DEVELOPMENTAL

BIOLOGY

66-79 (1976)

49,

Cell Cycle Changes

during

Neural Crest Cell Differentiation

in Vitro

GERALD D. MAXWELL' Biology

Department,

University

of Oregon, Eugene, Oregon 97403

Accepted October 8, 1975 Chick trunk neural tubes containing neural crest cells were cultured in vitro. Cell outgrowth from these neural tube explants consists primarily of a small stellate cell population. After 3 days in culture the small stellate cell population undergoes a remarkable change in morphology that is characterized by a more refractile appearance in the phase contrast microscope. Subsequent to this change in morphology, pigment granules become visible in the cytoplasm after 4 days in culture. After 6 days in culture, virtually all of the small stellate cells are pigmented. The cell cycle parameters of the small stellate cell population are: S = 4.4 i 1.2 hr (SD). G2 = 1.5 ? 1.0 hr (SD). M = 1.7 t 0.6 hr (SD). and Gl = 3.6 ? 1.0 hr (SD). Continuous label experiments demonstrate that (Gl+GS+M) increases from 7 hr in Day 4 cells, as yet unpigmented, to 12 hr in Day 5 cells that have become pigmented. This change is consistent with an increase in Gl and/or G2 that is closely correlated with the appearance of pigment granules. It is of interest that this cell cycle change is correlated with a rather late event in the developmental program of these neural crest cells rather than with the earlier morphological change observed after 3 days in culture. INTRODUCTION

neurons as early as 12 hr after the formation of the neural crest (Hamburger and Levi-Montalcini, 1949). This neuronal differentiation involves dramatic alterations in the mitotic behavior of these cells. Yates (1961) has quantitated the decline in proliferation rate in the developing sensory ganglion of the chick and has shown that the proliferation rate decreases greatly between the third and seventh day of development. On the basis of histological identification, Yates attributes the small amount of proliferation seen beyond Day 7 to supportive cells. Hamburger and Levi-Montalcini (1949) have demonstrated that grafting extra limb buds onto chick embryos results in more mitotic activity in the sensory ganglion at the level of the graft. This result suggests that control of proliferation in at least some neural crest derivatives is not totally intrinsically programmed very early in development. The proliferation in uiuo of neural crest cells early in development is difficult to study because of the small number of neural crest cells present at any given axial level and the difficulty of distinguish-

A number of adult structures including the pigment cells of the skin, the sensory and autonomic neurons, chromafIin cells of the adrenal medulla, some glial elements, and portions of the cranial skeleton arise in the embryo from neural crest cells (Horstadius, 1950; Weston, 1970; Weston, 1963; LeDouarin and Teillet 1974; Johnston, 1966; Noden, 1975). Prior to their differentiation neural crest cells undergo a period of extensive migration from their origin in the neural folds and dorsal neural tube to their ultimate sites of localization. During and after this migratory phase, neural crest cells also undergo extensive proliferation (Weston, 1963; Hamburger and Levi-Montalcini, 1949; Yates, 1961). As it is for other embryonic structures, precise control of cell migration and proliferation is essential for the normal development and morphogenesis of neural crest derivatives. Some chick neural crest cells in viva differentiate into sensory ganglion ’ Present address: Department of Neurobiology, Harvard Medical School, 25 Shattuck Street, Boston, Mass. 02115. 66 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

GERALD D. MAXWELL

Neural

ing these cells from their environment prior to their differentiation. Dorris (1936, 1938) demonstrated that cultured cranial neural folds of the chick give rise to melanocytes after several days in culture. In the present study, culture methods have been employed that allow the differentiation of melanocytes in vitro from a characteristic cell population derived from trunk neural tubes containing the neural crest. The morphological changes occurring in these cultures during differentiation are described in some detail. As a first step toward a quantitative understanding of the control of neural crest cell proliferation in early development, the cell cycle of this in vitro population of trunk neural crest cells was analyzed and the duration of each component in the cycle determined. Since a culture system is available in which identifiable neural crest cells differentiate into pigment cells after several days in culture, changes in the cell cycle of neural crest cells could be followed as a function of time in culture and state of differentiation. The relatively small number of cells present in early primary neural crest cultures limited the accuracy of cell cycle analysis using the fraction of labeled mitoses method of cell cycle analysis (Quastler and Sherman, 1959). Therefore, variations of traditional approaches were applied to obtain the desired information. MATERIALS

AND

METHODS

Tissue culture. Stage 14 (Hamburger and Hamilton, 1951) White Leghorn chick embryos were removed from the egg aseptically and transferred by widemouth pipette to 60-mm petri dishes containing 5 ml of Hanks’ balanced salt solution (Gibco) buffered to pH 7.2 with 10 mM N-2-hydroxyethylpiperazine -N’ - 2-ethanesulfonic acid, HEPES (Sigma). Vitelline membranes were removed with watchmakers forceps and the embryos transferred to agar albumin plates (Spratt, 1947) for dissection. Embryos were oriented with the

Crest Cell Differentiation

67

ventral surface facing up. Sharpened tungsten needles (Dossel, 1958) were used to remove the neural tube and notochord including and posterior to the last three somites. Significant neural crest cell migration has not yet begun at this axial level of the embryo (Fox, 1949; Weston and Butler, 1966). Neural tubes were not cleaned with trypsin, but mesodermal contamination was minimal (see Results). The neural tubes were then cut into pieces 1 mm long with the notochord attached and were cultured in a nutrient medium containing 50 ml H-17 special Ham’s F12 modified (Gibco), 35 ml Hanks’ balanced salt solution (Gibco), 10 ml fetal calf serum (Gibco), 5 ml chick embryo extract (Cahn et al., 1967), 3.0 mg L-glutamine, 1 ml penicillinstreptomycin (Gibco, 10,000 units penicillin and 10,000 pg streptomycin per ml), and 6.3 ml 5% sodium bicarbonate. The medium was filtered with a sterilized 0.45-pm pore size filter (Millipore). Two or three neural tubes were added to a 35-mm culture dish (Falcon) that had been previously wetted with medium. The neural tubes were then allowed to attach for 25 min in a 10% CO,90% air atmosphere at 37°C before additional medium was added. Cultures were fed every other day by replacing the medium in the dish with 2 ml of fresh medium. Isotopes. Isotopes used were: [methyl3Hlthymidine (6.7 Ci/mmole, New Eng[methyZ-14C]thymidine land Nuclear), (52.7 mCi/mmole, New England Nuclear) and 14,5-3H(N)lleucine (36.6 Ci/mmole, New England Nuclear). Labeling procedures. For continuous labeling experiments solutions of 1.0 $X/ml of 13Hlthymidine were prepared in fresh medium. Labeling was begun by removing the original medium from the dish and replacing it with 1.5 ml of fresh medium containing radioactive thymidine. The labeling medium remained in the cultures until the time of fixation in continuous labeling experiments. For experiments in which a brief expo-

68

DEVELOPMENTAL

BIOLOGY

sure to label was used, a solution of 4.0 &i/ml of 13Hlthymidine was prepared in fresh medium. The cultures were exposed to the radioactive medium for 0.5 hr, the radioactive medium was carefully removed, the cultures gently washed once with Hanks’ balanced salt solution buffered with HEPES to pH 7.2, and then fed with nonradioactive medium. The cultures were allowed to grow in nonradioactive medium until fixation. Histological procedures. In the labeling index experiments, cultures that had been incubated with L3H]thymidine were washed once with Hanks’ balanced salt solution containing 0.5 mg/ml thymidine buffered to pH 7.2 with HEPES and fixed in 10% form01 saline. Cultures were then subjected to autoradiography. After the autoradiographs were developed, cultures were stained with one-third strength Harris hematoxylin (Thompson, 1966). Visualization of mitotic figures was achieved by gently washing cultures in Hanks’ balanced salt solution buffered to pH 7.2 with HEPES followed by fixation in 3 parts absolute ethanol: 1 part glacial acetic acid and cultures were then processed using the Feulgen staining procedure (Thompson, 1966). In experiments to determine the fraction of labeled mitoses, the Feulgen procedure preceded the autoradiography step. Labeled cultures Autoradiography. were subjected to autoradiography using Kodak NTB-2 emulsion diluted 1:l with distilled water at 40°C. Cultures were stored in sealed metal dessicators at 4°C for 1 to 3 weeks, depending upon experimental conditions. Kodak D-19 developer was used for 6 min at 20°C to develop the autoradiographs.

VOLUME

49,

1976

neural tube tends to curve back on itself while attaching to the culture dish, so that the dorsal part of the neural tube is at the circumference. After 10 to 12 hr, cells begin to migrate away from the neural tube, and by 24 hr in culture about 200-400 cells surround the explant in a radially symmetric fashion (Fig. 1). These cells are a characteristic population of small stellate cells that tend to overlap one another at random, especially in regions closer to the neural tube. The radial distribution of the cells in the outgrowth is due both to the folding of the neural tube and to cell migration around the explant to fill uncolonized spaces near the tube. The possibility that some cells migrate between the neural tube and the substrate before migrating into view on the dish cannot be excluded. In some cultures, cells other than the characteristic small stellate cells are seen. These cells are often epithelial or large fibroblastic cells and are distinguishable from the population of smaller stellate cells. In these experiments, the presence of

RESULTS

Growth and Morphology

of

Cultures

Neural tubes cultured on tissue culture plastic show no cell outgrowth during the first 10 to 12 hr of culture. Often, the

FIG. 1. Neural tube explant after 1 day in culture. Phase contrast photograph of a neural tube explant (N) after 1 day in culture. The explant is surrounded by a uniform small stellate cell population. Bar, 200 pm.

GERALD D. MAXWELL

Neural

Crest Cell Differentiation

69

epithelial and fibroblastic cells in the cultures was minimized when neural tubes were carefully dissected and cultured without trypsinization of the neural tubes prior to culturing. Cultures showing extensive epithelial or fibroblastic outgrowth were discarded. Differentiation

of Cultures

As growth of the cultures continues, an intriguing characteristic change in the morphology of the small stellate cells occurs. On Day 3 in culture the cells round up, becoming somewhat less closely associated with the culture dish, and exhibit a more refractile appearance in the phase contrast microscope (Fig. 2). It is important to understand that during the growth of primary cultures the neural tube explant tends to flatten and contribute some epithelial and fibroblastic cells to the culture. These epithelial and fibroblastic cells in general remain isolated from the stellate cell elements and borders between them can be distinguished without difficulty (Fig. 3). By the fourth day in culture, small numbers of pigmented cells can be observed among the small stellate population. These pigmented cells are often near the periphery of the explant. When regions of the culture undergoing pigmentation are examined the number of cells with pigment granules in their cytoplasm increases dramatically between Day 4 and Day 5 in culture (Fig. 4). It is possible to remove the neural tube after the first day in culture and observe that virtually all the cells eventually differentiate into pigment cells. This procedure often tended to impair the health of the cultures; hence, the neural tube was not removed from the cultures in the experiments described below. In a heavily pigmented culture (Fig. 51, there are two important points to note. First, the not yet pigmented cells and lightly pigmented cells are interspersed with the more heavily pigmented cells. The shape of the unpigmented and lightly

FIG. 2. Morphology of prospective pigment cells. Phase contrast photograph of a region of a neural tube culture after 3 days in uitro. The cells appear more rounded and refractile than cells seen in Day 1 cultures. Bar, 50 pm.

FIG. 3. Neural tube culture showing epitheliumprospective pigment cell border. Phase contrast photograph of a neural tube culture after 4 days in culture showing the distinguishable border between an epithelium (E) and a population of neural crest cells (NC) that have undergone the morphological change characteristic of prospective pigment cells. Bar, 200 pm.

70

DEVELOPMENTAL BIOLOGY

1234567 Days in vitro FIG. 4. Percentage of cells with visible pigment granules in regions of the culture undergoing melanogenesis versus number of days in u&o. Neural tube explants were cultured for 1 to 7 days in uitro. After fixation and staining with hematoxylin (see Materials and Methods), regions of the cultures containing the characteristic small stellate cell population were scored for the percentage of cells containing visible pigment granules using bright field optics at a magnification of 494X. Each point represents the mean k standard deviation of at least two separate cultures. At least 500 cells were counted for each point. Regions of the cultures containing epithelia or larger fibroblastic cells were not included in these counts.

pigmented cells is very similar to the more heavily pigmented cells. Second, although the unpigmented and lightly pigmented cells are not separated from the pigmented cells in any obvious pattern over the twodimensional surface of the culture there does seem to be a significant relationship in the third dimension, the cell layering of the culture. Often the unpigmented or lightly pigmented cells act as a substrate for the more heavily pigmented cells. This relationship is seen even at the periphery of the culture where there is ample free tissue culture plastic in the immediate vicinity of the cells. There is also a strong tendency for pigment cells to associate with one another in characteristic two-dimensional arrays made by contacts between adjacent cell processes. It should be noted that neither the char-

VOLUME 49, 1976

acteristic association between pigment cells on a two-dimensional surface nor the cell layering described above are necessary conditions for the expression of the pigment cell phenotype. Pigment cells do express their phenotype as isolated, single cells on tissue culture plastic or on nonneural crest cell substrates such as epithelia from the neural tubes (Fig. 6). Association with other pigment cells or nonpigmented cells is not a requirement for either the establishment or maintenance of the pigment cell phenotype. When White Leghorn embryos are used as the source of neural crest cells, very dark pigment cells begin to form clumps in the cultures on Day 5 (Fig. 7). This clumping appears to be antecedent to the onset of a pattern of pigment cell death in the culture. This phenomenon is genetically determined and has been analyzed at the ultrastructural level (Jimbo et al., 1974; Nichols, personal communication). Due to this phenomenon it is difficult to use White Leghorn cultures beyond Day 7 when clumping becomes quite pronounced

FIG. 5. Melanocytes in a neural tube culture. Pigmented region of a neural tube culture after 6 days in vitro, fixed and stained with hematoxylin (see Materials and Methods). Note that the more heavily pigmented cells are using the less pigmented cells as substrate. Bar, 50 pm.

GERALD

D. MAXWELL

Neural

cyte using an epithelium produced by the neural tube as a substrate. Culture was fixed and stained with hematoxylin after 5 days in uitro. Bar, 50 Frn.

ment cells with a time course similar to that of White Leghorn cultures (Maxwell unpublished observations; Rosenthal, 1971; Zimmerman et al., 1974). The degenerative phenomenon, including extensive cell clumping, that occurs at a specific stage in the development of the White Leghorn pigment cells, does not appear when pigmented breeds are used. Cells that have just begun to make pigment in a White Leghorn culture will independently proceed through a normal sequence of development even in the presence of more mature, already clumped, cells. Thus, the degeneration phenomenon appears to be a part of the genetic program of each cell, and cells in an advanced state of degeneration do not appear to influence adversely the course of development of developmentally younger cells. Extent of Potential gration

FIG. 7. Clumping behavior melanocytes. Photograph of a tube culture after 6 days in vitro of melanocytes that occur when bryos are used (see text). Culture stained with hematoxylin. Bar,

of White Leghorn region of a neural showing the clumps White Leghorn emhas been fixed and 100 pm.

and affects a large percentage of the pigment cells in the culture. Cultures of neural tubes and somites already populated by neural crest cells of pigmented breeds have been tested and produce pig-

71

Crest Cell Differentiation

Pigment

Cell

Emi-

In the cultures described above, numerous cells are seen that differentiate into pigment cells. The question arises as to whether or not all cells capable of making pigment leave the neural tube during the first few days of culture. To examine this question neural tubes were removed from culture after they had been grown in vitro a specified number of days. The original explant was then transferred to a new culture dish and allowed to produce a new outgrowth. The outgrowth was examined for pigment cells during the culture period. The results are shown in Table 1. These results indicate that most, but not all, potential pigment cells leave the neural tube after 2 days in culture. The time necessary for pigment cells to appear in the transferred explant is consistent with the notion that the neural crest cells that initially remained associated with the explant were on the same timetable of pigment differentiation as the cells that migrated substantial distances from the explant. Specifically, cells containing pigment granules in the transferred explant

72

DEVELOPMENTAL BIOLOGY TABLE

1

NEURAL TUBE TRANSFER EXPERIMENT Hours in vitro when neural tube transferred to new dish

Number of cultures eventually containing pigment cellsD 515 415 214 313

24 48 72 96

Number of pigment cells per cultureb

100-400 5-50 l-10 l-10

” Transferred neural tube cultures were observed on each day after transfer. Pigment cells were usually seen by Day 5 from original culturing. In these transferred cultures more fibroblastic cells were seen than in ordinary nontransferred cultures. b The number of pigment cells was scored on Day 6 after explant transfer. In general, no characteristic small stellate cells without pigment granules were observed. Control cultures in which there is no explant transfer in general contain several thousand pigment cells after 6 days in culture.

outgrowth are often observed close to or occasionally on the neural tube itself. This indicates that association with the neural tube does not prohibit pigmentation. The nature of the association of the prospective pigment cells with the neural tube when it is transferred is unknown. They may still be on the dorsal portion of the neural tube, having never migrated, or they may have migrated along the neural tube surface to another location and been temporarily detained. Nutrient

VOLUME 49, 1976

Sephadex G-25) stimulated the differentiation of cloned retinal melanocytes and cartilage cells. Thus, embryo extract contains substances that in some way modulate the quantitative aspects of pigment cell differentiation. Cell Synchrony The presence of synchrony of cell division in a population of cells can influence analysis of the cell cycle by producing values for labeling and mitotic indices that are not representative averages for the entire cell cycle. For this reason the extent of synchrony in the neural crest cultures under study was determined. Parallel cultures were labeled at 3-hr intervals during the day with a 30-min pulse of 1.0 pC!i/ml of 13H]thymidine in the standard culture medium. After 30 min of labeling, the cultures were washed, fixed, and processed for autoradiography. The results are shown in Fig. 8. The data show that there is no signifir 100 k

I I

90 c

Conditions

In all cultures discussed above, the culture medium contained 5% chick embryo extract. This component was found to be extremely beneficial in the differentiation and proliferation of pigment cells in the culture. If embryo extract is omitted from the medium, the number of pigment cells per culture is reduced approximately XL to loo-fold compared to cultures in which embryo extract is used. This observation is consistent with that of Coon and Cahn (19661, who observed that a low molecular weight fraction of embryo extract (that portion of embryo extract not excluded by

8AM

IIAM

2PM

5PM

8PM

IIPM

Time of day 30 minute pulse of 3H-Thym~d~ne odded FIG. 8. Synchrony of DNA synthesis. After 1 day in vitro parallel neural tube cultures were labeled with 1.0 &i/ml [3Hlthymidine for 0.5 hr at times of day shown in the figure. The cultures were then fixed and processed for autoradiography (see Materials and Methods). The percentage of cells with silver grains over their nuclei was determined. Cells with more than 10 silver grains were counted as labeled. At least 150 cells were counted per culture. Each point represents the average of at least two cultures.

GERALD D. MAXWELL

Neural

Crest Cell Differentiation

73

curve plateaus by about 7 hr with about 90% of the cells labeled. The remaining 10% of the cells become labeled by 24 hr. There is no obvious morphological difference in the 10% of cells that require 24 hr to become labeled. These cells do not appear in any particular spatial arrangement with respect to the more rapidly labeling cells. The length of (Cl +G2+M) was taken as the time necessary for the labeling index curve to reach its first plateau, in this case 7 hr. The second minor population has a (Gl +G2+M) of about 24 hr. To determine whether a minor population of cells could be identified by another metabolic criterion, cultures were labeled with 5.0 &i/ml of [3H]leucine for 8 hr and Cell Cycle Parameters then processed for autoradiography. The results show that all of the cells in the Length of (Gl +G2+M). The length of culture are covered quite uniformly with (Gl + G2 + M) was determined by constructsilver grains. No subpopulations with reing a curve of labeling index as function spect to intensity of silver grains could of time the cultures were exposed to be distinguished. Droz and Warshowsky [3Hlthymidine (Fig. 9). In this case, the (1963) have shown that 90% of the silver grains in experiments similar to this represent amino acid incorporated into protein. It appears therefore that the 10% of the cells that take longer to become labeled with [3Hlthymidine are not moribund or reduced in their ability to take up and utilize all low molecular weight precursors. This is consistent with the hypothesis that the observed difference in the ability of the cells to take up L3Hlthymidine reflects an authentic alteration in a process specific to the cell cycle. 2 4 6 8 IO 12 14 16 18 20 22 24 Length of S. Since a convenient measure Time after oddltlon of 3H-Thym~d~ne of the doubling time for cells in primary (hours) culture was not available, it was not possiFIG. 9. Percentage labeled cells versus length of ble to go directly from the labeling index to exposure to r3Hlthymidine. After 1 day in uitro, an actual value for the length of S. To neural tube cultures were labeled with 1.0 &/ml of circumvent this difficulty, three independf3H]thymidine in the culture medium for 0.5 to 24 hr. After the cultures were processed for autoradioent estimates of the length of S were obgraphy the percentage of cells with silver grains tained. The first estimate employed the over their nuclei was determined. Cells with greater fraction of labeled mitoses method of than 10 silver grains were counted as labeled. Each Quastler and Sherman (1959). The results point represents the mean + standard deviation of of this experiment are shown in Fig. 10. at least three cultures. A total of at least 500 cells were counted for each data point. The length of S is estimated by the dis-

cant peak in the percentage of cells with silver grains over their nuclei at any one time over a 15-hr period. The average percentage of cells with labeled nuclei, or label index, is 35 + 6% (mean 2 standard deviation). Thus, there is no apparent synchrony in the population and about onethird of the cells are engaged in DNA synthesis at any given time. No spatial pattern of labeled cells with respect to unlabeled cells was observed in these cultures, indicating an absence of any relationship between DNA synthesis and the spatial proximity of cells in the culture. Subsequent analyses and comparisons were restricted to cultures prepared and labeled at the same time of day.

“C

74

DEVELOPMENTAL

1 I I I2345678

I,

I

I

BIOLOGY

I.

Time (hours) FIG. 10. Fraction of labeled mitosis curve. After 1 day in vitro neural tube cultures were labeled with 4.0 &i/ml of [3HIthymidine in the culture medium for 0.5 hr. The radioactive medium was then removed and the cultures were gently washed once with Hanks’ balanced salt solution buffered to pH 7.2 with HEPES. The cultures were then fed with medium without radroactive thymidine. After the times indicated in the figure cultures were stained for mitotic figures and processed for autoradiography. The cultures were then scored for the percentage of cells in mitosis that had silver grains over their nuclei. Five silver grains per mitosis was scored as a labeled mitosis. Each data point is the mean + standard deviation of at least two cultures. A total of at least 100 mitotic figures was counted for each data point.

tance along the abscissa between the 50% labeled mitoses value on the rising and falling arms of the curve. The value for S by this method is about 4.0 * 1.0 hr (median * standard deviation). The standard deviation was determined by the method of Takahashi ( 1966). The second method used to determine the length of S was the double label method of Wimber and Quastler (1963). This method takes advantage of the fact that 3H and 14C decays can de distinguished autoradiographically when a thick emulsion layer is used. The 3H grains are found in one focal plane, while the more energetic 14C decays produce grains in several planes of focus. Cultures were exposed to [3Hlthymidine at 4 &i/ml for 50 min, after which the medium was

VOLUME

4% 1976

supplemented with 0.04 ml (4 &i) of [14Clthymidine. After an additional 20 min of incubation, in the simultaneous presence of both [14Clthymidine and L3Hlthymidine, the cultures were processed for autoradiography. Cells overlaid only by 3H grains must have passed out of DNA synthesis sometime during the 50 min before the [‘“Clthymidine was added to the culture. This gives an absolute number of cells that have passed out of S in 50 min. The cells with 14Cgrains as well as “H grains represent all the cells in the S during the 20-min label with [‘4Clthymidine. The length of time it would take for all cells with 14Cgrains to pass out of S is the length of S plus the time of exposure to the [‘“Clthymidine. One can then set up the following ratio in which the only unknown is the length of S: number

of cells :lH grains alone

number

cells 14C grains time in [“Hlthymidine

alone

(length of S + time in [‘“Clthymidine)

Thus Length

of S = (time in :‘H alone) x (number (number

with

14C!grains)

- time in 14C.

with “H grains)

The results of these experiments are shown in Table 2. The length of S is 4.3 * 1.2 hr (mean * standard deviation). A third estimate of the length of S is given by extrapolating the initial slope of the label index curve (Fig. 9) back to its intercept on the abscissa. This extrapolation of the label index curve to the intercept on the abscissa represents the average length of time any given cell can incorporate L3Hlthymidine during its cycle which, of course, is the length of S. The estimate of S with this third technique is 4.8 hr, which is in good agreement with the double label method estimate and the fraction of labeled mitoses estimate. This estimation depends, as do other cell cycle methods, on the somewhat idealized assumption that cells move through the cycle at a

GERALD

D. MAXWELL

Neural TABLE

DETERMINATION

Experiment numbeln 1 2 3 4 5

Time 3H alone (min) 50 40 40 40 40

OF THE LENGTH

2

OF S BY THE DOUBLE

Time 14C(min)

20 20 20 20 15

75

Crest Cell Differentiation

Number

3H alone

17 12 16 8 9

LABEL

METHOD”

Number

103 76 68 59 86

‘“C

Length

of S (hr)

4.6 3.8 2.4 4.6 6.0 4.3 2 1.2’

(I Parallel cultures after 1 day in vitro were exposed to [“Hlthymidine at 4 pCi/ml for the designated time, after which the medium was supplemented with 0.04 ml (4 &I) of [Wlthymidine. After additional incubation indicated in the table, the cultures were processed for autoradiography using two or three emulsion layers. Cells with silver grains in only one focal plane were scored “3H alone” positive while cells with grains in more than one focal plane were scored “W’ positive. The poor optical quality of the tissue culture plastic, resulting from the curvature of the plastic, limits the regions of the culture that can be accurately scored at 1250x magnification. This accounts for the relatively small number of cells scored per experiment. ’ Each experiment represents one culture. c Value expressed as mean 2 standard deviation.

constant rate (see Smith and Martin, 1973). Since values for (Gl+ G2 + M) and S have been determined, the total cell cycle time is the sum of their two values. The total cell cycle time is 11.4 * 2.0 hr (mean & standard deviation). Length of M. When cultures were stained by the Feulgen procedure the relative proportion of mitotic figures was 10.6 percent. The length of M was calculated to be 1.7 hr from the relationship:

(1966). Since the M has been calculated above, G2 is simply 2.3 - l/2(1.7), or about 1.5 hr. Length of G1. Gl is the only remaining cell cycle parameter to be determined. By subtraction the length of Gl is about 3.8 hr. Changes in the Cell Cycle as a Function State of Differentiation in Culture

of

In order to detect changes in neural crest cell cycle parameters associated with the morphological differentiation of neural Length of M = crest cells into melanocytes, the labeling (proportion of cells in mitosis) x (cell cycle length) index after a 0.5 hr pulse of [3Hlthymidine In 2 was examined as a function of time in This formula corrects for the fact that in a culture. These data are shown in Table 3. cell population that is growing exponenIt can be observed that the labeling index tially the age distribution of cells around remains quite constant from Day 1 to Day the cell cycle is not uniform (Cleaver, 4 in culture. When the labeling index is 1967). examined in Day 5 cultures, however, Length of G2. The fraction of labeled counting only cells with pigment granules mitosis curve shown in Figure 10 was used in their cytoplasm, the labeling index is to estimate the length of G2 + l/zM. G2 + greatly reduced compared to the labeling l/2M was taken as time necessary for 50% index observed on Days l-4. The presence of the mitoses to become labeled. This of pigment granules in a cell does not invalue is 2.3 ? 1.0 hr (median & standard terfere with the interpretation of autoradideviation). The standard deviation was ographs because the silver grains are localculated by the method of Takahashi calized over the nuclei in a different focal

76

DEVELOPMENTAL

TABLE LABELING

INDEX

3

AS A FUNCTION CULTURE

Days in vitro

1 2 3 4 (unpigmented cells only) 5 (pigment cells only)

BIOLOGY

OF TIME

IN

VOLUME

49,

1976

a large majority of the melanocytes on day five are actually proliferating in vitro (Table 4).

Percentage labeled cells” 35 2 32 2 38 -e 39 t5*4

6 8 4 7

a Cultures were labeled with 1.0 &i/ml of [“Hlthymidine in the culture medium for 0.5 hr after the appropriate number of days in culture. The cultures were then processed for autoradiography. Cells with more than 10 silver grains were counted as labeled. At least three cultures were counted for each day. A total of at least 500 cells was counted for each day. The result is expressed as the mean + standard deviation. All cells included in Day 4 counts are identical to those shown in Fig. 2.

plane than the pigment granules in the cytoplasm. In addition, the silver grains over the nuclei are black, whereas pigment granules in the cytoplasm are brown in preparations stained with hematoxylin. To examine directly changes in the cell cycle apparently occurring between Day 4 and Day 5 when many cells are accumulating visible pigment granules, labeling index curves were constructed for Day 4 and Day 5 cultures (Fig. 11). The results show that while the curve for Day 4 cultures is virtually identical to that for Day 1 cultures (Fig. 9), the curve for Day 5 melanocytes shows a 70% increase in (Gl + G2 + M) to about 12 hr. In obtaining these data, the small number of pigmented cells seen in Day 4 cultures were not included in the determinations. All cells included in the Day 4 determination exhibited the pre-melanocyte morphology seen in Fig. 2. A second important fact provided by Fig. 11 is that about 80% of the Day 5 melanocytes are still capable of incorporating [3Hlthymidine, strongly suggesting that these cells are still in the pool of proliferating cells. The fact that Day 5 melanocytes have a substantial mitotic index is additional evidence in support of the view that

day 5 melanocytes

2 4 6 8 IO 12 14 16 18 20 22 24 Time after addition of ‘H -Thymldine (hours) . . . . .. . . FIG. 11. Percentage labeled cells versus length of exposure to f3H]thymidine for Day 4 and Day 5 cultures. Parallel neural tube cultures after 4 and 5 days in vitro were labeled with 1.0 &i/ml [3H]thymidine in the culture medium or 0.5 to 24 hr. After the cultures were processed for autoradiography (see Materials and Methods) the percentage of cells with silver grains over their nuclei was determined. For Day 4 cultures, only cells similar to those shown in Fig. 2 were counted. The few cells in Day 4 cultures with visible pigment in their cytoplasm were not included in this count. For Day 5 cultures only cells with pigment granules in their cytoplasm were counted. Cells with more than 10 silver grains were counted as labeled. Each data point represents the mean -t standard deviation of at least two cultures. At least 1000 cells were counted for each data point. TABLE COMPARISON

4

OF MITOTIC INDEX OF DAY 5 CULTURES

Mitotic

Culture Day 1 Day 5 (pigment

cells only)

1 AND DAY

index”

10.6 ? 4.4b (N = 14)’ 4.0 + 1.5b (N = 9)’

(1Mitotic index = [(number of cells in mitosis)/(total number of cells)] x 100. For Day 1 cultures at least 150 total cells were counted in each culture. For Day 5 at least 400 melanocytes were counted per culture. b Value expressed as mean k standard deviation. c N = number of cultures examined in determination.

GERALD

D. MAXWELL

Neural

DISCUSSION

Under the conditions used in this study, the trunk neural tube of the chick gives rise to a rapidly proliferating population of cells after 1 day in culture. The identity of these cells as neural crest cells is established by their characteristic morphology and the fact that these cells differentiate into melanocytes while in culture. These cells have the following average cell cycle parameters: S = 4.4 hr, Gl = 3.8 hr, G2 = 1.5 hr, and M = 1.7 hr. It must be emphasized that these values are averages for a population of cells and that some distribution around these values exists. The estimated standard deviations of these parameters are about one hour for each of the parameters. The differentation of these neural crest cells into melanocytes is preceded by a characteristic morphological change in the cells. This change occurs on Day 3 in vitro and is manifested by a more refractile appearance of the cells. This change in cell morphology may result in part from a transient decrease in affinity for the substrate. In addition to the more refractile appearance exhibited by the cells, processes can be seen to develop at the apices of the cells. These processes appeared to be the precursors of the processes seen in mature melanocytes. The events that initiate this change in cell morphology, its relation to biosynthetic events in the melanin pathway, and the mechanism by which the change in cell morphology is affected are not known at present and bear further investigation. Evidence from work on cell lines indicates that CAMP may play a direct role in the regulation of cell shape and cell adhesiveness (Hsie and Puck, 1971; Willingham and Pastan, 1975; Johnson and Pastan, 1972). The possible relevance of cyclic nucleotides to the change in cell morphology observed in neural crest cells prior to the appearance of visible pigment granules should be investigated. Ultrastructural studies on cellular cytoskeletal elements may also provide information

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about this morphological change. The close correlation between the appearance of cells with visible pigment granules in their cytoplasm and the increase in (Gl + G2 + M) is of particular interest (Figs. 4 and 11). The data indicate that (Gl + G2 + M) increases from 7 to 12 hr from Day 4 to Day 5 in culture. The fact that the increase in (Gl + G2 + M) does not appear to be linked to the earlier morphological change seen on Day 3 in culture is also of interest. The actual appearance of visible melanosomes appears coupled to the cell cycle change observed in this system, although the nature and extent of coupling between these two events is not known. The relationship of these in vitro changes in cell morphology and the cell cycle to the in uiuo differentiation of neural crest-derived melanocytes is difficult to assess due to the lack of detailed information about melanocyte differentiation in uiuo. The in vitro results of the present work are, however, consistent with report of Jimbo et al. (1975) that dividing melanocytes can be observed in the feathers of chicks at Days 9-11 of embryonic development. The observations of Cohen and Konigsberg (1975) on the temporal aspects of pigment appearance in quail neural crest cells are consistent with the data presented here. On the basis of preliminary data they suggest that an increased doubling time may be associated with the appearance of visible pigment in their system. The method of analysis employed in the present study allows only the conclusion that the increase in the cell cycle occurs in (Gl + G2 + M). In other developing systems, including cells of the chick neural tube, however, differentiation is often closely coupled with an increase in the duration of Gl with the other cell cycle parameters remaining relatively constant (Wilson, 1973; Buckley and Konigsberg, 1974a). Buckley and Konigsberg (1974b) have demonstrated that different results in cell

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BIOLOGY

cycle analysis experiments may be obtained when labeling studies are done using fresh culture medium as opposed to culture medium that has become “conditioned” in the presence of cell populations. Such an effect seems unlikely in the present work for three reasons. First, the cultures were fed with fresh medium every other day. Second, the number of cells in the cultures, especially during the initial days of culture, was small. Third, preliminary [3Hlthymidine experiments comparing fresh medium to medium “conditioned” from Day 3 to Day 5 in culture show no stimulation of 13Hlthymidine incorporation with fresh medium when Day 5 cultures were labeled for 10 hr (data not shown). In the present study one apparently anomalous fact is that the magnitude of the increase seen in (Gl + G2 + M) in Day 5 melanocyte cultures is not sufficient to account for the decrease seen in the labeling index after a 0.5 hr exposure to [3H]thymidine even when the total percentage of cells capable of incorporating [3Hlthymidine is taken into account. Two possible explanations for this fact exist. The first is that the length of S may be reduced in Day 5 cultures. Although this possibility has not been directly tested in the present work it seems unlikely in view of work on other developing systems in which S remains quite constant (Wilson, 1973; Buckley and Konigsberg, 1974a). A second possible explanation is that cells do not make visible pigment granules in S and since many cells make visible pigment between Day 4 and Day 5 one may be examining the labeling index at a time when cells have acquired pigment granules and an expanded (Gl + G2 + M), but have not yet moved into DNA synthesis again. It must be emphasized that the present experiments do not address the question of the state of phenotypic determination of the cells of the neural crest at the time they are explanted to an in vitro environ-

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ment. The one readily observable neural crest derived phenotype seen in the cultures used in these experiments is the melanocyte. The apparent absence of other neural crest derived phenotypes in vitro is consistent with experiments that indicate that interaction with somitic mesenthyme, conditioned by previous exposure to ventral neural tube, is apparently necessary for neural crest cells to differentiate into sympathetic neurons (Cohen, 1972; Nor-r, 1973). The possibility also exists that other cell interactions, not yet duplicated in culture, are required for the differentiation and maintenance of other neural crest phenotypes. This research was supported by PHS Grant HD05395 to Dr. James A. Weston and NIH Predoctoral Research Fellowship GM-47392 to Gerald D. Maxwell. The author thanks Dr. Donald Wimber for helpful discussions on the cell cycle. REFERENCES P. A., and KONIGSBERG, I. R. (1974a). Myogenic differentiation and the duration of the postmitotic gap (Gl). Deuelop. Biol. 37, 193212. BUCKLEY, P. A., and KONIGSBERG, I. R. (1974b). Avoidance of stimulatory artifacts in cell cycle determinations in vitro. Develop. Biol. 37,186192. CAHN, R. C., COON, H. G., and CAHN, M. B. (1967). Cell culture and cloning techniques. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 493-530. Crowell, New York. CLEAVER, J. (1967). Thymidine metabolism and cell kinetics. Wiley, New York. COHEN, A. M. (1972). Factor directing the expression of sympathetic nerve traits in cells of neural crest origin. J. Exp. Zool. 179, 167-182. COHEN, A. M., and KONIGSBERG, I. R. (19751. A clonal approach to the problem of neural crest determination. Develop. Biol. 46, 262-280. COON, H. G., and CAHN, R. (1966). Differentiation in vitro: Effects of Sephadex fractions of chick embryo extract. Science 153, 11161119. DORRIS, F. (1936). Differentiation of pigment cells in tissue cultures of chick neural crest. Proc. Sot. Erp. Biol. Med. 34, 448-449. DORRIS, F. (1938). The production of pigment in. vitro by chick neural crest. W. Roux Archiv. Ent. Organ. 138, 323-335. DOSSEL, W. E. (1958). Preparation of tungsten microneedles for use in embryological research. Lab. Invest. 7, 171-173. DROZ, B., and WARSHAWSKY, H. (1963). Reliability of the radioautographic technique for the detection BUCKLEY,

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