/. Embryol. exp. Morph. Vol. 55, pp. 17-31, 1980
Printed in Great Britain © Company of Biologists Limited 1980
\J
Translocation of neural crest cells within
a hydrated collagen lattice
By EDWARD M. DAVIS 1
From the Department of Biology, Yale University, New Haven
SUMMARY
Chick neural tubes were cultured either on planar substrata of collagen-coated Falcon
plastic in growth medium with serum or within a hydrated collagen lattice (HCL) in growth
medium either with or without serum. Using time-lapse cinemicrography, neural crest cells
were observed emigrating from neural tubes over the collagen substrata. Once separated
from the neural tube, they seldom reunite with it. Though the average rate at which the
neural crest cells translocate was the same in the different culture conditions, approximately
.l-0/<m/min, distinct differences in morphology and mode of translocation were observed.
Neural crest cells on collagen-coated culture dishes have a flattened fibroblastic morphology
and mode of translocation; in an HC1 with serum, they have a bipolar shape and translocate
by advancing a long, narrow leading protrusion and by periodically retracting the attenuated
trailing portion of the cell; and in a serum-free HCL, they have a unipolar shape and translocate by advancing a long, narrow, branched leading protrusion and by periodically transferring the cytoplasm of the large, rounded trailing cell body forward, past a bulbous structure,
and into the leading protrusion.
INTRODUCTION
Some embryos are sufficiently translucent to allow observation of cell
behavior in their interiors, but the majority are opaque and thus restrict our
view to their surfaces. How then can we observe the translocation (i.e. locomotion from one place to another) of cells within these opaque embryos?
Since we cannot observe them in vivo, we may study their movement in vitro.
Culturing, however, often alters the morphology and mode of translocation,
presumably because of dissimilarities in the in vivo and in vitro environments.
It seems possible that the culture environment may be modified to simulate
the fibrillar nature of the living environment. In such an attempt, Bard & Hay
(1975) cultured chick corneal mesenchyme cells in a hydrated collagen lattice
(Elsdale & Bard, 1972) and found their locomotory behavior and morphology
to be similar to that in situ, although the collagenous lattice lacks glycosaminoglycans and other intercellular components normally found in vivo. In the hope
that use of this culture technique might give us some insight into the mode of
locomotion of other kinds of cells within opaque embryos, neural crest cells of
1
Author's address: Department of Biology, Yale University, New Haven, Connecticut
06520, U.S.A.
18
E. M. DAVIS
the chick embryo were cultured in a similar way. Neural crest cells were selected
for this study because they engage in intensive locomotion as individual cells
during development (Weston, 1970) and, in part, utilize a fibrillar extracellular
matrix as the substratum for their emigration from the neural tube (Ebendal,
1977).
The methods were to culture neural tubes on a flat substratum in growth
medium with serum and within a hydrated collagen lattice, either with or
without serum in the growth medium. Serum was omitted from the medium in
order to determine what effects the presence of serum may have on the form and
migratory behavior of neural crest cells. For at this stage of the development
the circulatory system is still rudimentary (Hamilton, 1952) and the neural tube
is not yet vascularized (Feeney & Watterson, 1946), hence there may be little or
no serum in the normal environment of the migrating neural crest cells.
Rat tail collagen was used to simulate the collagenous portion of the extracellular matrix utilized by translocating neural crest cells in vivo (see Noden,
1978 for review). The locomotory behavior of the cslls was recorded with
time-lapse cinemicrography and compared to other cells that have been observed
translocating within translucent embryos.
MATERIALS AND METHODS
Preparation of collagen substrata
Stock solutions of rat tail collagen were prepared (Konigsberg, 1971) and
diluted with 0-1 M acetic acid to a final concentration of 2-1 mg/ml. Estimates of
the collagen concentration were made, assuming that 10% of the dry weight of
collagen is hydroxyproline. The concentration of hydroxyproline was determined colormetrically (Bergman & Loxley, 1963) by hydrolysing lyophilized
samples of collagen in 6 N-HC1 (azotropic) at 110 °C in a N 2 atmosphere for
24 and 48 h. The hydrated collagen lattice, HCL (Elsdale & Bard, 1972) was
prepared by adding 50 [A. of the collagen stock solution to 4-0 ml of the growth
medium (Dulbecco's Modified Eagle Medium containing 100 i.u./ml penicillin,
2-5 mg/ml Fungizone, and 1-0 mg/ml streptomycin), either with or without
10% fetal serum (Gibco), and adjusting the pH to 7-4 with approximately
20 /d of 0-2 M-NaOH. Falcon tissue culture dishes were coated with collagen as
described by Konigsberg (1971).
Primary cultures of neural crest cells
Neural tubes were isolated from stage-14 (Hamilton, 1952) White Leghorn
chick embryos (Spafas, Norwich, Conn.) by digesting with 0-1% trypsin
(Trypsin 1:250, Gibco) in calcium- and magnesium-free phosphate-buffered
saline, pH 7-4 for 45 min at 4 °C (Cohen, 1972). Though the neural tube could
often be freed from the seventh to the most posterior somite with tungsten
needle after digestion, any somite tissue remaining attached to the neural tube
Neural crest cell locomotion
19
Fig. 1. A frame from a time-lapse film of neural crest cells migrating from a neural
tube (at the bottom of the field) suspended in an HCL with serum and cultured in
sealed coverglass culture chambers at 37 °C. Note that some cells are in focus while
others are not. This results from cells emigrating from the dorsal portion of the tube
at various depths within the three-dimensional substratum of collagen. The culture
was incubated over night before filming. Nomarski interference optics, x 230.
after treatment was excised and discarded. The neural tubes were then cultured
directly on collagen-coated surfaces of Falcon plastic in growth medium with
serum and incubated at 37 °C in a 5 % CO2, 95 % air atmosphere, or were
suspended within an HCL, either with or without serum in the growth medium,
sealed in coverglass culture chambers (Bellco Glass), and incubated at 37 °C.
Cultures were incubated overnight before filming to allow time for the neural
crest cells to begin their emigration from the neural tube.
Time-lapse cinemicrography
Time-lapse films of neural crest cells migrating from the neural tubes were
made with a Bolex Camera and Sage Intervalometer. The camera was mounted
on Nikon Model M and Zeiss inverted microscopes. Phase contrast and
Nomarski interference optics were used throughout. The interval between
frames was 8 or 12 sec. The film used was Kodak 16 mm Plus X Reversal
Movie Film, type 7276, and was developed commercially. Films were analysed
with the aid of a Photo-Optical Data Analyzer, model 224 A, L-W Photo, Inc.
Van Nuys, California.
20
E. M. DAVIS
Fig. 2. A frame from a time-lapse film of neural crest cells cultured on a planar
substratum consisting of collagen-coated Falcon plastic at 37 °C. The culture was
incubated over night before filming. The neural tube is outside the field at the right.
Phase contrast optics, x 460.
RESULTS
Collagen and neural crest cell migration
As previously observed, neural crest cells emigrate readily from explanted
neural tubes on to the surface of collagen-coated plastic tissue culture dishes
(Maxwell, 1976). An HCL also serves as a substratum for translocating neural
crest cells. Single cells and streams of cells emigrate from and seldom reunite
with neural tubes suspended in HCL cultures (Fig. 1). Thus, outward migration
is encouraged but backward migration is somehow inhibited.
Identification of the cells emigrating from trypsin-treated neural tubes
A scanning electron microscopic examination of neural tubes cleaned with
trypsin and freed of somites with a tungsten needle, as in these experiments,
has shown them to be free of somitic tissue (Davis, 1979). Though small
quantities of contaminating flbroblasts may remain undetected, it is unlikely
they affect the analysis presented below, since the emigration of cells near the
dorsal part of the neural tube is so extensive. Two distinct kinds of cells were
observed at the dorsal surface of the neural tube: (1) neural crest cells, which
are similar to those shown by Bancroft & Bellairs (1975 and 1976), Ebendal
(1977) and Tosney (1978), and (2) an occasional small patch of ectoderm. The
small amount of ectoderm presents no problem to the analysis of neural crest
cell translocation for it grows as discrete patches of tissue in HCL cultures that
are easily distinguished from neural crest cells.
Neural crest cell locomotion
21
Table 1. The rates of translocation of chick neural crest cells*
Substrata
Rate of
translocationf
(//m/min)
Number
of cells
observed
Collagen-coated culture dishj
117 ±0-38
9
HCL withserum§
0-83±0-34
12
HCL without serum||
102±013
6
* The rates of translocation were estimated from time-lapse films of single neural crest
cells. Locomotion was measured as the distance traversed by the center of each cell, i.e. the
point midway between the leading and trailing edges.
t The values are given as the mean and standard deviation.
% Duration of observations ranged from 39 to 243 min.
§ Duration of observations ranged from 28 to 83 min.
II Duration of observations ranged from 40 to 130 min.
Additional evidence supporting the contention that the primary tissue of
emigration from trypsin-cleaned neural tubes is neural crest comes from three
sources: (1) Neural tubes were isolated from chick embryos utilizing techniques
previously developed and employed by others in their study of neural crest cell
growth and differentiation in culture (Cohen, 1972 and Cohen & Konigsberg,
1975). (2) The morphology of the cells emigrating from neural tubes explanted
onto a planar substratum of collagen-coated glass (Fig. 2) is very similar to that
of cells cultured under similar condition and shown to be neural crest cells by
other criteria (Cohen & Kongisbsrg, 1975). (3) No cells were observed to
emigrate from the ventral portion of cornally bisected neural tubes suspended in
HCL cultures during a 36 h culturing period, whereas many cells emigrated
from their dorsal counter part (Davis, unpublished results).
Morphology of chick neural crest cells in culture
Single neural crest cells have a morphology similar to fibroblasts (Fig. 2),
when cultured on a plane surface of collagen-coated Falcon plastic. In contrast,
neural crest cells in HCL cultures have a variety of shapes, but generally
maintain a bipolar symmetry. In an HCL with serum, single neural crest cells
have a spindle shape with a long, narrow leading protrusion, a constriction
segment composed of a pair of small constrictions near the center of the cell,
and long trailing protrusion (Figs. 1 and 3). Morphologically, single neural
crest cells in an HCL free of serum differ. They have a long, narrow leading
protrusion with a highly branched leading edge, a bulbous structure composed
of a bulging area between a pair of constrictions located just posterior to the
leading protrusion, and a large, round trailing cell body (Figs. 4 and 5).
Rate of translocation
In contrast to the morphology of neural crest cells, their average rates of
translocation were not greatly altered by the different culture conditions.
22
E. M. DAVIS
Neural crest cell locomotion
23
Single neural crest cells on flat collagen-coated substratum and in HCL cultures
with and without serum translocate at nearly the same average rate of
1-0/«n/min (Table 1).
Mode of translocation
The mode of translocation of neural crest cells is dependent on the culture
conditions. On a flat collagen-coated substratum, neural crest cells translocate
like fibroblasts. In an HCL with serum, however, they translocate by advancing
a narrow, leading protrusion and by periodically retracting a long, attenuated
trailing edge (Fig. 3). Upon retraction, the trailing edge apparently loses its
adhesions to the collagen substratum and within 10 sec the cell length shortens
and a rounded trailing cell body is formed (Fig. 3, frames 64:20 through 64:30).
The protoplasmic extension of the trailing protrusion which had been part of
the extended process in contact with the substratum can be seen as a phase-dark
tail and is quickly incorporated into the rounded cell body (Fig. 3, frames
64:30 through 65:00), and retraction fibers form at the trailing edge of the
rounded cell body (Fig. 3, frames 64:40 through 65:00). The rounded cell body
slowly combines with the leading protrusion so that a uniform bipolar spindleshaped cell is formed (Fig. 3, frame 75:00). Thus, by the continuous extension of
the leading edge and the periodic retraction of the trailing edge, the translocatory process continues.
When serum is omitted from the HCL cultures, neural crest cells translocate
with much cytoplasmic flow. In an HCL without serum, neural crest cells
extend a highly branched leading protrusion and periodically transfer the
cytoplasm of the large, rounded trailing cell body through the bulbous structure
and into the leading protrusion. As the cytoplasm flows, the diameter of the
trailing cell body decreases and the diameter of the leading protrusion increases
(Figs. 4 and 5, frames 12:20 through 17:00). Once the transfer of cytoplasm is
complete, the old trailing cell body and bulbous structure are incorporated into
FIGURE 3
A sequence from time-lapse films of a neural crest cell translocating in an HCL with
serum. Chick neural tubes were suspended in an HCL with serum and cultured in
sealed coverglass culture chambers at 37 °C over night before filming to allow time
for the neural crest cells to emigrate from the neural tube. Time is indicated in
minutes and seconds. The predominant morphology of locomoting cells in this
environment is shown in this sequence. Both leading and trailing edges advance
during cellular translocation. As the cell elongates, constrictions near the center of
the cell develop (arrows). These constrictions divide the cell into three segments: a
leading protrusion (LP), a constriction segment (CS), and a trailing protrusion (TP).
At the onset of the retraction phase, the trailing protrusion retracts, forms a sphere,
and is incorporated into the leading protrusion. Progress in the translocation of the
cell toward the lower left can be measured by reference to the stationary particle in
the gel labeled by the triangle at the lower right starting with frame 57:05. Note
that the orientation of the cell corresponds to that of the collagen fibers. Phase
contrast optics, x 580.
24
E. M. DAVIS
:40
2:05
•' " . t
2:30
3:20
Fig. 4 For legend see opposite.
Neural crest cell locomotion
25
the new trailing cell body and a new bulbous structure develops anteriorly
(Fig. 4, frame 3:20). Occasionally, the leading protrusion shortens (Fig. 5,
frames 16:10 through 17:00); however, this does not alter the shape of the
trailing cell body or the advancement of the branched leading edge.
The rate of advancement of the leading edge of neural crest cells in HCL
cultures with and without serum is nearly uniform, albeit it fluctuates in a series
of small extensions and retractions (Fig. 6). In contrast, the trailing edge advances
at two velocities, one slower and the other greater than the rate of advancement
of the leading edge. The slow advancement of the trailing edge may result from
distortions elicited in the collagen fibrils by the translocating cells. The rapid
advancement of the trailing edge of neural crest cells in an HCL with serum
occurs as the trailing edge loses its adhesions to the collagen substratum and
springs forward. In an HCL without serum, the fast rate of advancement of the
trailing edge of neural crest cells occurs as the cytoplasm of the cell body pours
forward through the bulbous structure and into the leading protrusion.
Advancement of the leading edge during translocation
The advancement of the leading edge of neural crest cells in an HCL is not
restricted by the rate of advancement of the trailing edge. In Fig. 6 A, the extension of the leading edge is not constant but fluctuates from the average rate
of advancement which is represented by the straight line. This variability in rate,
however, does not coincide with the rate of advancement of the trailing edge.
Note that during periods just prior to the onset of the rapid advancement of the
trailing edge (Fig. 6 A, 10 and 40 min), when the cell is near its maximum length,
the rate of advancement of the leading edge is slightly greater than its average
rate. This observation suggests that even at times when the cell is approaching
its maximum length and is presumably under the greatest tension, the advancement of the leading edge is not impeded.
Cell length changes during translocation
The mode of translocation of neural crest cells in an HCL produces continuous variations in their cell length. These changes in cell length are produced
Fig. 4. A sequence from a time-lapse film of a neural crest cell translocating within
an HCL without serum in the growth medium. Chick neural tubes were suspended
in an HCL without serum and cultured in sealed coverglass culture chambers at
37 °C over night before filming to allow time for the neural crest cells to emigrate
from the neural tube. This sequence demonstrates the changes that occur in the
cell shape during translocation. As the cell translocates towards the right, the
leading edge is extended and periodically the cytoplasmic content of the trailing
cell body (CB) is transferred past the bulbous structure (BS) and into the leading
protrusion (LP). A new cell body and bulbous structure develop anteriorly, and the
translocatory process is repeated. Time is indicated in minutes and seconds. Phase
contrast optics, x 1660.
E. M. DAVIS
Fig. 5. A sequence from a time-lapse film of a neural crest cell translocating in an
HCL without serum in the growth medium. Chick neural tubes were suspended in
an HCL without serum and cultured in sealed coverglass culture chambers at 37 °C
overnight before filming to allow time for the neural crest cells to emigrate from the
neural tube. Time is indicated in minutes and seconds. Typical morphology of
single neural crest cells translocating within this environment is shown in this
sequence. Note the rounded cell body and the branched leading protrusion. Both
leading and trailing edges advance continuously during cellular translocation. The
trailing edge advances in part by cytoplasmic flow, as in frames 12:20 through
17:00. Phase contrast optics. x460.
Neural crest cell locomotion
27
by the different rates of advancement of the leading and trailing edges. Thus as
neural crest cells translocate in an HCL, the cell length increases when the
trailing edge advances at a slow rate and shortens when the trailing edge advances at a fast rate.
DISCUSSION
Behavior of neural crest cells in culture
Collagen clearly promotes rapid outgrowth of cells from the neural tube
(Maxwell, 1976), whether coated on flat substrata of glass or plastic or in the
form of an HCL. On a flat collagen-coated substratum, neither the morphology
nor mode of translocation of neural crest cells appear to differ from that of
fibroblast-type cells cultured in similar conditions (for summary see Trinkaus,
1976). As on flat substrata, there are many similarities between neural crest cells
and fibroblasts of different tissue origins in HCL cultures with serum in the
growth medium. Human embryonic lung fibroblasts (Elsdale & Bard, 1972),
corneal mesenchyme (Bard & Hay, 1975), and neural crest cells all possess a
bipolar, spindle shape, as they utilize the collagen fibrils as substrata for
translocation. In contrast to the attenuated trailing process of fibroblasts on flat
substrata and in an HCL with serum, however, neural crest cells in an HCL
without serum maintain a rounded trailing cell body containing much of the
cytoplasm of the cell, even when the cell is fully extended, and an attenuated
branched leading protrusion. In the absence of serum, the cells translocate in
part by pouring the cytoplasm of the large, rounded trailing cell body into the
leading protrusion.
Although the morphology and mode of translocation of neural crest cells on
planar and three-dimensional collagen substrata with and without serum in the
growth medium differ greatly, their average rates of translocation are nearly
equal and about the same as for other fibroblast-like cells cultured under
similar conditions (Trinkaus, 1976), about 1 fim per min. Why neural crest cells
in the three different cultures translocate at nearly the same average rate is
unclear. In particular, it is puzzling that neural crest cells in an HCL without
serum translocate at the same low rate, for their mode of translocation has been
associated with rates of cellular translocation eight to ten times greater than
that of fibroblasts. For example, Fundulus deep cells in vivo (Trinkaus, 1973) and
polymorphonuclear leukocytes in vitro (de Bruyn, 1946; Ramsey, 1972) both
show much cytoplasmic flow during locomotion and both translocate at an
average rate of 8-10 jum/m'm. The significance of a rounded cell body and
cytoplasmic flow for the rate of locomotion of neural crest cells is, therefore,
unclear. Apparently, the association between rapid rates of translocation and
cytoplasmic fluidity previously observed with other cells (cf. Trinkaus, 1976)
cannot be generalized.
A second similarity between neural crest cells in HCL cultures with and
without serum, is the rates at which neural crest cells advance their leading and
28
E. M. DAVIS
50-
30-
10-
10
'
30
'
50
Fig. 6. For legend see opposite.
Neural crest cell locomotion
29
trailing edges. In both cases, the leading edge advances at a nearly uniform rate
and the trailing edge retracts at two different rates, one greater and the other
slower than the rate of advancement of the leading edge. I have no explanation at
present for this similarity.
Similarities between neural crest cells in HCL cultures and embryonic cells in vivo
Although this study has been concerned with the behavior of neural crest
cells in vitro, there are some remarkable similarities between neural crest cells
in HCL cultures and other types of embryonic cells observed directly in vivo.
In an HCL with serum, neural crest cells appear and translocate like corneal
mesenchyme cells migrating within an HCL with serum or through the
collagenous stroma between the endothelium and epithelium of the developing
eye of a chick embryo in situ (Bard & Hay, 1975). Both neural crest cells and
corneal mesenchyme cells have spindle-shaped bodies and long, branched
filopodia. In contrast, neural crest cells in an HCL without serum appear much
like the primary mesenchyme of echinoderms (Gustafson & Wolpert, 1961) and
Fundulus deep cells translocating over the enveloping layer and yolk syncytial
layer of gastrula embryos (Trinkaus, 1973) in that they possess a rounded cell
body and a long, extended leading protrusion. In addition, the mode of transloction of neural crest cells and Fundulus deep cells is similar, with each translocating, in part, by pouring the cytoplasm of the large, rounded trailing cell
body into the leading protrusion.
The purpose of studying neural crest cells in HCL cultures was to simulate
Fig. 6. Abscissa: time (min) cells were observed in locomotion; ordinate: distance
traversed C«m) by the leading edge (O) and the trailing edge (A). The translocating
activity of neural crest cells in HCL cultures with and. without serum in the growth
medium was recorded with time-lapse cinemicrography and the relationship between
the rates of advancement of the leading and trailing edges during locomotion was
compared. (A) Neural crest cells in an HCL with serum. This figure represents the
translocatory behavior of one neural crest cells. The leading edge advances at
nearly a constant rate (approximately 1 -0 /tm/min) which equals the average rate of
cellular translocation, whereas the trailing edge advances at two different velocities
that are either slower or faster than the average rate of cellular translocation.
Though many cells were observed for shorter periods of time, only two cells were
observed to translocate for nearly 60 min and in each case, the leading edge advanced
at a nearly constant rate, whereas the advancement of the trailing edge was biphasic.
The longer periods of observation were necessary to assure that the advancement of
the trailing edge would repeat the slow and fast rates of advancement. In both
cases, the advancement of the leading edge was nearly constant, whereas the
advancement of the trailing edge was biphasic. (B) Neural crest cells in an HCL
without serum. This figure represents the translocatory behavior of one neural crest
cell. The rates of advancement of the leading and trailing edges on neural crest cells
in an HCL without serum do not differ from the advancement rates of cells in serum.
The leading edge advances at nearly a constant rate, whereas the trailing edge
advances at two different rates. Three cells were observed to translocate for nearly
60 min and in each case, the leading edge advanced at nearly a constant rate,
whereas the advancement of the trailing edge was biphasic.
3
EMB
55
30
E. M. DAVIS
the collagenous component of their fibrillar, in vivo environment in order to
gain some insight into the possible appearance and mode of translocation of
neural crest cells in vivo. It was found, interestingly, that the morphology of the
trailing end and mode of translocation of neural crest cells in HCL cultures are
both dependent on the presence or absence of serum in the growth medium.
It is, of course, possible that serum in the growth medium selects for different
cell types. This seems unlikely, however, since cells in the presence or absence of
serum in HCL cultures are similar in many ways, including the morphology of
their leading protrusion, constriction near the center of the cell, and the rate at
which they translocate. It seems likely, therefore, that the same types of cells
are being observed in the presence and absence of serum.
This study has shown that the mode of translocation of neural crest cells is
related to their shape. Since their translocation in vivo cannot be observed
because of the opacity of the chick embryo, it may be possible to deduce how
neural crest cells translocate in vivo by observing their form. One can observe
their form by fixing embryos during times when neural crest cells are known to
be translocating and examining the neural crest cells with SEM. This has been
done recently in three independent studies (Bancroft & Bellairs, 1976; Ebendal,
1977; Tosney, 1978). The works of Bancroft & Bellairs (1976) and Tosney
(1978) show that neural crest cells near the edge of the outgrowth are elongated.
These cells appear similar to that of neural crest cells in HCL cultures with
serum. In contrast, however, no one using SEM to observe neural crest form
has reported observing neural crest cells with a rounded trailing cell body, such
as found in an HCL lacking serum. In consequence, it seems reasonable to
predict that at least some of the neural crest cells in vivo translocate like cells
in HCL cultures with serum, that is without massive cytoplasmic flow. If this is
so, an important matter in future investigation of the mechanism of the translocation of neural crest cells will be to determine which components of the
extracellular matrix or serum are responsible.
I am indebted to Professor J. P. Trinkaus for his assistance in preparing this manuscript
and to Dr Ray Keller and Mr Wen-Tien Chen for their discussion and assistance. This
research has been supported by grants from the NSF (BMS 70-00610) and the NIH (USPHSHD-07137) to J. P. Trinkaus and by postdoctoral fellowships from the American Cancer
Society (PF-1330) to E. M. Davis and a NIH Cell and Developmental Biology Training
Grant (USPHS-HD-00032-13) to the Department of Biology of Yale University.
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3-2