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The localization of ribonucleic acid in the egg of Ilyanassa obsoleta
Experimental
Cell Reseurch
OF RIBONUCLEIC
ACID
126
THE
LOCALIZATION EGG
OF ILYANASSA J. R.
Department
of Zoology, Marine
Biological
IN
(1860)
THE
OBSOLETA’
COLLIER2
Louisiana State University, Baton Rouge, Laboratory, Woods Hole, Massachusetts, Received
21, 126-136
October
Louisiana, U.S.A.
and the
2, 1959
this paper the results of a study of biochemical cell-lineage are presented. The hope for this work is to relate the biochemistry of the early blastomeres to the partial embryos which they form when isolated. ‘I’he chemical studies of today arc indebted to the classical cell-lineage studies begun by Conklin, \\‘ilson, and Lillie, and to the analysis of the development of isolated blastomeres which they initiated. ;\lorc recently, the careful studies of Costello [ 1 l] and Clement [5, li] on the differentiation of partial embryos have provided additional information needed for this approach. The role generally attributed to the ribonucleoproteins as a template molecule for protein synthesis centers attention on this nucleic acid as a determining factor in embryonic differentiation. Levcnbook ef al. [15] have correlated differences in the base ratios of th RNA of Drosophila eggs and the chromosomal composition of the egg. The present work is concerned with the distribution of RNA to cells with the same genetic constitution but with a difl’crent capacity for differentiation. n’hilc a study of the quantitative distribution of substances to the early blastomeres is in some ways of limited value, many functions vital to the differentiation of the embryo occur hcforc synthctic mechanisms come into play, and in this sense the initial concentration of substances in a given cell may be important. Berg [l] dealing with the same general problem and working with the egg of Ciona intestinalis, has studied the distribution of RNA to the anterior and posterior blastomeres. In the following experiments the localization of RNA has been determined in the egg, polar lobe, AR and CD blastomeres of the marine snail Z1yann.s.w obsolefn. IN
1 This work was supported in part by a research and Metabolic Diseases of the National Institutes 2 Present address: Marine Biological Laboratory, Experimental
Cell Research
21
grant (A-3554) of Health. Woods Hole,
from
the Division
Massachusetts,
of Arthritis U.S.A.
Localizafion MATERIALS Biological
of RNA
in the Ilyanassa AND
127
egg
METHODS
Material
obsoleta eggs were obtained from snails kept in an aquarium of sea water. The snails were fed on alternate days and an ample supply of freshly deposited egg capsules were collected daily. Each capsule contained from 80 to 120 eggs. The eggs were removed from the capsule and washed in pasteurized sea water; blastomeres and polar lobes were isolated by agitation in calcium-low sea water as described previously 181. The required number of cells, 200 eggs or 400 isolated blastomeres, were counted and transferred in 30~1 of sea water to a small glass vessel. The cells were homogenized by introducing a small vibrating glass rod into the drop of sea water containing the eggs. In the analysis of the yolk and cytoplasmic fractions the yolk platelets were removed from the homogenate by centrifugation in sea water at a very low speed for 30 seconds. The supernatant was withdrawn, and the yolk platelets were washed three times with 50 microliters of pasteurized sea water. The washes were combined with the original supernatant to make up the cytoplasmic fraction. Zlyanassa
General
Procedure
The chemical analysis of isolated blastomeres required the development of micromethods capable of separating and detecting amounts of material of the order of onehalf to one microgram. Several general procedures for chemical fractionation were tried and found unsuccessful with this material before the final adoption, with modification, of the Ogur and Rosen [18] procedure. The Schmidt-Thannhauser [22] scheme was unsatisfactory because the separation of the ribonucleic acid (RNA) from protein and deoxyribonucleic acid (DNA) was incomplete following alkaline hydrolysis of the RNA. Neither extraction of the protein from the hydrolyzate with chloroform or precipitation of DNA in the presence of added egg albumin was satisfactory in overcoming this difficulty. The Schneider [23] scheme for tissue fractionation has been adapted to a micro level by Patterson and Dackerman [19]. This scheme was not satisfactory because the calorimetric methods for detection of ribose could not be used with the Zlyanassa egg; however, the general technique of Patterson and Dackerman [lo] was valuable in working out the procedures finally adopted. Vsing a modified Ogur-Rosen [lg] fractionation scheme it was possible to separate and quantitate the following tissue fractions: (1) acid-soluble components, (2) lipids, (3) RNA, (4) DNA, and (5) protein. Homogenization and extraction:-All cells were transferred in 30 to 50 ,~l of sea water to a small centrifuge tube (3 x 36 mm) and homogenized by introducing a small vibrating glass rod. All homogenization was done in an ice bath at 2 to 4 degrees C. Originally all extractions were carried out in these centrifuge tubes, and the supernatant was withdrawn after centrifuging the precipitated protein. Later, it was found that the homogenate could be transferred to a depression in a Pyrex spot plate and that after heat drying at 85-90°C for 15 minutes the protein would stick to the slide; thus, all subsequent extractions could be carried out on the spot plates. In Experimental
Cell ICesearch
21
J. I?. Collier extractions requiring heat the depressions were covered with a glass slide, and the ,depression plate was placed in an oven at the required temperature. The same results were obtained with both procedures of extraction, and the technique using the spot plate was ultimately adopted because it was much less laborious and resulted in smaller losses of material. Acid-soluble fraction.-The homogenate was cooled to 4°C and washed three times, for 5 minutes each wash, with 100 microliters of 0.2 N perchloric acid (PCA), and all three washes were collected quantitatively and stored in a small glass vessel. These washes constituted the acid-soluble fraction, and after determining the ultraviolet absorption of this fraction, the phosphorus content was determined. When the washes were collected and analyzed separately, 99.0 per cent of the total acid-soluble phosphorus was removed in the first two washes. Lipid fraction.-After the removal of the acid-soluble fraction, lipids were extracted by two washes with ethanol-ether (3: 1) for 15 minutes at 35-40°C followed by one chloroform extraction for 15 minutes at 35540°C. These extractions were combined as the lipid fraction. Analysis of separate ethanol-ether extracts showed that 96 per cent of the lipid phosphorus was removed in the first wash. Only 3.8 per cent of the lipid phosphorus was found in the chloroform extract. Extraction of RNA.-The RNA was removed from the precipitated protein and DNA by hydrolyzing the RNA with 100 ~1 of 1 N PCA for 15 hours at 4°C. After hydrolysis the residue was washed with an additional 90 ~1 of 1 N PCA, and the extract and wash were combined and diluted to a volume of 200 ,ul. The amount of RNA was ‘determined from the optical density at 260 rnp and phosphorous content as described below. All determinations of RNA were calculated from the optical density at 260 rnp except in those cases where other methods of analysis are specifically indicated. After removal of the RNA, the DNA was extracted with hot 0.5 N PCA, and the protein residue was solubilized in 1 N NaOH. Results on these fractions are not included in this paper, and the methods for analysis are omitted. Analytical
Procedure
SpecfrophofomeQ-All samples for spectrophotometry were diluted to 200 ~1 in small calibrated vessels. Aliquots were transferred to Lowry and Bessey [16] microcells, and the optical density was determined with a Beckman DU spectrophotometer. The coefficient of variation for the estimation of RNA from the optical density at 260 rnp was 8.6 per cent. Phosphorus deferminafions.-Phosphorus was determined by Patterson and Dackerman’s [19] micro adaptation of King’s [14] modifications of the Fiske and Subbarow method for phosphorus analysis. The range of phosphorus determined was from 0.051.2 pg per sample. Ribose determinations.-The ribose content of the RNA fraction was determined by Drury’s 1121 modification of the orcinol reaction. A 75-fold reduction in the volume used by Drury permitted the measurement of from 0.37 to 1.5 pg of RNA. Yeast RNA from Schwarz was used as a standard. The ribose reaction carried out on the cold PCA hydrolyzate of RNA gave an amber color indicating interference from protein and amino acids. If the hydrolyzate was previously extracted with chloroform, these interfering substances were removed. Experimental
Cell Research
21
Localization
of RNA
in the llyanassa
129
egg
RESULTS
The ultraviolet absorption curve, Fig. 1, of the RSA separated by the Ogur-Rosen procedure from the Zlynntrsscr egg has a typical nucleic acid absorption peak at 260 m/d \vith minimum absorption at ‘230 m,u. The lox\ point at 230 rnp and the absence of an\r inflection at 280 rnp indicates a lo\\- lewl of protein contaminants. The 260/280 optical density ratio of 1.51 also indicates that the RNA fraction is moderately free from protein. Estraction of the RSA h~cirol~zate with chloroform gave a slightly larger ratio; halvevtr, this additional step often resulted in loss of material anti was not used routinely-. There is no significant clifkrence in the amount of RNA per egg (Table I) for the hydrolysis times of 18, 3‘2, and 40 hours. This indicates that h~drol~sis for 18 hours gives a maximal separation of RX-4 from the l>XA. However, the coefficient of variation for the RXh content increases with longer hydrolysis times, 8.6 per cent for 18 hours, 11.7 per cent for 32, anti 18.4 per cent for 10 hours. This suggests that longer periods of hytlrol~sis tend to h;vclrolyze some DNA, though not enough to alter significantly the determination of RSA. Also, using so fe\v cells it is not espected that the amount of l)SA present would interfere with the RNA determinations. Fig. 2 shows the correlation hetjvcen the absorption at 260 mp and the
0.01 220
. 240
260 Wovelength-
Fig. Fig.
I.--Ultraviolet
Fig.
Z.--Optical
9 - 60173254
5
0.0001
300
of RNA
* a 4,
4co
6CO
Number of Eggs
1.
absorption density
t 280 mp
Fig. curve
of the RNA
versus
number
2.
fraction. of eggs. Experimenlcl
Cell Research
21
130
J. R. Collier
number of eggs used. Table II summarizes the results obtained for the RN\‘A content of the egg as calculated from the optical density at 260 m,u phosphorus and ribose determinations. The agreement between the amounts calculated from the ultraviolet absorption and phosphorus determination show that the lipid and acid-soluble phosphorus have been adequately separated from the RNA fraction. The RNB content calculated from the ribose determinaTABLE
I. Relation of hydrolysis time (1 N PCA, 4°C) to RiVA content. 18hours 4.40+0.13
32 hours
40 hours
4.6OiO.32
4.30+0.18
II. RLVA content
TABLE
of the
Ilyanassa egg.
,ugx IO-3 per egg. Number of eggs per determination
4.50 4.00 4.70 4.70 4.00 4.10 4.30 4.80 4.50 -
200 200 400 400 400 600 600 200 200 200 200 Mean,
Calculated from D,,,
4.4OkO.13
S.E.
Calculated from P content
Calculated ribose
from
4.90
4.30 4.00 4.00 4.3050.27
6.60 6.40 7.40 6.80 6.80+0.20
Cons was consistently higher than those obtained from the ultraviolet absorption and phosphorus determinations; these high values probably resulted from the incomplete removal of interfering substances from the RNA hydrolyzate. The RNA content of the non-yolk cytoplasm was determined by removing the yolk platelets from the egg homogenate as described above. The recovery of all of the RNA in the yolk-free cytoplasm (Table III) indicates that the yolk platelets do not contain any RKA. Analysis of the isolated yolk platelets confirmed the absence of RN,4 in this fraction. Experimental
Cell Research
21
Localization
of RNA
in the Ilyanassa
131
egg
Table IV summarizes the data for the RNA content of the egg, AB and CD blastomeres, the loheless egg and the third polar lobe. The values for the RNA given in Table IV are somewhat higher than those previously reported, Collier [9]; however, the percentage distribution of RNA to the blastomeres is essentially the same. This difference may be in part a result of the variability of the biological material from one year to the next, but chiefly it results TABLE
III.
RNA content of the non-yolk cytoplasm. ,ug RNA
Whole
x 1O-3. Yolk-free cytoplasm
egg
4.50 4.50 4.4OkO.13
5.10
II=30
4.50 4.30 3.80
Mean,
TABLE
S.E.
4.40t0.1s
IV. RNA content of the egg, polar lobe, lobelessegg, AR and CD blastomeres. ,ug RNA AB blastomerc
4.40-to.13
n= 30
Mean,
S.E.
x 10-3.
CD blastomere
Lobeless egg
Polar lobe
1.70
2.30
3.90
0.50
2.20
2.60
3.40
1.00
2.00
2.40
4.00
0.40
2.00
2.90
2.00 1.80
2.70
2.20
2.70
1.90
2.20
2.10
2.40
3.so+o.20
O.SOi.O.20
2.50
2.10
2.70
2.00
2.70
2.00~0.05
2.50
F0.07
Experimental
Cell Research
21
132
J. K. Collier
from significant improvements in the technique of analysis as discussed above. The coefficient of variation for the RNA content of each of the isolates studied ranged betkveen 7.7 and 8.8 per cent; a comparison of these values with the coefficient of varitaion of 8.8 per cent for the RNA content of the lvhole egg indicates that there jvcre no additional errors, e.g. cvtolvsis of ~ . isolates, introduced by the process of hlastomere isolation. The values for ‘J’aBLl<
I’.
Concentration of RNA in the egg cud blastomeres. pg RNA. Per pl nonyolk cytoplasm
Egg
2.97 Ifr O.loa 3.28 I!Z 0.09 2.88 kO.15 3.02&0.15 2.73 k 0.87
AB blastomere CD blastomere Lobeless egg Polar lobe a All activity.
standard
Per pl total volume
errors
represent
the
composite
Per unit of dipeptitlase activity x 1OF
2.02 i 0.08 2.63 + 0.09 l.SS-tO.07 2.25 i 0.12 1.22 -t 0.22 errors
of the
HN4,
1.33 F 0.06 1.82+0.17 1.17+0.06 1.31 i 0.08 1.20 i 0.14 volume,
or tlipeptitlasc
the lobeless egg and the polar lobe are given only as estimates because of the relatively few determinations made on these isolates. The RNA content of the polar lobe was obtained by subtracting the RNA contained in the lobeless egg from that of the whole egg. The Cl> blastomere contains 20.0 per cent more RNA than the A13 blastomere, and this difference is significant at the 1 per cent level. However, the volume of the CD blastomere is 89.-f per cent greater than the volume of the AR hlastomere, and this size difference prevents a direct comparison of these cells from the vielvpoint of concentration of materials. Further, the CD cell has proportionatley more yolk than the AR blastomere. In Table 1’ the concentration of RNA in the egg and isolates is calculated on the basis of the total volume, volume of non-yolk cytoplasm, and dipeptidase activity. From the data in Table III it has been sho\vn that the yolk does not contain any RNA; therefore, the volume of non-yolk cytoplasm is a better reference unit than total volume for estimating the concentration of RN-4 per cell. The values for the volume of non-yolk cytoplasm and the dipeptidase activity were taken from previous work [8]. The dipeptidasc Experimental
Cell
Research
21
Localization
of RNA
in the Ilyanassa
egg
133
activity is included as a reference unit on the basis of the conclusion from earlier work, Collier [t(], that the distribution of the dipeptidase activity in early cleavage is proportional to the volume of hyaline protoplasm. Comparing only the concentration in the egg, AR and CD blastomeres, it is seen that in the case of all three reference units the concentration of RNA is greater in the AB blastomere and that the concentration in the CD blastomerc is slightly less than in the egg. ‘The differences in concentration between the AR and CD blastomcres are significant at the 1 per cent level for each reference unit used in calculating the concentrations. \I’hilc the data given for the RNA content of the lobeless egg and the polar lobe represent only an estimate because of the small number of determinations, it should be noted that the concentration of RNA in the polar lobe is approximately the same as the concentration in the CD blastomere. The discrepancy in the concentration of RSA, calculated on the basis of total volume. of the Cl) blastomcre and the polar lobe probably results from the fact that the yolk platelets occupy 5j.l per cent of the polar lobe volume and only 35 per cent of the volume of the CD cell. ,4lso the concentration of RNA in the lobeless egg is less than that of the AI3 blastomere, which is an expected result because part of the CD cytoplasm contributed one half of the cytoplasmic composition of the lobelcss egg. DISCUSSION
The yolk platelets of the Ilymnssrz egg do not contain RNA; however, the presence of RNA in the yolk granules of the egg of Limnnen stfrgnnlis has been reported from c;vtorhemical observations by Raven [TM]. Using chemical methods Cleveland [1] found RNA in the “protein yolk” granules of the egg of Ostrtw cwminer&lis. Cleland points out, however, that it is difficult to consider the “protein granules” of the oyster egg as classical yolk granules because of their enzymatic activity anti functional resemblance to mitochondria. The observation of Raven in I,imnrrecr may have been due to a shell of RNA granules adhering to the surface as has been reported by Rerthier [2] for the yolk granules in the Planorbis egg. It is possible that the particulate yolk materials in other molluscan eggs have more than a passive role of storage; ho\vcver, the yolk platelets of Ilytrnnsm appear, on the basis of the absence of RS;\ and proteolytic enzymes, Collier [8], to serve primarily as a site for the storage of materials. Berg [l j found that after the second cleavage of the egg of Cionrr intestinrrlis that there \vas a slight localization of RNA in the posterior cells. These results
134
J. R. Collier
are different from the situation found for the Ilyannssn egg as it is the anterior or AB blastomere which contains the greater concentration of RNA. Berg found that the difference in RNA content between the anterior and posterior blastomeres was not due to a difference in the total volume of the two sets of cells, and that they both had the same protein content. However, he did not consider the distribution of the formed particles to each blastomere and their relation to the volume of the anterior and posterior blastomeres. Whether the results of these two studies represent a basic difference bctwen the molluscan and the acidian eggs cannot be decided from the two forms studied. Working tvith an annelid Raven [21] applied cytochemical methods to a study of the egg of Sabellrrris rrloeolntcr, and he found that there were no peculiarities in the distribution of RNA which could be related to the determination of the cells. From the analysis of the anterior and posterior cells of the Ilyantrssn egg it is seen that the distribution of RNA follows an anterior-posterior gradient and that there is no correlation bet\veen the concentration of RNA and the segregation of the mesodermal components in the CD blastomere. However, it is necessary to study other cells and older embryos before the existence of a gradient can be firmly established. In general, no clear cases of a gradient, in the sense of Child’s axial gradients, have been found in the early cleavage stages of the mosaic eggs. Strelin [24], working with Nasscc reticulate, found that the D macromere reduced oxidation-reduction indicators at a slower rate than other sections of the embryo; however, he found that the polar lobe and the D macromere were more sensitive to cytolytic agents than other cells in the embryo. From the lack of correlation between these t\vo methods of observation Strelin concluded that no axial gradients are detectable during embryonogenesis of the molluscan egg. The lower concentration of RNA in the polar lobe and CD blastomere does not rule out the possibility that RNA may be concentrated in the descendants of the CD blastomere. The precision with which oiiplasmic substances are segregated suggests that a significantly large percentage of the RNA of the CD blatomere could be transferred to one or more micromeres with the result that a marked concentration of RNA would occur in these smaller cells. That such a localization of some materials does occur is shown by the results of Clement [6] in which he found that the 4LI macromere could be removed after the formation of the 4 d cell without interfering with the production of a prcfectly normal veliger larva. Another example of a similar type of localization has been described in the Zlyunassa egg by Clement and Lehman [7] in which numerous mitochondria are localized in the mesentoExperimental
Cell Research
21
Localization
of RNA
in the Ilyanassa
egg
135
blast cell and the fourth quartette entoblasts, leaving the 44, I3, C, and D macromeres relatively few mitochondria. Raven [‘LO] has described in Limnnen the transfer of RNA from the 3A-3D macromeres to the fourth quartette micromeres by means of RNA-rich which accumulate in the central ends of the granules, the “ectosomes,” macromeres prior to the formation of the micromeres. hlinganti [17], also working with Linmnen, suggests that protoplasmic bridges connecting the macromeres with the micromeres may be of special importance in transferring materials. The ribonucleoprotein (RKAP) molecule fits the necessary conditions described by Costello [lo] in his theory of oiiplasmic segregation based on the Teorell “diffusion effect.” This mechanism is particularly pertinent to the segregation of RSAP because a large portion of the RNAP in embryonic material is generally in the soluble phase as shown for the frog egg by Brachet [3], the oyster egg by Cleland [4], and for the sea urchin egg by Harvey and Lavin [13]. \\‘hatever the final decision might be concerning the quantitative localization of RSA one disconcerting feature is that d8erentiation is probably more influenced by the presence of specific ribonucleoprotein molecules than by the total amount of RNA per cell. Further, the number of RNAP molecules functional in determining the fate of a cell is probably so low as to be undetectable by present methods. This rather obvious fact suggests the importance of identifying particular molecular species of RNAP in differentiating cells, a task \vhich requires considerable refinement in present methods before they can be successfully applied to small quantities of material.
SUMMARY
Using quantitative less egg, polar lobe,
microchemical methods AB and CD blastomeres
the RSA content was determined
of the egg, lobein the egg of
Ilymassa obsoletn. The concentration of RNA, calculated from three different reference units, was significantly greater in the AB than in the CD blastomere. The CD cell and the polar lobe had approximately the same concentration of RX\‘A. ?;o R?;A was found in the yolk platelets of the Ilynnassn egg. The author wishes to express his appreciation to Marjorie M. Collier for her excellent assistance and especially for her skill in the difficult task of isolating blastomeres, to Mr. T. P. Hawes, Jr. for his very capable assistance during the early part of this work. Appreciation is also extended to Professor G. H. Mickey for his generous help in making available both time and facilities for research. Experimental
Cell
Research
21
J. R. Collier REFERENCES 1%‘. I?., Biol. Hull. 113, 365 (1957). J., Bull. Biol. France et Rely. 82, 61 (1948). 3. RRACIIET. .JEAN, Ann. 1Y. 1.. Acad. Sci. 50, X61 (1950). 4. (:LELAND, K. IV., Australian .J. Exptl. Bid. Med. Sci.‘29, 1.
IkRG,
2.
~JISRTHIER,
5. 6.
7. 8. 9.
10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22. 23. 24.
35
(19.51).
A. C., J. bhptl. ZOO/. 121, 593 (1952). ~ibid. 132, 427 (1956). CLEMENT, A. C. and LEHMAS, F. E., Srcturccrissellschcrften 43, 478 (1956). COLLIER, J. R.. Embryologic 3, 243 (1957). -~ Bid. Bull. 115, 348 (1958). (COSTELLO, I>. P., Ann. S.1’. Acad. Sci. 49, 663 (1948). -~ J. apt/. Zool. 100, 19 (1945). DRUHY, H. F., Arch. Biochem. 19, 455 (1948). HARVEY, E. H. and LAVIN, G. I., Viol. Hull. 86, 163 (1944). KING, E. .J., Biochem. .I. 26, 292 (1932). I.EVEK.BOOK, I,., TIIA~AGLINI, E. (:. and SCHULTZ, .J., Exptl. Cell Resewch I.OWRY, 0. H. and UESSRY, 0. A., J. Bid. Chem. 163, 633 (1946). nfINGANTI, A., Riu. Biol. 42, 295 (1950). OGUR, 31. and ROSES, G., Arch. Biochrm. 25, 262 (1950). PAI-TERSOS, E. K. ant1 DACKERMAX, RI. E., Arch. Hiochem. Biophys. 36, RAVEX, CHR. I’., Arch. SCerl. Zool. 7, 353 (1946). -- Proc. Koninkl. Std. Akcrd. IVetenschop 57, 1 (1950). SCHMIDT, G. and THANSAAUSER, S. .J., J. Hiol. Chem. 161, 83 (1945). SCHNEIDER, I%'. (:., 1. Bid. Chem. 161. 293 (1945). STRELIX, G., C. 13. (JIoklod!y) Acrrrl. Sri. C’. R.S.S. 24, 942 (1939). C:LEMENT.
Experimental
Cell
Research
21
15, 43 (195X).
97
(1952).
Cell Reseurch
OF RIBONUCLEIC
ACID
126
THE
LOCALIZATION EGG
OF ILYANASSA J. R.
Department
of Zoology, Marine
Biological
IN
(1860)
THE
OBSOLETA’
COLLIER2
Louisiana State University, Baton Rouge, Laboratory, Woods Hole, Massachusetts, Received
21, 126-136
October
Louisiana, U.S.A.
and the
2, 1959
this paper the results of a study of biochemical cell-lineage are presented. The hope for this work is to relate the biochemistry of the early blastomeres to the partial embryos which they form when isolated. ‘I’he chemical studies of today arc indebted to the classical cell-lineage studies begun by Conklin, \\‘ilson, and Lillie, and to the analysis of the development of isolated blastomeres which they initiated. ;\lorc recently, the careful studies of Costello [ 1 l] and Clement [5, li] on the differentiation of partial embryos have provided additional information needed for this approach. The role generally attributed to the ribonucleoproteins as a template molecule for protein synthesis centers attention on this nucleic acid as a determining factor in embryonic differentiation. Levcnbook ef al. [15] have correlated differences in the base ratios of th RNA of Drosophila eggs and the chromosomal composition of the egg. The present work is concerned with the distribution of RNA to cells with the same genetic constitution but with a difl’crent capacity for differentiation. n’hilc a study of the quantitative distribution of substances to the early blastomeres is in some ways of limited value, many functions vital to the differentiation of the embryo occur hcforc synthctic mechanisms come into play, and in this sense the initial concentration of substances in a given cell may be important. Berg [l] dealing with the same general problem and working with the egg of Ciona intestinalis, has studied the distribution of RNA to the anterior and posterior blastomeres. In the following experiments the localization of RNA has been determined in the egg, polar lobe, AR and CD blastomeres of the marine snail Z1yann.s.w obsolefn. IN
1 This work was supported in part by a research and Metabolic Diseases of the National Institutes 2 Present address: Marine Biological Laboratory, Experimental
Cell Research
21
grant (A-3554) of Health. Woods Hole,
from
the Division
Massachusetts,
of Arthritis U.S.A.
Localizafion MATERIALS Biological
of RNA
in the Ilyanassa AND
127
egg
METHODS
Material
obsoleta eggs were obtained from snails kept in an aquarium of sea water. The snails were fed on alternate days and an ample supply of freshly deposited egg capsules were collected daily. Each capsule contained from 80 to 120 eggs. The eggs were removed from the capsule and washed in pasteurized sea water; blastomeres and polar lobes were isolated by agitation in calcium-low sea water as described previously 181. The required number of cells, 200 eggs or 400 isolated blastomeres, were counted and transferred in 30~1 of sea water to a small glass vessel. The cells were homogenized by introducing a small vibrating glass rod into the drop of sea water containing the eggs. In the analysis of the yolk and cytoplasmic fractions the yolk platelets were removed from the homogenate by centrifugation in sea water at a very low speed for 30 seconds. The supernatant was withdrawn, and the yolk platelets were washed three times with 50 microliters of pasteurized sea water. The washes were combined with the original supernatant to make up the cytoplasmic fraction. Zlyanassa
General
Procedure
The chemical analysis of isolated blastomeres required the development of micromethods capable of separating and detecting amounts of material of the order of onehalf to one microgram. Several general procedures for chemical fractionation were tried and found unsuccessful with this material before the final adoption, with modification, of the Ogur and Rosen [18] procedure. The Schmidt-Thannhauser [22] scheme was unsatisfactory because the separation of the ribonucleic acid (RNA) from protein and deoxyribonucleic acid (DNA) was incomplete following alkaline hydrolysis of the RNA. Neither extraction of the protein from the hydrolyzate with chloroform or precipitation of DNA in the presence of added egg albumin was satisfactory in overcoming this difficulty. The Schneider [23] scheme for tissue fractionation has been adapted to a micro level by Patterson and Dackerman [19]. This scheme was not satisfactory because the calorimetric methods for detection of ribose could not be used with the Zlyanassa egg; however, the general technique of Patterson and Dackerman [lo] was valuable in working out the procedures finally adopted. Vsing a modified Ogur-Rosen [lg] fractionation scheme it was possible to separate and quantitate the following tissue fractions: (1) acid-soluble components, (2) lipids, (3) RNA, (4) DNA, and (5) protein. Homogenization and extraction:-All cells were transferred in 30 to 50 ,~l of sea water to a small centrifuge tube (3 x 36 mm) and homogenized by introducing a small vibrating glass rod. All homogenization was done in an ice bath at 2 to 4 degrees C. Originally all extractions were carried out in these centrifuge tubes, and the supernatant was withdrawn after centrifuging the precipitated protein. Later, it was found that the homogenate could be transferred to a depression in a Pyrex spot plate and that after heat drying at 85-90°C for 15 minutes the protein would stick to the slide; thus, all subsequent extractions could be carried out on the spot plates. In Experimental
Cell ICesearch
21
J. I?. Collier extractions requiring heat the depressions were covered with a glass slide, and the ,depression plate was placed in an oven at the required temperature. The same results were obtained with both procedures of extraction, and the technique using the spot plate was ultimately adopted because it was much less laborious and resulted in smaller losses of material. Acid-soluble fraction.-The homogenate was cooled to 4°C and washed three times, for 5 minutes each wash, with 100 microliters of 0.2 N perchloric acid (PCA), and all three washes were collected quantitatively and stored in a small glass vessel. These washes constituted the acid-soluble fraction, and after determining the ultraviolet absorption of this fraction, the phosphorus content was determined. When the washes were collected and analyzed separately, 99.0 per cent of the total acid-soluble phosphorus was removed in the first two washes. Lipid fraction.-After the removal of the acid-soluble fraction, lipids were extracted by two washes with ethanol-ether (3: 1) for 15 minutes at 35-40°C followed by one chloroform extraction for 15 minutes at 35540°C. These extractions were combined as the lipid fraction. Analysis of separate ethanol-ether extracts showed that 96 per cent of the lipid phosphorus was removed in the first wash. Only 3.8 per cent of the lipid phosphorus was found in the chloroform extract. Extraction of RNA.-The RNA was removed from the precipitated protein and DNA by hydrolyzing the RNA with 100 ~1 of 1 N PCA for 15 hours at 4°C. After hydrolysis the residue was washed with an additional 90 ~1 of 1 N PCA, and the extract and wash were combined and diluted to a volume of 200 ,ul. The amount of RNA was ‘determined from the optical density at 260 rnp and phosphorous content as described below. All determinations of RNA were calculated from the optical density at 260 rnp except in those cases where other methods of analysis are specifically indicated. After removal of the RNA, the DNA was extracted with hot 0.5 N PCA, and the protein residue was solubilized in 1 N NaOH. Results on these fractions are not included in this paper, and the methods for analysis are omitted. Analytical
Procedure
SpecfrophofomeQ-All samples for spectrophotometry were diluted to 200 ~1 in small calibrated vessels. Aliquots were transferred to Lowry and Bessey [16] microcells, and the optical density was determined with a Beckman DU spectrophotometer. The coefficient of variation for the estimation of RNA from the optical density at 260 rnp was 8.6 per cent. Phosphorus deferminafions.-Phosphorus was determined by Patterson and Dackerman’s [19] micro adaptation of King’s [14] modifications of the Fiske and Subbarow method for phosphorus analysis. The range of phosphorus determined was from 0.051.2 pg per sample. Ribose determinations.-The ribose content of the RNA fraction was determined by Drury’s 1121 modification of the orcinol reaction. A 75-fold reduction in the volume used by Drury permitted the measurement of from 0.37 to 1.5 pg of RNA. Yeast RNA from Schwarz was used as a standard. The ribose reaction carried out on the cold PCA hydrolyzate of RNA gave an amber color indicating interference from protein and amino acids. If the hydrolyzate was previously extracted with chloroform, these interfering substances were removed. Experimental
Cell Research
21
Localization
of RNA
in the llyanassa
129
egg
RESULTS
The ultraviolet absorption curve, Fig. 1, of the RSA separated by the Ogur-Rosen procedure from the Zlynntrsscr egg has a typical nucleic acid absorption peak at 260 m/d \vith minimum absorption at ‘230 m,u. The lox\ point at 230 rnp and the absence of an\r inflection at 280 rnp indicates a lo\\- lewl of protein contaminants. The 260/280 optical density ratio of 1.51 also indicates that the RNA fraction is moderately free from protein. Estraction of the RSA h~cirol~zate with chloroform gave a slightly larger ratio; halvevtr, this additional step often resulted in loss of material anti was not used routinely-. There is no significant clifkrence in the amount of RNA per egg (Table I) for the hydrolysis times of 18, 3‘2, and 40 hours. This indicates that h~drol~sis for 18 hours gives a maximal separation of RX-4 from the l>XA. However, the coefficient of variation for the RXh content increases with longer hydrolysis times, 8.6 per cent for 18 hours, 11.7 per cent for 32, anti 18.4 per cent for 10 hours. This suggests that longer periods of hytlrol~sis tend to h;vclrolyze some DNA, though not enough to alter significantly the determination of RSA. Also, using so fe\v cells it is not espected that the amount of l)SA present would interfere with the RNA determinations. Fig. 2 shows the correlation hetjvcen the absorption at 260 mp and the
0.01 220
. 240
260 Wovelength-
Fig. Fig.
I.--Ultraviolet
Fig.
Z.--Optical
9 - 60173254
5
0.0001
300
of RNA
* a 4,
4co
6CO
Number of Eggs
1.
absorption density
t 280 mp
Fig. curve
of the RNA
versus
number
2.
fraction. of eggs. Experimenlcl
Cell Research
21
130
J. R. Collier
number of eggs used. Table II summarizes the results obtained for the RN\‘A content of the egg as calculated from the optical density at 260 m,u phosphorus and ribose determinations. The agreement between the amounts calculated from the ultraviolet absorption and phosphorus determination show that the lipid and acid-soluble phosphorus have been adequately separated from the RNA fraction. The RNB content calculated from the ribose determinaTABLE
I. Relation of hydrolysis time (1 N PCA, 4°C) to RiVA content. 18hours 4.40+0.13
32 hours
40 hours
4.6OiO.32
4.30+0.18
II. RLVA content
TABLE
of the
Ilyanassa egg.
,ugx IO-3 per egg. Number of eggs per determination
4.50 4.00 4.70 4.70 4.00 4.10 4.30 4.80 4.50 -
200 200 400 400 400 600 600 200 200 200 200 Mean,
Calculated from D,,,
4.4OkO.13
S.E.
Calculated from P content
Calculated ribose
from
4.90
4.30 4.00 4.00 4.3050.27
6.60 6.40 7.40 6.80 6.80+0.20
Cons was consistently higher than those obtained from the ultraviolet absorption and phosphorus determinations; these high values probably resulted from the incomplete removal of interfering substances from the RNA hydrolyzate. The RNA content of the non-yolk cytoplasm was determined by removing the yolk platelets from the egg homogenate as described above. The recovery of all of the RNA in the yolk-free cytoplasm (Table III) indicates that the yolk platelets do not contain any RKA. Analysis of the isolated yolk platelets confirmed the absence of RN,4 in this fraction. Experimental
Cell Research
21
Localization
of RNA
in the Ilyanassa
131
egg
Table IV summarizes the data for the RNA content of the egg, AB and CD blastomeres, the loheless egg and the third polar lobe. The values for the RNA given in Table IV are somewhat higher than those previously reported, Collier [9]; however, the percentage distribution of RNA to the blastomeres is essentially the same. This difference may be in part a result of the variability of the biological material from one year to the next, but chiefly it results TABLE
III.
RNA content of the non-yolk cytoplasm. ,ug RNA
Whole
x 1O-3. Yolk-free cytoplasm
egg
4.50 4.50 4.4OkO.13
5.10
II=30
4.50 4.30 3.80
Mean,
TABLE
S.E.
4.40t0.1s
IV. RNA content of the egg, polar lobe, lobelessegg, AR and CD blastomeres. ,ug RNA AB blastomerc
4.40-to.13
n= 30
Mean,
S.E.
x 10-3.
CD blastomere
Lobeless egg
Polar lobe
1.70
2.30
3.90
0.50
2.20
2.60
3.40
1.00
2.00
2.40
4.00
0.40
2.00
2.90
2.00 1.80
2.70
2.20
2.70
1.90
2.20
2.10
2.40
3.so+o.20
O.SOi.O.20
2.50
2.10
2.70
2.00
2.70
2.00~0.05
2.50
F0.07
Experimental
Cell Research
21
132
J. K. Collier
from significant improvements in the technique of analysis as discussed above. The coefficient of variation for the RNA content of each of the isolates studied ranged betkveen 7.7 and 8.8 per cent; a comparison of these values with the coefficient of varitaion of 8.8 per cent for the RNA content of the lvhole egg indicates that there jvcre no additional errors, e.g. cvtolvsis of ~ . isolates, introduced by the process of hlastomere isolation. The values for ‘J’aBLl<
I’.
Concentration of RNA in the egg cud blastomeres. pg RNA. Per pl nonyolk cytoplasm
Egg
2.97 Ifr O.loa 3.28 I!Z 0.09 2.88 kO.15 3.02&0.15 2.73 k 0.87
AB blastomere CD blastomere Lobeless egg Polar lobe a All activity.
standard
Per pl total volume
errors
represent
the
composite
Per unit of dipeptitlase activity x 1OF
2.02 i 0.08 2.63 + 0.09 l.SS-tO.07 2.25 i 0.12 1.22 -t 0.22 errors
of the
HN4,
1.33 F 0.06 1.82+0.17 1.17+0.06 1.31 i 0.08 1.20 i 0.14 volume,
or tlipeptitlasc
the lobeless egg and the polar lobe are given only as estimates because of the relatively few determinations made on these isolates. The RNA content of the polar lobe was obtained by subtracting the RNA contained in the lobeless egg from that of the whole egg. The Cl> blastomere contains 20.0 per cent more RNA than the A13 blastomere, and this difference is significant at the 1 per cent level. However, the volume of the CD blastomere is 89.-f per cent greater than the volume of the AR hlastomere, and this size difference prevents a direct comparison of these cells from the vielvpoint of concentration of materials. Further, the CD cell has proportionatley more yolk than the AR blastomere. In Table 1’ the concentration of RNA in the egg and isolates is calculated on the basis of the total volume, volume of non-yolk cytoplasm, and dipeptidase activity. From the data in Table III it has been sho\vn that the yolk does not contain any RNA; therefore, the volume of non-yolk cytoplasm is a better reference unit than total volume for estimating the concentration of RN-4 per cell. The values for the volume of non-yolk cytoplasm and the dipeptidase activity were taken from previous work [8]. The dipeptidasc Experimental
Cell
Research
21
Localization
of RNA
in the Ilyanassa
egg
133
activity is included as a reference unit on the basis of the conclusion from earlier work, Collier [t(], that the distribution of the dipeptidase activity in early cleavage is proportional to the volume of hyaline protoplasm. Comparing only the concentration in the egg, AR and CD blastomeres, it is seen that in the case of all three reference units the concentration of RNA is greater in the AB blastomere and that the concentration in the CD blastomerc is slightly less than in the egg. ‘The differences in concentration between the AR and CD blastomcres are significant at the 1 per cent level for each reference unit used in calculating the concentrations. \I’hilc the data given for the RNA content of the lobeless egg and the polar lobe represent only an estimate because of the small number of determinations, it should be noted that the concentration of RNA in the polar lobe is approximately the same as the concentration in the CD blastomere. The discrepancy in the concentration of RSA, calculated on the basis of total volume. of the Cl) blastomcre and the polar lobe probably results from the fact that the yolk platelets occupy 5j.l per cent of the polar lobe volume and only 35 per cent of the volume of the CD cell. ,4lso the concentration of RNA in the lobeless egg is less than that of the AI3 blastomere, which is an expected result because part of the CD cytoplasm contributed one half of the cytoplasmic composition of the lobelcss egg. DISCUSSION
The yolk platelets of the Ilymnssrz egg do not contain RNA; however, the presence of RNA in the yolk granules of the egg of Limnnen stfrgnnlis has been reported from c;vtorhemical observations by Raven [TM]. Using chemical methods Cleveland [1] found RNA in the “protein yolk” granules of the egg of Ostrtw cwminer&lis. Cleland points out, however, that it is difficult to consider the “protein granules” of the oyster egg as classical yolk granules because of their enzymatic activity anti functional resemblance to mitochondria. The observation of Raven in I,imnrrecr may have been due to a shell of RNA granules adhering to the surface as has been reported by Rerthier [2] for the yolk granules in the Planorbis egg. It is possible that the particulate yolk materials in other molluscan eggs have more than a passive role of storage; ho\vcver, the yolk platelets of Ilytrnnsm appear, on the basis of the absence of RS;\ and proteolytic enzymes, Collier [8], to serve primarily as a site for the storage of materials. Berg [l j found that after the second cleavage of the egg of Cionrr intestinrrlis that there \vas a slight localization of RNA in the posterior cells. These results
134
J. R. Collier
are different from the situation found for the Ilyannssn egg as it is the anterior or AB blastomere which contains the greater concentration of RNA. Berg found that the difference in RNA content between the anterior and posterior blastomeres was not due to a difference in the total volume of the two sets of cells, and that they both had the same protein content. However, he did not consider the distribution of the formed particles to each blastomere and their relation to the volume of the anterior and posterior blastomeres. Whether the results of these two studies represent a basic difference bctwen the molluscan and the acidian eggs cannot be decided from the two forms studied. Working tvith an annelid Raven [21] applied cytochemical methods to a study of the egg of Sabellrrris rrloeolntcr, and he found that there were no peculiarities in the distribution of RNA which could be related to the determination of the cells. From the analysis of the anterior and posterior cells of the Ilyantrssn egg it is seen that the distribution of RNA follows an anterior-posterior gradient and that there is no correlation bet\veen the concentration of RNA and the segregation of the mesodermal components in the CD blastomere. However, it is necessary to study other cells and older embryos before the existence of a gradient can be firmly established. In general, no clear cases of a gradient, in the sense of Child’s axial gradients, have been found in the early cleavage stages of the mosaic eggs. Strelin [24], working with Nasscc reticulate, found that the D macromere reduced oxidation-reduction indicators at a slower rate than other sections of the embryo; however, he found that the polar lobe and the D macromere were more sensitive to cytolytic agents than other cells in the embryo. From the lack of correlation between these t\vo methods of observation Strelin concluded that no axial gradients are detectable during embryonogenesis of the molluscan egg. The lower concentration of RNA in the polar lobe and CD blastomere does not rule out the possibility that RNA may be concentrated in the descendants of the CD blastomere. The precision with which oiiplasmic substances are segregated suggests that a significantly large percentage of the RNA of the CD blatomere could be transferred to one or more micromeres with the result that a marked concentration of RNA would occur in these smaller cells. That such a localization of some materials does occur is shown by the results of Clement [6] in which he found that the 4LI macromere could be removed after the formation of the 4 d cell without interfering with the production of a prcfectly normal veliger larva. Another example of a similar type of localization has been described in the Zlyunassa egg by Clement and Lehman [7] in which numerous mitochondria are localized in the mesentoExperimental
Cell Research
21
Localization
of RNA
in the Ilyanassa
egg
135
blast cell and the fourth quartette entoblasts, leaving the 44, I3, C, and D macromeres relatively few mitochondria. Raven [‘LO] has described in Limnnen the transfer of RNA from the 3A-3D macromeres to the fourth quartette micromeres by means of RNA-rich which accumulate in the central ends of the granules, the “ectosomes,” macromeres prior to the formation of the micromeres. hlinganti [17], also working with Linmnen, suggests that protoplasmic bridges connecting the macromeres with the micromeres may be of special importance in transferring materials. The ribonucleoprotein (RKAP) molecule fits the necessary conditions described by Costello [lo] in his theory of oiiplasmic segregation based on the Teorell “diffusion effect.” This mechanism is particularly pertinent to the segregation of RSAP because a large portion of the RNAP in embryonic material is generally in the soluble phase as shown for the frog egg by Brachet [3], the oyster egg by Cleland [4], and for the sea urchin egg by Harvey and Lavin [13]. \\‘hatever the final decision might be concerning the quantitative localization of RSA one disconcerting feature is that d8erentiation is probably more influenced by the presence of specific ribonucleoprotein molecules than by the total amount of RNA per cell. Further, the number of RNAP molecules functional in determining the fate of a cell is probably so low as to be undetectable by present methods. This rather obvious fact suggests the importance of identifying particular molecular species of RNAP in differentiating cells, a task \vhich requires considerable refinement in present methods before they can be successfully applied to small quantities of material.
SUMMARY
Using quantitative less egg, polar lobe,
microchemical methods AB and CD blastomeres
the RSA content was determined
of the egg, lobein the egg of
Ilymassa obsoletn. The concentration of RNA, calculated from three different reference units, was significantly greater in the AB than in the CD blastomere. The CD cell and the polar lobe had approximately the same concentration of RX\‘A. ?;o R?;A was found in the yolk platelets of the Ilynnassn egg. The author wishes to express his appreciation to Marjorie M. Collier for her excellent assistance and especially for her skill in the difficult task of isolating blastomeres, to Mr. T. P. Hawes, Jr. for his very capable assistance during the early part of this work. Appreciation is also extended to Professor G. H. Mickey for his generous help in making available both time and facilities for research. Experimental
Cell
Research
21
J. R. Collier REFERENCES 1%‘. I?., Biol. Hull. 113, 365 (1957). J., Bull. Biol. France et Rely. 82, 61 (1948). 3. RRACIIET. .JEAN, Ann. 1Y. 1.. Acad. Sci. 50, X61 (1950). 4. (:LELAND, K. IV., Australian .J. Exptl. Bid. Med. Sci.‘29, 1.
IkRG,
2.
~JISRTHIER,
5. 6.
7. 8. 9.
10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22. 23. 24.
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(19.51).
A. C., J. bhptl. ZOO/. 121, 593 (1952). ~ibid. 132, 427 (1956). CLEMENT, A. C. and LEHMAS, F. E., Srcturccrissellschcrften 43, 478 (1956). COLLIER, J. R.. Embryologic 3, 243 (1957). -~ Bid. Bull. 115, 348 (1958). (COSTELLO, I>. P., Ann. S.1’. Acad. Sci. 49, 663 (1948). -~ J. apt/. Zool. 100, 19 (1945). DRUHY, H. F., Arch. Biochem. 19, 455 (1948). HARVEY, E. H. and LAVIN, G. I., Viol. Hull. 86, 163 (1944). KING, E. .J., Biochem. .I. 26, 292 (1932). I.EVEK.BOOK, I,., TIIA~AGLINI, E. (:. and SCHULTZ, .J., Exptl. Cell Resewch I.OWRY, 0. H. and UESSRY, 0. A., J. Bid. Chem. 163, 633 (1946). nfINGANTI, A., Riu. Biol. 42, 295 (1950). OGUR, 31. and ROSES, G., Arch. Biochrm. 25, 262 (1950). PAI-TERSOS, E. K. ant1 DACKERMAX, RI. E., Arch. Hiochem. Biophys. 36, RAVEX, CHR. I’., Arch. SCerl. Zool. 7, 353 (1946). -- Proc. Koninkl. Std. Akcrd. IVetenschop 57, 1 (1950). SCHMIDT, G. and THANSAAUSER, S. .J., J. Hiol. Chem. 161, 83 (1945). SCHNEIDER, I%'. (:., 1. Bid. Chem. 161. 293 (1945). STRELIX, G., C. 13. (JIoklod!y) Acrrrl. Sri. C’. R.S.S. 24, 942 (1939). C:LEMENT.
Experimental
Cell
Research
21
15, 43 (195X).
97
(1952).