline probably occurs prior to the point at al. (1982) have found that the newly synthesized tran- which most of the mass of transcript present is newly scripts belonging to high abundance classes character- synthesized; and (c) a late developmental increase in istically display low decay rates. Poly(A) RNAs of this prevalence due to accumulation of stable, newly synthesized transcripts. category begin to flow into the cytoplasm at different It is interesting that the final result of this process times in development and at different rates. Thus the point at which the accumulation of these stable, newly is the reestablishment of approximately the same tranDISCUSSION

34

DEVELOPMENTALBIOLOGY

script sequence concentration (e.g., per ribosome) as in the unfertilized egg. This suggests that the sequence concentration of the abundant cytoplasmic poly(A) RNA is physiologically important. The rates of maternal poly(A) RNA degradation must be sufficiently low that the embryo has time to generate enough nuclei to replace the maternal transcripts without suffering a decline in the total transcript level (per species) of more than three- or fourfold on the average (Fig. 2). Table 1 shows that in a minority of cases (8%) the decrease of transcript concentration in middevelopment is far more severe. It would be interesting to determine whether, in these examples, the new transcripts accumulating later in development derive from related genes whose expression is confined to late embryonic stages, rather than from the same genes that were active during oogenesis. A small fraction of the less abundant sequences is expressed significantly in embryos but not in maternal RNA. This fraction is about 3% of the sample of cDNA clones screened, and since a little more than half of these clones belong to the very rare sequence class (Lasky et aZ., 1980), the fraction of sequences sufficiently prevalent to be detected by colony screening which display this form of regulation could be 6-8%. Only moderate numbers of transcripts of these regulated genes are apparently required during embryogenesis, and thus they can be supplied by new synthesis even before many hundreds of nuclei have arisen. This argument is not sufficient to explain their apparent absence from the maternal RNA, however, since most of the very rare embryo mRNA species are known to be included in the maternal RNA (Galau et ah, 1976; Hough-Evans et aZ., 1977). However, transcription of such sequences could represent important control functions in development. By expressing these sequences the embryo may significantly alter its macromolecular constitution in a temporally and perhaps spatially specific way. In other studies carried out on the same cDNA libraries as used in this work, Bruskin et ah (1981) isolated several cDNA clones that are localized to the late embryo ectoderm. These clones behave like those shown in Fig. 3; that is, they are represented at very low levels if at all in the maternal poly(A) RNA, and later by transcripts whose prevalence increases rapidly during development. Brandhorst and his associates have analyzed the patterns of protein synthesis throughout embryonic development by two-dimensional electrophoresis (Brandhorst, 1976; Bedard and Brandhorst, unpublished results). Out of nearly 1000 visible polypeptides, the relative synthesis rate of 15-20% changes sharply during development, i.e., by a factor >lO-fold. While some of these changes may be due to translational regulatory events, most directly reflect corresponding changes in

VOLUME91, 1982

the prevalences of their respective messages. The most active period of changes in protein synthesis occurs between hatching of the blastula and the beginning of gastrulation; these changes include the appearance of many newly synthesized polypeptides not detected in earlier embryos. Bedard and Brandhorst (unpublished results) have calculated that most of these proteins are probably translated from mRNAs that are of low to moderate prevalence when expressed. The set of clones included in Figs, 3 and 4 and those described by Bruskin et al (1981) probably represent mRNAs for such developmentally changing proteins. In summary, though we have investigated only a fraction of the many regulatory patterns that no doubt exist, this study reveals that in the sea urchin embryo the vast majority of very abundant late embryo sequences are also very abundant maternal sequences. However, most of these sequences are regulated during the life cycle, since only a few percent of prevalent embryonic transcript species are represented detectably in the poly(A) RNA of adult sea urchin coelomocytes (Xin et al., 1982). Sequences that are markedly regulated during embryonic development tend to belong to less prevalent transcript classes. Some examples of rare mRNAs that are sharply stage specific have already been described (Galau et aZ.,1976; Lev et al, 1980). The present results, and those of Bruskin et al. (1981), identify several moderately prevalent sequences which are also regulated during embryogenesis, and which have the advantage that their translation products can be directly detected. This research was supported by NIH Grant HD-05753. C.N.F. was supported by a fellowship from the Deutsches Krebsforschungszentrum, Heidelberg, West Germany. B.P.B. was aided by a travel grant from the National Sciences and Engineering Research Council of Canada. REFERENCES AVIV, H., and LEDER, P. (1972). Purification of biologically active globin mRNA by chromatography on oligothymidilic acid-cellulose. Proc. Nat. Acad. Sci. USA 69, 1408-1412. 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 eetoderm of the sea urchin pluteus. Dewebp. Biol 87, 308-318. CABRERA,C. V., ELLISON,J. W., MOORE,J., BRITTEN, R. J., and DAVIDSON,E. H. (1982). Regulation of cytoplasmic mRNA prevalence in sea urchin embryos: Measurement of rates of synthesis and turnover for specific sequences. Submitted for publication. CHIRGWIN,J. M., PRZYBYLA,A. E., MACDONALD, R. J., and RU?TER, W. J. (1979). Isolation of biologically active RNA from sources enriched in ribonucleases. Biochemistry 18, 5294-5299. CRAIN, W. R., DURICA, D. S., and VAN DOREN,K. (1981). Actin gene expression in developing sea urchin embryos. Mel Cell. Biol 1,711720.

FLYTZANIS ET AL.

Sea Urchin Embryo

DEVLIN, R. (1976). Mitochondriall poly(A) RNA synthesis during early sea urchin development. Develop. Biol. 50,433-456. GALAU, G. A., BRITTEN, R. J., amd DAVIDSON, E. H. (1974). A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos. Cell 2, 9-21. 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. GLISIN, V., CRKVENJAKOV, R., and BYUS, C. (1974). Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 26332637. GRUNSTEIN, M., and HOGNESS, .D. S. (1975). Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Nat. Acad Sci. USA 72, 3961-3965. 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 development. Develop. Biol. 60, 25% 277. LASKY, L. A., LEV, Z., XIN, J.-H., BRITTEN, R. J., and DAVIDSON, E. H. (1980). Messenger RN,4 prevalence in sea urchin embryos measured with cloned cDNAs. Proc. Nat. Acad Sci. USA 77,53175321.

Cytoplasmic

Transcript

Prevalence

35

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. SCHELLER, R. H., MCALLISTER, L. B., CRAIN, W. R., DURICA, D. S., POSAKONY, J. W., THOMAS, T. L., BRITTEN, R. J., and DAVIDSON, E. H. (1981). Organization and expression of multiple actin genes in the sea urchin. Mol. Cell Biol. 1, 609-628. SMITH, M. J., HOUGH, B. R., CHAMBERLIN, M. E., and DAVIDSON, E. H. (1974). Repetitive and non-repetitive sequence in sea urchin hnRNA. .I MoL Biol. 85, 103-126. THOMAS, T. L., POSAKONY, W. J., ANDERSON, D. M., BRITTEN, R. J., and DAVIDSON, E. H. (1981). Molecular structure of maternal RNA. Chrmnosoma 84,319-335. XIN, J. B., BRANDHORST, B. P., BRITTEN, R. J., and DAVIDSON, E. H. (1982). Cloned embryo in mRNAs not detectably expressed in adult sea urchin coelomocytes. Develop. Biol. 89, 527-531. ZAIN, S., SAMBROOK, J., ROBERTS, R. J., KELLER, W., FRIED, M., and DUNN, A. R. (1979). Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA. Cell 16, 851861.