DEVELOPMENTAL
BIOLOGY
The Distribution
58, l-10
of Soluble
TERRY Department
(1977)
L. MOEN
of Zoology,
Received
March
Proteins along the Animal-Vegetal Frog Eggs AND MARION
University
31,1976;
of Wisconsin,
accepted
in revised
Axis of
NAMENWIRTH’ Madison, form
Wisconsin
January
53706
25,1977
We describe a procedure for rapidly dividing hundreds of frog eggs into transverse slices along the animal-vegetal axis. We have used this method to study the spatial distribution of soluble proteins in fertilized uncleaved eggs and late blastula embryos of Xenopus laevis. Approximately 25% of the protein bands we resolve by electrophoresis are present along only part of the egg’s animal-vegetal axis. INTRODUCTION
Gurdon, 1968; Donohoo and Kafatos, 1973; Lutzeler and Malacinski, 1974; Newrock and Raff, 1975). We describe here an efficient method for dividing large numbers of fertilized frog eggs into transverse slices along the animal-vegetal axis. With this procedure, groups of 50-100 eggs that have been artificially inseminated are placed in a monolayer inside a fluid-filled chamber and are permitted to undergo their normal rotation to the animal-pole-up position. The eggs are then quickly frozen into a single block of saline solution. This block is sectioned with a cryostat microtome to give the desired number of transverse slices from vegetal to animal pole. Comparable slices from many such blocks are pooled and used as starting material for a biochemical extraction. We have used this procedure to study the spatial distribution of soluble proteins along the animal-vegetal axes of fertilized uncleaved eggs and late blastula embryos of Xenopus laevis, the South African clawed frog.
In studying the molecular events that accompany early embryonic development and differentiation, numerous investigators have compared macromolecules synthesized or activated at successive stages of embryonic development (e.g., Crippa and Gross, 1969; Whiteley et al., 1966; Terman, 1970). Experiments of this sort almost always begin with the homogenization of entire eggs or embryos, so that all biochemical events are monitored at once regardless where they occur in the embryo. Very little information has yet been gathered on the spatial distribution of molecules within eggs and early embryos? primarily because this type of study is cumbersome to do. The most widely used biochemical procedures require large amounts of embryonic starting material. Yet, except for the velocity sedimentation technique adapted by Hynes and Gross (1970) for separating the macromeres, mesomeres, and micromeres of the 16-cell sea urchin embryo, virtually the only MATERIALS AND METHODS methods in use for dividing early embryos Obtaining synchronously developing, into different regions involve dissection or jelly-free embryos. Eight to twelve hours sorting of embryos one at a time (e.g., after injection of 800-1000 units of human Flickinger, et al., 1966; Woodland and chorionic gonadotropin (Antuitrin-S, Parke, Davis and Co.), Xenopus Zaevis fe’ To whom all correspondence should be admales were gently squeezed to obtain sevdressed. Copyright All
rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
2
DEVELOPMENTAL
BIOLOGY
eral hundred unfertilized eggs. These were artificially inseminated, using methods adapted from Wolf and Hedrick (1971) and were permitted to develop for either 30 min or 7 hr (late blastula stage). The embryos were then dejellied by treatment for 3-5 min in a solution containing 15 g of cysteine-HCUliter of 10% Steinberg’s solution (Steinberg, 1957), adjusted to a final pH of 8.0-8.5. Residual cysteine was removed by rinsing embryos repeatedly in 10% Steinberg’s solution. Papain, a common ingredient of dejellying solutions, was not used once it became clear that traces of papain remain, even after extensive rinsing, and lead to subsequent protein degradation. Freezing embryos into blocks. A chamber suitable for freezing embryos was assembled by placing a brass ring (19-mm o.d., 16.5-mm id., 7.5-mm high), the lower surface of which had been lightly coated with silicone grease, upon an aluminum plate, the upper surface of which bore a thin film of silicone grease (Fig. la>. The chamber was filled three-fourths full with 10% Steinberg’s solution, and 50-100 fertilized dejellied embryos were pipetted in.
d
.
Q
h
FIG. 1. Schematic representation of the method we use to prepare blocks of frozen saline solution containing 50-100 fertilized frog eggs. The blocks are later sectioned with a cryostat microtome SO that all the eggs are divided simultaneously into transverse slices from vegetal to animal pole. For details see Materials and Methods.
VOLUME
58, 1977
The chamber was left undisturbed while the embryos completed their rotation to the animal-pole-up position. Any embryos exhibiting faulty rotation were removed by pipet and discarded. Liquefied Freon 22 (CHClF,) was collected in a small dish placed in a liquid nitrogen bath inside an insulated container (Fig. lb). The copper coil which was attached to the Freon container was repeatedly bathed in liquid nitrogen to lower the temperature of the Freon as it passed through the coil into the dish. Once the embryos had reached the desired stage of development, the entire embryo chamber was frozen by lowering it into the dish of liquihed Freon (Fig. lc). When the chamber had been partially frozen, 10% Steinberg’s solution was added until it overflowed. Once the solution was completely frozen, the chamber was removed from the Freon, and the brass ring containing the frozen block of eggs could be easily lifted from the underlying plate. Blocks still surrounded by their brass rings were then stored at -70°C to await further processing. Sectioning frozen eggs and blastulae. To prepare frozen blocks for mounting on the cryostat specimen holder, the original upper surface of each frozen block was scraped with a razor blade until it was perfectly flush with the upper surface of the brass ring that still surrounded it (Fig. Id). These blocks were then stored in a petri plate on dry ice with the original lower surface of each sample in contact with the plate. Note that the lower surface of the sample is almost flush with the vegetal poles of the frozen eggs, but there are several millimeters of ice between the upper surface of the sample and the animal poles of the frozen eggs. A specimen holder was prechilled on dry ice for a few seconds only. A drop of 10% Steinberg’s solution was placed on the cooled holder, and a frozen egg sample still ensheathed in its brass ring was quickly inverted and pressed down on the holder, so that the
MOEN
AND NAMENWIRTH
Soluble Protein Localization
vegetal poles of the eggs faced up (Fig. le). The loaded specimen holder was placed on dry ice for about 30 set and was then removed. A pair of heated forceps was held in contact with the metal ring (Fig. lf, g> until it could be lifted away from the frozen egg sample which adhered to the specimen holder (Fig. lh). The frozen egg samples were sectioned at 20 pm using a cryostat microtome prechilled to -30°C. Our objective was to divide each block of eggs into 10 transverse fractions. Since there is substantial variability in the diameters of eggs spawned by different females (1.2-1.5 mm), a test block of eggs from each spawning was first sectioned to determine the number of 20-pm sections necessary to section completely through an egg or blastula from that spawning. This calibration of egg diameter was repeated for each new spawning so that variability in egg diameters between different spawnings did not introduce unnecessary error. Nonetheless, even eggs within a single spawning may show slight variability in diameter. We estimate that this introduces a maximum error equivalent to one-tenth the diameter of an egg. Protein extraction. A set of 10 transverse fractions obtained from several thousand sectioned embryos provided the starting material for each series of experiments. All procedures were carried out at 4°C. The 10 fractions obtained from eggs frozen in 10% Steinberg’s solution (which contains 6 m&I NaCl + CO.1 mM KCl, CaCl,, and MgSO,) were thawed and homogenized with 30 strokes of a motor-driven ground-glass homogenizer. MgCl, was added to a concentration of 10 mIk?, and the samples were overlaid with mineral oil and were centrifuged at 20,OOOgfor 45 min. The mineral oil and associated lipid were aspirated and discarded. Part of each 20,OOOgsupernatant was immediately extracted (see ahead), whereas the remainder was again overlaid with mineral oil and centrifuged in a 50 Ti rotor at 200,OOOg for 8 hr.
in Eggs
3
The high-speed supernatant and the remaining low-speed supernatant from each of the 10 transverse fractions were treated as follows. Solid urea was added to a final concentration of 0.45 g/ml, and the samples were made 0.1% in SDS. A 30-~1 aliquot was removed from each sample and frozen. These were used later for protein determinations (Lowry et al., 1951). pMercaptoethanol was added to the remainder of each sample to a final concentration of 20 &ml. The samples were incubated at 65°C for 15 min and then were stored at -70°C until analyzed by acrylamide gel electrophoresis. Our extracts contained approximately 5 pg of protein/egg. Estimating the total amount of nonyolk protein in a Xenopus egg to be about 120 pg (R. Wallace, personal communication), our extracts contained 4% of the egg’s nonyolk protein. Yolk proteins and other proteins associated with large particulates, as well as proteins insoluble in dilute saline, were removed by centrifugation of the initial homogenates. SDS-Acrylamide gel electrophoresis. SDS-Acrylamide slab gels, 0.15-cm thick, were prepared according to the Laemmli formula (Laemmli, 1970) with a 9.75-cmhigh 10% acrylamide running gel, overlaid by a 0.75-cm-high 3% acrylamide stacking gel. A group of samples, each containing approximately 35 pg of protein, was thawed and then incubated for 2 min at 90°C. When the samples had cooled, 1 ~1 of 0.1% bromphenol blue tracking dye was added to each, and the samples were then loaded into the slots in the stacking gel. Electrophoresis was carried out at 100 V until the tracking dye reached the bottom of the gel (approximately 4 hr). The gel was stained for an hour in a freshly prepared filtered solution of 0.05% Coomassie brilliant blue + 0.05% buffalo black dissolved in 5 parts methanol, 1 part acetic acid, and 5 parts water. Diffusion destaining was carried out in 7% acetic acid. When the staining was judged to be
4
DEVELOPMENTAL BIOLOGY
too light, staining was repeated for another hour, and the gel was destained again. RESULTS
Fertilized
Eggs
Groups of eggs that had been artificially inseminated were allowed to develop for 1 hr at 21°C and then were frozen into blocks for cryostat sectioning. (Control eggs that continued developing at 21°C initiated first cleavage 1 hr and 20 min after fertilization.) The frozen eggs were divided into 10 transverse fractions from vegetal to animal pole. After thawing and homogenization, each fraction was centrifuged at 20,OOOgfor 45 min in order to pellet yolk platelets, mitochondria, nuclei, and other large particulates. The pellets were disand proteins were prepared carded, from a portion of each 20,OOOg supernatant. The proteins present in the 10 transverse egg fractions were then compared by electrophoresis in SDS-acrylamide slab gels (Fig. 2a). Approximately 50 protein bands were resolved in each transverse egg fraction, and about 80% of these bands appeared to be constant across the entire egg. The remaining protein bands change as one follows them along the egg axis from vegetal to animal pole. The changes are summarized in Fig. 2b. Included are seven protein bands that occur in the animal half and equatorial region of the egg but are absent from the vegetal region, two bands that are confined to the area near the animal pole, a faint band present only near the vegetal pole, and two bands that occur along the entire egg axis but show a pronounced change in concentration as one moves across the egg. Considering the very large number of soluble proteins present in Xenopus embryos, it is clear that each band on the gel must consist of many proteins of similar electrophoretic mobilities. To achieve better resolution we sought to reduce the number of proteins in our samples by subjecting the 20,OOOgsupernatants to furth-
VOLUME 58, 1977
er centrifugation at 200,OOOgfor 8 hr. Proteins prepared from the high-speed supernatants were then examined by SDSacrylamide slab gel electrophoresis. Figure 3A shows a gel separation of the proteins present in the high-speed supernatants of each of the 10 transverse egg fractions. Of the approximately 44 bands visible per fraction, about 75% seem to be constant along the animal-vegetal axis of the egg. The changing bands (Fig. 3b) include seven that extend from the animal pole through the equatorial region of the egg, two bands present only near the animal pole, one band confined to a narrow region near the equator, two bands present everywhere except near the vegetal pole, one band extending from the vegetal pole to the equatorial region, and one band extending across the entire egg but showing a markedly greater protein concentration in the vegetal half of the egg than in the animal half. Thus, in both the low-speed and highspeed supernatants of eggs, roughly 25% of the protein bands resolved on SDS gels occur along only part of the animal-vegetal axis. Most of these changing bands extend over half or more of the egg’s diameter, and most are represented in the animal and equatorial regions of the egg while being absent from the vegetal region. Blastula Embryos Further studies showed that the soluble proteins present in late blastula stage embryos have a quite analogous distribution along the animal-vegetal axis. Eggs fertilized by artificial insemination were permitted to develop to stage 9 (Nieuwkoop and Faber, 1967) before being dejellied, frozen, and sectioned from vegetal to animal pole. Figures 4a and 5a show gel separations of the blastula proteins contained in the 20,OOOgand 200,OOOgsupernatants, respectively. Figures 4b and 5b indicate the locations of the changing bands. Approximately 55 bands were resolved in each
MOEN
AND
NAMENWIRTH
Soluble
fraction of the 20,OOOg supernatant compared to 50 bands per fraction in the 200,OOOg supernatant. About one-third of these bands change along the animal-vegetal axis, and, as was observed also in fertilized uncleaved eggs, most of the changing bands extend from the animal pole through the equatorial region, disappearing in the vegetal region of the blastula. There are, of course, exceptions to this pattern. The high-speed supernatant of blastula embryos, for example, has three bands that are present in the vegetal half of the blastula, but which are absent from at least part of the animal half. Reproducibility The electrophoretic comparison of each set of protein samples was repeated several times to insure that the results were reproducible. In addition, we duplicated the entire experiment, beginning with different batches of frog eggs. We found remarkably little variation, other than an occasional one-fraction difference in the distribution of a band; e.g., a band that extended from the animal pole through fraction 6 in one experiment might extend only as far as fraction 7 in the second experiment. DISCUSSION
We have described
a method for rapidly
Protein
Localization
in Eggs
5
dividing hundreds of frozen frog eggs into transverse slices from animal to vegetal pole, making it possible, with conventional biochemical techniques, to study the distribution of macromolecules along the animal-vegetal axis. Using this procedure we observed that about 25% of the dilute saline-soluble protein bands resolved by electrophoresis on SDS-acrylamide slab gels occurred along only a portion of the embryo’s animal-vegetal diameter. These results certainly underestimate the actual extent of heterogeneity in the distribution of soluble proteins for, in view of the large number of different protein species present in eggs, one must expect that frequently a stained gel band consists of numerous proteins of similar electrophoretie mobilities. In such a compound band, individual proteins could be added or subtracted along the animal-vegetal axis without always altering the appearance of the stained band on the gel. In this way, some limited-distribution proteins would go undetected. Similarly, while most of our “changing” bands extended over half or more of the egg’s animal-vegetal diameter, some of these bands might contain component proteins having a much narrower distribution. Many of these uncertainties can be clarified by higher resolution two-dimensional gel separations. Yet,
FIG. 2. (a) SDS-Acrylamide slab gel of the soluble proteins detectable in the 20,OOOg supernatant of Xenopus laeuis eggs 1 hr postfertilization. Groups of 50 fertilized eggs were frozen and sliced simultaneously into 10 transverse fractions from vegetal to animal pole. Aliquots consisting of 35 pg protein prepared from the 20,OOOg supernatants of each of the 10 transverse egg fractions were then compared by electrophoresis in a 10% SDS-acrylamide slab gel. Proteins were visualized by staining the gel with Coomassie brilliant blue and buffalo black. Proteins from the vegetal pole slice are shown in the left channel. The second channel from the left shows proteins from the transverse slice just above the vegetal pole, and so on proceeding in order to the channel at the right which carried proteins from the animal pole slice. Migration occurred from top to bottom of the gel. Electrophoresis of marker proteins suggested the following rough relationship between molecular weight and the distance traveled along the centimeter ruler at the right: MW 135,000 (P-galactosidase) = 2.2 cm; MW 67,500 (bovine serum albumin) = 3.5 cm; MW 43,000 (ovalbumin) = 5.0 cm; MW 27,500 (chymotrypsinogen) = 7.3 cm; MW 18,400 (P-lactoglobulin) = 9.8 cm. (b) Diagram of those protein bands shown in gel (a), which change as one moves across the egg from vegetal to animal pole. The location of each changing band (given in the left column) indicates the approximate distance along the centimeter ruler in (a) that these proteins migrated. The gel bands that appeared constant along the animal-vegetal axis were omitted from the diagram. FIG. 3. Electrophoretic analysis of the proteins present in the 200,OOOg supernatant of Xenopus laeuis eggs 1 hr postfertilization. Eggs were frozen and divided into 10 transverse slices from vegetal to animal pole. Soluble proteins present in these 10 slices were compared. Details as in Fig. 2.
DEVELOPMENTAL
BIOLOGY
EGG - 20,OOOg
LOCATION km1
Vegetal Pa’e I
0.25
2
3
4
58,
VOLUME
5
1977
supernate
6
7
Animal Pale 9 10
8
-----m-
0.45
--
0.65
-
0.70
----
1.50
-----
1.55 1.80
~-----
2.00
----
2.35
----------
2.40
---m------
3.30
~-----
5.30
------
b FIGURE
it must be recognized that some proteins occur inside eggs in quantities so small as to make detection with conventional electr ophoretic procedures impossible. We are th lerefore observing only the more preva-
2
lent protein species in each egg fractia In. Although there were exceptions, it is interesting that, in both eggs and late blastula embryos, most of the limiter d-distribution bands were present in the : ani-
MOEN
Soluble
AND NAMENWIRTH
EGG
LOCATION (cm)
Protein
- 200,
OOOg
Localization
in Eggs
supernote Animal Pale
Vegetal Pale I
2
3
4
5
6
7
0
-----
0.5
--m
0.0 1.2 -__--__~
1.6 ~_---___
I.9
__------
2.1 --------
2.3 -----__
2.4 2.5
---~-~~~-~
2.7
--------
2.8
------
3.25 3.4
v-p--
5.9
--s-I
FIGURE
3
9
10
7
BLASTULA
-
20,ooog
WW~CI~~
!getar Pole I
LOCATION km1
2
Ininml
3
4
5
6
7
8
9
Poll? 10
0 55 0 62
----
0 75
-
1 00 I 05 I I5 I .40 I 45 1.65
--------mm
I 80
-----e-m---------A---mm
2 00
-
2 IO
-----
2 I5 2 20
------WC-
------_-
2 30 2 40
------A-_-
2 45
-
2 55
-----
_---------
2 80
----A---
5 95
b FIG. embryos. Soluble
Electrophoretic analysis of the proteins present in the 20,OOOg supernatants Stage 9 blastulae were frozen and divided into 10 transverse slices from vegetal proteins present in these 10 slices were compared. Details as in Fig. 2.
4.
8
of late blastula to animal pole.
MOEN
AND
Soluble
NAMENWIRTH
BLASTULAegem Pyle
LOCATlOll ICrnJ
Protein
Localization
200,OOOg
9
in Eggs
supernote Animal Pp2
23456769
0.45
----
0.70
----
I.60 I.85 I 95
--------__
2 IO
---
2.35
---_---_
2.40 m----------------__
2.80 3 30
-------
4.30 4.70 5.25
----__
6.30
b FIG. 5. Electrophoretic analysis of the proteins embryos. Stage 9 blastulae were frozen and divided Soluble proteins present in these 10 fractions were
present in the 200,OOOg supernatant into 10 transverse fractions from vegetal compared. Details as in Fig. 2.
ma1 and equatorial regions of the embryo, but were absent from the vegetal region. This cannot be attributed to the smaller quantity of cytoplasm present in the vegetal region, since we adjusted our samples
of late blastula to animal pole.
so that an equivalent number of micrograms of protein were loaded into each channel of the gel. Nor could the pattern have been caused by the nuclear proteins present in the animal hemisphere within
10
DEVELOPMENTAL
BIOLOC Y
the single fertilization nucleus, since a similar pattern occurs in the multinucleate late blastula embryo. Our conclusion that the frog egg is heterogeneous along its animal-vegetal axis will raise few eyebrows among experimental embryologists, for several forms of animal-vegetal differentiation in frog eggs are already familiar. The cortex of the egg is thicker in the animal and dorsal regions than in the ventral and vegetal regions. Yolk platelets and other cytoplasmic particulates vary in size and concentration, showing a complex distribution pattern from animal to vegetal pole (Nieuwkoop and Faber, 1967). In the eggs of various amphibian species, animal-vegetal as well as dorso-ventral gradients have been described for oxygen consumption, reducing activity, RNA concentration, and sulfhydry1 protein concentration (reviewed in Brachet, 1960; see also Czolowska, 1969). Germinal cytoplasm, necessary for the differentiation of germ cells, is localized near the vegetal pole of frog eggs at first cleavage (Blackler, 1958, Czolowska, 1969, 1972). We add to these observations the result that, in fertilized eggs as well as in late blastula embryos, a substantial proportion of the proteins detectable in SDSacrylamide gels show a localized distribution along the animal-vegetal axis. We are extremely grateful to Dr. Pamela Barald, Dr. Richard Burgess, Dr. Hans Ris, and Dr. Pallaiah Thammana for their expert counseling on matters of technique. We also thank Dr. Robert Briggs and Dr. Jack Lilien for a critical reading of the manuscript, Donald Chandler for assisting with the photography, and Cheryle Hughes for preparing the drawings. This work was supported by NSF Grant GB-33806, NIH Developmental Biology Training Grant HD 00409, and by funds provided by the Graduate School of the University of Wisconsin. REFERENCES BLACKLER, A. W. (1958). Contribution to the study of germ cells in the Anuru. J. Embryol. Exp. Morphol. 6, 491-503. BRACHET, J. (1960). “The Biochemistry of Development.” Pergamon Press, New York. CRIPPA, M., and GROSS, P. R. (1969). Maternal and
VOLUME
58,
1977
embryonic contributions to the functional messenger RNA of early development. Proc. Nut. Acad. Sci. USA 62, 120-127. CZOLOWSKA, R. (1969). Observations on the origin of the germinal cytoplasm in Xenopus 1aeui.s. J. Embryol. Exp. Morphol. 22, 229-251. CZOLOWSKA, R. (1972). The tine structure of the “germinal cytoplasm” in the egg of Xenopus laevis. Wilhelm Roux Arch. Entwicklungs mech. Organismen 169, 335-344. DONOHOO, P., and KAFATOS, F. C. (1973). Differences in the proteins synthesized by the progeny of the first two blastomeres ofllyunassa, a “mosaic” embryo. Develop. Biol. 32, 224-229. FLICKINGER, R. A., GREENE, R., KOHL, D. M., and MIYAGI, M. (1966). Patterns of synthesis of DNAlike RNA in parts of developing frog embryos. Proc. Nat. Acad. Sci. USA 56, 1712-1718. HYNES, R. O., and GROSS, P. R. (1970). A method for separating cells from early sea urchin embryos. Develop. Biol. 21, 383-402. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Ilondon) 227, 680-685. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. LUTZELER, I. E., and MALACINSKI, G. M. (1974). Modulations in the electrophoretic spectrum of newly-synthesized protein in early axolotl (Ambystoma me&unum) development. Differentiation 2, 287-297. NEWROCK, K. M., and RAFF, R. A. (1975). Polar lobe specific regulation of translation in embryos of Zlyanassa obsoleta. Develop. Biol. 42, 242-261. NIEUWKOOP, P. D., and FABER, J. (eds.) (1967). “Normal Table of Xenopus Laevis (Daudin),” 2nd ed. North-Holland, Amsterdam. STEINBERG, M. S. (1957). Report by J. D. Ebert. In Carnegie Znst Washington Yearb. 56,347. TERMAN, S. A. (1970). Relative effect of transcription-level and translation-level control of protein synthesis during early development of the sea urchin. Proc. Nat. Acad. Sci. USA 65, 985-992. WHITELEY, A. H., MCCARTHY, B. J., and WHITELEY, H. R. (1966). Changing populations of messenger RNA during sea urchin development. Proc. Nat. Acad. Sci. 55, 519-525. WOLF, D. P., and HEDRICK, J. L. (1971). A molecular approach to fertilization. II. Viability and art%cial fertilization of Xenopus laevis gametes. Develop. Biol. 25, 348-359. WOODLAND, H. R., and GURDON, J. B. (1968). The relative rates of synthesis of DNA, sRNA and rRNA in the endodermal region and other parts of Xenopus laevis embryos. J. Embryol. Exp. Morphol. 19, 363-385.
BIOLOGY
The Distribution
58, l-10
of Soluble
TERRY Department
(1977)
L. MOEN
of Zoology,
Received
March
Proteins along the Animal-Vegetal Frog Eggs AND MARION
University
31,1976;
of Wisconsin,
accepted
in revised
Axis of
NAMENWIRTH’ Madison, form
Wisconsin
January
53706
25,1977
We describe a procedure for rapidly dividing hundreds of frog eggs into transverse slices along the animal-vegetal axis. We have used this method to study the spatial distribution of soluble proteins in fertilized uncleaved eggs and late blastula embryos of Xenopus laevis. Approximately 25% of the protein bands we resolve by electrophoresis are present along only part of the egg’s animal-vegetal axis. INTRODUCTION
Gurdon, 1968; Donohoo and Kafatos, 1973; Lutzeler and Malacinski, 1974; Newrock and Raff, 1975). We describe here an efficient method for dividing large numbers of fertilized frog eggs into transverse slices along the animal-vegetal axis. With this procedure, groups of 50-100 eggs that have been artificially inseminated are placed in a monolayer inside a fluid-filled chamber and are permitted to undergo their normal rotation to the animal-pole-up position. The eggs are then quickly frozen into a single block of saline solution. This block is sectioned with a cryostat microtome to give the desired number of transverse slices from vegetal to animal pole. Comparable slices from many such blocks are pooled and used as starting material for a biochemical extraction. We have used this procedure to study the spatial distribution of soluble proteins along the animal-vegetal axes of fertilized uncleaved eggs and late blastula embryos of Xenopus laevis, the South African clawed frog.
In studying the molecular events that accompany early embryonic development and differentiation, numerous investigators have compared macromolecules synthesized or activated at successive stages of embryonic development (e.g., Crippa and Gross, 1969; Whiteley et al., 1966; Terman, 1970). Experiments of this sort almost always begin with the homogenization of entire eggs or embryos, so that all biochemical events are monitored at once regardless where they occur in the embryo. Very little information has yet been gathered on the spatial distribution of molecules within eggs and early embryos? primarily because this type of study is cumbersome to do. The most widely used biochemical procedures require large amounts of embryonic starting material. Yet, except for the velocity sedimentation technique adapted by Hynes and Gross (1970) for separating the macromeres, mesomeres, and micromeres of the 16-cell sea urchin embryo, virtually the only MATERIALS AND METHODS methods in use for dividing early embryos Obtaining synchronously developing, into different regions involve dissection or jelly-free embryos. Eight to twelve hours sorting of embryos one at a time (e.g., after injection of 800-1000 units of human Flickinger, et al., 1966; Woodland and chorionic gonadotropin (Antuitrin-S, Parke, Davis and Co.), Xenopus Zaevis fe’ To whom all correspondence should be admales were gently squeezed to obtain sevdressed. Copyright All
rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
2
DEVELOPMENTAL
BIOLOGY
eral hundred unfertilized eggs. These were artificially inseminated, using methods adapted from Wolf and Hedrick (1971) and were permitted to develop for either 30 min or 7 hr (late blastula stage). The embryos were then dejellied by treatment for 3-5 min in a solution containing 15 g of cysteine-HCUliter of 10% Steinberg’s solution (Steinberg, 1957), adjusted to a final pH of 8.0-8.5. Residual cysteine was removed by rinsing embryos repeatedly in 10% Steinberg’s solution. Papain, a common ingredient of dejellying solutions, was not used once it became clear that traces of papain remain, even after extensive rinsing, and lead to subsequent protein degradation. Freezing embryos into blocks. A chamber suitable for freezing embryos was assembled by placing a brass ring (19-mm o.d., 16.5-mm id., 7.5-mm high), the lower surface of which had been lightly coated with silicone grease, upon an aluminum plate, the upper surface of which bore a thin film of silicone grease (Fig. la>. The chamber was filled three-fourths full with 10% Steinberg’s solution, and 50-100 fertilized dejellied embryos were pipetted in.
d
.
Q
h
FIG. 1. Schematic representation of the method we use to prepare blocks of frozen saline solution containing 50-100 fertilized frog eggs. The blocks are later sectioned with a cryostat microtome SO that all the eggs are divided simultaneously into transverse slices from vegetal to animal pole. For details see Materials and Methods.
VOLUME
58, 1977
The chamber was left undisturbed while the embryos completed their rotation to the animal-pole-up position. Any embryos exhibiting faulty rotation were removed by pipet and discarded. Liquefied Freon 22 (CHClF,) was collected in a small dish placed in a liquid nitrogen bath inside an insulated container (Fig. lb). The copper coil which was attached to the Freon container was repeatedly bathed in liquid nitrogen to lower the temperature of the Freon as it passed through the coil into the dish. Once the embryos had reached the desired stage of development, the entire embryo chamber was frozen by lowering it into the dish of liquihed Freon (Fig. lc). When the chamber had been partially frozen, 10% Steinberg’s solution was added until it overflowed. Once the solution was completely frozen, the chamber was removed from the Freon, and the brass ring containing the frozen block of eggs could be easily lifted from the underlying plate. Blocks still surrounded by their brass rings were then stored at -70°C to await further processing. Sectioning frozen eggs and blastulae. To prepare frozen blocks for mounting on the cryostat specimen holder, the original upper surface of each frozen block was scraped with a razor blade until it was perfectly flush with the upper surface of the brass ring that still surrounded it (Fig. Id). These blocks were then stored in a petri plate on dry ice with the original lower surface of each sample in contact with the plate. Note that the lower surface of the sample is almost flush with the vegetal poles of the frozen eggs, but there are several millimeters of ice between the upper surface of the sample and the animal poles of the frozen eggs. A specimen holder was prechilled on dry ice for a few seconds only. A drop of 10% Steinberg’s solution was placed on the cooled holder, and a frozen egg sample still ensheathed in its brass ring was quickly inverted and pressed down on the holder, so that the
MOEN
AND NAMENWIRTH
Soluble Protein Localization
vegetal poles of the eggs faced up (Fig. le). The loaded specimen holder was placed on dry ice for about 30 set and was then removed. A pair of heated forceps was held in contact with the metal ring (Fig. lf, g> until it could be lifted away from the frozen egg sample which adhered to the specimen holder (Fig. lh). The frozen egg samples were sectioned at 20 pm using a cryostat microtome prechilled to -30°C. Our objective was to divide each block of eggs into 10 transverse fractions. Since there is substantial variability in the diameters of eggs spawned by different females (1.2-1.5 mm), a test block of eggs from each spawning was first sectioned to determine the number of 20-pm sections necessary to section completely through an egg or blastula from that spawning. This calibration of egg diameter was repeated for each new spawning so that variability in egg diameters between different spawnings did not introduce unnecessary error. Nonetheless, even eggs within a single spawning may show slight variability in diameter. We estimate that this introduces a maximum error equivalent to one-tenth the diameter of an egg. Protein extraction. A set of 10 transverse fractions obtained from several thousand sectioned embryos provided the starting material for each series of experiments. All procedures were carried out at 4°C. The 10 fractions obtained from eggs frozen in 10% Steinberg’s solution (which contains 6 m&I NaCl + CO.1 mM KCl, CaCl,, and MgSO,) were thawed and homogenized with 30 strokes of a motor-driven ground-glass homogenizer. MgCl, was added to a concentration of 10 mIk?, and the samples were overlaid with mineral oil and were centrifuged at 20,OOOgfor 45 min. The mineral oil and associated lipid were aspirated and discarded. Part of each 20,OOOgsupernatant was immediately extracted (see ahead), whereas the remainder was again overlaid with mineral oil and centrifuged in a 50 Ti rotor at 200,OOOg for 8 hr.
in Eggs
3
The high-speed supernatant and the remaining low-speed supernatant from each of the 10 transverse fractions were treated as follows. Solid urea was added to a final concentration of 0.45 g/ml, and the samples were made 0.1% in SDS. A 30-~1 aliquot was removed from each sample and frozen. These were used later for protein determinations (Lowry et al., 1951). pMercaptoethanol was added to the remainder of each sample to a final concentration of 20 &ml. The samples were incubated at 65°C for 15 min and then were stored at -70°C until analyzed by acrylamide gel electrophoresis. Our extracts contained approximately 5 pg of protein/egg. Estimating the total amount of nonyolk protein in a Xenopus egg to be about 120 pg (R. Wallace, personal communication), our extracts contained 4% of the egg’s nonyolk protein. Yolk proteins and other proteins associated with large particulates, as well as proteins insoluble in dilute saline, were removed by centrifugation of the initial homogenates. SDS-Acrylamide gel electrophoresis. SDS-Acrylamide slab gels, 0.15-cm thick, were prepared according to the Laemmli formula (Laemmli, 1970) with a 9.75-cmhigh 10% acrylamide running gel, overlaid by a 0.75-cm-high 3% acrylamide stacking gel. A group of samples, each containing approximately 35 pg of protein, was thawed and then incubated for 2 min at 90°C. When the samples had cooled, 1 ~1 of 0.1% bromphenol blue tracking dye was added to each, and the samples were then loaded into the slots in the stacking gel. Electrophoresis was carried out at 100 V until the tracking dye reached the bottom of the gel (approximately 4 hr). The gel was stained for an hour in a freshly prepared filtered solution of 0.05% Coomassie brilliant blue + 0.05% buffalo black dissolved in 5 parts methanol, 1 part acetic acid, and 5 parts water. Diffusion destaining was carried out in 7% acetic acid. When the staining was judged to be
4
DEVELOPMENTAL BIOLOGY
too light, staining was repeated for another hour, and the gel was destained again. RESULTS
Fertilized
Eggs
Groups of eggs that had been artificially inseminated were allowed to develop for 1 hr at 21°C and then were frozen into blocks for cryostat sectioning. (Control eggs that continued developing at 21°C initiated first cleavage 1 hr and 20 min after fertilization.) The frozen eggs were divided into 10 transverse fractions from vegetal to animal pole. After thawing and homogenization, each fraction was centrifuged at 20,OOOgfor 45 min in order to pellet yolk platelets, mitochondria, nuclei, and other large particulates. The pellets were disand proteins were prepared carded, from a portion of each 20,OOOg supernatant. The proteins present in the 10 transverse egg fractions were then compared by electrophoresis in SDS-acrylamide slab gels (Fig. 2a). Approximately 50 protein bands were resolved in each transverse egg fraction, and about 80% of these bands appeared to be constant across the entire egg. The remaining protein bands change as one follows them along the egg axis from vegetal to animal pole. The changes are summarized in Fig. 2b. Included are seven protein bands that occur in the animal half and equatorial region of the egg but are absent from the vegetal region, two bands that are confined to the area near the animal pole, a faint band present only near the vegetal pole, and two bands that occur along the entire egg axis but show a pronounced change in concentration as one moves across the egg. Considering the very large number of soluble proteins present in Xenopus embryos, it is clear that each band on the gel must consist of many proteins of similar electrophoretic mobilities. To achieve better resolution we sought to reduce the number of proteins in our samples by subjecting the 20,OOOgsupernatants to furth-
VOLUME 58, 1977
er centrifugation at 200,OOOgfor 8 hr. Proteins prepared from the high-speed supernatants were then examined by SDSacrylamide slab gel electrophoresis. Figure 3A shows a gel separation of the proteins present in the high-speed supernatants of each of the 10 transverse egg fractions. Of the approximately 44 bands visible per fraction, about 75% seem to be constant along the animal-vegetal axis of the egg. The changing bands (Fig. 3b) include seven that extend from the animal pole through the equatorial region of the egg, two bands present only near the animal pole, one band confined to a narrow region near the equator, two bands present everywhere except near the vegetal pole, one band extending from the vegetal pole to the equatorial region, and one band extending across the entire egg but showing a markedly greater protein concentration in the vegetal half of the egg than in the animal half. Thus, in both the low-speed and highspeed supernatants of eggs, roughly 25% of the protein bands resolved on SDS gels occur along only part of the animal-vegetal axis. Most of these changing bands extend over half or more of the egg’s diameter, and most are represented in the animal and equatorial regions of the egg while being absent from the vegetal region. Blastula Embryos Further studies showed that the soluble proteins present in late blastula stage embryos have a quite analogous distribution along the animal-vegetal axis. Eggs fertilized by artificial insemination were permitted to develop to stage 9 (Nieuwkoop and Faber, 1967) before being dejellied, frozen, and sectioned from vegetal to animal pole. Figures 4a and 5a show gel separations of the blastula proteins contained in the 20,OOOgand 200,OOOgsupernatants, respectively. Figures 4b and 5b indicate the locations of the changing bands. Approximately 55 bands were resolved in each
MOEN
AND
NAMENWIRTH
Soluble
fraction of the 20,OOOg supernatant compared to 50 bands per fraction in the 200,OOOg supernatant. About one-third of these bands change along the animal-vegetal axis, and, as was observed also in fertilized uncleaved eggs, most of the changing bands extend from the animal pole through the equatorial region, disappearing in the vegetal region of the blastula. There are, of course, exceptions to this pattern. The high-speed supernatant of blastula embryos, for example, has three bands that are present in the vegetal half of the blastula, but which are absent from at least part of the animal half. Reproducibility The electrophoretic comparison of each set of protein samples was repeated several times to insure that the results were reproducible. In addition, we duplicated the entire experiment, beginning with different batches of frog eggs. We found remarkably little variation, other than an occasional one-fraction difference in the distribution of a band; e.g., a band that extended from the animal pole through fraction 6 in one experiment might extend only as far as fraction 7 in the second experiment. DISCUSSION
We have described
a method for rapidly
Protein
Localization
in Eggs
5
dividing hundreds of frozen frog eggs into transverse slices from animal to vegetal pole, making it possible, with conventional biochemical techniques, to study the distribution of macromolecules along the animal-vegetal axis. Using this procedure we observed that about 25% of the dilute saline-soluble protein bands resolved by electrophoresis on SDS-acrylamide slab gels occurred along only a portion of the embryo’s animal-vegetal diameter. These results certainly underestimate the actual extent of heterogeneity in the distribution of soluble proteins for, in view of the large number of different protein species present in eggs, one must expect that frequently a stained gel band consists of numerous proteins of similar electrophoretie mobilities. In such a compound band, individual proteins could be added or subtracted along the animal-vegetal axis without always altering the appearance of the stained band on the gel. In this way, some limited-distribution proteins would go undetected. Similarly, while most of our “changing” bands extended over half or more of the egg’s animal-vegetal diameter, some of these bands might contain component proteins having a much narrower distribution. Many of these uncertainties can be clarified by higher resolution two-dimensional gel separations. Yet,
FIG. 2. (a) SDS-Acrylamide slab gel of the soluble proteins detectable in the 20,OOOg supernatant of Xenopus laeuis eggs 1 hr postfertilization. Groups of 50 fertilized eggs were frozen and sliced simultaneously into 10 transverse fractions from vegetal to animal pole. Aliquots consisting of 35 pg protein prepared from the 20,OOOg supernatants of each of the 10 transverse egg fractions were then compared by electrophoresis in a 10% SDS-acrylamide slab gel. Proteins were visualized by staining the gel with Coomassie brilliant blue and buffalo black. Proteins from the vegetal pole slice are shown in the left channel. The second channel from the left shows proteins from the transverse slice just above the vegetal pole, and so on proceeding in order to the channel at the right which carried proteins from the animal pole slice. Migration occurred from top to bottom of the gel. Electrophoresis of marker proteins suggested the following rough relationship between molecular weight and the distance traveled along the centimeter ruler at the right: MW 135,000 (P-galactosidase) = 2.2 cm; MW 67,500 (bovine serum albumin) = 3.5 cm; MW 43,000 (ovalbumin) = 5.0 cm; MW 27,500 (chymotrypsinogen) = 7.3 cm; MW 18,400 (P-lactoglobulin) = 9.8 cm. (b) Diagram of those protein bands shown in gel (a), which change as one moves across the egg from vegetal to animal pole. The location of each changing band (given in the left column) indicates the approximate distance along the centimeter ruler in (a) that these proteins migrated. The gel bands that appeared constant along the animal-vegetal axis were omitted from the diagram. FIG. 3. Electrophoretic analysis of the proteins present in the 200,OOOg supernatant of Xenopus laeuis eggs 1 hr postfertilization. Eggs were frozen and divided into 10 transverse slices from vegetal to animal pole. Soluble proteins present in these 10 slices were compared. Details as in Fig. 2.
DEVELOPMENTAL
BIOLOGY
EGG - 20,OOOg
LOCATION km1
Vegetal Pa’e I
0.25
2
3
4
58,
VOLUME
5
1977
supernate
6
7
Animal Pale 9 10
8
-----m-
0.45
--
0.65
-
0.70
----
1.50
-----
1.55 1.80
~-----
2.00
----
2.35
----------
2.40
---m------
3.30
~-----
5.30
------
b FIGURE
it must be recognized that some proteins occur inside eggs in quantities so small as to make detection with conventional electr ophoretic procedures impossible. We are th lerefore observing only the more preva-
2
lent protein species in each egg fractia In. Although there were exceptions, it is interesting that, in both eggs and late blastula embryos, most of the limiter d-distribution bands were present in the : ani-
MOEN
Soluble
AND NAMENWIRTH
EGG
LOCATION (cm)
Protein
- 200,
OOOg
Localization
in Eggs
supernote Animal Pale
Vegetal Pale I
2
3
4
5
6
7
0
-----
0.5
--m
0.0 1.2 -__--__~
1.6 ~_---___
I.9
__------
2.1 --------
2.3 -----__
2.4 2.5
---~-~~~-~
2.7
--------
2.8
------
3.25 3.4
v-p--
5.9
--s-I
FIGURE
3
9
10
7
BLASTULA
-
20,ooog
WW~CI~~
!getar Pole I
LOCATION km1
2
Ininml
3
4
5
6
7
8
9
Poll? 10
0 55 0 62
----
0 75
-
1 00 I 05 I I5 I .40 I 45 1.65
--------mm
I 80
-----e-m---------A---mm
2 00
-
2 IO
-----
2 I5 2 20
------WC-
------_-
2 30 2 40
------A-_-
2 45
-
2 55
-----
_---------
2 80
----A---
5 95
b FIG. embryos. Soluble
Electrophoretic analysis of the proteins present in the 20,OOOg supernatants Stage 9 blastulae were frozen and divided into 10 transverse slices from vegetal proteins present in these 10 slices were compared. Details as in Fig. 2.
4.
8
of late blastula to animal pole.
MOEN
AND
Soluble
NAMENWIRTH
BLASTULAegem Pyle
LOCATlOll ICrnJ
Protein
Localization
200,OOOg
9
in Eggs
supernote Animal Pp2
23456769
0.45
----
0.70
----
I.60 I.85 I 95
--------__
2 IO
---
2.35
---_---_
2.40 m----------------__
2.80 3 30
-------
4.30 4.70 5.25
----__
6.30
b FIG. 5. Electrophoretic analysis of the proteins embryos. Stage 9 blastulae were frozen and divided Soluble proteins present in these 10 fractions were
present in the 200,OOOg supernatant into 10 transverse fractions from vegetal compared. Details as in Fig. 2.
ma1 and equatorial regions of the embryo, but were absent from the vegetal region. This cannot be attributed to the smaller quantity of cytoplasm present in the vegetal region, since we adjusted our samples
of late blastula to animal pole.
so that an equivalent number of micrograms of protein were loaded into each channel of the gel. Nor could the pattern have been caused by the nuclear proteins present in the animal hemisphere within
10
DEVELOPMENTAL
BIOLOC Y
the single fertilization nucleus, since a similar pattern occurs in the multinucleate late blastula embryo. Our conclusion that the frog egg is heterogeneous along its animal-vegetal axis will raise few eyebrows among experimental embryologists, for several forms of animal-vegetal differentiation in frog eggs are already familiar. The cortex of the egg is thicker in the animal and dorsal regions than in the ventral and vegetal regions. Yolk platelets and other cytoplasmic particulates vary in size and concentration, showing a complex distribution pattern from animal to vegetal pole (Nieuwkoop and Faber, 1967). In the eggs of various amphibian species, animal-vegetal as well as dorso-ventral gradients have been described for oxygen consumption, reducing activity, RNA concentration, and sulfhydry1 protein concentration (reviewed in Brachet, 1960; see also Czolowska, 1969). Germinal cytoplasm, necessary for the differentiation of germ cells, is localized near the vegetal pole of frog eggs at first cleavage (Blackler, 1958, Czolowska, 1969, 1972). We add to these observations the result that, in fertilized eggs as well as in late blastula embryos, a substantial proportion of the proteins detectable in SDSacrylamide gels show a localized distribution along the animal-vegetal axis. We are extremely grateful to Dr. Pamela Barald, Dr. Richard Burgess, Dr. Hans Ris, and Dr. Pallaiah Thammana for their expert counseling on matters of technique. We also thank Dr. Robert Briggs and Dr. Jack Lilien for a critical reading of the manuscript, Donald Chandler for assisting with the photography, and Cheryle Hughes for preparing the drawings. This work was supported by NSF Grant GB-33806, NIH Developmental Biology Training Grant HD 00409, and by funds provided by the Graduate School of the University of Wisconsin. REFERENCES BLACKLER, A. W. (1958). Contribution to the study of germ cells in the Anuru. J. Embryol. Exp. Morphol. 6, 491-503. BRACHET, J. (1960). “The Biochemistry of Development.” Pergamon Press, New York. CRIPPA, M., and GROSS, P. R. (1969). Maternal and
VOLUME
58,
1977
embryonic contributions to the functional messenger RNA of early development. Proc. Nut. Acad. Sci. USA 62, 120-127. CZOLOWSKA, R. (1969). Observations on the origin of the germinal cytoplasm in Xenopus 1aeui.s. J. Embryol. Exp. Morphol. 22, 229-251. CZOLOWSKA, R. (1972). The tine structure of the “germinal cytoplasm” in the egg of Xenopus laevis. Wilhelm Roux Arch. Entwicklungs mech. Organismen 169, 335-344. DONOHOO, P., and KAFATOS, F. C. (1973). Differences in the proteins synthesized by the progeny of the first two blastomeres ofllyunassa, a “mosaic” embryo. Develop. Biol. 32, 224-229. FLICKINGER, R. A., GREENE, R., KOHL, D. M., and MIYAGI, M. (1966). Patterns of synthesis of DNAlike RNA in parts of developing frog embryos. Proc. Nat. Acad. Sci. USA 56, 1712-1718. HYNES, R. O., and GROSS, P. R. (1970). A method for separating cells from early sea urchin embryos. Develop. Biol. 21, 383-402. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Ilondon) 227, 680-685. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. LUTZELER, I. E., and MALACINSKI, G. M. (1974). Modulations in the electrophoretic spectrum of newly-synthesized protein in early axolotl (Ambystoma me&unum) development. Differentiation 2, 287-297. NEWROCK, K. M., and RAFF, R. A. (1975). Polar lobe specific regulation of translation in embryos of Zlyanassa obsoleta. Develop. Biol. 42, 242-261. NIEUWKOOP, P. D., and FABER, J. (eds.) (1967). “Normal Table of Xenopus Laevis (Daudin),” 2nd ed. North-Holland, Amsterdam. STEINBERG, M. S. (1957). Report by J. D. Ebert. In Carnegie Znst Washington Yearb. 56,347. TERMAN, S. A. (1970). Relative effect of transcription-level and translation-level control of protein synthesis during early development of the sea urchin. Proc. Nat. Acad. Sci. USA 65, 985-992. WHITELEY, A. H., MCCARTHY, B. J., and WHITELEY, H. R. (1966). Changing populations of messenger RNA during sea urchin development. Proc. Nat. Acad. Sci. 55, 519-525. WOLF, D. P., and HEDRICK, J. L. (1971). A molecular approach to fertilization. II. Viability and art%cial fertilization of Xenopus laevis gametes. Develop. Biol. 25, 348-359. WOODLAND, H. R., and GURDON, J. B. (1968). The relative rates of synthesis of DNA, sRNA and rRNA in the endodermal region and other parts of Xenopus laevis embryos. J. Embryol. Exp. Morphol. 19, 363-385.
