Nucleic acid synthesis in the normal and lobeless embryo of Ilyanassa obsoleta

NUCLEIC LOBELESS

ACID

SYNTHESIS EMBRYO

IN

THE

OF ILYANASSA

NORMAL

AND

OBSOLETA

J. R. COLLIER

SUMMARY The RNA and DNA content of the normal and measured at several stages ofembryogenesis. In the of RNA and a decrease in the rate of DNA synthesis some of the lobe dependent organs to differentiate of determined embryonic ceils.

The failure of normal embryogenesis to occur in many spiralian eggs following the removal of a portion of the cytoplasm is a classical observation of embryology. Following the early experiments of Crampton [I] with the prosobranch gastropod Ilyrrnassa, cytoplasmic deletion experiments have been extensively studied by Wilson [2], Costello [3], Clement [4, 5, 61 and Verdonk [7-lo]. Clement’s detailed and careful deletion and blastomere isolation experiments with ZIyanassa have clearly established that the removal of the polar lobe, a pre-cleavage protrusion of vegetal cytoplasm, results in the formation of a partial embryo that lacks axial organization and most of the normal organs of the veliger larva. Atkinson [I I] has contributed a valuable cytological study of the lobeless embryo of Zlyanassa, and Cather [12] and Clement [13] have reviewed the experimental embryology of spiralian embryos. Berg & Kato [14] have found an enrichExptl Cdl Rr.r 95

(1975)

lobeless embryo of the I/ynussu embryo was lobeless embryo a delay in the net accumulation were observed. It is suggested that the failure of may be caused by a reduced rate of proliferation

ment of nucleic acid precursors in the polar lobe of Ilyanassa; Collier [15] reported a similar concentration of acid soluble phosphorus in the polar lobe. From these observations on nucleic acid precursors arises the question of whether the lobeless embryo has the capability to synthesize and accumulate nucleic acids. Davidson’s [16] observations on the incorporation of uridine into RNA established that some RNA synthesis occurs in both the normal and lobeless embryo, but they do not pertain to the synthesis and accumulation of the bulk of the cellular RNA. While preliminary reports on this point have been made previously [17, 181 and the multicellular nature of the partial embryo shows a capability for DNA synthesis, the data presented in this paper provide a quantitative answer to this question. These data are for total RNA and DNA content and as such do not bear on the question of dRNA transcription.

Nucleic acid synthesis in the Ilyanassa embryo

255

operations were performed. Prior to extraction and determination of nucleic acid content embryos were stored frozen at -40°C.

Extraction

of nucleic acids

Embryos were homogenized with a rotating glass rod driven by a Moto-Tool (Dremel, Inc.) and extracted three times with 0.2 ml of 0.2 N cold perchloric acid (PCA). The acid precipitated homogenate was defatted by two ethanol-ether (3 : I) extractions at 4O”C, dried and extracted with 0.5 N PCA at 85°C for I5 min. The hot PCA extract was then quantitated for RNA and DNA as described below. (Exceptions to this procedure were made when DNA was determined by fluorescence.) All pellets were collected by centrifuging the homogenate in a refrigerated Sorvall centrifuge at I7 000 g for IO min; adapters for the small glass tubes were drilled from lucite or aluminium cylinders cut to tit the SS-34 Sorvall head.

Quantitation 012345678

Fig.

1.

12

days of development at 19°C; ng of nucleic acid/embryo. - ., RNA of normal embryo; - - - ., DNA of normal embryo; O-O, RNA of lobeless embryo: 0- - -0, DNA of lobeless embryo. Nucleic acid synthesis and accumulation during development. Upper panel: Number of cells versus days of development; Lower panel: nucleic acid content versus days of development. Abscissa:

ordinate:

MATERIALS

AND METHODS

Snails were collected from mud flats in the vicinity of Woods Hole, MA, and kept in a tank of recirculating sea water. The snails were given a raw clam for food on alternate days and egg capsules deposited on the sides of the glass tank were collected, staged and reared at 19°C in pasteurized sea water (PSW). Polar lobes were isolated by removing the eggs from the capsule and incubating them in a mixture of 80 % of Van’t Hoff’s calcium-magnesium free sea water and 20% PSW. No osmotic compensation was made in preparing the artificial sea water, but it was buffered to a pH of 8.2 with 0.05 M Tris buffer. As the eggs reached the peak of the trefoil stage, i.e. the point of maximal separation of the polar lobe from the egg, they were sucked into a capillary pipet (operated by a mouth-hose) and gently expelled onto the bottom of the dish. This operation was repeated two or three times until the polar lobes separated from the egg. Generally, IO to I5 eggs from a capsule of 80 to 120 eggs reached the peak of trefoil simultaneously. Immediately after separation of the polar lobe the lobeless eggs were transferred to a dish of PSW containing 0.5 mg each of penicillin and streptomycin G. Eggs or embryos were counted and placed into a small glass tube (4x34 mm) in which all subsequent

of nucleic acids

The total nucleic acid content of the hot PCA extract was measured by its absorbance at 260 nm (I pg/ml of hydrolyzed nucleic acid in a I cm light path gave an absorbance of 0.030) and the DNA content, which was determinated by Burton’s [19] modification of the diphenylamine reaction, was subtracted from the total nucleic acid value to obtain the RNA content. In one series of measurements Ceriotti’s [20] modification of the orcinol reaction was also used to measure the RNA content of the hot PCA extract. In all cases the final reaction volume was either 0. I or0.2 ml, and the optical density was measured in spectrophotometric microcells. The DNA content was also measured by fluorometry using the diaminobenzoic acid reaction with DNA f21, 221. ]n this determination the acid washed pellet was extracted once with ethanol-ether at 60°C for 15 min, once with absolute ethanol at room temperature, air dried and reacted with freshly prepared diaminobenzoic acid. Calf thymus DNA was used as standards and fluorescence was measured with a Turner fluorometer. Although the results obtained by different methods for both RNA (absorbancy at 260 nm and the orcinol reaction) and DNA (diphenylamine and diaminobenzoic acid) were not significantly different, a complete series of DNA determinations were made by fluorometry for both normal and lobeless embryos.

RESULTS The mature ovarian egg of Zlyanassa contains 0.432kO.038 (S.E.) ng of DNA [35]. This value has been confirmed using the fluorometric DNA assay described above. The germinal vesicle of the ovarian egg is in the diplotene stage of the first meiotic division and therefore has a 4 N compleExptl

Cell Res 95 (1975)

256

J. R. Collier

Fig. 2. Developmental stages of I/ycrnasscr. Sketches were drawn from sections of embryos reared at 19°C; numbers l-7 are days of development. The animal pole or dorsal side is toward the top of the page, and for stages 3-7 the anterior end of the embryo is to the right. Abbreviations: e, eye; ect, ectoderm; mt, entoderm;

es,

foot; gr, germinal vesicle; h, heart; or inr, intestine; mat, macromere; nucleolus; op, operculum; of, otosh, shell: si, stomodeal insg, shell gland; st, stomach; V, velum; vc, y. yolk. (From Collier, 1965; reprinted with from Academic Press.)

esophagus;S,

hv, head vesicle; i mir, micromere; n, cyst: sd, stomodeum; vagination; velar cilia; permission

ment of chromosomes, which is equivalent 49-cell stage in Limnaeu there is no G 1 to 0.012 ng of DNA. This value is based phase in the cell cycle. The number of cells may be readily on a haploid DNA content of 3.1 pg [23, 26, 35-j. Thus, there is 0.420 ng of non- counted by phase microscopy of the comchromosomal DNA, presumably mitopressed embryo. Cell counts of l-day (34+ chondrial, in the Zlya~a.s~a egg. This value 1.5 cells), 2-day (8Ok1.4 cells) and 3-day has been used to estimate the number of (2Olk8 cells) embryos agree with the cell cells per embryo by subtracting the non- numbers calculated from the DNA content chromosomal DNA content from the total (fig. 1). This agreement supports the use of the 4 N amount of DNA in estimating cell DNA and dividing by twice the diploid number for early stages of development and DNA content which is 12.4 pg. The 4 N DNA content was used to cal- suggests that rapid synthesis of DNA may culate cell number on the assumption that continue up to about 200 cells or for the during early cleavage stages DNA replicafirst three days of development in Zlyation occurs early in the cell cycle and re- nassa. That this pattern of DNA synthesis sults in the 4 N amount of DNA in each probably does not extend beyond this stage cell. DNA replication during telophase has is indicated by the larger discrepancy bebeen demonstrated for the first two cleav- tween cell counts and calculations based on ages of the sea urchin [27], and Biggelaar DNA content in 3-day embryos than ob[2X] has shown that up to and including the served in either the l- or 2-day embryos. In

Nucleic

Table 1. Analysis

of variance

acid synthesis

in the Ilyanassa embryo

257

of RNA content of normal and lobeless embryos

ng of RNA/embryo Significance among groups

Significance between normal and lobeless

Lobeless

Normal Stage

n

Mean”

0 I 2 3 4 5 6 7 8

I5 I4 I1 I4 31 26 21 30 I9

6.58 8.79 8.72 8.18 14.72 19.73 22.80 26.00 3 I .OO

Normal (6.17-7.02) (8.X-9.02) (8.27-9.19) (7.50-8.91) (13.95-15.53) (18.70-20.82) (20.64-25.18) (24.47-27.63) (28.90-33.30)

Lobeless

Stage

n

Mean

*z+z* ns ns r*,; ** z+* ***

ns ns *** * ns

3 4 5 6 7 8

4 II 6 ; 5

7.37 7.94 9.97 18.83 25.16 24.80

(6.34-8.58) (6.71-9.40) (8.43-l I .79) (14.80-23.96) (23.44-27.00) (23. I l-26.62)

ns *** *** ns ns *

p The mean values are followed by the lower and upper (P>O.OS): *, P-cO.05; **, P
confidence computed

limits at the 95 % level. ns, not significant from the F ratio. All data in tables I and

the absence of data on the cell cycle and cell counts for older embryos it is not possible to accurately estimate the cell number for older stages. For this reason the cell numbers, calculated by using the 4 N DNA complement, for embryos older than 3 days in fig. 1, are estimates useful only for a relative comparison of normal and lobeless embryos. The cytoplasmic DNA content of the polar lobe could alter the calculation of cell number of the lobeless embryo. However, since no DNA was detected in a sample of 988 polar lobes it seems unlikely that the removal of the polar lobe creates a significant error. Again, cell counts of l-day lobeless embryos (26k3.6 cells) agree with the computation of cell number from the DNA content (fig. 1). Davidson [16] has reported similar cell counts of normal and lobeless embryos. Further, electron microscopy of the polar lobe [29] showed relatively few mitochondria, the presumed source of the non-chromosomal DNA, in this region of the egg. In fig. 1 are the RNA and DNA contents of the normal and lobeless embryos. The

RNA content of lobeless embryos was not measured prior to the third day of development because of (1) the large number of embryos required and (2) with the exception of the first day of development, the absence of a net increase in RNA content until after the third day of embryogenesis. In fig. 2 are sketches that show the major stages of the development of the Zlyanassa embryo. In tables 1 and 2 are the statistical analyses for the data shown in figs 1, 3 and 4. It should be re-emphasized that these data refer only to the synthesis and accumulation of the bulk RNA, i.e. rRNA and tRNA. However, it is also important to stress that rRNA, tRNA and dRNA are synthesized as early as the 4-cell stage and at all other stages of development. This has been demonstrated by pulse-labelling with uridine and fractionation by methylated albumin kieselguhr chromatography [30] and by gel electrophoresis [32]. The amount of RNA in the egg reported in table 1 is significantly greater than previously reported [ 151. This resulted from the incomplete hydrolysis of RNA Evpprl Cdl

Rcs ‘2.5 (197.5)

2

258

J. R. C’ollic~r

Fig.

3. Abscissa: ng of DNA/embryo; RNA/embryo. -, Normal embryo; embryo. Accumulation of RNA/nucleus.

0-

ordinate: - -0.

ng of lobeless

by the Ogur & Rosen procedure used in the earlier work; this point was subsequently corrected by the extraction procedure described above in the section on materials and methods. During the first day of embryogenesis, which involves the formation of about 29 cells and the embryonic determination of many of the major organ anlagen, there is a 33.6% net increase in RNA content (fig. 1, table 1). During the second and third days there is no further net change in the accumulation of RNA. This period includes gastrulation which, as judged by the maximal closure of the blastopore, is completed between 57 and 60 h at 18°C. By the fourth day of development there is an 80% increase in RNA; this is correlated with the appearance of the shell gland, which, aside from the formation of the stomodeal invagination in the 3-day embryo, is the first organ anlage to appear. Organogenesis progresses rapidly throughout all subsequent periods of development (fig. 2) and is correlated with a linear increase in the total RNA content of the embryo.Exprl

Cell

Res 95 (1975)

In the lobeless embryo there is no significant difference in RNA content among the 3. 4 and 5-day embryos (fig. I. table I). The first significant post-gastrular increase in RNA occurs on the sixth day. This increment (88.9 %I) is comparable to the postgastrular increase in RNA of the normal embryo (80.0%), which occurs during the fourth day of development. The 7-day lobeless embryo shows a further 33.6% increase in RNA; there is no significant change in the RNA content of the g-day embryo. I am hesitant to emphasize this last point, although statistically valid, because it is not supported by measurements of embryos older than eight days. The regression of RNA content on days of development (from 3 to 7 days) for both the normal and lobeless embryo is approximately linear with a coefficient of determination of 98.27 and 98.01 %, respectively. (These regression lines were computed from the data in table 1; for the lobeless embryo the RNA content of stages 3-5 was averaged as they were not significantly different.) The slopes of these lines were 4.31kO.286 and 4.02kO.572 for the normal and lobeless embryo, respectively, and were not significantly different at the 95% level. Thus, the rate of accumulation of total RNA is the same for both the normal and lobeless embryo. Further, the total amount of RNA (table 1) synthesized by these two classes of embryos is quite comparable, and there is no difference between their RNA content by day seven, at which time the normal embryo is nearly fully differentiated. Fig. 3 shows the relation of RNA accumulation to DNA content (the points plotted are mean values of each nucleic acid for a given stage and the regression of this plot was done by Bartlett’s three group method for Model II regression [24]).

Nucleic

Table 2. Analysis

of variance

of DNA

acid synthesis

in the Ilyanassa embryo

259

content of normal and lobeless embryos

ng of DNA/embryo Significance among groups Normal n

Mean”

I 2 3 4 5 6

I3 I3 I2 25 I9 18

i 12

t: 6

0.78 1.42 2.47 3.38 6.23 12.03 15.78 18. I5 19.93

Stage

(0.71-0.85) (1.24-1.64) (2.19-2.78) (3.14-3.64) (5.97650) (11.44-12.65) (14.54-17.13) (16.68-19.75) (18.53-21.44)

Normal

Lobeless

Stage

n

Mean

Significance between normal and lobeless

**z,c **z+ *** *** *** *** **

** *** * *z,c *** *** **

1 2 3 4 5 6 7 8

5 5 5 6 9 I1 I6 I2

0.71 (0.67-0.76) 1.10(1.02-1.18) 2.43 (2.27-2.59) 3.14 (2.97-3.31) 4.19 (3.66-4.82) 5.79 (4.73-7. I I) 7.93 (7.12-8.82) 9.89 (9.31-10.50)

ns * ns ns *** *** *** ***

ns

Lobeless

a The mean values are followed by the lower and upper confidence limits at the 95% level. ns, not significant (IQO.05); *, P
During normal embryogenesis there is between stages 5 and 6 a significant change in the amount of RNA accumulated per unit of DNA (slope 2.769 versus 0.679; P~0.05 ~0.01). The accumulation of RNA versus DNA content is a single line for the lobeless embryo from stages 5-7 and is not significantly different from the corresponding plot for the normal embryo during early stages (slope 3.852 versus 2.769; P>O.O5). There is no change in this plot for the lobeless embryo between early and late stages of development. Consideration of these data show that the amount of RNA accumulated by the normal embryo does not increase uniformly as a function of genomic replication and that the accumulation of RNA by the lobeless embryo continues to be proportional to genomic replication throughout all stages. Because DNA, in contrast to RNA, is stable and conserved from each cell generation, changes in DNA content are taken to represent the rate of DNA synthesis in the following account. The following statistical comparisons of the rates of DNA synthesis

are readily apparent from the semilogarithmic plot (fig. 4) of the data in table 2. The synthesis of DNA is logarithmic with respect to stage of development (fig. 4 and table 2) for stages l-6 of the normal embryo, which have a 99.25 coefficient of determination; this is a single component plot with a slope of 0.22940.0099. The corresponding plot for the DNA content of the lobeless embryo has two logarithmic components, stages l-4 have a 96.64 coefficient of determination with a slope of 0.228+ 0.0301, and stages 5-8 have a 99.29 coefficient of determination and a slope of 0.1262 0.0075. The rates of DNA synthesis, as estimated from the slopes of the plot of DNA content versus stage, for early and late stages of the lobeless embryo are significantly different (P0.01). The rate of DNA synthesis for stages 14 of the lobeless embryo is not significantly different from that of the normal embryo (slope 0.228+0.0301 versus 0.229?0.0084; PBO.05). However, the rate of DNA synthesis by the lobeless embryo during stages 5-8 is significantly different from the Exptl CeURes 95 (1975)

260 2oc

I 0.c

5.0

I .o

05 1

2

3

4

5

6

7

8

Fig. 4. Ahscissn: days of development ordinate; logarithm of DNA content/embryo. mal embryo; 0 . 0, lobeless embryo. Rate of DNA synthesis during development.

at -,

normal embryo (slope 0.126+0.0075

versus

19°C; Nor-

0.229+0.0099; FKO.01). That the decreased rate of DNA synthesis by the lobeless embryo is not a mimic of the normal pattern of DNA synthesis, which decreases between stages 6 and 8, is evident from the following points: (I) the change in rate of DNA synthesis by the lobeless embryo occurs earlier-between the fourth and fifth days of developmentand (2) the decreased rate of DNA synthesis by the lobeless embryo is logarithmic, whereas the decrease in DNA synthesis by older normal embryos is not logarithmic. Thus, there is a significant difference between these two types of embryos in the total amount of DNA synthesized and in the rate of DNA synthesis. DISCUSSION The results reported in this paper show a 33.6% increase in bulk RNA (tRNA and rRNA) during the first 24 h of development. The lack of a further net increase in RNA until day 4 despite the synthesis of rRNA, Expd Cell Res 95 (1975)

which began at the 4-cell stage ]30]. suggests that some of the bulk RNA of the egg is being degraded and replaced. Accordingly, the net increase in RNA, principally rRNA, during the first day would have resulted from the accumulation of newly synthesized rRNA before a substantial amount of egg rRNA had degraded. The change in slope of the plot of RNA versus DNA during the course of normal development (fig. 3) is a result of the DNA synthetic rate being constant and exponential (stages l-6), while the accumulation of RNA is approximately linear (actually slight curvilinear) for stages 3-7. The RNA accumulation not being logarithmic means either (1) that the rRNA and tRNA cistrons are not transcribed at a constant rate at all stages of development or in all cells of the embryo or (2) that the rate of degradation of these RNAs is not the same for all stages or cells of the embryo. In the absence of data on the absolute rates of RNA synthesis neither of these alternatives can be eliminated; however, the relative stability of rRNA in growing cells [33] supports the first possibility. The lobeless embryo does not accumulate any additional RNA after the first seven days of development. RNA content was not measured on lobeless embryos older than 8 days, at which time there was no increase in RNA over that measured in the 7-day embryo. I have reared many lobeless embryos up to 23 days, and in no case did they show any signs of further differentiation, even though they were active swimmers and capable of the usual contractility observed in the lobeless embryo. Thus, the developmental stages studied in this work are sufficient to detect virtually all RNA synthesis that is likely to occur. There are three principal effects on the synthesis and accumulation of nucleic acids

Nucleic caused by the removal of the polar lobe at first cleavage. They are: (1) a delay in the post-gastrular increase in the accumulation of RNA, (2) a constant rate of accumulation of RNA per nucleus (fig. 3) throughout the development of the lobeless embryo, and (3) a decrease in the rate of DNA synthesis during later stages of embryogenesis of the lobeless embryo. Effects (1) and (2) are explicable in terms of a qualitative and quantitative change, respectively, in the cleavage pattern (reflected by the rate of DNA synthesis) of the lobeless embryo. My interpretation is that the delay in RNA accumulation results, in part, from the failure of the shell gland to differentiate and the absence of the mesodermal band cells, which result from the removal of the polar lobe and the subsequent abnormal development of the mesentoblast (4d) cell. In the 4-day embryo the shell gland is composed of a number of large columnar cells whose cytoplasm is rich in RNA, as illustrated by the photographs in figs 1 and 2 of Collier & McCann-Collier [34]. Similarly, Cather [3 l] has reported the presence of a well developed endoplasmic reticulum in these cells. Even though the lobeless embryo develops a number of scattered shell fragments [5], it never produces an amount of shell material that is comparable to the normal shell. These facts support the interpretation that a significant portion of the RNA accumulated by the 4-day normal embryo is in the cells of the shell gland, which are absent in the lobeless embryo. It is unlikely that the failure of cellular differentiation in the lobeless embryo results from regulatory mechanisms operating solely or chiefly on the accumulation of bulk RNA. Accordingly, one may expect that the delay in RNA accumulation by the lobeless embryo is a result, rather

acid synthesis in the Ilyanassa embryo

261

than a cause, of the failure of this embryo to differentiate. The observation that the slope of the plot of RNA versus DNA (fig. 3) for the lobeless embryo is, unlike the normal embryo, constant throughout development is predictable from the decreased rate of DNA synthesis (fig. 4) by the lobeless embryo during later stages of development and the continued linear accumulation of RNA (fig. 3). That the slope in this plot (fig. 3) is not significantly different from that of the normal embryo shows that removing the polar lobe does not impair the ability of the lobeless embryo to synthesize and to accumulate RNA. Thus, the decrease in the rate of DNA synthesis is the most significant effect of removing the polar lobe on nucleic acid synthesis and accumulation. This is reflected by the altered cleavage pattern and the failure of certain cell lines to proliferate in the lobeless embryo. The continued division of ectodermal, endodermal and mesenchymal cells and the data on DNA synthesis presented in this paper suggest that the effect of the polar lobe cytoplasm on cell proliferation is highly specific for certain cell types and does not result from a general inability to synthesize DNA. I suggest that most of the morphogenetic deficiencies, with the possible exception of heart, intestine and shell gland, result from the failure of stem cells of lobe dependent organs to undergo sufficient proliferation for normal morphogenesis. It is possible that these stem cells do not proliferate because extirpation of the polar lobe reduces the level of certain metabolites that are essential for the normal rate of cell division and that the undifferentiated ectodermal, endodermal and mesenchymal cells of the embryo require a lower level of these materials for extensive cell division Exptl

Cell

Res 95 (1975)

262

J. R. Collirr

than determined stem cells. What these materials may be or why stem cells may require more of them for cell division is not clear at this time. I am not implying that the polar lobe or other localized egg cytoplasms are lacking in specific morphogenetic effects [2, 3, 5, 311, rather that there may be superimposed upon the specific events of determination a generalized effect on differentiation that makes the problem much more complicated. This work was supported number GB-1529-O). 1 thank excellent technical assistance.

in part Miss

by the NSF Alice Grebanier

(grant for

13.

14. IS. 16.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

REFERENCES I. Crampton, lungsmech 2. Wilson, 3. Costello, 4. Clement, 5. - Ibid 6. - Ibid 7. Verdonk, 33. 8. - Ibid 9. Verdonk, mermans, 57. 10. Verdonk, 47. Il. Atkinson, 12. Cather.

Exptl

H E, Wilhelm Roux Arch organ 3 ( 1896) I. E B, J exp zool 1 (1904) I. D P, J exp zool 100 (1945) 19. A C, J exp zool 121 (1952) 593. 149 (1962) 193. I66 (1967) 77. N H, J embryo1 exp morphol 20 (1968) N H, L PM,

J W, J morphol J N, Advances

Cell Res 95 (1975)

28. 29. 30. 31. 32.

I9 (1968)

33.

101. Geilenkirchen, J embryo1

N H & Cather.

Entwick-

exp

W L M morph0125

J N, .l exp

in

zool

133 (1968) 339. morphogenesis

&

Tim(1971)

34.

186 (1973)

35.

(ed

M

Ahercrombie. J Brachet & I .[ king) p. 67 Academic Press. New York (I97 I ). Clement. A C, Experimental embryology ofmarine and fresh-water invertebrates (ed G Reverheri) p. 188. North-Holland, London (1971). Berg. W E & Kato, Y, Acta embryo1 moi-phol exp 2 (1959) 227. Collier, J R. Exp cell res 21 (1960) 126. Davidson, E H, Haslett, G W. Finney. R J. Allfrey. VG&Mirsky,AE.ProcnatlacadsciUS54(1965) 696. Collier, J R, Exp cell res 24 (1961) 320. - The biochemistry of animal development (ed R Weber) p. 203. Academic Press. New York (1965). Burton, K, Biochem j 62 (1956) 3 IS. Ceriotti, G. J hiol them 214 (1955) 59. Kissance, J M & Robins, E. J hiol them 233 (1958) 184. Hinegardner, R T, Anal hiochem 39 (1971) 197 - Personal communication. Sokal, R R, Rohlf, J, Biometry. W H Freeman and Co. San Francisco, CA (1969). Bliss, C I, Statistics in biology, vol. I. McGrawHill, New York (1967). Collier, J R, Exp cell res 69 (1971) 181. Hinegardner. R’T, Rao. B & Felman. D E, Exp cell res 36 (1964) 53. Biggelarr, J A M van den, J embryo1 exp morphol 26 (1971) 367. Pucci-Minafra, I, Minafra, S & Collier. J R, Exp cell res 57 (1969) 167. Collier, J R, Weinstein, H M, J cell hiol39( 1968) 27. Cather, J N, J exp zool 166 (1967) 205. Koser, R B, Doctoral thesis. City Univ of New York (1974). Abelson, H T, Johnson, L F, Penman, S &Green, H, Cell I(l974) 161. Collier, J R, McCann-Collier. M. Exp cell res 34 (1964) 512. - Ibid 27 (1962) 553.

Received Revised

December 30, version received

1974 March

25,

1975