Neural tubes whose neural crest had just begun migration were isolated from stage-14 chick embryos, cleaned with 0·1% trypsin, and cultured in transparent hydrated collagen lattices (HCL) in an effort to stimulate in part the three-dimensional environment through which neural crest cells migrate in situ, in the embryo. The concentration of collagen in the lattices varied from 50 μ g/ml to 390 μ g/ml. The mode of movement and contact behaviour of neural crest cells migrating from the neural tube under these conditions were recorded directly with time-lapse cinemicrography. Both their shape and their rate of translocation were dependent on the concentration of collagen in the HCL. In low concentrations (50 μ g/ ml to 105 μ g/ml), neural crest cells have elongate spindle shapes and translocate at an average rate of 1 μ m/min, whereas in high concentrations (190μ g/ml to 390μ g/ml), their shape is rounded, and they translocate at an average rate of only 0·5 μ m/min. Neural crest cells migrate from neural tubes in these preparations principally in loose clusters, with a few single cells in the lead. The cells in these groups display leading-to-trailing edge adhesions and form tongues or streams of cells directed away from the neural tube. The paths of migration of both individual cells and groups of cells are aligned with the collagen fibrils of the HCL, which radiate from the neural tube. The classical visible characteristic of contact inhibition of movement, change in direction of cell movement after contact with other cells, was not observed; neither the rate of translocation nor the time spent migrating away from the tube is dependent on the number of contacts between cells. It is concluded that the directional movement of neural crest cells in HCL cultures does not depend on contact inhibition of movement.

Neural crest cells migrate from the neural tube during embryogenesis in a highly directional manner (Hörstadius, 1950; Weston, 1970; Noden, 1978). Evidence presented by Weston & Butler (1966), Le Douarin & Teillet (1974) and Noden (1975) indicates that the environment through which these cells pass plays a crucial role in this directionality. Löfberg (1976) and Ebendal (1977) have suggested that within this environment it is the extracellular matrix that influences directional movement by a kind of contact guidance (Weiss, 1961). However, since alignment of the substratum in and of itself can only give such moving cells orientation, not directionality (Trinkaus, 1976), other factors must be involved. Tosney (1978) and Noden (1978) suggest that one such factor could be contact inhibition of cell movement (Abercrombie & Heaysman, 1966). If neural crest cells show contact inhibition, this, combined with an aligned substratum, could provide the directionality required, at least during the initial phases of their migration, (see Trinkaus, 1980).

To determine if this is so, it is necessary to observe the movement of these cells directly as they leave the neural crest and migrate into the extracellular matrix of the embryo. Unfortunately, however, the favoured materials for studying the migratory behaviour of neural crest cells, amphibian and chick embryos, are so opaque that direct observation of cell movement in situ is precluded. We therefore deemed it necessary to construct a three-dimensional quasi in vivo environment in vitro that would at the same time encourage cell movement and be of sufficient transparency to permit direct observation of it. Elsdale & Bard (1972), have provided just such an environment in the hydrated collagen lattice (HCL) and Bard & Hay (1975) have shown that the form and movement of one kind of cell in an HCL in vitro - chick corneal mesenchyme - is remarkably similar to the form and movement of these same cells in the cornea in vivo. And Yang et al. (1979) have recently confirmed these favourable features by showing that epithelial cells of a primary mammary tumor maintain sustained growth and three-dimensional organization in such a gel. In consequence, we decided to study the directional movement and social interaction of neural crest cells in such hydrated collagen lattices in an effort to observe whether contact inhibition of movement is at work and, if so, is related to their directional movement. Cell contact behaviour was recorded with time-lapse cinemicrography and the films thus obtained provided the basis for our analyses.

Preparation of collagen for hydrated collagen lattice

Rat tail collagen was isolated and the collagen concentration estimated, as previously described (Davis, 1980). Hydrated collagen lattices, as described by Elsdale & Bard (1972), containing 26μ g/ml to 394μ g/ml of collagen, were prepared by mixing equal volumes of collagen stock solution and twice concentrated growth medium, adjusting the pH to 7·4 with 1 N-NaOH, and diluting with normal strength growth medium (Delbecco’s Modified Eagle’s Medium with 10% fetal calf serum, 100 U/ml Penicillin, 0·25 mcg/ml fungizone, and 100 mcg/ml streptomycin) to the desired concentration of collagen.

Primary cultures of neural crest cells

Neural tubes, 15 to 20 somites long, were isolated from the seventh to the most posterior somite of stage-14 (Hamburger & Hamilton, 1951) White Leghorn chick embryos (Spafas, Norwich, Conn.) and stripped of clinging mesenchyme cells by digestion with 0·1% trypsin (Trypsin 1:250, Gibco) (Davis, 1980). At this stage, neural crest cells have begun their migration. These neural tubes, with their neural crest, were then suspended in an HCL and incubated, as previously described (Davis, 1980).

Scanning electron microscopic examination of trypsin-treated neural tubes

Freshly isolated, trypsin-treated neural tubes that had not been cultured in HCL cultures were fixed overnight in 2% glutaraldehyde in PBS, postfixed in 1% osmium buffered with PBS, dehydrated through a series of ethanol dilutions, and dried in a Sorvall critical-point drier, according to Anderson (1951). They were then coated with 20 nm of gold, using a rotary shadower (Ladd), and examined with an ETEC Scanner operating at 10 kV.

Time-lapse cinemicrography

Time-lapse films of migrating neural crest cells were made as previously described (Davis, 1980), except that the interval between frames was 5 or 7-5 seconds. Filming was begun about 12 h after culturing.

Polarization microscopy

Polarization microscopy was used to examine the orientation of the collagen fibrils of the HCL. A 35 mm camera was mounted to a Zeiss Universal microscope modified for polarization optics with a Polanochromatic 16×/0·035 objective and 5 x ocular. A neural tube cultured in an HCL was placed on a graduated rotating stage and the analyzer was adjusted to 86·8°. Photographs of the negative and positive retardation images were taken for every 3(F of stage rotation with Kodak SO 115 film and were developed in HC110 developer (Dilution D for 8 min), fixed with Kodak rapid fixer, and processed normally.

Rate of translocation

Analysis of the rate of translocation that used longer intervals than frame-by-frame analysis would facilitate the analysis of larger amounts of data. Increasing the interval, however, might also obscure changes in minute-to-minute activity. To test for this, the rates of translocation were determined both frame-by-frame and at 5 min intervals, using photographic prints from the films (see Table 1). Since the rates of translocation estimated with the two different techniques were nearly the same, it appears that increasing the time intervals for analysis to 5 min is approximately as accurate a method for determining rate of movement as is frame-by-frame analysis. Accordingly, all subsequent determinations of rate of cell movement (Tables 2, 3, 5, 7, 8, 9, 10) are based on observations made at 5 min intervals.

Table 1

Shape and locomotory behaviour of neural crest cells in HCL cultures containing different concentrations of collagen

Shape and locomotory behaviour of neural crest cells in HCL cultures containing different concentrations of collagen
Shape and locomotory behaviour of neural crest cells in HCL cultures containing different concentrations of collagen
Table 2

The average rate of translocation of neural crest cells with increasing time in culture

The average rate of translocation of neural crest cells with increasing time in culture
The average rate of translocation of neural crest cells with increasing time in culture
Table 3

Average rate of translocation of neural crest cells at various distances from the neural tube

Average rate of translocation of neural crest cells at various distances from the neural tube
Average rate of translocation of neural crest cells at various distances from the neural tube
Table 4

Relative amount of time spent by neural crest cells moving away from and towards the neural tube

Relative amount of time spent by neural crest cells moving away from and towards the neural tube
Relative amount of time spent by neural crest cells moving away from and towards the neural tube
Table 5

Average rate of translocation of neural crest cells migrating away from and toward the neural tube

Average rate of translocation of neural crest cells migrating away from and toward the neural tube
Average rate of translocation of neural crest cells migrating away from and toward the neural tube
Table 6

Correlation of contact number with the migratory activity of neural crest cells

Correlation of contact number with the migratory activity of neural crest cells
Correlation of contact number with the migratory activity of neural crest cells
Table 7

The effect of cell-to-cell contact on the average rate of translocation of neural crest cells

The effect of cell-to-cell contact on the average rate of translocation of neural crest cells
The effect of cell-to-cell contact on the average rate of translocation of neural crest cells
Table 8

Effect of contact number on the rate of translocation of neural crest cells moving away from the neural tube

Effect of contact number on the rate of translocation of neural crest cells moving away from the neural tube
Effect of contact number on the rate of translocation of neural crest cells moving away from the neural tube
Table 9

Effect of contact number on the rate of translocation of neural crest cells moving toward the neural tube

Effect of contact number on the rate of translocation of neural crest cells moving toward the neural tube
Effect of contact number on the rate of translocation of neural crest cells moving toward the neural tube
Table 10

Changes in contact number and the associated rate of translocation

Changes in contact number and the associated rate of translocation
Changes in contact number and the associated rate of translocation

Evidence that the cells studied are derived from the neural crest

Since our interest was solely in the emigration of neural crest cells, we had to be certain that the cultured neural tubes were free of contaminating somitic mesenchyme. An SEM examination of neural tubes treated with trypsin and dissected free of somitic mesenchyme revealed no somitic tissue (Fig. 1). Other evidence has already been presented by one of us (Davis, 1980, p. 20–21).

Fig. 1

(a) Scanning electron micrograph of a neural tube isolated from a stage-14 chick embryo (see Methods) placed in 2% glutaraldehyde approximately 15 min after isolation. The tube is clean, with only the notochord (NCH) and neural crest remaining attached, to the ventral and dorsal surfaces, respectively. (b) An enlargement of the part of the tube included in the square of Figure 1 a. In this region, the crest cells (NC) have already begun to move down the surface of the neural tube. Neural crest cells appear highly spread, in close apposition to one another, and have few blebs on their surfaces. In contrast, cells of the neural tube are covered with many blebs. Occasionally, some cells of the overlying ectoderm (EC) remain attached to the isolated tube. ‘

Fig. 1

(a) Scanning electron micrograph of a neural tube isolated from a stage-14 chick embryo (see Methods) placed in 2% glutaraldehyde approximately 15 min after isolation. The tube is clean, with only the notochord (NCH) and neural crest remaining attached, to the ventral and dorsal surfaces, respectively. (b) An enlargement of the part of the tube included in the square of Figure 1 a. In this region, the crest cells (NC) have already begun to move down the surface of the neural tube. Neural crest cells appear highly spread, in close apposition to one another, and have few blebs on their surfaces. In contrast, cells of the neural tube are covered with many blebs. Occasionally, some cells of the overlying ectoderm (EC) remain attached to the isolated tube. ‘

Form, orientation, and social relations of migrating neural crest cells

Using the collagen fibrils of the HCL as a substratum, neural crest cells begin to emigrate within 2-4 h. During this process, the majority of the cells are in contact with each other (Fig. 2). Only a few single cells have been observed. Characteristically, groups of cells become organized into elongate streams that extend far away from the neural tube. In general, after 36 h in culture, a stream consists of about ten cells and extends approximately 150 to 200 μ m from the neural tube. Cells which compose the streams line up largely in single file; however, at the base of the stream, they may be three to four abreast. Each cell is elongate and oriented along the axis of migration, with the most distal cells generally the most elongate of all (Fig. 2).

Fig. 2

A frame from a time-lapse film of neural crest cells emigrating as streams of cells from a neural tube (at the bottom and out of view) suspended in an HCL containing 50μ g/ml of collagen, after 18 h in culture. Many cells are out of focus because of the three-dimensional character of the preparation. Nomarski interference optics, × 230.

Fig. 2

A frame from a time-lapse film of neural crest cells emigrating as streams of cells from a neural tube (at the bottom and out of view) suspended in an HCL containing 50μ g/ml of collagen, after 18 h in culture. Many cells are out of focus because of the three-dimensional character of the preparation. Nomarski interference optics, × 230.

Locomotion of neural crest cells

Obviously, as neural crest cells move in an HCL, they frequently adhere to other cells. The stability of these adhesions depends in part on cell locomotor behaviour. When cells in contact are moving in the same direction, they stay together, suggesting either that their adhesions are quite stable or that their identical direction of movement does not subject their adhesions to enough stress to cause them to rupture. When two adhering cells move in opposite directions, however, their adhesions rupture and the cells separate. Separation also occurs when one cell of a moving adhering pair stops while the other continues to move. In either case, before the cells separate they become more elongate in the direction of movement and the collagen fibrils about them become reoriented (Fig. 3). Note how one of the cells in Fig. 3 rounds up upon separation and begins to bleb, as often occurs when cells de-adhere and retract (Harris, 1973; Chen, 1981). When more than two cells are in contact and begin to separate, neither the morphological distortion nor the rate of separation appears as great.

Fig. 3

A sequence from a time-lapse film of two neural crest cells emigrating from a neural tube (out of view at the left) suspended in an HCL culture containing 50μ /g/ml of collagen, after 20 h in culture. This sequence demonstrates the recoil of collagen fibrils and the fast rate of separation when two cells break with one another. Times are in minutes and seconds. 0:00 As the two cells begin to separate, three collagen fibrils can be seen. The parts of fibrils a and b which are visible in the plane of focus are: 20 μ m and 24 μ m long, respectively. Fibril c was not measured. The length of the cell at the left is approximately 61 μ m and that of the cell at the right approximately 37 μ m. 2:38 The apparent lengths of fibrils a, and b and of the cell at the left have not changed; however, the length of the cell at the right has increased to approximately 49 μ m. 2:45 Within seconds, the cells separate and the cell at the right retracts approximately 14 μ m to a length of about 23 μ m and rounds up considerably. The cell at the left has retracted little but is clearly less taut. The apparent length of fibril a

Fig. 3

A sequence from a time-lapse film of two neural crest cells emigrating from a neural tube (out of view at the left) suspended in an HCL culture containing 50μ /g/ml of collagen, after 20 h in culture. This sequence demonstrates the recoil of collagen fibrils and the fast rate of separation when two cells break with one another. Times are in minutes and seconds. 0:00 As the two cells begin to separate, three collagen fibrils can be seen. The parts of fibrils a and b which are visible in the plane of focus are: 20 μ m and 24 μ m long, respectively. Fibril c was not measured. The length of the cell at the left is approximately 61 μ m and that of the cell at the right approximately 37 μ m. 2:38 The apparent lengths of fibrils a, and b and of the cell at the left have not changed; however, the length of the cell at the right has increased to approximately 49 μ m. 2:45 Within seconds, the cells separate and the cell at the right retracts approximately 14 μ m to a length of about 23 μ m and rounds up considerably. The cell at the left has retracted little but is clearly less taut. The apparent length of fibril a

Dependence of cell shape and rate of movement on the density of the HCL

Although emigration is not inhibited, there are distinct morphological differences between cells in HCL cultures containing low concentrations of collagen (26 μg/ml to 130 μg/ml) and cells in cultures with higher concentrations (200 μg/ml to 390 μg/ml) (Figs. 4, 5). In low concentrations, cells are usually long and bipolar in shape (see Davis, 1980), whereas in high concentrations, cells are more rounded. The average ratio of the length to width of cells was used to quantify the differences in their morphology (Table 1). Increasing the concentration of collagen in HCL cultures to 200 μg/ml or more causes a two-fold reduction in the length-to-width ratio of moving neural crest cells.

Fig. 4

A frame from a time-lapse film of neural crest cells emigrating from the neural tube (lower left) in a low density hydrated collagen lattice, containing 26 μg/ml of collagen, after 21 h in culture, × 480.

Fig. 4

A frame from a time-lapse film of neural crest cells emigrating from the neural tube (lower left) in a low density hydrated collagen lattice, containing 26 μg/ml of collagen, after 21 h in culture, × 480.

Fig. 5

A frame from a time-lapse film of neural crest cells emigrating from the neural tube (lower left) in a high density hydrated collagen lattice, containing 394μg/ml of collagen, after 21 h in culture. ×480. increases to about 24μm, as the cell at the left moves slightly to the left. Fibril b decreases in length to about 22 μm, when the protrusion of the cell at the left loses contact with the cell at the right, shortens, and retracts toward the left. Fibril c, previously slanted towards the right (times 0:00-2:38), now has moved nearly 6 μm to the left. 2:53 The visible length of fibril a has now increased to about 27 μm, while fibril b apparently shortens and becomes curved (distance between reference points 18 μm). The cell at the left has clearly retracted and its length has decreased to approximately 57 μm. 3:45 The apparent length of fibril a is now about 29 μm and fibril b has become curved, with the distance between its two reference points decreasing to 16 μm. The cell at the left retracted an additional 8 μm and its length has decreased to approximately 49 μm. The cell at the right is now fully retracted and is blebbing actively. ×480.

Fig. 5

A frame from a time-lapse film of neural crest cells emigrating from the neural tube (lower left) in a high density hydrated collagen lattice, containing 394μg/ml of collagen, after 21 h in culture. ×480. increases to about 24μm, as the cell at the left moves slightly to the left. Fibril b decreases in length to about 22 μm, when the protrusion of the cell at the left loses contact with the cell at the right, shortens, and retracts toward the left. Fibril c, previously slanted towards the right (times 0:00-2:38), now has moved nearly 6 μm to the left. 2:53 The visible length of fibril a has now increased to about 27 μm, while fibril b apparently shortens and becomes curved (distance between reference points 18 μm). The cell at the left has clearly retracted and its length has decreased to approximately 57 μm. 3:45 The apparent length of fibril a is now about 29 μm and fibril b has become curved, with the distance between its two reference points decreasing to 16 μm. The cell at the left retracted an additional 8 μm and its length has decreased to approximately 49 μm. The cell at the right is now fully retracted and is blebbing actively. ×480.

The rates of translocation of neural crest cells in different concentrations of collagen are shown in Table 1. The average rate of translocation was found to be clearly dependent on the collagen concentration. In cultures containing low concentrations, the average rate was approximately 1 μm/min, whereas at higher concentrations, it was reduced to approximately 0·5 μm/min.

Possible variation in the rate of translocation with time in culture and distance from the neural tube

To test for variations in the rate of translocation with time and distance, estimates of the rate of translocation were made at 60 min intervals from the beginning to the end of filming, i.e. during a period of nearly 6 h. The differences between the rates during the first hour and the succeeding hours were then compared. They were found to be statistically insignificant; no correlation was observed between the average rates of translocation and the duration of migration (Table 2). Thus, estimates of the average rate of translocation of neural crest cells are not significantly biased by the time in culture at which the estimates are made.

Variations in the rate of translocation with increasing distance from the neural tube could be significant in HCL cultures containing low concentrations of collagen, since the average rate of translocation and the distance the cells travel are both great. To test for this possibility, estimates of the rate of trans-location made at 20 μm intervals of distance from the neural tube were compared. The difference between the rates of translocation at various distances from the neural tube were found to be statistically insignificant; no correlation was observed between the average rate of translocation and the distance cells have migrated from the neural tube (Table 3).

Directionality of migration of neural crest cells

Once neural crest cells are moving within an HCL, the paths of migration are largely perpendicular to the neural tube (Fig. 6). Cells were seldom observed to move to the left or right and never observed to move above or below the plane of focus. Since the route of migration is so direct, the time spent moving away from and towards the neural tube and the rate of cell movement must be the main factors determining the distance they travel. Analysis revealed that they move faster and spend more time moving away from the neural tube than toward it (at least in low concentrations of collagen). Statistically significant differences were observed in both instances (Tables 4, 5).

Fig. 6

The course traversed by neural crest cells emigrating from neural tubes suspended in hydrated collagen lattices was plotted from time-lapse films. The initial position of each cell, the course of its migration, and changes in its direction Of translocation are designated by solid lines and arrows, respectively. The curvaceous line on the left side of each tracing represents the edge of the neural tube. In both low and high density hydrated collagen lattice cultures, neural crest cell locomotion from the neural tube appears to be directional. Their paths of migration leads almost always away from the neural tubes. (A) The neural tube was suspended in a low-density hydrated collagen lattice containing 26 μg/ml of collagen. The illustratéd sequence represents an elapsed time of 100 min. (B) The neural tube was suspended in a high-density hydrated-collagen lattice containing 394μg/ml of collagen. The illustrated sequence represents an elapsed time of 500 min.

Fig. 6

The course traversed by neural crest cells emigrating from neural tubes suspended in hydrated collagen lattices was plotted from time-lapse films. The initial position of each cell, the course of its migration, and changes in its direction Of translocation are designated by solid lines and arrows, respectively. The curvaceous line on the left side of each tracing represents the edge of the neural tube. In both low and high density hydrated collagen lattice cultures, neural crest cell locomotion from the neural tube appears to be directional. Their paths of migration leads almost always away from the neural tubes. (A) The neural tube was suspended in a low-density hydrated collagen lattice containing 26 μg/ml of collagen. The illustratéd sequence represents an elapsed time of 100 min. (B) The neural tube was suspended in a high-density hydrated-collagen lattice containing 394μg/ml of collagen. The illustrated sequence represents an elapsed time of 500 min.

The next question is that of directionality itself. What factors contribute to directional movement of neural crest cells in an HCL ? One could be the orientation of the collagen fibrils. If these fibrils were aligned perpendicular to the neural tube, they might provide a kind of contact guidance and by reducing or preventing sideward movements contribute to directionality. The fibrils were examined with polarization microscopy to deteimine their orientation. As can be seen in Fig. 7, the majority of the fibres were aligned perpendicular to the neural tube. Since the cells are similarly aligned, this would appear to be a clear case of contact guidance. However, since contact guidance can only provide orientation, toward or away from the tube (see Trinkaus, 1976), some other factor must be added to give directionality, i.e. movement away from the tube. It is possible that this directionality could come from cell-to-cell interactions.

Fig. 7

Orientation of the fibrils of the HCL with respect to the neural tube and the direction of migration of emigrating neural crest cells. An isolated neural tube with adhering neural crest was suspended in an HCL culture containing 50 μ g/ml of collagen and cultured over night to permit time for the neural crest cells to migrate away from the neural tube. Polarization microscopy was used to examine the orientation of the collagen fibrils of the HCL (see Methods). Photographs of the positive (A’, B’, etc.) and negative (A, B, etc.) retardation images were taken as the culture was rotated 0, 60, 90, 120 and 180°. (A, A′) Retardation images of the collagen lattice before the culture was rotated. The majority of the biréfringent collagen fibrils radiate from the neural tube; however, upon close examination, some biréfringent fibrils appear oriented perpendicular to the longer fibres of the HCL. (B, B’) The culture was rotated 60°. Note that the intensity of the retardation images radiating from the neural tube has decreased and that no new biréfringent collagen fibrils appear. (C, C’) The culture was rotated 90°. As in A, A’, the biréfringent images radiate from the neural tube; however, their polarity is reversed. (D, D′) The culture was rotated 120°. The intensity of the retardation images has decreased; however, the orientation of the collagen fibrils with respect to the neural tube remains unchanged. (E, E′) The culture was rotated 180°. The retardation images appear as in A, A′. Since most of the birefringent images of the HCL radiate from the neural tube like the spokes from the hub of a wheel, regardless of the degrees of rotation, it may be concluded that much of the HCL is highly ordered. In addition, it is important to point out that the orientation of the HCL is nearly identical to that of the path of migration of emigrating neural crest cells.

Fig. 7

Orientation of the fibrils of the HCL with respect to the neural tube and the direction of migration of emigrating neural crest cells. An isolated neural tube with adhering neural crest was suspended in an HCL culture containing 50 μ g/ml of collagen and cultured over night to permit time for the neural crest cells to migrate away from the neural tube. Polarization microscopy was used to examine the orientation of the collagen fibrils of the HCL (see Methods). Photographs of the positive (A’, B’, etc.) and negative (A, B, etc.) retardation images were taken as the culture was rotated 0, 60, 90, 120 and 180°. (A, A′) Retardation images of the collagen lattice before the culture was rotated. The majority of the biréfringent collagen fibrils radiate from the neural tube; however, upon close examination, some biréfringent fibrils appear oriented perpendicular to the longer fibres of the HCL. (B, B’) The culture was rotated 60°. Note that the intensity of the retardation images radiating from the neural tube has decreased and that no new biréfringent collagen fibrils appear. (C, C’) The culture was rotated 90°. As in A, A’, the biréfringent images radiate from the neural tube; however, their polarity is reversed. (D, D′) The culture was rotated 120°. The intensity of the retardation images has decreased; however, the orientation of the collagen fibrils with respect to the neural tube remains unchanged. (E, E′) The culture was rotated 180°. The retardation images appear as in A, A′. Since most of the birefringent images of the HCL radiate from the neural tube like the spokes from the hub of a wheel, regardless of the degrees of rotation, it may be concluded that much of the HCL is highly ordered. In addition, it is important to point out that the orientation of the HCL is nearly identical to that of the path of migration of emigrating neural crest cells.

Effect of cell-to-cell contact on cell migration

One possibility is that the greater time spent by neural crest cells moving away from the neural tube (Table 4) is related to cell-to-cell contact. Following the procedure of Abercrombie & Heaysman (1966), we found no correlation between the number of cell-to-cell contacts per cell and duration of emigration (Table 6). These results suggest that the increased time neural crest cells spend moving away from the neural tube is not influenced by cell-to-cell contacts.

Although the duration of directional migration is unaffected, cell contacts might nevertheless influence the rate at which neural crest cells translocate (Abercrombie & Heaysman, 1953; Martz, 1973), and this, in turn, could have a significant effect on the distance cells migrate, since the chance of collisions between cells would be greater for cells migrating toward the neural tube than away from it. Analysis of the effect of cell-to-cell contact on rate of translocation, however, revealed no correlation between these two variables (Table 7). Regardless of the contact number, the average rate of translocation remained unaltered.

Since the duration and rate of translocation of neural crest cells is greater when moving away from the neural tube, cell-to-cell contacts might influence cells differently, depending on their direction of movement. Such a difference might be obscured by analyses that incorporated data from cellular movement in both directions (as in Table 7). To test for this possibility, the rate of translocation, as related to contact number, was determined separately for cells moving in only one direction. It is clear from the results, summarized in Tables 8 and 9, that no correlation is evident; regardless of the contact number, the rate of directional translocation remained unaltered. Thus, the net directional displacement of neural crest cells away from the neural tube in an HCL is not influenced by cell-to-cell contacts and, in consequence, other factors must influence their directional migration.

Changes in cell-to-cell contact and their effect on the rate of translocation

It was noted above (p. 34) that the rate of separation between two cells losing contact with each other appeared to depend in part on the number of remaining adhesions with neighbouring cells. To test this impression, the average rate of translocation for cells (1) making, (2) breaking, or (3) maintaining a constant contact number was determined, as described by Abercrombie & Heaysman (1954). Only cells losing contacts had a statistically greater rate of movement (Table 10). Since only cells losing contacts translocate at a significantly greater rate, the effect of cell-to-cell contact on this form of movement was determined.

The correlation between the rate of separation of two cells losing mutual contacts and the remaining number of cell-to-cell contacts retained after separation was equivocal. The average rate of separation tends to decrease as contact number increases; however, no statistically significant correlation was observed between contact number and the rate of separation of cells losing contact (Table 11).

Table 11

Contact number and the rate of separation of cells losing mutual contacts

Contact number and the rate of separation of cells losing mutual contacts
Contact number and the rate of separation of cells losing mutual contacts

This study has explored some of the factors that might influence the migratory behaviour of chick neural crest cells. Since the movement of these cells cannot be observed directly within the embryo, neural tubes with intact neural crest were suspended in a transparent hydrated collagen lattice in vitro (Elsdale & Bard, 1972). This culture system was chosen not only because it permits direct observation of cell movement, but because it resembles, in part, the threedimensional fibrillar matrix through which neural crest cells migrate normally in vivo (see Noden, 1978, for review). It should be emphasized, however, that although such an HCL consists primarily of collagen, a normal constituent of the matrix, it lacks other normal constituents, including glycosaminoglycans.

Neural crest cells were found to translocate in a highly directional manner in an HCL, spending 74% of their time, on the average, moving away from the neural tube. Analysis of the effect of cell-to-cell contacts on cell movement revealed no correlation between the number of contacts between cells and their rate or direction of movement. Since neural crest cells are perfectly capable of forming firm adhesions with neighbouring cells under these conditions, as shown by their physical distortion when two adhering cells move apart, it seems unlikely that this could be attributed to weak intercellular adhesions. In fact, it has been shown (Johnston & Listgarten, 1972; Ebendal, 1977) that neural crest cells form cell-to-cell contacts in vivo that are similar to those formed in vitro on a planar substratum by chick heart fibroblasts (Heaysman & Pegrum, 1973), whose rate and direction of translocation is clearly modulated by cell-to-cell contacts. It seems clear from these observations that neural crest cells moving within an HCL do not show contact inhibition of movement, as originally defined by Abercrombie & Heaysman (1953, 1954). Thus, other factors must be responsible for the directional movement of these cells under these conditions.

It goes without saying that these results do not tell us whether the striking directional migration of neural crest cells as they leave the neural tube in situ during normal development is independent of contact inhibition or not. The contact behaviour of cells in vitro does not necessarily reflect their normal behaviour in vivo (see, for example, Lesseps, Lapeyre & Hall, 1979). These results do suggest, however, that contact inhibition may not operate for these cells in situ within the embryo and they compel us to pay more attention to other possible causes of the directionality of neural crest cells (and of other directionally moving cells as well).

One possibility is that directional migration is determined intrinsically for each cell; i.e. once cells begin to move in a certain direction they will tend to persist in that direction. This is not implausible. Two different forms of fibroblasts, primary cultures of chick heart fibroblasts (Abercrombie & Gitlin, 1964; Abercrombie & Heaysman, 1966) and 3T3 cells (Gail & Boone, 1970), have been observed to continue their directional, non-random translocation in vitro for long periods of time, ranging from 2·5 to 5 h, in the absence of cell contact and orientation of the substratum. Clusters of fish melanoma cells also show such persistence in vitro (Kolega, 1981). If neural crest cells in an HCL similarly maintained their initial direction of movement, it would be away from the neural crest, exactly what we have observed. Retraction induced spreading (Chen, 1979; Dunn, 1980), whereby the protrusive activity of the advancing leading edge of a fibroblast is augmented by the retraction of its trailing edge, could be the mechanism (Trinkaus, 1980). However, our observations of neural crest Cells in an HCL offer no support for this hypothesis. No spurt of protrusive activity at the leading edge was observed when the trailing edge of these cells retracted (Davis, 1980). This does not eliminate persistence as a possible explanation of neural crest directionality, but is does suggest that if it occurs it is by some other mechanism.

The other possibility, of course, is that factors in the cellular environment (other than contacts with other cells) have a strong directional influence on the migration of neural crest cells. The experiments of Weston & Butler (1966), Le Douarin & Teillet (1974) and Noden (1975) all have provided evidence that transplanted neural crest cells tend to follow the pathways normal for that level of the neural tube; i.e. the direction of their migration is strongly influenced by the tissue environment.

In the present study, one element of the environment - the collagen fibrils - appears to have a strong influence on the migratory behaviour of the cells. Polymerization of these fibrils apparently begins soon after a neural tube is placed into the solution of collagen, and they soon become aligned perpendicular to the neural tube, radiating from it like spokes of a wheel. The cause of this alignment is unknown, but it seems probable that the neural crest cells themselves are in part responsible, as they pull and tug on these fibrils during their migration (see Harris, Wild & Stopak, 1980; Stopak & Harris, 1980). Markwald, Fitzharris, Bolender & Bernanke (1979) have shown, for example, that microfibrils of the cell-free cardiac jelly matrix of the chick embryo are unoriented prior to cellular invasion. But, with entrance of the cushion cells, those fibrils that are associated with motile cellular extensions become oriented. As the authors point out, this association suggests but does ‘not prove’ that these oriented microfibrils constitute the natural in vivo substratum of the cell. However the collagen fibrils become oriented in an HCL in our experiments, neural crest cells quickly become oriented and aligned with them in what appears to be typical contact guidance. It is not known how the cells align in this manner. Of course, the simplest explanation, and perhaps the most probable, would be that the aligned collagen fibrils physically restrain the cells from moving tangentially. In any case, as already emphasized (see p. 29), such an orienting influence cannot in and of itself give directionality (see Trinkaus, 1976). Further, a crucial element in this demonstration of possible contact guidance is unfortunately lacking. We have not been able to construct an HCL containing a neural tube in which the collagen fibrils are unoriented. Until such a control can be provided, these remarks can only be regarded as speculative.

Since we chose an HCL for these studies in order to try to simulate, though crudely, certain aspects of the normal environment within the embryo, it is of interest that the fibrillar matrix through which neural crest cells normally migrate in the embryo is likewise highly aligned, at least in amphibians (Löfberg, 1976; Löfberg & Ahlfors, 1978). It is not clear whether this is so for the avian embryo. Ebendal (1977) has observed some fibre alignment near the neural crest with SEM, but Bancroft & Bellairs (1976) and Tosney (1978) have not. Tosney (1978) points out that this failure to observe alignment of the fibrillar extracellular matrix may be due in part to fixation problems. She may well be correct; for an HCL fixed with glutaraldehyde collapses upon critical-point drying, forming a flat mat, in which both the orientation and three-dimensional distribution of the collagen fibres is destroyed (Davis, unpublished results).

Another environment factor that has an important influence on the locomotor behaviour of neural crest cells in an HCL is the concentration of the collagen. Although it does not modify the direction of cell movement, it has important quantitative effects on cell shape, rate of translocation, distance of migration, and the rate of separation of two adhering cells. These responses are not understood. Perhaps cells move more readily in lower concentrations because these provide sufficient substrata for movement but little impediment, whereas higher concentrations are so dense that they physically impede movement.

Mutual negative chemotaxis might also be a factor in this directional migration, as for amphibian melanoblasts, which are a derivative of the neural crest (Twitty, 1944; Twitty & Niu, 1954). This seems unlikely, however, since neural crest cells in HCL cultures tend to remain in contact with one another during migration. Moreover, continuous perfusion of growth medium through these cultures does not alter the directionality of the cells (Davis, unpublished results).

An important feature of neural crest cells within an HCL is their tendency to move in streams, the leading edge of one cell adhering to the trailing edge of another cell. This is no doubt an inevitable consequence of their moving in the same direction. Not surprisingly, this same phenomenon has been observed elsewhere, both in vivo (Bancroft & Bellairs, 1976) and in vitro. Chick heart fibroblasts, for example, form similar cell streams occasionally in vitro (Ambrose, 1961), albeit, unlike neural crest cells in an HCL, they show contact inhibition of movement. Apparently, leading-to-trailing edge collisions do not elicit contact inhibiting responses from either neural crest or chick heart fibroblasts in vitro. t

What then is the mechanism of the directional migration of neural crest cells, either in an HCL in vitro or normally in vivo? There is no evidence for contact inhibition, once they have left the crest. By elimination, it seems possible that the intrinsic resistance of the cells themselves to changes in direction is a primary force in their directional movement. They no doubt move away from the crest initially, because there is no other direction available to them. What causes them to persist in this path of movement is the question. All we can say at present is that retraction-induced protrusive activity appears not to be at work and that contact guidance may prevent them from moving sideways.

We are indebted to Raymond E. Keller and Wen-Tien Chen for discussion and assistance. This research has been supported by a grant from the NIH (CA22451) to J.P.T. and by postdoctoral fellowships from the American Cancer Society (PF-1330) and a Cell and Developmental Biology Training Grant (USPHS-HD-00032-13) to the Department of Biology of Yale University to E.M.D.

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