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Ruffling and locomotion in Rana pipiens gastrula cells
Printed
rn Swedrn
Experimental
RUFFLING
Cell Research 101 (1976) 71-77
AND LOCOMOTION GASTRULA
IN RANA
PZPZENS
CELLS
K. E. JOHNSON Department
of Anatomy,
Duke University
Medical
Center, Durham, NC 27710, USA
SUMMARY Embryonic amphibian cells move during gastrulation, even though they are in contact with many neighboring cells. The behavior of these cells in vitro with respect to cell movement and contact inhibition is thus of interest. Cultures of isolated presumptive mesodermal cells of early Rana pipiens gastrulae were sealed with a coverslip and filmed under phase contrast at 16frameslmin. At the end of 30 mm in vitro, cells settle to the substratum and form fan-like lamellipodia which are sites of cell attachment. Ruffling is qualitatively similar to that seen in many chick and mammalian cell types in vitro. Ruffles lift up and move back from marginal extensions of cells. When lamellipodiaare symmetrically arranged around the cell periphery, no net translocation of the cell occurs. In contrast, when cells have a dominant lamellipodium (larger and/or more active), movement occurs in that direction. Cells may exhibit complex margins composed of microspikes, ruffles, and hyahne extensions of the cell draped between microspikes. When cells come in contact there is a local paralysis of ruffling. When cells lose contact, a broad ruffling lamellipodium often appears immediately at the former sites of contact.
One of the cardinal features of morphogenesis in vertebrate embryos is cell movement. In amphibian gastrulation, sheets of some cells move from the surface of the early gastrula to occupy an internal position (invagination) and sheets of others spread to occupy a greater portion of the outer surface of the embryo (epiboly). Epiboly occurs in a sheet of presumptive ectodermal cells without the formation of patent gaps between cells [14] but gaps do occur between invaginating presumptive mesodermal cells in toad [15] and frog embryos as shown below. Except for the pioneering studies of Holtfreter [7, 81 we know little about the factors which initiate and control cell migration in amphibian gastrulae. Studies on the locomotion of fibroblasts
in vitro have revealed that cells attach to, and move upon, solid substrata by means of hyaline extensions of the cell periphery, known as ruffling lamellae or lamellipodia [2, 5, 6, 8, 221. Also, for fibroblasts and epithelial cells, cell contact restricts cell movement by contact paralysis of the ruffled membrane [ 1, 5, 20, 221. Several authors have suggested that the contact inhibition of cell locomotion may also serve as an important guiding force for morphogenetic cell movements [19, 201. The present study was undertaken to examine the contact inhibition of cell locomotion of presumptive mesodermal cells from Rana pipiens embryos in vitro at the time of invagination. A preliminary report of this work has been published elsewhere [ 131. Exp Cell Rrs 101 (1976)
72
K. E. Johnsorl
1 Fig. 1. Tracing from a film of a cell with a radially symmetrical lamellipodium. The solid line is a tracing of the cell at 0 time and the dotted line is 28 min later. The small central ellipse is the outline of the nucleus. The larger ellipse is the boundary between the granular endoplasm and the hyaline ectoplasm. Notice that the cell does not move appreciably in any direction. Bar 50 pm.
MATERIALS
AND METHODS
Frogs and embryos pipiens were obtained from Nasco (Ft Atkinson, Wise.). Females were ovulated, eggs were fertilized and jelly coats were removed as described previously [IO]. Preliminary studies indicated that a modification of the Steam’s & Kostellow (S-K) [I81 medium promoted cell movement. The published formula was modified by substituting bovine serum albumin, fraction V (Sigma, A-4503), for human serum albumin and by adding phenol red (8 mg/l), penicillin-G (lOUtI lU/l) and streptomycin sulfate (50 mgll). Albumin was present in cell culture medium but not in solutions for rearing embryos, removing jelly, washing, or dissociating embryos. Embryos were reared in 10% S-K until ihey were early gastrulae (Shumway, Stage 10
Rana
2 Fig. 2. Tracing from film of a cell with a dominant
lamellipodium. The solid line is a tracing at 0 min, the dotted line is 5 min later, and the dashed and dotted line is 10 min later. Notice that as the lamellipodium on the top of the cell increases in area, the cell moves in that direction and that the two lamellipodia on the bottom of the cell simultaneously become smaller. Bar 30 pm.
micromanipulator. Cells were drawn into the pipette by gentle suction applied with a microinjection apparatus, and expelled with a gentle stream of EDTA into a reservoir of S-K with 0.5% bovine serum albumin. The reservoir of culture medium was contained by a rectangle of Vaseline on a standard microscope slide. Cells were allowed to settle to the substratum, then washed 3 times with fresh culture medium to remove EDTA carried over from the dissociation step. Finally, cultures were sealed with a coverslip and viewed with phase contrast optics. Cultures were photographed on I6 mm Kodak Plus-X Reversal movie film at I6 framesimin. Commercially developed films were then analysed with a film analyser. For illustrations, individual frames from films were projected and traced on white paper. Fixation of embryos has been described previously [12].
[171).
RESULTS Isolation and observation mesodermal cells
of presumptive
Stage 10 embryos were transferred to Ca’+-, Mgz+-free S-K and the roof of the blastocoel was cut away using sharpened Dumont no. 5 watchmaker’s forceps. The remaining vegetal portion of the embryo was rinsed 3 times with CaZ*-, Mg*+-free S-K with I mM disodium ethylenediamine tetraacetate (EDTA) and left undisturbed for 30 min. At-the end of the dissociation period, a group of medium sized, moderately pigmented cells, located above and behind the blastopore and in front of the endodermal mass, were collected in a pipette (100 pm tip inside diameter) held in a Leitz
Cell attachment
Immediately after cultures have been sealed, cells are round and not attached to Fig. 3. Tracing from film of two cells which make and
break contacts and show local inhibition of ruffling at contact sites. In each figure, KS indicate locations where ruffling is occurring and NRs indicate locations were ruffling is not occurring. In what fotIows, all times refer to elapsed time from 0 time: -, (a) 0; (6) 15; (c) 23; (d) 30; (e) 38; v) 54 min; -- -, (a) 10; (b) 19; (c) 25; (d) 36; (e) 48; (f, 64 min. Bar 30 Nrn.
Motility
of frog gastrula cells
73
d
Exp Cell Res 101 (1976)
74
K. E. Johnson
Fig. 4. Photograph of large gaps between invaginating cells in a stage lO$ Rana pipiens early gastrola. Notice the large gaps between cells in the center of the photo-
graph. This group of cells is located near the tip of the archenteron close to the uninvaginated cells of the outer layer of the embryo. Bar 50 pm.
the substratum. By gently depressing the coverslip on the preparation, the culture medium can be moved. When this is done, rounded cells also move with the medium, indicating that they are unattached. Rounded cells have a few spiky phase-dark protrusions from the cell surface which move as if they are feeling about for a substratum. Thirty minutes later, many cells have flattened on the glass and formed fan-like lamellipodia in agreement with previous findings [lo]. Flattened cells with lamellipodia are attached to the substratum. If the coverslip is depressed, medium flows but cells do not flow with it. Also, when a ruffle detaches suddenly from the substratum, a portion of the cell snaps back from the site of attachment, sometimes leaving behind retraction fibers. This sudden retraction of an extended portion of the cell periphery
also exhibits graphically the radial tension in a stretched cell. In addition, expansion of the cell margin away from the central portion of the cell invariably involves a sudden expansion and attachment of the lamellipodium. Sometimes cells which are actively ruffling would stop, round up (leaving numerous retraction fibers still attached to the substratum) and then divide. Soon after cell division is completed, the daughter cells flatten on the substratum and begin to ruffle again.
Exp Cell Res 101 (1976)
Lamellipodial
morphology
and behavior
Lamellipodia represent flattened fan-like extensions of the cell periphery. They are dark in phase contrast, hyaline, and represent extremely thin portions of the cell cortex. They are draped between rigid microspikes which project away from the cell.
Motility of frog gastrula cells It is evident from analysis of many films that small portions of lamellipodia are constantly rising up from the substratum and moving toward the center of the cell. Occasionally , particulate matter becomes attached to the surface of the lamellipodium and this appears to be swept rapidly on the upper surface towards the central portions of the cell as well. It was not possible to make measurements of rates of particle transport on the cell surface because in the few instances where debris in the culture was picked up on a lamellipodium, it rapidly became invisible, either because it moved out of the plane of focus or was engulfed by the cell. Marginal uplift was not viewed from the side, but movement of these parts of the lamellipodia relative to immobile particulate markers on the substratum made it apparent that marginal folds were moving away from the cell margins. In addition, focusing up and down showed that these parts of the lamellipodium were above the substratum on the upper surface of the lamellipodium.
75
and arrows with an NR indicate no ruffling. At the beginning of this sequence, two cells are clearly separated (fig. 3a). Ten minutes later, the cells have come into contact at one point (fig. 3 a). Soon after the cells come into contact, active ruffling ceases and the contact region broadens from 2 pm (10 min) to 22 pm (15 min) and then narrows again to 7 pm (19 min) (fig. 3b). By 23 min, the contact region has broadened again to 18 pm and developed a small hole which enlarges to give two separate contact regions at 25 min (fig. 3~). The upper contact then breaks (30 min) and reforms (36 min) (fig. 3 d). During this short period, ruffling continues at the former site of upper contact. At 38 min, the lower contact breaks and the upper contact broadens. As the lower contact breaks, ruffling commences anew (fig. 3 e). Finally, the upper contact breaks and ruffling begins again there (fig. 3fl as well. Contact relations in vivo Near the blastopore, presumptive mesodermal and endodermal cells are packed tightly together. There are few if any spaces between cells. Further away from the blastopore, however, presumptive mesodermal cells have large spaces between them, often S-10 pm in greatest dimension (fig. 4). These spaces often have portions of cells in them which can be seen, in some cases, to be projections from the cell surface. These projections are thin, devoid of organelles such as mitochondria and yolk platelets [ 111, and may represent fixed ruffles.
Inhibition of rufj7ing by cell contact When two cells are moving toward one another they often meet and begin to interact. At points of contact, there is a local dimunition of both the size and the activity of the lamellipodia and ruffling ceases at the site of contact. RuMing activity continues unabated, however, at other parts of the periphery which are not in contact with other cells. When cells in contact detach from one another (contacts retract rapidly soon after breaking) ruffling becomes more active and,lamellipodia grow larger near the former site of contact. For example, in fig. 3, two cells repeatedly make and break conDISCUSSION tacts and alter their ruffling behavior with changes in contact relations. In this se- Results presented here show that presumpquence arrows with an R indicate ruffling tive mesodermal cells from Rana pipiens Exp Cell Res IO1 (1976)
76
K. E. Johnson
early gastrulae behave in many respects like many other kinds of cells in vitro. They form fan-like hyaline attachments to the substratum which ruffle and are the locomotory organelle of the cell. Furthermore, when lamellipodia of adjacent cells collide, ruffling and cell movement in that part of the cell ceases locally. Similar behavior has been described for a variety of cell types including embryonic chick heart and mouse skeletal muscle 121,myoblasts from embryonic chick heart [22], HiK21 cells and 3T3 cells [6], and epithelial cells [5]. Cells which show a paralysis of ruffling activity upon contact will tend to spread out to fill gaps between cells, since lamellipodia will only form at free edges. The problem that we must deal with here is that groups of cells in an amphibian embryo move during invagination and epiboly without any obvious free edge. During epiboly in Fundulus embryos, the yolk mass is engulfed by a layer with a free edge, the spreading enveloping layer. During epiboly, cells at the margin of the enveloping layer show undulating or ruffling behavior [20] while cells away from the margin do not unless they break contact with their neighbors. In such cases, cells appear to undulate at their margins and move to close the gap. Although there is no experimental evidence presented to support the notion, it has been suggested that the lateral emigration of mesoblast cells from the primitive streak in chick embryos is also a phenomenon which results from the tendency of cells to occupy spaces or to spread only when there is a free edge [19]. In amphibian embryos at the early gastrula stage, cells near the blastopore are packed tightly together, with no spaces between them [3, 12, 161.Further away from the blastopore, however, the presumptive pharyngeal endoderm and mesoderm cells Exp Cell Res 101 (I 976)
are often separated from one another by gaps of 5-10 pm and show formation of pseudopodia [ 151and spiky processes [14] in these spaces. Invaginating cells near the blastocoel may form a functional “free edge” along the front where the migrating mass of endodermal and mesodermal cells meets the inner surface of the ectodermal layer. As this free edge advances, gaps may form between cells which then allows movement of cells until the gaps are closed. This mechanism, which would generate progressive invagination, would require that the endodermal-mesodermal mass recognize the ectodermal surface as something to adhere to (to gain traction for movement) without adhering in such a way as to prevent the formation of lamellipodia and pseudopodia. Detailed fine-structural studies of the junction between the invaginating endodermal-mesodermal mass and the overlying ectodermal surface, especially with the scanning electron microscope, are needed to see if lamellipodia are present here. The spreading behavior of cells in vitro may be a reflection of their spreading tendency in vivo [7]. We do not know what signals the onset of locomotion in amphibian embryos, but once set in motion, the tendency of cells to move awav from regions of frequent cell contact when they find a suitable substratum for cell movement could account for invagination. Analysis of films revealed several examples where an actively ruffling cell suddenly stopped, rounded up, divided, daughter cells flattened out again, and cells reformed ruffling lamellipodia. The blastula period in amphibian embryos is marked by rapid cell division. In blastula stage embryos, mitotic indices are high and decrease rapidly by the mid-gastrula stage [4, 141. The decrease in the rate of cell division may also be an important signal for the onset of
Motility
lamellipodium formation and consequently active cell locomotion. This work was supported by research grant HD-07082 from USPHS, Health Sciences Advancement Award 35S04RRO6148, and by agrant from the United Health Services of North Carolina. I would like to thank Dr S. J. Counce and Dr J. P. Trinkaus for reading the manuscript.
Note added in proof Monroy et al. (Dev biol 49 (1976) 250) have recently used the scanning electron microscope to show that newly invaginated chordamesodermal cells in gastrula stage Xenopus embryos are “. . . fibroblast-like with long and thin interwoven filopodia.” This observation suggests that ruffling may occur in vivo, since many of the processes seen extending from the cells may be lamellipodia.
REFERENCES 1. Abercrombie, M, Exp cell res, suppl. 8 (1961) 188. 2. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 60 (1970) 437. 3. Baker, P C, J cell bio124 (1965) 95. 4. Bragg, AN, Z Zellforsch mikros Anat 28 (1938) 154.
of frog gas&a
cells
17
5. DiPasquale, A, Exp cell res 94 (1975) 191. 6. Harris, A K, Locomotion of tissue cells, Ciba symposium 14, new series, p. 3. Elsevier, Amsterdam (1973). 7. Holtfreter, J, Arch exp Zellforsch 23 (1939) 169. 8. -J morph01 80 (1947) 25. 9. Ingram, V M, Nature 222 (1969) 641. 10. Johnson, K E, J exp zoo1 170 (1969) 325. 11. -Ibid 175 (1970) 391. 12. -Ibid 179 (1972) 227. 13. - J cell biol 63 (1974) 158a. 14. - Unpublished observations. 15. Nakatsuji, N, J embryo1 exp morph01 32 (1974)795. 16. Perry, M W & Waddington, C H, J embryo1 exp morph01 15 (1966) 317. 17. Shumway, W, Anat ret 78 (1940) 139. 18. Stearns, RN & Kostellow, A B, The chemical basis of development (ed W D McElroy & B Glass. p. 448. Johns Hopkins, Baltimore, Md (1958). 19. Trelstad. R L. Hav. E D & Revel. J-P. Dev biol 16 (1967) 78. I 20. Trinkaus, J P, Cells into organs, the forces that shape the embryo, p. 22. Prentice-Hall, Englewood Cliffs, NJ (1969). 21. - Dev biol30 (1973) 68. 22. Trinkaus. J P. Betchaku. T & Krulikowski. L S. Exp cell res 64 (1971) 29i. Received October 13, 1975 Accepted December 10, 1975
Exp Cell Res 101 (1976)
rn Swedrn
Experimental
RUFFLING
Cell Research 101 (1976) 71-77
AND LOCOMOTION GASTRULA
IN RANA
PZPZENS
CELLS
K. E. JOHNSON Department
of Anatomy,
Duke University
Medical
Center, Durham, NC 27710, USA
SUMMARY Embryonic amphibian cells move during gastrulation, even though they are in contact with many neighboring cells. The behavior of these cells in vitro with respect to cell movement and contact inhibition is thus of interest. Cultures of isolated presumptive mesodermal cells of early Rana pipiens gastrulae were sealed with a coverslip and filmed under phase contrast at 16frameslmin. At the end of 30 mm in vitro, cells settle to the substratum and form fan-like lamellipodia which are sites of cell attachment. Ruffling is qualitatively similar to that seen in many chick and mammalian cell types in vitro. Ruffles lift up and move back from marginal extensions of cells. When lamellipodiaare symmetrically arranged around the cell periphery, no net translocation of the cell occurs. In contrast, when cells have a dominant lamellipodium (larger and/or more active), movement occurs in that direction. Cells may exhibit complex margins composed of microspikes, ruffles, and hyahne extensions of the cell draped between microspikes. When cells come in contact there is a local paralysis of ruffling. When cells lose contact, a broad ruffling lamellipodium often appears immediately at the former sites of contact.
One of the cardinal features of morphogenesis in vertebrate embryos is cell movement. In amphibian gastrulation, sheets of some cells move from the surface of the early gastrula to occupy an internal position (invagination) and sheets of others spread to occupy a greater portion of the outer surface of the embryo (epiboly). Epiboly occurs in a sheet of presumptive ectodermal cells without the formation of patent gaps between cells [14] but gaps do occur between invaginating presumptive mesodermal cells in toad [15] and frog embryos as shown below. Except for the pioneering studies of Holtfreter [7, 81 we know little about the factors which initiate and control cell migration in amphibian gastrulae. Studies on the locomotion of fibroblasts
in vitro have revealed that cells attach to, and move upon, solid substrata by means of hyaline extensions of the cell periphery, known as ruffling lamellae or lamellipodia [2, 5, 6, 8, 221. Also, for fibroblasts and epithelial cells, cell contact restricts cell movement by contact paralysis of the ruffled membrane [ 1, 5, 20, 221. Several authors have suggested that the contact inhibition of cell locomotion may also serve as an important guiding force for morphogenetic cell movements [19, 201. The present study was undertaken to examine the contact inhibition of cell locomotion of presumptive mesodermal cells from Rana pipiens embryos in vitro at the time of invagination. A preliminary report of this work has been published elsewhere [ 131. Exp Cell Rrs 101 (1976)
72
K. E. Johnsorl
1 Fig. 1. Tracing from a film of a cell with a radially symmetrical lamellipodium. The solid line is a tracing of the cell at 0 time and the dotted line is 28 min later. The small central ellipse is the outline of the nucleus. The larger ellipse is the boundary between the granular endoplasm and the hyaline ectoplasm. Notice that the cell does not move appreciably in any direction. Bar 50 pm.
MATERIALS
AND METHODS
Frogs and embryos pipiens were obtained from Nasco (Ft Atkinson, Wise.). Females were ovulated, eggs were fertilized and jelly coats were removed as described previously [IO]. Preliminary studies indicated that a modification of the Steam’s & Kostellow (S-K) [I81 medium promoted cell movement. The published formula was modified by substituting bovine serum albumin, fraction V (Sigma, A-4503), for human serum albumin and by adding phenol red (8 mg/l), penicillin-G (lOUtI lU/l) and streptomycin sulfate (50 mgll). Albumin was present in cell culture medium but not in solutions for rearing embryos, removing jelly, washing, or dissociating embryos. Embryos were reared in 10% S-K until ihey were early gastrulae (Shumway, Stage 10
Rana
2 Fig. 2. Tracing from film of a cell with a dominant
lamellipodium. The solid line is a tracing at 0 min, the dotted line is 5 min later, and the dashed and dotted line is 10 min later. Notice that as the lamellipodium on the top of the cell increases in area, the cell moves in that direction and that the two lamellipodia on the bottom of the cell simultaneously become smaller. Bar 30 pm.
micromanipulator. Cells were drawn into the pipette by gentle suction applied with a microinjection apparatus, and expelled with a gentle stream of EDTA into a reservoir of S-K with 0.5% bovine serum albumin. The reservoir of culture medium was contained by a rectangle of Vaseline on a standard microscope slide. Cells were allowed to settle to the substratum, then washed 3 times with fresh culture medium to remove EDTA carried over from the dissociation step. Finally, cultures were sealed with a coverslip and viewed with phase contrast optics. Cultures were photographed on I6 mm Kodak Plus-X Reversal movie film at I6 framesimin. Commercially developed films were then analysed with a film analyser. For illustrations, individual frames from films were projected and traced on white paper. Fixation of embryos has been described previously [12].
[171).
RESULTS Isolation and observation mesodermal cells
of presumptive
Stage 10 embryos were transferred to Ca’+-, Mgz+-free S-K and the roof of the blastocoel was cut away using sharpened Dumont no. 5 watchmaker’s forceps. The remaining vegetal portion of the embryo was rinsed 3 times with CaZ*-, Mg*+-free S-K with I mM disodium ethylenediamine tetraacetate (EDTA) and left undisturbed for 30 min. At-the end of the dissociation period, a group of medium sized, moderately pigmented cells, located above and behind the blastopore and in front of the endodermal mass, were collected in a pipette (100 pm tip inside diameter) held in a Leitz
Cell attachment
Immediately after cultures have been sealed, cells are round and not attached to Fig. 3. Tracing from film of two cells which make and
break contacts and show local inhibition of ruffling at contact sites. In each figure, KS indicate locations where ruffling is occurring and NRs indicate locations were ruffling is not occurring. In what fotIows, all times refer to elapsed time from 0 time: -, (a) 0; (6) 15; (c) 23; (d) 30; (e) 38; v) 54 min; -- -, (a) 10; (b) 19; (c) 25; (d) 36; (e) 48; (f, 64 min. Bar 30 Nrn.
Motility
of frog gastrula cells
73
d
Exp Cell Res 101 (1976)
74
K. E. Johnson
Fig. 4. Photograph of large gaps between invaginating cells in a stage lO$ Rana pipiens early gastrola. Notice the large gaps between cells in the center of the photo-
graph. This group of cells is located near the tip of the archenteron close to the uninvaginated cells of the outer layer of the embryo. Bar 50 pm.
the substratum. By gently depressing the coverslip on the preparation, the culture medium can be moved. When this is done, rounded cells also move with the medium, indicating that they are unattached. Rounded cells have a few spiky phase-dark protrusions from the cell surface which move as if they are feeling about for a substratum. Thirty minutes later, many cells have flattened on the glass and formed fan-like lamellipodia in agreement with previous findings [lo]. Flattened cells with lamellipodia are attached to the substratum. If the coverslip is depressed, medium flows but cells do not flow with it. Also, when a ruffle detaches suddenly from the substratum, a portion of the cell snaps back from the site of attachment, sometimes leaving behind retraction fibers. This sudden retraction of an extended portion of the cell periphery
also exhibits graphically the radial tension in a stretched cell. In addition, expansion of the cell margin away from the central portion of the cell invariably involves a sudden expansion and attachment of the lamellipodium. Sometimes cells which are actively ruffling would stop, round up (leaving numerous retraction fibers still attached to the substratum) and then divide. Soon after cell division is completed, the daughter cells flatten on the substratum and begin to ruffle again.
Exp Cell Res 101 (1976)
Lamellipodial
morphology
and behavior
Lamellipodia represent flattened fan-like extensions of the cell periphery. They are dark in phase contrast, hyaline, and represent extremely thin portions of the cell cortex. They are draped between rigid microspikes which project away from the cell.
Motility of frog gastrula cells It is evident from analysis of many films that small portions of lamellipodia are constantly rising up from the substratum and moving toward the center of the cell. Occasionally , particulate matter becomes attached to the surface of the lamellipodium and this appears to be swept rapidly on the upper surface towards the central portions of the cell as well. It was not possible to make measurements of rates of particle transport on the cell surface because in the few instances where debris in the culture was picked up on a lamellipodium, it rapidly became invisible, either because it moved out of the plane of focus or was engulfed by the cell. Marginal uplift was not viewed from the side, but movement of these parts of the lamellipodia relative to immobile particulate markers on the substratum made it apparent that marginal folds were moving away from the cell margins. In addition, focusing up and down showed that these parts of the lamellipodium were above the substratum on the upper surface of the lamellipodium.
75
and arrows with an NR indicate no ruffling. At the beginning of this sequence, two cells are clearly separated (fig. 3a). Ten minutes later, the cells have come into contact at one point (fig. 3 a). Soon after the cells come into contact, active ruffling ceases and the contact region broadens from 2 pm (10 min) to 22 pm (15 min) and then narrows again to 7 pm (19 min) (fig. 3b). By 23 min, the contact region has broadened again to 18 pm and developed a small hole which enlarges to give two separate contact regions at 25 min (fig. 3~). The upper contact then breaks (30 min) and reforms (36 min) (fig. 3 d). During this short period, ruffling continues at the former site of upper contact. At 38 min, the lower contact breaks and the upper contact broadens. As the lower contact breaks, ruffling commences anew (fig. 3 e). Finally, the upper contact breaks and ruffling begins again there (fig. 3fl as well. Contact relations in vivo Near the blastopore, presumptive mesodermal and endodermal cells are packed tightly together. There are few if any spaces between cells. Further away from the blastopore, however, presumptive mesodermal cells have large spaces between them, often S-10 pm in greatest dimension (fig. 4). These spaces often have portions of cells in them which can be seen, in some cases, to be projections from the cell surface. These projections are thin, devoid of organelles such as mitochondria and yolk platelets [ 111, and may represent fixed ruffles.
Inhibition of rufj7ing by cell contact When two cells are moving toward one another they often meet and begin to interact. At points of contact, there is a local dimunition of both the size and the activity of the lamellipodia and ruffling ceases at the site of contact. RuMing activity continues unabated, however, at other parts of the periphery which are not in contact with other cells. When cells in contact detach from one another (contacts retract rapidly soon after breaking) ruffling becomes more active and,lamellipodia grow larger near the former site of contact. For example, in fig. 3, two cells repeatedly make and break conDISCUSSION tacts and alter their ruffling behavior with changes in contact relations. In this se- Results presented here show that presumpquence arrows with an R indicate ruffling tive mesodermal cells from Rana pipiens Exp Cell Res IO1 (1976)
76
K. E. Johnson
early gastrulae behave in many respects like many other kinds of cells in vitro. They form fan-like hyaline attachments to the substratum which ruffle and are the locomotory organelle of the cell. Furthermore, when lamellipodia of adjacent cells collide, ruffling and cell movement in that part of the cell ceases locally. Similar behavior has been described for a variety of cell types including embryonic chick heart and mouse skeletal muscle 121,myoblasts from embryonic chick heart [22], HiK21 cells and 3T3 cells [6], and epithelial cells [5]. Cells which show a paralysis of ruffling activity upon contact will tend to spread out to fill gaps between cells, since lamellipodia will only form at free edges. The problem that we must deal with here is that groups of cells in an amphibian embryo move during invagination and epiboly without any obvious free edge. During epiboly in Fundulus embryos, the yolk mass is engulfed by a layer with a free edge, the spreading enveloping layer. During epiboly, cells at the margin of the enveloping layer show undulating or ruffling behavior [20] while cells away from the margin do not unless they break contact with their neighbors. In such cases, cells appear to undulate at their margins and move to close the gap. Although there is no experimental evidence presented to support the notion, it has been suggested that the lateral emigration of mesoblast cells from the primitive streak in chick embryos is also a phenomenon which results from the tendency of cells to occupy spaces or to spread only when there is a free edge [19]. In amphibian embryos at the early gastrula stage, cells near the blastopore are packed tightly together, with no spaces between them [3, 12, 161.Further away from the blastopore, however, the presumptive pharyngeal endoderm and mesoderm cells Exp Cell Res 101 (I 976)
are often separated from one another by gaps of 5-10 pm and show formation of pseudopodia [ 151and spiky processes [14] in these spaces. Invaginating cells near the blastocoel may form a functional “free edge” along the front where the migrating mass of endodermal and mesodermal cells meets the inner surface of the ectodermal layer. As this free edge advances, gaps may form between cells which then allows movement of cells until the gaps are closed. This mechanism, which would generate progressive invagination, would require that the endodermal-mesodermal mass recognize the ectodermal surface as something to adhere to (to gain traction for movement) without adhering in such a way as to prevent the formation of lamellipodia and pseudopodia. Detailed fine-structural studies of the junction between the invaginating endodermal-mesodermal mass and the overlying ectodermal surface, especially with the scanning electron microscope, are needed to see if lamellipodia are present here. The spreading behavior of cells in vitro may be a reflection of their spreading tendency in vivo [7]. We do not know what signals the onset of locomotion in amphibian embryos, but once set in motion, the tendency of cells to move awav from regions of frequent cell contact when they find a suitable substratum for cell movement could account for invagination. Analysis of films revealed several examples where an actively ruffling cell suddenly stopped, rounded up, divided, daughter cells flattened out again, and cells reformed ruffling lamellipodia. The blastula period in amphibian embryos is marked by rapid cell division. In blastula stage embryos, mitotic indices are high and decrease rapidly by the mid-gastrula stage [4, 141. The decrease in the rate of cell division may also be an important signal for the onset of
Motility
lamellipodium formation and consequently active cell locomotion. This work was supported by research grant HD-07082 from USPHS, Health Sciences Advancement Award 35S04RRO6148, and by agrant from the United Health Services of North Carolina. I would like to thank Dr S. J. Counce and Dr J. P. Trinkaus for reading the manuscript.
Note added in proof Monroy et al. (Dev biol 49 (1976) 250) have recently used the scanning electron microscope to show that newly invaginated chordamesodermal cells in gastrula stage Xenopus embryos are “. . . fibroblast-like with long and thin interwoven filopodia.” This observation suggests that ruffling may occur in vivo, since many of the processes seen extending from the cells may be lamellipodia.
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Exp Cell Res 101 (1976)