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Cell electrophoresis of amphibian blastula and gastrula cells; the relationship of surface charge and morphogenetic movement
DEVELOPMENTAL
BIOLOGY
34, 66-76
(1973)
Cell Electrophoresis of Amphibian Blastula and Gastrula Cells; the Relationship of Surface Charge and Morphogenetic Movement BARRY Department
of Biology,
E. SCHAEFFER,’ Graduate
School
E. SCHAEFFER,’
HELENE of Arts
Accepted
and
Science, April
New
York
AND IRVING BRICK Uniuersity,
New
York,
New
York
4, 1973
By use of a novel modification in cell electrophoresis technique, an “electrophoretic map” has been made of selected embryonic regions at four different stages of embryogenesis in Rana pipiens. Hitherto, it had been impossible to make surface charge determinations for cells from small, selected regions of early amphibian embryos. Embryonic regions were dissected, and the tissue was disaggregated in EDTA. For cell electrophoresis, cells were suspended in low ionic strength medium with 15% Ficoll, and timed over 14.6 micrometers in a voltage gradient of 20 V/cm. The data reveal a correlation between three parameters: that of surface charge, morphogenetic movement, and adhesiveness. Embryonic regions that undergo a rapid increase in surface charge with the onset of gastrulation, are the same regions that initiate morphogenetic activity. Regions in which surface charge remains constant tend to be morphogenetically less active. Adhesive estimates were based on observations of degree of rounding of cells in situ. There is an apparent correlation, in ectoderm, mesoderm, and endoderm, of increased surface charge with decreased adhesiveness (and vice versa).
tafson and Wolpert, 1967) and sorting out of different cell types in reaggregates The period of embryonic development (Townes and Holtfreter, 1955: Steinberg, embraced by this study is one of the most 1964) points toward changes (or differinteresting, because the blastula, basically ences) in intercellular adhesiveness as an undifferentiated ball of cells, undergoes causes of specific morphogenetic events. an amazing transformation with the onset of gastrulation. Sheets of cells begin to Since certain regions of the gastrula unchange, i.e., migrate as though responding to some sig- dergo rapid morphogenetic dorsal lip, while other regions are relatively nal, and some cells, previously on the surquiescent, one might expect to find reface, come to reside inside the embryo as a gional differences in cellular adhesiveness. new cell layer. It is not known why cells The measurement of cellular adhesivein the dorsal lip region begin the migration ness poses a difficult technical problem, inward, nor is it known why they do so at and it may be said that no method in use that precise time. Since gastrulation is today accurately measures the strength of not an entirely unique morphogenetic intercellular attachments (Weiss, 1967; event, but involves principles that operate Curtis, 1967; Gershman, 1970). throughout development, an understanding Nevertheless, the study of such cell surof the mechanism of gastrulation would be face properties as net negative surface invaluable. charge may lead to an understanding of Changes in cellular adhesiveness might adhesive phenomena. Surface charge be of great importance in the control of morphogenesis. Evidence based on obser- measurements have in some cases shown a vations of echinoderm development (Gus- correlation between changes in net surface charge and altered states of cellular adhesiveness. Dan (1936) showed that when ‘Present address: Department of Cell Biology, surface charge is increased, the adhesiveUniversity of Glasgow, Glasgow, Scotland. INTRODUCTION
66 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form resewed.
SCHAEFFER,
SCHAEFFER,
AND BRICK
ness of sea urchin eggs to a glass surface is reduced. Garrod and Gingell (1970) and Lee (1972) have shown a linear reduction in cell surface charge during the early stages of differentiation in the slime mold Dictyostelium discoideum, which can be correlated with a progressive increase in agglutinability and adhesiveness of the cells. Lee concludes that this change in electrophoretic mobility may be essential for cell adhesion and subsequent development. Weiss (1968) suggested that contact interactions between cells may in part be regulated by electrostatic repulsion between their surfaces, but could obtain no correlation between surface charge and adhesion. It has been suggested that negative charges originating on the cell surface give rise to electrostatic repulsive forces, which reduce cell adhesiveness by preventing close approximation of cell surfaces (Pethica, 1961). It is also possible that a sufficiently high surface charge may effect a reduction in the strength of existing adhesive bonds. Average surface charge densities can be estimated from measurements of cell electrophoretic mobility, and were obtained here for amphibian blastula and gastrula cells. Until now, possibly because of the special technical difficulties encountered in electrophoresing rapidly sedimenting amphibian blastula and gastrula cells, only one previous attempt has been made to measure their mobilities (MacMurdo-Harris and Zalik, 1971). However, because the technique used for isolation of embryonic regions was based on differential densities of cells, they were unable to obtain data on selected, morphogenetically active regions. The present study presents what may be described as an “electrophoretic map” of various regions of the blastula and gastrula at four different stages of embryogenesis. A change in surface charge with time, within the same group of cells during the course of normal development is demonstrated, and the possible relevance of these surface changes and regional differences to cellular
Surface
Charge
adhesiveness cussed.
in Ranu
and
MATERIALS
Gastrulation
morphogenesis AND
67 is dis-
METHODS
Artificial ovulation and fertilization. Ovulation was induced in sexually mature Rana pipiens by injecting females with pituitary extract (Ward’s Natural Science Establishment, Rochester, New York) equivalent to three female pituitary glands, plus 3-5 mg of progesterone (50 mg/ml, Gotham Pharmaceutical, Brooklyn, New York). Eggs can be consistently obtained all year, including the summer months with this technique. Eggs were stripped directly into a previously prepared sperm suspension, where fertilization occurs (Rugh, 1962). Dissection of embryos. Late blastula, and early, mid and late gastrula stages were used, corresponding to Shumway stages 9, 10, 11, and 11% (Shumway, 1940). At appropriate times after fertilization, eggs were removed from the incubator (18”C), but maintained in cool spring water to retard further development of embryos. Jelly was removed, and dejellied eggs were collected in precooled standard Holtfreter’s (1944) solution with 0.75 A4 sucrose for removal of the vitelline (fertilization) membrane. The eggs were then immediately replaced into standard Holtfreter’s solution. That this procedure does not harm the embryo is illustrated by the fact that embryos whose membranes were removed in this fashion undergo normal development if permitted to do so. Specific areas of the embryo to be utilized were dissected free and trimmed to standard size. Embryonic regions utilized in this study included inner ectoderm, dorsal lip, head endoderm, and endodermal floor of archenteron, in all three gastrula stages. Other regions studied were “coated” (outer) ectoderm, gray crescent, and chordamesoderm (see Table 1 and Fig. 4). Preparation of cells for electrophoresis. Dissected embryonic pieces were disaggregated in calcium-free Holtfreter’s solution
68
DEVELOPMENTAL
BIOLOGY
with 15 mg/lOO ml EDTA, at pH 8.9. Endoderm and chordamesoderm were usually disaggregated after 25 min, while inner ectoderm was disaggregated after 45 ,min in the above solution. However, the more heavily pigmented “coated” ectoderm was not disaggregated by this treatment, even if greatly prolonged. Attempts to disaggregate “coated” ectoderm with up to 60 mg/lOO ml EDTA proved to be unsuccessful even after 1.5 hr. However, a similar procedure employing 0.034 M potassium oxalate successfully disaggregated “coated” ectoderm after 45 min at room temperature. Viability studies (see results) done on cells so disaggregated revealed a 98% viability, and cells of known electrophoretic mobility gave the expected readings after treatment with potassium oxalate. After 45 min in disaggregation medium at room temperature all cells were washed in calcium-free Holtfreter’s solution. Cells were then washed in electrophoresis medium (see below). Measurement of electrophoretic mobility. The fluid used to suspend amphibian cells in the electrophoresis chamber was a calcium and bicarbonate free salt solution (modified Ko strength Holtfreter’s solution) to which Ficoll (Pharmacia Fine Chemicals, New Jersey) and sucrose were added to increase density and to adjust osmotic pressure, respectively. Phenol red was added to indicate pH change. Its ionic strength was 0.0065, and the calculated absolute viscosity as measured in a Cannon-Fenske reverse-flow viscometer (No. 450) at 25°C was 12.71 centipoises. Density was determined with a pycnometer to be 1.0599 relative to distilled water, and the osmotic pressure was calculated to be 3000 cm H,O. pH was adjusted to 7.0 immediately before experimental use with 0.05 normal sodium hydroxide. The time it took a cell to travel, in a voltage gradient of 20 V/cm, an arbitrary distance of 14.6 micrometers, which is the
VOLUME
34, 1973
distance between two lines of a previously calibrated ocular reticle, was recorded by an electronic timer to the nearest 0.1 sec. The polarity was then reversed, and the same cell was timed in the opposite direction. A single cell measurement consisted of the average time in both directions. This minimized the effect of any local drift, thermal convection, turbulence, or timing error. In addition, it was imperative that the direction of current flow be reversed every few seconds to prevent polarization of electrodes. Raw data were converted into electrophoretic mobility, p, defined as the electrophoretic velocity per unit field strength, expressed as &ec/V/cm. Electrophoresis apparatus. Curtis (1967) stated that it is impossible to suspend embryonic amphibian cells in the conventional type of electrophoresis apparatus because of their high density, and cited this as the main reason why few measurements have been made. The apparatus used was a cylindrical cell electrophoresis unit, made by Rank Brothers, Bottisham, Cambridge, England, and is similar to the one designed by Bangham (Bangham et al., 1958). Several modifications in design and procedure had to be worked out before this unit could be used on amphibian gastrula cells. Since only very small regions of the gastrula were dissected out for use, cells from many embryos were pooled to provide a sufficient number of cells with which to work. Maximum use was made of the available cells by placing them in the center of the optical flat of the electrophoresis chamber. This was achieved by the insertion of a fine flexible polyethylene tube through one electrode side well into the capillary until the center of the optical flat was reached. If the chamber is first filled with solution and one electrode is sealed in place, the solution cannot flow even though the second electrode is not yet in place. This permits accurate placement of cells. The technique
SCHAEFFE~
SCHAEFFE~
AND BRICK
was further refined by using a micrometercontrolled syringe to control flow through the tubing, thereby permitting the injection of minute quantities of cells directly into the microscopic field of view. Embryonic amphibian cells have an unusually high density due to the presence of large numbers of yolk platelets, and settle so rapidly as to prevent accurate mobility determinations. In addition, accumulation of sedimented cells within the chamber destroys its symmetry, distorts electroendosmotic flow, and creates turbulence. After testing many high density nonionic solutions, it was found that Ficoll (15%) prevented cells from sedimenting from suspension and maintained them in healthy condition. In order to overcome the reduction in cell velocity produced by the high viscosity of Ficoll, the ionic strength of the solution was reduced. To further increase cell mobility rates, the chamber was modified to reduce its interelectrode distance from 16 cm in the commerically available chamber, to 5 cm, which resulted in a proportionate increase in field strength. RESULTS
Electrophoresis
of Red Blood Cells
Freshly drawn human blood diluted in phosphate buffer (ionic strength 0.172) was poured into the electrophoresis chamber, and in a voltage gradient of 4 V/cm and 1.8 mA the mean mobility in terms of /*/ set/V/cm was -1.28 & 0.01 (SEM) at 25°C. This agrees with the value of -1.28 reported in the literature by Bangham et al. (1958). Erythrocytes suspended in phosphate buffer containing 15% Ficoll had a mean mobility of -0.466 &ec/Vlcm. The high viscosity of ficoll can account for much of the observed decrease in electrophoretic mobility. Uncertainty regarding the effects of Ficoll on dielectric constant, and on the “double-layer” surrounding the cells make calculations of absolute values for zeta
Surface
Charge
in Ranu
69
Gastrulation
potential and surface charge density of doubtful value. However, as both zeta potential and surface charge density are directly proportional to electrophoretic mobility, our data enable us to compare these parameters. Cell Viability EDTA-disaggregated cells, obtained from early and late gastrula stages, were tested for their ability to exclude dye (Kaltenbach, 1958) after a 30-min incubation period in electrophoresis medium at pH 7.0 and 25”, since it was under these conditions that electrophoresis runs were performed. Repeated testing indicated that under the given conditions a maximum of 4% of the cells could be considered as nonviable. Embryos whose vitelline membranes were removed at the early gastrula stage and left to develop in electrophoresis medium underwent normal gastrulation. Further confirmation that the electrophoresis medium was physiological and well tolerated by the cells was the observation that cells rapidly adhered to the glass surface of the depression slide and began to flatten and spread against it, as did control cells in Holtfreter’s solution. The same cells in a less favorable milieu retain their spherical shape. Electrophoretic Cells
Mobility
of
Embryonic
Frequency distribution of cell populations. The frequency histograms presented in Figs. 1 and 2 illustrate the accumulated data for each region at each developmental stage studied. It can be seen that each histogram follows what is essentially a normal distribution pattern, and each is characteristically unimodal. This, plus the rather narrow dispersion indicated by a small standard error for each population, indicates that each histogram represents an electrophoretically homogeneous population.
70
DEVELOPMENTAL
BIOLOGY
VOLUME
34, 1973
Dorrol lip Edy gartrula
I I Chordamemderm Mid gartrulo 74 cells
Chordmeroderm
Seconds
FIG. different
1. Electrophoretic mobility stages of development.
Differences Studied
in Mobility
frequency
to
Three regions of the late blastula were studied, namely, presumptive dorsal lip, gray crescent, and inner ectoderm. The electrophoretic mobilities of all three were essentially the same, the differences being statistically insignificant as judged by the Student’s t test (see Table 2). Additionally, the mobilities of cells from the late blastula are slower than those of most cells
Dorrol
lip
Mid gartrvla
+
travel
histograms
of the Regions
I
_
14.6
for gray
Dord lip Late gortrula 72 cells
microns
crescent-chordamesoderm
and
dorsal
lip at
from any region of subsequent stages (i.e., they have the least surface charge). With 5 hrs of additional development at 18”C, the early gastrula stage is reached. In contrast with the blastula, each region has now become electrophoretically different from every other (see Table 1). At this, and subsequent gastrula stages, the archenteron floor (endoderm) maintains the highest surface charge of any region, while ectoderm maintains the lowest surface charge. Gray crescent and dorsal lip have
SCHAEFFER,
SCHAEFFE~
Surface
AND BRICK
I
LATE
Charge
in Ram
71
Gastrulation
BLASTULA
Inner ectoderm Head endodcrm
I
EARLY
GASTRULA
GASTRULA
Seconds
FIG. 2. Electrophoretic floor at different stages
mobility frequency of development.
surface charges falling extremes.
between
to
travel
histograms
these two
The electrophoretic mobilities of the same regions in the mid and late gastrula show much the same pattern, endoderm having the highest surface charge, chordamesoderm having less, and ectoderm having the lowest (see Fig. 3). The difference in surface charge between these regions is highly significant (P < 0.001). “Coated” (outer) ectoderm cells, studied only in the late gastrula, were observed to have the slowest migration rate of any cell type, even slower than cells of the late blastula.
14.6
for inner
microns
ectoderm,
head
endoderm,
and
Changes in Mobility as a Function velopmental Stage
archenteron
of De-
In the section above it has been shown that within each stage of gastrulation, cells from different embryonic regions have significantly different electrophoretic mobilities. There also appears to be an electrophoretic hierarchy of embryonic regions, with the magnitude of surface charge diminishing from endoderm to mesoderm and ectoderm. An exception to this was the late blastula, where the three regions studied had mobilities that closely approximated each other. If one now follows the cell population of
72
DEVELOPMENTAL TABLE
Embryonic
REGIONS
region
OF
Mobility ‘+%Z
Late blastula (Shumway stage 9) Early gastrula (Shumway stage 10)
Mid-gastrula (Shumway stage 11)
Late gastrula (Shumway stage 11%)
Inner ectoderm Gray crescent Presumptive dorsal lip Inner ectoderm Dorsal lip Gray crescent Head endoderm Presumptive floor of archenteron Inner ectoderm Dorsal lip Chordamesoderm Head endoderm Floor of archenteron “Coated” ectoderm Inner ectoderm Dorsal lip Chordamesoderm Head endoderm Floor of archenteron
VOLUME
0.292 0.295 0.296 0.296
0.433 0.358 0.442 0.506 0.300 0.408 0.427 0.442 0.472 0.221 0.302 0.406 0.439 0.406 0.511 TABLE
COMPARISON
Mean Inner ectoderm
electrophoretic
Gray crescent
Dorsal lip
Chordamesoderm
0.005 11 O.l
1
0.007 0.012 P < 0.001 1 1 P
0.300 'p
gastrula
0.302 zt 0.004
P < 0.001
0.006 1 IO.05
P < 0.001 <
P < 0.001
OF THE Rana
pipiens
+ SEM Head endoderm -
Floor of archenteron -
0.015 1
gastrula
Mid-gastrula
(-&ec/V/cm)
-
0.003
Late
mobility
blastula
0.004 1 O.l
2
OF THE DIFFERENCES IN ELECTROPHORETIC MOBILITY OF VARIOUS REGIONS EMBRYO WITHIN EACH OF FOUR DEVELOPMENTAL STAGES
Developmental Stage
Late
34, 1973
particular regions through progressive developmental stages, it is observed that for some regions, great changes in electrophoretic mobility occur, while for other regions, mobilities remain fairly constant throughout (see Fig. 3). A great increase in electrophoretic mobility occurs in the gray crescent cells of the blastula as they progress to become the chordamesoderm cells which first appear in the mid-gastrula. During this time the gray crescent region migrates downward and sinks to the embryonic interior at the dorsal lip. Thus, in the early gastrula, cells comprising the dorsal lip are really gray crescent cells migrating inward, where they become chordamesoderm. The greatest increase in electrophoretic mobility is seen in the dorsal lip region as the blastula progresses to the early gastrula. The increase in mobility is more than 46% at this time. It is important to note that although the dorsal lip is a permanent structure in the gastrula, it exists only as a pathway through which the
1
ELECTROPHORETIC MOBILITY OF VARIOUS THE Rana pipiens EMBRYO Developmenta Stage
BIOLOGY
0.001 0.406 + 0.008 ] I 0.001
0.442 zt 0.506 * 0.011 0.029 1 IO.05 < P < 0.11
+ 0.442 ztz 0.472 + 0.012 0.018 0.009 < 0.11 1 0.1
o.a39 l 0.406 * 0.511 f 0.007 O.CO8 0.018 < P < 0.01~~0.001 < P < 0.01) I P < 0.001 1
SCHAEFFE~
SCHAEFFER,
Surface
AND BRICK
0..
*...
Charge
in Ranu
Gastrulation
73
Archenteron . ..*0 floor I... ...*
‘...&.”
*..f. ..**
,&------A
Inner ectoderm
“Cooted” @ ectoderm -0.20
’
I Blastul0
I Early g0strul0 Stage
I Mid gostrulo of development
I Lote gostrulo
FIG. 3. Electr Ithoretic mobility as a function of developmental stage. Statistically significant increases in mobility occur with onset of gastrulation in dorsal lip and gray crescent. Inner ectoderm shows no change in mobility with development. No data could be obtained for the archenteron floor and head endoderm at the blastula stage.
cells migrate to the interior. Thus, the cell population that comprises the dorsal lip at one- stage is not the same population as in the later stage. Further examination of Table 3 and Fig. 3 reveals that the mobilities of cells from inner ectoderm and also the archenteron floor do not change significantly between any of the developmental stages studied. The mobility of inner ectoderm remains remarkably constant, maintaining the same low migration rate found for blastula cells. Appraisal of Adhesiveness in Situ Gross observation while dissecting the gastrula gives the impression that cells of some gastrula regions do not cohere as tightly as others (Fig. 4). The large yolky endoderm cells of the archenteron floor appear almost spherical, and are very
weakly cohesive as judged by the ease with which individual cells may be displaced when gently pushed with a hair loop. Their spherical shape indicates a minimal contact area between cells. In contrast, inner ectoderm cells may be characterized as a sheet of strongly cohesive, flattened cells. Individual cells cannot be separated with a hair loop. Chordamesoderm appears to be more cohesive than endoderm and less than ectoderm. The cells are sufficiently cohesive to be removed as a sheet, but individual cells are more rounded (have less intercellular contact) than those of ectoderm. Differences in contact area between cells and ease of separation are suggestive of differences in their adhesive strengths. Although quantitative data are not available, these parameters suggest an adhesive hierarchy descending from ectoderm to mesoderm and endoderm. Such
74
DEVELOPMENTALBIOLOGY
FIG.
VOLUME
34, 1973
4. A late gastrula dissected along the mid-sagittal plane. The large, yolky endoderm cells (end) ares #een very weakly cohesive mass. In contrast, ectoderm cells (ect) form a highly cohesive sheet. Dorsal lip plug (yp); ventral lip (ul).
to form a (a’1); yolk
TABLE 3 MOBILITY WITH TIME IN VARIOUS REGIONS AT FOUR DEVELOPMENTAL STAGES
CHANC :E IN ELECTROPHORETIC
Embryonic region
Mean Late
Inner
ectoderm
0.292
blastula
Early
lip
0.296
crescent
0.295
(-&e&‘/cm)
gastrula
ss 0.015
f 0.005
0.358
* 0.012
0.408
1 1 0.05
+ 0.067
0.1
Late
gastrula
0.302
! 0.004
+ 0.006
< 0.1 1 1
EMBRYO
+ SEM
* 0.004
1 1
0.1
Ranu pipiens
Mid-gastrula
< p < :.ioo
P < 0.001 Gray
mobility
*o.oo40.296;t~ 0.1
Dorsal
electrophoretic
OF THE
I 0.406
+ 0.008
0.1
I
-
-
P < 0.001 Chordamesoderm
Head
endoderm
-
-
p
I L, -
0.442
0.427
+ 0.011
+ 0.009 -0.1
0.442 0.1
1
+ 0.012 0.01
0.439
* 0.007
0.406
+ 0.008
< 0.02
1
I Floor
of archenteron
-
0.506
i 0.029
0.472 0.1
i 0.018
0.511 0.1
i 0.018
SCHAEFFER,
SCHAEFFER,
AND BRICK
Surface
Charge
in Ranu
Gmtrulation
75
an adhesive hierarchy is compatible with overlooked that the surface charge increase the previously described electrophoretic may be the natural consequence of morhierarchy. Thus, a large surface charge is phogenetic activity or perhaps of a bioassociated with a weakly adhesive cell type chemical change which follows morphogenetic movement. It is conceivable that the (endoderm), and a low surface charge with increase in surface charge observed here a strongly adhesive type (ectoderm). may be related to increased metabolism. DISCUSSION Mayhew and Weiss (1968) showed this The data presented here reveal an in- relationship for some cultured cells. The apparent correlation, in ectoderm, triguing correlation between three parameand endoderm, of increased ters: surface charge, morphogenetic move- mesoderm, surface charge with decreased adhesiveness ment, and adhesiveness. Those embryonic regions that undergo a rapid increase in (and vice versa), must be considered causurface charge are the very same regions to tiously. It is possible that this correspondAlso, quantitainitiate morphogenetic activity. With the ence might be fortuitous. onset of gastrulation, cells of the presumptive studies on cell adhesion have not been tive dorsal lip region are the first to become done on the amphibian gastrula. Relative active (forming bottle cells which migrate differences in adhesion in situ have been estimated based upon observations of coninward), and also show the most dramatic increase in electrophoretic mobility. When tact area, degree of rounding, and ease of gray crescent cells begin to migrate down- separation. That these parameters are to ward toward the dorsal lip, they too show a some extent a measure of adhesiveness is supported by the studies of Axelrad and corresponding increase in electrophoretic mobility. Thus, those embryonic regions McCulloch (1958) and of Terasima and which will be the most active are observed Tolmach (1963), who showed that cells to greatly increase their surface charge at undergoing mitosis round up and adhere the precise time they begin morphogenetic less firmly to the substratum. In fact, mitotic cells may be selectively collected activity. It is noteworthy that these changes derive from a blastula which is by a technique based upon their decreased electrophoretically, as well as morability to adhere. Nevertheless, a more phogenetically, undifferentiated. The less formal, quantitative estimation of adheactive embryonic regions, such as ectoderm siveness is desirable. and archenteron floor, do not undergo a We have observed in the amphibian change in electrophoretic mobility. Ec- gastrula that increased surface charge is toderm spreads somewhat, to replace gray associated with decreased adhesiveness crescent as it migrates inward, but stops and the initiation of morphogenetic activspreading when it reaches the dorsal lip ity. It is a well known fact that negative and apparently lacks the morphogenetic charges on the cell surface are responsible capacity for inward migration. for electrostatic forces of repulsion (PeThe association of morphogenetic activ- thica, 1961). Although speculative, it is ity with increased surface charge is dif- conceivable that a sufficiently large repulficult to interpret at this time. Although sive force may be generated by an increase the data are suggestive of a causal relationin surface charge so that cell adhesion is ship with an increase in surface charge reduced. The resulting alteration in cell triggering morphogenesis, more work must contact may in some way influence the be done before other possibilities can be initiation of morphogenetic movements. ruled out. The possibility should not be Thus, differences in cell surface charge
76
DEVELOPMENTAL
BIOLOGY
appear to provide a physical basis for the adhesive differences postulated by Gustafson and Wolpert (1967), and Steinberg ( 1964). REFERENCES A. A., and MCCULLOCH, E. A. (1958). Obtaining suspensions of animal cells in metaphase from cultures propogated on glass. Stain Technol. 33, 67-71. BANGHAM, A. D., FLEMANS, R., HEARD, D. H., and SEAMAN, G. V. F. (1958). An apparatus for microelectrophoresis of small particles. Nature (London), 182, 642-644. CURTIS, A. S. G. (1967). “The Cell Surface: Its Molecular Role in Morphogenesis.” Academic Press, New York. DAN, K. (1936). Electrokinetic studies of marine ova. III. Physiol. 2001. 9, 43-57. GARROD, D. R., and GINGELL, D. (1970). A progressive change in the electrophoretic mobility of preaggregation cells of the slime mould, Dictyostelium discoideum. J. Cell Sci. 6, 277-284. GERSHMAN, H. (1970). On the measurement of cell adhesiveness. J. Exp. Zool. 174, 391-406. GUSTAFSON, T., and WOLPERT, L. (1967). Cellular movement and contact in sea urchin morphogenesis. Biol. Rev. Cambridge Phil. Sot. 42, 442-498. HOLTFRETER, J. (1944). A study of the mechanics of gastrulation. Part II. J. Exp. Zool. 95, 171-212. KALTENBACH, J. P., KALTENBACH, N. H., and LYONS, W. B. (1958). Nigrosin as a dye for differentiating live and dead ascites cells. Exp. Cell Res. 15, 112-117. AXELRAD,
VOLUME
34, 1973
LEE, K. C. (1972). Cell electrophoresis of the cellular slime mould, Dictyostelium discoideum. II. Relevance of the changes in cell surface charge density to cell aggregation and morphogenesis. J. Cell Sci. 10, 249-265. MACMURDO-HARRIS, H., and ZALIK, S. E. (1971). Microelectrophoresis of early amphibian embryonic cells. Develop. Biol. 24, 335-347. MAYHEW, E., and WEISS, L. (1968). Ribonucleic acid at the periphery of different cell types, and effect of growth rate on ionogenic groups in the periphery of cultured cells. Exp. Cell Res. 50, 441-453. PETHICA, B. A. (1961). The physical chemistry of cell adhesion. Exp. Cell Res., Suppl. 8, 1233140. RUGH, R. (1962). “Experimental Embryology.” Burgess, Minneapolis, Minnesota. SHUMWAY, W. (1940). Stages in the normal development of Rana pipiens. Anat. Rec. 78, 139-148. STEINBERG, M. S. (1964). The problem of adhesive selectivity in cellular interactions. In “Cellular Membranes in Development”(M. Locke, ed.), pp. 321-366. Academic Press, New York. TERASIMA, T., and TOLMACH,L. J. (1963). Growth and nucleic acid synthesis in a synchronously dividing population of HeLa cells. Z&p. Cell Res. 30, 344-362. TOWNES, P. L., and HOLTFRETER, J. (1955). Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 128, 53-118. WEISS, L. (1967). “The Cell Periphery, Metastasis and Other Contact Phenomena.” Wiley, New York. WEISS, L. (1968). Studies on cellular adhesion in tissue culture. IX. Electrophoretic mobility and contact phenomena. Exp. Cell Res. 51,609-625.
BIOLOGY
34, 66-76
(1973)
Cell Electrophoresis of Amphibian Blastula and Gastrula Cells; the Relationship of Surface Charge and Morphogenetic Movement BARRY Department
of Biology,
E. SCHAEFFER,’ Graduate
School
E. SCHAEFFER,’
HELENE of Arts
Accepted
and
Science, April
New
York
AND IRVING BRICK Uniuersity,
New
York,
New
York
4, 1973
By use of a novel modification in cell electrophoresis technique, an “electrophoretic map” has been made of selected embryonic regions at four different stages of embryogenesis in Rana pipiens. Hitherto, it had been impossible to make surface charge determinations for cells from small, selected regions of early amphibian embryos. Embryonic regions were dissected, and the tissue was disaggregated in EDTA. For cell electrophoresis, cells were suspended in low ionic strength medium with 15% Ficoll, and timed over 14.6 micrometers in a voltage gradient of 20 V/cm. The data reveal a correlation between three parameters: that of surface charge, morphogenetic movement, and adhesiveness. Embryonic regions that undergo a rapid increase in surface charge with the onset of gastrulation, are the same regions that initiate morphogenetic activity. Regions in which surface charge remains constant tend to be morphogenetically less active. Adhesive estimates were based on observations of degree of rounding of cells in situ. There is an apparent correlation, in ectoderm, mesoderm, and endoderm, of increased surface charge with decreased adhesiveness (and vice versa).
tafson and Wolpert, 1967) and sorting out of different cell types in reaggregates The period of embryonic development (Townes and Holtfreter, 1955: Steinberg, embraced by this study is one of the most 1964) points toward changes (or differinteresting, because the blastula, basically ences) in intercellular adhesiveness as an undifferentiated ball of cells, undergoes causes of specific morphogenetic events. an amazing transformation with the onset of gastrulation. Sheets of cells begin to Since certain regions of the gastrula unchange, i.e., migrate as though responding to some sig- dergo rapid morphogenetic dorsal lip, while other regions are relatively nal, and some cells, previously on the surquiescent, one might expect to find reface, come to reside inside the embryo as a gional differences in cellular adhesiveness. new cell layer. It is not known why cells The measurement of cellular adhesivein the dorsal lip region begin the migration ness poses a difficult technical problem, inward, nor is it known why they do so at and it may be said that no method in use that precise time. Since gastrulation is today accurately measures the strength of not an entirely unique morphogenetic intercellular attachments (Weiss, 1967; event, but involves principles that operate Curtis, 1967; Gershman, 1970). throughout development, an understanding Nevertheless, the study of such cell surof the mechanism of gastrulation would be face properties as net negative surface invaluable. charge may lead to an understanding of Changes in cellular adhesiveness might adhesive phenomena. Surface charge be of great importance in the control of morphogenesis. Evidence based on obser- measurements have in some cases shown a vations of echinoderm development (Gus- correlation between changes in net surface charge and altered states of cellular adhesiveness. Dan (1936) showed that when ‘Present address: Department of Cell Biology, surface charge is increased, the adhesiveUniversity of Glasgow, Glasgow, Scotland. INTRODUCTION
66 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form resewed.
SCHAEFFER,
SCHAEFFER,
AND BRICK
ness of sea urchin eggs to a glass surface is reduced. Garrod and Gingell (1970) and Lee (1972) have shown a linear reduction in cell surface charge during the early stages of differentiation in the slime mold Dictyostelium discoideum, which can be correlated with a progressive increase in agglutinability and adhesiveness of the cells. Lee concludes that this change in electrophoretic mobility may be essential for cell adhesion and subsequent development. Weiss (1968) suggested that contact interactions between cells may in part be regulated by electrostatic repulsion between their surfaces, but could obtain no correlation between surface charge and adhesion. It has been suggested that negative charges originating on the cell surface give rise to electrostatic repulsive forces, which reduce cell adhesiveness by preventing close approximation of cell surfaces (Pethica, 1961). It is also possible that a sufficiently high surface charge may effect a reduction in the strength of existing adhesive bonds. Average surface charge densities can be estimated from measurements of cell electrophoretic mobility, and were obtained here for amphibian blastula and gastrula cells. Until now, possibly because of the special technical difficulties encountered in electrophoresing rapidly sedimenting amphibian blastula and gastrula cells, only one previous attempt has been made to measure their mobilities (MacMurdo-Harris and Zalik, 1971). However, because the technique used for isolation of embryonic regions was based on differential densities of cells, they were unable to obtain data on selected, morphogenetically active regions. The present study presents what may be described as an “electrophoretic map” of various regions of the blastula and gastrula at four different stages of embryogenesis. A change in surface charge with time, within the same group of cells during the course of normal development is demonstrated, and the possible relevance of these surface changes and regional differences to cellular
Surface
Charge
adhesiveness cussed.
in Ranu
and
MATERIALS
Gastrulation
morphogenesis AND
67 is dis-
METHODS
Artificial ovulation and fertilization. Ovulation was induced in sexually mature Rana pipiens by injecting females with pituitary extract (Ward’s Natural Science Establishment, Rochester, New York) equivalent to three female pituitary glands, plus 3-5 mg of progesterone (50 mg/ml, Gotham Pharmaceutical, Brooklyn, New York). Eggs can be consistently obtained all year, including the summer months with this technique. Eggs were stripped directly into a previously prepared sperm suspension, where fertilization occurs (Rugh, 1962). Dissection of embryos. Late blastula, and early, mid and late gastrula stages were used, corresponding to Shumway stages 9, 10, 11, and 11% (Shumway, 1940). At appropriate times after fertilization, eggs were removed from the incubator (18”C), but maintained in cool spring water to retard further development of embryos. Jelly was removed, and dejellied eggs were collected in precooled standard Holtfreter’s (1944) solution with 0.75 A4 sucrose for removal of the vitelline (fertilization) membrane. The eggs were then immediately replaced into standard Holtfreter’s solution. That this procedure does not harm the embryo is illustrated by the fact that embryos whose membranes were removed in this fashion undergo normal development if permitted to do so. Specific areas of the embryo to be utilized were dissected free and trimmed to standard size. Embryonic regions utilized in this study included inner ectoderm, dorsal lip, head endoderm, and endodermal floor of archenteron, in all three gastrula stages. Other regions studied were “coated” (outer) ectoderm, gray crescent, and chordamesoderm (see Table 1 and Fig. 4). Preparation of cells for electrophoresis. Dissected embryonic pieces were disaggregated in calcium-free Holtfreter’s solution
68
DEVELOPMENTAL
BIOLOGY
with 15 mg/lOO ml EDTA, at pH 8.9. Endoderm and chordamesoderm were usually disaggregated after 25 min, while inner ectoderm was disaggregated after 45 ,min in the above solution. However, the more heavily pigmented “coated” ectoderm was not disaggregated by this treatment, even if greatly prolonged. Attempts to disaggregate “coated” ectoderm with up to 60 mg/lOO ml EDTA proved to be unsuccessful even after 1.5 hr. However, a similar procedure employing 0.034 M potassium oxalate successfully disaggregated “coated” ectoderm after 45 min at room temperature. Viability studies (see results) done on cells so disaggregated revealed a 98% viability, and cells of known electrophoretic mobility gave the expected readings after treatment with potassium oxalate. After 45 min in disaggregation medium at room temperature all cells were washed in calcium-free Holtfreter’s solution. Cells were then washed in electrophoresis medium (see below). Measurement of electrophoretic mobility. The fluid used to suspend amphibian cells in the electrophoresis chamber was a calcium and bicarbonate free salt solution (modified Ko strength Holtfreter’s solution) to which Ficoll (Pharmacia Fine Chemicals, New Jersey) and sucrose were added to increase density and to adjust osmotic pressure, respectively. Phenol red was added to indicate pH change. Its ionic strength was 0.0065, and the calculated absolute viscosity as measured in a Cannon-Fenske reverse-flow viscometer (No. 450) at 25°C was 12.71 centipoises. Density was determined with a pycnometer to be 1.0599 relative to distilled water, and the osmotic pressure was calculated to be 3000 cm H,O. pH was adjusted to 7.0 immediately before experimental use with 0.05 normal sodium hydroxide. The time it took a cell to travel, in a voltage gradient of 20 V/cm, an arbitrary distance of 14.6 micrometers, which is the
VOLUME
34, 1973
distance between two lines of a previously calibrated ocular reticle, was recorded by an electronic timer to the nearest 0.1 sec. The polarity was then reversed, and the same cell was timed in the opposite direction. A single cell measurement consisted of the average time in both directions. This minimized the effect of any local drift, thermal convection, turbulence, or timing error. In addition, it was imperative that the direction of current flow be reversed every few seconds to prevent polarization of electrodes. Raw data were converted into electrophoretic mobility, p, defined as the electrophoretic velocity per unit field strength, expressed as &ec/V/cm. Electrophoresis apparatus. Curtis (1967) stated that it is impossible to suspend embryonic amphibian cells in the conventional type of electrophoresis apparatus because of their high density, and cited this as the main reason why few measurements have been made. The apparatus used was a cylindrical cell electrophoresis unit, made by Rank Brothers, Bottisham, Cambridge, England, and is similar to the one designed by Bangham (Bangham et al., 1958). Several modifications in design and procedure had to be worked out before this unit could be used on amphibian gastrula cells. Since only very small regions of the gastrula were dissected out for use, cells from many embryos were pooled to provide a sufficient number of cells with which to work. Maximum use was made of the available cells by placing them in the center of the optical flat of the electrophoresis chamber. This was achieved by the insertion of a fine flexible polyethylene tube through one electrode side well into the capillary until the center of the optical flat was reached. If the chamber is first filled with solution and one electrode is sealed in place, the solution cannot flow even though the second electrode is not yet in place. This permits accurate placement of cells. The technique
SCHAEFFE~
SCHAEFFE~
AND BRICK
was further refined by using a micrometercontrolled syringe to control flow through the tubing, thereby permitting the injection of minute quantities of cells directly into the microscopic field of view. Embryonic amphibian cells have an unusually high density due to the presence of large numbers of yolk platelets, and settle so rapidly as to prevent accurate mobility determinations. In addition, accumulation of sedimented cells within the chamber destroys its symmetry, distorts electroendosmotic flow, and creates turbulence. After testing many high density nonionic solutions, it was found that Ficoll (15%) prevented cells from sedimenting from suspension and maintained them in healthy condition. In order to overcome the reduction in cell velocity produced by the high viscosity of Ficoll, the ionic strength of the solution was reduced. To further increase cell mobility rates, the chamber was modified to reduce its interelectrode distance from 16 cm in the commerically available chamber, to 5 cm, which resulted in a proportionate increase in field strength. RESULTS
Electrophoresis
of Red Blood Cells
Freshly drawn human blood diluted in phosphate buffer (ionic strength 0.172) was poured into the electrophoresis chamber, and in a voltage gradient of 4 V/cm and 1.8 mA the mean mobility in terms of /*/ set/V/cm was -1.28 & 0.01 (SEM) at 25°C. This agrees with the value of -1.28 reported in the literature by Bangham et al. (1958). Erythrocytes suspended in phosphate buffer containing 15% Ficoll had a mean mobility of -0.466 &ec/Vlcm. The high viscosity of ficoll can account for much of the observed decrease in electrophoretic mobility. Uncertainty regarding the effects of Ficoll on dielectric constant, and on the “double-layer” surrounding the cells make calculations of absolute values for zeta
Surface
Charge
in Ranu
69
Gastrulation
potential and surface charge density of doubtful value. However, as both zeta potential and surface charge density are directly proportional to electrophoretic mobility, our data enable us to compare these parameters. Cell Viability EDTA-disaggregated cells, obtained from early and late gastrula stages, were tested for their ability to exclude dye (Kaltenbach, 1958) after a 30-min incubation period in electrophoresis medium at pH 7.0 and 25”, since it was under these conditions that electrophoresis runs were performed. Repeated testing indicated that under the given conditions a maximum of 4% of the cells could be considered as nonviable. Embryos whose vitelline membranes were removed at the early gastrula stage and left to develop in electrophoresis medium underwent normal gastrulation. Further confirmation that the electrophoresis medium was physiological and well tolerated by the cells was the observation that cells rapidly adhered to the glass surface of the depression slide and began to flatten and spread against it, as did control cells in Holtfreter’s solution. The same cells in a less favorable milieu retain their spherical shape. Electrophoretic Cells
Mobility
of
Embryonic
Frequency distribution of cell populations. The frequency histograms presented in Figs. 1 and 2 illustrate the accumulated data for each region at each developmental stage studied. It can be seen that each histogram follows what is essentially a normal distribution pattern, and each is characteristically unimodal. This, plus the rather narrow dispersion indicated by a small standard error for each population, indicates that each histogram represents an electrophoretically homogeneous population.
70
DEVELOPMENTAL
BIOLOGY
VOLUME
34, 1973
Dorrol lip Edy gartrula
I I Chordamemderm Mid gartrulo 74 cells
Chordmeroderm
Seconds
FIG. different
1. Electrophoretic mobility stages of development.
Differences Studied
in Mobility
frequency
to
Three regions of the late blastula were studied, namely, presumptive dorsal lip, gray crescent, and inner ectoderm. The electrophoretic mobilities of all three were essentially the same, the differences being statistically insignificant as judged by the Student’s t test (see Table 2). Additionally, the mobilities of cells from the late blastula are slower than those of most cells
Dorrol
lip
Mid gartrvla
+
travel
histograms
of the Regions
I
_
14.6
for gray
Dord lip Late gortrula 72 cells
microns
crescent-chordamesoderm
and
dorsal
lip at
from any region of subsequent stages (i.e., they have the least surface charge). With 5 hrs of additional development at 18”C, the early gastrula stage is reached. In contrast with the blastula, each region has now become electrophoretically different from every other (see Table 1). At this, and subsequent gastrula stages, the archenteron floor (endoderm) maintains the highest surface charge of any region, while ectoderm maintains the lowest surface charge. Gray crescent and dorsal lip have
SCHAEFFER,
SCHAEFFE~
Surface
AND BRICK
I
LATE
Charge
in Ram
71
Gastrulation
BLASTULA
Inner ectoderm Head endodcrm
I
EARLY
GASTRULA
GASTRULA
Seconds
FIG. 2. Electrophoretic floor at different stages
mobility frequency of development.
surface charges falling extremes.
between
to
travel
histograms
these two
The electrophoretic mobilities of the same regions in the mid and late gastrula show much the same pattern, endoderm having the highest surface charge, chordamesoderm having less, and ectoderm having the lowest (see Fig. 3). The difference in surface charge between these regions is highly significant (P < 0.001). “Coated” (outer) ectoderm cells, studied only in the late gastrula, were observed to have the slowest migration rate of any cell type, even slower than cells of the late blastula.
14.6
for inner
microns
ectoderm,
head
endoderm,
and
Changes in Mobility as a Function velopmental Stage
archenteron
of De-
In the section above it has been shown that within each stage of gastrulation, cells from different embryonic regions have significantly different electrophoretic mobilities. There also appears to be an electrophoretic hierarchy of embryonic regions, with the magnitude of surface charge diminishing from endoderm to mesoderm and ectoderm. An exception to this was the late blastula, where the three regions studied had mobilities that closely approximated each other. If one now follows the cell population of
72
DEVELOPMENTAL TABLE
Embryonic
REGIONS
region
OF
Mobility ‘+%Z
Late blastula (Shumway stage 9) Early gastrula (Shumway stage 10)
Mid-gastrula (Shumway stage 11)
Late gastrula (Shumway stage 11%)
Inner ectoderm Gray crescent Presumptive dorsal lip Inner ectoderm Dorsal lip Gray crescent Head endoderm Presumptive floor of archenteron Inner ectoderm Dorsal lip Chordamesoderm Head endoderm Floor of archenteron “Coated” ectoderm Inner ectoderm Dorsal lip Chordamesoderm Head endoderm Floor of archenteron
VOLUME
0.292 0.295 0.296 0.296
0.433 0.358 0.442 0.506 0.300 0.408 0.427 0.442 0.472 0.221 0.302 0.406 0.439 0.406 0.511 TABLE
COMPARISON
Mean Inner ectoderm
electrophoretic
Gray crescent
Dorsal lip
Chordamesoderm
0.005 11 O.l
1
0.007 0.012 P < 0.001 1 1 P
0.300 'p
gastrula
0.302 zt 0.004
P < 0.001
0.006 1 IO.05
P < 0.001 <
P < 0.001
OF THE Rana
pipiens
+ SEM Head endoderm -
Floor of archenteron -
0.015 1
gastrula
Mid-gastrula
(-&ec/V/cm)
-
0.003
Late
mobility
blastula
0.004 1 O.l
2
OF THE DIFFERENCES IN ELECTROPHORETIC MOBILITY OF VARIOUS REGIONS EMBRYO WITHIN EACH OF FOUR DEVELOPMENTAL STAGES
Developmental Stage
Late
34, 1973
particular regions through progressive developmental stages, it is observed that for some regions, great changes in electrophoretic mobility occur, while for other regions, mobilities remain fairly constant throughout (see Fig. 3). A great increase in electrophoretic mobility occurs in the gray crescent cells of the blastula as they progress to become the chordamesoderm cells which first appear in the mid-gastrula. During this time the gray crescent region migrates downward and sinks to the embryonic interior at the dorsal lip. Thus, in the early gastrula, cells comprising the dorsal lip are really gray crescent cells migrating inward, where they become chordamesoderm. The greatest increase in electrophoretic mobility is seen in the dorsal lip region as the blastula progresses to the early gastrula. The increase in mobility is more than 46% at this time. It is important to note that although the dorsal lip is a permanent structure in the gastrula, it exists only as a pathway through which the
1
ELECTROPHORETIC MOBILITY OF VARIOUS THE Rana pipiens EMBRYO Developmenta Stage
BIOLOGY
0.001 0.406 + 0.008 ] I 0.001
0.442 zt 0.506 * 0.011 0.029 1 IO.05 < P < 0.11
+ 0.442 ztz 0.472 + 0.012 0.018 0.009 < 0.11 1 0.1
o.a39 l 0.406 * 0.511 f 0.007 O.CO8 0.018 < P < 0.01~~0.001 < P < 0.01) I P < 0.001 1
SCHAEFFE~
SCHAEFFER,
Surface
AND BRICK
0..
*...
Charge
in Ranu
Gastrulation
73
Archenteron . ..*0 floor I... ...*
‘...&.”
*..f. ..**
,&------A
Inner ectoderm
“Cooted” @ ectoderm -0.20
’
I Blastul0
I Early g0strul0 Stage
I Mid gostrulo of development
I Lote gostrulo
FIG. 3. Electr Ithoretic mobility as a function of developmental stage. Statistically significant increases in mobility occur with onset of gastrulation in dorsal lip and gray crescent. Inner ectoderm shows no change in mobility with development. No data could be obtained for the archenteron floor and head endoderm at the blastula stage.
cells migrate to the interior. Thus, the cell population that comprises the dorsal lip at one- stage is not the same population as in the later stage. Further examination of Table 3 and Fig. 3 reveals that the mobilities of cells from inner ectoderm and also the archenteron floor do not change significantly between any of the developmental stages studied. The mobility of inner ectoderm remains remarkably constant, maintaining the same low migration rate found for blastula cells. Appraisal of Adhesiveness in Situ Gross observation while dissecting the gastrula gives the impression that cells of some gastrula regions do not cohere as tightly as others (Fig. 4). The large yolky endoderm cells of the archenteron floor appear almost spherical, and are very
weakly cohesive as judged by the ease with which individual cells may be displaced when gently pushed with a hair loop. Their spherical shape indicates a minimal contact area between cells. In contrast, inner ectoderm cells may be characterized as a sheet of strongly cohesive, flattened cells. Individual cells cannot be separated with a hair loop. Chordamesoderm appears to be more cohesive than endoderm and less than ectoderm. The cells are sufficiently cohesive to be removed as a sheet, but individual cells are more rounded (have less intercellular contact) than those of ectoderm. Differences in contact area between cells and ease of separation are suggestive of differences in their adhesive strengths. Although quantitative data are not available, these parameters suggest an adhesive hierarchy descending from ectoderm to mesoderm and endoderm. Such
74
DEVELOPMENTALBIOLOGY
FIG.
VOLUME
34, 1973
4. A late gastrula dissected along the mid-sagittal plane. The large, yolky endoderm cells (end) ares #een very weakly cohesive mass. In contrast, ectoderm cells (ect) form a highly cohesive sheet. Dorsal lip plug (yp); ventral lip (ul).
to form a (a’1); yolk
TABLE 3 MOBILITY WITH TIME IN VARIOUS REGIONS AT FOUR DEVELOPMENTAL STAGES
CHANC :E IN ELECTROPHORETIC
Embryonic region
Mean Late
Inner
ectoderm
0.292
blastula
Early
lip
0.296
crescent
0.295
(-&e&‘/cm)
gastrula
ss 0.015
f 0.005
0.358
* 0.012
0.408
1 1 0.05
+ 0.067
0.1
Late
gastrula
0.302
! 0.004
+ 0.006
< 0.1 1 1
EMBRYO
+ SEM
* 0.004
1 1
0.1
Ranu pipiens
Mid-gastrula
< p < :.ioo
P < 0.001 Gray
mobility
*o.oo40.296;t~ 0.1
Dorsal
electrophoretic
OF THE
I 0.406
+ 0.008
0.1
I
-
-
P < 0.001 Chordamesoderm
Head
endoderm
-
-
p
I L, -
0.442
0.427
+ 0.011
+ 0.009 -0.1
0.442 0.1
1
+ 0.012 0.01
0.439
* 0.007
0.406
+ 0.008
< 0.02
1
I Floor
of archenteron
-
0.506
i 0.029
0.472 0.1
i 0.018
0.511 0.1
i 0.018
SCHAEFFER,
SCHAEFFER,
AND BRICK
Surface
Charge
in Ranu
Gmtrulation
75
an adhesive hierarchy is compatible with overlooked that the surface charge increase the previously described electrophoretic may be the natural consequence of morhierarchy. Thus, a large surface charge is phogenetic activity or perhaps of a bioassociated with a weakly adhesive cell type chemical change which follows morphogenetic movement. It is conceivable that the (endoderm), and a low surface charge with increase in surface charge observed here a strongly adhesive type (ectoderm). may be related to increased metabolism. DISCUSSION Mayhew and Weiss (1968) showed this The data presented here reveal an in- relationship for some cultured cells. The apparent correlation, in ectoderm, triguing correlation between three parameand endoderm, of increased ters: surface charge, morphogenetic move- mesoderm, surface charge with decreased adhesiveness ment, and adhesiveness. Those embryonic regions that undergo a rapid increase in (and vice versa), must be considered causurface charge are the very same regions to tiously. It is possible that this correspondAlso, quantitainitiate morphogenetic activity. With the ence might be fortuitous. onset of gastrulation, cells of the presumptive studies on cell adhesion have not been tive dorsal lip region are the first to become done on the amphibian gastrula. Relative active (forming bottle cells which migrate differences in adhesion in situ have been estimated based upon observations of coninward), and also show the most dramatic increase in electrophoretic mobility. When tact area, degree of rounding, and ease of gray crescent cells begin to migrate down- separation. That these parameters are to ward toward the dorsal lip, they too show a some extent a measure of adhesiveness is supported by the studies of Axelrad and corresponding increase in electrophoretic mobility. Thus, those embryonic regions McCulloch (1958) and of Terasima and which will be the most active are observed Tolmach (1963), who showed that cells to greatly increase their surface charge at undergoing mitosis round up and adhere the precise time they begin morphogenetic less firmly to the substratum. In fact, mitotic cells may be selectively collected activity. It is noteworthy that these changes derive from a blastula which is by a technique based upon their decreased electrophoretically, as well as morability to adhere. Nevertheless, a more phogenetically, undifferentiated. The less formal, quantitative estimation of adheactive embryonic regions, such as ectoderm siveness is desirable. and archenteron floor, do not undergo a We have observed in the amphibian change in electrophoretic mobility. Ec- gastrula that increased surface charge is toderm spreads somewhat, to replace gray associated with decreased adhesiveness crescent as it migrates inward, but stops and the initiation of morphogenetic activspreading when it reaches the dorsal lip ity. It is a well known fact that negative and apparently lacks the morphogenetic charges on the cell surface are responsible capacity for inward migration. for electrostatic forces of repulsion (PeThe association of morphogenetic activ- thica, 1961). Although speculative, it is ity with increased surface charge is dif- conceivable that a sufficiently large repulficult to interpret at this time. Although sive force may be generated by an increase the data are suggestive of a causal relationin surface charge so that cell adhesion is ship with an increase in surface charge reduced. The resulting alteration in cell triggering morphogenesis, more work must contact may in some way influence the be done before other possibilities can be initiation of morphogenetic movements. ruled out. The possibility should not be Thus, differences in cell surface charge
76
DEVELOPMENTAL
BIOLOGY
appear to provide a physical basis for the adhesive differences postulated by Gustafson and Wolpert (1967), and Steinberg ( 1964). REFERENCES A. A., and MCCULLOCH, E. A. (1958). Obtaining suspensions of animal cells in metaphase from cultures propogated on glass. Stain Technol. 33, 67-71. BANGHAM, A. D., FLEMANS, R., HEARD, D. H., and SEAMAN, G. V. F. (1958). An apparatus for microelectrophoresis of small particles. Nature (London), 182, 642-644. CURTIS, A. S. G. (1967). “The Cell Surface: Its Molecular Role in Morphogenesis.” Academic Press, New York. DAN, K. (1936). Electrokinetic studies of marine ova. III. Physiol. 2001. 9, 43-57. GARROD, D. R., and GINGELL, D. (1970). A progressive change in the electrophoretic mobility of preaggregation cells of the slime mould, Dictyostelium discoideum. J. Cell Sci. 6, 277-284. GERSHMAN, H. (1970). On the measurement of cell adhesiveness. J. Exp. Zool. 174, 391-406. GUSTAFSON, T., and WOLPERT, L. (1967). Cellular movement and contact in sea urchin morphogenesis. Biol. Rev. Cambridge Phil. Sot. 42, 442-498. HOLTFRETER, J. (1944). A study of the mechanics of gastrulation. Part II. J. Exp. Zool. 95, 171-212. KALTENBACH, J. P., KALTENBACH, N. H., and LYONS, W. B. (1958). Nigrosin as a dye for differentiating live and dead ascites cells. Exp. Cell Res. 15, 112-117. AXELRAD,
VOLUME
34, 1973
LEE, K. C. (1972). Cell electrophoresis of the cellular slime mould, Dictyostelium discoideum. II. Relevance of the changes in cell surface charge density to cell aggregation and morphogenesis. J. Cell Sci. 10, 249-265. MACMURDO-HARRIS, H., and ZALIK, S. E. (1971). Microelectrophoresis of early amphibian embryonic cells. Develop. Biol. 24, 335-347. MAYHEW, E., and WEISS, L. (1968). Ribonucleic acid at the periphery of different cell types, and effect of growth rate on ionogenic groups in the periphery of cultured cells. Exp. Cell Res. 50, 441-453. PETHICA, B. A. (1961). The physical chemistry of cell adhesion. Exp. Cell Res., Suppl. 8, 1233140. RUGH, R. (1962). “Experimental Embryology.” Burgess, Minneapolis, Minnesota. SHUMWAY, W. (1940). Stages in the normal development of Rana pipiens. Anat. Rec. 78, 139-148. STEINBERG, M. S. (1964). The problem of adhesive selectivity in cellular interactions. In “Cellular Membranes in Development”(M. Locke, ed.), pp. 321-366. Academic Press, New York. TERASIMA, T., and TOLMACH,L. J. (1963). Growth and nucleic acid synthesis in a synchronously dividing population of HeLa cells. Z&p. Cell Res. 30, 344-362. TOWNES, P. L., and HOLTFRETER, J. (1955). Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 128, 53-118. WEISS, L. (1967). “The Cell Periphery, Metastasis and Other Contact Phenomena.” Wiley, New York. WEISS, L. (1968). Studies on cellular adhesion in tissue culture. IX. Electrophoretic mobility and contact phenomena. Exp. Cell Res. 51,609-625.