While I suspect that intercellular communication by propagated signals may be common indeveloping systems, there is no hard evidence except for the cellular slime molds, whose phylogcnetic position is ambiguous. Indeed, it is difficult to avoid the feeling that the slime moldsrepresent a cul-de-sac in evolution. Nonetheless, their development is intriguing in its own right, and there is good precedent for the usefulness ofunderstanding thoroughly a process in a simpleorganism, even though it appears exceptional. There is fortunately no doubt that slime moldsare Eucaryotes and that the majority of species possess a multicellular stage in their developmental cycle!
With this apology I shall devote the bulk of my article to the development of Dictyostelium discoideum and in particular to an analysis of intercellular communication during its development. At the end I shall try to put this analysis into the wider context of intercellular communication indeveloping embryos.
D.discoideum was discovered by Raper (1935), and his description of its morphogenesis and of experiments he performed is a classic paper (Raper, 1940). In the intervening years the popularity of D.discoideum has grown dramatically. While it was possible for Bonner (1967) to refer to all published work on D.discoideumin his book, that would now be beyond the scope of any one author. Early work was concentrated on descriptions and investigations of morphology and differentiation at or above the cellular level. More recent publications show a heavy bias toward biochemistry and the molecular biology of differentiation. Extensive reviews have been published by Shaffer (1962), Bonner (1967), Ashworth (1971), Robertson and Cohen (1972), Newell (1971), Sussman and Sussman (1969), Wright (1973), and Olive (1975), among others.
The single most provocative discovery was the probable identification of cyclic adenosine monophosphate (c-Amp) as the "Acrasin" or chemotactic attractant for aggregation in D. discoideum (Konijn et al., 1968).
Copyright © 1977 by Anthony Robertson.
Life Cycle of D. discoideum
D. discoideum amoebae hatch from elliptical spores about 6µ long, which germinate in moist places in the soil and on decaying vegetation (Cotter and Raper, 1966, 1968a, 1968b). The amoebae feed on bacteria and other microorganisms, which they find by Chemotaxis (Potts, 1902; Bonner, et al., 1970). Recently strains that will grow axenically have been produced. As long as food is available they divide about every four hours, somewhat less frequently in axenic media. When the food is exhausted the amoebae enter a period of differentiation called interphase (Bonner, 1963), which lasts for nine hours (Gerisch, 1968; Cohen and Robertson, 1972). During interphase the amoebae develop the competences that are required for aggregation, including the ability to respond to an extracellular signal, which is probably cyclic adenosine monophosphate (C-AMP) by Chemotaxis (Konijn et al., 1968); the ability to relay a pulsatile signal ofC-AMP when stimulated by a suprathreshold extracellular C-AMP concentration (Robertson, Drage, and Cohen, 1972); the ability to form EDTA stable intercellular contacts (DeHaan, 1959); and the ability, for a small proportion of the cells, to release periodic pulses of C-AMP autonomously (Cohen and Robertson, 1972). Development of each of these competences must involve changes in many cellular properties, in particular in the cell membrane. Such changes are being investigated by many workers (see Newell, 1971, for a review).
At the end of interphase some amoebae begin to release pulses of C-AMP autonomously. Their neighbors are competent to respond to autonomous signals by Chemotaxis and by signal relaying. Each signal can travel only a limited distance because its amplitude is reduced by both diffusion and the action of an extracellular phosphodiesterase (PDE) (Chang, 1968). Just as there is a threshold concentration for signal relaying, there is also a critical density of amoebae below which a signal cannot be propagated (Cohen and Robertson, 1971a). This density corresponds to a distance of approximately 70µ between amoeba centers. After receiving a signal, an amoeba becomes refractory to further stimulation (Shaffer, 1957; Gerisch, 1968; Cohen and Robertson, 1971a). The refractory period is a function of age, decreasing from about nine or ten minutes at the beginning of aggregation to approximately two minutes within four hours. The refractory period ensures that signals propagate unisensally from a center and that a territorial boundary will form between neighboring centers as colliding signals annihilate each other. These properties of the system lead to the outward propagation of periodic waves of C-AMP concentration from centers, marked by inward waves of cell movement (Gerisch, 1968; Cohen and Robertson, 1971a, 1971b; Robertson, 1974).
Because each cell relays the signal, amoebae outside a center tend to move toward their nearest central neighbors, forming streams radiating from the center (Shaffer, 1957; Cohen and Robertson, 1972; Nanjundiah, 1973). An early aggregate therefore consists of a central mass of cells with randomly branching streams leading away from it. During the later stages of aggregation the central cell mass develops a nipple shaped tip (Raper, 1940; Robertson, 1972). Cells within the tip remain there during the rest of morphogenesis (Takeuchi, 1969; Farnsworth, 1973). In late aggregation, cells in the aggregate secrete a mucopolysaccaride slime, which is liquid at the highest, most central region of the aggregate, but hardens as it flows downward (Shaffer, 1965). As cells are still entering the aggregate within streams, and as the slime is liquid only at the center, pressure in the center can be relieved only by the erection of a cylindrical column, bearing the tip and covered in slime. It grows until it becomes unstable and falls over to form the slug, or migrating pseudoplasmodium. Cells within the slug are joined by antero-post- erior contacts and by lateral membrane inter- digitations. They migrate as a unit within the slime sheath, which remains stationary with respect to the substrate and collapses behind the slug. Raper showed that the tip controls slug migration (Raper, 1940) and that extra tips grafted onto the side of a slug will take over cells posterior to the graft site. He concluded that the tips acted as though they were organizers, controlling cell movement and defining the developmental axis of the slug.
The distance that the slug migrates depends on environmental conditions (Raper, 1940; Newell, Telser, and Sussman, 1969). At the end of migration the cell mass rounds up and rotates so that the tip again moves to the top. At this stage the cells in the anterior third of the slug have become "pre-stalk" cells, showing histological and biochemical changes, while those in the posterior two thirds are "pre-spore" cells, again with a characteristic histology (Bonner, Chiquoine, and Kolderie, 1955; Gregg, 1965; Gregg and Badman, 1970; Hohl and Hamamoto, 1969; Maeda and Takeuchi, 1969) that has been developing since late aggregation. Their axial organization is retained on rotation of the cell mass. The tip is therefore on top of a mass of pre-stalk cells, which is on top of the pre-spore cells. Formation of the fruiting body ensues. Raper and Fennell (1952) have described this process in great detail. A tube of cellulose fibrils is formed with its top at the base of the tip. It extends downward until it makes contact with a group of cells at the base of the cell mass which will form the base-plate of the mature fruiting body.
At this stage the outer cells in the tip are organized radially, like a columnar epithelium, and it has been suggested that the stalk cellulose is secreted from their central faces (Bonner, 1967). Pre-stalk cells, which move inside the cellulose tube, vacuolate and increase in volume in addition to producing cellulose walls. This differentiation may be triggered by enclosure in the cellulose tube (Farnsworth, 1973). Cells are continually added to the top of the stalk, and the cellulose fibrils are continually laid down at the top of the cellulose cylinder. The stalk, therefore, elongates until the stock of pre-stalk cells is exhausted. We have noticed that the tip of the fruiting body shows periodic jerks, while elongation of the stalk itself by vacuolation of cells within it is continuous (Robertson, 1972). The period of tip jerks is between six and seven minutes, and its distribution is similar to that of signal periods from autonomous cells in early aggregation (Durston, 1974). We have therefore speculated that cell movement to the top of the stalk is periodic and under the control of the same signaling system that is responsible for cell movement during aggregation and during slug migration which also shows a periodic component although it is less well defined (Robertson and Cohen, 1972).
This description of the life cycle of D. discoideum shows that there is good reason to assume that the tip retains a special role throughout. Rubin and Robertson (Robertson et al., 1972; Rubin and Robertson, 1975) therefore repeated Raper's grafting experiments and further made grafts of tips from all developmental stages into all other stages. They also assayed tip function by grafting tips into fields of amoebae at different stages of interphase. Their experiments showed that tips from all stages have qualitatively similar properties. The signal a tip supplies is apparently not a function of its developmental age, but the response it evokes is characteristic of the recipient structure. Tip function remains constant until all tip cells have been used up in the process of fruiting body formation. The presence of a tip in a pseudoplasmodium inhibits further tip formation. Removal of a tip leads to the determination of a new tip within forty four minutes and its visible emergence within about an hour and a half, confirming Farnsworth's observation for the conus (Farnsworth, 1973) and Bonner's film (Bonner, 1959). Other pseudoplasmodial portions cannot replicate tip function until, as in the case of slug midportions, tip function reappears when, after a significant delay, the midportion regulates to produce its own tip. The signal from a tip corresponds in all respects to a continuous C-AMP signal.
The tip controls morphogenetic movement at all stages in the D. discoideum life cycle from late aggregation onward. The tip defines the direction of the developmental axis and controls a field of cells. It is able to do this by steadily secreting an attractant, presumably C-AMP, at concentrations above the threshold for signal relaying (Rubin and Robertson, unpublished results; Durston, 1974). This feature of its signal, fundamental to the tip's role as an organizer, allows it to "take over" and dominate centers of any type whose signals are initiated by autonomous cells or pulsing microelectrodes. Finally, a new tip or organizer can be produced by a regulative process when a tip is removed. A further implied, but not explicitly demonstrated, role of the tip is the control of differentiation. Regulation following tip renewal involves regulation of the proportions of pre-stalk and prespore cells (Raper, 1940; Bonner, 1967; Robertson, 1972). It is possible that the tip supplies positional information (Wolpert, 1969) by way of the gradient of signal, probably C-AMP, that it produces.
Thus the tip, like the autonomous cell in early aggregation, is the center of communication in the developing organism.
This rather formal description of the life cycle emphasizes the importance of the early stages of development during interphase. It is during this period that the amoebae undergo a sequence of differentiations, expressed finally as the ability to communicate by intercellular signaling, and leading inevitably to aggregation and later morphogenesis. We have therefore examined interphase in great detail in order to gain a suitable background for biochemical and genetic studies of differentiation.
First, we wished to measure the rate of emergence of each competence: chemotactic sensitivity to C-AMP, the ability to relay a C-AMP signal, and the ability to release C-AMP signals autonomously. The last two have been measured exactly and the first roughly.
At the beginning of interphase amoebae are relatively insensitive to C-AMP, but within four hours all cells can respond chemotactically to a C-AMP signal provided it exceeds a threshold concentration of about 10"9 M. This is only a rough estimate, as all the amoebae begin to secrete an extracellular phosphodiesterase at the beginning of interphase and we have not yet taken the enzyme activity into account in our measurements. Its effect is to give artificially high threshold measurements, so 109 M may be taken as an upper limit. We made the measurements by observing the area of attraction toward a microelectrode that was releasing pulses of C-AMP into fields of amoebae going through interphase on an agar surface. The relaying threshold was measured by the same technique; it is approximately 108 M, which is significantly greater than the chemotactic threshold.
The technique was also used to measure the fraction of cells, X2(t)9 in a population capable of relaying a C-AMP signal. This measurement is more complicated. As cells in a given population differentiate, more and more become capable of relaying. However, a relayed signal cannot be propagated until the density of competent cells exceeds a critical value because the signal has a finite range. Thus the time at which relaying is first observed is a function of initial cell density. Measuring this time for a range of densities gives the crude rate of emergence of the relaying competence. However, critical density is itself a function of initial cell density and time, through the density and time dependence of PDE activity as PDE is continuously secreted by the amoebae. Thus a correction to the raw data must be made to take account of the reduction of signal by increasing PDE activities. This has been done elegantly by Gingle (1976), who mixed populations of wild-type amoebae with cells of Dl, a non relaying mutant. Dl has the same PDE activity of the wild-type, but never contributes to the population of relaying-competent cells. Using such mixtures, Gingle obtained not only a corrected X2(t) curve but also the time and density dependence of PDE secretion. X2(t) itself is only time dependent; there is no evidence for its enhancement by cellular interaction.
Very different results were obtained for X3(t),the proportion of cells, as a function of time, capable of signaling autonomously. For one thing, as was well known, only a few cells ever show this competence (Sussman and Ennis, 1959; Konijn and Raper, 1961). The technique for measuringX3(t) was to observe the initiation time for signaling in small populations of amoebae. X3(t) begins to increase from zero between seven and eight hours from the beginning of interphase, and saturates at different levels, which depend on both cell density and cell number, implying a cellular interaction for the development of this competence, at least for relatively long times. The most important results are that no more than one percent of cells ever become autonomous and that no cells, as far as we can judge, become autonomous unless they are already capable of relaying signals. In normal populations, aggregation begins at about nine hours because the first autonomous cells release signals into a field that is capable of relaying. It is only by the artificial confinement of small populations that we can follow the differentiation of autonomy beyond the tenth or eleventh hours, when cellular interactions become apparent. These interactions thus may represent an adaptation allowing efficient aggregation in sparse populations of cells that have been starved for a long time, but they are not important under normal circumstances.
To repeat, interphase is a period of differentiation during which the four competences essential for normal aggregation emerge (Cohen and Robertson, 1972):
(1) The ability to respond to a suprathreshold extracellular concentration of C-AMP by Chemotaxis toward a C-AMP source, (2) the ability to relay a pulsatile signal of C-AMP when stimulated by a suprathreshold extracellular concentration of C-AMP higher than that for Chemotaxis, (3) the ability to release periodic pulses of C-AMP autonomously, and (4) the ability to form EDTA stable polar intercellular contacts. The first three competences provide the basis for morphogenetic movement and its control during aggregation. Knowledge of the rates at which these competences appear during interphase is essential. Without it, biochemical and genetic studies of differentiation for aggregation are severely hampered (Cohen and Robertson, 1972), as are mathematical analyses of wave propagation and aggregation. This analysis now allows a much more detailed understanding of cellular behavior during the aggregation process, in particular wave propagation and the behavior of centers.
THE SIGNALING RANGE
The cellular event basic to the control of morphogenetic movement during aggregation and to the communication between cells in D. discoideum is the release of a burst of C-AMP molecules into the aqueous film surrounding each amoebae on, e.g., agar. The C-AMP molecules diffuse into the agar and, concurrently, become converted into linear AMP by phosphodiesterase (PDE), both bound to the plasma membrane and released into the agar. We have worked out the resulting somewhat complicated mathematical problem of determining the C-AMP concentration C(r, t) at a distance r on the agar surface from the point of release and a time t after release. The result is qualitatively the same as found earlier, assuming a homogenous background of PDE in the agar (Cohen and Robertson, 1971a). The important points to note at present are that C(r, t) has a maximum value Cm(r) and that Cm(r) is a monotonically decreasing function of distance, as sketched in Fig. 1 of Cohen and Robertson (1971a). Thus at distances larger than R, the signaling range (Cohen and Robertson, 1972), the value of C(r, t) never rises above C*, the threshold for the stimulation of signal relaying.
Cm(R) = C*
Because C(r, t) depends on amoeba density Nand on time into interphase through the action of thePDE, so does R. Determination ofR vs.Ncan lead in principle to determination of the PDEactivity both in membrane-bound and in free form.
The significance of R is that an amoeba cannot stimulate another amoeba at a greater separation to relay its signal. R imposes a scale of distance on the mean amoeba separation. In the high-density case, when there are many amoebae within the range of a single signal, on average, i.e., when the field of amoebae may be regarded as a continuous medium for the propagation of signaling waves.
After ten hours, when X2(t) has reached unity, the field is a sensitive medium (Robertson and Cohen, 1972; Robertson, 1972; Durston, 1973), in a condition to propagate waves in response to any suprathreshold signal of external origin. Such sensitive media are well understood (Wiener and Rosenblueth, 1946; Krinskii, 1968). Heart tissue, certain solutions of chemical reactants (Zaikin and Zhabotinsky, 1970; Winfree, 1972), multivibrator networks, and models of neural networks are known to be sensitive media. Wave propagation in such media is well understood (Krinskii, 1968; Goodwin and Cohen, 1969; Winfree, 1972). It follows the eikonal 'equation, but in addition possesses a Huygens construction without a superposition principle. Because of the existence of the refractory period, the boundary conditions are absorbing for propagation into a boundary. The eikonal equation admits the propagation of kinks in wave fronts. However, there are no shadows, as occur in geometric optics, because of the Huygens construction, and beams spread out to fill the medium.
A point source produces circular wave fronts propagating at a velocity, v, dependent on cell density and culture age. Spiral propagation occurs (Winfree, 1972, 1973) with the inner end of the most stable spiral describing a circle of perimeter VTR with v the propagation speed and TR the refractory period.
What further remains to be understood is the propagation speed v. We have constructed a theory of v that relates it to the parameters of the signaling system: the number of C-AMP molecules released, n; the diffusion coefficient of C-AMP in agar or water, D; the threshold concentration for signal relaying, C*; the delay time between signaling and receiving a suprathreshold signal, TD ; and the free and membrane-bound PDE activity. We have measured D, and TD can be obtained by other measurements (see below). The density dependence of v can therefore in principle give values for n/C* and the PDE activities.
For low-density fields condition (1) does not apply. The field is patchy, density fluctuations are important, and the continuum treatment can no longer be used. The range of a signal is enhanced by cooperative effects within a wave front. That is, when several amoebae in a wave front signal together, the concentration produced near one of the signaling amoebae is larger than that produced by an individual amoeba alone. As the density is reduced, however, the randomness of the amoeba distribution breaks up the regular wave fronts so that cooperative effects are reduced. They are also reduced by the increasing separation of amoebae. Thus, as shown directly by R. P. Futrelle (unpublished) in computer simulations, simultaneous signaling by several amoebae does not have any significant quantitative effect on wave propagation at low densities. Under those circumstances, wave propagation can be considered a percolation process on a random medium (Shante and Kirk- patrick, 1971; Cohen and Robertson, 1972). Long-range propagation of signals cannot be sustained by the field unless the amoeba density exceeds a critical density N* given by
πR(N*)2N* = 4.5. (2)
For N less than N*, signal propagation is restricted within isolated clusters of amoebae, the mean size of which falls rapidly with decreasing density. The criterion (2) for N* derives from percolation theory (Shante and Kirkpatrick, 1971) and is only approximately applicable to our problem. However, the form of equation (2) should remain correct, the only change being in the numerical value of the right-hand side, which should decrease. Futrelle's simulations show that the decrease should be small, so I shall ignore it here.
The current best estimate of N* from the literature (Konijn and Raper, 1961) and from our own measurements is about 2.5X104cm¯2, which yields a value of 70µ for R (Gingle and Robertson, 1976).
For densities just above N*, wave propagation is restricted to narrow channels within the field. Indeed, some portions of these channels degenerate to strings of individual cells each one signaling only the next cell along the string as propagation proceeds. It is possible to identify signaling cells visually because they round up during the delay period between signaling and being signaled (Drage and Robertson, unpublished). The delay time, TD, and the signaling range, R(N), can thus be measured directly. Preliminary values of 15 sec and 70µ are consistent with earlier results.
Propagation along strings occurs whenever the number of sensitive cells just exceeds the critical density N*. This happens during interphase when X2(t)N becomes greater than N*. Identification of the onset of the capability of long-range wave propagation during interphase thus permits determination of X2(t), R(N), and TD. Similarly, the refractory period, TR, has a fairly broad distribution of values at the time aggregation normally starts. Thus if a pulse is followed by another at an interval within the distribution of Tr such that the density of cells is again just above N*, propagation along strings occurs. This permits the determination of the probability distribution of TR, R(N), and TD. Such experiments are under way.
In the preceding section, I have sketched the properties of a field of amoebae as a continuum or discrete sensitive medium. Without a source of cyclic AMP to initiate wave propagation, however, wave propagation simply does not occur in such a medium. This is observed in small-drop experiments (Raman, Hashimoto, Cohen, and Robertson, 1976). For example, a certain percentage of drops containing a small enough number of amoebae does not aggregate even though the density is well above N*. Sources of C-AMP can be external, e.g., a microelectrode (Robertson, Drage, and Cohen, 1972), an autonomous cell, or a more complex entity consisting of a group of cells. We call entities of this sort, which elicit a periodic response from the field, pacemakers. Durston (1973, 1974) has made a detailed study of natural pacemakers in D. discoideum.
He finds two geometries for wave propagation away from pacemakers, concentric and spiral. The latter have been observed to be single or double. Spiral to concentric switches, correlated with the emergence of the tip, have been observed.
Histograms were constructed for the intervals between successive waves for all waves observed, for various geometries, and for various stages of the life cycle. Time courses of intervals were determined for individual centers.
Durston's observations and analyses taken together with other observations and theoretical analyses suggested the following conclusions:
Autonomous cells contain an autonomous oscillator having a fairly sharply defined five minute period, which is independent of developmental age. This oscillator is linked to the C-AMP release mechanism but is independent of it. Differentiation into autonomy could therefore take place in two ways. First, the oscillator could be present in all cells, e.g., a metabolic oscillator such as the glycolytic oscillator in yeast, and only the link need be constructed. Second, both the oscillator and the link must be constructed.
The refractory period decreases from about nine minutes at the onset of wave propagation to about two minutes after several hours. Initially, the refractory period is broadly distributed; the width of its distribution decreases markedly with time.
This rather broad initial probability distribution of values of refractory period means that the field is then spatially heterogeneous with regard to refractivity. Consequently, spirals are initiated in an otherwise homogeneous field at local maxima of refractivity by waves propagating from autonomous cells.
Finally, tips are steady sources of C-AMP.
Communication Systems in the Cellular Slime Molds
I have described a communication system that controls aggregation in the cellular slime mold D. discoideum. A small fraction of the cells are competent to release pulses of an acrasin (most likely C-AMP) autonomously with a period of about five minutes. Essentially all the cells are competent to relay a suprathreshold C-AMP signal. After releasing a signal itself a cell is refractory to further C-AMP signals for a period of about nine minutes, decreasing to two minutes, depending on developmental history. Cells respond chemotactically to a C-AMP signal above another threshold lower than the relaying threshold by a movement step toward the signal source. The step duration, about 100 seconds, substantially exceeds the duration of the signal itself. We have visual evidence from time-lapse films that the movement step is preceded by an approximately fifteen-second interval during which the cell tends to become hemispherical and all movement ceases, even the weak random movements common between successive movement steps. The agreement between the duration of fifteen second period of stationarity and rounding with that of the delay period between signaling and being signaled (Cohen and Robertson, 1971a) as well as its preceding the movement step suggest that the two are coincident. This, in turn, implies a complementarity between morphogenetic movement and secretion in D. discoideum cells (Robertson, 1974).
Such a complementarity exists in all species of cellular slime molds for which the information is available. Before discussing the point further, however, reference should be made to Table 1, in which a classification of some of the cellular slime molds according to the known features of their aggregation control system, i.e., their intercellular communication system is presented (Cohen and Robertson, 1972). The various species are classified according to two main characteristics: whether they possess founder cells, i.e., source cells specialized and morphologically distinct from the remaining cells in the field, which act as centers of aggregation; and whether almost every cell in the field is a local source of acrasin (aggregative signal). The cells can be steady local sources, as in D. lacteum, or periodically relaying, as in D. discoideum, and this is indicated in a third column.
What is remarkable is that the founder cells are hemispherical in shape and do not move (Bonner, 1967: plate 2), precisely as for D. discoideum cells during the stage tentatively identified as the period between signaling and being signaled. It thus appears that in all cases where there is information, cells have similar morphologies and remain stationary while signaling. This suggests that founder cells in P. violaceum and P. pallidum are steady sources of acrasin and that the observed periodicity in their signal propagation derives from the refractory period of the field. Experiments to test this possibility are under way.
It can be seen from Table 1 that the diversity of known communication systems present in the cellular slime molds derives from only three binary choices: founder cells or no founder cells, local signal sources in the field or no local signal sources, steady local sources or pulsatile local relaying. These observations have considerable relevance to evolutionary and genetic questions.
What has this very detailed analysis of a developmental control system in one rather obscure organism to tell us about the control of development in general and intercellular communication in particular? It is intriguing to realize how many phenomena that are considered typical in multicellular development imply the existence of control systems that depend on intercellular communication. For example, regulation is a feature of all Metazoan embryos; by regulation, we mean the production of form independent of linear dimension, as is seen when normal tadpoles develop from the separated blastomeres of an amphibian embryo. I have reviewed many similar examples; see Robertson and Cohen (1972), Wolpert (1969) for a summary of the various theories that have been adduced to account for regulative processes. Here it is enough to say that both reliable development and regulation itself imply that cellular behavior in embryos may be subject to control by intercellular communication. While it is known that there are anatomical bases for such communication, nothing is known about how information is passed from cell to cell or what molecular species are involved.
Communication systems of some cellular slime molds.
In the slime molds we have a beautiful system in which the role of intercellular communication is clear and in which the molecule used is known. Further, the control of pseudoplasmodial morphogenesis depends on the tip, which is suspiciously like an "organizer." If the analogy between slime mold tips and Metazoan organizers can be sustained, we have a unique opportunity to investigate organizer action at every level of function, from the multicellular down, ultimately, to the control of gene expression. The most promising feature of the slime mold control system is that it can be subjected to a classical genetical analysis because mutations that disrupt the control system are not necessarily lethal. It is hard to imagine that analogous mutations would be either easy to obtain and study or relatively benign in, say, a chick embryo. Thus, if it is ever possible to analyze a control system in a vertebrate it is unlikely that classical genetics will be useful. Therefore, the more we can learn about the function of developmental control systems in simple organisms and the patterns of cell behavior to be expected from different kinds of intercellular communication, the easier it will be to classify cell behavior in Metazoan embryos and to guess the kinds of intercellular interaction on which they depend. The subject has become more exciting with our recent work which has shown evidence for an almost identical signaling system controlling cell movement in the early chick embryo (Robertson and Gingle, unpublished results).
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