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The hyaline layer: Its isolation and role in echinoderm development
DEVELOPMENTAL
BIOLOGY
24, 348-362
(1971)
The Hyaline Layer: Its Isolation and Role in Echinoderm Development ELENA Department
CITKOWITZ 1
of Biological
and Marine
Sciences, Columbia University, Biological Laboratory, Woods Hole, Accepted
November
New York, Massachusetts
New York 02543
10027
2, 1970
INTRODUCTION
The hyaline layer is an extracellular coat surrounding echinoderm embryos. It arises from the cortical granules during a complex series of events following fertilization or artificial activation. In the unfertilized sea urchin egg, the cortical granules lie beneath the vitelline and plasma membranes. These granules are formed by the Golgi complex during oogenesis (Anderson, 1968). Using the electron microscope, Endo (1961) clearly established the role of the cortical granules in forming both the hyaline layer and fertilization membrane. The fertilization membrane can be removed without altering the developmental process and is, therefore, of little consequence for normal development in the laboratory. There are no physical connections between the fertilization membrane and the embryo which, during the blastula stage, hatches out of this membrane. The hyaline layer, on the other hand, is firmly attached to the embryo and is not lost until metamorphosis of the larva (Dan, 1960). According to Moore (1928) and Harvey (1934), if the hyaline layer is removed, it is rapidly reformed by the embryo. As early as 1900, Herbst showed that the hyaline layer is necessary for adherence of the blastomeres. Many investigators (Sugiyama, 1951; Hagstriim and Hagstrom, 1954; Nakano, 1956) have implicated the hyaline layer in the inhibition of polyspermy; and others (Chambers, 1940; Dan, 1960; Gustafson and Wolpert, 1967) believe that the hyaline layer plays an essential role in changes of the external form of the developing embryo. Herbst (1900) demonstrated the dependency of the structural integrity of the hyaline layer on the presence of divalent cations, and hyaline layer material has been isolated by dissolving it from the surface of eggs in media devoid of these cations (Nakano and Ohashi, 1954; ‘Present address: Division Crw, California 95060.
of Natural
Sciences 348
I, University
of California,
Santa
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Faust et al., 1959; Vacquier, 1969; Kane, 1970). Hyaline layer material can also be obtained from whole egg homogenates (Yazaki, 1968) or isolated cortices (Kane and Stephens, 1969). This protein has been studied recently in more detail than previous studies (Stephens and Kane, 1970). The hyaline layer was isolated as an intact structure from cleaving eggs (Vacquier, 1969) and from blastulae and gastrulae (Citkowitz-Hinegardner, 1969). The present paper describes, in greater detail than the previous report (Citkowitz-Hinegardner, 1969), a new method for isolating large numbers of clean, intact hyaline layers from embryos at the l-cell to late gastrula stages, and examines the role of the hyaline layer in development. MATERIALS
AND
METHODS
Isolation of the hyaline layer. Eggs and sperm of Strongylocentrotus purpuratus were spawned by injection of 0.5 M KC1 and collected in the normal manner. The eggs were washed several times in Milliporefiltered seawater and fertilized; excess sperm were removed by several more washings. Only eggs showing 95% fertilization or greater were used for subsequent experiments. Embryos were raised at 15°C in concentrations of 1 to 2 X 10” eggs per 100 ml and stirred at 30 rpm. The embryos were collected by centrifugation at 2500 rpm, and the seawater was removed. Approximately 15 x lo6 embryos were then suspended in 40 ml of a solution containing 4 M NaCl, 0.2 M MgCl,, and 0.05 M Tris, pH 8. The suspension was homogenized with a tightfitting (type B) Dounce homogenizer (Kontes Glass Co.) which forced most of the cells out of the hyaline layers. Cells and debris were then sucked through nylon monofilament bolting cloth (Nitex, 35-p mesh opening) which was attached to one end of a short length of plastic tubing 3 cm in diameter. All but a few hyaline layers were stopped by the cloth, but the cells, nuclei, and debris passed through. The hyaline layer fraction was resuspended in fresh salt solution, and the filtrate was again drawn off. This process was repeated until the filtrate sucked through the bolting cloth was clear. If necessary, the hyaline layers were rehomogenized briefly, and the debris was again removed as above. Since the nuclei followed the hyaline layers during subsequent purification, it was particularly important to free the hyaline layers of nuclei at this stage of the procedure. A French press may be used in place of the Dounce homogenizer to strip the cells from the hyaline layers. Once isolated, the hyaline layers were further purified on a discon-
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tinuous sucrose gradient of 7 ml each of 90, 84, and 777; 2.5 M sucrose and 3 ml of 74 and 70% 2.5 M sucrose made up by diluting 2.5 M SUcrose with a salt solution containing 4 M NaCl, 0.8 M MgCl,, and 0.05 M Tris, pH 8. The partially purified hyaline layers were mixed with an equal volume of 70% 2.5 M sucrose and layered over the gradient. Fresh homogenizing solution was then layered on top. The tubes were centrifuged at 80,000 g for 1.5 hours at 4°C in an International ultracentrifuge, Model B-60, using an SB 110 swinging-bucket rotor. The clean hyaline layers accumulated between 74 and 77% 2.5 M sucrose. The hyaline layers were then stored in homogenizing solution at - 15°C. Yields averaged 45%. If the embryos were still surrounded by a fertilization membrane at the stage when the hyaline layers were isolated, most of the fertilization membranes in the gradient were found at the bottom of the tube. This method for isolating hyaline layers has also been applied to the sea urchins Lytechinus pictus and Arbacia punctuluta, the sand dollar Echinarachnius parma, and the starfish Asterias forbesi. Treatment of the hyaline layer. Experiments on blastomere interaction were carried out according to the procedures developed by Vacquier and Mazia (1968a,b). Embryos were treated with dithiothreitol (Calbiochem) because of reports (Vacquier and Mazia, 1968a,b) that this agent separates blastomeres at the 2-cell stage. Embryos were also treated with pronase which, as Vacquier and Mazia described (1968b), digests the hyaline layer of S. purpuratus and L. pictus. When the hyaline layer was partially digested or weakened, it became extremely difficult to see. Addition of a drop of either 0.1 M sodium dodecyl sulfate or 10% Triton X to a drop of embryos dispersed the cells and revealed the hyaline layer, although a sufficiently weak hyaline layer was itself often dispersed shortly after the cells had disintegrated. Labeling. S. purpuratus was used for labeling experiments. For pulse labeling, one million embryos were cultured at 15°C in 50 ml of Millipore-filtered seawater and stirred with a 30 rpm motor. At 18 hours, just prior to hatching, 30 &I of L-valine-3H (0.161 Ci/mmole; Schwartz BioResearch, Inc.) was added along with 50 u/ml penicillin and 50 pg/ml streptomycin. Puromycin (100 pg/ml; Nutritional Biochemical Corp.) was added 1.25 hours before addition of label. Actinomycin D (25 @g/ml; Sigma Chemical Co.) was added at fertilization, 18 hours before the label. Embryos for the two inhibitor studies were taken from the same suspension as the controls and exposed to the same concentration of label at the same time and for the same dura-
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tion. Subsequent treatment of the cultures was identical. After 1 hour in label, samples of embryos and supernatant were taken and quickfrozen. Cold L-valine (1 mg/ml) was added to the remaining embryos, which were then kept at 4°C during the isolation of the hyaline layers. Purification was carried out as already described. The procedure for long-term labeling was basically like that for the pulse. Autoradiography. For autoradiography, purified hyaline layers were spread on slides, air-dried, and fixed in Baker’s formaldehyde-calcium fixative (Baker, 1944). The slides were dipped in Kodak liquid emulsion NTB2 and exposed for 8 days at room temperature. The slides were developed in D-19 developer. Determination of radioactivity. Hyaline layer concentration was determined on a Model A Coulter counter using a 100-p aperture. Purified hyaline layers, 15,000-100,000 per sample, were centrifuged at 2500 rpm, the supernatant was removed, and the hyaline layers were washed once with 95(‘; ethanol. Samples of 2000 whole embryos were first treated with 0.5?L sodium dodecyl sulfate and then 15% cold trichloroacetic acid (TCA) to ensure the release of trapped amino acids (Hynes and Gross, 1970). Hyaline layers and embryos were then washed once in cold 5% TCA, heated for 30 minutes at 80-90°C in 5r, TCA, and washed twice with methanol. Samples were then mixed with 0.1 ml of hydroxide of Hyamine and heated for 5 minutes at 60°C. Samples were counted in either 10 ml of Kinard’s solution (Kinard, 1957) or 5 gm PPO and 100 gm naphthalene brought to 1 liter with dioxane (formula from Beckman Instruments, Inc.). Radioactivity was counted on a Beckman liquid scintillation counter. RESULTS
HYALINE
LAYER MORPHOLOGY
Hyaline layers isolated from fertilized eggs and blastulae of S. purpurutus have the same appearance. They are both clear, round sacs, intact except for a hole or tear through which the cells were forced during homogenization. They are easily visible using phase microscopy (Fig. 1). Beginning with early gastrulation, there is a distinct change in the morphology of the hyaline layer. Where the cells of the embryo are beginning to invaginate, there is a corresponding inpocketing of the hyaline layer (Fig. 2). This inpocketing, or invagination, remains in close contact with the archenteron cells and increases in length as gastrulation proceeds. When the gut forms the coelomic pouches, the hyaline layer invagination shows similar protrusions. The hyaline layer
FIG. 1. Isolated blastulahyaline layers of Strongylocentrotuspurpuratus. FIG. 2. An isolated mid-gastrula hyaline layer of S. purpuratus tion. X 360. Figures l-10, phase contrast.
showing
x 90. invagina-
in the region underlying the gut appears thicker than the rest of the hyaline layer. Hyaline layers of E. parma, L. pi&us, A. punctulata, and A. forbesi show the same change in morphology from blastula to gastrula stage. ROLE OF THE HYALINE
LAYER
Blastomere Interaction The goal of the following experiments was to correlate loss of adhesion of the blastomeres with significant changes in the state of the hyaline layer. Dithiothreitol. After exposure of E. parma eggs to 0.05 M dithiothreitol for 10 minutes during first division, the two blastomers remained spherical (Fig. 3) as reported by Vacquier and Mazia (1968a) for a similar sand dollar, Dendraster excentricus. The blastomeres of normal 2-cell embryos were closely apposed to one another (Fig. 4). When the blastomeres of dithiothreitol-treated embryos were dispersed with sodium dodecyl sulfate, for some embryos a separate hyaline layer could be seen surrounding each blastomere (Fig. 5); and for others, the hyaline layer retained the shape of the furrow as shown for L. pictus (Fig. 7). In contrast, the hyaline layer of normal 2-cell embryos became oval as soon as the cells were disrupted by sodium dodecyl sulfate (Fig. 6). Dithiothreitol-treated eggsdeveloped into twins, partial twins, and normal embryos. Vacquier and Mazia first demonstrated this phenomenon for D. excentricus. Treatment of L. pictus eggs with 0.08 M dithiothreitol during cleavage had the same effect on the hyaline layer and blastomeres as described above for E. parma; some hyaline layers were pinched in two, and others retained
ISOLATION
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a furrow (Fig. 7). Twins, as well as elongated blastulae and normal embryos, were obtained (Fig. 8). Pronuse. Table 1 summarizes the effects of pronase on the hyaline layer and the interaction of the blastomeres. When the hyaline layer was present, the blastomeres were closely apposed to one another; and when the hyaline layer had been dispersed, the blastomeres separated from one another. Because the results for L. pictus were contrary to those of Vacquier and Mazia (1968b), experiments with this species will be considered in more detail. When eggs were fertilized and pronase added within 90 seconds, the fertilization membrane did not form; but the hyaline layer, although extremely thin, was visible at the 2-cell stage (Fig. 9). The hyaline layer of normal embryos at this stage was distinctly thicker (Fig. 10). If removed from pronase at the 2-cell stage, the embryos developed normally. Treatment with 0.067 M dithiothreitol for 14 minutes during mid-cleavage after digestion in pronase caused disruption of the hyaline layer and separation of the blastomeres in many embryos. Those cells which did not separate from one another were invariably held together by a thin hyaline layer. Vacquier and Mazia (196813)reported that in the presence of calcium-free seawater, the blastomeres of pronase-treated L. pictus embryos fell apart. Because they believed that pronase prevented hyaline layer formation, Vacquier and Mazia concluded that calcium is required to sustain cell surface interaction. The following experiment was performed to distinguish between the necessity of the hyaline layer as opposed to calcium in the maintenance of cell contact. Eggs were taken from pronase after 30 minutes, washed 3 times with calcium- and magnesium-free seawater (CMFSW), and cultured in CMFSW for an additional 25 minutes. Before first cleavage had begun, the eggs were returned to normal seawater, where calcium and magnesium were again present. Because the integrity of the hyaline layer is dependent on divalent cations, the hyaline layer was no longer present; and the blastomeres dissociated after division was complete, indicating that at this stage the hyaline layer and not calcium-mediated intercellular adhesion is necessary for normal development. Occasionally the spindle remnant maintained a loose association between the two cells until second cleavage. Control embryos, left in pronase or washed and returned to seawater, were continuously in the presence of divalent cations. These embryos remained intact and had a thin hyaline layer. Extreme care had to be taken with eggs treated with CMFSW. Damage to the cell surface would result’in nonspecific adhesion.
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FIG. 3. An Echinarczchnius parma embryo at the 2-cell stage exposed to dithiothreitol during cleavage. The blastomeres are separated from one another. X 460. FIG. 4. A normal E. parma embryo at the 2-cell stage. The blastomeres are closely apposed. X 460. FIG. 5. The hyaline layer of an E. parma embryo at the 2-cell stage exposed to dithiothreitol during cleavage and later treated with sodium dodecyl sulfate. Two separate hyaline layers can be seen. X 460. FIG. 6. The hyaline layer of a normal E. parma embryo at the 2-cell stage treated with sodium dodecyl sulfate. x 460. FIG. 7. The hyaline layer of a Lytechinus pictus embryo exposed to DTT during cleavage and later treated with sodium dodecyl sulfate. The shape of the furrow is retained. In this figure, the cytoplasm is particularly well dispersed. X 460. FIG. 8. A twinned L. pictus embryo at the 4.cell stage exposed to dithiothreitol during first cleavage. x 460.
ISOLATION
AND
EFFECT OF 1 MIS/ML
Species
Time
Echinaruchnius parma
Strongylocentrotus purpuratus Lytechinus pictus
ROLE
OF
THE
HYALINE
TABLE 1 PRONASE ON BLASTOMERE THE HYALINE LAYER
in pronase
Fertilization mid-cleavage Fertilization S-cell stage Fertilization mid-cleavage Fertilization mid-cleavage Fertilization &cell stage
to to to to to
355
LAYER
INTERACTION
AND ON
Position of blastomeres
State of hyaline layer
Closely apposed Partially separated Separated
Thin, but intact Partially dispersed Dispersed
Closely apposed Partially separated
Thin, but intact Partially dispersed
-
FIG. 9. A Lytechinus pictus embryo exposed to pronase from fertilization through first cleavage. A thin hyaline layer can be seen. X 460. FIG. 10. An untreated L. pictus embryo at the 2-cell stage showing the normal appearance of the hyaline layer. X 460.
Gastrulution Table 2 summarizes the effects of pronase digestion on the hyaline layer and on the ability of the embryo to gastrulate. Weakening of the hyaline layer was correlated, in most cases, with blocked invagination. Amino
Acid Incorporation
Using autoradiography, Immers (1961) showed that the hyaline layer incorporates amino acids as well as sulfate during development. Amino acid incorporation was investigated further in the following experiments. After exposure of the embryos to valine-3H for 1 hour, the isolated hyaline layer accounted for somewhat more than 1% of the counts in-
356
CITKOWITZ TABLE EFFECT
Species
OF PRONASE
con~e~~~ion
AND
ON THE ~~YALINE
LAYER
Time in pronase and stage treated
Effect on gastrulation
0.1
Continuously
Blocked
Very
1.0
l-2
Blocked
Extremely thin Very thin
(ma/ml) Echinumchnius parma Strongylocentrotus purpuratus Lytechinus picks
2
ON GASTRULATION
1.0
hours, late blastula 9-10 hours, blastula
Blocked or abnormal
State of hyaline layer thin
corporated by the whole embryo at hatching. At that time, valine was being added to the hyaline layer at the rate of 1.4 x lo-l2 mM per hour. This is approximately 0.04% of the total valine in the hyaline layer (Citkowitz, unpublished results). Pretreatment with puromycin reduced incorporation into the hyaline layer and the whole embryo by 96%. Continuous exposure to actinomycin reduced incorporation of valine into the hyaline layer by 35 %, although incorporation into the whole embryo remained unchanged. Autoradiographs of blastula hyaline layers labeled continuously from fertilization to hatching showed a fairly even distribution of grains over the surface. Hyaline layers of embryos grown in valine-3H from hatching to mid-gastrula or from beginning to late gastrula stage were labeled over most of their surface, except for the invagination and extreme vegetal region where there was a greatly reduced number of grains (Fig. 11). DISCUSSION
The Hyaline
Layer Invagination
The results above indicate that the hyaline layer remains associated with the archenteron throughout gastrulation by means of the hyaline layer invagination. Gustafson observed a similar structure (Gustafson and Wolpert, 1967); however, he thought that the hyaline layer remained outside and that a new structure of similar appearance was established later in the gut. In view of the present results, origin of the hyaline layer invagination by this mechanism appears unlikely. Dan has suggested that the hyaline layer and the amphibian embryo’s surface coat may play analogous roles. If the surface coat is dissolved, the blastomeres fall apart; and if dissolved at a later stage, the embryo will not gastrulate. This similarity is made even more striking by the fact that both structures follow the archenteron during gastrulation. The
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FIG. 11. An isolated hyaline layer of a Strongylocentrotus purpurutus embryo exposed to valine-3H from early to late gastrula stage. Fewer grains are present over the protruding invagination and the extreme vegetal pole. x 460. Bright field.
presence of the sea urchin’s hyaline layer or the amphibian embryo’s surface coat in the archenteron probably provides an anchorage for the cells while they are undergoing the stresses and changes in shape taking place during invagination. Blastomere
Interaction
There are a number of ways that the blastomeres could be held together. There may be a component other than the hyaline layer which is also sensitive to proteolytic enzymes and to the absence of divalent cations, and whose sensitivity varies from species to species in the same way as the sensitivity of the hyaline layer. Although the existence of such a component cannot be altogether dismissed, the present experiments suggest that the hyaline layer is an absolute requirement for the adhesion of the blastomeres in early cleavage stages. When blastomeres adhered to one another after digestion in pronase, they did so because they were still surrounded by a hyaline layer. Moreover, in all circumstances that gave the appearance of weakened or disrupted cellular adhesions, alterations of the hyaline layer were found which alone were sufficient to explain the results. Thus, when eggs divided in the presence of dithiothreitol, many blastomeres remained spherical; and the hyaline layers of these embryos were furrowed or cleaved in two. Dithiothreitol apparently alters the hyaline layer rather than cell adhesion. Although the effect may be direct, there are indications that the appearance of dithiothreitol-treated hyaline layers is caused by alterations of the connections between the hya-
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line layer and the surface of the dividing egg. A normal hyaline layer is, to a degree, plastic; if stretched into a given shape, it retains that shape. The normal hyaline layer of a fertilized egg is round after dispersing the egg with sodium dodecyl sulfate; the hyaline layer of the 2cell stage is oval. If in the presence of dithiothreitol the cell retains its connections with the hyaline layer and pulls it down into the furrow, when normally it would be released in this region, the hyaline layer might retain the shape of that furrow; and if pulled far enough, it could fuse with itself, forming two individual hyaline layers. This interpretation requires no direct action of dithiothreitol on the hyaline layer. On the other hand, retention of the furrow might be caused by increased plasticity of the hyaline layer. Whatever the cause, the altered shape of the hyaline layer is sufficient to explain the resulting twins, partial twins, and elongated embryos following exposure to dithiothreitol during cleavage. The argument can be raised that the reagents used are breaking down intercellular adhesions which are important and that loss of the hyaline layer is only secondary. This could be tested if the hyaline layer were removed in some other way. Harvey (1934) centrifuged eggs of several species of sea urchin just before cleavage in a solution of seawater and sucrose which was both isotonic and isodense with the eggs. This treatment caused the hyaline layer to be thrown off. If the eggs were not surrounded by a fertilization membrane, the blastomeres separated when the eggs divided. If held together by a fertilization membrane, the hyaline layer could regenerate and normal development ensued. However, if embryos at the 2-cell stage with fertilization membranes were centrifuged and the hyaline layer was thrown off, there were all degrees of cellular interaction ranging from single embryos to complete twins; the apparent cause for the differences was the time at which the new hyaline layer was regenerated (Harvey, 1935). It would, therefore, seem that the hyaline layer is the critical structure that normally holds the blastomeres together at the 2-cell stage. This conclusion is also supported by the finding of Tupper et al. (1970) that there is no conductivity between the cells from completion of first cleavage until the 32-cell stage. The onset of conductivity coincides with the appearance of intercellular adhesions in the form of desmosomes (Wolpert and Mercer, 1963). Gastrulation Although there is variation in the response of embryos at later stages to treatment with pronase, the overall conclusion appears justified that
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a strong hyaline layer or a firm attachment of the cells to the hyaline layer is necessary for gastrulation. For S. purpuratus, whose hyaline layer is very sensitive to pronase, exposure to this enzyme for only 1 hour is enough to prevent any invagination. But for L. picks, whose hyaline layer is more resistant to pronase, treatment for 10 hours results in blocked invagination in some, but not all, embryos. These results are consistent with the theory that the hyaline layer is necessary for gastrulation and that embryos with hyaline layers, or connections to the hyaline layer, that are more sensitive to pronase will show more pronounced effects than embryos whose hyaline layers are less easily digested. For example, Moore (1952) found that embryos of the sand dollar D. excentricus grown in 1 mg/ml trypsin still invaginated. He believed that the hyaline layer had been dissolved; and yet the initial inturning of gastrulation could take place, though he did not observe any further invagination. It is likely that, because of the extreme difficulty if not impossibility of seeing a partially digested hyaline layer at this stage without dispersing the cells, the embryos did have a hyaline layer which was still strong enough to permit initial invagination. Experiments with pronase reported in the present paper and experiments with trypsin (unpublished observation) support this conclusion. Synthesis The incorporation of amino acids into the hyaline layer is a further indication that this structure is intimately involved in developmental events. Confirmation that incorporation is taking place in the hyaline layer and is not simply due to contamination is drawn from two sources. First, autoradiographs show a pattern of incorporation in gastrula hyaline layers. This would not be possible if the radioactivity was caused by randomly adhering cell debris. Second, if radioactive hyaline layers are run on acrylamide gels, the bulk of the radioactivity is found in the bands of hyaline layer protein. These results will be published separately. The inhibition by puromycin indicates that this protein is being synthesized and that the incorporation is not the result of terminal addition. A somewhat unexpected finding was the depression in incorporation after treatment with actinomycin. One possible interpretation of both these results is that the hyaline layer protein is translated from maternal messenger which is gradually replaced by a newly synthesized messenger during development. Another explanation is that actinomycin adversely affected the connections between the hyaline
360
CITKOWITZ
layer and the embryo, and that newly synthesized protein could not be transferred to the hyaline layer as efficiently. It is difficult to interpret the pattern of incorporation demonstrated in the autoradiographs of gastrulae. These results suggest either that the original hyaline layer material which was laid down at fertilization is used preferentially over newly synthesized material for the invagination, or that the invagination is formed from preexisting material stored in the egg before fertilization and secreted during gastrulation. To examine this and other questions, the biochemistry of the hyaline layer is now being investigated. SUMMARY
A method is described for isolating large numbers of clean, intact hyaline layers from embryos at the one-cell to late gastrula stage. The hyaline layers of embryos through the late blastula stage are clear, round sacs. Beginning with early gastrulation, an invagination is present corresponding to the invaginating archenteron. The hyaline layer is indispensable for normal development. The interaction of blastomeres at the Z-cell stage and the process of gastrulation both require the presence of an intact hyaline layer. During development, amino acids are incorporated into the hyaline layer. This incorporation is blocked by puromycin but not actinomycin. Autoradiographs of blastula hyaline layers show a uniform distribution of grains. Gastrula hyaline layers show a greatly reduced number of grains over the invagination and extreme vegetal region compared to the rest of the surface. ACKNOWLEDGMENTS This investigation was supported by Predoctoral Research Fellowship GM-34,173 and Training Grant GM-216 from the National Institutes of Health, U. S. Public Health Service and Grant GB6950 to Dr. E. Holtzman from the National Science Foundation. I am grateful to my husband, Dr. R. Hinegardner, for his advice and encouragement during this investigation and preparation of the manuscript; and I would like to thank Dr. E. Holtzman for his thoughtful criticism of the manuscript. REFERENCES E. (1968). particular reference cortical reaction. J. BAKER, J. R. (1944). Quart. J. Microsc.
ANDERSON,
Oocyte differentiation in the sea urchin, Arbaciapunctuhtu, to the origin of cortical granules and their participation Cell Biol. 37, 514-539. The structure and chemical composition of the Golgi Sci. 85, 1-71.
with in the element.
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CHAMBERS, R. (1940). The relation of extraneous coats to the organization and permeability of cellular membranes. Cold Spring Harbor Symp. Quant. Biol. 8, 144-153. CITKOWITZ-HINECARDNER, E. (1969). Characteristics of the isolated hyaline layer. Biol. Bull. 137, 403. DAN, K. (1960). f&to-embryology of echinoderms and amphibia. Int. Reu. Cytol. 9, 321367. ENDO, Y. (1961). Changes in the cortical layer of sea urchin eggs at fertilization as studied with the electron microscope. 1. Clypeasterjuporzicus. Exp. Cell Res. 25, 383397. FAUST, R. G., JONES, R. F., and PARPART, A. K. (1959). Isolation and characterization of cortical granule-hyaline material of the Arbacia egg. Biol. Bull. 117, 394. GUSTAFSON, T., and WOLPERT, L. (1967). Cellular movement and contact in sea urchin morphogenesis. Biol. Reu. Cambridge Phil. Sot. 42, 442-498. HAGSTR~M, B., and HAGSTR~M, B. E. (1954). Re-fertilization of the sea urchin egg. Exp. Cell. Res. 6, 491-496. HARVEY, E. B. (1934). Effects of centrifugal force on the ectoplasmic layer and nuclei of fertilized sea urchin eggs. Biol. Bull. 66, 228-245. HARVEY, E. B. (1935). Some surface phenomena in the fertilized sea urchin egg as influenced by centrifugal force. Biol. Bull. 69, 298-304. HERBST, C. (1900). mer das Auseinandergehen von Forchungs-und Gewebezellen in kalkfreiem Medium. Wilhelm Roux’ Arch. Entwicklungsmech. Organ. 9, 424-463. HYNES, R.. O., and GROSS, P. R. (1970). A method for separating cells from early sea urchin embryos. Develop. Biol. 21, 383-402. IMMERS, J. (1961). Comparative study of the localization of incorporated “C-labeled amino acids and ““SO, in the sea urchin ovary, egg and embryo. Exp. Cell Res. 24, 356-378. KANE, R. E. (1970). Direct isolation of the hyaline layer protein released from the cortical granules of the sea urchin egg at fertilization. J. Cell Biol. 45, 615-622. KANE, R. E., and STEPHENS, R. E. (1969). A comparative study of the isolation of the cortex and the role of the calcium-insoluble protein in several species of sea urchin egg. J. Cell Biol. 41, 133-144. KINARD, F. E. (1957). Liquid scintillator for the analysis of tritium in water. Reu. Sci. Instrum. 28, 293-294. MOORE, A. R. (1928). On the hyaline membrane and hyaline droplets of the fertilized egg of the sea urchin, Strongylocentrotus purpurutus. Protoplasma 3, 524-530. MOORE, A. R. (1952). The process of gastrulation in trypsin embryos of Dendruster excentricus. J. Exp. Zool. 119, 37-46. NAKANO, E. (1956). Physiological studies on re-fertilization of the sea urchin egg. Embryologia 3, 139-165. NAKANO, E., and OHASHI, S. (1954). On the carbohydrate component of the jelly coat and related substances of eggs from Japanese sea urchins. Embryologia 2, 81-85. STEPHENS, R. E., and KANE, R. E. (1970). Some properties of hyalin. The calcium-insoluble protein of the hyaline layer of the sea urchin egg. J. Cell Biol. 44, 611-617. SUGIYAMA, M. (1951). Re-fertilization of the fertilized eggs of the sea urchin. Biol. Bull. 101, 335-344. TUPPER, J., SAUNDERS, J. W., JR., and EDWARDS, C. (1970). The onset of electrical communication between cells of the developing starfish embryo. J. Cell Biol. 46, 187191.
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VACQUIER, V. D. (1969). The isolation and preliminary analysis of the hyaline layer of sea urchin eggs. Exp. Cell Res. 54, 140-142. VACQUIER, V. D., and MAZIA, D. (1968a). Twinning of sand dollar embryos by means of dithiothreitol. The structural basis of blastomere interactions. Ezp. Cell Res. 52, 209219. VACQUIER, V. D., and MAZIA, D. (1968b). Twinning of sea urchin embryos by treatment with dithiothreitol. Roles of cell surface interactions and of the hyaline layer. Exp. Cell Res. 52, 459-468. WOLPERT, L., and MERCER, E. H. (1963). An electron microscope study of the development of the blastula of the sea urchin embryo and its radial polarity. Exp. Cell Res. 30, 280-300. YAZAKI, I. (1968). Immunological analysis of the calcium precipitable protein of sea urchin eggs. 1. Hyaline layer substance. Embryologiu 10, 131-141.
BIOLOGY
24, 348-362
(1971)
The Hyaline Layer: Its Isolation and Role in Echinoderm Development ELENA Department
CITKOWITZ 1
of Biological
and Marine
Sciences, Columbia University, Biological Laboratory, Woods Hole, Accepted
November
New York, Massachusetts
New York 02543
10027
2, 1970
INTRODUCTION
The hyaline layer is an extracellular coat surrounding echinoderm embryos. It arises from the cortical granules during a complex series of events following fertilization or artificial activation. In the unfertilized sea urchin egg, the cortical granules lie beneath the vitelline and plasma membranes. These granules are formed by the Golgi complex during oogenesis (Anderson, 1968). Using the electron microscope, Endo (1961) clearly established the role of the cortical granules in forming both the hyaline layer and fertilization membrane. The fertilization membrane can be removed without altering the developmental process and is, therefore, of little consequence for normal development in the laboratory. There are no physical connections between the fertilization membrane and the embryo which, during the blastula stage, hatches out of this membrane. The hyaline layer, on the other hand, is firmly attached to the embryo and is not lost until metamorphosis of the larva (Dan, 1960). According to Moore (1928) and Harvey (1934), if the hyaline layer is removed, it is rapidly reformed by the embryo. As early as 1900, Herbst showed that the hyaline layer is necessary for adherence of the blastomeres. Many investigators (Sugiyama, 1951; Hagstriim and Hagstrom, 1954; Nakano, 1956) have implicated the hyaline layer in the inhibition of polyspermy; and others (Chambers, 1940; Dan, 1960; Gustafson and Wolpert, 1967) believe that the hyaline layer plays an essential role in changes of the external form of the developing embryo. Herbst (1900) demonstrated the dependency of the structural integrity of the hyaline layer on the presence of divalent cations, and hyaline layer material has been isolated by dissolving it from the surface of eggs in media devoid of these cations (Nakano and Ohashi, 1954; ‘Present address: Division Crw, California 95060.
of Natural
Sciences 348
I, University
of California,
Santa
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Faust et al., 1959; Vacquier, 1969; Kane, 1970). Hyaline layer material can also be obtained from whole egg homogenates (Yazaki, 1968) or isolated cortices (Kane and Stephens, 1969). This protein has been studied recently in more detail than previous studies (Stephens and Kane, 1970). The hyaline layer was isolated as an intact structure from cleaving eggs (Vacquier, 1969) and from blastulae and gastrulae (Citkowitz-Hinegardner, 1969). The present paper describes, in greater detail than the previous report (Citkowitz-Hinegardner, 1969), a new method for isolating large numbers of clean, intact hyaline layers from embryos at the l-cell to late gastrula stages, and examines the role of the hyaline layer in development. MATERIALS
AND
METHODS
Isolation of the hyaline layer. Eggs and sperm of Strongylocentrotus purpuratus were spawned by injection of 0.5 M KC1 and collected in the normal manner. The eggs were washed several times in Milliporefiltered seawater and fertilized; excess sperm were removed by several more washings. Only eggs showing 95% fertilization or greater were used for subsequent experiments. Embryos were raised at 15°C in concentrations of 1 to 2 X 10” eggs per 100 ml and stirred at 30 rpm. The embryos were collected by centrifugation at 2500 rpm, and the seawater was removed. Approximately 15 x lo6 embryos were then suspended in 40 ml of a solution containing 4 M NaCl, 0.2 M MgCl,, and 0.05 M Tris, pH 8. The suspension was homogenized with a tightfitting (type B) Dounce homogenizer (Kontes Glass Co.) which forced most of the cells out of the hyaline layers. Cells and debris were then sucked through nylon monofilament bolting cloth (Nitex, 35-p mesh opening) which was attached to one end of a short length of plastic tubing 3 cm in diameter. All but a few hyaline layers were stopped by the cloth, but the cells, nuclei, and debris passed through. The hyaline layer fraction was resuspended in fresh salt solution, and the filtrate was again drawn off. This process was repeated until the filtrate sucked through the bolting cloth was clear. If necessary, the hyaline layers were rehomogenized briefly, and the debris was again removed as above. Since the nuclei followed the hyaline layers during subsequent purification, it was particularly important to free the hyaline layers of nuclei at this stage of the procedure. A French press may be used in place of the Dounce homogenizer to strip the cells from the hyaline layers. Once isolated, the hyaline layers were further purified on a discon-
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tinuous sucrose gradient of 7 ml each of 90, 84, and 777; 2.5 M sucrose and 3 ml of 74 and 70% 2.5 M sucrose made up by diluting 2.5 M SUcrose with a salt solution containing 4 M NaCl, 0.8 M MgCl,, and 0.05 M Tris, pH 8. The partially purified hyaline layers were mixed with an equal volume of 70% 2.5 M sucrose and layered over the gradient. Fresh homogenizing solution was then layered on top. The tubes were centrifuged at 80,000 g for 1.5 hours at 4°C in an International ultracentrifuge, Model B-60, using an SB 110 swinging-bucket rotor. The clean hyaline layers accumulated between 74 and 77% 2.5 M sucrose. The hyaline layers were then stored in homogenizing solution at - 15°C. Yields averaged 45%. If the embryos were still surrounded by a fertilization membrane at the stage when the hyaline layers were isolated, most of the fertilization membranes in the gradient were found at the bottom of the tube. This method for isolating hyaline layers has also been applied to the sea urchins Lytechinus pictus and Arbacia punctuluta, the sand dollar Echinarachnius parma, and the starfish Asterias forbesi. Treatment of the hyaline layer. Experiments on blastomere interaction were carried out according to the procedures developed by Vacquier and Mazia (1968a,b). Embryos were treated with dithiothreitol (Calbiochem) because of reports (Vacquier and Mazia, 1968a,b) that this agent separates blastomeres at the 2-cell stage. Embryos were also treated with pronase which, as Vacquier and Mazia described (1968b), digests the hyaline layer of S. purpuratus and L. pictus. When the hyaline layer was partially digested or weakened, it became extremely difficult to see. Addition of a drop of either 0.1 M sodium dodecyl sulfate or 10% Triton X to a drop of embryos dispersed the cells and revealed the hyaline layer, although a sufficiently weak hyaline layer was itself often dispersed shortly after the cells had disintegrated. Labeling. S. purpuratus was used for labeling experiments. For pulse labeling, one million embryos were cultured at 15°C in 50 ml of Millipore-filtered seawater and stirred with a 30 rpm motor. At 18 hours, just prior to hatching, 30 &I of L-valine-3H (0.161 Ci/mmole; Schwartz BioResearch, Inc.) was added along with 50 u/ml penicillin and 50 pg/ml streptomycin. Puromycin (100 pg/ml; Nutritional Biochemical Corp.) was added 1.25 hours before addition of label. Actinomycin D (25 @g/ml; Sigma Chemical Co.) was added at fertilization, 18 hours before the label. Embryos for the two inhibitor studies were taken from the same suspension as the controls and exposed to the same concentration of label at the same time and for the same dura-
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tion. Subsequent treatment of the cultures was identical. After 1 hour in label, samples of embryos and supernatant were taken and quickfrozen. Cold L-valine (1 mg/ml) was added to the remaining embryos, which were then kept at 4°C during the isolation of the hyaline layers. Purification was carried out as already described. The procedure for long-term labeling was basically like that for the pulse. Autoradiography. For autoradiography, purified hyaline layers were spread on slides, air-dried, and fixed in Baker’s formaldehyde-calcium fixative (Baker, 1944). The slides were dipped in Kodak liquid emulsion NTB2 and exposed for 8 days at room temperature. The slides were developed in D-19 developer. Determination of radioactivity. Hyaline layer concentration was determined on a Model A Coulter counter using a 100-p aperture. Purified hyaline layers, 15,000-100,000 per sample, were centrifuged at 2500 rpm, the supernatant was removed, and the hyaline layers were washed once with 95(‘; ethanol. Samples of 2000 whole embryos were first treated with 0.5?L sodium dodecyl sulfate and then 15% cold trichloroacetic acid (TCA) to ensure the release of trapped amino acids (Hynes and Gross, 1970). Hyaline layers and embryos were then washed once in cold 5% TCA, heated for 30 minutes at 80-90°C in 5r, TCA, and washed twice with methanol. Samples were then mixed with 0.1 ml of hydroxide of Hyamine and heated for 5 minutes at 60°C. Samples were counted in either 10 ml of Kinard’s solution (Kinard, 1957) or 5 gm PPO and 100 gm naphthalene brought to 1 liter with dioxane (formula from Beckman Instruments, Inc.). Radioactivity was counted on a Beckman liquid scintillation counter. RESULTS
HYALINE
LAYER MORPHOLOGY
Hyaline layers isolated from fertilized eggs and blastulae of S. purpurutus have the same appearance. They are both clear, round sacs, intact except for a hole or tear through which the cells were forced during homogenization. They are easily visible using phase microscopy (Fig. 1). Beginning with early gastrulation, there is a distinct change in the morphology of the hyaline layer. Where the cells of the embryo are beginning to invaginate, there is a corresponding inpocketing of the hyaline layer (Fig. 2). This inpocketing, or invagination, remains in close contact with the archenteron cells and increases in length as gastrulation proceeds. When the gut forms the coelomic pouches, the hyaline layer invagination shows similar protrusions. The hyaline layer
FIG. 1. Isolated blastulahyaline layers of Strongylocentrotuspurpuratus. FIG. 2. An isolated mid-gastrula hyaline layer of S. purpuratus tion. X 360. Figures l-10, phase contrast.
showing
x 90. invagina-
in the region underlying the gut appears thicker than the rest of the hyaline layer. Hyaline layers of E. parma, L. pi&us, A. punctulata, and A. forbesi show the same change in morphology from blastula to gastrula stage. ROLE OF THE HYALINE
LAYER
Blastomere Interaction The goal of the following experiments was to correlate loss of adhesion of the blastomeres with significant changes in the state of the hyaline layer. Dithiothreitol. After exposure of E. parma eggs to 0.05 M dithiothreitol for 10 minutes during first division, the two blastomers remained spherical (Fig. 3) as reported by Vacquier and Mazia (1968a) for a similar sand dollar, Dendraster excentricus. The blastomeres of normal 2-cell embryos were closely apposed to one another (Fig. 4). When the blastomeres of dithiothreitol-treated embryos were dispersed with sodium dodecyl sulfate, for some embryos a separate hyaline layer could be seen surrounding each blastomere (Fig. 5); and for others, the hyaline layer retained the shape of the furrow as shown for L. pictus (Fig. 7). In contrast, the hyaline layer of normal 2-cell embryos became oval as soon as the cells were disrupted by sodium dodecyl sulfate (Fig. 6). Dithiothreitol-treated eggsdeveloped into twins, partial twins, and normal embryos. Vacquier and Mazia first demonstrated this phenomenon for D. excentricus. Treatment of L. pictus eggs with 0.08 M dithiothreitol during cleavage had the same effect on the hyaline layer and blastomeres as described above for E. parma; some hyaline layers were pinched in two, and others retained
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a furrow (Fig. 7). Twins, as well as elongated blastulae and normal embryos, were obtained (Fig. 8). Pronuse. Table 1 summarizes the effects of pronase on the hyaline layer and the interaction of the blastomeres. When the hyaline layer was present, the blastomeres were closely apposed to one another; and when the hyaline layer had been dispersed, the blastomeres separated from one another. Because the results for L. pictus were contrary to those of Vacquier and Mazia (1968b), experiments with this species will be considered in more detail. When eggs were fertilized and pronase added within 90 seconds, the fertilization membrane did not form; but the hyaline layer, although extremely thin, was visible at the 2-cell stage (Fig. 9). The hyaline layer of normal embryos at this stage was distinctly thicker (Fig. 10). If removed from pronase at the 2-cell stage, the embryos developed normally. Treatment with 0.067 M dithiothreitol for 14 minutes during mid-cleavage after digestion in pronase caused disruption of the hyaline layer and separation of the blastomeres in many embryos. Those cells which did not separate from one another were invariably held together by a thin hyaline layer. Vacquier and Mazia (196813)reported that in the presence of calcium-free seawater, the blastomeres of pronase-treated L. pictus embryos fell apart. Because they believed that pronase prevented hyaline layer formation, Vacquier and Mazia concluded that calcium is required to sustain cell surface interaction. The following experiment was performed to distinguish between the necessity of the hyaline layer as opposed to calcium in the maintenance of cell contact. Eggs were taken from pronase after 30 minutes, washed 3 times with calcium- and magnesium-free seawater (CMFSW), and cultured in CMFSW for an additional 25 minutes. Before first cleavage had begun, the eggs were returned to normal seawater, where calcium and magnesium were again present. Because the integrity of the hyaline layer is dependent on divalent cations, the hyaline layer was no longer present; and the blastomeres dissociated after division was complete, indicating that at this stage the hyaline layer and not calcium-mediated intercellular adhesion is necessary for normal development. Occasionally the spindle remnant maintained a loose association between the two cells until second cleavage. Control embryos, left in pronase or washed and returned to seawater, were continuously in the presence of divalent cations. These embryos remained intact and had a thin hyaline layer. Extreme care had to be taken with eggs treated with CMFSW. Damage to the cell surface would result’in nonspecific adhesion.
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FIG. 3. An Echinarczchnius parma embryo at the 2-cell stage exposed to dithiothreitol during cleavage. The blastomeres are separated from one another. X 460. FIG. 4. A normal E. parma embryo at the 2-cell stage. The blastomeres are closely apposed. X 460. FIG. 5. The hyaline layer of an E. parma embryo at the 2-cell stage exposed to dithiothreitol during cleavage and later treated with sodium dodecyl sulfate. Two separate hyaline layers can be seen. X 460. FIG. 6. The hyaline layer of a normal E. parma embryo at the 2-cell stage treated with sodium dodecyl sulfate. x 460. FIG. 7. The hyaline layer of a Lytechinus pictus embryo exposed to DTT during cleavage and later treated with sodium dodecyl sulfate. The shape of the furrow is retained. In this figure, the cytoplasm is particularly well dispersed. X 460. FIG. 8. A twinned L. pictus embryo at the 4.cell stage exposed to dithiothreitol during first cleavage. x 460.
ISOLATION
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EFFECT OF 1 MIS/ML
Species
Time
Echinaruchnius parma
Strongylocentrotus purpuratus Lytechinus pictus
ROLE
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HYALINE
TABLE 1 PRONASE ON BLASTOMERE THE HYALINE LAYER
in pronase
Fertilization mid-cleavage Fertilization S-cell stage Fertilization mid-cleavage Fertilization mid-cleavage Fertilization &cell stage
to to to to to
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AND ON
Position of blastomeres
State of hyaline layer
Closely apposed Partially separated Separated
Thin, but intact Partially dispersed Dispersed
Closely apposed Partially separated
Thin, but intact Partially dispersed
-
FIG. 9. A Lytechinus pictus embryo exposed to pronase from fertilization through first cleavage. A thin hyaline layer can be seen. X 460. FIG. 10. An untreated L. pictus embryo at the 2-cell stage showing the normal appearance of the hyaline layer. X 460.
Gastrulution Table 2 summarizes the effects of pronase digestion on the hyaline layer and on the ability of the embryo to gastrulate. Weakening of the hyaline layer was correlated, in most cases, with blocked invagination. Amino
Acid Incorporation
Using autoradiography, Immers (1961) showed that the hyaline layer incorporates amino acids as well as sulfate during development. Amino acid incorporation was investigated further in the following experiments. After exposure of the embryos to valine-3H for 1 hour, the isolated hyaline layer accounted for somewhat more than 1% of the counts in-
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CITKOWITZ TABLE EFFECT
Species
OF PRONASE
con~e~~~ion
AND
ON THE ~~YALINE
LAYER
Time in pronase and stage treated
Effect on gastrulation
0.1
Continuously
Blocked
Very
1.0
l-2
Blocked
Extremely thin Very thin
(ma/ml) Echinumchnius parma Strongylocentrotus purpuratus Lytechinus picks
2
ON GASTRULATION
1.0
hours, late blastula 9-10 hours, blastula
Blocked or abnormal
State of hyaline layer thin
corporated by the whole embryo at hatching. At that time, valine was being added to the hyaline layer at the rate of 1.4 x lo-l2 mM per hour. This is approximately 0.04% of the total valine in the hyaline layer (Citkowitz, unpublished results). Pretreatment with puromycin reduced incorporation into the hyaline layer and the whole embryo by 96%. Continuous exposure to actinomycin reduced incorporation of valine into the hyaline layer by 35 %, although incorporation into the whole embryo remained unchanged. Autoradiographs of blastula hyaline layers labeled continuously from fertilization to hatching showed a fairly even distribution of grains over the surface. Hyaline layers of embryos grown in valine-3H from hatching to mid-gastrula or from beginning to late gastrula stage were labeled over most of their surface, except for the invagination and extreme vegetal region where there was a greatly reduced number of grains (Fig. 11). DISCUSSION
The Hyaline
Layer Invagination
The results above indicate that the hyaline layer remains associated with the archenteron throughout gastrulation by means of the hyaline layer invagination. Gustafson observed a similar structure (Gustafson and Wolpert, 1967); however, he thought that the hyaline layer remained outside and that a new structure of similar appearance was established later in the gut. In view of the present results, origin of the hyaline layer invagination by this mechanism appears unlikely. Dan has suggested that the hyaline layer and the amphibian embryo’s surface coat may play analogous roles. If the surface coat is dissolved, the blastomeres fall apart; and if dissolved at a later stage, the embryo will not gastrulate. This similarity is made even more striking by the fact that both structures follow the archenteron during gastrulation. The
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FIG. 11. An isolated hyaline layer of a Strongylocentrotus purpurutus embryo exposed to valine-3H from early to late gastrula stage. Fewer grains are present over the protruding invagination and the extreme vegetal pole. x 460. Bright field.
presence of the sea urchin’s hyaline layer or the amphibian embryo’s surface coat in the archenteron probably provides an anchorage for the cells while they are undergoing the stresses and changes in shape taking place during invagination. Blastomere
Interaction
There are a number of ways that the blastomeres could be held together. There may be a component other than the hyaline layer which is also sensitive to proteolytic enzymes and to the absence of divalent cations, and whose sensitivity varies from species to species in the same way as the sensitivity of the hyaline layer. Although the existence of such a component cannot be altogether dismissed, the present experiments suggest that the hyaline layer is an absolute requirement for the adhesion of the blastomeres in early cleavage stages. When blastomeres adhered to one another after digestion in pronase, they did so because they were still surrounded by a hyaline layer. Moreover, in all circumstances that gave the appearance of weakened or disrupted cellular adhesions, alterations of the hyaline layer were found which alone were sufficient to explain the results. Thus, when eggs divided in the presence of dithiothreitol, many blastomeres remained spherical; and the hyaline layers of these embryos were furrowed or cleaved in two. Dithiothreitol apparently alters the hyaline layer rather than cell adhesion. Although the effect may be direct, there are indications that the appearance of dithiothreitol-treated hyaline layers is caused by alterations of the connections between the hya-
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line layer and the surface of the dividing egg. A normal hyaline layer is, to a degree, plastic; if stretched into a given shape, it retains that shape. The normal hyaline layer of a fertilized egg is round after dispersing the egg with sodium dodecyl sulfate; the hyaline layer of the 2cell stage is oval. If in the presence of dithiothreitol the cell retains its connections with the hyaline layer and pulls it down into the furrow, when normally it would be released in this region, the hyaline layer might retain the shape of that furrow; and if pulled far enough, it could fuse with itself, forming two individual hyaline layers. This interpretation requires no direct action of dithiothreitol on the hyaline layer. On the other hand, retention of the furrow might be caused by increased plasticity of the hyaline layer. Whatever the cause, the altered shape of the hyaline layer is sufficient to explain the resulting twins, partial twins, and elongated embryos following exposure to dithiothreitol during cleavage. The argument can be raised that the reagents used are breaking down intercellular adhesions which are important and that loss of the hyaline layer is only secondary. This could be tested if the hyaline layer were removed in some other way. Harvey (1934) centrifuged eggs of several species of sea urchin just before cleavage in a solution of seawater and sucrose which was both isotonic and isodense with the eggs. This treatment caused the hyaline layer to be thrown off. If the eggs were not surrounded by a fertilization membrane, the blastomeres separated when the eggs divided. If held together by a fertilization membrane, the hyaline layer could regenerate and normal development ensued. However, if embryos at the 2-cell stage with fertilization membranes were centrifuged and the hyaline layer was thrown off, there were all degrees of cellular interaction ranging from single embryos to complete twins; the apparent cause for the differences was the time at which the new hyaline layer was regenerated (Harvey, 1935). It would, therefore, seem that the hyaline layer is the critical structure that normally holds the blastomeres together at the 2-cell stage. This conclusion is also supported by the finding of Tupper et al. (1970) that there is no conductivity between the cells from completion of first cleavage until the 32-cell stage. The onset of conductivity coincides with the appearance of intercellular adhesions in the form of desmosomes (Wolpert and Mercer, 1963). Gastrulation Although there is variation in the response of embryos at later stages to treatment with pronase, the overall conclusion appears justified that
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a strong hyaline layer or a firm attachment of the cells to the hyaline layer is necessary for gastrulation. For S. purpuratus, whose hyaline layer is very sensitive to pronase, exposure to this enzyme for only 1 hour is enough to prevent any invagination. But for L. picks, whose hyaline layer is more resistant to pronase, treatment for 10 hours results in blocked invagination in some, but not all, embryos. These results are consistent with the theory that the hyaline layer is necessary for gastrulation and that embryos with hyaline layers, or connections to the hyaline layer, that are more sensitive to pronase will show more pronounced effects than embryos whose hyaline layers are less easily digested. For example, Moore (1952) found that embryos of the sand dollar D. excentricus grown in 1 mg/ml trypsin still invaginated. He believed that the hyaline layer had been dissolved; and yet the initial inturning of gastrulation could take place, though he did not observe any further invagination. It is likely that, because of the extreme difficulty if not impossibility of seeing a partially digested hyaline layer at this stage without dispersing the cells, the embryos did have a hyaline layer which was still strong enough to permit initial invagination. Experiments with pronase reported in the present paper and experiments with trypsin (unpublished observation) support this conclusion. Synthesis The incorporation of amino acids into the hyaline layer is a further indication that this structure is intimately involved in developmental events. Confirmation that incorporation is taking place in the hyaline layer and is not simply due to contamination is drawn from two sources. First, autoradiographs show a pattern of incorporation in gastrula hyaline layers. This would not be possible if the radioactivity was caused by randomly adhering cell debris. Second, if radioactive hyaline layers are run on acrylamide gels, the bulk of the radioactivity is found in the bands of hyaline layer protein. These results will be published separately. The inhibition by puromycin indicates that this protein is being synthesized and that the incorporation is not the result of terminal addition. A somewhat unexpected finding was the depression in incorporation after treatment with actinomycin. One possible interpretation of both these results is that the hyaline layer protein is translated from maternal messenger which is gradually replaced by a newly synthesized messenger during development. Another explanation is that actinomycin adversely affected the connections between the hyaline
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layer and the embryo, and that newly synthesized protein could not be transferred to the hyaline layer as efficiently. It is difficult to interpret the pattern of incorporation demonstrated in the autoradiographs of gastrulae. These results suggest either that the original hyaline layer material which was laid down at fertilization is used preferentially over newly synthesized material for the invagination, or that the invagination is formed from preexisting material stored in the egg before fertilization and secreted during gastrulation. To examine this and other questions, the biochemistry of the hyaline layer is now being investigated. SUMMARY
A method is described for isolating large numbers of clean, intact hyaline layers from embryos at the one-cell to late gastrula stage. The hyaline layers of embryos through the late blastula stage are clear, round sacs. Beginning with early gastrulation, an invagination is present corresponding to the invaginating archenteron. The hyaline layer is indispensable for normal development. The interaction of blastomeres at the Z-cell stage and the process of gastrulation both require the presence of an intact hyaline layer. During development, amino acids are incorporated into the hyaline layer. This incorporation is blocked by puromycin but not actinomycin. Autoradiographs of blastula hyaline layers show a uniform distribution of grains. Gastrula hyaline layers show a greatly reduced number of grains over the invagination and extreme vegetal region compared to the rest of the surface. ACKNOWLEDGMENTS This investigation was supported by Predoctoral Research Fellowship GM-34,173 and Training Grant GM-216 from the National Institutes of Health, U. S. Public Health Service and Grant GB6950 to Dr. E. Holtzman from the National Science Foundation. I am grateful to my husband, Dr. R. Hinegardner, for his advice and encouragement during this investigation and preparation of the manuscript; and I would like to thank Dr. E. Holtzman for his thoughtful criticism of the manuscript. REFERENCES E. (1968). particular reference cortical reaction. J. BAKER, J. R. (1944). Quart. J. Microsc.
ANDERSON,
Oocyte differentiation in the sea urchin, Arbaciapunctuhtu, to the origin of cortical granules and their participation Cell Biol. 37, 514-539. The structure and chemical composition of the Golgi Sci. 85, 1-71.
with in the element.
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CHAMBERS, R. (1940). The relation of extraneous coats to the organization and permeability of cellular membranes. Cold Spring Harbor Symp. Quant. Biol. 8, 144-153. CITKOWITZ-HINECARDNER, E. (1969). Characteristics of the isolated hyaline layer. Biol. Bull. 137, 403. DAN, K. (1960). f&to-embryology of echinoderms and amphibia. Int. Reu. Cytol. 9, 321367. ENDO, Y. (1961). Changes in the cortical layer of sea urchin eggs at fertilization as studied with the electron microscope. 1. Clypeasterjuporzicus. Exp. Cell Res. 25, 383397. FAUST, R. G., JONES, R. F., and PARPART, A. K. (1959). Isolation and characterization of cortical granule-hyaline material of the Arbacia egg. Biol. Bull. 117, 394. GUSTAFSON, T., and WOLPERT, L. (1967). Cellular movement and contact in sea urchin morphogenesis. Biol. Reu. Cambridge Phil. Sot. 42, 442-498. HAGSTR~M, B., and HAGSTR~M, B. E. (1954). Re-fertilization of the sea urchin egg. Exp. Cell. Res. 6, 491-496. HARVEY, E. B. (1934). Effects of centrifugal force on the ectoplasmic layer and nuclei of fertilized sea urchin eggs. Biol. Bull. 66, 228-245. HARVEY, E. B. (1935). Some surface phenomena in the fertilized sea urchin egg as influenced by centrifugal force. Biol. Bull. 69, 298-304. HERBST, C. (1900). mer das Auseinandergehen von Forchungs-und Gewebezellen in kalkfreiem Medium. Wilhelm Roux’ Arch. Entwicklungsmech. Organ. 9, 424-463. HYNES, R.. O., and GROSS, P. R. (1970). A method for separating cells from early sea urchin embryos. Develop. Biol. 21, 383-402. IMMERS, J. (1961). Comparative study of the localization of incorporated “C-labeled amino acids and ““SO, in the sea urchin ovary, egg and embryo. Exp. Cell Res. 24, 356-378. KANE, R. E. (1970). Direct isolation of the hyaline layer protein released from the cortical granules of the sea urchin egg at fertilization. J. Cell Biol. 45, 615-622. KANE, R. E., and STEPHENS, R. E. (1969). A comparative study of the isolation of the cortex and the role of the calcium-insoluble protein in several species of sea urchin egg. J. Cell Biol. 41, 133-144. KINARD, F. E. (1957). Liquid scintillator for the analysis of tritium in water. Reu. Sci. Instrum. 28, 293-294. MOORE, A. R. (1928). On the hyaline membrane and hyaline droplets of the fertilized egg of the sea urchin, Strongylocentrotus purpurutus. Protoplasma 3, 524-530. MOORE, A. R. (1952). The process of gastrulation in trypsin embryos of Dendruster excentricus. J. Exp. Zool. 119, 37-46. NAKANO, E. (1956). Physiological studies on re-fertilization of the sea urchin egg. Embryologia 3, 139-165. NAKANO, E., and OHASHI, S. (1954). On the carbohydrate component of the jelly coat and related substances of eggs from Japanese sea urchins. Embryologia 2, 81-85. STEPHENS, R. E., and KANE, R. E. (1970). Some properties of hyalin. The calcium-insoluble protein of the hyaline layer of the sea urchin egg. J. Cell Biol. 44, 611-617. SUGIYAMA, M. (1951). Re-fertilization of the fertilized eggs of the sea urchin. Biol. Bull. 101, 335-344. TUPPER, J., SAUNDERS, J. W., JR., and EDWARDS, C. (1970). The onset of electrical communication between cells of the developing starfish embryo. J. Cell Biol. 46, 187191.
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VACQUIER, V. D. (1969). The isolation and preliminary analysis of the hyaline layer of sea urchin eggs. Exp. Cell Res. 54, 140-142. VACQUIER, V. D., and MAZIA, D. (1968a). Twinning of sand dollar embryos by means of dithiothreitol. The structural basis of blastomere interactions. Ezp. Cell Res. 52, 209219. VACQUIER, V. D., and MAZIA, D. (1968b). Twinning of sea urchin embryos by treatment with dithiothreitol. Roles of cell surface interactions and of the hyaline layer. Exp. Cell Res. 52, 459-468. WOLPERT, L., and MERCER, E. H. (1963). An electron microscope study of the development of the blastula of the sea urchin embryo and its radial polarity. Exp. Cell Res. 30, 280-300. YAZAKI, I. (1968). Immunological analysis of the calcium precipitable protein of sea urchin eggs. 1. Hyaline layer substance. Embryologiu 10, 131-141.