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AMER. ZOOL., 18:81-93 (1978). Liquid-Tissue Mechanics in Amphibian Gastrulation: Germ-Layer Assembly in Rana Pipiens HERBER'I M. PHILLIPS Department of Anatomy, Wayne State University School of Medicine, Detroit, Michigan 48201 AND GRAYSOX S. DAVIS Department of Biology, University of Virginia, Charlottesville, Virginia 22901 SYNOPSIS During amphibian gastrulation, migrating subsurface germ layers may flow past one another like immiscible viscous liquids in response to tissue surface tension forces. We describe here two physical tests for liquid-tissue morphogenesis in cultured aggregates of subsurface ectoderm (E), mesoderm (M) and endoderm (N) excised from mid-yolk-plug Rana pipiens gastrulae. (i) Liquids are coherent substances in which subunits can slip past one another to relax internal shear stresses. We find, in cross-sections of cell aggregates fixed during compression, that cells within flattened aggregates are intially deformed, but do, as predicted, gradually reassume their original, undistorted shapes, (ii) Surface tensions (y's) govern ordinary liquid-droplet spreading; e.g., if equal-sized droplets A and B fuse in medium 0, B spreads around A when yA0 > yao- When pairs of subsurface aggregates are cultured together, N surrounds M and E, and M surrounds E. To see if yE0 > "YMO > TNO. we flatten aggregates with quartz fibers calibrated to measure the force of compression. As predicted, under the same flattening force, E aggregates are rounder than M aggregates, which are rounder than N aggregates. Furthermore, a second surface tension relationship can account for the autonomous involution of M between E and N; and these surface tension relationships can also explain the inversion of E, M and N by coated ectoderm to produce normal gastrular germ-layer arrangements. We conclude that, combined with active cell shape changes in solid-like surface cell layers and also with autonomous elongation of dorsal lip mesoderm, tissue surface tension control of liquidtissue flow in subsurface germ layers is a key morphogenetic mechanism in amphibian gastrulation which might be regulated by changes in intercellular adhesiveness. IXTRODL'CTIOX „ , . £ . , , , • r Studies or the molecular rproperties or „ , f „, c c c cell surfaces are often Jjustified by the as. . • • a sumption that those properties influence tissue morphogenesis by regulating inter... iu • w-u• cellular adhesiveness. This assumption, . . . . . . . . . , , ,.rcr , while plausible, is remarkably difficult to r ,... , , • ii J L • prove. With the various cell adhesion asr ., , , , says now available, one can detect many .; . c ,.«• • ii u tissue-specific differences in embryonic cell ; c p .. . c surfaces. Moreover, specihc morphogeneK ' . u u tic events may ; even correlate with obWe gratefully acknowledge the excellent technical assistance of Ms. Sandra Mitchell and Ms. Margaret MacQueen in our experiments described here, which were supported at the University of Virginia, De- served changes in cell surfaces. But d o such manifestations of cell surface dif• • ,, , r ferentiation cause and/or control — or , merely accompany — tissue rearrange? . '. . , • , An alternative approach is to ask, not „ , . , i• r what is the ultimate cause or a mor, . , , t „ , „• • phogenetic movement?v but what is its r *> .„ _. .. . . , immediate cause? Since all motion in the . . • , • r r r rpresence of friction requires the action of , c ^ , , , u l unbalanced forces, unbalanced fforces , , . ,. ,, ,, . must be the immediate cause ofr all cell and • • • -,-. r . • tissue migrations. Therefore, whatever the molecular interactions regulating tissue morphogenesis, they must function to synthesize and activate force-generating mechanisms in embryonic tissue systems, „ , • • • .i partment of Biology, by NSF grant GBg40041 to R a t h e r , t h a n attempting to inventory the H.M.P. G.S.D. is supported by NIH traineeship myriad changes in differentiating cell surHD00430. faces, we have sought to characterize the 81 82 HERBERT M. PHILLIPS AND GRAYSON S. DAVIS mesoderm and endoderm in late blastula and gastrula stages by cell size (smaller, intermediate and larger, respectively) and color (dark gray, light gray and white, respectively). We have not attempted to determine the precise fates of these cell populations, and simply avoid using cells wherever the above morphological differences are not perfectly distinct under the dissecting microscope. However, the three cell populations so defined lie precisely in the positions, both in blastula and gastrula stages, assigned to presumptive ectoderm, mesoderm and endoderm by Vogt (1929) in his dye-marking experiments on Rana esculenta and Ranafusca. As Holtfreter (1943a,6; 1944) did, we can peel away with dissecting needles a continuous, single-cell layer from the surface of the late blastula, and from external, archenteric and blastopore lip surfaces in gastrula stages, thus uncovering multilayered subsurface populations of presumptive ectoderm, mesoderm and endoderm. Under the dissecting microscope, subsurface cells of each type appear to be adhering uniformly, although sometimes quite loosely, to other cells on all sides. By contrast, the outer faces of the surface cell populations appear to be shiny—"coated" in Holtfreter's terminology. We find, as he did (op. cit.), that the shiny sides of these surface cells are non-adhesive with respect to blastula and gastrula cells of all types. (By contrast, their "uncoated" lateral and basal sides do adhere to other uncoated ment, certain embryonic tissues may become cell surfaces.) temporarily converted into liquid-like cell masses We observe, as Holtfreter did, that durwhich flow from one configuration into another ing gastrulation shiny surface ectoderm guided by tissue surface tension forces. While plus underlying subsurface ectoderm those tests involve only artificial combina- gradually spread over the entire surface of tions of morphogenetically remote chick the embryo. In late (mid-yolk-plug— cell populations, this hypothesis may also Shumway [1940] stage 11+) gastrulae, be applied to contiguous, migrating popu- after dissecting away surface ectoderm, we lations of subsurface ectoderm, mesoderm routinely obtain samples' of subsurface ecand endoderm in amphibian gastrulation. toderm from the anterior hemisphere of the embryo opposite the receding yolk plug (including all of the roof of the now greatly LOCATING AND DISSECTING COATED AND SLBdiminished blastocoel) by peeling it off of SLRFACE GERM LAYERS underlying mesoderm and endoderm with In our analysis of tissue rearrangements a hairloop. We also confirm Holtfreter's in Northern Rana pipiens embryos, we op- conclusion that in these late gastrula stages erationally define presumptive ectoderm, coated endoderm cells cover the plug and morphogenetic forces immediately responsible for tissue movements, avoiding, insofar as is possible, artifacts due to in vitro tissue manipulation. The monumental studies of Holtfreter (1939; 1943a,6; 1944) and of Townes and Holtfreter (1955) demonstrate that small groups of cells excised from amphibian gastrulae and neurulae and placed in organ culture can move autonomously i.e., independently of the rest of the embryo, to form miniature copies of in vivo embryonic structures. Therefore, these germ-layer migrations are evidently driven by intrinsic morphogenetic forces originating within the tissues themselves. Moreover, these forces seem to operate normally, and thus can be reliably examined, in in vitro culture conditions. Steinberg (1963, 1964, 1970, 1975) has pointed out that the tissue-spreading and cell-sorting movements observed in vitro in amphibian germ-layer (as well as laterstage embryonic chick) tissue masses are analogous to rounding-up, droplet coalescence and the break-up of emulsions into homogeneous phases in systems of immiscible liquids. The intrinsic forces governing these latter processes are the surface tensions at immiscible liquid interfaces. Steinberg and I and co-workers (Phillips, 1969, and in preparation; Phillips and Steinberg, 1969, 1978, and in preparation; Phillips et al., 1973, 1977a; Phillips et al., 1977ft) have been refining and testing the hypothesis that, during develop- LIQUID-TISSUE MECHANICS IN GASTRULATION the inner lip of the blastopore, and continue inward to line the floor, sides and anterior roof of the archenteron. We dissect subsurface endoderm out of the region underneath the coated endoderm lining the floor of the archenteron. Similarly, coated mesoderm covers the outer lip of the blastopore, and dorsal coated mesoderm may provide the lining for the posterior roof of the early archenteron. (According to Vogt [1929] and Mayer [1931], in anurans this dorsally involuted surface mesoderm soon becomes displaced by coated endoderm migrating up to line the entire archenteron roof. Others argue that all surface cells which in the anuran blastula are located above subsurface mesoderm will actually become part of subsequent endodermal structures [Keller, 1975].) We note that the surface cells lining the outer lip of the blastopore (they cover subsurface layers of involuting mesoderm cells and thus up to this stage would be classified as "coated mesoderm" cells on morphological grounds) seem to lose their shiny coats once they have moved a few cell diameters forward into the embryo away from the outer margin of the lip. In late gastrula stages, after dissecting away surface (coated) ectoderm cells and then peeling off one multilayered group of subsurface cells (subsurface ectoderm in anterior regions, yet-to-be-involuted subsurface medoserm in posterior regions), we can peel a several-cell-thick layer of lateral (and, if desired, ventral) involuted subsurface mesoderm off of the underlying endoderm. Moreover, if we avoid freshly involuted mesoderm close to the blastopore, our mesoderm aggregates have no shiny, coated cells and will adhere on all sides to other subsurface cell aggregates. (For reasons to be explained below, we avoid using subsurface dorsal mesoderm in our tests for tissue liquidity.) 83 microfilaments evidently produce active cell shape changes which cause tissue thickening and bending. Similar cell shape changes have been observed in coated amphibian germ layers—e.g., bottle-cell formation in surface cells at the blastopore lip (see Holtfreter, 1943a,6; Baker, 1965; Perry and Waddington, 1966). Therefore this intracellular force-generating mechanism may be responsible for external, archenteric and blastopore lip morphologies during amphibian gastrulation. It requires, however, that tissues behave like deformable solids. Cells must be tightly bound to one another to prevent cell shuffling movements that would disrupt coordinated tissue deformations. While useful in thin-tissue morphogenesis, this prohibition of cell shuffling movements would cause severe cell distortions in subsurface, thick-tissue masses during gastrulation (Fig. 1). In fact, coordinated cell shape changes in gastrulating germ layers seem to be confined to surface layers; cells in subsurface layers appear to maintain relatively undistorted shapes as they migrate (e.g., Holtfreter, 19436; Baker, 1965; Nakatzuji, 1975). We have therefore been exploring the hypothesis that subsurface germ layers instead flow as liquid-like masses during gastrulation. Also, since subsurface tissue movements seem to be governed by intrinsic morphogenetic forces (see above), we have been examining the further possibility that subsurface germ-layer flow is directed by tissue surface tension forces. TESTS FOR CELL SLIPPAGE PERMITTING LIQUIDTISSUE FLOW A liquid is defined (Symon, 1971) as a cohering substance in which internal shear stresses are spontaneously dissipated as the subunits of which it is composed slip past one another. Consider two models for originally spherical cell aggregates in cul"SOLID" AND "LIQUID" TISSUES IN AMPHIBIAN ture medium that are deformed by being CASTRULATION compressed between parallel glass In single-cell-thick epithelia in a variety coverslips (Fig. 2). In the elastic-solid of embryonic systems (Wessels et al., 1971; model, cell slippage movements are prohibBurnside, 1973; Spooner, 1973; Karfun- ited. Therefore, initial horizontal stretchkel, 1974), cytoplasmic microtubules and ing of interior cells should continue as long HERBERT M. PHILLIPS AND GRAYSON S. DAVIS (A) N (B) / FIG. 1. In anuran amphibian gastrulae, thick prospective germ layers migrate past one another as cohering, multilayered cell masses, as schematically diagrammed here. A, A blastula with monolayered surface and multilayered subsurface ectoderm (E and E), mesoderm (M — but see text — and M) and endoderm (N and N) will form a trilayered gastrula. B, If strong cross-linking were to prevent cell slippage, these solid-tissue redistributions would result in major cell distortions (although not necessarily in the directions shown simply for illustrative purposes here). C, In fact, most subsurface cells are not markedly distorted during gastrulation. as the aggregate remains compressed. By contrast, in the viscous-liquid model, aggregates would behave like slow-flowing liquids composed of elastic subunits (cells). Thus, during prolonged aggregate compression, initially stretched cells should be free to slip past one another and return to their original, undistorted shapes, even though the aggregate would remain flattened. To test for cell slippage in deformed cell aggregates, we allowed excised fragments of subsurface ectoderm, mesoderm or endoderm to round up for several hours in Steinberg's medium (Hamburger, 1962) in dishes coated with agar (Colab Ionagar #2). We compressed each one, immersed in medium, between parallel glass surfaces in a miniature vise. We fixed aggregates during compression by gently pipetting LIQUID-TISSUE MECHANICS IN GASTRULATION A. Elasticsolid model 85 B. Viscous liquid model I. Before compression II. Initial compression I. Continued compression FIG. 2. Liquids are cohering substances in which internal shear forces are relaxed by subunit slippage movements. A, Elastic-solid model for compressed aggregates: cell stretching but no cell slippage. B, Viscous-liquid model (i.e., elasticoviscous-liquid model — see Phillips and Steinberg, 1978): initial cell stretching, then slow cell slippage. HERBERT M. PHILLIPS AND GRAYSON S. DAVIS 2% formalin in culture medium under the coverslip. Fifteen minutes later the coverslips were removed and aggregates were prepared for scanning electron microscopy by standard techniques. After critical-point drying, we broke compressed aggregates in planes perpendicular to their flattened sides to observe cell shapes in cross-section in the scanning electron microscope. Most of our experiments to date have been performed on subsurface endoderm aggregates (18 samples), although the few samples of subsurface ectoderm and mesoderm which we have examined have yielded similar results. Loosely packed, initially rounded cells in uncompressed aggregates (Fig. 3A) become markedly flattened during the first minute of compression (Fig. 3B). After five minutes of compression, interior cells are still quite flattened, although surface cells may begin to return to their original rounded shapes (Fig. 3C). However, after fifteen minutes of compression, virtually all cell stretching disappears as shear stresses become relaxed, leaving rounded cells within these flattened aggregates in their original undistorted shapes (Fig. 3D). These observations are consistent with predictions derived from the viscousliquid model for cell aggregates and clearly contradict those derived from the elasticsolid model. Similar cell slippage movements occur in centrifugally deformed embryonic chick liver aggregates (Phillips et.al., 1977a) and in whole, intact chick limb buds compressed with tiny surgical clamps (Daggy and Phillips, in preparation). However, cell slippage in these later-staged embryonic chick tissues seems to require at least several hours, which is consistent with the slower rate of rounding-up of chick aggregates (1-2 days) compared to amphibian germ-layer aggregates (2-6 hours). By liquid-tissue flow we mean changes in the configurations of cohering cell masses that leave cells undistorted thereafter. Therefore, the spontaneous relaxation of internal shear forces reflected in these cell shape changes is the sole physical prerequisite for liquid-tissue flow. Tissue 0 1 MM FIG. 3. Scanning electron micrographs of subsurface N aggregates fixed before compression (A), and during compression after one (B), five (C) and 15 minutes (D), and then broken to reveal interior cell shapes in cross-section. (Variations in compressed aggregate heights are due to differing distances set between the rigid compression plates in our miniature clamping vise.) liquidity is in principle compatible with, yet need not depend upon, active cell motility. Cells in liquid tissues could dissipate shear forces by self-propelled translocations or LIQUID-TISSUE MECHANICS IN GASTRULATION 87 by passive slippage movements or both. Also, tissue liquidity, of course, neither assumes nor requires that individual cells must be liquid droplets, any more than individual molecules in ordinary liquids need themselves be liquids. other [Phillips, 1969, and in preparation]. The correlation between completeenvelopment configurations and aggregate-medium surface tensions has been confirmed for embryonic chick cell aggregates in previous aggregatecentrifugation experiments [op. cit.; Phillips and Steinberg, 1969, and in preparation; TESTS FOR SURFACE TENSION CONTROL OF TISSUE et al, 19776J. Phillips FLOW Holtfreter (1944) and Townes and Surface tension is the force within (tan- Holtfreter (1955) reported that, in fused gent to) a liquid interface which opposes pairs of fragments of gastrula and neurula any increase in its surface area, if cell subsurface germ layers, endoderm tends to aggregates behave like liquid droplets, surround ectoderm and mesoderm. Steinthen, in the absence of external deforming berg and Kelland (1967) confirmed these forces, their behavior should be deter- findings and also reported that subsurface mined simply by tissue surface tensions. mesoderm tends to surround subsurface For example, two immiscible but mutually ectoderm. We have obtained similar readhering droplets A and B immersed in sults in our laboratory (Phillips, Mitchell, medium O form three liquid-liquid inter- MacQueen and Davis, in preparation). Acfaces, A-O, B-O and A-B, with surface cordingly, if these subsurface germ-layer tensions yA0, yBn a n d TAB respectively (Fig. movements are determined by tissue sur4). When pairs of similarly-sized droplets face tensions, as predicated above, then with approximately spherical interfaces the surface tensions of subsurface eccoalesce, the surrounding droplet will have toderm (E), mesoderm (M) and endoderm a lower surface tension with respect to the (N) in medium (O) should fall into the medium than does the surrounded drop- sequence let: i.e., (Fig. 4), if droplet B spreads around droplet A, yB0 is less than yA0 (Phillips, 1969, and in preparation; Torza and Mason, 1969). (Special cases of this relationship for coalescing droplets of greatly differing size are discussed in Heintzelman, Phillips and Davis fin preparation]. However, this relationship holds regardless of dropletsize if one droplet completely envelops the TEO > 7MO > TNO- (1) To test this prediction, we have again resorted to aggregate compression. However, in this case, one compressing glass chip is mounted on the end of a flexible quartz fiber (Fig. 5). Using preweighed bits of aluminum foil, we determine the bending constant of the fiber. If we then observe how much the fiber bends during compression, we can calculate how much upward force is flattening the aggregate. Once internal cell stretching within a deformed aggregate has been dissipated by cell slippage, its surface tension is the sole force resisting deformation. Thus, the greater that surface tension, the rounder should be the shape of the aggregate under a given force of compression. In FIG. 4. For comparably-sized immiscible droplets A and B fusing with approximately spherical interfaces principle, quantitative measurements of in medium 0, spreading configurations are deter- droplet-medium surface tensions can be mined by the three surface tensions yA0, yB0 and yAB. determined by this technique (e.g., YoneIf B spreads around A (rather than A around B), yB0 da, 1964, but see Hiramoto, 1967; Phillips, < yA0. (yAB determines immiscibility, and in this case affects the extent of spreading but not the direction of in preparation). envelopment.) In practice, we allow freshly excised HERBERT M. PHILLIPS AND GRAYSON S. DAVIS 88 ///7/ffA V////II 0 FIG. 5. An aggregate (A) in culture medium (O) is compressed between two glass chips, one of which (GC) is glued to a quartz fiber (F) which can be raised or lowered at its other end. The greater the surface tension at the A-O interface, the rounder will be the equilibrium shape of the compressed aggregate. (Preliminary analysis of aggregate shapes suggests that these aggregates form small contact angles with the compressing glass surfaces.) samples of subsurface ectoderm, mesoderm and endoderm from stage 11 + Rana pipiens gastrulae to round up in organ culture 2-8 hours. Initially round aggregates are placed on the moveable glass chip, which is then raised until the aggregate becomes flattened against the coverslip mounted on the roof of the chamber. Within ten minutes after the final turn of our compression dial, these aggregates adopt stable ("equilibrium") shapes which remain constant for at least another 20 minutes thereafter. (By this time, of course, internal stretching forces have been dissipated by cell slippage — see Fig. 3). In qualitative terms, our results reveal that, when flattened by the same compressing force, subsurface ectoderm aggregates adopt much rounder equilibrium shapes than subsurface mesoderm aggregates, which adopt slighdy rounder shapes than subsurface endoderm aggregates (Fig. 6). (The height-to-width ratios of mesoderm and endoderm aggregates compressed by the same force partially overlap, but preliminary estimates of their aggregate-medium surface tensions are nevertheless statistically different.) Precise quantitative determinations of aggregate-medium surface tensions are still in progress. However, our preliminary results are consistent with relation (1) above and thus tentatively confirm the predicted correlation between aggregate behavior and germ-layer surface tensions. TISSUE-TISSUE SURFACE TENSIONS AND MESODERMAL INVOLUTION When subsurface samples of all three germ layers are combined in triplet-fusion and cell-sorting experiments, endoderm surrounds mesoderm, which surrounds ectoderm (Townes and Holtfreter, 1955). The mechanism by which coated germlayer tissues invert this configuration to form the normal germ-layer arrangement is discussed below. Here we wish to point out that a second surface tension relationship, in addition to (1) above, is required to explain triplet-fusion behavior. The spreading of mesoderm between ectoderm and endoderm implies (Fig. 7) that 7 EN > 7 EM + (2) E M N 0.1mm FIG. 6. Representative profiles of subsurface aggregates flattened by equal compressing forces until they adopted stable shapes (see text and Fig. 5). As predicted, aggregate resistance to expansion of surface area falls into the sequence E > M > N. 89 LIQUID-TISSUE MECHANICS IN GASTRULATION FIG. 7. When all three subsurface tissues fuse, forming inverse germ-layer configurations, M spreads between E and N. In a liquid-tissue model, the surface tensions driving this process can be represented by vectors drawn tangent to the E-M, E-N and M-N interfaces. It is evident by inspection that the point at which all three meet will move to the right in the above diagram when y^M + ^ N <1YEN- This relationship would account for the spontaneous flow of M into E-N interfaces observed in in vitro experiments and for autonomous involution of lateroventral mesoderm during gastrulation in vivo. that is, the E-N interface resists expansion more than the E-M and M-N interfaces combined. We are only now attempting to make the tissue-tissue surface tension measurements required to test this relationship directly. However, tissue behavior, both in vitro and in vivo, suggests that the flow of subsurface mesoderm between subsurface ectoderm and endoderm consistent with this relationship is a major force in amphibian gastrulation. For example, Holtfreter (1944) described an experiment (Fig. 8) in which Lehmann removed the coated and subsurface ectoderm from a small area in the ventral region of a gastrula, thereby exposing underlying subsurface endoderm. Into that wound he placed a piece of dorsal lip mesoderm, only part of which was covered by coated mesoderm. Subsurface mesoderm fused with the exposed subsurface endoderm; but in addition, at the edge of the wound, the subsurface mesoderm went on to spread between subsurface ectoderm and endoderm. This movement dragged the coated mesoderm cells on the exterior surface of the aggregate inward to form an archenteron-like cavity. Of course, such transplanted dorsal lips go on to induce secondary axes in the- host embryos (Spemann and Mangold, 1924). Schechtman (1942) mechanically isolated various portions of the blastopore lip in intact Hyla regilla gastrulae using cellophane inserts, and found that lateral and ventral mesoderm involute autonomously, but dorsal mesoderm involutes only when connected with lateral mesoderm. Recall that in early gastrulae, while bottle cell formation in surface mesodermal layers can produce some initial blastopore lip invagination, there is no dorsal subsurface ectoderm-endoderm interface into which the subsurface mesoderm can then spread. By contrast lateral and ventral lip mesoderm is confronted with such an E-N interface. Given the capacity of dorsal lip mesoderm to spread in such an interface (as demonstrated by Lehmann's experiment described above), Schechtman's results support the hypothesis that mesodermal involution in vivo is driven by the flow of subsurface lateroventral mesoderm M B. M N \ M M N E E FIG. 8. Diagram by Holtfreter (1944) of experiments by Lehmann (our labels added). When implanted into a wound in the ventral ectoderm of a host embryo, a graft of dorsal lip mesoderm spontaneously flows into the subsurface E-N interface. The coated surface of the graft forms an archenteron-like cavity, and can give rise by induction to an entire secondary axis. Tissues here are labeled as in Figure 1. 90 HERBERT M. PHILLIPS AND GRAYSON S. DAVIS into the E-N interface. We propose that this autonomous tissue flow is driven by intrinsic tissue-tissue surface tension forces according to relation (2) above. GERM-LAYER INVERSION BY COATED ECTODERM Given surface tension relations (1) and (2) for subsurface tissues, and given the additional fact that the shiny side of coated ectoderm is non-adhesive towards other cells, one can explain how the presence of coated ectoderm would cause subsurface germ layers to form their normal in vivo arrangement (ectoderm surrounding mesoderm surrounding endoderm). In qualitative terms, "non-adhesive" in this case means, of course, that the coated sides of cells adhere more strongly to the culture medium than to other cells. This would explain observations by Holtfreter (1943a, 1944) and Townes and Holtfreter (1955) that in tissue-spreading and cell-sorting experiments coated cells usually tend to collect at and spread over the surfaces of cell aggregates. The following argument, diagrammed in Figure 9, first formulated by Steinberg and Kelland (1967), is restated here in terms of tissue surface tensions, (i) Once on the outside of an aggregate (or embryo), coated ectoderm forms an wncoated ectodermal boundary facing the subsurface cell populations which it has enveloped, (ii) Since their surface tensions cause germ-layers to be immiscible (e.g., as demonstrated graphically by their cellsorting behavior), subsurface ectoderm will adhere to "its own kind," and thus will displace subsurface mesoderm and endoderm along the inner (uncoated, adhesive) boundary of the surface layer of coated ectoderm, (iii) Tissue-tissue surface tenions (relation (2) and Fig. 7 above) causes subsurface mesoderm to spread into the E-N interface, (iv) This displaces subsurface endoderm to the most interior position in this tissue system. Steinberg and Kelland (op. cit.) performed a direct behavioral test of this explanation in their "sandwich experiments" (Fig. 10). They simply excised whole chunks of tissue from the sides of FIG. 9. Our diagram of the sequence of events proposed by Steinberg and Kelland (1967) as a mechanism by which coated ectoderm could assemble subsurface germ layers into their normal gastrular arrangement (see text). This mechanism requires only surface tension relations (1) and (2) plus the observed fact that the coated faces of surface ectoderm cells are non-adhesive. Rana pipiens gastrulae at sufficiently late stages so that lateral mesoderm had already spread between ectoderm and endoderm. Each excised fragment then contained portions of all three subsurface germ layers. On some fragments Steinberg and Xelland left the surface layer of coated ectoderm intact; from others they peeled it LIQUID-TISSUE MECHANICS IN GASTRULATION A. With coated ectoderm -E M N B. Without coated ectoderm M N FIG. 10. Our diagram of the "sandwich" experiments by Steinberg and Kelland (1967), which were designed to test the effect of removing coated ectoderm from samples of subsurface E, M and N. As they predicted, excised pieces of lateral tissue from late gastrulae (containing all three subsurface germ layers) in organ culture form (A) the normal arrangement when coated ectoderm has been left intact and (B) an inverted arrangement when coated ectoderm has been removed from otherwise identical tissue samples. away. Organ culture fragments with coated ectoderm formed round, multilayered aggregates with the normal gastrular germ-layer arrangement. In otherwise identical fragments with the coated ectoderm removed, the resulting configurations of subsurface germ layers were reversed. This demonstrates that coated ectoderm is the sole additional factor required to cause subsurface germ layers to adopt their proper orientations during in vitro tissue spreading. Holtfreter (1944) and Townes and Holtfreter (1955) obtained similar results in various combinations of subsurface germ layers with and without coated ectoderm. This explanation further implies that, in the absence of coated ectoderm, the normal distribution of subsurface germ layers should be unstable. An early observation by Holtfreter (1944) provides an elegant in vivo confirmation of this prediction. He placed a whole, intact late-gastrula in dissociating medium. After many coated ectoderm cells had fallen off the embryo, but 91 before further cell dissociation could proceed, he returned the embryo to normal culture medium. At first the endoderm from the archenteron began to emerge from the blastopore and spread over the denuded, now largely uncoated surface of the embryo. However, the remaining coated ectoderm soon spread out to cover the rest of the surface of the embryo, then halted the advance of the endoderm and eventually forced it to recede back to its interior position! Thus, in the normal embryo, coated ectoderm may confine endoderm to its proper internal location as gastrulation proceeds. (By means of this analysis one can also account for the envelopment of mesoderm by endoderm in the absence of ectoderm in vivo, e.g., as Holtfreter [1933] observed in whole embryos following exogastrulau'on.) CONCLUSION Liquid-tissue flow is clearly not the sole morphogenetic mechanism operating in amphibian gastrulation. For example, active cell shape changes evidently cause elastic-solid deformations in surface cell layers which initiate blastopore lip imagination and maintain the external contours of the lip during gastrulation (see above). Another apparently solid-like forcegenerating movement is the spontaneous elongation of dorsal lip mesoderm, which Schechtman (1942) described in Hyla, and which one of us also confirmed in Rana pipiens (Phillips, in preparation). For example, excised pieces of dorsal lip mesoderm in organ culture will at first round up like aggregates of lateroventral lip mesoderm. Unlike the latter, however, a dorsal lip aggregate (with or without coated mesoderm) will then form a cylindrically-shaped protrusion several cell diameters in thickness and many cell diameters long—an obvious departure from a simple minimum-surface-area configuration characteristic of isolated liquid droplets. The significance of this dorsal lip mesodermal elongation is shown by Schechtman's experiments {op. cit.) in which he mechanically isolated dorsal lip 92 HERBERT M. PHILLIPS AND GRAYSON S. DAVIS mesoderm or removed it entirely from otherwise intact, whole embryos. (As explained above, when mechanically isolated from lateral lip mesoderm, dorsal lip mesoderm does not involute. However, it does still elongate, so dorsal lip elongation occurs independently of its own involution.) In the absence of normally involuted dorsal lip mesoderm, Schechtman obtained "ring" embryos, which were greatly compressed in an anterior-posterior direction, but which still exhibited involution of mesoderm along the lateral and ventral lips of the blastopore. Thus, dorsal mesoderm elongation, while not required for lateroventral mesodermal involution, is evidently necessary for the maintenance of overall embryonic shape (and, in addition, he found, for dorsal convergence of involuting mesoderm and for subsequent closure of the blastopore over the yolk plug). While the mechanism of dorsal mesoderm elongation remains entirely unknown (this occurs long before the notochordal elongation due to cell vacuolization described by Mookerjierfa/. [1953]), suffice it to say that it is clearly a significant force in amphibian gastrulation which is evidently different from and independent of subsurface germ-layer flow. While recognizing the need for several different force-generating mechanisms to combine in producing amphibian gastrulation, we nevertheless wish to emphasize the importance of liquid-tissue flow as a major factor in this process, not only for mesodermal involution (discussed above), but perhaps also for ectodermal epiboly and for the anterior spreading of subsurface endoderm on the inner margin of blastocoel-roof ectoderm. (This seals off the blastocoel and creates the subsurface E-N interface into which involuting mesoderm then flows.) Amphibian gastrulation has been a subject of scientific investigation for nearly a century, but has to this day remained a mechanically puzzling process. We suggest that this has been in large part due to a failure to appreciate conceptually and to explore experimentally the mechanism of liquid-tissue flow governed by tissue surface tensions as a potential explanation for subsurface germ-layer movements. (Credit should be given, however, to Holtfreter [1943a,6; 1944] and to Rhumbler [quoted therein] for preliminary explorations in this direction, as well as to Steinberg and Kelland [1967] and Steinberg [1970] for partial formulation of this hypothesis.) We are now investigating this mechanism in other thick-tissue morphogenetic processes, specifically limb bud formation in chick embryos (Daggy and Phillips, in preparation; Heintzelman et al., in preparation). Physical studies of morphogenetic movements at the cell-and-tissue level of analysis are not only essential in providing a complete mechanical explanation of these phenomena, but may also prove to be useful in molecular approaches to these problems. For example, in liquid-tissue systems, those molecular interactions which can be shown to affect tissue surface tensions would be morphogenetically significant in the regulation of tissue flow; those which do not—interesting as they may be for other reasons — would in this context be morphogenetically irrelevant. Moreover, regulation of intercellular adhesiveness, e.g., via changes in cell-cell bonding strengths and/or patterns (Steinberg, 1963, 1964, 1970, 1975; Phillips, 1969, and in preparation), constitutes the simplest, although not the only, means of generating and modulating the tissue surface tensions which direct liquid-tissue flow. REFERENCES Baker, P. C. 1965. Fine structure and morphogenetic movements in the gastrula of the tree Frog, Hyla regilta.J. Cell Biol. 24:95-1 16. Burnside, B. 1973. Microtubules and microfilaments in amphibian neurulation. Amer. Zool. 13:9891006. Hamburger, V. 1962./} maunal ofexperimental embryol- ogy, p. 36. 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