partial characterization of the component from normal eggs which corrects the maternal effect of...

Partial Characterization of the Component from Normal Eggs which Corrects the Maiernal Effect of Gene o in the Mexican Axolotl (Ambystoma mexicanum)

ROBERT BRIGGS AND J. T . JUSTUS’ Department of Zoology, Indiana University, Bloomington, Indiana

ABSTRACT Axolotl females homozygous for o produce eggs which cleave normally, but never develop beyond gastrulation. This cessation of development is due to a cytoplasmic deficiency which can be corrected by injecting eggs of o/o females with cytoplasm from normal eggs. When so injected, the recipient eggs develop beyond gastrulation and may attain larval stages. The corrective component of normal cytoplasm has the following characteristics: 1. It remains in the supernatant of normal egg homogenates after two hours of centrifugation at 105 g, but is gradually sedimented thereafter. 2. The corrective activity persists for at least eight days when preparations are stored at 0°C. It is abolished on heating for one hour at 50” to 55’, and on incuba- tion with crystalline trypsin. 3. The active material is customarily extracted from egg homogenates in 0.1 M or 0.2 M KC1, with 0.01 M Tris, pH. 7.6. It can be precipitated by 2M to 3M ammonium sulfate and retains its corrective activity. It is also precipitated by distilled water, but is then irreversibly denatured. 4. The corrective component is found mainly within the germinal vesicle of large oocytes; then in the egg cytoplasm or extracts thereof following germinal vesicle breakdown. Comparable extracts of blastulae show a reduced corrective activity. Attempts to extract the active material from older embryos and from organs of adults have so far failed. These results suggest that the corrective material, presumably a product of the normal allele of 0, depends upon a protein or proteins for its activity. It is produced during oogenesis and later plays an indispensible role in early organogenesis.

Gene o, recently described by Humphrey (’66), exerts an interesting combination of effects in the axolotl. The gene acts as a simple recessive, heterozygotes ( + / o ) being indistinguishable from wild type animals. When heterozygotes are mated with each other they produce offspring of the expected genotypes, as illustrated in figure 1. Of these, the +/+ and + / o are completely normal. The homozygous re- cessives ( o / o ) also are normal through embryonic and early larval life, but later, at about two months of age, begin to show the effects of the gene. They grow somewhat more slowly than their normal sibs, and become somewhat darker in pigmen- tation. However, their most striking feature is a drastic reduction in capacity to regenerate amputated limbs. Normal larvae can, of course, regenerate perfect limbs; by contrast, o/o larvae two months old or older display a delayed and abnormal regeneration. This reduction in capacity for regeneration is a reliable diagnostic feature of the O/O’S. About 25% of the offspring of

J. Em. ZOOL., 167: 105-116.

heterozygotes wiU show the reduced and abnormal regeneration. The animals so selected, almost without exception, later show the other effects of the o/o genotype. The most important of these are the effects on the germ cells. About one-half of the affected animals remain more or less juvenile with respect to secondary sexual characteristics, and on further examination are found to be sterile males with small testes in which the germ cells remain predominantly in the spermatogonial stage and display degeneration, also usually in spermatogonial stage. The remaining half of the o/o’s are females with ovaries of normal appearance. These females can be successfully mated with either +/+ or + / o males and will then spawn fertile eggs. The cleavage of these eggs appears completely normal until late blastula stage

IContribution no. 805 from the Department of Zoology Indiana University. This investigation was supportAd by research grant R01 GM 05850, Research Grants Division, National Institutes of Health, U. S. Public Health Service.

2 Present address: Roswell Park Memorial Institute. Springville Laboratories, Springville, New York.

105

106 ROBERT BRIGGS AND J. T. JUSTUS

+/o x +/o

normsi notma I

?74+ % YO

normal narmal poor regeneratiin males-ster ~ l e ~ a l e s - m a T e t n a l

effect

%8 x %$!

+/O ;q i

arrested a r rested gast r h . g ast'r u 1 a

Diagram illustrating the effects of the o gene in the axolotl. See text (Introduction) for description.

is reached, toward the end of the first day of development. The cleavage rate is reduced at this time, and the embryos then enter gastrulation with cells noticeably larger than those of normal embryos of the same stage. The embryos proceed part way through gastrulation, usually not beyond the crescentic blastopore stage, and then cease development. In rare spawnings they may proceed to the end of gastrulation before they stop, but in no case have they ever formed neural folds. This cessation of development has occurred without exception in thousands of embryos derived from eggs of o/o females, and is observed whether or not the normal aIlele of gene o is introduced by the sperm at fertilization. Thus, the block to development beyond gastrulation must be due to a maternal effect of gene o-a modification of the egg cytoplasm produced by the o/o genotype during oogenesis and later expressed in the manner described above, regardless of the genotype established at fertilization.

Fig. 1

Since the egg cytoplasm is known from classical experimental embryology to set the pattern for early organogensis, any gene which modifies it is of potential interest, especially if the modification results in a cessation of development just at the beginning of organogenesis, as is the case with gene 0. For this reason we have under- taken an investigation of this gene-induced cytoplasmic change. Work already pub- lished has shown that this change is a deficiency which can be corrected by a component or components from normal (+/+ or +/o) oocytes (Briggs and Cas- sens, '66). The corrective component (s) is accumulated first within the germinal vesicle (nuclear sap) of ovarian oocytes. After the breakdown of the germinal vesicle at meiosis the corrective component is found in the egg cytoplasm. The material must be injected directly into the recipient o/o eggs in order to exert its corrective effect, leading to development beyond the gastrula stage. If only one of the first two blastomeres is injected, then only that part of the embryo which is derived from the injected blastomere shows the correction (Cassens, '65) . Thus, the corrective component appears to be incapable of passing through the plasma membrane in effective amounts. To this information we may now add the results of the experiments to be described below, indicating that the material correcting the maternal effect of o is macromolecular and depends on protein for its activity.

MATERIALS AND METHODS

In attempting to characterize the component of normal cytoplasm which corrects the deficiencies of the o/o's we usually fractionated mature normal eggs in various ways, and then tested the fractions for activity by injecting them into fertilized eggs of o/o females. The details concerning the preparation of the various fractions will be given in the appropriate sections of the main part of the paper. We will de- scribe here only those parts of the procedure common to all experiments.

Source of test eggs from o/o females. These eggs, as well as those of normal females, were generously provided by Dr. R. R. Humphrey. In the case of the o/o's, the females were mated with normal males,

MATERNAL EFFECT OF GENE o IN THE AXOLOTL 107

usually of +/+ genotype. Occasionally +/o males were used. As mentioned in the Introduction, the genotype of the male has no influence on the expression of the maternal effect. In either case, when the matings were successful the females began to spawn (at ca. 20” C) about 20 hours after the animals had first been put to- gether. The spawned eggs were collected at hourly intervals, approximately, and examined for sperm pits. The fertile ones (those with sperm pits) were demembranated at about two hours following spawning, and were injected at 2-4 hours, while still uncleaved, or at about six hours when they were beginning the first cleavage.

In order to ob- tain large numbers of normal eggs, females of +/+ (occasionally + / o ) genotype were injected with 250-350 Interna- tional units of FSH (Armour Co.), sometimes with 2-3 mg LH added. The spawnings began about 20 hours later and continued for another 18 to 24 hours. The eggs were collected and stored in their membranes in dechlorinated tap water at about 4” C until the spawning was completed or nearly so. They were then manually demembranated, and fractionated according to procedures to be described below.

Injection procedure. Injections of o/o eggs were carried out in a divided Petri dish, both sides of which contained a layer of 2% agar. The recipient eggs were placed, submerged in Steinberg’s (’57) solution, in depressions in the agar on one side of the dish. A small droplet of the material to be tested was then placed on the surface of the “dry” agar in the other half of the operating dish. The tip of a micropipette was immediately lowered into the droplet, filled to the desired level, raised to clear the barrier between the two sides of the dish, lowered again, inserted into an o/o egg, and its contents injected. Eggs were usually injected in groups of about ten eggs each, the whole series taking about five minutes to complete. The most important technical detail, upon which the success of the injections mainly depends, concerns the construction of the micropipettes. These were made from precision drawn, thin-walled, 1 mm (O.D.), Pyrex capillary tubing, provided

Source of normal eggs.

by Drummond Scientific Company, Broomall, Pa. From this stock the micropipettes were drawn with the Livingston Micropipette Puller (Otto Hebel, Swarth- more, Pa.), to an outside diameter of 10 to 15 IA. The advantage of the Livingston machine is that, once it is adjusted to pull pipettes of a particular size, large numbers of such pipettes can be made rapidly. The pipettes come out of the machine with their tips closed. The tips were broken off with watchmaker’s forceps just before use, to give a sharp leading edge which enters the egg readily. A second technical detail concerns the choice of a micromanipulator. Many types might be satisfactory, but we have found the Leitz micromanipulator and injection apparatus to be convenient because of the ease and rapidity with which pipettes can be changed and po- sitioned on this apparatus.

Rearing of injected o/o eggs. After being injected, the o/o eggs were transferred into agar-bottomed small Petri dishes containing 20% Steinberg‘s solution with antibiotics (see notes to table 1). They were reared throughout in this solution, at a temperature of about 20” C.

RESULTS Fractionation of normal cytoplasm by

centrifugation. Undiluted whole cytoplasm from normal mature eggs, when injected into eggs of o/o females, corrects the maternal effect of gene 0. The effect of the injected cytoplasm is unmistakable. Untreated o/o eggs never develop beyond gastrulation while those receiving normal cytoplasm, in amounts equal to about 2% of the recipient egg volume, neurulate and proceed to later stages of development (Briggs and Cassens, ’66). Egg cytoplasm is, of course, a very complex mixture- consisting of several types of organelles (some peculiar to eggs) suspended in an amorphous ground substance. Thus, an obvious first step in the characterization of the active component of normal eggs was to fractionate egg homogenates centrifugally, and then to determine whether the capacity to correct the maternal effect of o is associated with any of the sedi- mentable components. Two sets of experiments of this type were carried out, with similar but not identical results. In the


TABLE 1 Fractionation of undiluted cytoplasm of f/+ eggs

Material injected

Control - nothing injected Whole cytoplasm S1 (2,000 g-7 minutes) Sz (20,000 g-20 minutes) S3 (105 g-2 hours) S4 ( 105 g-7 hours) P4 + s 4 P4 + 0.1 M KC1 (0.01 M WS, p H 7.6)

Number of Cleavage recipient o/o eggs Partial Complete

477 477 45 8 34 18 14 1 9 2 7

82 13 61 33 4 29 30 4 26 18 14

Number of o/o showing correction (Develop to neurulation and beyond)

0 34 8 7

66 0

27 10

~~

1. Preparation of cytoplasm fractions: Donor eggs were obtained from hormone-injected females (see Materials and Methods). The capsule and outer jelly layers were removed with forceps leaving the egg surrounded only by the thin vitelline. membrane.. In each experiment three hundred’or more eggs were prepared in this way, washed twice m t h Stemberg‘s (’57) solution, and pipetted into a “Corex” centrifuge tube. The eggs settled immediately to the bottom of the tube and excess finid was then removed as completely as possible mth a Pastew Pipette. The packed eggs were covered with a layer of mineral oil (Squibb), homogenized gently with a loose fitting plunger and run through the series of centrifugations listed in the table. The first of these, a low speed kpb, gives a large pellet (yolk and pigment) and a supernatant (Si). This S1 is centrifuged at intermediate speed to give a second pellet (mitochopdr?al) and the secqnd supernata?t (SZ) which in turn is centrifuged at high speed for the times indicated. All operatzons were carried out at 00 tn AW

2. Iniection pfocedure: Approximately 0.04 PI of each fraction was injected- i&o‘<ach-oi $e test eggs. Injections were sometxmes done abou! 1 to 3 hours Prior to first cleavage. More frequently the eggs were injected just after the b e m m g of the first cleavage. In this case each nf tho firrt two biastomeres was injected with about one-half of the total dose- Injections were L>Z,,Z--ZX the recipient were submkrged in Steinberg’s solution containing 0.01% peniciJ&, and 0.05% sodium elkosin. After the injections were completed the eggs were transferred into 20% Steinberg’s solution contaming 0.016% Ca (N03)~.4HsO and antzbiotics as listed above.

&st of these we packed eggs into a centrifuge tube and removed as much as possible of the surrounding fluid so as to avoid diluting the cytoplasm. The packed eggs were covered with mineral oil, homogenized very gently, the homogenate fractionated centrifugally, and the fractions tested by injection into o/o eggs. The results are summarized in table 1. They show that the activity remains in the supernatant following centrifugation at high forces (lo5 g) for as long as two hours. An additional five hours of centrifugation gives an inactive supernatant and a pellet which, on being taken up in either the supernatant or in KC1-Tris, can be shown to contain the active component.

In the second set of experiments we added to the packed eggs an equal volume of an aqueous medium. The medium first used was 0.25 M sucrose (with l o 3 M MgCL), with or without Tris buffering (pH 7.5). For reasons as yet unknown to us, homogenates prepared with this medium were completely inactive. We then used 0.1 M KC1 (with 0.01 M Tris, pH 7.6) as an homogenizing medium, with results summarized in table 2. These are similar

to the results obtained with undiluted homogenates in showing that the active component remains in the supernatant after centrifugation for two hours at lo5 g. However, on continued centrifugation a difference appears between the two sets of experiments. As already noted, an additional five hours of centrifugation sediments the active material completely from homogenates of undiluted cytoplasm. In the case of the diluted homogenates prepared with KC1-Tris, an equivalent period of centrifugation gives a small pellet with some corrective activity, but most of the active component remains in the supernatant.

The main facts emerging from the experiments described above are: (1 ) that centrifugations (lo5 g, two hours) com- monly used to sediment the smallest organelles (ribosomes) fail to sediment the active component of nomal cytoplasm, and (2) that the active component is gradually sedimented on long continued centrifugation, the rate of sedimentation being greater in the more concentrated homogenates. It would appear, then, that the component of normal cytoplasm which


TABLE 2 Fractions of +/+ egg cytoplasm in equal volume of KC1 (0.1 M) + TrLs pH 7.5 (0.01 M)

No correction Correction

Number of Cleavage recipient o/o eggs Partial Complete

Material injected

Control - Whole cytoplasm 18 9 9 2 5 3 7 Pz (22,000 g-20 minutes) 9 9 9 P3 (105 g-2 hours) 39 15 5 17 1 53 (105 g-2 hours) 30 6 21 3 16 8 Pq ( 10s 8-7 hours) 15 4 8 3 5 4 54 ( 105 g-7 hours ) 13 2 11 3 3 2 5

nothing injected 136 2 134 136

Normal eggs were obtained and demembranated according to the procedure outlined in the notes to table 1. They were allowed to settle to the bottom of Corex centrifuge tubes, after which an equal volume of KCI- Tris was added, the eggs gently homogenized, and then fractionated by centrifugation. Supernatants (SS and S4) were concentrated against carbowax 4000 (50% w/v in KC1-Tris) to 1/5 to 1/10 of initial volume, or until slight cloudiness appeared. Pellet material (Pz, Pa, P4) was suspended in approximately the same volumes fi.e., the same as the final supernatant volumes) in KCI-Tris.

Injection procedure: As described in the notes to table 1. Development of recipient o/o eggs: Diagrams illustrate the extent of development of control and experi-

mental eggs. The controls all stopped in early gastrula stage, with cells larger than normal for this stage. Experimental eggs which showed correction of the maternal effect developed to late gastrula, neurula, or post- neurula stages. The embryos which stopped in late gastrula or neurula stages usually displayed a persistent yolk plug, and deficiencies in Fxial organs, but were none the less clearly superior to the controls in their development. Embryos developlng to post-neurula stages were sometimes normal or nearly so, but more frequently displayed deficiencies such as persistent yolk plugs and smaller than normal head structures.

corrects the maternal effect of o is a macromolecule, not necessarily associated with any of the cytoplasmic organelles. That the activity is sedimented more readily from the undiluted homogenates suggests that the macromolecules may be forming aggregates in the more concentrated preparations.

Table 3 summarizes the results of experiments on the stability of the corrective material at various temperatures. Since very little material was required for these tests, they were usually carried out with germinal vesicles isolated from normd ovarian eggs. The active material is found in its most concentrated and effective form in the nuclear sap of the germinal vesicle-an ideal source provided only very small volumes are required (see Introduction).

The germinal vesicles were isolated according to procedures given in the notes to table 3, and exposed to various temperatures for various periods of time. The nuclear sap was then injected into o/o eggs, with the results given in the table. These results show that the active material is quite stable in the cold. Its capacity to correct the maternal effect of o appears undiminished after eight days at -0.5" C. After 19 days at this temperature the ac-

Stability of the corrective factor.

tivity is reduced but still detectable. At elevated temperatures, on the other hand, the corrective activity is rapidly lost. At 40" and 45" the germinal vesicles retain their activity for one hour; at 50" the activity is definitely reduced; at 52" and 55" a white precipitate forms which is insoluble in our standard medium (0.1 M KCl-0.01 M Tris, pH 7.6). The soluble material that is left when this precipitate is removed has no trace of corrective activity.

Effects of enzymes on the corrective factor from normal eggs. A standard procedure in the characterization of bio- logical macromolecules is to determine the effects of various enzymes on their activity. In the present study this involved incu- bating supernatants of normal egg homogenates with the enzymes, and then injecting the treated supernatants into o/o eggs to determine whether their corrective activity was affected. The results of these tests, along with the experimental details, are given in table 4. The following points can be made from the evidence presented in this table: (1) In these experiments, as in others, the untreated control o/o eggs were without exception arrested in gastrulation. (2) When they were injected with supernatant from nor-

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TABLE 4 Effects of enzymes on normal egg supernatant

No correction Correction

Cleavage Treatment of Tz$$:$f o / o eggs Abort. Partial Complete

Untreated 73 12 13 48 8 9 31 16 Trypsin; then

Trypsin inhibitor

DNAase 15 3 12 5 5 5

RNAase; then bentonite or macaloid 40 40

trypsin inhibitor 63 19 16 28 39 31

alone 29 6 23 3 3 11 12

Controls - nothing injected 199 199 1. Experiments with trypsin and trypsin inhibitor: Normal egg supernatant was prepared according to the

procedure described in the notes to table 1. It was then mixed with 1/10 volume of trypsin ( 2 x crystallized, salt free, Worthington or Sigma) to give final concentrations of 10 fig/ml and 50 &g/mf. The combination was incubated at 20°C for 70 minutes. It was then mixed with 1/10 volume of soybean trypsin inhibitor ( 5 x crystallized, N.B.C., in Steinberg's solution without Ca, pH 7.4) to give final inhibitor concentrations of 40 fig/ml and 100 &g/ml. These mixtures were incubated an additional 70 minutes at 20°C, then placed in ice Unt11 they were injected mto fertilized eggs of o / o females. Each egg received a volume of about 0.04 &I.

Normal egg supernatant was mixed with 1/4 volume of DNAase I (Worth- ington, crystallized) to give an enzyme concentration of 0.2 mg/ml, with 0.001 M Mg, at pH 7.4. The mixture was. incubated at 20°C for 50 to 90 minutes, then injected into fertilized eggs from o/o females. Each egg received about 0.04 pl. 3. Experiments with RNAase: Normal egg supernatant was mixed with 1/10 volume of RNAase (Worth-

ington, crystallized from EtOH, in Steinberg's solution) to give enzyme concentrations of 1 fig/ml and 10 &g/ml. The mixtures were incubated for 45 to 60 minutes at 20°C. One-fifth volume of a suspension of maqaloid or bentonite was then added to give final concentrations of 2 mg/ml and 10 mg/ml. After an additional 40 to 60 minutes at 20°C the mixtures were centrifuged for 15 minutes at 37,000 g and the supernatants then injected into fertilized eggs of O/O females in volumes ranging from 0.005 to 0.04 &l per egg.

2. Experiment with DNAase:

ma1 egg homogenates, the large majority of the recipient o/o eggs developed to neurula stages and beyond. This simply demonstrated that the particular supernatant used in these tests was active in correcting the maternal effect of gene 0. (3) This supernatant was incubated with trypsin for one hour at 20". Trypsin inhibitor was then added to stop the action of the enzyme. This step was necessary for other- wise the preparation would interfere later with the cleavage of the injected eggs. The supernatant was then injected into o/o eggs. These eggs cleaved normally and formed blastulae, but failed to develop beyond gastrulation. Trypsin inhibitor alone had no such effect-supernatants treated with it retained their corrective activity. It would appear from this experiment that trypsin abolishes the corrective activity of normal egg supernatant. (4) Active supernatant was treated with DNA- ase, then injected into o/o eggs. The recipient eggs developed to advanced gas-

trula and neurula stages, indicating that the enzyme had not abolished the corrective activity. (5) We were anxious to test the effect of RNAase on the corrective activity of normal egg supernatant, but were frustrated in this effort by the pro- nounced inhibitory effect of this enzyme on the cleavage of the recipient eggs. Our plan was to incubate the supernatant with RNAase, then add bentonite or macaloid and subsequently remove the adsorbed enzyme by centrifugation before injecting the supernatant into o/o eggs. Preliminary tests showed that macaloid (and to a lesser degree, bentonite) could remove the enzyme from solution when supernatant was not present. However, in the presence of supernatant the absorbent was less effective, and enough RNAase re- mained in the supernatant to stop all injected eggs in early cleavage. For this reason we were not able to determine what effect, if any, the enzyme might have on the component of normal eggs which corrects the maternal effect of o.

112 ROBERT BRIGGS

Salting out of the corrective component with ammonium sulfate. The studies so far described show that the active component of normal cytoplasm is non-particulate, sediments slowly in the ultra- centrifuge, and is inactivated by trypsin and by moderate heating (52"). These findings encouraged us to try isolating the active material by one of the classical methods of protein chemistry-salting out with ammonium sulfate. The details of the procedure are given in the notes to table 5. It involved successive additions of the salt to a water-clear, high speed supernatant from normal eggs. The precipitates that formed at each salt concentration were dissolved in KCl-Tris, dialysed: concentrated, and Snally injected into o/o eggs. The results of these tests showed that the corrective activity was always in the 2M or 3M fractions-usually in both. (see table 5). In one experiment the 2M and 4M fractions were active, and the 3M fraction inactive. However, this was the only experiment (out of 6) in which the 4M fraction showed any activity. Possibly

AND J. T. JUSTUS

the 3M and 4M fractions, or the test eggs, were mis-labelled at some point in the whole procedure in this one experiment. With this one dubious exception, the activity was limited to the 2M-3M fractions. These fractions produced dehite corrections of the maternal effect of gene o, leading to neurulation and in some cases to limited abnormal post-neurula development on the part of the injected o/o eggs. These corrections were as extensive or more extensive than those obtained with the supernatants from which the fractions were isolated. However, it should be pointed out that the activity of these fractions, as well as that of the diluted and reconcentrated supernatants, is definitely lower than that of the most active material available-undiluted nuclear sap of germinal vesicles of ovarian eggs. The reasons for this difference are not clear. The corrective activity of the starting materid (mature egg cytoplasm) varies to begin

JDialysis was always carried out against 0.1 M KC1 + 0.01 M Tris, pH 7.6. If the supernatants. or ammonium sulfate fractions thereof. were dialysed against distilled water, a precipitate formed which appeared to be completely insoluble in KC1-Tris.

TABLE 5 Corrective activity of ammonium sulfate fractions of normal egg supernatant

Corrective effect on o/o eggs

Exp. SS Ammonium sulfate fraction of Sa no. (normal egg supernatant) 1 M 2M 3M 4 M Saturated Supernatant

~-~~ -

1. Preparation of normal egg supernatant: Unfertilized, demembranated eggs of normal axolotl females were homogenized gently in an equal volume of 0.1 M KC1 + 0.01 M Tris, pH 7.6. The homogenate was centrifuged at low speeds to remove the larger particulates; then at 100,000 g for two hours. This gave a water-clear supernatant - the S3 referred to in the table above. Part of this supernatant was concentrated to about 10% of its starting volume against carbowax 4000 (50% w/v in KCI-Tris, pH 7.6) and later tested for corrective activity by injection into fertilized eggs of o/o females. The .remainder of the supernatant was set aside for fractionation with ammomum sulfate. All operahons were carried out at Oo to 4"C.

2. Ammonium sulfate fractionation: Solid ammonium sulfate, recrystallized and pulverized, was added to the supernatant to give a 1 M concentration. This was allowed to remain for 60 to 90 minutes at O', during which time a light, white precipitate formed. The precipitate was removed by centrifugation, and more ammonium sulfate was then added to the supernatant to give a 2 M concentration. After 90 minutes the rather heavy white precipitate that had formed was collected by centrifugation. The process was repeated to give 3 M and 4 M precipitates, both rather heavy, and a light precipitate with saturated ammonium sulfate. All of these precipitates were dissolved in 0.1 M KC1 + 0.01 M Tris, pH 7.6, and dialysed a ainst the same solution overnight to remove the .ammonium sulfate. They were then concentrated against carbowax 4000 (see above) usually unu slight traces of cloudiness appeared. If significant precipitation occurred, small volumes of KCI-Tris were added to brin the recipitate back into solution. The fractions so prepared were injected into fertilized eggs of o / o &males in volumes ranging from 0.006 to 0.04 fil per egg. Each fraction, in each experiment, was injected into 10 to 35 eggs.

3. Controls: A-total of 365 uninjected fertilized control eggs were available in the experiments summarized m this table. All were arrested u1 early gastrula stage, in the manner typical of eggs of o/o females.


with. Further variations, presumably losses, may occur at one or another of the steps in the preparative procedure outlined above. We have attempted to improve the yield of active material by starting the isolation procedure with germinal vesicles rather than with whole mature eggs. This requires a method for mass isolation of germinal vesicles, and we have not yet succeeded in devising such a method for axolotl eggs. Attempts have also been made to separate the active material on Sepha- dex G 75, but all fractions coming off the column were completely inactive. We mention these problems to indicate that much remains to be done to improve the isolation of the material that corrects the maternal effect of gene o. But despite these reservations concerning the present experiments, they do show that the ammonium sulfate precipitates are at least as active as the supernatants from which they were isolated, indicating again that a protein is involved in the correction.

Effect of extracts of older embryos and of adult organs on o/o eggs. For both practical and theoretical reasons it is of considerable interest to know whether the active compound of normal eggs is present only in eggs, or can be found in other cell types of normal animals as well. One

set of experiments was done to test this point. Older embryos, and certain adult organs, were homogenized and centrifuged to give high speed supernatants comparable to the supernatants prepared from eggs. These supernatants were injected into o/o eggs, with the results given in table 6. These results show that blastula supernatants give a slight correction of the maternal effect, but one that is definitely inferior to the corrections obtained with egg supernatants. Supernatants from older embryos, and from adult testis, liver, and spleen, all were completely inactive. From this experiment it appears that the active material rapidly disappears from the soluble, non-particulate, component of embryonic cells in the course of early development.

DISCUSSION

Humphrey’s (’66) discovery of gene o provides us with a rare opportunity to study an ooplasmic component, which o/o eggs lack, and which is absolutely essential for early organogenesis. We know that the o/o eggs are deficient in this component because it can be transferred into them from normal eggs and will correct the deficiency. The effect is striking. Without the corrective component the eggs

TABLE 6 Effects of extracts of older embryos and of adult organs on o/o eggs

Number of Cleavage No Partia! recipient Partial Complete correction correchon o/o eggs

Source of extract

Control - nothing injected 145 6 139 139 Blastula 10 10 3 7 Gastrula 20 5 15 20 Tailbud 30 2 28 30 Adult organs:

Testis 16 16 16 Liver 35 7 22 29 Sdeen 12 1 2 3

The extracts listed in this table were prepared as follows: 1. Blastulae: Several hundred normal late bastulae were placed in a centrifuge tube. All

excess fluid was removed with a fine pipette a layer of mineral oil was added, and the blastulae gently homogenized. Large particulates were’ removed by a low speed centrifugation (20pO g, . lo minutes). The supernatant was spun at 105 g for two hours. The supernatant of thzs centnfugahon was tested on ferhhzed eggs of o/o females, each egg receiving an injechon .of about 0.04 ~ 1 .

Two hundred and sixty-seven early gastrulae were homogenized m an equal volume of 0.1 M KC1 + 0.01 M W s pH 7.6. Larger particulates were removed by centrifugations at 2000 g and 22,000 g. The supernatAnt from the 22,000 g centrifugation was spun at 105 g for five hours. The supernatant was concentrated against carbowax (50% w/v, in KC1-Tris) from 2.0 ml to 0.3 ml. The pellet was taken up in 0.1 ml KCI-Tris. Both fractions were injected (ca. 0.04 81 per egg) into fertilized eggs of o/o females. 3. Tailbud embryos: 4. Adult organs were homogenized, in a ‘Tertis” homogenizer, in equal volumes of either 0.25 M

sucrose or KCI-Tris containing 12% hexylene glycol, pH 7.5. The homogenates were centrifuged (30,000 g, 30 minutes), the svpernatants dialysed overnight against KC1-Tris, centrifuged again (105 g 5 hours) and the resulhng supernatant and pellet fractions assayed for activity by injection into fgrtilized e&s of o/o females. Injections were done into both cells at the early two cell stage. Volumes per egg ranged from 0.005 to 0.1 d.

2. Gastrulae:

The procedure was the same as that given above for the gastrulae.


of o/o females invariably stop in late blastula or in gastrula stages and never show any differentiation. With it the development proceeds to later embryonic stages, and may give viable larvae with the complete array of fully differentiated func- tional cell types. The component in ques- tion is thus involved in some essential way in the transition from the early phase of development, characterized by rapid cell division without dserentiation, to the later phases in which mitotic rate drops off and cell differentiations occur. Sev- eral questions now arise concerning the nature, origin, and mode of action of the corrective substance. As to its nature, earlier work showed only that it fails to pass through the plasma membrane, and is therefore probably macromolecular (Cassens, '65). This expectation has been borne out in the experiments reported in this paper, in which we show that the active component of normal cytoplasm sediments slowly at lo5 g, and has prop- erties characteristic of proteins. In particular, the heat lability and trypsin sen- sitivity of normal egg supernatants indicate that their capacity to correct the maternal effect of o depends upon a protein or proteins. Other types of substances could also be involved, but we have no evidence of this at present.

Concerning the origin of the active material we have as yet very little information. The material must, of course, be a product of the normal allele of 0. Evi- dence of the activity of the gene can be obtained by assaying extracts of normal eggs, embryos, and adult organs for its product-the material which corrects the maternal effect of 0. So far, we have found the corrective substance in large oocytes and mature eggs, but not in embryos at gastrula stage or older, nor in adults. On the basis of this evidence we can draw a limited conclusion, which is that the + gene must synthesize a store of the material during oogenesis. The fact that we do not detect the active material in extracts of older embryos and adults is more difficult to interpret. It might actually be absent, or it might be present but not in an extractable form. For ex- ample, it might be transferred from the soluble phase of the cytoplasm to cyto-

plasmic or nuclear organelles during cleavage, and would then not appear in the supernatants we prepare of older embryos. More work must be done on this problem, but meanwhile we can get some clues concerning the function of the + gene from other types of observations, as follows: ( 1 ) The results of matings of o/o females with + / o males indicate that the + gene is inactive during early development. One half of the zygotes from such a mating will be o/o and the other half +/o. Yet, all embryos stop developing at gastrula stage (Humphrey, '66). Thus, there is no indication that the + gene can function during these early stages of development. (2) Matings between heterozygotes also tell us something about the function of the normal allele of o. In such matings all eggs will have been produced under the influence of the + /o genotype. After fertilization 25% of the zygotes will be o/o in genotype. These zygotes are indistinguishable from the +/+ and + / o siblings through embryonic development, and reach feeding larval stages before any effects of the absence of the + gene can be detected. Thus, the + substance(s) stored during oogenesis must be sufficient by itself to promote normal development to larval stages without any requirement for additional synthesis. Later in larval development these o/os (from + /o females) do show deficiencies, especially in regeneration and spermatogenesis, indicating that these processes require that the + gene be present and active.

Humphrey ('66) has discussed possible modes of action of gene o in his paper re- porting the discovery of the gene. Here we would like to consider briefly just the maternal effect of the gene. As we have already noted, the eggs of o/o females cleave normally until mid- to late blastula stage when the cell division rate slows down. The embryos then enter gastrulation with cells somewhat larger than normal, and are later invariably arrested prior to neurulation. Recently these embryos have been studied cytologically. Cassens ('65) and Carroll ('67) have both found a very sharp drop in mitotic frequency in blastulae at about Harrison stage 8+. This reduction occurs in all parts of the blastula. Carroll also fmds that the embryos possess


normal complements of chromosomes, and that the complements remain normal throughout the time during which analys- able mitoses occur. Thus, the cessation of development is not associated with the appearance of karyotypic abnormalities, nor does it appear to be localized in any spe- cial embryonic region. An interesting ques- tion, which has not yet been explored, concerns RNA synthesis in o/o embryos. Recently it has been established that nuclear RNA synthesis undergoes a rapid and striking increase in normal mid- to late blastulae of Xenopus (Bachvarova and Davidson, '67; Brachet, '65; Brown and Littna, '64). The stage of development at which this increase occurs is the same as that at which the maternal effect of o first becomes apparent in the axolotl. It might be of real interest to determine whether embryos from o/u females lack this activation of nuclear RNA synthesis, and to ex- plore what effects might be exerted on their synthetic activity by the material from normal eggs, which corrects the maternal effect of 0.

ACKNOWLEDGMENTS We wish to thank Dr. R. R. Humphrey

for having provided all of the mutant and

normal axolotl eggs used in this investigation, and for a critical reading of the man- uscript. We also wish to thank Mrs. Carolyn Huffman and Mrs. Dorothy Barone for valued assistance in the laboratory.

LITERATURE CITED Bachvarova, R., and E. H. Davidson 1966 Nu-

clear activation at the onset of amphibian gastrulation. J. Exp. Zool., 163: 285-296.

Brachet, J. 1965 The role of nucleic acids in morphogenesis. Progress in Biophysics and Molecular Biology, 15: 99-127.

Briggs, R., and Gloria Cassens 1966 Accumula- tion in the oiicyte nucleus of a gene product essential for embryonic development beyond gastrulation. P.N.A.S., 55: 1103-1109.

Brown, D. D., and E. Littna 1964 RNA synthesis during the development of Xenqpus laevis, the South African clawed toad. Jour. Mol. Biol., 8: 669-695.

Carroll, Carole 1967 Personal communication. Cassens, Gloria 1965 Personal communication. Humphrey, R. R. 1966 A recessive factor ( 0 ,

for ova deficient) determining a complex of abnormalities in the Mexican axolotl (Amby- stoma mexicanum). Develop. Biol., 13: 57- 76.

Steinberg, M. 1957 Carnegie Institute Wash- ington Year Book. 56: 347 (Report by J. D. Ebert ).

partial characterization of the component from normal eggs which corrects the maternal effect of...

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