Migration-Directing Liquid Properties of Embryonic Amphibian Tissues 1

Transcription

1 AMER. ZOOL., 24: (1984) Migration-Directing Liquid Properties of Embryonic Amphibian Tissues 1 GRAYSON S. DAVIS Department of Biology, Valparaiso University, Valparaiso, Indiana SYNOPSIS. Deep ectoderm, mesoderm and endoderm excised from gastrulating amphibian embryos spontaneously undergo liquid-like movements in organ culture. Cell populations of these tissues on nonadhesive substrata will round up into spheres, spread over one another and segregate (sort out) from one another just as immiscible liquid droplets do. In ordinary liquids, movements like these are controlled by surface tensions; perhaps surface tensions also control the similar movements of liquid-like tissues. One necessary condition for tissue surface tension analysis is that the tissue must be able (just as ordinary liquids are able) to spontaneously relax internal stretching forces (shear stresses). When cellular aggregates of the germ layers were deformed by gentle compression between parallel glass plates, cells within the aggregates were initially stretched. However, the cells soon returned to their original undistorted shapes. Thus, cell stretching forces were gradually relaxed by cell rearrangements. The in vitro spreading movements of the deep germ layers imply that the surface tension of ectoderm should be greater than the surface tension of mesoderm which should be greater than the surface tension of endoderm. Quantitative measurements of tissue surface tensions made by parallel plate compression confirm precisely that relationship. Furthermore, the surface tensions of these tissues remain constant regardless of the amount of aggregate flattening another necessary condition for valid surface tension measurements. These results demonstrate that amphibian deep germ layers possess fundamental liquid properties which are sufficient to direct their liquid-like rearrangements in organ culture. Furthermore, I also report that one of these properties, surface tension, displays a preliminary correlation with density of cell surface charge (assessed by electrophoretic mobility) and with the onset of in vivo mesodermal involution. INTRODUCTION A morphogenetic event can be analyzed at many different levels. One can attempt to determine which genes are active, which proteins are being synthesized, which cell organelles are functioning and which cells are growing, dividing, changing shape or moving. However, information about microscopic events may not be sufficient for a complete explanation of morphogenesis. Often, a macroscopic analysis of the movement-generating mechanism is required. This paper reviews a macroscopic analysis of certain in vitro tissue movements (which often resemble normal morphogenesis) and then examines certain microscopic events which could produce those movements. According to Newton's laws, all move- ' From the Symposium on Castrulation presented at the Annual Meeting of the American Society of Zoologists, December 1982, at Louisville, Kentucky. 649 ments which occur where friction is present must be the result of unbalanced forces. Therefore, for every tissue movement which we observe, we should be able to identify an unbalanced force which acts to make those tissues move. This kind of physical analysis has been especially fruitful for certain embryonic tissues which display liquid-like movements in organ culture. When irregularly-shaped explants of these tissues are cultured on nonadhesive substrata (agar, e.g.), the tissue fragments will round up to form spheres (Holtfreter, 1939). When two such spheres composed of different liquid-like tissues are touched together, they will fuse as one tissue spreads about the other in a characteristic envelopment pattern (Holtfreter, 1939). When two different, liquid-like tissues are disaggregated into single cells, and the cells are mixed and reaggregated, the cell types will sort out from one another, eventually forming the same stable configuration attained by fragment fusion (Townes and Holtfreter, 1955).

2 650 GRAYSON S. DAVIS In ordinary liquids, movements like these are controlled by the surface tensions of liquid interfaces (Adamson, 1967). Perhaps surface tensions can also direct similar movements of liquid-like tissues. The control mechanism of such movements is significant because the final arrangement attained by some tissue combinations resembles complex in vivo morphology. Townes and Holtfreter (1955), for example, found that certain tissue fragments or even dissociated cells from an early amphibian neurula could autonomously rearrange to form miniature replicas of late amphibian neurulae. However, these authors suggested that the different cell types of the embryo were able to segregate themselves by cell-specific adhesion and by chemotaxis. Noting that the time course of such cellular rearrangements is very similar to types of liquid behavior controlled by intermolecular adhesions, but quite different from that expected from chemotaxis, Steinberg proposed that rounding up, aggregate fusion and cell sorting could be explained by differential cellular adhesion alone (Steinberg, 1962a, b, 1963). To define strength of adhesion in physical terms, Steinberg (1964) referred to "works of adhesion" (the amounts of reversible work performed in forming or separating a unit area of adhesion). Unfortunately, because of the difficulties in measuring the changes in free energy and in cell contact area, works of adhesion have never been quantified for any tissues (Steinberg, 1964). Phillips (1969) has pointed out that, for liquid substances, surface tensions are physically equivalent to works to adhesion and offer the further advantage of being measurable for living tissues. A macroscopic analysis of liquid-like tissue movements could address the following questions. Do those embryonic amphibian tissues which behave like liquids actually possess fundamental liquid properties which make surface tension analysis appropriate? If so, are their surface tensions of the right strengths to direct tissues in their in vitro (and perhaps in vivo) migrations? What microscopic cell properties contribute to tissue surface tensions? FIG. 1. Diagram by Holtfreter (1943) of cells dissected from a morula. In contrast to the deep cells, the outermost cells possess a more heavily pigmented, nonadhesive, exteriorly-directed surface. FUNDAMENTAL LIQUID PROPERTIES OF THE DEEP TISSUES In addition to the usual distinctions between germ layers, Holtfreter found that the surface cells of the amphibian embryo are significantly different from the deep cells (Holtfreter, 1943) (Fig. 1). He proposed that the exterior cells form a "surface coat" because their more heavily pigmented, exteriorly-directed surfaces were nonadhesive to other cells. During gastrulation, cells of the surface coat do exchange neighbors laterally, but they do not sink into the deep layers, nor do deep cells come out onto the surface (Keller, 1978). It is important to remember that liquidlike behavior is a property of the deep tissues but may not be characteristic of coated cells. In all of our experiments with midgastrula Rana pipiens embryos, we peeled away and discarded the coated surface cells and then dissected out the underlying deep or uncoated tissues. In these experiments, deep ectoderm, mesoderm and endoderm excised from embryos exhibited fundamental liquid behavior. First, when excised pieces of these tissues were cultured on a nonadhesive substratum, they rounded up like liquids, adopting nearly spherical shapes in 4-5 hr. Scanning electron micrographs showed that the shapes of the cells inside these tissues were nearly isodiametric both before and after rounding up

3 LIQUIDITY OF EMBRYONIC AMPHIBIAN TISSUES A third liquid property is demonstrated by the fusion behavior of deep ectoderm, mesoderm and endoderm aggregates. When two such aggregates touch, one spreads around the other in a tissue-specific pattern of envelopment. Deep ectoderm always goes to the inside, deep FIG. 2. Diagram of the shapes of cells within initially round aggregates before and after differing durations of constant compression. A. Before compression, interior cells are isodiametric. B. During initial compression, the overall shape change experienced by the aggregate creates internal stresses which stretch all the cells. C. However, after continued compression, the stretched cells are able to rearrange themselves and thereby relax the stress. Cells gradually return to their original undistorted shapes, even though the aggregate remains under constant compression. endoderm always goes to the outside, and deep mesoderm is intermediate since it surrounds ectoderm and is itself surrounded by endoderm (Phillips and Davis, 1978). In a similar experiment, Steinberg and Kelland (1967) excised chunks of tissue from the sides of amphibian gastrulae. Each tissue fragment contained coated ectoderm, deep ectoderm, deep mesoderm and deep endoderm. If the surface layer of coated ectoderm cells was peeled away, the remaining deep layers rounded up into a single sphere and rearranged such that endoderm was outermost, mesoderm in the middle and ectoderm innermost. This result is not unexpected given the behavior of fused pairs of aggregates, but it is the opposite of the gastrula's normal germ layer (Davis and Phillips, submitted for publication). This implies that rounding up proceeds by a process of cell rearrangement rather than by shape changes of immobile cells. Ordinary liquid droplets round up because their subunits can move with respect to one another while trying to maximize their adhesions to each other (Symon, 1971). Furthermore, this type of subunit redistribution permits liquids to relax internal shear stresses which would otherwise accompany overall shape changes. Do cells of liquid-like tissues also display this second fundamental liquid property? To find out, we gently flattened spherical aggregates of deep ectoderm, mesoderm and endoderm between two parallel, agarcoated coverslips and fixed them at various times during compression so that the shapes of their interior cells could be observed with the scanning electron microscope (Phillips and Davis, 1978). The interior cells of uncompressed aggregates are loosely packed and rounded (Fig. 2A). When the aggregate's overall shape is changed by compression, internal stresses are initially created which stretch all the cells (Fig. 2B). Stretched cells then gradually rearrange themselves to relieve those internal stresses. By the end of fifteen minutes of compression, no orientated cell stretching was apparent in the aggregate's interior, even though the aggregate remained under constant compression (Fig. 2C). Although the cells were initially stretched (like the subunits of an elastic solid), they gradually rearranged themselves (like the coherent, mobile subunits of a viscous liquid) to relax stretching forces inside the aggregate. Substances which display this type of shortterm solid behavior and long-term viscous liquid behavior are appropriately termed elasticoviscous liquids (Phillips et al., 1977; Phillips and Steinberg, 1978). 651

4 652 GRAYSON S. DAVIS 'AO organization. Moreover, this inside-out architecture raises the question of what, if anything, liquid behavior in general and fragment fusion in particular have to do with normal, in vivo morphogenesis. However, Steinberg and Kelland also found that if the excised fragment was allowed to round up with the coated ectoderm cells in place, that sheet of coated cells seemed to be held at the exterior by its nonadhesive surface. As the coat spread over the surface of the aggregate, the deep tissues rearranged to form the normal germ layer architecture (Fig. 3). This observation suggests that when coated ectoderm is present, liquid-like fusion behavior of the deep germ layers is sufficient to direct tissue explants to form the normal germ layer configuration (Phillips and Davis, 1978). SURFACE TENSION AND AGGREGATE FUSION BEHAVIOR But what of the fusion behavior of the deep tissues? What does the "insidedness" hierarchy of ectoderm > mesoderm > endoderm imply about the liquid properties of those tissues? When two ordinary liquid droplets fuse, the one which goes to the inside has the higher surface tension (Fig. 4). By analogy, that tissue which goes inside in a fragment fusion should have the higher tissue surface tension. The aggregate fusion behavior of deep ectoderm, mesoderm and endoderm predicts that the FIG. 4. Diagram of two similarly-sized, immiscible liquid droplets (A and B) fusing in medium O. Their final arrangement is determined by the surface tensions (<T'S) of the AO, BO and AB interfaces. If B surrounds A, then <jao > <rbo. The surface tension of the AB interface determines the degree, but not the direction, of envelopment. surface tensions, er's, will be in the following sequence: crect > o-mes > crend (1) How can the surface tension of living aggregates be measured to see if this prediction is valid? Of course, surface tension is that force which promotes droplet rounding up and opposes increases in surface area. To measure tissue surface tension then, one can deform initially spherical aggregates with a known force, wait for them to reach mechanical equilibrium and monitor the increase in aggregate surface area. Figure 5 is a diagram of a device which facilitates such measurements. Here an aggregate is gently compressed between two parallel, agar-coated glass plates. The top plate is immobile and the bottom plate is attached to one end of a flexible quartz fiber. The opposite end of the fiber is held by a simple micromanipulator. The compressing force applied to the aggregate can be increased by turning a dial on the micromanipulator. FIG. 3. Diagram of an experiment by Steinberg and Kelland (1967). A piece of tissue containing coated ectoderm (black), deep ectoderm (dark gray), deep mesoderm (medium gray) and deep endoderm (light gray) was excised from the flank of a gastrula. When this fragment was allowed to round up in organ culture, the tissues rearranged to form the normal germ layer architecture.

5 LIQUIDITY OF EMBRYONIC AMPHIBIAN TISSUES 653 FIG. 5. Diagram of a device for compressing cellular aggregates. An aggregate (a), surrounded by medium, is compressed between two agar-coated glass plates. The lower plate (gc) is attached to one end of a flexible quartz fiber (f). The other end of the fiber is held by a simple micromanipulator. Turning the dial (d) will increase or decrease the force applied to the aggregate and the flexure of the fiber. Since the bending constant of the flexible quartz fiber has been previously determined, measurement of the fiber's degree of deflection will permit a calculation of the force applied to the aggregate. The shape of the aggregate may be monitored by side view photographs. If the applied force is increased, the aggregate will be flattened more and the quartz fiber will be bent more. Since the fiber has been calibrated, a simple measurement of its degree of deflection will permit a calculation of the force (F) applied to the aggregate. The deforming pressure (AP) experienced by the aggregate is equal to the force applied divided by the area of contact between the aggregate and glass plate. The sides of the aggregate can be described by two radii of curvature. The horizontal radius (parallel to the compressing plates) is called R,, and the vertical radius (perpendicular to the compressing plates) is called R 2. These variables can be measured from side view photographs of aggregates undergoing compression. Their values may then be substituted into the Young-Laplace equation (Adamson, 1967), (2) to compute the value of surface tension (a). This method gives the correct value for the surface tension of water (i.e., air-water interface) when small air bubbles are compressed between the plates (Davis and Phillips, submitted for publication). Furthermore, the surface tension value obtained is constant regardless of the degree of compression. All liquid substances must have area-invariant surface tension. By contrast, the surface tension of a solid substance always increases as the degree of compression increases (Zemansky, 1957). Therefore, if the liquid behavior of deep ectoderm, mesoderm and endoderm is directed by their surface tensions, then their germ layer surface tensions must not only satisfy equation (1), they must also be area-invariant. Measurements of deep ectoderm, lateral mesoderm and endoderm excised from mid-gastrula Rana pipiens embryos do satisfy equation (1) (Table 1). The probability that any two of these values are equal is less than Furthermore, these values are area-invariant, which could not be true if these tissues were solid-like. Deep ectoderm, mesoderm and endoderm do resemble liquids. They have classically defined (area-invariant) surface tensions, and furthermore, the values of their surface tensions are appropriate to direct their observed rounding up, sorting out and fusion behavior. PRELIMINARY CORRELATIONS OF SURFACE TENSION, SURFACE CHARGE AND MESODERMAL INVOLUTION Tissue surface tension measurements have provided quantitative data supporting the above macroscopic analysis of liquid-tissue behavior. Those same measurements may now be used to help identify TABLE 1. Area-invariant surface tension values (in ergs / centimeter 2 ± standard errors) of tissues excised from midyolk plug stage Rana pipiens. Tis! Number of cases Surface tension Ectoderm Mesoderm Endoderm 2.92 ± ± ± 0.03 Probability that surface tension values are inconsistent with the predicted sequence ect mes: P < ect : end: mes end: P < P < 0.021

6 654 GRAYSON S. DAVIS those microscopic (e.g., ultrastructural) properties of cells which produce tissue specific germ layer surface tensions. Those cell characteristics which are shown to correlate with surface tension would then be implicated in the control of liquid-like tissue movements. Some authors have proposed that negative charges on the surfaces of cells produce electrostatic forces which may reduce cell-cell adhesiveness by preventing the close approach of cell surfaces (Pethica, 1961; Garrod and Gingell, 1970; Lee, 1972). If this is true, then tissues with greater densities of surface charge would be less adhesive and so have lower surface tensions. Therefore, according to equation (1), the surface charge of deep ectoderm should be lower than that of deep mesoderm which should be lower than that of deep endoderm. In fact, measurements of cell surface charge (using the rates of electrophoretic mobility of germ layer cells) found precisely this relationship (Schaeffer et al., 1973a). Furthermore, there is a correlation between surface charge and adhesion. Deep tissues in organ culture will dissociate if the ph of the surrounding medium is increased (Townes and Holtfreter, 1955). Schaeffer e/az. (19736) found a corresponding increase in cell electrophoretic mobilities as the ph increased. This is consistent with the proposed inverse relationship between surface tension and surface charge. Changes in surface charge also correlate with certain morphogenetic movements of gastrulating germ layers. At the blastula stage, dorsal mesoderm possesses an electrophoretic mobility very similar to that of blastula ectoderm. However, at the early gastrula stage, shortly before the involution of dorsal mesoderm, that tissue's electrophoretic mobility decreases to became intermediate between ectoderm and endoderm (Schaeffer et al., 1973a), suggesting that this surface charge relationship, and therefore the surface tension relationship in equation (1) may be developed in preparation for mesodermal involution. Is there a corresponding change in the surface tension of blastula mesoderm (from ectoderm-like to intermediate between ectoderm and mesoderm)? To answer this question, we have made surface tension measurements of several tissues excised from early gastrula stage embryos (Davis and Phillips, in preparation). Preliminary results for dorsal mesoderm indicate that, after its involution, this tissue may no longer possess a liquid-like (area-invariant) surface tension. This nonliquid behavior makes dorsal mesoderm an inappropriate tissue to test for a correlation between surface tension change and morphogenesis. However, lateral and ventral mesoderm excised from the early gastrula did possess area-invariant surface tensions. Lateral mesoderm, which was nearly ready to involute, had a surface tension somewhat less than that of ectoderm of the same stage. Ventral mesoderm, which would not begin to involute for several hours, had a surface tension greater than that of ectoderm. The surface tension of lateral mesoderm was significantly lower than the surface tension of ventral mesoderm, suggesting that the surface tension of mesoderm does decrease in preparation for its involution. Further analysis and additional experiments are now underway to examine this correlation more carefully. An evaluation of the covariance between the surface tension of a tissue and the surface charge of its cells could permit an assessment of the potential role of cell surface charge as a primary determinant of the morphogenesis of liquid-like tissues. Moreover, quantitative measurements of surface tension provide a criterion to appraise the contributions of other ultrastructural properties to the control of liquid-like tissue flow. ACKNOWLEDGMENTS I am grateful to Dr. Herbert M. Phillips for his help and guidance and to Ms. Sandra Mitchell and Ms. Margaret MacQueen for their expert technical assistance. This research was supported by N.S.F. grants GB and PCM to H.M.P., N.I.H. traineeship HD00430 and a Valparaiso University Research Fellowship to G. S. D.

7 LIQUIDITY OF EMBRYONIC AMPHIBIAN TISSUES 655 REFERENCES Adamson, A. W Physical chemistry of surfaces. John Wiley & Sons, New York. Garrod, D. R. and D. Gingell A progressive change in the electrophoretic mobility of preaggregation cells of the slime mould, Dictyostehum discoideum. J. Cell Sci. 6: Holtfreter, J Gewebeaffinitat, ein Mittel der embryonalen Formbildung. Arch. Exp. Zellforsch. Besonders Gewebeznecht. 29, Revised and reprinted in English, In B. H. Wilher and J. M. Oppenheimer (eds.), Foundations of experimental embryology, pp Prentice-Hall, Englewood Cliffs,' NJ. Holtfreter, J Properties and functions of the surface coat in amphibian embryos. J. Exp. Zool. 93: Keller, R. E Time-lapse cinemicrographic analysis of superficial cell behavior during and prior to gastrulation in Xenopus laevis. J. Morph. 157: Lee, K. C Cell electrophoresis of the cellular slime mould, Dictyostehum discoideum. II. Relevance of the changes in cell surface charge density to cell aggregation and morphogenesis. J. Cell Sci. 10: Pethica, B. A The physical chemistry of cell adhesion. Exp. Cell Res. Suppl. 8: Phillips, H. M Equilibrium measurements of embryonic cell adhesiveness: Physical formulation and testing of the differential adhesion hypothesis. Ph.D. Diss., Johns Hopkins University, Baltimore. Phillips, H. M. and G. S. Davis Liquid-tissue mechanics in amphibian gastrulation: Germ-layer assembly in Rana pipiens. Amer. Zool. 18: Phillips, H. M., M. S. Steinberg, and B. H. Lipton Embryonic tissues as elasticoviscous liquids. II. Direct evidence for cell slippage in centrifuged aggregates. Dev. Biol. 59: Phillips, H.M. and M.S. Steinberg Embryonic tissues as elasticoviscous liquids. I. Rapid and slow shape changes in centrifuged cell aggregates. J. Cell Sci. 30:1-20. Schaeffer, B. E., H. E. Schaeffer, and I. Brick. 1973a. Cell electrophoresis of amphibian blastula and gastrula cells; the relationship of surface charge and morphogenetic movement. Dev. Biol. 34: SchaefFer, H. E., B. E. Schaeffer, and I. Brick. 1973*. Electrophoretic mobility as a function of ph for disaggregated amphibian gastrula cells. Dev. Biol. 35: Steinberg, M. S. 1962a. On the mechanism of tissue reconstruction by dissociated cells. I. Population kinetics, differential adhesiveness, and the absence of directed migration. Proc. Natl. Acad. Sci., U.S.A. 48: Steinberg, M. S. 1962A. Mechanism of tissue reconstruction by dissociated cells. II. Time-course of events. Science 137: Steinberg, M. S Tissue reconstruction by dissociated cells. Science 41: Steinberg, M. S The problem of adhesive selectivity in cellular interactions. In Cellular membranes in development. Symp. Soc. Study Develop. Growth 22: Steinberg, M. S. and J. L. Kelland Cellular adhesive differentials in the determination of the structure of the amphibian gastrula. Paper presented at symposium: Control mechanisms in morphogenesis, 134th annual meeting Am. Assoc. Adv. Sci., New York, December Symon.K.R Mechanics Addison-Wesley Publ. Co., Reading, Mass. Townes, P. L. and J. Holtfreter Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 128: Zemansky, M. W Heat and thermodynamics. McGraw-Hill Book Company, Inc., New York.

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