DEVELOPMENTAL

BIOLOGY

46, 262-280 (1975)

A Clonal Approach ALAN Department

ofAnatomy, Department

to the Problem M. COHEN

of Neural

AND IRWIN

Crest Determination1

R. KONICSBERG

The Johns Hopkins University School of Medicine, Baltimore, Mayland of Biology, The Uniuersity of Virginia, Charlottesuille, Virginia 20293

21205 and

Accepted May 12.1975 A fundamental question regarding neural crest development is the possible pluripotential nature of this embryonic tissue. As a first step in examining this problem, clonal techniques are used to produce homogeneous populations of crest cells. Primary cultures of these cells are obtained by explanting neural tubes from Japanese quail in uitra. and allowing crest cells to migrate away. The explant is removed, the outgrowth is isolated, dissociated with trypsin, and the cells plated at clonal density. Colonies derived in this manner fall into the following categories: all cells of the colony pigmented; none of the cells pigmented; and some of the cells pigmented, the remainder unpigmented. Pigmented colonies generally arise from small, round cells whereas the non-pigmented colonies usually originate from large, flattened polymorphous cells. Differentiation of melanocytes does not preclude their continued proliferation. The pigment phenotype, in addition, is stable through at least 25 generations. That the mixed colonies, in fact, are clonally derived is shown by physically isolating single cells. The identity of the non-pigment cells was not established in the present work. A possible neural fate is suggested, however, since nerve-like cells develop after the petri plates become overgrown. Neural clones did not form even though nerve growth factor activity is present as a normal constituent of the culture medium and was added as a supplement in some instances. These techniques permit the preparation of large, homogeneous populations of neural crest cells and afford an opportunity to examine aspects of crest determination heretofore impossible to study:

tion (Cohen, 1972; Norr, 1973). For example, the difference in abilities of head and trunk crest to differentiate into cartilage (Raven, 19361 suggests a diversity inherent among crest cells. Also, the appearance of pigment in cultures of developing sensory ganglia has been interpreted similarly to represent the expression of determined but unexpressed melanocytes migrating past the ganglia to their definitive site in the mesenteries (Peterson and Murray, 1955). On the other hand, evidence also exists which suggests that diversity among crest derivatives is due to localized, special conditions acting on cells with diverse developmental potentialities. For instance, dorsal ectoderm implanted into the flank in Amblystoma recruits crest cells there to form a fin. Bodenstein (1952) suggests that these cells were destined by their location to synthesize melanin, but could be induced by dorsal ectoderm to secrete connective tissue components. Likewise, Cowell and Weston (19701 submit that the

INTRODUCTION

The neural crest represents a population of cells which, although initially contiguous and localized, migrate extensively from their origin and eventually populate diverse locations in the organism. In different definitive sites these cells differentiate into specialized cells with distinctly different functions. It has been variously suggested that this diversity is: a) present initially before migration, but unexpressed (Hamburger et al., 1966; Mintz, 1967; Holtfreter, 1968); b) is imposed by the environment provided by the final localization (Horstadius and Sellman, 1946; Silvers and Russell, 1955; LeDouarin and Teillet, 1974); c) or represents responses to influences encountered along the path of migra*Supported by United States Public Health Service Grant (HD 07083) and a National Science Foundation Grant (GB 5963X3 to I. R. Konigsberg, and United States Public Health Service Grant (HD 07389) and a grant from The National Foundation -March of Dimes to A. M. Cohen. 262 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

COHEN

AND

KONIGSBERG

appearance of pigment in cultured sensory ganglia is due to the influence of culture conditions on pluripotent crest cells. Any examination of neural crest potentialities as well as the mechanisms involved in the expression of the definitive phenotypes would be aided considerably by the ability to work with uniform populations of cells whose response to environmentally provided stimuli could be tested and measured. The objective of the present study is to apply clonal techniques for the purpose of obtaining homogeneous populations of crest cells. In this study, crest cells are isolated before they migrate and interact with those tissues thought to affect their differentiation. The cells are plated at clonal density and their behavior under these conditions is described. MATERIALS

AND

METHODS

Japanese quail (Coturnix coturnix japonica) eggs incubated in a forced draft, humidified incubator at 37.8”C for 47 hr were used throughout this study. Quail embryos incubated for this period have morphological features comparable to chick embryos at stages 14-15 based on the criteria of Hamburger and Hamilton (19511. Cell outgrowth from neural tubes. Neural tubes from quail embryos were isolated using the same techniques developed for preparing chick neural tubes (Cohen. 1972). Embryos were removed sterilely, placed in Pannett-Compton’s solution (Pannett and Compton, 1924) and the trunk region consisting of the posterior 6-9 segments was dissected out by means of electrolitically-sharpened tungsten needles. The trunks were dissociated at 5°C with a l’% solution of crude trypsin (Trypsin l-300, Nutritional Biochemicals) (Rawles. 19631 in saline G (Puck et al., 19581 for 25 min. Trypsinization was stopped by replacing the enzyme with a 10% solution of horse serum in saline G after which the tissue was gently pipetted

Clonal Studies

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263

with a small-bore Pasteur pipette to separate the neural tube from other components of the trunk. Purity of the neural tubes was checked by careful visual inspection. Chick neural tubes prepared in this manner are free of basal laminae as well as adhering mesenchymal cells (Cohen and Hay, 19711. The purpose of the primary culture is to allow crest cells to migrate away from the neural tube after which the tubes were discarded. Up to four neural tubes were transferred to 35 mm Falcon tissue culture petri plates coated with collagen (Hauschka and Konigsberg. 19661 purified as described (Konigsberg. 19711. The acid collagen had been previously polymerized by the addition of 6c(’ NaCl, spread on the floor of the culture dish with a bent glass applicator, and the dishes stored in a humidified chamber. After transferring the neural tubes, a small amount of growth medium (see below). not enough to float the tissue, was added to prevent dessication. The tissue was incubated at 36.5”C in a humidified atmosphere of 5’; CO, in air as were all the cultures in this report. After 1 hr. the neural tubes adhered sufficiently and 1 ml of medium was added. After 20-24 hr the neural tubes were carefully detached from the dishes with tungsten needles and discarded. The cells that had emigrated from the explants were fed with I.5 ml of fresh medium. Clonal cell cultures. The primary outgrowth was cultured for an additional 48 hr by which time sufficient numbers of cells were available to allow dissociation and counting. Clones of crest cells were established using modifications of techniques designed for cloning muscle cells (Konigsberg, 19631. The primary cultures were rinsed with saline G, and a Pyrex cylinder (5 mm inside diameter, 7 mm outside diameter) with a continuous rim of silicone grease on its lower edge was pressed around each outgrowth. About 0.4 ml of a crude preparation of 0.025% collagenase (Worth-

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ington CLS) in saline G was added to each cylinder and the culture dishes were maintained at 37°C on a warming bar for 20 min with intermittent pipetting to dislodge the cells from the plate. The cell suspension was added to an equal volume of cold medium and was centrifuged for 5 min at 800 rpm in a clinical centrifuge. The supernatant was removed by aspiration and the pellet was resuspended in a total of 0.4 ml growth medium by pipetting with a hypodermic syringe mounted with a 20-gauge spinal-tap needle. The cell suspension without filtering was counted in a PetroffHeuser bacterial cell counter and was diluted appropriately in growth medium. About 70%’ of the cells were single cells; the remainder were mostly doublets with larger clumps present occasionally. Clonal cultures were inoculated with 200 cells into 60 mm Falcon petri plates coated with gelatin and preincubated with 2.0 ml of medium. After 24 hr the medium was replaced with 3 ml fresh medium and the cultures were fed every third day thereafter. Growth medium. This consisted of 10 parts embryo extract, 15 parts horse serum, 74 parts Eagles MEM (with Earle’s salts but with 1.2 g NaHCO,/liter), one part antibiotic mixture (10,000 units penicillin and 5 mg streptomycin per ml of stock solution) and 0.25 ml Fungizone (Squibb) (0.8 mg/ml of distilled water) for every 100 ml of complete medium. The medium was sterilized by filtration through a Millipore filter of 0.45 pm pore size. Fixation and staining. The cultures were fixed for 10 min in 1.6% glutaraldehyde

VOLUME 46, 1975

buffered by 0.05 M sodium cacodylate at pH 7.2-7.4. The fix was replaced with buffer for 10 min, and the plates were rinsed briefly in distilled water and stained with Carazzi’s (1911) alum hematoxylin. The surface of the culture dish was protected by a t.hin plastic coating applied by rinsing with 5%, aqueous polyvinyl alcohol and was dried on a warming bar at 60°C. RESULTS

Behavior

of Neural

Tube Explants

The observation that crest cells freely migrate from a neural tube explanted in vitro was first reported by Dorris (1936) and has been exploited here as a first step in isolating crest cells. Neural tubes were removed from posterior regions of axial trunks where it is known that crest’ cell migration at the stage considered has progressed but little if at all (Fox, 1949; Nawar, 1956). This insures a full complement of potential crest derivatives in each neural tube isolated. The neural tubes readily attach to collagen coated petri plates and within 3-6 hr, mesenchymal cells begin migrating from the explant. The sequence of events characterizing this phenomenon is illustrated in Fig. 1 A-D. For purposes of orientation, carbon particles were affixed to the basal surface of the tube in several experiments (arrows, Fig. 1). One striking behavioral feature of the explanted neural tube is that the emigration of cells occurs initially only from its dorsal surface, much as crest cells do in vivo. Mesenchymal cells occasionally migrate from foci on the basal surface during this early period but these cells seem to arise

FIG. 1. The migration of neural crest cells from the neural tube is shown in this series of composite phase contrast photomicrographs of a single living neural tube explanted in uitro. Blood charcoal (arrows) was applied to the ventral (notochordal) surface of the neural tube before it was placed in vitro to indicate polarity. One of the lateral surfaces of the neural tube is in contact with the collagen substratum of the petri plate. (A) During the first 2 hr no cells migrate from the explant. (B) After 8 hr mesenchymal cells begin to migrate away from the dorsal aspect of the tube. (C) Within 20 hr hundreds of cells have left the neural tube, primarily from the dorsal surface. A few mesenchymal cells are seen along the ventral surface, arising from an area damaged while explanting the tube. In addition, one end of the tube has flattened to form an epithelial sheet. tD) The mesenchymal outgrowth has been isolated by carefully dissecting away the neural tube and its tlattened epithelial end. The remaining primary outgrowth is grown for an additional 2 days, until enough cells are available for routine cloning procedures. x 65.

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Clona~ Studies

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Crest

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DEVELOPMENTALBIOLOGY

only from areas accidently damaged during explantation of the neural tube. Such an area is shown (Fig. lB, C) and serves to contrast the morphogenetic tendencies of the dorsal and ventral surfaces of the neural tube. The transected ends of the neural tube either remain intact or spread as an epithelial sheet. The assumption that the mesenchymal cells leaving the roof of the neural tube in fact represent exclusively the neural crest cells, predicates the next step in the isolation. After 1 day in uitro, at which time there are some 500-1000 mesenchymal cells (Fig. lC), the neural tube and flattened epithelial areas are carefully detached and discarded. The cells left behind constitute the primary culture (Fig. lD), consisting of fibroblast-like cells, indistinguishable from one another. Cytodifferentiation begins 3-4 days after the neural tube is removed at which time pigment granules are first detected in a few cells. Pigmentation increases both in intensity and number of cells during the next few days. The pigmented cells represent a variety of morphological forms, some of them polygonal and smooth in outline, while others are highly branched. Description

of Neural

Crest Clones

In order to determine whether the progeny of single crest cells are capable of differentiating into more than one definitive type of cell (that is, for example melanocytes as well as neurons) secondary cultures were established at clonal density. The immediate goals of these experiments were to establish homogeneous populations of crest cells and to determine with what frequency, if any, colonies of multiple cell types arise. Cell suspensions were prepared from primary cultures on day 3, i.e., 2 days after the original neural tube explant had been removed. By this time frank cytodifferentiation has not begun, although shortly thereafter, by day 4, pigment granules

VOLUME 46,1975

would have been present in some cells (see above). Series of petri plates were inoculated with 200 cells each and within 5 days, small foci of pigmented cells are observed macroscopically. On closer examination, however, it is clear that three different colony types are present. The frequency of each colony type is compared in cultures fixed and stained 8 days after seeding (first line, Table 1). By then the melanocytes are heavily pigmented and the colonies of nonpigmented cells are relatively large (Fig. 2). Scoring older, 2-week cultures is complicated by the marginal overlap of colonies resulting from continued growth in area. These older cultures are not qualitatively different from the &day cultures; therefore 8 days has been chosen as an end point when comparing colonies. Some colonies consist of cells all of which are pigmented (Fig. 3); others are composed of uniformly unpigmented cells (Fig. 5); and in still a third type the center of the colony consists of pigmented cells surrounded by a fringe of non-pigmented “fibroblast-like” cells (Fig. 4). The colonies derived from the neural tube outgrowths differ not only in pigmentation but also in size. The pigment colonies are compact and smaller in diameter than either the non-pigment or mixed colonies (Table 2). The morphology of cells in a pigment colony varies from closely packed epithelial cells (Fig. 3) to irregular dendritic cells, with both forms often present in the same colony. In older pigment colonies (11-15 days) the epithelial configuration predominates giving a highly regular arrangement, resembling a cobblestone pavement (Fig. 6). Although the pigment colonies consist entirely of pigment cells, the degree of pigmentation is not uniform from cell to cell throughout the colony. Punctate and dendritic cells appear black in color and more densely pigmented whereas epithelial and lightly pigmented cells are usually brown. Observation with oil immersion objectives show only brown

TABLE BEHAVIOR

Medium

1

OF CLONED CREST CELLS~

Mean plating efficiency0 Pigment

Nutrient medium Nutrient medium + NGF

26’i

CIonal Studies of Neural Crest

COHEN AND KONIGSBERG

Mean ‘; differentiation’ Non-pigment

Mixed

48.4 * 10.2 (N = 22, 49.8+4.4(N=6)

67.5 r 11.6 IN = “21 67.1 2 5.6 (N = 6)

29.1 r 11.4 tN = 22, 27.7 T 5.9 (N = 6,

3.4 I 2.4 (N = 2”) 5.” zc 1.21N = 6,

P > 0.7Od

P > 0.90

P > 0.40

P > 0.05

“Petri plates were seeded with 200 cells per dish and scored atter :I days. bValues are presented as mean plating efficiency [(total colonies per platet/tnumber of cells seeded per plate) x 1001 per petri plate + standard error of the mean. ‘Values are presented as mean ‘7 differentiation for each colony type [(number of particular colonies)/ttotal colonies per plate) Y 1001 per plate + standard error of the mean d Significance of the differences hetween plating efficiency and ‘7 differentiation of cells grown in nutrient medium with and without NGF were determined by Student’s t test. Addition of NGF did not significantly affect the behavior of cloned crest cells in any of the ahove categories.

FIG. 2. Macrophotograph of a petri plate seeded with 200 cells prepared from a mesenchymal outgrowth similar to that shown in Fig. 1D. Eight days after plating, two types of colonies are obvious at this magnification, although a third type can he seen at higher power (Fig. 4). The pigmented colonies seen here are smaller. more compact and appear black whereas the non-pigmented colonies are larger in diameter. diffuse and although stained with hematoxylin appear less dark. Hematoxylin. , 1.

granules in both the black and brown cells. The granules (Fig. 6B inset) are round to oval and generally are distributed uniformly throughout the cell. The non-pigment colonies are larger, both in area and number of cells than

pigment colonies (compare Figs. 3 and 5 and see Table 2). The non-pigment cells are polymorphous in outline tending to be fibroblast-like in appearance (Fig. 51. In some colonies the cells are closely packed, while in others the cells are more flattened and more widely dispersed. The behavior and appearance of these colonies gives no clue as to their identity although they show nerve-like processes if cultivated for longer periods. Such arborized cells are characterized by a perikaryon that stains intensely with hematoxylin; branched processes terminating in structures similar to growth cones also are observed. Additional work is required to characterize more fully the potentialities of non-pigment colonies. The third colony type in the 8 day cultures consists of both pigment and nonpigment cells (Fig. 41. The pigment cells are located centrally and are similar in form and degree of pigmentation to cells in homogeneous pigment colonies. The peripheral cells of such mixed colonies are unpigmented and fibroblast-like in appearance. By 13-15 days, the mixed colonies consist primarily of pigment cells with onl! a small border of non-pigment cells. Often even these peripheral cells have a few melanosomes in their cytoplasm. Whether the sparsely pigmented cells at the periph-

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DEVELOPMENTAL

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A

VOLUME 46, 1975

COHEN AND KONIGSEERG

Clonal Studies TABLE

of Neural Crest

2

SIZE COMPARISONOF PIGMENT AND NON-PIchwwr Mean diameter

Colony type

t mm)

0.86 * 0.31 (N = 611 3.21 + 1.31(N = 561 P i O.OOlb

Pigment Non-pigment

269

COLONIES~ Mean no. cells/colony "52.4 zt "83.7 (N = 60) 1901.8 zt 3567.2 (N = 38) P < 0.005

“Petri plates seeded with 200 cells per dish were scored after 8 days. Values are determined from 6 experiments and are expressed as mean * standard error. b Probabilities (P) were determined by Student’s t test. The differences between the means for diameter and number of cells per colony were significantly different between pigment and non-pigment colonies. Analysis of variance indicates no significant difference between the mean number of pigment cells/colony between experiments [F(5.54) = 1.60 (P > 0.10) ]. nor between the mean number of non-pigment cells/colony between experiments [F(5.41) = 0.71 tP > O.lO)].

ery of the mixed clones the act either of losing pigment phenotype has lished (cf. Cahn and Cahn, 1966). Pattern

of Pigment

represent cells in or acquiring the not been estabWhittaker, 1967

Colony Formation

In order to identify the pattern of colony formation and differentiation in pigment clones, sequential observations were made on single cells, identified and marked shortly after attachment. Single cells separated from their neighbors by at least 4 mm were chosen and their location marked by pressing an inked marker to the under* surface of the petri plate. These cells were photographed within 4-6 hr after plating and periodically thereafter during the culture period. A photographic history of a typical pigment clone is shown in Fig. 7. After an initial lag of some 6-12 hr the presumptive pigment cells divide with a generation time of from 17 to 24 hr for the first two days, followed by an increase in

the doubling time to approximately 42-60 hr for at least the next 6 days. The first sign of pigment formation occurs on the third day; generally all cells in the clone then become pigmented within the following 24 hr. The relationship between the increased generation time after 2 days and the appearance of pigment at 3 days requires more detailed analysis. It is important to note, however, that these clones, even though they consist entirely of moderately to intensely pigmented cells, continue to increase in number logarithmically for at least 8 days (unpublished data). It should also be pointed out that initiation of pigment formation in the clones lags 2-3 days behind primary cultures which are not dispersed and plated at clonal density. Close examination of developing pigment clones (Fig. 7) indicates that the more compact appearance of such clones at 8 and 12 days reflects the inability of cell migration to keep pace with the increase in cell number rather than a secondary rear-

FIG. 3-5. Light micrographs of the three types of colonies present in crest cultures shown at lower power in Fig. 2, fixed and stained 8 days after plating. Hematoxylin. (3., 4-, and 5A) * 28; (3-, 4, and 5B) x 109. FIG. 3. Pigmented colony. (A) Pigment cells form compact colonies of closely arrayed cells. (B) At higher power the cells appear tightly packed with overlapping processes. All the cells contain moderate to large numbers of pigment granules. FIG. 4. Mixed colony. (A) The mixed colonies consist of closely packed pigment cells centrally positioned and surrounded by more widely separated non-pigment cells. (B) Note the differences in cell distribution and degrees of pigmentation here. Isolation experiments have shown that mixed colonies form as clones from single cells. FIG. 5. Non-pigmented colony. (A) These colonies contain more cells than pigmented colonies (Table 2): the cells. in addition are also more widely dispersed. (B) The non-pigment cells are polymorphic in outline with no obvious indication of differentiation.

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FIG. 6. Pigment colonies form a highly ordered, epithelium after 11-15 days in vitro. (A) Low power light photomicrograph of such a colony showing the different distribution of cells at the periphery and in the center of the clone. (B) At higher power the tightly packed, cobblestone-like arrangement of cells is seen. Compare the smooth outline of these cells with that of younger pigment cells (Fig. 3). The inset illustrates the round to oval melanosomes typical of these cells. Hematoxylin. (A) k 52; (B) z 170; Inset: :%736.

rangement of the cells. Often the daughter cells of the first few divisions remain contiguous and seldom at any stage, are separated by a distance greater than two cell diameters. In cant rast , the progeny of those cells that will form non-pigment clones have a much greater tendency to move away from one another. Pigment colonies, by the eighth day, are a mixture of arboreal and polygonal cells, but by 12 days most of the pigment colonies consist of cells whose lateral bounda-

ries are closely apposed and resemble a pavement epithelium (Fig. 6). With additional growth, highly dendritic pigment cells appear in the center of the clone and begin to overlap adjacent cells to form a multilayer. This creates a centrally positioned tangled skein of overlapping processes and cell bodies which is encircled by a monolayer of epithelial pigment cells.

Stability of the Pigment Phenotype The stability

of the pigment

phenotype

FIG. 7. This sequence of phase contrast photomicrographs depicts the growth and differentiation of a pigment clone from a single cell. Cells attach shortly after plating and proliferate during the first 2 days but do not synthesize pigment granules. By 4 days a few pigment cells differentiate although several non-pigment cells (arrows) are still present in the clone. By 8 days all the cells contain pigment granules, nevertheless. they continue to proliferate and by 12 days, the clone has increased in number of cells and diameter. (6 hr and 1 day) x 330; (2- and 4 days) x 165; (8- and 12 days) < 100.

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AND KONICSBERC

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DEVELOPMENTALBIOLOGY

was examined by serially passing homogeneous pigment colonies. Cells were serially subcloned three times from primary pigment colonies and the pigment phenotype was maintained for at least 25 generations in vitro. The cells continued to breed true during these additional three passages and gave rise exclusively to pigment colonies (Fig. 8). The colonies in the first and second passages were intensely pigmented whereas the cells in the last passage were moderately to intensely pigmented but grew much more slowly. Some colonies in the third passage contained cells at their edges which were only faintly pigmented. Epithelial pigment colonies similar to the one shown in Fig. 6 were also subcloned to examine whether this morphological trait would be irreversibly passed on to subsequent generations. After dispersing and plating such an epithelial group, the cells were initially polygonal and smooth in outline for several divisions but after 3-5 days assumed an irregular dendritic appearance. Some of the colonies retained the dendritic form as the colony grew, while others reestablished the cobblestone pattern. It is clear that the polygonal and dendritic shapes reflect modulations

FIG. 8. A culture derived from a pigmented clone that had been serially subcloned three times over an 8-week period. During that time and after approximately 25 generations the colonies retain their pigmented phenotype: no unpigmented colonies are present (compare with Fig. 2). Hematoxylin. x 1.

VOLUME 46,1975

(Weiss, 1939) of a single phenotype and these characteristics are not irreversibly determined. The conditions that effect the modulations, however, are not immediately obvious from the present experiments. Potency Tube

of First

Emigrants

from Neural

The expression of both pigmentation and neurogenesis (see below) in the primary crest cultures raises the possibility that various modes of crest differentiation are determined prior to migration from the neural tube. One possible corollary of this hypothesis is that covertly different cells migrate from the neural tube sequentially, possibly beginning with primordial pigment cells which normally migrate furthest. To test this question, the first cells to leave the neural tube were immediately isolated, rather than allowed the usual 24 hr for additional cell outgrowth. To accomplish this, the neural tube was removed from the petri dish within 4 hr at which time some two to four cells had migrated away at any particular point, and a total of approximately 75-100 cells were distributed along the entire 2 mm length of neural tube. These few cells were allowed to proliferate for 3 days as a primary culture, dissociated, and seeded at 200 cells per plate. Progeny of the first crest cells to leave the neural tube exhibit virtually the same distribution of clonal types as the cells t.hat emigrate during the usual 24-hr period of outgrowth. If the neural crest is a heterogeneous population of covertly determined cell types, this is not reflected in a sequential departure of pigment and nonpigment producing progenit.ors. These results confirm those of Weston and Butler (1966) who examined in uiuo the potency of crest cells after various periods of outgrowth. Origin of the Mixed

Colonies

The presence of colonies containing both pigmented and non-pigmented cells (Fig.

COHEN AND KONIGSBEKG

4) raises the possibility that some crest cells are pluripotent. On the other hand, mixed colonies could also originate from a doublet of predetermined but different cells or through contamination of a pure colony by migrating cells or “floaters”. A few spherical. unattached cells can in fact be observed floating in the medium of cultured crest cells. The “floaters” may reflect a high rate of cell proliferation because cells during mitosis round up and are more apt to detach from the plate. Alternatively, the “floaters” may represent cells with poor adhesivity throughout their mitotic cycle. By transferring this culture medium to a fresh petri plate it can, in fact, be demonstrated that some of these cells reattach and give rise both to pigment and non-pigment clones (Fig. 9). To examine the origin of mixed colonies, single cells were physically isolated 4-6 hr after seeding the dish. After carefully surveying the vicinity surrounding the cell to determine that there were no near neighbors, the cell was photographed and its position marked (see above). To preclude contamination by either floating or wandering cells, porcelain penicyclinders (Fisher Scientific) were

Clod

placed around the cells. The cylinders were pressed against and attached to the petri plate by a ring of vacuum grease applied to the flattened edge of the cylinder. The interior of the cylinder was carefully examined again after removing the black ink mark to insure the presence of only one cell. In only this experiment, the cells were suspended and cloned immediately after removing the neural tube at 24 hr, rather than allowing the usual 48 hr period for additional proliferation. Consequently, the clones start from chronologically younger cells enhancing, theoretically, the likelihood of selecting a precursor capable of divergent expressions. Such isolated crest cells produced mixed clones as well as homogeneous pigmented and non-pigmented clones. Of the 30 single cells isolated that were viable, 11 produced pure pigment, nine non-pigment and 10 mixed clones. The pigment clones were compact and otherwise similar to those clones derived from older crest cells that had not been physically isolated. Similarly, the non-pigment clones appeared no different from those obtained by conventional cloning. The data also clearly demonstrate that mixed clones can originate from single crest cells. Most (8 of 10) of the mixed clones consisted of dispersed non-pigmented cells with centrally located pigment cells mixed among them. This situation is different than the mixed colonies from older crest where the pigment cells formed a contiguous core surrounded by non-pigment cells. In two instances, a single cell produced adjacent pigment and non-pigment clones. Classification

FIG. 9. This petri plate contains both pigment and non-pigment colonies derived from cells floating in the nutrient medium of clonal cultures. Medium removed on day 5 from a culture such as shown in Fig. 2, was transferred to a fresh petri plate and grown for 10 days. Hematoxylin. x 1.

273

Studies of Neural Crest

of Progenitor

Cells

If the neural crest is comprised of subpopulations of already determined phenotypes, such differences might be reflected in morphologic characteristics of the progenitor cells. To examine this possibility, single cells marked by an inked circle and identified by number were photographed.

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DEVELOPMENTALBIOLOC’~

The photographs are grouped according to whether the clones derived from such cells became pigmented or remained unpigmented (Fig. 101. Among the 52 progenitor cells photographed in this series, the forms range from small dense, rounded cells to flattened stellate cells with little contrast in phase optics. Also present are cells intermediate in shape and density including bi- and multipolar progenitor cells. Inspection of the grouped cells (Fig. 101 indicates that the majority (23/25) of the small round dense cells (Fig. 10, upper rowi produce pigment clones whereas the flattened cells (Fig. 10, lower row) generally (13/161 initiate non-pigment clones. The cells intermediate in shape contribute to both pigmented, non-pigmented and mixed clones.

VOLUME 46. 1975

This data shows that the melanotic progenitor can be identified with good confidence, but does not resolve the issue of neural crest potentialities. One of the possibilities still to be considered is that crest cells are pluripotential with only the pigmented phenotype expressed under these clonal conditions. Alternatively, the neural crest may incorporate previously determined and different cell types with only the pigmented cells differentiating and the other derivatives latent and represented by the unpigmented clones.

Developmental Colonies

Potency of Non-Pigment

A reasonable possibility is that the nonpigment colonies are presumptive neuroblasts, either adrenergic or sensory. How-

FIG. 10. Phase contrast photomicrographs of single cells shortly after plating. These were followed during the culture period. The micrographs were grouped according to the eventual fate the upper row containing cells which formed pigment clones, the lower row destined to formed clones. The pre-pigment cells are distinguished by their small size and dense, refractile properties. in the pre-nonpigment cells are not visible under bright-field optics and are probably mitochondria. represents 50 pm.

marked and of these cells, non-pigment The granules x 280. Bar

COHEN AND KONIGSBERG

ever, due to inadequacies of the clonal situation or culture medium they fail to differentiate. This possibility is supported by the observation that nerves develop in the primary outgrowths grown for 2 weeks rather than the 2 days usually allowed in this study. In these older cultures, bi- and multipolar neurons with long processes rest on a pavement of tightly packed epithelial cells. In addition, aggregates of cells extend above the petri plate surface and are interconnected by long, thin nerve-like processes having varicosities along their length (Fig. 11). These clusters resemble embryonic avian and rodent dorsal root ganglion and spinal cord neuroblasts grown in vitro (Cavanaugh, 1955; Scott et al.. 1969; Peacock et al., 1973). A preliminary examination using formaldehyde-induced fluorescence techniques indicates that the neurons in the primary outgrowth do not contain catecholamines. Under clonal con-

Clonul Studies of Neural

Crest

275

ditions, nerves and neural aggregates are not. observed until after several weeks when the cultures become overgrown. To examine whether changes in clonal conditions might facilitate expression of a covert nervous phenotype, nerve growth factor (NGF) was added to the medium. NGF promotes growth and axon formation in crest derived neuroblasts in situ and in vitro (Levi-Montalcini and Angeletti, 1968). Its absence might explain the failure of axonation under clonal situations. NGF (sedimentation coefficient = 2.5S), generously provided by Dr. Levi-Montalcini, was added to the cultures at a concentration of 10 pm/ml. This did not change either the gross cytologic characteristics or the distribution of clonal types (Table 1) even after a period of 11 days. the longest period examined. In addition, the first stages of neurogenesis in sensory ganglia are presumably not dependent upon NGF

FIG. 11. Primary outgrowth from the neural tube similar to Fig. 1D but allowed to grow for a total of 11 days. Clusters of intensely staining cells. similar to sensory and spinal neuroblasts grown in oitro, are connected by nerve like processes. These aggregates, although attached, extend above the surface of the petri plate and the other cells. Hematoxylin. x 336.

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(Winick and Greenberg, 1965; Luduefia, 1973). Because NGF is known to promote the differentiation of crest derived neuroblasts, we examined the possibility that NGF or material with NGF activity might normally be present in our nutrient medium. NGF activity was determined by minor modifications of the bioassays described by Levi-Montalcini et al. (1954) and Hier et al. (1972). The response of 7-day sensory ganglia grown for 24 hr in various test media is shown in Fig. 12. Neurite outgrowth in routine nutrient medium was virtually identical to that supplemented with 10 pm/ml NGF. Medium containing embryo extract which was fractionated to remove molecules greater than 1000 MWZ did not support neurogenesis. When such medium was supplemented with NGF, however, neurite promoting activity was restored. Therefore, NGF or NGF-like material is a normal constituent of embryo extract and thus of our routine medium. The failure of nerves to differentiate at clonal densities cannot be attributed t.o absence of NGF. DISCUSSION

The immediate goal of this investigation was to develop procedures for obtaining uniform populations of neural crest cells. This was simplified to a large extent by one of the striking properties of this embryonic cell type. In the organism, shortly after the neural fold closure stage these cells disperse and migrate from their location in the roof of the neural tube. Although, in culture, rapid cell migration occurs from explants of virtually any embryonic tissue, one might anticipate that crest cells, because of their intrinsic migratory habit, * Levi-Montalcini and Angeletti (1963) and others have reported that horse serum does not have NGF activity. By elimination, embryo extract would be the only complex component likely to support neurogenesis. (N.B. the active subunit of NGF is reported to have a M.W. between 24,000 and 29.000 (Zaimis, 1972).

VOI.VME 46, 1975

might leave the explant earlier. Indeed this advantage has been exploited in several earlier studies (viz., Dorris, 1938; Twitty and Bodenstein, 1939). In the present study cell migration was observed as early as 4 hr after explantation and usually from only one edge of the explant. That these “pioneering cells” are, in fact, crest cells is reinforced by using carbon marks as reference points and observing that migration invariably occurs from the dorsal surface, where crest cells are known to be localized (Ris, 1940). After the first 12-18 hr during which time cell migration is almost exclusively restricted to the dorsal edge, migration can be observed from the cut ends and ventral edge, as well. These later cells migrate as an epithelial sheet, however, differing in appearance from the early mesenchymal cells suggesting again, that the early migrators are predominately of crest origin. Evidence that the early cells which migrate from the neural tube in vitro are in fact neural crest cells is provided by cloning this populat.ion. Large numbers of these cells give rise to clones which eventually exhibit one of the crest traits, that is pigment formation. The initiation of melaninization under clonal situations provides an ideal system for examining the transition from melanoblast to frank melanocyte. Clones of retinal pigment cells that maintain their epithelial characteristics and pigmentation have been described by Cahn and Cahn (1966) but these were derived from cells already containing pigment. Colonies of crest derived melanocytes have also been obtained from monolayers of dissociated somites containing crest cells (Zimmerman et al., 1974). Under these conditions, however, the presumptive melanocytes cannot be distinguished from somite cells. In the present study, presumptive melanocytes can be identified by their appearance and behavior. As early as the first day, crest cells plated at clonal density exhibit differences in their shape, appearance in phase optics and migratory activ-

FIG. 12. Bioassay for nerve growth factor (NGF) in nutrient medium. Seven day embryonic dor sal root mglia were grown for 24 hr in test media with and without NGF. (AI Abundant axon growth is suppclrted by rtrienr medium (NM) normally supplied to the primary and clonal cultures. (BI Addttron of 1tJMm/ml NtiY to rtrisnt medium does not noticcnbly incrcosc axon&ion. tC) Deletion of the high molecular weight (71000 W’t fraction of embryo extract t HMW EEI virtually eliminate< axon formation. (III Adding 10~m/ml NGF to edium deprived of HMW EE results in exuberant axon outgrowth. Hematoxylin. y 120.

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ity. These differences are more striking in cells obtained from older primary cultures. The cells vary, ranging from small, spherical, highly refractile cells that are virtually sessile to cells that are flattened, have pseudopods and are highly mot.ile. The small, round, stationary cells invariably produce clones which differentiate into melanocytes within 72 hr whereas the flattened cells usually produce unpigmented clones. The fate of those cells intermediate in appearance cannot be confidently predicted. The relationship between loss of cell motility and appearance of pigmentation has been noted by other investigators (Twitty and Bodenstein? 1939; Hamilton, 1940). In addition, Model and Dalton (1968) report that amphibian melanoblasts first acquire the ability to incorporate DOPA-3H, an intermediate in melanin synthesis, only after migration ceases but before granules can be visualized. The compact nature and close association of cells in the pigment colonies reflects, perhaps, the inability of pigment cells to translocate. In clonal culture, using the conditions which we have described here, the only crest potentiality which is expressed and immediately recognized is melanin synthesis. Microscopic observation alone gives no clue to the identity of the cells which comprise the unpigmented colonies. Other criteria (viz., cytochemical or biochemical) may be more informative. There may, however, be cells which, in fact, fail to differentiate either because they have been altered (damaged) by our culture procedures or because the culture conditions do not provide an adequate environment. The failure of neurogenesis to occur in these clones may result from interference with heterotypic, trophic or inductive interactions required for differentiation. Dissociated neurons from sympathetic and dorsal root ganglia are more likely to survive and extend processes if grown with glia or other non-neuronal elements in uitro, even in the presence of NGF (Burn-

VOLUME 46, 1975

ham et al., 1972; Luduena, 1973). The homogenity of clonally derived colonies would rule out these interactions of course. The importance of such events is suggested perhaps by the observation that nerve-like cells appear in clonal plates only after several weeks, at which time the plate is overgrown and the clones are no longer solitary. The absence of nerves in crest clones also cannot be explained on the basis of inadequate levels of NGF. Even though NGF is not thought to be needed for axon formation at the stages examined in the present study (Winick and Greenberg, 1965; Ludueria, 19731, its presence in our culture medium was ascertained to be adequate to support outgrowth of dorsal root ganglion neurons. Neither endogenous NGF-like activity in the medium nor exogenously administered purified factor stimulated nerve differentiation in clones. In addition, collagen or gelatin, substances that promote differentiation of sensory neuroblasts in vitro (Luduena, 1973) are also provided here. Certain pathways of differentiation may, for example, require limits of cell density which are not reached in these cultures. Weston (1971) finds that neurogenesis of cultured spinal ganglia is promoted by conditions that restrict cell dispersion. The fact that neuronal differentiation is seen in primary crest cultures but not in clonal cultures fixed at 8-12 days, may reflect a requirement for cell density. This could explain why cultures allowed to reach confluency exhibit neural characteristics. Whether the confluent conditions promote neurogenesis via cell-cell interactions or modification of the culture medium is unknown. It should be pointed out that the neurons observed in the primary and secondary cultures might be derived from the presumptive dorsal horn region of the spinal cord and comigrate with and contaminate the crest cells, a.possibility not yet ruled out. Earlier studies suggest that certain

COHEN AND KON~GSBERG

modes of crest cell differentiation require inductive influences provided by other cell types associated wit,h crest cells either transitorily or permanently. For instance, differentiation of sympathetic and sensory ganglia in the trunk normally involve cues from somitic mesoderm (Detwiler, 1934: Cohen, 1972). Presumptive sympathetic neurons of the trunk in vitro. also require the presence of somites in order to differentiate (Norr, 1973). In addition, cranial crest in cells which synthesize catecholamines oitro in the absence of somites, may require the presence of some mesodermal mesenthyme (Bjerre, 1973). The crest cells cloned in the present study, however, are isolated in vitro before they have an opportunity to interact with somites. This ma> explain in part, our failure to detect catecholamine-containing cells in the cultures. The identity of the non-pigmented crest clones and the possibility that neural crest cells prior to migration harbor previously determined and different descendants will be examined in future work. We are grateful to Dr. Rita Levi-Montalcini generous gift of nerve growth factor.

for her

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