A method for separating cells from early sea urchin embryos

DEVELOPMENTAL.

BIOLOGY

A Method

21, 383-402

(1970)

for Separating Urchin

Cells from

Early Sea

Embryos’

RICHARD 0. HYNES AND PAUL R. GROSS Department

of Biology,

Massachusetts Institute of Technology, Massachusetts 02139

Cambridge,

Accepted August 20, 1969

INTRODUCTION

Most biochemical studies on early embryogenesis have been concerned with temporal changes in the metabolic activities of the whole embryo. If one wishes to understand the way in which the different parts of the early embryo differentiate into a great variety of tissues, one must study the molecular events in these different sections. In order to do this, it is necessary to devise a method for isolating cells from distinct regions of an embryo. This has been done by dissection in amphibians (Flickinger et al., 1966, 1967; Woodland and Gurdon, 1966). These authors have studied the nucleic acid contents and rates of synthesis in endoderm and other tissues. Early development in the sea urchin provides an interesting opportunity for studying this problem. The classical work of Horstadius (1939) showed that, as early as the 16-cell stage, there exist cells whose developmental fate is to some extent determined. More recent work has shown that sea urchin eggs inherit, from the period of oogenesis, a stock of stable mRNA (Gross, 1967). Since the embryos cleave apparently normally in the presence of actinomycin D (Gross and Cousineau, 1963), or when enucleated by irradiation (Neifakh and Krigshaber, 1968), it is clear that the early events of cleavage are not under immediate nuclear control. Their course must therefore be determined by cytoplasmic agents, perhaps including the stable mRNA’s. In particular, Giudice and Horstadius (1965) have shown that there is no actinomycin-sensitive step in the development of the ability of 16-cell stage micromeres to vegetalize animal half-embryos. It thus appears that the 16-cell stage of these embryos represents a collection of cell types whose developmental ‘This research was supported (GM -13560).

by a grant from the National 383

Institutes

of Health

384

HYNES

AND

GROSS

properties are already partially delineated, apparently by cytoplasmic influences. Fortunately, the different cell types also differ in size, which provides a means for their separation. With the aim of investigating the molecular bases for the differences between the cells and the mechanism of their determination by the cytoplasm, we have developed a method for separating the three cell types. The purpose of this paper is to describe the technique. We also present some data on the properties of the isolated cells. MATERIALS

Preparation

AND

METHODS

of Embryos

Arbacia punctuluta were obtained from Mr. Glendle Noble, Panama City, Florida, and Strongylocentrotus purpuratus from Pacific Bio-Marine, Venice, California. The work to be reported concerns Arbacia, but the separation has been applied to S. purpuratus also and works in that species essentially as described. Collection of eggs and sperm was by dissection of the gonads. Ovaries were filtered through cheesecloth and the eggs were washed by gentle sedimentation from Millipore-filtered seawater (MPFSW) until clean. This frees them of contaminating oocytes, cell debris, and bacteria. Eggs were fertilized and demembranated in 0.04% papain + 0.2% cysteine in seawater, pH 7.8, (Tyler and Spiegel, 1956). Papain (Nutritional Biochemicals, Inc.) and neutralized cysteine solutions were prepared fresh each day, and the solutions plus sperm were mixed immediately prior to fertilization. The eggs were kept suspended for 4-5 minutes. During this time the fertilization membrane rises as usual, although it is much thinner. It subsequently dissolves. Thus, one can check the percentage fertilization, and polyspermy is low. The eggs were then spun down gently and the supernatant was removed. They were washed twice with MPFSW by gentle centrifugation and resuspended in MPFSW + 50 units of penicillin/streptomycin per milliliter (Microbiological Associates, Inc., Bethesda, Maryland). They were incubated on a magnetic stirrer at a temperature close to that of the seawater from which the adults came, usually 18°C. The whole procedure takes lo-15 minutes. The embryos develop normally, cleaving at the same rate as controls and form normal plutei. Bacterial contamination is undetectable, as judged by the

CELL

385

SEPARATION

absence of 16 S and 23 S labeled peaks after incubation “H and extraction of RNA (unpublished results).

with uridine-

Cell Separation Embryos were harvested, chilled, and centrifuged down at 250 g. They were washed twice with cold calcium-magnesium-free seawater (CMFSW)’ and once with CMFSW + 2 mM EDTA. They were resuspended in about lo-20 times their volume in CMFSW + EDTA and disaggregated by shaking in the cold-usually for 30 minutes. Generally, there remained very few embryos that did not disaggregate, and there was very little cytolysis. The suspension of cells (2-5 ml) was layered over 30-ml linear 5-15s density gradients of Ficoll (Pharmacia) made up in CMFSW and spun at 250 g for 60 seconds. Bands of cells were removed with Pasteur pipettes. This must be done slowly since Ficoll solutions are viscous and contamination of the bands occurs if one is not careful. An alternative procedure, occasionally used when large numbers of cells were required, was to layer over a 120 ml 5-15s Ficoll gradient made in a beaker and allow to stand in the cold until the bands were well separated (2-4 hours). Ficoll gradients were made using a Buchler polystaltic pump. Incorporation

Procedures

All radioactive precursors were obtained from New England Nuclear, Boston, Massachusetts. Thymidine-methyl-“H, > 15 Ci/ mmole; uridine-5-“H, > 20 Ci/mmole; r,-leucine-14C (U), > 250 mCi/mmole; L-leucine-4,5-‘H, 30-50 Ci/mmole. Incubations were carried out at the temperatures stated and always in the presence of 50 units/ml each of penicillin and streptomycin. Incorporation was terminated by pelleting the embryos or cells in a hand centrifuge (10 seconds), removing the supematant, and adding 1 ml of 0.5% sodium dodecyl sulfate (SDS) to lyse the cells. Two volumes of cold 15% trichloroacetic acid (TCA) were then added to precipitate macromolecules. The precipitates were filtered on Millipore filters with 5 washes with 2 ml of cold 5% TCA + 1 mg/ml of the relevant precursor. On occasion, 100 rg of bovine serum albumin were added to ensure complete precipitation when ’ CMFSW: 0.5 M NaCl; 9 m&f KCl; 2 mM NaHC&; EDTA: ethylenediaminetetraacetic acid. disodium salt.

29 mM Na2S0,;

pH 7.8.

HYNES AND GROSS

only small amounts of material were present. This method was found to give good removal of acid-soluble radioactivity. Amino acid incorporations were also measured after incubation at 80-90°C for 30 minutes in 5% TCA. The results were the same. The SDS/ TCA method also reduces the self-absorption, which is considerable for tritium remaining inside whole cells precipitated with TCA. All values were corrected for self-absorption by comparison with a correction curve derived using serum albumin standard. Samples were counted in a Beckman LS-250 liquid scintillation counter using toluene and PPO-POPOP scintillant (Spectrafluor, Nuclear Chicage). RESULTS

Validity

of the Method

The techniques are described in detail under Materials and Methand the fertilization membrane removed. The embryos cleave, the cells being held together by the hyaline layer. At the desired stage, the latter is dissolved by removal of divalent cations and the cells fall apart. The mixture of disaggregated cells is then separated by velocity sedimentation in a density gradient composed of Ficoll in calcium-magnesium-free seawater. Ficoll was chosen as an osmotically neutral density medium (mol. wt. -400,000). The whole process from harvesting of the embryos to collection of the fractions takes place in the cold and is completed in about 1 hour. During disaggregation, the cells swell somewhat but do not appear to be damaged. Their fine structure, as observed in the electron microscope, is the same as that of cells ods. In essence, eggs are fertilized

in situ.

Fractionations have been carried out on all stages up to morula (Fig. 1). At the stage of particular interest, the 16-cell stage, three bands result (Figs. Id, 2). As will be discussed later, these correspond to micromeres, mesomeres, and macromeres. It should be noted that the three-band pattern is entirely stage specific (Fig. 1). At earlier stages (2-, 4-, and 8-cell, Figs. la-c) only a single band is found, its sedimentation velocity decreasing with each cleavage. At later stages (32- and 64-cell) only two bands are found: micromeres and others. Only at the X-cell stage are three bands found and then only in synchronous populations. In general it is essential to use embryos from a single female in order to obtain good separation. If cleavage is not entirely regular, the two faster bands are

CELL SEPARATION

0

b

d

e

f

FIG. 1. A batch of embryos was incubated, samples being removed at each stage and disaggregated. Gradients were run in parallel on each stage: (a) 2-cell stage; (b) 4cell; (c) S-cell; (d) X-cell; (e) 16/32-cell-a minority of the embryos had reached 32-cell stage; (f) 32/64-cell stage. The narrow band near the bottom in each case contains uncleaved eggs and a few embryos which are not d&aggregated, apparently because they retain their fertilization membranes. Bands are numbered from the top of the gradient.

not resolved and a single nonmicromere fraction results. The band pattern gives one a sensitive index of synchrony. If some embryos lag behind the rest and are in the ES-cellstage at the time of separation, then band 3 becomes thicker than band 2, whereas, in good populations, the reverse is true, since there are more mesomeres than macromeres. Figure le shows the result obtained when some embryos are ahead of the rest. The slowest moving band at all stages from 16cell onward is white, while the others contain echinochrome, the red pigment found in Arbaciu eggs. This correlates with the fact that the micromeres in this species are relatively colorless, containing few or no pigment granules. All subsequent studies to be reported here were carried out at the 16-cell stage. Figure 3 shows embryos from this stage in various states of disaggregation. Figure 4 shows photomicrographs of unfractionated cells and of each of the three fractions. It can be seen clearly that considerable separation on the basis of size has been achieved. This is seen more readily from the diameter histograms shown in Fig. 5. From these histograms one can determine the diameters of the predominant cell type in each fraction and some measure of the degree of crosscontamination between the fractions.

388

FIG. 2. Photograph of the bands.

HYNES

of a separation

AND

GROSS

at 16cell stage. The arrows mark the positions

Frc. 3. Embryos at 16cell stage were washed in CMFSW, causing partial disaggregation. The arrow in (a) points to an embryo showing no disaggregation, while that in (b) points to one which has opened out almost completely, showing the three cell types.

CELL

SEPARATION

389

FIG. 4. Cells were fixed in 3% glutaraldehyde in phosphate buffer pH 7.6, + 2% NaC12 + 0.5% MgClz and photographed at magnification of $120. The large amount of shrinkage (50%) was probably caused by incomplete removal of seawater before addition of the fixative. (a) Unfractionated mixture of cells; (b) band 1, micromeres; (c) band 2, mesomeres; (d) band 3, macromeres.

390

HYNES

AND

GROSS b

J 0.6

T.a d

50

IMAGE DIAMETER

(CM)

FIG. 5. Image diameters were measured on prints to the nearest millimeter. (a) histograms for all three fractions plotted together to show degree of overlap. White, band 1; cross-hatches, band 2; stippled, band 3; (b) band 1, micromeres; (c) band 2, mesomeres; (d) band 3, macromeres.

Table 1 shows the values obtained for the diameters of the separated cells and of cells still partially aggregated in embryos. It is clear that the cells swell slightly on removal of the constraint imposed by the hyaline layer. It is also clear that the diameters of the predominant cell types in the three bands are equivalent to those of micromeres, mesomeres, and macromeres, respectively. Histograms of the unfractionated mixture of cells show peaks at the same three diameters found for the separated fractions. Volumes, calculated from these diameters and summed in the proportions (4 micromeres + 8 mesomeres + 4 macromeres) found in the embryo,

CELL

391

SEPARATION

give a calculated volume for the embryo which agrees well with the known one. Also shown in Table 1 are the protein contents of the three classes as determined by the method of Lowry et al. (1951). In summary, the evidence that the fractions correspond to micromeres, mesomeres and macromeres is as follows: 1. The three-band pattern is stage specific, appearing only when these three cell types are present. 2. Measured diameters of the separated cells correspond with those of the cells in the whole embryo. 3. The cells in band 1 are relatively colorless as are micromeres. 4. The bands are unchanged by recentrifugation (see below). Cross-Contamination This was investigated in two ways. The first used the diameter histograms shown in Fig. 5. In the case of bands 1 and 2, contamination is easily determined. Band 3, which appears to be less homogeneous, is less easily analyzed. However, if one assumes a symmetrical distribution about the modal value (16 mm), one can calculate that band 3 contains approximately 20% mesomeres. The contaminations of each cell class by the other two, as estimated in this way, are shown in Table 2. TABLE CELL

1

SIZES

Measurement

Micromeres

Mesomeres

Macromeres

Diameters” of cells associated in embryosb (p) Diameters” of separated cells’ (p) Relative volumes calculated from diameters Protein contentd (mpg per cell) Relative sizes from protein contents

20.2

27.2

31.2

21.3 1.00

29.7 2.71

34.0 4.07

0.83 f 0.02 1.00

2.35 f 0.12 2.85

3.60 i 0.13 4.35

a Diameters were calculated from photographic image diameters, measured on a single preparation (see Fig. 5). Microscopic magnification: X 120. Photographic magnification: X 7.9. The values were corrected for shrinkage in the fixative by comparing the diameter of eggs in the same preparation (36.8 w) with that given by Harvey (1956) for Arbacia eggs (74 p). ‘Measured from photographs of partially disaggregated embryos such as in Fig. 3b. ’ The diameters for the separated cells were taken to be those of the most prominent class, as shown in the histograms. d Mean and standard error of seven analyses on different preparations.

392

HYNES AND GROSS TABLE 2 CROSSCONTAMINATION

Band 1 Band 2

Percentage of cells with image diameter:

Band 3 Percentage of mesomeres in band 3 assuming macromere diameters show symmetrical distribution about 16 mm

>12mm <13 mm >15mm <13 mm

4.6 2.6 2.8 2.0 22.0

Secondly, a mixing experiment was performed as follows. Two batches of embryos from a single female were harvested at 16-cell stage, one having been pulse-labeled with leucine-‘4C the other with leucine-“H. The cells were separated and the fractions collected. The counts per minute/cell for each one were determined by plating an aliquot for scintillation counting and measuring cell counts in a hemacytometer. The fractions were then pooled, as shown below, to give all the possible combinations: X--“H micromeres + 14Cmesomeres; Y--“H mesomeres + 14Cmacromeres;Z“H macromeres + 14Cmicromeres. The mixtures were then separated again on Ficoll gradients, and the resulting bands were removed and plated on filters for doublelabel counting. From the “H: 14C ratios the contamination of each band could be determined. The results are shown in Table 3. With one exception, only the two bands expected were found. In Z, there was a faint band in the mesomere position, although there should have been only micromeres and macromeres. This suggests that the macromere band may contain some mesomeres. Such a conclusion is supported by the data for mixture Y. Band Y2 shows approximately 20% contamination with 14C-labeled cells. In contrast, Y3 shows very little contamination with “H-labeled cells. There is, consequently, a one-way movement of cells, from band 3 to band 2, and not in the other direction. Mixture X showed no visible evidence of a macromere band. One can thus conclude that band 3 of the 14Cpreparation contained some mesomeres which, on mixing and recentrifugation, shifted to band 2 (i.e., Y2). All bands except Y2 show very low cross-contamination. Therefore, with the exception of the contamination of band 3 by mesomeres, it can be concluded that the bands constitute discrete cell classes with little contamination of one another. The fact that mixing and recentrifu-

CELL

393

SEPARATION

gation reproduces the expected band patterns with very little switching of cells from one fraction to another provides further evidence that the banding pattern is a property of the cells and is not an artefact of the sedimentation. The contaminations estimated by this method are only an indication of the actual values since it is not clear whether contaminating cells found in a given band after mixing and recentrifugation are there because they were in the wrong band initially and have now found their proper location or whether they are introduced into the wrong band in the procedure of refractionation. Furthermore, the very recentrifugation procedure is likely to introduce some spurious cross-contamination. In conclusion, bands 1 and 2 are, respectively, micromeres and mesomeres, and each is better than 95% pure. Band 3 consists mostly of macromeres but may be as much as 20% contaminated with mesomeres. Incorporation

Rates

There have been a number of reports concerning the relative rates of RNA and protein synthesis, or rather incorporation of radioactive precursors, in different parts of sea urchin embryos. These have been of three kinds: using autoradiography (Immers, 1959; Markman, 1961a; Czihak, 1965), half-embryos (Markman, 1961b, TABLE MIXING

AND

3

SEPARATION

OF

BANDS

Band

SH (cd

Xl x2

68,030 0

179,200 0

8784 401,250

5260 236,900

2.88

Y2 Y3

70,500 5,434

76,800 5,900

43,332 105,314

16,400 40,000

21.40 1.48

Zl 23

88 37,770

124 53,100

86,728 1524

93.200 1035

0.13 1.95

Number of cells

“C

(cnm)

Number of cells

Percent contamination

‘The number of cells labeled with each isotope in each fraction was calculated using the specific activity in counts per minute per cell of the relevant fraction, determined before mixing. These were as follows: 0.92 Band1 JH: Band3 1.47 Band 2 0.38 ‘T: Band1 Band2 Band3 2.64 0.71 1.70

394

HYNES AND GROSS

1967; Berg, 1965, 1968), or separated cells (Spiegel and Tyler, 1966). The results have been conflicting. It would be of interest to know whether any one cell class shows a markedly different rate of synthesis of any of the major classes of macromolecule. To this end, we have studied the rates of incorporation of radioactive precursors to DNA, RNA, and protein in these cell classes. The experiments were of two kinds: 1. Incorporation was carried out in a short pulse ending at the 16cell stage. The cells were then separated and the counts per minute per cell and per milligram of protein for each cell class were determined (Table 4). 2. The kinetics of incorporation were studied on whole embryos, starting at the 16-cell stage, and on the three cell fractions prepared from this stage (Tables 5 and 6, and Figures 6-8). These experiments also give some indication of the state of health of the cells after disaggregation and separation. The relative incorporations occurring before separation are shown in Table 4. As might have been expected, a thymidine-“H label lasting through three cycles of DNA synthesis gives equal incorporation per cell for all three cell types. In contrast, in a short pulse with uridine-“H, the micromeres are more active than the other two cell classes in incorporation per cell (i.e., per genome). The leucine results show that the larger the cell, the higher the incorporation per cell, as expected. But on a per milligram of proTABLE 4 RELATIVE

INCORPORATION

BEFORE SEPARATION

Per milligram

Per cell Micro Thymidine-3H” Uridine3Hb Leucine’

Meso

1.00 1.00

Macro 0.97

1.09 0.57

f

Micro

0.09

1.00 3.38hO.16

0.58

i

1.06 0.12

4.78~0.41

1.00

1.06

Meso

Macro

0.39

0.27 0.13 * 0.03 l.lo+O.O7

0.03 1.18+0.05

0.20

protein

f

’ Thymidine-3H pulse began late in first division cycle and ended at X-cell stage. Pulse length was about 2 hours. Thymidine-‘H was at 5 &i/ml. bUridine-“H pulses began at 8-X-cell stage and ended when all embryos were at X-cell stage. Pulse length varied from 20 to 30 minutes in different experiments. Uridine-“H was at 100 &i/ml. ‘Leucine pulses were for 10 minutes at 16ceh stage. Leucine-“C was at 0.5 pCi/ml; Leucine-“H at 1 &i/ml. Rasulta for uridine and leucine are given as mean and standard error of analyses performed on several different batches of cells.

CELL

395

SEPARATION

TABLE 5 RELATIVE INCORPORATIONAFFER SEPARATION Per milligram

Per cell Label

Thymidine-‘H Uridine-‘H Leucine-14C

Micro

Meso

Macro

Micro

Meso

Macro

1.00 1.00 1.00

1.23 1.62 2.46

1.78 1.87 3.35

1.00 1.00 1.00

0.43 0.57 0.87

0.41 0.43 0.77

‘These figures were calculated sizes shown in Table 1.

from the rates shown in Table 6 and the relative

tein basis, all three cell types are essentially equal in activity. If one compares these results with those obtained after separation and shown in Table 5, it is clear that, after separation, the micromeres are, relative to the other cell types, less active in incorporation of uridine-“H and thymidine-“H and more active in leucine‘C incorporation than was the case before separation. It should be noted here that differences in incorporation rates do not necessarily reflect differences in absolute rates of synthesis. This point will be discussed more fully later. Table 6 and Fig. 6-8 compare the incorporation rates of the separated cells with those of the whole embryo. In all cases, the separated cells are less active per mg protein. Giudice (1962), comparing rates of incorporation of amino acids by whole and disaggregated embryos at postblastula stages, found the cells to be about 20% as active as the whole embryos. This agrees well with our results. Guidice found that carrying out the incorporation into disaggregated cells in calcium-free seawater raised their activity to 50% of that of the embryos. The difference may be caused by aggregation occurring during the incubation in seawater. However, the fact remains that the separated cells are capable of incorporation rates at least 25% of those of the whole embryo. The higher relative rates obtained for thymidine-“H incorporation may be an artefact. As can be seen from the inset of Fig. 6, the whole embryos show a biphasic curve during a 2-hour incorporation starting at early E-cell stage. The rate given for whole embryos in Table 6 is that occurring after 90 minutes (i.e., after the plateau). Since all thymidine incorporation curves show a continuously increasing slope, this estimated rate may be low if one assumes that the separated cells start, so to speak, from after the pause in DNA

HYNES

AND

GROSS

synthesis and, thus, that their incorporation rates should be compared with incorporation by whole embryos in a 2-hour period following the plateau. Of course, it is possible that some control mechanisms are operating in the separated cells, causing them to show a greater emphasis on DNA synthesis than before separation, but the data do not warrant speculation along these lines. The main point is that the cells show active incorporation when returned to seawater after fractionation, which suggests that they have not been seriously damaged in the process. DISCUSSION

The method described allows fractionation of M-cell embryos of sea urchins into the three cell classes present at this stage. An earlier paper (Spiegel and Tyler, 1966) reported the isolation of pure micromeres, but the authors’ method did not purify the other two classes as has been possible here. The micromeres and mesomeres are > 95% pure while macromeres are at least 7580% pure, being contaminated by mesomeres. The technique is also capable of separating micromeres from other cells at the cleavage stages following 16-cell stage. The cells appear not to be damaged to any great extent. They appear morphologiTABLE

6

INCORPORATION RATESAFTERSEPARATION Rates (cpm/mg protein/hour”) ..~ Label Embryos

Thymidine-JH, 10 &i/ml Uridine-“H, 20 &i/ml Leucine-“C, 0.5 j&i/ml

250,000 167,000 940,ooo

Micro

Meso

240,000 (96) 76,500

103,000 (41) 43,600

(46)

(26)

282,000 (30)

244,000

(26)

Macro

97,500 (39) 32,800

Reconstituted embryosb 111,800 (45) 43,860

cm

(26)

217,006

237,000

(23)

(25)

‘Embryo time courses started at 16-cell stage. Time courses for the separated cells were carried out in MPFSW: precursors were added after preincubation of the cells. All preparations were preincubated with 50 units/ml each of penicillin and streptomycin. The values shown in parentheses are percentages of the rates for whole embryos. b Calculated, from the measured rates for the individual cell classes allowing for their relative contributions to the mass of the embryo.

CELL

397

SEPARATION

1

200

z iz r2 a 0 I n\ 100 ‘0

; i-5

-0

60 MINUTES

120

F’IG. 6. Incorporation of thymidine-‘H (10 &i/ml). Inset shows whole embryos. Filled circles, embryos; open circles, micromeres; triangles, mesomeres; squares, macromeres.

tally normal by electron microscopic observation (unpublished results) and are capable of incorporation rates at least 25% of those of the whole embryo. In attempting to analyze the incorporation data, it is important to remember that these may not represent true rates of synthesis. The incorporation rate depends both on the absolute rate of synthesis and on the specific activity of the precursor pool. Particularly in short pulses, the latter may change with time and it may differ between cell types. When the cells are of different sizes, the differences in surface to volume ratio may affect the rates of uptake of radioactive precursors per unit volume of pool. Without assay of the pool specific activities, one is unable to eliminate these possible complications.

398

HYNES AND GROSS

With these reservations in mind, the following conclusions can be drawn concerning the incorporation data. In a period covering three cycles of DNA synthesis, all the cell types incorporate equal amounts of thymidine-3H per cell (i.e., per genome). This is what one would expect if each cell had a diploid gene content and there were no interference by pool effects. There has been a claim (Lindahl, 1953) that micromeres are haploid. Our data do not support this. While the thymidine-3H incorporation per cell (Table 5) of separated micromeres is somewhat less than that of the other cell types, the data for rates before separation suggest that 16-cell stage micromeres are diploid. If they were to become haploid by 32-cell stage, as Lindahl proposes, they would show no incorporation for a cycle. We do not see such a lag. While our experiments were not designed to examine this problem closely and our results do not completely eliminate the possibility of haploid micromeres, they make it seem unlikely.

a

0

0

A A

0

0 LL 0

8

60 MINUTES FIG. 7. Incorporation of uridine-“H (20 &i/ml).

120 Symbols as Fig. 6.

CELL SEPARATION

r

MINUTES FIG, 8. Incorporation of leucine-“C (0.5 &i/ml). Symbols as Fig. 6. The 30-min point for mesomeres is aberrant and is not repeatable in other experiments.

Concerning the uridine incorporation before separation, there are several possible explanations for the fact that micromeres are more active than the others. 1. They are more active, per genome, in RNA synthesis. 2. Their pools acquire a greater specific activity during the pulse. In a short pulse, pool problems are particularly acute, especially in the case of uridine and thymidine, which are taken up very slowly, as is shown by the lag in the incorporation curves. Thus, the specific activities of the pools are changing constantly during the pulse, and if the micromeres have either (a) a smaller pool or (b) a greater rate of uptake per unit volume of pool, then their pools would be more radioactive. We are unable to decide among these possibilities without further work. In the same way, we cannot say whether the differences between the relative incorporation rates before and after

406

HYNES AND GROSS

separation are developmentally related changes in rates of synthesis or are due to artefacts of separation or differences between a pulse before and a longer label after separation. In any case, under all conditions, the rates of incorporation per cell of uridine-“H, and thymidine-“H are within a factor of two in the three cell types. Similarly, both before and after separation, the rates of incorporation of leucine per milligram of protein are close to equal in the three cell types. Both Markman (1961b) and Berg (1965, 1968) claim that during cleavage, animal halves (mesomeres) are slightly more active on a per embryo basis than vegetal halves (macromeres + micromeres) in amino acid incorporation, but Berg points out that the animal halves are larger (our results show them to be 54% of the embryo) and in essence their results agree with ours, that on a per milligram of protein basis all the regions of the embryo are equally active in protein synthesis. This was also found by Spiegel and Tyler (1966) comparing micromeres with an unfractionated mixture of cells. Czihak (1965) has presented autoradiographic data showing strongly preferential incorporation of uridine-3H in micromeres at the E-cell stage. This is in conflict with our results. Although micromeres are 5-8 times as active (Table 4) on a per milligram basis (which is more directly related to grain density than per cell considerations) this alone does not explain the manyfold excess in grain density which he shows. Markman (1961a) showed no regional differences in incorporation before 64-cell stage, also by autoradiography. Experiments with half-embryos are also in conflict on this point. Berg (1968) showed essentially equal incorporation of valine14C, adenine-‘4C, and guanosine-“H by animal and vegetal halves at all stages, after correction for size differences, with some suggestion of greater incorporation of uridine-14C by vegetal halves. Markman (1961b, 1967) showed equal incorporation of adenine-14C per half embryo at cleavage stages, but animal halves were more active at later stages, when uridine-14C was also incorporated more actively by animal halves. A report by Runnstrijm et al. (1964) showed that after the mesenchyme blastula stage, animal halves are also significantly more active than vegetal in incorporating amino acids. It is possible that differences exist between cleavage and later stages, and between species, but it appears that most of the evidence on early stages is in agreement with our data, viz., the various parts of the embryo are essentially equivalent with respect to incorporation rates.

CELL

SEPARATION

401

While there appear to be no gross differences between the cell classes in overall synthesis of DNA, RNA, and proteins, this is hardly surprising. The interesting possibility remains that the various cell types contain different complements of the mRNA made during oogenesis and/or that they are making different populations of RNA and proteins. Work is under way in our laboratory to investigate these possibilities. SUMMARY

A method is described for obtaining in quantity preparations of micromeres, mesomeres, and macromeres from 16-cell sea urchin embryos. The rates of DNA, RNA, and protein synthesis in each cell class are examined by incorporation of radioactive precursors. No marked differences exist between the cells in these activities. REFERENCES BERG, W. E. (1965). Rates of protein synthesis in whole and half embryos of the sea urchin. Exptl. Cell Res. 40, 469-489. BERG, W. E. (1968). Rates of protein and nucleic acid synthesis in half embryos of the sea urchin. Erptl. Cell Res. 50, 679-683. CZIHAK, G. (1965). Evidences for inductive properties of the micromere-RNA in sea urchin embryos. Naturwissenschuften 52, 141-142. FLICKMGER, R. A., GREENE, R. KOHL, D. M., and MNAGI, M. (1966). Patterns of synthesis of DNA-like RNA in parts of developing frog embryos. fioc. Natl. Acad. Sci. U.S. 56, 1712-1718. FLICKINGER, R. A., MNAGI, M., MOSER, C. R., and ROLLINS, E. (1967). The relation of DNA synthesis to RNA synthesis in developing frog embryos. Deoelop. Biol. 15, 414-431. GIUDICE, G. (1962). Amino acid incorporation into the proteins of isolated cells and total homogenates of sea urchin embryos. Arch. Biochem. Biophys. 99, 447-450. GIUDICE, G., and H~RSTADIUS, S. (1965). Effect of actinomycin D on the segregation of animal and vegetal potentialities in the sea urchin egg. Exptl. Cell Res. 39, 117-120. GROSS, P. R. (1967). RNA metabolism in embryonic development and differentiation. New Engl. J. Med. 276, 1230-1247, 1297-1305. GROSS, P. R., and COUSINEAU, G. H. (1963). Effects of actinomycin D on macromolecule synthesis and early development in sea urchin eggs. Biochem. Biophys. Res. Commun. 10, 321-326. H~RSTADIUS, S. (1939). The mechanics of sea urchin development studied by operative methods. Biol. Rev. Cambridge Phil. Sot. 14, 132-179. IMMERS, J. (1959). Autoradiographic studies on incorporation of “C-labelled algal protein hydrolysate in the early sea urchin development. Exptl. Cell Res. 18, 585588. LINDAHL, P. E. (1953). On a normally occurring reduction division in the somatic cells of the sea-urchin embryo. Exptl. Cell Res. 5, 416. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

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MARKMAN, B. (1961a). Regional differences in isotopic labelling of nucleic acid and protein in early sea urchin development: an autoradiographic study. Exptl. Cell Res. 23, 118-129. MARKMAN, B. (1961b). Differences in isotopic labelling of nucleic acid and protein in sea urchin embryos developing from animal and vegetal halves. Erptl. Cell Res. 25, 224-227. MARKMAN, B. (1967). Isotopic labelling of nucleic acids in sea urchin embryos de-

veloping from animal and vegetal halves in relation to protein and nucleic acid content. Exptl. Cell Res. 46, 1-18. NEIFAKH, A. A., and KRIGSHABER, M. R. (1968). Synthesis of protein in the embryological development of the sea urchin egg after inactivation of the nucleus. Dokl. Akad. Nauk. SSSR 183, 493-496. RUNNSTR~M, J., H~RSTADIUS, S., IMIMERS, J., and FUDGE-MASTRANGELO, M. (1964). An analysis of the role of sulfate in the embryonic differentiation of the sea urchin (Paracentrotus liuidus). Rev. Suisse Zool. 71, 21-54. SPIEGEL, M., and TYLER, A. (1966). Protein synthesis in micromeres of the sea urchin egg. Science 151, 1233-1234. TYLER, A., and SPIEGEL, M. (1956). Elevation and retraction of the fertilization membrane of echinoderm eggs fertilized in papain solutions. Biol. Bull. 110, 196200. WOODLAND, H. R., and GURDON, J. B. (1968). The relative rates of synthesis of DNA, sRNA and rRNA in the endodermal region and other parts of Xenopus lneuis embryos. J. Embryol. Exptl. Morphol. 19, 363-385.