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Role of the substratum, supracellular continuity, and differential growth in morphogenetic cell movements
DEVELOPMENTAL
BIOLOGY,
7, 51-63
Role of the Substratum, Differential
(1963)
Supracellular
‘Growth Cell
Continuity,
and
in Morphogenetic
Movements’
NELSON T. SPRATT, JR.: Department
of Zoology, University of Minnesotu, Minneapolis 14, Minnesotu Accepted
September
18, 1962
“
some familiarity with the real phenomena that are to be explained is indispensable, for our supply of explanations for imaginary concepts of cell movement and cell interactions is already superabundant.” P. Weiss ( 1961) .
.
INTRODUCTION
A basic yet little understood phenomenon characteristic of the early development of most animal embryos is the morphogenetic streaming of layers or sheets of cells by which the germ layer architecture of the system is achieved. Since an exhaustive and systematic presentation of even our scant knowledge of the mechanisms underlying these cell movements is out of the question in this brief essay, we shall simply examine some of the evidence gleaned from recent studies of the early chick embryo in my laboratory for the seemingly important roles played by the substrata on which the cells move, by a supracellular (noncellular) continuum and by differential growth (localized, rapid cell proliferation). MATERIALS
AND
METHODS
With the exception of a few experiments in which the vitelline membrane of turkey and duck eggs was used as a substratum, all the results described below were obtained with unincubated chick ‘The research described in this paper has been supported in part by grants from the National Science Foundation. ’ Much of the experimental work on which this paper is based has been possible only through the expert assistance of my associate, Doctor Hermann Haas. To him I owe a debt of gratitude for his diligent and painstaking efforts. 51
52
SELSON
T. SPHATT,
JH.
blastoderms removed from freshly laid eggs and explanted in vitro to the surface of an agar-chick egg extract medium, to the surface of a polypore filter disk floating on a liquid chick egg extract medium or to a piece of chick vitelline membrane placed on the agar medium (modification of a method described by New, 1955 and 1959). In general, explantation, operative, carbon and carmine marking, and culture preparation procedures were like those previously described ( Spratt and Haas, 1960a,b,c, 1961a,b). RESULTS
AND
DISCUSSION
Pattern of Morphogenetic Cell Movements Examination of hundreds of living and sectioned unincubated chick blastoderms, along with observations of the movements of marked cell groups and extensive studies of the regulative and integrative
FIG. 1. Diagram of a longitudinal half pell = pellucid area; op = opaque area.
of an unincubated
chick
blastoderm.
BOLE
OF
THE
SUBSTRATUM,
SUPRACELLULAH
CONTINUITY
Fj3
mechanisms of explanted blastoderms (Spratt and Haas, 1960a,b,c, 1961a,b, 1962a) has made possible an analysis of the system into the several basic morphological components illustrated by the diagrams of Fig. 1. It should be noted that although the blastoderm at the time of egg laying is a population of approximately 60,000 cells, only the upper germ layer ( epiblast ) is complete ( = upper layer substratum). Completion of the middle and lower germ layers (mes:)blast and hypoblast) is accomplished by a pattern of cell movements indicated by the arrows in Fig. 1 and shown in lower surface view in Fig. 3A. Extensive descriptions of these cell movements have been previously reported (Spratt and Haas, 1960a,b, 1961a,b) and will thus not be reviewed here. Germ Layer Mozjements and Substrate Specificity The critical role played by the structural (and macromolecular) properties of the substratum in controlling the movement of multicellular sheets (tissues), as well as of single cells cultivated in &To, has long been recognized [see especially Weiss ( 1961) and numerous earlier papers (Rosenberg, 1960; Taylor, 1961) 1. What degree of specificity, if any, exists in the relation of the lower cells (including both prospective mesoderm and endoderm cells) to the substratum over which they move ? The normal substratum is the lower surface of the uppermost epithelial cell layer (Figs. 1 and 2’A’). Could the upper surface of the upper cell layer serve as a substratum for these morphogenetic movements? An answer to the above question was obtained by an experiment in which a transverse strip near the middle level of 36 unincubated, explanted blastoderms was cut out and inverted such that the lower cells of the strip were in contact with the surface of the agar-egg extract medium, the upper surface of the remainder of the blastoderm lying, as in control explants, next to the surface of the culture medium (Fig. 3C). Transverse bands of lower cells, one in the anterior and another in the posterior part of the blastoderm, were marked with carbon powder. A band of upper layer cells in the inverted strip was marked with carmine powder. Since explants are always covered with a thin fluid film, some of the carbon or carmine particles stick in the latter, others become more intimately stuck to the surfaces of the cells. In all 36 of the above explants the carbon-marked bands of lower
54
NELSON
T.
SPRATT,
JR.
cells (Fig. 3C) failed to continue movement in the typical pattern illustrated in Fig. 3A. The marked cells just posterior to the inverted strip were thus unable to move over the upper surface of the strip. Further evidence that the movements of the lower cell layers can only occur on the lower surface of the upper layer is provided by numerous observations that these movements always cease where no upper layer substratum is present as, for example, in certain isolated pieces of the blastoderm (Spratt and Haas, 196Oc, 1961a). Where upper layer substratum is present, movements continue to its edge (Fig. 3E), never beyond, as for example over an agar surface, and will resume only along with extension of the upper layer by regenerative growth (Fig. 3F) and later stages in regulative development of an isolated posterior half (Fig. 3E ) . These observations suggest a very specific substratum requirement for the movement of the middle and lower cells (hereafter referred to collectively as the lower cell group). Among several substrates tested, namely, agar, glass, inner or outer surface of the vitelline membrane, and lower or upper surface of the epiblast, movements of cells continued only over the lower surface of the epiblast. Perhaps this surface possesses a specific colloidal exudate or “ground mat” (Weiss, 1961 and earlier papers) making movements possible but certainly not orienting the cell movements since the collective and unified cell movement pattern can be made to traverse the substratum in any direction simply by changing the position of the growth center (Spratt and Haas, 1961a,b, 1962a). Integration
of Movements
by a Supracellular
Continuum
The role of an extracellular or cell product continuum (e.g., a “surface coat” or “surface gel layer”) in unifying the cell movements of gastrulation has been described at some length in amphibia by Holtfreter (1943) and in fish by Lewis (1949), Trinkaus (1951), and Devillers (1961). The presence and function of a similar continuum in early chick development has not been described in any detail to date. In fact, the presence of a surface coat in the chick blastoderm has been denied on the basis of electron microscopic studies (Balinsky and Walther, 1961). Evidence for the presence of and role played by an extracellular continuum in unifying the movements of the lower cell group was provided by the following experiments. Unincubated blastoderms
ROLE
OF THE
SUBSTRATUM,
SUPRACELLULAR
CONTINUITY
55
were explanted and marked on the lower surface with carbon powder in the positions, a, b, c, and d denoted in Fig. 2A,A’. As indicated in the diagram of a median longitudinal section, A’, some of the particles were stuck to the surfaces of lower cells forming the coherent, diskshaped posterior cell group, a; others were stuck to the surfaces of isolated groups of more anteriorly located cells, c; and many others were stuck in the fluid film between the groups of cells, b and d. To
0
10 Hrs.
Hrs.
-
d’
FIG. 2. Diagrams illustrating the role of the fluid continuum, F, in integrating the movement of nonadjacent lower cell groups and the posterior, coherent cell sheet. Stippling represents carbon-marked areas.
what extent the fluid film contains a “cellular exudate” (Weiss, 1961; Rosenberg, 1960) has not been determined. However, continuity of the subgerminal fluid (below the blastoderm in o~o ) and of the liquid phase of the culture medium (above the explant in &TO) with the underside of the blastoderm is incontestable. Even in hematoxylin-stained and sectioned blastoderms, a thin film can be seen adhering to and traversing spaces between groups of lower cells (indicated by the thin line in Fig. 2A’,B’). Results summarized in Fig. 2B,B’ are based on observations and recordings of cell movements in over 100 explanted blastoderms. In all explants which developed embryos later (about 95-100%) the midline distances between the two bands and groups of carbon par-
56
NELSON
T. SPRATT,
JR.
titles (positions a, h, c, and d) remained essentially unchanged during the first IO-15 hours of incubation (positions a’, b’, c’, and d’). Indeed, the parallel bands remained parallel until the anterior band reached the marginal zone where the particles (including those still embedded in the film) began the typical circumferential movement (Fig. 3A). The a band, of course, also eventually reaches the anterior and anterior-lateral marginal zone. The supracellular fluid continuum not only integrates the movements of nonadjacent cells, cell groups, and the coherent sheet of posterior cells into a single, fountainlike streaming pattern, but, as demonstrated below, is actually necessary for imparting motion to the isolated cells and cell groups.
Differential Growth As A Motive Force One of the simplest questions one may ask about morphogenetic movements is: “What makes the cells move?” The observations and experiments summarized in the diagrams of Fig. 3 are based upon the study of over 200 explanted blastoderms (cf. similar results reported earlier by Spratt and Haas, 1960a,b,c, 1961a,b) and constitute evidence for the important role played by a center of growth” (center in which cells are rapidly proliferated and from which they move out radially) eccentricalhy located within the rapidly growing marginal zone ring. More accurately (cf. Weiss, 1961), cell movements in the patterns under discussion should perhaps be described as dislocations and translocations of cells and cell sheets. Moreover, neither the results indicated in Fig. 3 nor any other observations or experiments, to date, require the postulation of chemotactic, galvanotactic, or mechanotactic (thigmotactic) mechanisms as motive or guiding forces in the germ layer-forming, cell movement patterns of the early chick blastoderm. Actually, the ability to control at will the ” Evidence for the reality of a center and marginal zone ring of differentially rapid cell proliferation is provided by numerous types of cell-marking exprriments and by the differential growth in vitro of isolated parts of the blastoderm (Spratt and Haas, 1960a,b,c, 1961a,b, 1962a). Thus, explanted parts containing the growth center or a segment of the marginal zone ring exhibit a strikingly greater rate of increase in cell number than do even larger parts not containing the growing zones. Furthermore, unpublished autoradiographic studies demonstrate a markedly greater incorporation of tritiated thymidine and uridine by cells of the growing zones, an observation indicative of a pattern of differential nucleic acid synthesis in the blastodenn.
ROLE
OF
THE
SUBSTRATUM,
SUPRACELLULAR
CONTINUITY
57
position of the anterior-posterior axis of the lower cell movement pattern, and hence that of the embryo body, by changing the position of the growth center (Spratt and Haas, 196Oc, 1961a,b, and especially, 1962a) makes any kind of mechanism involving the attraction of the cells highly improbable (cf. Abercrombie, 1961) .
D
+f+
..,._,;.:,w,,..j.~
MOVEMENT
,.
MOVi%NT bs),,
MOVEMENT
OPPOSITE DIRECTION
STOPS
CONTINUES
TO CUT EDGE
STOPS
FK. 3. Diagrams illustrating the consequence of interrupting the continuity between the growth center, 6, and the remainder of the unincubated blastoderm. LL = lower layer; UL = upper layer.
As indicated in Fig. 3, any procedure which interrupted the continuity between the growth center, G, and the remainder of the unincubated, explanted blastoderm stopped the lower cell movements in the portion separated from the growth center. Thus, in B, when the movements of cells from the growth center were mechanically blocked by the weight of large clumps of carmine powder covering the posterior half of the lower surface (lined area in the figure), a transverse band of carbon-marked cells and intercellular film in the lower surface of the anterior half failed to continue movement as it would in a control explant (Fig. 3A and Fig. 2). In Fig. 3C, as noted above, inversion of a transverse band of cells blocked the movement of carbon particles in the anterior band. In D, marked cells in an isolated anterior half of the blastoderm ceased moving almost immediately after cutting. When and only when a
58
NELSON
T. SPRATT,
JR.
new growth center, G, was initiated from the anterior or anterolateral marginal zone ring, F, did the marked cells resume movement, always away from the new growth center and toward the geometrical center of the reconstituted, circular shaped blastoderm (cf. Abercrombie, 1961, for examples of similar behavior in fibroblast explants). The fountainlike pattern of movement of the lower cell group is thus entirely dependent upon continuity between the cells and the cell-producing growth center. No evidence has been found for the existence of any autonomous, intrinsic, and directional movement properties in the lower cell group of either the intact or subdivided blastoderm. The above demonstration that the motive force for the translocation of the middle and lower germ layer cells resides in a center of cell production suggests the possibility that a similar mechanism may underlie the expansion of the blastoderm over the yolk. Indeed, recent studies, still in progress, have revealed that a striking feature of the first day of incubation of the chick blastoderm is a change in the geometric pattern of its growth, involving changes in the positions of differentially, rapidly growing regions and in the direction of growth. Description of these studies is beyond the scope of this report, but it is noteworthy that observations of cell movements in carbon- and carmine-marked blastoderms indicate that the motive force for outward movement of the edge of the opaque area resides in a subperipheral, cell-producing ring (Spratt and Haas, 1962b). Blustoderm
Expansion
and Substrate Specificity
We may now examine briefly the cell-substratum relation during growth of the opaque area and outward movement of its edge over the yolk mass and lower surface of the vitelline membrane. Studies of New (1959) and Bellairs and New (1962) indicate that an important and special relation exists between the inner surface of the vitelline membrane and the margin of the 24-36 hour incubated blastoderm. In general, the observations described below extend and confirm the conclusions of New and Bellairs but raise some interesting problems in respect to the nature and degree of specificity in the relation under discussion. The problem of substrate specificity was approached by explanting unincubated chick blastoderms, lower surface up, to various types of substrates (Fig. 4). The approximate rate of growth in area of the
ROLE
OF
THE
SUBSTRATUM,
SUPRACELLULAR
CONTINUITY
59
CHICK INNER SURFACE
(20’1
/
P
CHICK INNE TRYPSIN 9 ZED SUliFAiE
J/.0-
0 0
I IO
I 20 HOURS
/
I 30 IN VITRO
-4’
I 40
P’
P,“Z3 SURFACE
I 50
FIG. 4. Increase in area and outward movement of the edge of unincubated blastoderms explanted on various substrates in uitro. “Inner surface” or “outer surface” refers to that of the vitelline membrane.
blastoderm was obtained from measurements of camera-lucida tracings. Aside from the striking acceleration in the rate of blastoderm expansion around 20 hours of incubation (i.e., after the definitive primitive streak stage) it is clear that the inner surface of the vitelline membrane of the unincubated chick egg (the normal substratum)
60
NELSON
T. SPRATT,
JR.
supported the most rapid expansion. The inner surface of the 48-hour vitelline membrane appears slightly less effective in supporting expansion, an observation that may be significant in view of studies in progress involving successive explantation of unincubated blastoderms to the same area of a piece of vitelline membrane in vitro. The suggestion is that the blastoderm cells may in some way change the nature of the substratum as they move over it. Shalumovich (1960) has concluded that nucleic acids and nucleoproteins in the membrane are utilized by the blastoderm cells, but it is far from certain, in view of the results described below, that this is actually true or that specific nutrients, if present in the membrane, are a significant feature of its expansion promoting nature. Might it not be equally probable that specific structural properties of the lower surface of the membrane are just as, or even more important? Particularly interesting is the observation that the surface of a pol!/pore filter disk-l floating in a liquid egg extract medium supported a rate of expansion of the unincubated blastoderm almost equivalent to that of the normal, lower vitelline membrane surface. This rather surprising discovery thus seems to minimize the importance of any special nutrients in the lower surface of the vitelline membrane and maximizes the importance of the structure of this surface-a structure presumably rather closely simulated by the network of polyconnected cellulose ester filaments of the polypore filter but altered by trypsinization (1% for 3 hours) and absent from the outer surface of the vitelline membrane and from an agar surface (Fig. 4). The significant difference in chick blastoderm expansion supporting properties of the inner surface of the unincubated turkey membrane and that of the unincubated duck membrane (Fig. 4) may well reside in structural differences, and it will have been fortuitous if further studies (including electron microscopic examination of the membranes of different species by Mrs. Cynthia Jensen) reveal a closer structural similarity between the surface of the type of polypore filter used here and the inner surface of the chick membrane than between the latter and either the turkey or duck membrane. Yet, some degree of structural specificity that may be taxonomically significant is certainly suggested by the experiments, and it does not now seem improbable that additional artificial surfaces, simulating the natural, may soon be ’ Gelman Instrument Co., Chelsea, Michigan. 0.45 k 0.02 p was used in these experiments.
Type AM-6
with
n pore size of
ROLE
OF
THE
SUBSTRATUM,
SUPRACELLULAR
CONTINUITY
found, i.e., porous and fibrous surfaces approximating ture of the lower surface of the vitelline membrane.
61
the architec-
SUMMARY AND CONCLUSIONS I hope that my attempt to describe some of the real phenomena of morphogenetic cell movements in the early chick blastoderm constitutes a degree of compliance with the admonition of Doctor Paul Weiss with which this essay was introduced. I am convinced that some of the real phenomena have been at least identified. In taking stock of our knowledge, it appears highly probable that special structural (mechanical) properties of the cellular or extracellular substrates over which cells move during germ layer formation and expansion are important to this growth and movement. However, little can be concluded as to the detailed nature of the specificity in the cell-substratum relation. A prime feature of the type of cell movements described above is that they consist in large part of a dislocation or translocationpassive as regards the majority of the cells whether components of a coherent cell sheet (tissue) or as members of scattered groups of cells. There is rather convincing evidence that the motive force behind these translocations resides in the cell-producing activities of localized growth regions, e.g., the growth center and marginal zone ring. It is interesting, nevertheless, that the cells or cell sheets can be “pushed” only over an “appropriate” substratum, e.g., one permitting cells to attach and spread. Consideration was also given to the role played by the noncellular continuum in integrating (actually guiding) the movements of nonadjacent cells into the single, fountainlike pattern in which many of the cells of the lower cell group participate. Although the exact nature of this integrating continuum is not known, perhaps the most important conclusion to be drawn is that the patterns of morphogenetic cell movements have relatively simple explanations in mechanical terms, i.e., obey principles of fluid flow. There seems to be little reason, at least for the present, to conjure up biochemical mechanisms, e.g., chemotaxes, as the agents immediately directing the collective movements of cells in the blastoderm population. This is certainly not to deny the important role of biochemical gradients or differentials as environmental agents controlling many other cellular activities, for example, growth, cytodifferentiation.
62
NELSON T. SPRATT, JH.
REFERENCES ABEHCROMBIE, M. ( 1961). The basis of the locomotory behavior of fibroblasts. Exptl. Cell Research, Suppl. 8, 188-198. BALINSKY, B. I., and WALTHER, H. ( 1961). The immigration of presumptive mesoblast from the primitive streak in the chick as studied with the electron microscope. Acta Embryol. Morphol. Exptl. 4, 261-283. BELLAIRS, R., and NEW, D. A. T. (1962). Phagocytosis in the chick blastoderm. Exptl. Cell Research 26, 275-279. DEVILLERS, CH. ( 1961). Mouvements cellulaires dans le developpement de l’embryon des amphibiens et des poissons. Exptl. Cell Research, Suppl. 8, 221-233. HOLTFRETER, J. ( 1943). Properties and functions of the surface coat in amphibian embryos. J. Exptl. Zool. 93, 251-323. LEWIS, W. H. (1949). Gel layers of cells and eggs and their role in early development. Lecture Ser. Roscoe B. Jackson Mem. Lab., pp. 59-77. NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. .l. Embryol. Exptl. Morphol. 5, 326-331. NEW, D. A. T. ( 1959). The adhesive properties and expansion of the chick blastoderm. J. Embryol. Exptl. Morphol. 7, 146-164. ROSENBERG, M. D. ( 1960). Microexudates from cells grown in tissue culture. Biophys. J. 1, 137-159. SHALUMOVICH, V. N. (1960). The origin and fate of the nucleic acids in the vitelline membrane of the chick egg. Doklady Akad. Nauk S. S. S. R. 130, 11261129 ( in Russian ) . SPRATT, N. T., JR., and HAAS, H. (1960a). Morphogenetic movements in the lower surface of the unincubated and early chick blastoderm. J. Exptl. 2001. 144, 139-157. SPHATT, N. T., JR., and HAAS, H. (1960b). Importance of morphogenetic movements in the lower surface of the young chick blastoderm. J. Exptl. Zool. 144, 257-275. SPRATT, N. T., JR., and HAAS, H. (196Oc). Integrative mechanisms in development of the early chick blastoderm. I. Regulative potentiality of separated parts. J. Exptl. Zool. 145, 97-137. SPRATT, N. T., JR., and HAAS, H. (1961a). Integrative mechanisms in development of the early chick blastoderm. II. Role of morphogenetic movements and regenerative growth in synthetic and topographically disarranged blastoderms. J. Exptl. Zool. 147, 57-93. SPRATT, N. T., JR., and HAAS, H. (196lb). Integrative mechanisms in development of the early chick blastoderm. 111. Role of cell population size and growth potentiality in synthetic systems larger than normal. J. Exptl. 2001. 147, 271-294. SPRATT, N. T., JR., and HAAS, H. (1962a). Integrative mechanisms in development of the early chick blastoderm. IV. Synthetic systems composed of parts of different developmental age. Synchronization of development rates. J. Exptl. 2001. 149, 75-102. SPRATT, N. T., JR., and HAAS, H. (196213). Unpublished studies.
ROLE
OF
THE
SUBSTRATUM,
SUPRACELLULAR
CONTINUITY
63
TAYLOR, A. C. ( 1961). Attachment and spreading of cells in culture. Exptl. Cell Research, Suppl. 8, 154-173. TRINJCAUS, J. P. ( 1951). A study of the mechanism of epiboly in the egg of Fund&s heteroclitus. J. Exptl. Zool. 118, 269-319. WEISS, P. ( 1961). Guiding principles in cell locomotion and cell aggregation. Exptl. Cell Research, Suppl. 8, 260-281.
BIOLOGY,
7, 51-63
Role of the Substratum, Differential
(1963)
Supracellular
‘Growth Cell
Continuity,
and
in Morphogenetic
Movements’
NELSON T. SPRATT, JR.: Department
of Zoology, University of Minnesotu, Minneapolis 14, Minnesotu Accepted
September
18, 1962
“
some familiarity with the real phenomena that are to be explained is indispensable, for our supply of explanations for imaginary concepts of cell movement and cell interactions is already superabundant.” P. Weiss ( 1961) .
.
INTRODUCTION
A basic yet little understood phenomenon characteristic of the early development of most animal embryos is the morphogenetic streaming of layers or sheets of cells by which the germ layer architecture of the system is achieved. Since an exhaustive and systematic presentation of even our scant knowledge of the mechanisms underlying these cell movements is out of the question in this brief essay, we shall simply examine some of the evidence gleaned from recent studies of the early chick embryo in my laboratory for the seemingly important roles played by the substrata on which the cells move, by a supracellular (noncellular) continuum and by differential growth (localized, rapid cell proliferation). MATERIALS
AND
METHODS
With the exception of a few experiments in which the vitelline membrane of turkey and duck eggs was used as a substratum, all the results described below were obtained with unincubated chick ‘The research described in this paper has been supported in part by grants from the National Science Foundation. ’ Much of the experimental work on which this paper is based has been possible only through the expert assistance of my associate, Doctor Hermann Haas. To him I owe a debt of gratitude for his diligent and painstaking efforts. 51
52
SELSON
T. SPHATT,
JH.
blastoderms removed from freshly laid eggs and explanted in vitro to the surface of an agar-chick egg extract medium, to the surface of a polypore filter disk floating on a liquid chick egg extract medium or to a piece of chick vitelline membrane placed on the agar medium (modification of a method described by New, 1955 and 1959). In general, explantation, operative, carbon and carmine marking, and culture preparation procedures were like those previously described ( Spratt and Haas, 1960a,b,c, 1961a,b). RESULTS
AND
DISCUSSION
Pattern of Morphogenetic Cell Movements Examination of hundreds of living and sectioned unincubated chick blastoderms, along with observations of the movements of marked cell groups and extensive studies of the regulative and integrative
FIG. 1. Diagram of a longitudinal half pell = pellucid area; op = opaque area.
of an unincubated
chick
blastoderm.
BOLE
OF
THE
SUBSTRATUM,
SUPRACELLULAH
CONTINUITY
Fj3
mechanisms of explanted blastoderms (Spratt and Haas, 1960a,b,c, 1961a,b, 1962a) has made possible an analysis of the system into the several basic morphological components illustrated by the diagrams of Fig. 1. It should be noted that although the blastoderm at the time of egg laying is a population of approximately 60,000 cells, only the upper germ layer ( epiblast ) is complete ( = upper layer substratum). Completion of the middle and lower germ layers (mes:)blast and hypoblast) is accomplished by a pattern of cell movements indicated by the arrows in Fig. 1 and shown in lower surface view in Fig. 3A. Extensive descriptions of these cell movements have been previously reported (Spratt and Haas, 1960a,b, 1961a,b) and will thus not be reviewed here. Germ Layer Mozjements and Substrate Specificity The critical role played by the structural (and macromolecular) properties of the substratum in controlling the movement of multicellular sheets (tissues), as well as of single cells cultivated in &To, has long been recognized [see especially Weiss ( 1961) and numerous earlier papers (Rosenberg, 1960; Taylor, 1961) 1. What degree of specificity, if any, exists in the relation of the lower cells (including both prospective mesoderm and endoderm cells) to the substratum over which they move ? The normal substratum is the lower surface of the uppermost epithelial cell layer (Figs. 1 and 2’A’). Could the upper surface of the upper cell layer serve as a substratum for these morphogenetic movements? An answer to the above question was obtained by an experiment in which a transverse strip near the middle level of 36 unincubated, explanted blastoderms was cut out and inverted such that the lower cells of the strip were in contact with the surface of the agar-egg extract medium, the upper surface of the remainder of the blastoderm lying, as in control explants, next to the surface of the culture medium (Fig. 3C). Transverse bands of lower cells, one in the anterior and another in the posterior part of the blastoderm, were marked with carbon powder. A band of upper layer cells in the inverted strip was marked with carmine powder. Since explants are always covered with a thin fluid film, some of the carbon or carmine particles stick in the latter, others become more intimately stuck to the surfaces of the cells. In all 36 of the above explants the carbon-marked bands of lower
54
NELSON
T.
SPRATT,
JR.
cells (Fig. 3C) failed to continue movement in the typical pattern illustrated in Fig. 3A. The marked cells just posterior to the inverted strip were thus unable to move over the upper surface of the strip. Further evidence that the movements of the lower cell layers can only occur on the lower surface of the upper layer is provided by numerous observations that these movements always cease where no upper layer substratum is present as, for example, in certain isolated pieces of the blastoderm (Spratt and Haas, 196Oc, 1961a). Where upper layer substratum is present, movements continue to its edge (Fig. 3E), never beyond, as for example over an agar surface, and will resume only along with extension of the upper layer by regenerative growth (Fig. 3F) and later stages in regulative development of an isolated posterior half (Fig. 3E ) . These observations suggest a very specific substratum requirement for the movement of the middle and lower cells (hereafter referred to collectively as the lower cell group). Among several substrates tested, namely, agar, glass, inner or outer surface of the vitelline membrane, and lower or upper surface of the epiblast, movements of cells continued only over the lower surface of the epiblast. Perhaps this surface possesses a specific colloidal exudate or “ground mat” (Weiss, 1961 and earlier papers) making movements possible but certainly not orienting the cell movements since the collective and unified cell movement pattern can be made to traverse the substratum in any direction simply by changing the position of the growth center (Spratt and Haas, 1961a,b, 1962a). Integration
of Movements
by a Supracellular
Continuum
The role of an extracellular or cell product continuum (e.g., a “surface coat” or “surface gel layer”) in unifying the cell movements of gastrulation has been described at some length in amphibia by Holtfreter (1943) and in fish by Lewis (1949), Trinkaus (1951), and Devillers (1961). The presence and function of a similar continuum in early chick development has not been described in any detail to date. In fact, the presence of a surface coat in the chick blastoderm has been denied on the basis of electron microscopic studies (Balinsky and Walther, 1961). Evidence for the presence of and role played by an extracellular continuum in unifying the movements of the lower cell group was provided by the following experiments. Unincubated blastoderms
ROLE
OF THE
SUBSTRATUM,
SUPRACELLULAR
CONTINUITY
55
were explanted and marked on the lower surface with carbon powder in the positions, a, b, c, and d denoted in Fig. 2A,A’. As indicated in the diagram of a median longitudinal section, A’, some of the particles were stuck to the surfaces of lower cells forming the coherent, diskshaped posterior cell group, a; others were stuck to the surfaces of isolated groups of more anteriorly located cells, c; and many others were stuck in the fluid film between the groups of cells, b and d. To
0
10 Hrs.
Hrs.
-
d’
FIG. 2. Diagrams illustrating the role of the fluid continuum, F, in integrating the movement of nonadjacent lower cell groups and the posterior, coherent cell sheet. Stippling represents carbon-marked areas.
what extent the fluid film contains a “cellular exudate” (Weiss, 1961; Rosenberg, 1960) has not been determined. However, continuity of the subgerminal fluid (below the blastoderm in o~o ) and of the liquid phase of the culture medium (above the explant in &TO) with the underside of the blastoderm is incontestable. Even in hematoxylin-stained and sectioned blastoderms, a thin film can be seen adhering to and traversing spaces between groups of lower cells (indicated by the thin line in Fig. 2A’,B’). Results summarized in Fig. 2B,B’ are based on observations and recordings of cell movements in over 100 explanted blastoderms. In all explants which developed embryos later (about 95-100%) the midline distances between the two bands and groups of carbon par-
56
NELSON
T. SPRATT,
JR.
titles (positions a, h, c, and d) remained essentially unchanged during the first IO-15 hours of incubation (positions a’, b’, c’, and d’). Indeed, the parallel bands remained parallel until the anterior band reached the marginal zone where the particles (including those still embedded in the film) began the typical circumferential movement (Fig. 3A). The a band, of course, also eventually reaches the anterior and anterior-lateral marginal zone. The supracellular fluid continuum not only integrates the movements of nonadjacent cells, cell groups, and the coherent sheet of posterior cells into a single, fountainlike streaming pattern, but, as demonstrated below, is actually necessary for imparting motion to the isolated cells and cell groups.
Differential Growth As A Motive Force One of the simplest questions one may ask about morphogenetic movements is: “What makes the cells move?” The observations and experiments summarized in the diagrams of Fig. 3 are based upon the study of over 200 explanted blastoderms (cf. similar results reported earlier by Spratt and Haas, 1960a,b,c, 1961a,b) and constitute evidence for the important role played by a center of growth” (center in which cells are rapidly proliferated and from which they move out radially) eccentricalhy located within the rapidly growing marginal zone ring. More accurately (cf. Weiss, 1961), cell movements in the patterns under discussion should perhaps be described as dislocations and translocations of cells and cell sheets. Moreover, neither the results indicated in Fig. 3 nor any other observations or experiments, to date, require the postulation of chemotactic, galvanotactic, or mechanotactic (thigmotactic) mechanisms as motive or guiding forces in the germ layer-forming, cell movement patterns of the early chick blastoderm. Actually, the ability to control at will the ” Evidence for the reality of a center and marginal zone ring of differentially rapid cell proliferation is provided by numerous types of cell-marking exprriments and by the differential growth in vitro of isolated parts of the blastoderm (Spratt and Haas, 1960a,b,c, 1961a,b, 1962a). Thus, explanted parts containing the growth center or a segment of the marginal zone ring exhibit a strikingly greater rate of increase in cell number than do even larger parts not containing the growing zones. Furthermore, unpublished autoradiographic studies demonstrate a markedly greater incorporation of tritiated thymidine and uridine by cells of the growing zones, an observation indicative of a pattern of differential nucleic acid synthesis in the blastodenn.
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57
position of the anterior-posterior axis of the lower cell movement pattern, and hence that of the embryo body, by changing the position of the growth center (Spratt and Haas, 196Oc, 1961a,b, and especially, 1962a) makes any kind of mechanism involving the attraction of the cells highly improbable (cf. Abercrombie, 1961) .
D
+f+
..,._,;.:,w,,..j.~
MOVEMENT
,.
MOVi%NT bs),,
MOVEMENT
OPPOSITE DIRECTION
STOPS
CONTINUES
TO CUT EDGE
STOPS
FK. 3. Diagrams illustrating the consequence of interrupting the continuity between the growth center, 6, and the remainder of the unincubated blastoderm. LL = lower layer; UL = upper layer.
As indicated in Fig. 3, any procedure which interrupted the continuity between the growth center, G, and the remainder of the unincubated, explanted blastoderm stopped the lower cell movements in the portion separated from the growth center. Thus, in B, when the movements of cells from the growth center were mechanically blocked by the weight of large clumps of carmine powder covering the posterior half of the lower surface (lined area in the figure), a transverse band of carbon-marked cells and intercellular film in the lower surface of the anterior half failed to continue movement as it would in a control explant (Fig. 3A and Fig. 2). In Fig. 3C, as noted above, inversion of a transverse band of cells blocked the movement of carbon particles in the anterior band. In D, marked cells in an isolated anterior half of the blastoderm ceased moving almost immediately after cutting. When and only when a
58
NELSON
T. SPRATT,
JR.
new growth center, G, was initiated from the anterior or anterolateral marginal zone ring, F, did the marked cells resume movement, always away from the new growth center and toward the geometrical center of the reconstituted, circular shaped blastoderm (cf. Abercrombie, 1961, for examples of similar behavior in fibroblast explants). The fountainlike pattern of movement of the lower cell group is thus entirely dependent upon continuity between the cells and the cell-producing growth center. No evidence has been found for the existence of any autonomous, intrinsic, and directional movement properties in the lower cell group of either the intact or subdivided blastoderm. The above demonstration that the motive force for the translocation of the middle and lower germ layer cells resides in a center of cell production suggests the possibility that a similar mechanism may underlie the expansion of the blastoderm over the yolk. Indeed, recent studies, still in progress, have revealed that a striking feature of the first day of incubation of the chick blastoderm is a change in the geometric pattern of its growth, involving changes in the positions of differentially, rapidly growing regions and in the direction of growth. Description of these studies is beyond the scope of this report, but it is noteworthy that observations of cell movements in carbon- and carmine-marked blastoderms indicate that the motive force for outward movement of the edge of the opaque area resides in a subperipheral, cell-producing ring (Spratt and Haas, 1962b). Blustoderm
Expansion
and Substrate Specificity
We may now examine briefly the cell-substratum relation during growth of the opaque area and outward movement of its edge over the yolk mass and lower surface of the vitelline membrane. Studies of New (1959) and Bellairs and New (1962) indicate that an important and special relation exists between the inner surface of the vitelline membrane and the margin of the 24-36 hour incubated blastoderm. In general, the observations described below extend and confirm the conclusions of New and Bellairs but raise some interesting problems in respect to the nature and degree of specificity in the relation under discussion. The problem of substrate specificity was approached by explanting unincubated chick blastoderms, lower surface up, to various types of substrates (Fig. 4). The approximate rate of growth in area of the
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CHICK INNER SURFACE
(20’1
/
P
CHICK INNE TRYPSIN 9 ZED SUliFAiE
J/.0-
0 0
I IO
I 20 HOURS
/
I 30 IN VITRO
-4’
I 40
P’
P,“Z3 SURFACE
I 50
FIG. 4. Increase in area and outward movement of the edge of unincubated blastoderms explanted on various substrates in uitro. “Inner surface” or “outer surface” refers to that of the vitelline membrane.
blastoderm was obtained from measurements of camera-lucida tracings. Aside from the striking acceleration in the rate of blastoderm expansion around 20 hours of incubation (i.e., after the definitive primitive streak stage) it is clear that the inner surface of the vitelline membrane of the unincubated chick egg (the normal substratum)
60
NELSON
T. SPRATT,
JR.
supported the most rapid expansion. The inner surface of the 48-hour vitelline membrane appears slightly less effective in supporting expansion, an observation that may be significant in view of studies in progress involving successive explantation of unincubated blastoderms to the same area of a piece of vitelline membrane in vitro. The suggestion is that the blastoderm cells may in some way change the nature of the substratum as they move over it. Shalumovich (1960) has concluded that nucleic acids and nucleoproteins in the membrane are utilized by the blastoderm cells, but it is far from certain, in view of the results described below, that this is actually true or that specific nutrients, if present in the membrane, are a significant feature of its expansion promoting nature. Might it not be equally probable that specific structural properties of the lower surface of the membrane are just as, or even more important? Particularly interesting is the observation that the surface of a pol!/pore filter disk-l floating in a liquid egg extract medium supported a rate of expansion of the unincubated blastoderm almost equivalent to that of the normal, lower vitelline membrane surface. This rather surprising discovery thus seems to minimize the importance of any special nutrients in the lower surface of the vitelline membrane and maximizes the importance of the structure of this surface-a structure presumably rather closely simulated by the network of polyconnected cellulose ester filaments of the polypore filter but altered by trypsinization (1% for 3 hours) and absent from the outer surface of the vitelline membrane and from an agar surface (Fig. 4). The significant difference in chick blastoderm expansion supporting properties of the inner surface of the unincubated turkey membrane and that of the unincubated duck membrane (Fig. 4) may well reside in structural differences, and it will have been fortuitous if further studies (including electron microscopic examination of the membranes of different species by Mrs. Cynthia Jensen) reveal a closer structural similarity between the surface of the type of polypore filter used here and the inner surface of the chick membrane than between the latter and either the turkey or duck membrane. Yet, some degree of structural specificity that may be taxonomically significant is certainly suggested by the experiments, and it does not now seem improbable that additional artificial surfaces, simulating the natural, may soon be ’ Gelman Instrument Co., Chelsea, Michigan. 0.45 k 0.02 p was used in these experiments.
Type AM-6
with
n pore size of
ROLE
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SUBSTRATUM,
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found, i.e., porous and fibrous surfaces approximating ture of the lower surface of the vitelline membrane.
61
the architec-
SUMMARY AND CONCLUSIONS I hope that my attempt to describe some of the real phenomena of morphogenetic cell movements in the early chick blastoderm constitutes a degree of compliance with the admonition of Doctor Paul Weiss with which this essay was introduced. I am convinced that some of the real phenomena have been at least identified. In taking stock of our knowledge, it appears highly probable that special structural (mechanical) properties of the cellular or extracellular substrates over which cells move during germ layer formation and expansion are important to this growth and movement. However, little can be concluded as to the detailed nature of the specificity in the cell-substratum relation. A prime feature of the type of cell movements described above is that they consist in large part of a dislocation or translocationpassive as regards the majority of the cells whether components of a coherent cell sheet (tissue) or as members of scattered groups of cells. There is rather convincing evidence that the motive force behind these translocations resides in the cell-producing activities of localized growth regions, e.g., the growth center and marginal zone ring. It is interesting, nevertheless, that the cells or cell sheets can be “pushed” only over an “appropriate” substratum, e.g., one permitting cells to attach and spread. Consideration was also given to the role played by the noncellular continuum in integrating (actually guiding) the movements of nonadjacent cells into the single, fountainlike pattern in which many of the cells of the lower cell group participate. Although the exact nature of this integrating continuum is not known, perhaps the most important conclusion to be drawn is that the patterns of morphogenetic cell movements have relatively simple explanations in mechanical terms, i.e., obey principles of fluid flow. There seems to be little reason, at least for the present, to conjure up biochemical mechanisms, e.g., chemotaxes, as the agents immediately directing the collective movements of cells in the blastoderm population. This is certainly not to deny the important role of biochemical gradients or differentials as environmental agents controlling many other cellular activities, for example, growth, cytodifferentiation.
62
NELSON T. SPRATT, JH.
REFERENCES ABEHCROMBIE, M. ( 1961). The basis of the locomotory behavior of fibroblasts. Exptl. Cell Research, Suppl. 8, 188-198. BALINSKY, B. I., and WALTHER, H. ( 1961). The immigration of presumptive mesoblast from the primitive streak in the chick as studied with the electron microscope. Acta Embryol. Morphol. Exptl. 4, 261-283. BELLAIRS, R., and NEW, D. A. T. (1962). Phagocytosis in the chick blastoderm. Exptl. Cell Research 26, 275-279. DEVILLERS, CH. ( 1961). Mouvements cellulaires dans le developpement de l’embryon des amphibiens et des poissons. Exptl. Cell Research, Suppl. 8, 221-233. HOLTFRETER, J. ( 1943). Properties and functions of the surface coat in amphibian embryos. J. Exptl. Zool. 93, 251-323. LEWIS, W. H. (1949). Gel layers of cells and eggs and their role in early development. Lecture Ser. Roscoe B. Jackson Mem. Lab., pp. 59-77. NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. .l. Embryol. Exptl. Morphol. 5, 326-331. NEW, D. A. T. ( 1959). The adhesive properties and expansion of the chick blastoderm. J. Embryol. Exptl. Morphol. 7, 146-164. ROSENBERG, M. D. ( 1960). Microexudates from cells grown in tissue culture. Biophys. J. 1, 137-159. SHALUMOVICH, V. N. (1960). The origin and fate of the nucleic acids in the vitelline membrane of the chick egg. Doklady Akad. Nauk S. S. S. R. 130, 11261129 ( in Russian ) . SPRATT, N. T., JR., and HAAS, H. (1960a). Morphogenetic movements in the lower surface of the unincubated and early chick blastoderm. J. Exptl. 2001. 144, 139-157. SPHATT, N. T., JR., and HAAS, H. (1960b). Importance of morphogenetic movements in the lower surface of the young chick blastoderm. J. Exptl. Zool. 144, 257-275. SPRATT, N. T., JR., and HAAS, H. (196Oc). Integrative mechanisms in development of the early chick blastoderm. I. Regulative potentiality of separated parts. J. Exptl. Zool. 145, 97-137. SPRATT, N. T., JR., and HAAS, H. (1961a). Integrative mechanisms in development of the early chick blastoderm. II. Role of morphogenetic movements and regenerative growth in synthetic and topographically disarranged blastoderms. J. Exptl. Zool. 147, 57-93. SPRATT, N. T., JR., and HAAS, H. (196lb). Integrative mechanisms in development of the early chick blastoderm. 111. Role of cell population size and growth potentiality in synthetic systems larger than normal. J. Exptl. 2001. 147, 271-294. SPRATT, N. T., JR., and HAAS, H. (1962a). Integrative mechanisms in development of the early chick blastoderm. IV. Synthetic systems composed of parts of different developmental age. Synchronization of development rates. J. Exptl. 2001. 149, 75-102. SPRATT, N. T., JR., and HAAS, H. (196213). Unpublished studies.
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TAYLOR, A. C. ( 1961). Attachment and spreading of cells in culture. Exptl. Cell Research, Suppl. 8, 154-173. TRINJCAUS, J. P. ( 1951). A study of the mechanism of epiboly in the egg of Fund&s heteroclitus. J. Exptl. Zool. 118, 269-319. WEISS, P. ( 1961). Guiding principles in cell locomotion and cell aggregation. Exptl. Cell Research, Suppl. 8, 260-281.