neural crest cell behavior in white and dark larvae of ambystoma mexicanum: time-lapse...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 229: 109-126 (1984)

Neural Crest Cell Behavior in White and Dark Larvae of Ambystoma mexicanum:Ti me-lapse C i nem icrograp h ic Analysis of Pigment Cell Movement In Vivo and in Culture

R E KELLER AND JOHN SPIETH Department of Zoology, University of California, Rerkeley, Rerkeley, California 94720 (R E K ) and Department ofBiology, Indiana University, Rloomington, Indiana 47402 (<JS i

ABSTRACT The pattern of migration and motile activity of developing pigment cells of the Mexican axolotl, Ambystoma mexicanum, were analyzed by time-lapse cinemicrography in vivo and in culture. In vivo, melanocytes of dark (DL) larvae migrate from dorsal to ventral in a highly directional manner. They are elongated and aligned parallel to the direction of migration. Nearly all protrusive activity occurs a t their ventral, leading edges. Translo- cation occurs a t a mean rate of 0.7 p d m i n and involves alternate or simultaneous advance of the leading and trailing edges of the cell. Indirect evidence suggests that cytoplasmic flow is common. Directional migration occurs in apparent absence of contact between melanocytes. In white (did) larvae, protrusive activity is infrequent and the melanocytes move slowly or not a t all. Explanted neural crest cells of dark and white larvae attach, spread, and differentiate into melanophores and xanthophores in culture. Individual cultured cells are unbiased in direction of protrusive activity and path of migration. Centrifugal spreading occurs by contacting inhibition of movement. Distribution of protrusive activity, polarity, and contact behavior changes with developmental age in vivo and in culture in ways that may be important in establishing the pigment pattern.

The important questions concerning the mechanism and control of movement of cells as individuals in early development are elo- quently posed by the migration of the verte- brate neural crest cells. The crest is formed as a discrete population of cells at the dorsal side of the neural tube, often by processes related to closure of the neural tube. They migrate as individuals or as cell streams along multiple and specific pathways to numerous sites throughout the embryo. There they stop and differentiate or complete differentiation to form numerous cell types [see review by Weston ('83); also see Horstadius ('50), Chibon ('741, LeDourain ('801, and No- den ('78).

There is evidence for the involvement of several factors in controlling the onset, direction, and cessation of neural crest cell migration. The composition and organization of the extracellular matrix is important in these processes but specific mechanisms are not yet known (Pratt et al., '75; Derby, '78; Pin-

tar, '78; Newgreen and Thiery, '80; Mayer et al., '81; Loring et al., '82). Structural alignment of fibrillar matrix may orient migration by contact guidance (Lofberg et al., '80). Contact inhibition of movement, which neural crest cells show in vitro (Epperlein, '74), may direct them from their site of origin in vivo. Chemotactic repulsion may be a factor in amphibians (Twitty and Niu, '54). There is evidence that neural crest cell differentiation and migration are determined by an interplay of factors intrinsic to the cells and by environmental factors (Lehman, '57; Erickson et al., '80; LeDourain, '80; Bronner- Fraser, '82).

It would be useful to know more precisely how neural crest cells move in vivo, what paths they take, and what social behavior they show en route. Here we analyze with

Address reprint requests to R. E. Keller, Department of Zool- ogy, University of California, Berkeley, CA 94720.

0 1984 ALAN R. LISS, INC

110 R.E. KELLER AND J. SPIETH

time-lapse cinemicrography the direction of movement, the process of translocation, and developmentally regulated changes in protrusive activity and contact behavior of pigment cell derivatives of the neural crest both in vivo and in cell culture. The results are correlated with a scanning electron micro- scopic analysis in a companion paper (Spieth and Keller, '83).

MATERIALS AND METHODS Use of axolotl mutants

Time-lapse cinemicrography of pigment cells in vivo was done through a pigmentless epidermis by using larvae that lack primary (egg) pigment. Such larvae were produced from albino mothers as described in Keller et al. (82). Pigment cell behavior was analyzed in dark (D/-) larvae, which show normal migration, and in white (dd) larvae, in which lateral migration of pigment cells is inhibited by the epidermis (Keller et al., '82). Larvae were obtained from the Axolotl Col- ony, Indiana University, Bloomington, Indiana.

Time-lapse cinemicrography (TLC) of pigment cells in vivo

Embryos were manually dejellied in 20% Steinberg's solution at about stage 30 [according to Detlaff ('7511, placed in a 35-mm plastic petri dish containing a 2% agar base and 20% Steinberg's solution with 1 part per 7500 of MS 222 (ethyl-aminobenzoate meth- anesulfonic acid) a t pH 7.4-7.6. Anesthetized larvae were wedged in depressions cut in the agar, and a coverslip, ringed with paraffin, was floated on the surface of the medium. Cinemicrography was done with a compound microscope with heat-filtered, epi-illumina- tion and an Arriflex time-lapse camera and intervalometer.

Cell isolation and culture Tailbud embryos were dejellied manually

and held overnight in 20% Steinberg's solution with antibiotics (10,000 units penicillin, 5 mg Gentamycin, and 25 pg Fungizone per liter). Stage 26 to 30 embryos were placed in 35-mm plastic culture dishes containing a Permoplast base and 100% Steinbergs with 1 mgiml collagenase (Worthington, 107 units/ mg). The epidermis was cut with glass needles, on both sides, from the tail forward, along the midlateral somite level, and dorsally, across the midline just posterior to the hindbrain. After 10 min the epidermis within

the cuts was peeled off with forceps. Then the neural crest was lifted off the dorsal neural tube with a hair loop, washed in culture medium, cut into explants, and cultured in 50% L-15 Liebowitz with 10% fetal calf serum, either on tissue culture plastic, collagen mats, or glass coverslips. Collagen mats were made in 35-mm culture dishes by drying hy- drated collagen lattices prepared by the method of Elsdale and Bard ('72). Cinemi- crography was done as above but with trans- mitted light and phase contrast optics.

Morphometrics The cellular length-width ratio was used

as a measure of cell elongation, and the projected length-width ratio was used as a measure of alignment (Fig. la,b,c,). The efficiency and directionality of migration was determined by comparing the total path traveled, the direct path length, and the net vertical or radial displacement (Fig. Id). The frequency of protrusive activity around the perimeter of the cell was determined by mea- suring the frequency of protrusions forming in each of eight segments of a circle centered on the cell. Because the method is biased in favor of segments containing extended parts of the cell (see Fig. 9 and the Results), the actual positions of protrusion formation were plotted on acetate overlays (Fig. 9).

RESULTS Time-lapse cinemicrography of pigment cells

in vivo The developing, pigmented melanocytes

can be seen through the pigmentless epidermis on the lateral surface of the larvae (Fig. 2). The first neural crest cells that migrate laterally into the subepidermal space form melanocytes (see Keller et al., '82; Lehman, '57). These become pigmented en route and usually reach the midlateral region of the somite before they can be seen through the pigmentless epidermis. In middle and late periods of migration, the cells are pigmented darkly enough to be visible on the dorsal part of the somite. Xanthophore-producing neural crest cells migrate later, at stage 36 and beyond. Nearly all the cells in the subepider- ma1 space in the period of filming (stages 33 to 37) are melanocytes (Keller et al., '82).

The migration paths of the melanocytes are traced through 10 hr of development in a stage 34 larva (Fig. 2, upper panel) and for 22 hr of development in a stage 36 larva (Fig. 2, lower panel). Higher magnification prints

AXOLOTL NEURAL CREST MIGRATION 111

a

b

Fig. 1. A diagram fa) shows how the length-width and projected length-width ratios were measured. The length-width ratio is 1, the maximum diameter within the cell, divided by w, the largest extent of the cell projected on a line perpendicular to 1. The projected length-width ratio is the maximum extent of a cell in some reference direction (L) divided by its maximum extent in the perpendicular direction (W). The reference directions in this work are the dorsoventral axis, in vivo (arrow in b) and the radii of the explants in culture

i C d (arrow in c). The length-width ratio reflects the shape of the cell and the projected length-width ratio reflects orientation of the cell with respect to the reference direction. Diagam (d) shows how the efficiency of movement in vivo and in culture was measured by comparing the total path length (TPL, heavy line), the net, linear displacement (NLD) between starting and final positions (light line), and the net vertical displacement (NVD) in vivo or the net radial displacement (NRD) in culture (dotted line) of cell over some period of time.

Fig. 2. Time-lapse cineframes show melanocytes migrating in vivo from stage 34 onward (top, 0 through 10 hr) and later, from stage 36 onward (bottom, 0 through 22 hr). The asterisk, small and large arrowheads, and

small and large arrows in each frame indicate corre- sponding cells. The light area, apparently free of pigment cells, is the region of the lateral line GL). Dorsal is above in all cases. Magnification is ~ 4 6 .


of time-lapse cineframes are shown in Figure jected length-width ratios (see stage 34, Fig. 3. Those cells leading the migration in 4). As migration proceeds, the cells slow in younger stages (Fig. 2, top panel; Fig. 3) are their movement and spread radially to oc- widely spaced, bipolar or multipolar, and cupy more area on the flank (Fig. 2, bottom). generally elongated and aligned parallel to They also loose their polarity and dorsoven- the dorsoventral axis of the embryo, as indi- tral alignment (see llw and L/W, stages 37- cated by their high length-width and pro- 39, Fig. 4). They form many fine protrusions

Fig. 3. High magnification (~180) time-lapse cineframes show the details of melanocyte translocation in vivo (see text). Five cells are identified by numbers in

the first and last Games. Elapsed time is given in minutes. The bar is 50 pm.


3 r In Vivo In Culture

'\day cultures

3 Sta 3R

I b I I r l 1 I I I

34 37 39 34 37 39 Fig. 4. Plots of length-width (llw, circles) and pro-

jected length-width (LAN, squares) ratios show the pattern of change in vivo and in culture with developmental stage (according to Detlaff, '75) or chronological age (in hours). The open and closed triangles indicate mean

values for cells from pairs of neural crest cell explants from different stage embryos, cultured for the same period of time in the same dish. The stage designations for cultures are determined from the stages reached by companion, intact, control animals.

(Fig. 2, bottom) that are connected by anas- tomosing strands [see Fig. 3n of Spieth and Keller ('8411.

The rate of movement varies between cells and for a given cell with time. A cell may move quickly-on the order of 1.5 pndmin- for a time, slow or stop, and then accelerate. The mean rate of movement and the ex- tremes observed are given in Table 1. At stages 36 to 37, when migration slows, cells in a given region do not cease movement at the same time. Individuals were observed to move past neighbors that were ahead and had stopped.

High magnification shows details of the process of translocation (Fig. 3). At time 0, cell No. 4 is extended dorsoventrally and bears two protrusions at the ventral end, a large one to the right and a smaller one to the left. Its dorsal end bears a small knob of dark pigment that invariably precedes tail retraction (see the same in Fig. 2). The tail retracts concurrently with extension of the leading (ventral) margin (9.3 to 17.7 min), and the cell advances with only slight shortening. About a half hour later (31 min), the tail has retracted in absence of additional extension of the leading margin, and the cell has rounded up in a more ventral position. In the next hour and a half, a new protrusion forms, again at the ventral side, and the cycle

TABLE 1. Rates of movement of axolotl m,elanophores in vivo and in culture

No. cells Cell hours Mean rate and Range analyzed analyzed S.D. (pmlmin) (pmlmin)

In vivo:

18 65 0.8 k 0.21 0.2 to 1.5 16 30 0.7 i 0.19 0.4 to 1.2 In culture:

stage 35 14 65 1.8 i 0.19 0.8 to 3.3

___-

stages 34-35

Means were calculated from total distance/total time measurements for the number of cells indicated. Rates in culture are for cells from stage 28 larvae, in the second day of culture a t a developmental age of stage 35.

of extension and retraction is repeated. Cell Nos. 1 and 3 undergo a similar process in this sequence of film frames.

The positions of the leading and trailing edges of two cells, plotted against time (Fig. 51, shows the cyclical process of lengthening during extension of a leading protrusion and shortening during tail retraction. Less com- monly, the leading and trailing edges will advance concordantly, without change in the cell length (Fig. 5 , cell No. 2, advancing from 8 to 11 hr).

The cells leading the migration are widely spaced and move directly ventrally. Thus col-


lisions are rare. An overlap of images of two

. . collision in which cell No. 1 stopped and : . . . . . . . . . . . . . . . ,~ ,, . . . . . . . . . . . . . ~ .+ .%a changed direction while cell No. 2 continued

to move. The resolution of the film is not sufficient to resolve contacts between the cells. They may have passed without contact in the depth of subepidermal space, though this dimension of the subepidermal "space"

G7.J ''.p.' ' '-.-.@' '...A'' +'" cells (Fig. 5, 4.3 to 6.5 hr) may represent a ... : ..+ i ; . . . . . . : . . . . . . . . . . . . . . . . . i .,

. . . . . . . . . . . . . . j . . . . . . . .

L..

* 'i Y L

0 2.7 4 4.3 6.5 . . . . . ..... ................ .......

p) $"!\ 4% .'% at...s ;,,$ ........% + 'h + 14 Z9 8.5 9.8 10.5- '84).

)i. .%( :" . . 2

.... ,,.-i . . . . . : :..: . . . . : . . . . . . . .:

V is very small (see Fig. 5 of Spieth and Keller, . . f . .

The resolution of the films is not sufficient to establish, by direct observation, that cytoplasmic flow occurs, but there is indirect evidence for it. Tail retraction often occurs with concurrent dilation and advance of a leading edge that is separated from the trailing edge by stationary, lateral protrusions on both sides (Fig. 6,O to 24 min). It appears that the contents of the cell are being moved through a constriction (dashed arrow, at 24 min, Fig. 6) just ahead of these lateral, stable protrusions (large arrows, 24 min, Fig. 6).

Lateral and dorsal protrusions that are not successful in leading the movement of the cell ventrally are retracted individually, to form a thick, dark, knob-like region that typ- ically precedes, and predicts, periods of rapid advance of the dorsal (posterior) margin of

Fig. 5. The paths of movement oftwo cells, Nos. 1 and 2. over 10.5 hr during stages 35 and 36 are shown in the top panel. Below, the positions of the leading and trailing edges of both are plotted against time.

the cell (0 to 88 min, Fig, 6). Retraction of the protrusions at trailing or lateral margins of the cell does not appear to be related to the formation of a tail or to the stretching of

0 24 41 48 62 88 Fig. 6. Tracings of an axolotl melanocyte migrating

in viva at stage 34 shows details of translocation (see text). Elapsed time, in minutes, is indicated at the hot-

tom of each tracing. The bar is 100 pm and magnification is x330.


the posterior region of the cell as is the case in cultured fibroblasts (Chen, '81). Instead, retraction of the trailing margin is usually preceded by the withdrawal of lateral protrusions at the rear of the cell (small, lateral protrusions, 0 min, Fig. 6). The dark, thick- ened knob may flatten and show a scalloped profile, partway through its advance (41 min, Fig. 6). Likewise, two or three protrusions may extend ventrally from the leading (ventral) margin of the cell, but often all but one are retracted dorsally, prior to a major advance of the cell body into the remaining protrusion (data not shown). In other cases, the cell will flow into one of several leading protrusions and thus move forward, leaving the others in lateral and posterior positions, with respect to the cell.

Pattern of pigment cell migration in vivo Continuous tracings of the paths of those

melanocytes that lead the migration show that these cells move from dorsal to ventral with high efficiency (Fig. 7). Occasionally a cell will move laterally (No. 7, Fig. 7) for a short distance but then continues ventrally. The direct path length and net ventral displacement are large fractions of the total path the cells travel (Table 2). The high values of these parameters are a quantitative valida- tion of what is visually apparent from the tracings-the movement is highly efficient and directed ventrally. Indeed, the mean deviation of the direct path length from the vertical (see Fig. Id) is 16" (k10.8, n=21), with an extreme of 40". In many cases, the pigment cells appear to follow one another along common paths that may anastomose or separate (Fig. 7).

Fig. 7. Continuous tracings show the paths of migration of 12 melanocytes in a stage 34 dark axolotl larva. The starting and final positions of each cell are indicated by outlines; symbols indicate the positions at 66 min (squares), 108 min (triangles), and 141 min (arrows). Dor- sal is above. Magnification is ~ 1 4 0 .

Directed protrusive activity in vivo Highly directed protrusive activity (Fig. 8)

underlies the highly directional paths of migration seen in vivo. Sixty percent of all protrusive activity occurs in the ventral quadrant, with an additional 18% occurring in each of the lateral quadrants, and very little in the dorsal quadrant (Fig. 8a). The ventral bias in frequency of protrusive events is greater if only successful protrusions are

TABLE 2. Expression o f the net vertical displacernent (NVL?) or net radial displacement (NRD) and the direct path length

(DPU as a fraction of the total path length iTPL) of axolotl melanoohores in ciivo and in culture

Mean DPL/TPL, Mean NVD/TPL Stage No. of' cells f S.E. or NRD/TPL f S.E. --

In vivo 34-35 51 0.9 i- 0.02 0.9 i- 0.09 36 17 0.9 * 0.10 0.7 f 0.19

35-36 14 0.3 f 0.04 0.2 f 0.04 In culture

The DPLrfPL is a measure of how efficiently a cell moves from star t ing to finishing positions in viva or in culture. The NVDiTPL and NRDiTPL a re measures of how efficiently a cell moves from dorsal to ventral or radially in viva or in culture, respcctively. Measurements in culture are from cells of a stage 28 larvae in the second day of culture and of developmental age of s tages 35 t o 36.

116 R.E. KELLER AND J. SPIETII


Fig. 9. Time-lapse cineframes of a melanocyte at- tempting to migrate laterally in a white larva at stage 35. The time elapsed is indicated in hours. The last frame shows one of the few melanocytes (pointer) that do

considered (Fig. 8b). A successful protrusion is one that is rewarded by having the cell move in its direction. However, measure- ment of the frequency of protrusions formed in segments of a circle centered on the cell is biased in favor of those segments containing extended parts of the cell, because these segments contain more of the perimeter of the cell. However, a plot of the actual positions of formation of protrusions around the cells in vivo bears out the extreme ventral bias (Fig. 8, dot diagrams at top).

Time-lapse cinemicrography of pigment cells in the white axolotl

Cinemicrography of pigment cells in white axolotl larvae demonstrates directly what is known from indirect evidence (Keller et al., '82)-melanocytes cannot migrate in the sub-

Fig. 8. The frequency distribution of protrusive activity in each of eight segments around the perimeter of pigment cells in vivo (squares) and in culture (circles) is shown. (a) Shows distribution of all protrusive activity; and 03) shows the distribution of successful protrusions (see Materials and Methods). The neural crest or the explant side is at 0 or 360", and the ventral or centripetal side is at 180". The actual positions of protrusion formation about the cell are shown in dot diagrams for cells in vivo (top left) and in culture (top right).

manage to migrate laterally in the intersomitic furrows of white larvae, Magnification is ~ 8 4 , except for the last, which is ~ 4 9 .

epidermal space of white larvae. Figure 9 shows the slow advance of the leading edge of a melanocyte in a white larva, and the retraction of its tail ventrally, away from contact with the crest (2.8 to 4.2 hr). How- ever, it subsequently renews contact with the crest (7.2 hr) and fails to change position or shape significantly in nearly 10 hr. Protru- sive activity anywhere on the perimeter of the melanocytes of white larvae is rare. The few melanocytes that do manage to migrate laterally in the white larvae, do so in the intersomitic grooves [see Fig. 3j of Spieth and Keller, ('84)], and they rarely go beyond the midlateral level of the somites. We were not able to film these cells because of their depth below the surface, though their outlines can be seen in light micrographs (pointer, 9.6 hr, Fig. 9).

Isolation and culture of neural crest cells The crest cells of the axolotl are extruded

from the neural tube during its closure and form a cord or "crest" of tightly packed, elongated, spindle-shaped cells, aligned parallel to one another and to the long axis of the neural tube; they remain in this codigura- tion for some time before the onset of migration [Fig. 4 of Spieth and Keller ('84)]. After collagenase-aided, mechanical removal of the epidermis (see Materials and Methods), the


Fig. 10. After mechanical and enzymatic removal of the epidermis (see Materials and Methods), the neural crest can be seen as a cord of cells (above pointer, a), which can he removed, intact, with a hair loop (arrow, b), and cut into pieces from specific somite levels (c) for culture.

cord of crest cells can be seen in the stereo- microscope (Fig. 10a), removed with a hair loop (Fig. lob), and cut into explants that represent the neural crest from a specific somite level (Fig. 1Oc). The explants can be reimplanted into hosts, cultured as explants, or dissociated in EGTA and cultured as individual cells.

Explants of neural crest cells of stage 28 larvae, white or dark, will attach to tissue culture plastic or a collagen mat within 2 to 4 hr and spread centrifugally from 6 to 8 hr onward (Fig. 11). By 3 days in culture, they

have spread to form a monolayer of cells and concurrently differentiate into melanophores and xanthophores on a schedule that is similar to that seen in vivo. Melanophore differentiation is evident in the second day of culture by the appearance of dark granules in the cell periphery, and in the next 5 days the granules accumulate progressively from the periphery toward the nucleus concurrent with the confinement of a decreasing number of yolk platelets toward the nucleus (small arrows, Fig. llb,dj. Xanthophores are first recognized by their retention of yolk platelets and absence of melanin. Later, in 5-7 day cultures, they appear as bright-yellow cells, fewer in number than the melanophores (large arrows, Fig. llb,d). Guano- phores or other cell types are not seen under these culture conditions. The cultured cells rarely divide, and this is consistent with their behavior in vivo for the first 5 days of migration. The isolation procedure is not trau- matic, and cell death rarely occurs. Small populations of dissociated cells can be cultured and individual cells monitored contin- uously, from explantation to differentiation, with video- or cinemicrography. No differences in behavior or differentiation were found between cultures on collagen and tissue culture plastic.

Pigment cell behavior in culture Young explants spread by migration of

their marginal cells centrifugally, followed by submarginal cells, and thus they show directed migration. Migration in culture dif- fers from that in vivo in one basic respect, however. The directionality observed in culture is derived from their contact behavior, whereas that seen in vivo is displayed in absence of contact with one another. In contrast to cells in vivo, cells in culture show no polarity in their protrusive activity (Fig. 8). Moreover, they show little persistence in their direction of movement (Fig. 12). Their net radial displacement and direct path length are small fractions of their total path length, in strong contrast to their behavior in vivo (Table 2). Marginal cells of the explants are polarized in their movement by contact inhibition of movement (Fig. 13). Turns are usually, but not always associated with contact with another cell (track of cell No. 1, Fig. 12). Marginal cells respond to contact by local paralysis of protrusive activity, followed by contact-induced retraction.


Fig. 11. Explants of neural crest cells from stage 28 dark axolotls on collagen (a, b) and plastic (c, d) at 1 day (a, c) and 3 days (b, d) of culture. The small arrows indicated melanin deposition in melanocytes, and the

large arrows indicate xanthophores, which are first char- acterized by lack of melanization and later by their bright-yellow color. Magnification is X 110.


Fig. 12. Continuous tracings show the paths of displacement of nuclei of marginal cells of a young (I-day) culture of stage 28 neural crest cells on plastic. The large arrows in tracing No. 1 indicate the position and direc-

tion of contacts with adjacent cells, The tracings are Positioned such that the parent explants are at the bottom with their radii vertical. The bar represents 100 pm.

Retraction is not usually followed by directed (centrifugal) migration for any distance, away from the site of contact. Instead the cell oscillates to and fro, within one cell diameter, with its movement dominated by first one combination of protrusions and then another (Fig. 13). The new marginal cell of the explant, centripetal to the detached cell, is polarized by having a free margin, and it soon catches up and contacts the detached cell. The combination of contact inhibition movement and lack of ability to polarize the protrusive activity (and thus show persistence of movement) results in an abrupt decrease in cell density at the margin of the explant, few free spaces within the explant, and few cells detached from the explant (Fig. 11).

Cultured neural crest cells of young developmental ages, equivalent to the migratory phase in vivo, are basically bipolar cells with an elongate shape (see peripheral cells, Fig. lla,b) comparable to their counterparts in vivo (Fig. 3). They maintain a length-width

ratio greater than two, comparable to that seen in vivo (Fig. 4). However, they are ori- ented at random, with respect to the radius of the explant, as reflected in a projected length-width ratio of unity (Fig. 4). The film record shows that cells pass from one bipolar configuration, dominated by two major protrusions, to another of different orientation, through an intermediate, multipolar configuration that arises by sprouting of lateral protrusions that grow and dominate the orig- inals (Fig. 1 3 , O to 2:22). Inability to stabilize and maintain protrusive activity a t specific, local sites results in a cell with minimal ability to translocate directionally without exter- nal agents to polarize or stabilize protrusive activity. It is possible that the culture conditions are unfavorable and prevent expression of any directional movement. Occasionally, however, a cell will demonstrate rapid, directional movement over several cell diameters by rapid advance of a broad lamellipodium and concurrent retraction of a blunt, appar-

Fig. 13. Time-lapse cineframes show the instability of protrusive activity, lack of persistence in direction, and contact inhibition that characterize axolotl neural crest cell movement in culture. The explant is at the bottom of each frame. Cell No. 1 (frame 0:OO) has three major protrusions. The two at the right pull the cell toward the upper right. The top protrusion recedes (arrows, 0:20, 0:46) and a new one sprouts nearby (arrow, 0:54). This one grows (curved arrow, 1:04), whereas the other on that end regresses (straight arrow, 1:04). The cell then moves to the right until a protrusion forms on the lower side (arrow, 1:16); the new protrusion grows and moves the cell toward the explant (1:26). Both right and left protrusions (1:04) have split in two parts; the left regresses, whereas one branch of the right forms a long protrusion on the trailing side of the cell (1:16 to 1:48).

The cell rotates clockwise as the end nearest the explant moves to the left and a new protrusion forms on the right side of the elongated cell (see in 2:22). It grows, moves to the right (straight arrow, 3:011, and becomes the leading edge of the cell (3:261; the long protrusion at the earlier, trailing edge retracts (curved arrow, 3:Ol). The protrusions a t the new trailing edge (left in 3:Ol) regresses (curved arrow, 3:43) as a new protrusion forms nearby (straight arrow, 3:43). This new one splits 14:12), and half of it regresses, while the other half becomes the leading edge and moves the cell toward the explant (4:12, 5:ll). There it contacts another cell (5:ll); ruMing of the leading edge ceases in the region of contact and retraction occurs (5:16). Time is indicated in hours:minutes. Magnification is x 105.


Fig. 14. Time-lapse cineframes show a rare, rapid, and directional translocation of a cultured neural crest

cell (arrow). Time is given in minutes. Magnification is x 140.

Fig. 15. High resolution microscopy reveals the mi- straight with bends at discrete points (curved arrows), crospikes and lamellipodia (L) at the advancing margins and they often meet to form what appear to be broader, of cultured neural crest cells. The microspikes are lamellar regions (straight arrow). Magnification is x 732.

ently weakly adherent tail (Fig. 14). This movement is similar to that seen in vivo. Moreover, movement is generally faster in culture than in vivo (Table 11, a fact that

argues against the culture conditions’ inhib- itingmovement.

High resolution objectives reveal the details of advance of the margin of cultured

AXOLOTL NEURAL

neural crest cells (Fig. 15). Filiform microspikes are extended beyond the margin of the lamellipodium, where they wave about, apparently as rigid, distally unattached struts.

Fig. 16. Tracings of the nuclei and adjacent margins of two neural crest cells in an older (3-day) culture shows the repeated, local retractions of the margins following contact and the relatively stable positions of the nuclei of the cells. Magnification is X 156.

I CREST MIGRATION 123

The lamellipodium advances as a concave margin between adjacent microspikes. The microspike-lamellipodial complex may col- lapse back into the lamella of the cell as the microspikes bend at their bases. Microspikes of adjacent cells appear to fuse to form inter- cellular bridges that are maintained in absence of retraction for long periods of time. Contact of the lamellipodia of young cells usually results in retraction, whereas contact between microspikes does not.

Changes in morphology and behavior with developmental age in culture and in vivo The length-width ratios of young cells in

vivo and in culture, a t equivalent developmental ages (stage 34) are about 2.5 and change coordinately with age to near unity by stage 39 (Fig. 4). In vivo (Fig. 2, bottom panel; also see Fig. 3n of Spieth and Keller (‘8411 and in culture (Fig. llb,c), the developing pigment cells progressively lose their asymmetry of shape, become multipolar, and spread over larger area. Culture of pairs of explants from different aged donors for the same period of time shows that developmental age, not time in culture, determines the progressive change in length-width ratio (Fig. 4) and appearance of melanin and mul- tipolarity (data not shown). The rate of translocation also decreases concordantly to zero in culture (see Fig. 16) and in vivo. Finally, the response to contact changes in old, fully differentiated cultures. Repeated contacts of marginal lamellipodia of old cells results in local, short-range retraction of the margins, but there is no general retraction response that might affect the position of the cell as whole (Fig. 16). In contrast, contact among young cells in culture results in retraction of the margin and movement of the whole cell some distance (Figs. 12, 13).

DISCUSSION Motile behavior of neural crest cell during

translocation in vivo

Several points should be made about neural crest cell translocation in vivo. First, they move slowly-about 1 pdmin . Second, movement may occur by simultaneous and equal advance of the leading and trailing edges of an elongate cell of uniform length, or by alternate advance of leading and trailing edges in the manner characteristic of fibroblastic cells in culture (see Chen, %l), but without the formation of a long, tapered tail. Third, advances of the first type seem to involve


cytoplasmic flow. Their movement resembles that of melanoblasts of Fundulus in their monopodial mode of translocation (Arm- strong, '80). They resemble the yolk sac melanocytes of Blenius in appearance (Trinkaus, '82), though the details of translocation may be different; the Blenius melanocytes have not yet been filmed.

Directional migration in vivo Migration in vivo is highly directional from

dorsal to ventral, with little deviation ante- riorly or posteriorly, and it is efficient. The total distance traveled is only 10 or 15% greater than the shortest route from origin to destination.

The basis of this directionality is a strong bias in the formation of protrusions toward the ventral, leading edges of the cells. Biased formation rather than biased effectiveness, argues against mechanisms such as hapto- taxis (Carter, '65), which would imply a uniform distribution in the formation of protrusions but a bias in their effectiveness (see Harris, '73). However, the distribution of effective protrusions is biased somewhat more than that of the formation of protrusions (Fig. 8), suggesting that some selection of protrusions occurs. Cells routinely chose between two or three large protrusions by moving into one and retracting the others, but it is rare that any of the choices available are directed any way other than ventrally.

There are several facts that argue against contact inhibition's playing a large role in this highly directed migration. First, the direct path of migration and high efficiency of movement are not characteristics that would be expected, in theory (see Abercrombie and Heaysman, '53, '54) to result from contact inhibition of movement, and in fact, contact inhibition in culture yields less than a third of the efficiency of directionality displayed in vivo (Table 2). Second, contact between the highly directional leaders of the migration is rare. Contact and the resulting inhibition of movement probably occur in the crowded region near the crest itself and they may con- tribute to directing the cells laterally in this region, but something in addition is needed to account for the highly directional migration of the leading cells.

Contact guidance by aligned fibrils of matrix (Lofberg et al., '80) may guide cell move- ments by contact guidance [see Dunn ('83) for review], but we were unable to find con-

sistent alignment of fibrils in advance of the neural crest migration in the axolotl (see Spieth and Keller, '84). Leading neural crest cells may align the fibrils of the matrix (Spieth and Keller, '84) and thus entrain those behind them to follow in their path. Stopak and Harris ('82) have shown that cells can align matrix fibrils by traction and thus guide movement of other cells. Traction alignment of the matrix by the leaders and subsequent contact guidance of the followers would explain the tendency of the pigment cells to follow one another. Oster et al. ('83) present a convincing argument that cell traction on a fibrillax matrix would not only align the fibrils but concentrate potential adhesion sites and thus generate a haptotactic gra- dient across the cell.

Neural crest cell behavior in culture The behavior of the neural crest cells in

culture sheds some light on their intrinsic motile properties, particularly when com- pared to their behavior in vivo. Young (migratory age) neural crest cells show the following characteristics in culture. They, iike their counterparts in vivo, show protrusive activity a t only part of their perimeter. Thus, their shape is determined by the activ- ities of two or more zones of protrusive activity. Like the cells in vivo, they are basically polar cells with a long axis and one or several major protrusions at either end. However, they are unable to stabilize and maintain a given array of zones of protrusive activity. Thus, they are in constant transition from one bipolar, elongate state to another. They are unable to form a zone of protrusive activity that will dominate the movement of the cell for any length of time. As a result, they show so little persistence in direction of movement (see Gail and Boone, '70) that translocation is negligible. Directional (centrifugal) translocation in culture is the result of contact inhibition and contact-induced retraction. The fact that they maintain, as a population, an elongate form (length-width ratio greater than unity), suggests that they are intrinsically bipolar cells. The fact that they cannot maintain any particular bipolar state suggests that their stability, in this respect, in vivo is due to environmental influ- ences. Moreover, the fact that they cannot maintain a dominant protrusion, even in the bipolar state, in culture, suggests that this too is environmentally induced in vivo.


Developmental changes in shape and contact behavior

Cultured neural crest cells of the axolotl undergo changes in shape and contact behavior that are correlated with developmental age and are independent of time in culture. In culture, they become increasingly multipolar and less elongate. They spread to oc- cupy a larger area. They slow and finallly stop in their movement. These changes in culture are parallel and coincident with those in vivo. Moreover, their contact behavior changes in culture. Contact between young cells results in contact inhibition of protrusive activity and contact-induced retraction. The cells pull apart. Old, multipolar cells do not show contact retraction of the whole cell, but only of a local region of the perimeter. The result is a stable monolayer of contiguous flattened, melanophores, comparable to the spread, contiguous monolayer of melanophores seen at comparable stages of development in vivo. It is likely that these changes reflect an internal developmental program of these neural crest cells, that is independent of the culture conditions.

Other properties may be sensitive to the conditions in culture. Only two cell types differentiated under the culture conditions use- melanophores and xanthophores-even when explants were made prior to the onset of migration and thus at a stage when the progen- itors of other neural crest derivatives would be expected in the crest. The same was found by Lehman (‘57) using different conditions and isolation procedures. These culture conditions do not support differentiation of all neural crest derivatives. It is unlikely that selection is occurring. These cultures have been made under conditions when all cells explanted have been followed, and only one or two cells among hundreds were observed to die. Conditions can modulate differentiation of neural crest cells (see Loring et al., ’821, and this is likely the explanation for the limited number of phenotypes expressed in culture.

Of the phenotypes expressed in these cultures, differentiation parallels their counterparts in vivo, in the morphology and contact behavior noted above, and in the temporal order and frequency of differentiation of melanophores and xanthophores (see Lehman, ’57).

ACKNOWLEDGMENTS

We wish to thank John Shih and Pam

Harty for technical assistance; Alan Warble, who helped in the early stages of the cinemicrography; the staff of the Axolotl Colony, Indiana University, for their determined ef- forts to provide the proper mutants; and es- pecially Fran Briggs, who drew our attention to the fact that the proper crosses would yield larvae in which the melanocytes could be seen in vivo. This work was supported by NSF Grant PCM81-10985 to R. E. Keller, American Cancer Society Postdoctoral Fel- lowship PF 1463 to J. Spieth, and U. S. Pub- lic Health Service Grant R 0 1 GM 05850-21 to the late Robert Briggs.

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neural crest cell behavior in white and dark larvae of ambystoma mexicanum: time-lapse...

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