Morphogenetic substances from sea urchin eggs. Isolation of animalizing and vegetalizing substances from unfertilized eggs of Paracentrotus lividus

DE’JJSLOPMENTAL BIOLOGY 20,

Morphogenetic Isolation

481-500

(1969)

Substances

from Sea Urchin

of Animalizing

Substances

LARS JOSEFSSON Department

and Vegetalizing

from Unfertilized

Paracentrotus

Eggs.

Eggs of

lividus

AND SVEN

H~RSTADIUS

of Biochemistry C, University of Copenhagen, DK-2200 Copenhagen, Denmark, and Department of Zoology, University of Uppsala, S-75122 Uppsala, Sweden Accepted May 27, 1969 INTRODUCTION

Specific morphogenetic substances have been assumed to form the basis of differentiation in the early development of sea urchins. No direct evidence for the existence of such morphogenetic agents has been available, however, until very recently, when fractions isolated from unfertilized eggs of Pamcentrotus lividus were shown to influence specifically the early differentiation of sea urchin larvae (HiSrstadius et al., 1967). One of the fractions had a strong animalizing activity when tested both on whole eggs and on animal and vegetal halves, isolated in the 16- and 32-cell stages. A second fraction, not completely separated from the animalizing one, caused a vegetalization under the same test conditions, although this effect was somewhat weaker. The demonstration of specific morphogenetic active substances in extracts of sea urchin eggs has now been followed by further studies directed against their isolation and their chemical identification. The present report describes the complete separation of animalizing and vegetalizing activities and the isolation of different extensively purified animalizing substances from unfertilized sea urchin eggs. EXPERIMENTAL

All chemicals were of analytical grade, and glass-distilled water was employed, unless otherwise stated. Dowex 50 W-X2, 100-200 mesh, was obtained from Kabi, Stockholm; Sephadex G-25, fine (lot No. 1062) was purchased from Pharmacia, Uppsala. Source of the morphogenetic substances. Mature, unfertilized eggs 481 Copyright

0 1969 by Ada&c

Press, Inc.

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JOSEFSSON

AND

HdRSTADIUS

of Paracentrotus lividus were collected at Stazione Zoologica, Naples. The eggs were rinsed in fresh seawater, and their jelly coat was removed. The eggs were washed by suspension in seawater; this was followed by slow centrifugation. From each batch of eggs, samples were taken after centrifugation and tested for fertilizability with an appropriate sperm suspension in seawater. The remaining eggs were then lyophilized and stored in closed bottles at -20° until used. Embryological tests. The tests were performed with developing whole eggs or animal and vegetal halves of eggs of Paracentrotus lividus according to the procedure previously described in detail (Hbrstadius et al., 1967). Solutions for the embryological tests were prepared immediately before use by dissolving samples from the different fractions under investigation in normal seawater and adjusting the pH to about 8.2 with 0.1 M NaOH. The halves were separated in the 32-cell stage and then immediately brought into test solutions or into normal seawater as controls. The following day, after a treatment of about 20-26 hours, the halves were examined individually, classified, and transferred to normal seawater. The size of the apical tuft formed the basis for the classification of the animal halves, 4/4-l/8 signifying the part of the surface covered by long stiff “stereocilia” (Fig. 5); l/8 corresponds to a normal tuft, 3/4-4/4 indicates a strong or complete extension of the tuft, which in turn reflects the size of the “acron” region of the embryo. In the fully differentiated animal halves (the second day after fertilization or sometimes later) the “stereocilia” have been replaced by shorter motile cilia. The halves are classified as uniformly ciliated blastulae with a wall of columnar epithelium (A), blastulae with less than half transformed to squamous epithelium (Ba), with a ciliary field smaller than half the surface (Bb), with a ciliary band (0, and with a ciliary band and also a stomodeum (D), the latter being the most vegetal type (Fig. 5). The vegetal halves with the strongest animal properties are recorded as Rad and are characterized by the presence in the early stages of an apical tuft, sometimes even enlarged, and a further development with a thick ectoderm covering about half the body and with the skeleton in *equatorial and radial position. Other halves have been recorded as Pl even if their shape deviates in several aspects from that of a normal pluteus. Less animal types of halves have been recorded as Pr, because they resemble a prism stage. Those of

MORPHOGENETIC

SUBSTANCES

FROM

SEA URCHIN

EGGS

483

ovoid shape, Ov, are normally provided with a tripartite digestive tract, but rarely have a mouth; their skeleton is irregular and rather poorly developed. The exogastrulae, Ext, represent the most vegetal way of differentiation of the vegetal halves (Fig. 5). Eggs within one batch raised in seawater show a certain variation in the type of differentiation, and there is also a variation between different batches. Control tests with the same batch of eggs were therefore always run in parallel with the sample tests. Ultraviolet absorption was measured in a Zeiss spectrophotometer, model PMQ II, using l.OO-cm quartz cells. Isolation

and firification

of the Morphogenetic

Substances

All operations were performed at room temperature, unless otherwise stated. Preparation of homogenate. Lyophilized eggs (40 gm) were homogenized with simultaneous cooling in an ice-bath for 3 minutes (MSEhomogenizer, 14,500 rpm) in 10 equal batches with precooled water (100 ml). The insoluble material was centrifuged off (20,000 g, 180 minutes, O°C) and the red-brown supematants were collected. The sediments were washed with an additional amount of cold water (125 ml), and the centrifugation was repeated. The combined supematants (825 ml, homogenate, Table 1) were lyophilized and a dry yellow-red powder were obtained (28.3 gm). Acid precipitation. The yellow-red powder (28 gm) was extracted with 110 ml of water for 30 minutes. The homogeneous red-brown suspension was then brought to pH 3.7 by careful addition of formic acid (98-lOO%, 1.1 ml). The heavy yellow-red precipitate formed was centrifuged off (27,000 g, 120 minutes, 0%) after 30 minutes of standing at 4O. The supematant was collected, and the sediment was washed with 10 ml of 0.1 M ammonium formate buffer, pH 3.7, before being discarded. The supernatants were combined to a slightly opalescent, red-yellow solution (84 ml, supematant after acid precipitation, Table 1). Chromatography on Dowex 50 W-X2. The combined supematants from step 2 were applied to a 5.3 cm x 55 cm column of Dowex 50 W-X2, prepared in and equilibrated with 0.1 M ammonium formate buffer, pH 3.7. The column was eluted with a 3-step elution system at a constant rate of 285 ml/hour using a pump. The 3-step elution system included 0.1 M ammonium formate buffer, pH 3.7, 0.1 M ammonium acetate buffer, pH 5.5, and 0.1 M ammonium bicarbonate

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buffer, pH 9.2. The effluent was continuously monitored at 254 rnp with an LKB 8300 A Uvicord II provided with a 3-mm measuring cell (LKB-Produkter AB, Stockholm). Fractions were collected for each 6 minutes. The fractions corresponding to the peaks indicated as A and V, respectively, in the elution diagram (Fig. 1) were pooled separately and lyophilized. Solution A gave a dry yellow preparation, which was dissolved in 10 ml of water. A small insoluble residue was centrifuged off (27,000 g, 30 minutes), washed twice with 1.5 ml of water and discarded. The supernatants were combined giving a clear yellow solution (11.4 ml, fraction A, Table 1). 1 ml of the solution was withdrawn and evaporated to dryness at 80% in vacua to remove the salts. After being redissolved in water it was lyophilized and taken for embryological tests (Table 2). Solution V gave a dry white powder, which readily dissolved in water, producing a clear brown-yellow solution (8.5 ml, fraction V, Table 1). One milliliter of the solution was withdrawn and its salts were removed as described above. After being redissolved in water, the preparation was lyophilized and used for embryological tests (Table 2). Chromatography of fraction A on Sephadex G-25. The clear yellow solution (fraction A, Table 1) was applied to a 2.4 cm X 52 cm column of Sephadex G-25, prepared in water according to the manufacturer. The column was eluted with water at a constant rate of 22.6 ml/hour using a pump. The column eluate was continuously monitored at 254 rnp as above, and fractions were collected for each 12 minutes. The fractions corresponding to ranges Al, Ax, AS, and A+ respectively, as indicated in the elution diagram (Fig. 2), were pooled separately and lyophilized. The four dry preparations (fraction AI-Ad, Tables 1 and 2) were analyzed for morphogenetic activity and for ultraviolet absorption. Chromatography of Fraction V on Sephadex G-25. The clear brown-yellow solution (fraction V, Table 1) was applied to a 2.4 cm x 50 cm column of Sephadex G-25, prepared in water. The column was eluted with water at a constant rate of 22 ml/hour and the eluate was continuously monitored at 254 rncc as above. Fractions were collected for each 15 minutes. The fractions corresponding to ranges VI, VP, V3, V4, Vs , and Vs , respectively, as indicated in the elution diagram (Fig. 8) were pooled separately. The six solutions were lyophilized, and the dry preparations (fractions VI-Vs, Tables

MORPHOGENETK!

SUBSTANCES

FROM

SEA URCHIN

EGGS

485

1 and 2) were analyzed for morphogenetic activity and for ultraviolet absorption. RESULTS

In Tables 1 and 2 results of a typical purification experiment are presented. Partial Separation of Animalizing

and Vegetalizing

Fractions

According to our previous findings (Horstadius et al., 1967), chromatography of partly purified egg homogenates on Dowex 50 W-X2 resulted in two morphogenetic active fractions. The two active fractions (fractions A and V), eluted when the pH was changed to an alkaline pH, were obtained as two partially separated ultraviolet absorbing peaks, as shown in Fig. 1. Fraction A had a strong animalizing activity when tested both on whole eggs and on animal and vegetal halves (Table 2), while fraction V, having a vegetalizing activity, influenced only the animal and the vegetal halves and had no effect when tested on whole eggs (Table 2). Isolation and Purification

of Different Animalizing

Substances

Although fraction A had a strong animalizing activity, it was evident that it contained mostly inactive material. Part of the animdizing activity showed a strong aflinity for Sephadex gel in water, and TABLE 1 ISOLATION OF ANIMALIZING AND VEGETALIZJNC SUBSTANCESFROM UNFERTILIZED EGGS OF THE SEA URCHIN Pamcentrotus lividus” Fraction

Homogenate Supematant after acid precipitation Fraction A Fraction V Fraction A1 Fraction & Fraction Vr Fraction V, Fraction Vs

Volume (ml) 825 84 11.4 8.5 15.0 1.5 22.7 1.8 0.5

A **O

A 110

107 251

115 290

9.20

11.4 57.3 6.72 0.650 1.63 2.19 6.88

57.5 4.35 0.295 1.54 2.02 2.38

’ Figures given are for 40 gm of lyophilized

eggs.

Am: A280

DW weight

(mg)

0.93

-

0.87

-

0.81 1.00 0.65 0.45 0.94 0.92 0.36

57 0.02 57 0.14 0.08

486

JOSEFSSON AND HijRSTADIUS

pH 332

--*I-

PH so

-

I.---pH9z

A,+”

loo

) 0

5

10

*I

15

EFFLUENT (I 1

FIG. 1. Separation of animalizing and vegetalizing activity on Dowex 50 W-X2. Supernatant after acid precipitation (10.5 ml) from 40 g of lyophilized unfertilized eggs was applied to a 5.3 cm x 55 cm column, equilibrated with 0.1 M ammonium formate buffer, pH 3.72. The column was eluted with 0.1 M ammonium formate buffer, pH 3.72, 0.1 M ammonium acetate buffer, pH 5.50, and 0.1 M ammonium bicarbonate buffer, pH 9.22, as shown. Flow rate 285 ml/hour. The curve indicates the transmission of the effluent at 254 m s. Effluent was collected in 28.5-ml fractions. Pooled fractions are indicated as A and V.

a further extensive purification was obtained by chromatography on Sephadex G-25. This activity was eluted as a single, much retarded peak (A4), as demonstrated in Fig. 2. The remaining material of fraction A was separated into two large, ultraviolet absorbing peaks, of which the front peak (fraction AI) also exhibited a strong animalizing activity (Table 2). The intermediate fractions (AZ and AZ) were inactive when assayed for morphogenetic activity (Table 2). Chromatography on Sephadex G-25 thus resulted in a complete separation of the animalizing activity into two different fractions. The ultraviolet absorption curves of the two active fractions are shown in Fig. 3. Fraction A1 has a maximum at 256 rnk and a minimum at 247 rnp, and fraction Aq has a maximum at 258 rnp and a minimum at 240 mp. The two animalizing fractions exhibited strong morphogenetic activity, and both were capable of inducing animalization in whole

MORPHOGENETIC

SUBSTANCES TABLE

MORPHOGENETIC

FROM

SEA URCHIN

2

ACTNITY OF FRACTIONS ISOLATED FROM UNFERTILIZED THE SEA URCHIN Paracentrotus lividus”

Morphogenetic Concentration (s,)

Fraction

- - Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction

A V A, A2 A3 Al V, V2 V3 V, Vg V6

0.1-0.025 0.1-0.025 0.1-0.025 0.2-0.012 0.1-0.025 0.00-0.0005 O.Ol-0.0025 0.01-0.0025 0.1-0.012 0.1-0.012 0.002-0.0005 0.0025-0.00015

487

EGGS

Animal halves An W An 0 0 An Veg Veg 0 0 An An

2X2’ AD Veg An 0 0 An Veg Veg 0 0 0 An

EGGS OF

activity Whole eggs An 0 An 0 0 An 0 0 0 0 0 An

‘Tests on whole eggs, and on animal and vegetal halves, isolated in the 16- and 32-cell stages.

Y 200 300 4al EffUJEM(mI) FTC. 2. Separation of animalizing substances on Sephadex G-25. Animalizing active material (10.4 ml), obtained from 40 g of lyophilized eggs and corresponding to fraction A in Table 1, was applied to a 2.4 cm x 52 cm column. The column was prepared in and eluted with water. Flow rate 22.5 ml/hour. The curve indicates the transmission of the efhuent at 254 rnp. EWuent was collected in 5%ml fractions, which were pooled as indicated (- +) to four separate solutions (AI-A,). 100, 0

I m

488

JOSEFSSON AND HdRSTADIUS

O%k--%-

Wavelength rnj~

Frc. 3. Ultraviolet absorption curves of animalizing active material. (a) 0.1% aqueous solution of lyophilized material of fraction Al. (b) 0.0015% aqueous solution of lyophilized material of fraction G .

MORF’HOGENETIC

SUBSTANCES

FROM

SEA URCHIN

EGGS

489

eggs, resulting in larvae with an enlarged apical tuft and radial arrangements of the ciliated band and skeletal spicules, as illustrated in Fig. 4, A-D. However, when they were tested on animal and vegetal halves, fraction A4 showed a much stronger and more definite animalization than fraction AI. Thus many animal halves developed stereocilia all around the blastula wall (4/4) after treatment with fraction Ad (Fig. 5), a feature never obtained in any control halves. Also the vegetal halves presented an extreme differentiation (Rad.), hitherto not yet described, after treatment with fraction Aq (Fig. 5). A series of vegetal halves obtained after exposure to fraction Aq are shown in Fig. 6. While some halves were of the usual ovoid type (0~. in Fig. 5, G, H in Fig. 6), as were most of the control halves, the main portion of the halves had reached various of the differentiation stages described below. With a slightly more animal trend, the organization becomes bilateral with a ventral ciliated band and two spicules in a more vegetal position (B. in Fig.

FIG. 4. Representative drawings of animalized whole eggs. A and B: from testa of sample from fraction G, added in 0.001% concentration; C and D: from tests of sample from fraction A,, added in 0.1% concentration; E and F: from tests of sample from fraction Vs , added in 0.0025% concentration.

490

JOSEFSSON

AND

HiiRSTADIUS

5, E in Fig. 6). A further step in animal direction is represented by the pluteus (PL), as certain qualities are required of the ectoderm in order to form arms and a skeleton of typical pattern (F in Fig. 6). The extreme animal type (Rad. in Fig. 5, A-D in Fig. 6), is characterized by the presence in the early stages of an apical tuft, sometimes even enlarged, and a further development with a thick ectoderm covering about half the body (cf. the small animal plate in ovoid larvae) and with the skeleton in equatorial and radial position because of the stronger animal forces in the an/veg relation (cf. the animal position of the spicules in ovoid larvae, G and H in Fig. 6).

nm 25 45 FIG. 5. Embryological

OEZ3 24

or 45

II

52

tests of samples from fraction A,, showing a strong animaliz. ing effect on both animal and vegetal halves. In the block diagrams the columns indicate the percentage distribution of different larval types. The numerals below each diagram state the total number of control halves (heavy lines) and treated halves (hatched columns) from a series of parallel experiments of assay of fraction A,, added in concentrations varying from 0.001 to 0.0005%. The diagram to the left gives the frequency of the types of animal halves (414 to l/8) on the first day after the operation; the diagram in the middle, of the types of animal halves (A to D) on the second day or later; and the diagram to the right, the frequency of the types of vegetal halves (Rod. to Ex.). For details about the classification of the various types of larvae, see Experimental.

MORPHOGENETIC

SUBSTANCES

D

FROM

F

SEA URCHIN

EGGS

491

L?

FIG. 6. Representative

drawings of individual vegetal halves, showing the various types of larvae obtained after treatment with a sample from fraction Ad. Younger stages (A and C) and fully differentiated (B and D) vegetal halves of radial type; prism larva (E); pluteus (F); ovoid larvae (G and H).

These radial halves in relation to the plutei halves therefore are real counterparts to the animalized whole eggs (Fig. 4) in relation to normal plutei. Fraction Al showed much weaker activity when tested on halves, as illustrated in Fig. 7a, which gives the results from several concentration tests. The weaker action was particularly marked in the treatment of the vegetai halves, as demonstrated in a series shown in Fig. 7b. The more animal differentiation is mainly expressed by a richer skeleton. Complete Separation and Isolation of Vegetalizing and Animalizing Substances The vegetalizing active material, obtained only partially separated from the animalizing activity in the alkaline effluent from the Dowex column (fraction V in Fig. 1 and Table l), separated into several well-defined ultraviolet absorbing peaks when chromatographed on Sephadex G-25. A typical elution pattern is given in Fig. 8, which shows that part of the material was retarded on the Sephadex gel and resulted in an extensive purification of those components

492

JOSEFSSON

H6RSTADIUS

nm

o-7 23

AND

33

21

32

r-r 20

27

(b)

FIG. varying halves. halves. female,

7. Embryological tests of samples from fraction A,, added in concentrations from 0.1 to 0.025%, which show animalization on both animal and vegeti (a) Diagrams (for explanation see Fig. 5). (b) Drawings of individual vegetal Upper row: control halves; lower row: treated halves from eggs of the same showing an animalization marked by a richer skeleton.

MORF’HOGENETIC

SUBSTANCES

FROM

SEA URCHIN

EGGS

493

(Table 1). When the different peaks were assayed for their morphogenetic activity, it was found that the vegetalizing activity was obtained in the front peaks (fractions V1 and V,), while the retarded components (Fractions Vs and V,) represented animalizing substances (Table 2). The intermediate eluate (fractions V3 and V,) exhibited no morphogenetic activity. The vegetalizing activity of fractions V1 and VP was strong when tested on both animal and vegetal halves. This is illustrated in Fig. 9, which shows diagrams from tests with samples of fraction VZ. No effect was observed, however, in parallel tests on whole eggs. The ultraviolet absorption curve of fraction VP is shown in Fig. 10. It has a maximum at 274 rnp and a minimum at 261 mr. The two retarded and extensively purified fractions with animalizing activity (V, and V,) showed ultraviolet absorption curves (Fig. ll), which indicated that they differed significantly in their chemical composition. The absorption curve of fraction VS has a maximum at 270 rnp and a minimum at 255 rnp, while the absorption curve of fraction Vs has a maximum at 260 mu, and a minimum at 240 rnp, and thus resembles the absorption curve of fraction Ad. The two fractions also exhibited differences in their morpho-

0

100

200 EFFLIENT

300

400

(ml)

FIG. 8. Separation of animalizing and vegetalizmg substances on Sephadex G-25. Vegetalizing active material (7.5 ml) obtained from 40 gm of lyophilized eggs and corresponding to fraction V in Table 1, was applied to a 2.4 cm X 50 cm column. The column was prepared in and eluted with water. Flow rate 22 ml/hour. The curve indicates the transmission of the effluent at 254 rnp. Effluent was collected in 5.5-ml fractions. Fractions were pooled as indicated ( t -1 to six separate solutions (VI-V,).

494

JOSEFSSON

AND

HORSTADIUS

FIG. 9. Diagrams obtained from an embryological test of samples from fraction VT, added in concentrations varying from 0.01 to O.O025C;, which show a strong vegetalization on both animal and vegetal halves. For explanation of the block diagrams see Fig. 5.

1.0 -

;

a5 -

W

t

CJ’

’ ’ 24a Wavelerqih

FIG. 10. Ultraviolet solution of lyophilized

’

’

’

280

I

mp

absorption curve of vegetalizmg material of fraction VL

active material;

0.08% aqueous

MORPHOGENETIC

SUBSTANCES

FROM

SEA URCHIN

EGGS

4%

1.0

Okii?-+F Wavkrgth

mp

FIG. 11. Ultraviolet absorption curves of animalizing active material. (a) 0.002c; aqueous solution of lyophilized material of fraction Vs. (b) 0.002% aqueous solution of lyophilized material of fraction V,

496

JOSEFSSON

AND

H&tSTADIUS

genetic activity. Fraction Vs possesseda much stronger activity than fraction Vs on whole eggs (E and F in Fig. 4) and on vegetal halves, where fraction Vs forces the vegetal halves very strongly in animal direction (Fig. 13). Figures 12 and 13 also show that exposure to fraction Vs as well as fraction Vs leads to strong animalization in the animal halves. The strong animalizing activity of fraction Vs, resulting in a great number of vegetal halves of the radial type, is presented more in full in Fig. 14. The control larvae were all of the pure ovoid type, while even low concentrations of fraction Vs caused a considerable delay of differentiation, which was more pronounced with higher concentrations. Some of the radial halves have a thick animal ectoderm, as described above, whereas ot.her halves have formed a ciliated band, in close conformity to many animalized whole eggs, as shown in Fig. 4. DISCUSSION

The presence of morphogenetic substances in the unfertilized eggs of the sea urchin capable of influencing the early differentiation

ABaBcC

rml 17

20

or

16

20

PI.Pr.oM Ex.

OY 17

FIG. 12. Diagrams from embryological tests of samples from fraction VA, added in concentrations varying from 0.002 to O.O005R, which show a strong animalization on the animal halves but no effect on the vegetal halves. For explanation of the block diagrams see Fig, 5.

MORPHOGENETIC

%

a+ ti

$6

r/s

SUBSTANCES

FROM SEA URCHIN EGGS

A BaBc C

497

R~P.C.Pp/ ol%

FIG. 13. Diagrams from embryological tests of samples from fraction V6, added in concentrations varying from 0.0025 to 0.00015%, which show a strong animalization on both animal and vegetal halves. For explanation of the block diagrams see Fig. 5.

of the sea urchin larvae into an animal or a vegetal direction has now been established by their separation and further purification. The isolation of several animalizing substances, different both in chemical and morphogenetic respects, has furthermore shown that the developmental processes can be influenced by various types of substances that may exist in the eggs prior to fertilization. Extensive purification of at least two of the animalizing substances was attained due to a marked af%nity of fraction Vs and fraction A4 or Vs for Sephadex G-25. The chemical and morphogenetic properties of the latter two fractions are very similar; fraction A4 and fraction Vs probably represent an identical substance. This result is also in conformity with an expected presence of some identical constituents in fraction A and fraction V, since they were obtained only partially separated in the Dowex eluate. Although no quantitative determination of the morphogenetic activity is possible as yet, the results obtained in the embryological

498

JOSEFSSON

C

AND

I

HCjRSTADIUS

D

FIG. 14. Drawings of individual

vegetal halves from an embryological test of sample from fraction Ve, showing the various types of larvae obtained. (A) Control half, 1 day after operation; (B) control halves, fully differentiated larvae; (C) treated halves, 1 day after operation; (D) treated halves, fully differentiated larvae. The larvae in the upper row of section C were treated with a 0.0012% solution of fraction V,, and are more retarded than those in the lower row of panel C, which were treated with an 0.0003% solution of the animalizing fraction. The four larvae to the left in panel D are representative examples of strongly animalized larvae; to the right are a pluteus, a prism stage, and two ovoid larvae.

tests obviously show that the purification of the two activities strongly increase their specific effects. Thus we found two different types of strongly animalized halves, the 414 type of the early differentiated animal halves, which never appears in control halves, and the radial type of the vegetal halves. The latter type has hitherto never been observed as a result of any treatment. The high specific activity of the purified animalizing substances is also demonstrated by their ability in very low concentrations to induce strong animalization on whole eggs, those representing a much more balanced system of regulation than the halves. The two animalizing substances, separated from an overall vegetalizing fraction (fraction V), differ not only in their chemical properties, but also in their morphogenetic activity. Fraction V5 with an absorption curve, which suggests a significant content of trypto-

MORPHOGENETIC

SUBSTANCES

FROM

SEA URCHIN

EGGS

4%

phan, induces strong animalization on animal halves, but fails completely to influence the vegetal halves or the whole eggs. In contrast, fraction Vs (A*) has an absorption curve, which indicates a nucleotide structure, and it induces strong animalization in all the test systems. These differences suggest that the two substances influence differentiation in separate ways. This has to be fukher studied on a molecular basis when they are available as pure substances. The purified vegetalizing activity (fractions V1 and V,), although considerably increased in force in comparison with the vegetalizing fraction from the Dowex eluate, still fails to affect whole eggs, These results could represent a real difference between the vegetalizing and the animalizing substances in their ability to influence the an/ veg balance of the eggs, or it might be a question of contamination by some animalizing material, which partly neutralizes the vegetalizing effect (Hiirstadius et al., 1967). This is at present impossible to decide. The strong animalizing activity exhibited by fraction A, obtained from the parallel Sephadex experiments, as well as the inevitable mixing of common constituents of fraction A and fraction V speak, however, in favor of the latter. It is therefore possible that further purification of the vegetalizing fractions will give substances with much stronger activity, able to induce a vegetalization on whole eggs and thereby also capable of matching the animalizing substances in their effect. SUMMARY

A method is described for the isolation of morphogenetic substances from mature, unfertilized eggs of the sea urchin Purucentrotus liuidus. A complete separation of several different substances with specific animalizing or vegetalizing effect on the early development of sea urchins, when tested on whole eggs and on isolated animal and vegetal halves, was achieved by chromatography on Dowex 50 W-X2 and on Sephadex G-25. The results suggest that developmental processes can be influenced by various types of substances that may preexist in the unfertilized eggs. Two of the animalizing substances were obtained extensively purified. According to their ultraviolet absorption curves, one may contain tryptophan and the other consist of a nucleotide. They both exhibit a very high specific activity but differ significantly in their morphogenetic specificity. One induces strong animalization when tested on animal halves but has no effect when tested on whole eggs

500

JOSEFSSON

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or on vegetal halves. In contrast, the other shows a strong animalizing activity in all three test systems. The high specific activity of the purified animalizing substances is demonstrated by their ability in very low concentrations to force the animal halves into their most extreme animal type of differentiation, i.e., with stereocilia all around the blastula wall, and to induce a differentiation on the vegetal halves not only to plutei but to a new extreme animalized larval type, the radial type. This type of vegetal half is characterized by the presence in the early stages of an apical tuft, and a further development with a thick ectoderm covering about half the body and with the skeleton in equatorial and radial position. We wish to express our thanks to Mrs. A. Runnstrcim for collecting and preparing the egg material in an excellent way. We also thank Professor J. Runnstriim for his continuous interest in the investigations. The collection of egg material and the embryological tests were carried out at the Stazione Zoologica, Naples. The isolation and purification were done at Division of Physiological Chemistry, Chemical Center, University of Lund. We acknowledge also the skillful technical assistance of Miss V. A. Andersson, Mrs. G. Hilrstadius, and Mrs. I. von Plenker-Tind. The investigation was aided by grants from the Swedish Cancer Society (Project No. 83) and from the Swedish Natural Science Research Council. REFERENCES H~RSTADIUS, S., JOSEFSSON,L., and RUNNSTR~M, J. (1967). Morphogenetic agenta from unfertilized eggs of the sea urchin Paracentrotus lividus. Develop. Biol. 16, 189-202.