Promotion of chromatophore differentiation in isolated premigratory neural crest cells by extracellular material explanted on microcarriers

DEVELOPMENTAL

BIOLOGY

113, 327-341 (1986)

Promotion of Chromatophore Differentiation in Isolated Premigratory Neural Crest Cells by Extracellular M a terial Explanted on M icrocarriers ROBERTO

PERRIS

AND JAN L~FBERG

Received Murch 26, 1985; accepted iv revised form October I, 1985 This study was undertaken to determine whether premigratory neural crest cells of the axolotl embryo differentiate autonomously into chromatophores, or whether stimuli from the environment, particularly from the extracellular matrix, are required for this process. Neural crest cells were excised from the dorsal part of the premigratory crest cord and cultured alone, either in a serum-free salt solution or in the presence of fetal calf serum (FCS), and together with explants of the neural tube or dorsal epidermis. A “microcarrier” technique was developed to assay the possible effects of subepidermal extracellular matrix (ECM) on chromatophore differentiation. ECM was adsorbed irr. Go onto microcarriers, prepared from Nuclepore filters, by inserting such carriers under the dorsolateral epidermis in the embryonic trunk. Neural crest cells were then cultured on the substrate of ECM deposited on the carriers. Melanophores were detected by DOPA incubation, revealing phenol oxidase activity, or by externally visible accumulation of melanin. Prospective xanthophores were visualized before they became overtly differentiated by alkali-induced pteridine fluorescence. Isolated premigratory neural crest cells did not transform autonomously into any of these phenotypes. Conversely, coculture with the neural tube or the dorsal epidermis, and also the initial presence or later addition of FCS during incubation, resulted in differentiation of neural crest cells into chromatophores. Both chromatophore phenotypes were also expressed on the ECM substrate deposited on the microcarriers. The results indicate that neural crest cells do not differentiate autonomously into melanophores and xanthophores, but that interactions with components of, or factors associated with the extracellular matrix surrounding the premigratory neural crest and present along the dorsolateral migratory pathway are crucial for the expression of these chromatophore phenotypes in the embryo. cl 1986 Academic

Press, Inc.

et nl., 1978; Loring

INTRODUCTION

et uh, 1982; Smith and Thorogood,

1983; Weston et a,l., 198413). In studies in which specific markers were used for the identification of differentiating melanophore and xanthophore phenotypes, Epperlein and Liifberg (1984) found that the expression of melanophores coincided with the initial neural crest cell migration, whereas xanthophores emerged somewhat after the onset of migration in clusters formed along the dorsal tube and containing both types of chromatophores. The present experiments were designed with the aim of establishing the degree to which premigratory neural crest cells of the axolotl embryo differentiate autonomously into chromatophores, and to what extent this process occurs in response to extrinsic signals, in particular from the extracellular matrix around the premigratory neural crest and along the subepidermal migratory route. For this purpose cells were excised from the premigratory crest cell cord (Liifberg et al., 1980; Keller and Spieth, 1984) at developmental stages preceding those at which chromatophore differentiation can be detected i?z.situ (Epperlein and Lofberg, 1984). Isolated neural crest cell populations were cultured both alone, either in a serum-free salt solution or in a medium containing fetal calf serum (FCS), from the start or

In the vertebrate embryo, a broad spectrum of cell types originates from the neural crest. After an extensive migratory phase, the neural crest gives rise to structurally and biochemically specialized cells of the peripheral nervous system, chromatophores, nerve-supporting cells, endocrine cells, and head mesenchyme (Horstadius, 1950; Le Douarin, 1982; Weston, 1982). One of the major problems in the development of the neural crest centers on the mechanisms whereby this transitory embryonic structure diverges into a variety of cell lines. Although there is evidence of a number of developmentally committed precursors of the peripheral nervous system in the premigratory crest (Barald, 1983; Cochard and Coltey, 1983; Ziller et al., 1983; Payette et ul., 1984; Sieber-Blum and Sieber, 1984; Weston, 1982), it has clearly been demonstrated that many initially undifferentiated neural crest cells attain different phenotypic characters in response to external influences (Noden, 1978; Teillet et al., 1978; Ziller et ul., 1979; Le Douarin, 1982). Extrinsic signals, which appear to elicit differentiation of neural crest cells, have recently been connected with the extracellular matrix present along the migratory pathways of the cells (Newsome, 1976; Weston 327

0012-1606/86 $3.00 Copyright All rights

8 1986 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

added later during incubation, and together with the neural tube or the dorsal epidermis. To disclose differentiated chromatophores, the same markers as had previously been applied for early detection of these cell types in the embryo (Epperlein and Liifberg, 1984) were used. The cell-free subepidermal space around the premigratory neural crest and along the dorsolateral migratory pathway of the axolotl embryo is known to be filled with a network of matrix fibrils, which are associated with the neural tube and dorsal epidermis and serve as a substrate for neural crest cell locomotion (Lofberg et al, 1980,1985). We assumed that this ECM could provide the source of information governing chromatophore differentiation. In order to examine this possibility, a “microcarrier” technique was developed, whereby embryonic extracellular matrix (ECM) can be adsorbed irl viva onto microcarriers, and isolated neural crest cells can be cultured on the ECM substrate deposited on the carriers. We report here data emphasizing the importance of interactions between neural crest cells and their immediate environment for the expression of chromatophores. MATERIALS

AND

METHODS

VOLIJME 113,1986

Epi

FIG. 1. Diagram showing the explantation procedures of: A, the premigratory neural crest cell cord; B, the entire neural primordium; C, the neural crest cord together with the dorsal epidermis. Epi = dorsal epidermis; NC = neural crest; NT = neural tube.

Culture Procedure Neural

crest cells. Axolotl embryos (Ambystoma mexfrom naturally spawning animals kept in our laboratory were collected at stages 25-27 (Bordzilovskaya and Dettlaff, 1979). They were dipped twice in 0.05% benzalconium chloride (Sigma) and 70% ethanol (2 min in each), dejellied, decapsulated, and rinsed in sterile Niu-Twitty solution (Flickinger, 1949). For the surgical procedure, they were transferred to sterilized solution (Steinberg, 1957) four times stronger than the original formula, which facilitates the removal of the epidermis from the underlying tissues (Lofberg et al., 1985). The operation solution was supplemented with 0.1% polyvinylpyrrolidone (PVP, mol wt 360,000), 250 U/ml benzylpenicillin, 25 pg/ml streptomycin sulfate and 0.1 pg/ml Fungizone (all chemicals purchased from Sigma). The PVP was added to the operation solution to prevent adhesion of cells to the walls of the micropipettes (see below). After removal of the epidermis, the dorzsal part of the premigratory neural crest cord between the 3rd and the 9th trunk segments was gently sucked into a micropipettes (Figs. 1A and 3-6) and transferred to Earl’s balanced salt solution (EBSS) for rinsing. This solution contained 0.1% PVP, 25 U/ml benzylpenicillin, and 25 pg/ml streptomycin sulfate. When rinsed, the isolated neural crest cell populations were deposited directly on the plastic surface of tissue culture dishes (35-mm, Nunc). The culture solution conicanum)

sisted of Nakatsuji’s modified Stearns’ balanced salt solution (Stearns and Kostellow, 1958). It was buffered with 5 mM Hepes (Sigma) according to Nakatsuji’s (1979) modification, but the bovine serum albumin was either omitted or replaced with 8% fetal calf serum (FCS, Flow Lab). The explants were grown on tissue culture plastics for up to 5-8 days at room temperature (20-22°C). Phasecontrast micrographs were taken throughout the culture period with an inverted microscope (Leitz). The behavior of cells in culture was recorded in phase contrast with a video time-lapse system (National: camera WV-1350 AE/G; time-lapse video tape recorder VTR NV-8030; time-date generator WJ 800; tape NV-P76H). Neural crest cells with neural tube. Following a procedure identical to that used for crest cell cultures, the dorsal epidermis of embryos at stages 25-27 was removed. The neural tube was then cut through at approximately the 3rd trunk segment level, lifted up, and separated from the underlying notochord in the craniocaudal direction (Fig. 1B). The excised, entire neural primordium, 7-8 somites long, was then rinsed once in the same EBSS as was employed for the isolated neural crest cells, and cultured in an identical serum-free solution on untreated tissue culture plastics (35-mm dishes, Nunc) for up to 7 days. The cultures were photographed intermittently in phase contrast during the culture period and video time-lapse taped. Cultures were examined

PERRIS AND L~FBERC

ChrorrLatophore

329

Differentiaticm

Explantation

of Extracellular

Matrix

on Microcarriers

NUCLEPORE

\ \

I I I I

ECM ON MICROCARRI

i ER

4 NEURAL CREST CELLS

FIG. 2. Diagram of the experimental procedure utilizing the microcarrier system. Microcarriers were cut from Nuclepore filters and inserted under the dorsolateral epidermis of stage 25 to 27 embryos for adsorption of extracellular matrix (ECM). The conditioning of the carriers lasted till the embryos had reached stages 29-30, i.e., just before the onset of neural crest cell migration. Removed from the embryo, the carriers were found to be covered by ECM, which remained attached to its surface. Subsequently, excised premigratory neural crest cells (NC) were deposited on the ECM substrate attached to the microcarriers. NC = neural crest.

and photographs were taken for documentation on the 3rd and 5th days. Neural tube explants. After removal of the neural crest, tube explants were isolated, cultured and video time-lapse recorded in the same way as the neural crestneural tube explants. Neural crest with dorsal epidermis. Incisions were made in the epidermis lateral to the neural tube and above the dorsal border of the somites. At approximately the 3rd trunk segment, the dorsal epidermis and the underlying neural crest cord were cut transversely and the epidermis was folded up, allowing the dorsal part of the crest cell cord to remain attached to its ventral side (Fig. 1C). Thus, when the epidermal sheet was excised from the embryo, the neural crest cells were removed with it simultaneously. The epidermis-crest explants were cultured analogously to the isolated neural crest cells and neural primordia on a plastic substrate. Phase-contrast photographs of the cultures were taken intermittently over a period of 7 days and the cultures were evaluated after 3 to 5 days.

Microcarriers were cut from Nuclepore filter membranes (pore size 0.4 pm), using iridectomy scissors (Liifberg et al., 1985). The carriers, which measured approximately 0.15 by 0.4 mm, were sterilized under UV light for 30 min (15 min for each side) and washed extensively in sterile PBS. Sterile carriers were then inserted subepidermally into embryos at stages 25-27, level with the 4th to the 8th segments, with the “rough” side of the carrier facing the basal side of the epidermis (Figs. 2 and 16). In this position the carriers were left overnight in the living embryo for adsorption of extracellular material. This conditioning period corresponded to the time taken for embryos to reach stages 29-30, i.e., the stages when neural crest cell migration is just about to start where the carriers had been inserted (Lofberg et al., 1980, 1985). After removal from the embryo, microcarriers that had become covered with ECM were immediately transferred to culture dishes containing the serum-free culture solution, to avoid possible detachment and consequent loss of adsorbed material. Neural

Crest Cells on Conditioned

Microcarriers

Neural crest cell populations were isolated as indicated above and shortly after rinsing deposited on the conditioned microcarriers (Figs. 2 and 22), or on untreated Nuclepore filters (controls). Cells were placed on the side of the carrier which during conditioning in the embryo had faced the basal side of the epidermis and which had adsorbed epidermis-produced ECM intended for testing. In some cultures, a number of crest cells were also placed directly on the plastic surface beside the carriers (Fig. 22). In separate cultures, excised neural crest cells were grown on tissue culture plastic for a period of up to 24 hr, during which time the cells spread and arranged themselves uniformly. Subsequently, conditioned microcarriers were carefully laid on the crest cell monolayers, with the ECM-covered side facing the cells, and incubated for up to 5 days. All cultures were phasecontrast photographed and evaluated on the 3rd and 5th day. DOPA

incubation

For incubation in DL-p-3,4-dihydroxyphenylalanine (DOPA; Sigma) explants cultured on glass coverslips were transferred to dishes filled with 2 ml of sterile NiuTwitty solution containing 0.1% DOPA. To prevent autooxidation of DOPA the sterilized Niu-Twitty solution was gassed beforehand at least 1 hr with Nz at room temperature. During incubation in the dark for 5 hr the dishes were tightly sealed with Parafilm to avoid has-

330

FIGS. 3-6. segments, of of a stage 25 NT = neural

DEVELOPMENTALBIOLOGY

VOLUME 113,1986

SEM and light micrographs showing the premigratory neural crest cell cord in the trunk region, between the 4th and the 6th a stage 25 axolotl embryo, before (Figs. 3 and 4) and after removal of the dorsal part of the cord (Figs. 5 and 6). Inset: Overview embryo in which the dorsal epidermis has been removed. EP = dorsal epidermis; NC = neural crest; -NC = neural crest removed; tube; S = somite. Figs. 3 and 5, SEM, X375 and X325; Figs. 4 and 6, LM, cross sections, X390 and X230.

tened oxidation. A similar modification of Mishima’s (1964) procedure has been applied on whole embryos (Epperlein and Lofberg, 1984). Following incubation, the cultures were washed extensively in 0.1 M PBS, left in buffer at least 24 hr at 4°C and subsequently photographed. Alkali-Induced

Pteridine

Flwrescence

(AIPF)

Cultures to be analyzed were placed under a microscope supplied with an epifluorescence system (Leitz Ploemopak with filter block A-BP 340-380 excitation filter and LP 430 barrier filter-and equipped with an MHO 200 mercury lamp). Approximately half of the original culture medium was replaced with a weak ammonia solution (prepared by adding one drop of 25%

ammonia to 10 ml of double-distilled water (Epperlein and Claviez, 1982) to obtain a high pH. The alkaline solution is known to release pteridines from their protein carriers in xanthophores and these pigment substances can then be visualized under UV light, where they emit a bright blue fluorescence (Epperlein and Claviez, 1982). The induced fluorescence faded within 5 to 10 min and the cultures were therefore photographed shortly after the addition of ammonia solution. Preparation jbr Light Microscopy Electron Microscopy (SEM)

and Scanning

Embryos. Normal embryos and embryos from which premigratory neural crest cells had been removed were rinsed in Niu-Twitty solution and fixed in cacodylate-

PERRIS

AND

L~FBERG

Chromatoph~ore

buffered (0.1 ill, pH 7.4) 1.5% glutaraldehyde + 1.5% paraformaldehyde containing 400 ppm ruthenium red (RR; Lofberg et uZ., 1980) for several hours to days at 4°C. The embryos were then rinsed in buffer and postfixed in 1% 0~0~ with the addition of RR for 4 hr at 4°C. After rinsing, the specimens were dehydrated in a graded series of ethanol. Some were subsequently embedded in Epon, serially sectioned at 5 pm between the 4th and 6th trunk segments and stained with methylene blue for light microscopic examination. Other embryos, some of them freeze-fractured (Lbfberg et al., 1980), were transferred via Freon TF (113) into liquid CO;? and critical point-dried. The dried specimens were coated with gold/palladium (60%/40%) in a sputter coater for SEM analysis. Newal crest cells on conditioned microcarriers. Immediately after removal from the embryo, ECM-covered microcarriers and crest cell cultures on such carriers were similarly fixed in RR-containing fixative and prepared for SEM. RESULTS

Eflects of Cu,ltzrre Conditions

on Social Behavior

Neural crest cells. On tissue culture plastics, the explanted neural crest cells condensed into spherical clusters before dispersing radially. Within 10 to 12 hr virtually all cells of the clusters had attached to the substrate, migrated out and formed a monolayer (Fig. 8). Cell locomotion was now observed mostly at the periphery of the confluent monolayer. Filopodial processes were alternately extended and retracted by cells, and often switched from one side to the other. Both blebbing and contact paralysis of pseudopodial activity occurred, but no mitotic activity was seen in the absence of serum. When FCS was included in the culture medium, however, occasionally dividing cells were observed. During the first 5 days of serum-free culture, all cells appeared perfectly healthy and not until the sixth day did the first signs of cell death become notable. Dying cells retracted their filopodial projections and detached from the substrate before, or concomitantly with cytolysis. Neural crest with neural tube. Attachment of the excised, entire neural primordia to the plastic surface occurred within 10 to 15 hr after isolation from the embryo. Shortly afterward, neural crest cells began to migrate away from the tube. Conversely, no cells migrated out from the neural crest-free, isolated neural tube explants, which remained attached to the substrate as distinct aggregates. During the first 2 days in culture, crest cells migrated extensively, but later there was a considerable decrease in directional movement away from the central explant. The neural crest cells became uniformly distributed around the tube explant and formed a mono-

Differentiation

331

layer. Mitotic activity was occasionally observed after 3 to 4 days. The area occupied by individual explants varied, but outgrowths tended to disperse more in the cranial part of the neural primordia than in the caudal one. Neural crest with dorsal epidermis. When explanted together with the dorsal epidermis, the neural crest cells became overgrown by the curling epidermal strands. The explants then tended to acquire a vesicle-like structure (not shown), but within a few hours the majority of the epidermal cells had attached to the substrate and the epidermal explants could progressively spread out. Most of the neural crest cells remained located under the epidermal outgrowths, but some highly migratory cells spread further beyond the edge of the sheets. Neural crest cells on microcarriers. SEM analysis of some of the cultures of neural crest cells on microcarriers revealed that isolated crest cells attached and spread out both on conditioned and on untreated microcarriers, with a somewhat more rapid spreading rate on the conditioned ones. When migrating crest cells reached the borders of the carriers, they continued to spread on the plastic substrate beyond them. On the ECM substrate covering conditioned carriers, the crest cells were found to have become enmeshed by the ECM fibrils (Fig. 23), but no obvious differences were observed between the morphology of crest cells grown on conditioned and unconditioned carriers. Assessment of Dilereutiution Individual cell counting was not performed in the cocultures (except in epidermis-crest cocultures with overtly differentiated melanophores). The reason for this was that in neural tube-neural crest explants, most of the crest cells became densely distributed within the monolayered outgrowths and the margins between two cells were not distinguishable. Similarly, in epidermiscrest cocultures the flattened epidermal sheets almost entirely covered the neural crest explants and individual crest cells could not be discerned unless they became overtly differentiated. Thus, as the exact percentage of differentiated neural crest cells was not assessable in our cocultures we define positive cultures here as only those that contained at least ten separate, expressed chromatophores. Each one of these cultures constitutes one individual tube-crest or epidermis-crest explant. Since the microcarriers were opaque, it was not possible to estimate by light microscopy the number of neural crest cells plated on their surface. We therefore counted the number of cultures showing phenotypic expression, relative to the total number of cultures studied. One culture constituted approximately one-third to one-half of the entire premigratory neural crest cord

332

DEVELOPMENTALBIOLOGY

excised between the 3rd and 9th trunk segment and deposited on one microcarrier. A culture was judged to show phenotypic expression when at least three separate chromatophores could be clearly identified on the carrier.

Chromatophore Diflerentiation in the Absence or Presence of Fetal Calf Serum In a total of 84 serum-free cultures of isolated neural crest cells, none of the chromatophore phenotypes could be identified during the culture period. When 8% FCS was added initially to the culture medium, however, virtually all neural crest cells differentiated into chromatophores. Overtly expressed melanophores and AIPFpositive xanthophores appeared on the 3rd day in all 43 cultures studied. Melanophores were scattered throughout the monolayer formed by the explanted crest cells. Xanthophores, on the other hand, usually aggregated and were located in the middle of the explants. Moreover, xanthophores mostly tended to assume a rounded, almost spherical shape and to be located on top of the melanophores, which flattened out and often became multipolar (Figs. 9 and 10). Addition of 8% FCS after 3, 4, and 5 days of culture (six cultures for each experimental case) induced within 24 hr of culture a differentiation sequence identical to that observed with the initial inclusion of serum. In comparison with serumfree cultures the lifetime of isolated neural crest cells in serum-supplemented ones was prolonged 2 to 3 days and cell death generally occurred in the sequence: xanthophores, mesenchymal cells, and eventually melanophores.

Chromatophore Diaerentiation in Cocultures Neural crest with neural tube. In neural crest cell cultures in the absence of FCS but in the presence of the neural tube, overtly expressed melanophores with a pronounced melanin content could be discerned on the 3rd day. Melanin-producing cells appeared in all cocultures studied, in total 44, but only crest cells adjacent to the tube explants became clearly melanized (Fig. 12). These cells also acquired the typical multipolar shape of larval melanophores emerging at the border of the dorsal fin (Fig. 7). Overtly differentiated melanophores did not aggregate, although they were frequently located close to one another. DOPA incubation, performed in 15 of the tube-crest cocultures, showed that even some of the outgrowths displayed phenol oxidase activity (Fig. 15). Those cells, however, still exhibited a fibroblastic shape typical of early migratory neural crest cells in culture, which differs from the patchy or dendritic morphology of larval melanophores. Intense fluorescence observed after alkali treatment in all the 48 cultures examined proved that neural crest cells differentiated into pteridine-containing xantho-

VOLUME 113.1986

phores when the neural tube was included. The majority of the AIPF-positive cells tended to coalesce into small groups, preferentially close to the tube explants (Fig. 13). Moreover, in this type of coculture, the fluorescent xanthophores showed a spherical shape comparable to that of premigratory neural crest cells in the embryo (Spieth and Keller, 1984; Lofberg et al., 1985). Unlike DOPA-positive cells, AIPF-positive xanthophores were never observed at the outermost periphery of the outgrowths. Cultures were first examined for the presence of chromatophores on the 3rd day, since this time in culture corresponded to the period in which companion control embryos reached developmental stages at which melanophores were clearly recognizable and prospective xanthophores were identifiable as fluorescent cells. The cell viability in these cocultures was comparable to that of neural crest cell cultures supplemented with FCS, in that a few dead cells surrounded the outgrowths already on the 3rd day of culture. Neural crest with dorsal epidermis. When cultured together with the overlying epidermis, neural crest cells differentiated into both melanophores and xanthophores. Melanophores first emerged on the 3rd day, beneath the epidermal sheets in all cultures in a total of 51, where they could be recognized as overtly melanized cells (Fig. 12). They were mostly multipolar, often dendritic and similar in morphology to the highly branched, subepidermal melanophores of the larva (Fig. 7). Melanophores expressed in these cocultures rarely occurred close to one another, but made occasional contacts via elongated processes. On individual cell counting in 48 comparable epidermis-crest cocultures it was found that the number of identifiable melanophores increased with time, being higher on the 5th day of culture (641 cells) than on the 3rd (299 cells). External administration of DOPA in nine of these cocultures disclosed that even some of the neural crest cells distributed immediately beyond the edge of the epidermal sheets showed phenol oxidase activity (Fig. 16). Those neural crest cells that were triggered to differentiate into xanthophores by the cocultured epidermis, always occurred under the sheet formed by this tissue. Even in this type of coculture, in total 43 tested, AIPFpositive cells tended to aggregate, but their morphology, especially as observed on the 5th day of culture, diverged from that of corresponding cells in tube-crest cocultures, showing a clearly branched shape (Fig. 14). No cell death seemed to occur before the 7th day.

Explantation

of Extracelbular Matrix (ECM)

As revealed by SEM, subepidermal ECM was adsorbed and remained attached to microcarriers that had been implanted and conditioned in living embryos (Figs. 17

PERRIS AND L~FBERG

Chromdophore

333

Dzfferenhccticm

FIG. 7. Melanophores in the trunk region of an axolotl larva at stage 40. At the base of the dorsal fin, these chromatophores assume a dense and compact shape (arrowheads), whereas when suspended in the mesenchyme of the fin (arrows) or down on the flank, they exhibit a multipolar, dendritic shape. The same features apply for xanthophores. X80. FIG. 8. Phase-contrast micrograph showing isolated neural crest cells after 3 days in serum-free solution. x1200. FIGS. 9 AND 10. Overtly expressed melanophores (Fig. 9) and AIPP-positive xanthophores (Fig. 19) in 3-day-old neural crest cell cultures supplied with 8% FCS. Fig. 9, X1900; Fig. 10, X1150.

and 18). The explanted ECM was scattered on the carriers as tufts of collagenous, granulated fibrils (Lofberg et ccl., 1980), which did not show any alignment that could be related to the pores of the carriers. The granules associated with the fibrils possibly represented precipitated proteoglycans (Luft, 19’71; Hay, 1978; Lofberg et ob, 19X3,1980). The material adsorbed onto the carriers appeared to be of the same structure and network-like organization as the extracellular matrix observed in the subepidermal space of the embryo and which seem to be used as a substrate for locomotion by the neural crest

cells (Figs. 19-21). No cells or larger cell fragments seemed to be included in the extracellular matrix covering the carriers, but some cell surface components may have been present. C/I romatophore Di,ferentiatio?l Conditioned Microca rriers

on

In 25 individual control cultures of isolated, premigratory neural crest cells grown on unconditioned microcarriers, none of the phenotypes investigated could

FIGS. 11-16. Expression of melanophores FIG. 11. Overtly expressed melanophores FIG. 12. Overtly expressed melanophores

and AIPF-positive xanthophores in cocultures with the neural tube and the dorsal epidermis. adjacent to neural tube tissue after 3 days in culture. X1900. covered by a cocuitured explant of dorsal epidermis; 3rd day of culture. X1900.

334

PERRIS AND L~FBERG

be identified with the markers employed. However, both melanophores and xanthophores were revealed in cultures of neural crest cells grown on conditioned carriers (Fig. 22). DOPA-positive melanophores were expressed in cxll23 cultures studied (Figs. 24 and 25), whereas AIPFpositive cells were found in 29 of 39 cultures (Figs. 26 and 27). Thus, in most cases (100% and 74%, respectively), chromatophore differentiation was strongly promoted by the explanted, subepidermal ECM. In 14 cases, DOPA incubation and the alkali treatment were performed during the first 2 days of culture. After one day, neither melanophores nor xanthophores were found, whereas on the second day, a few prospective melanophores were identified in six cultures after DOPA reaction. No AIPF-positive xanthophores, however, were identified on the conditioned carriers until the 3rd day of culture. In 37%’ of the cultures showing phenotypic expression, even some of the cells that had migrated away from their initial position on the carriers differentiated into melanophores and xanthophores. However, neural crest cells that were deposited directly on the plastic surface beyond the microcarriers (Fig. 22) and hence were not permitted contact with the ECM substrate, did not transform into melanophores and xanthophores. No difference in mitotic activity was found between neural crest cells cultured on conditioned and those cultured on unconditioned microcarriers. When isolated neural crest cells were first cultured for up to 24 hr on tissue culture plastic (13 cultures) and then offered contact with carrier-provided ECM from above, differentiation into melanophores and xanthophores was again disclosed in wll cultures. DISCUSSION

Since the purpose of this study was to determine the capability of isolated, premigratory neural crest cells to differentiate autonomously into chromatophores, and to investigate how environmental factors might affect this process, we aimed at obtaining as pure neural crest cell cultures as possible. This was accomplished by excising only the dorsal cells of the premigratory neural crest cord, thus avoiding simultaneous removal of tube tissue.

Chronlcrtophore

Dijfiirentiutim

335

In avian material, by explanting entire neural primordia, it has been possible to optimize culture conditions in order to obtain “homogeneous” neural crest cell populations with different developmental fates in vitro (Glimelius and Weston, 1981a,b; Glimelius and Pintar, 1981; Loring ef ul., 1981, 1982). Furthermore, by cloning single neural crest cells, a certain time after similar explantations, three separate, crest-derived progenies have been obtained: one comprising melanocytes alone; one containing mainly neuronal cells, which could be detected as catecholaminergic; and a third one showing a mixed population of these two phenotypes (Cohen and Konigsberg, 1975; Sieber-Blum and Cohen, 1980; SieberBlum and Sieber, 1984). However, our findings and those of other authors (Norr, 1973; Teillet et nl., 1978; Glimelius and Weston, 19811~;Derby and Newgreen, 1982) demonstrate that the neural tube influence the differentiation of neural crest cells, and tube tissue should therefore not be included in cultures aimed at elucidating the developmental potentialities of premigratory neural crest cells (see also Ziller et rrl., 1979). It has been claimed that pure neural crest cell populations have been obtained in the avian embryo by excising the tips of the neural folds alone (Smith et ul., 1977; Teillet et rrl., 1978; Smith and Fauquet, 1984), and traditionally similar excisions have also been carried out in the amphibian embryo (DuShane, 1935; Flickinger, 1949; Niu, 1954; Epperlein, 1974, 1978). At such excisions, however, neural crest cells cannot be totally isolated but are taken together with their overlying epidermis. In some cases, these excisions are performed even later, during the initial phase of migration. Even if the majority of the epidermal cells, which in culture tend to form aggregates, are removed within the first few days, possible initial inductive effects of these cells and effects of cells that might be retained in the cultures, on neural crest cell differentiation cannot be ruled out. Pure premigratory neural crest cell populations are also difficult to obtain from the avian embryo, since in contrast to the axolotl, the exclusion of the neural crest cells from the tube occurs almost concomitantly with the onset of migration (Tosney, 1978; Newgreen and Thiery, 1980; Newgreen ef al., 1982). Once moving from their original position on the dorsal aspect of the neural tube, the crest cells of avian embryos enter cell-free

FIG. 13. AIPF-positive xanthophores in a 5-day-old coculture with the neural tube (NT). Note the tendency of the xanthophores to group themselves near the edge of the tube. X620. FIG. 14. Branched, AIPF-positive xanthophores under a cocultured explant of dorsal epidermis; 5th day of culture. X1150. FIGS. 15 AND 16. Peripheral neural crest cells in the outgrowth of a neural tube-neural crest coculture (Fig. 15) and crest cells located at the edge of a cocultured epidermal explant (Fig. 16). Virtually all neural crest cells (exemplified by arrows) display phenol oxidase activity. The majority of the neural crest cells, which normally appear colorless (arrowheads), display after DOPA-incubation (arrows) phenol oxidase activity. Fig. 15, X950; Fig. 16, X1900.

FIG. 17. A microcarrier implanted for adsorption of ECM subepidermally is seen through the epidermis. X75. FIG. 18. Scanning electron micrograph of ruthenium red-stained ECM adsorbed on a microcarrier. X9000. FIG. 19. SEM micrograph of an axolotl embryo freeze-fractured at approximately the 5th trunk segment. Extracellular matrix fibrils are distributed in the subepidermal space around the neural crest cells and along their dorsolateral migratory pathway. ECM and migrating neural crest cells from similar regions as that indicated are shown in Figs. 5 and 6. X1680. 336

PERRIS AND L~FBERC

Ch rornafophow

spaces (Pratt ef al., 1975; Newgreen and Thiery, 1980), the extracellular matrix of which might affect their development and hinder the obtainment of uncommitted neural crest cell populations for in vitro studies. More recently, Keller and Spieth (1984) succeeded in excising the entire axolotl premigratory neural crest cell cord, but did not emphasize the fact that in doing this the presence of tube cells in cultures could largely be avoided. In the present study we have attempted to refine this technique by removing only neural crest cells from the dorsal purf of the premigratory cord (Figs. 3-6). We are confident that this technique ensures that inclusion of contaminating neural tube cells can be entirely prevented, and this assumption was also strengthened by our video time-lapse recordings, which showed that all cells in the neural crest cultures, unlike tube cells, were motile (see also Newgreen et uI., 1982). by Fetal Cdf Serwtr Differentiation into chromatophores occurred both in the constant presence of and after later addition (3rd5th day of culture) of FCS, indicating that initial culture in a serum-free environment did not greatly alter the capability of crest cells to differentiate into chromatophores. The effects of FCS in our system are consistent with a wealth of earlier data demonstrating that FCS affects melanophore differentiation (Nichols ef al., 1977; Greenberg and Schrier, 1977; Derby and Newgreen, 1982; Matsuda, 1983). While cell death in neural crest cell cultures more than 5 days old could probably cause an environmental selection for survival of committed cell phenotypes, the lack of melanophore differentiation in the serum-free crest culture does not seem to be attributable to a selection against cells able to undergo melanogenesis. This assumption is based on the finding that overtly expressed melanophores and AIPF-positive xanthophores emerged in FCS cultures on the 3rd day, and before the 5th day virtually all neural crest cells had become transformed into chromatophores. Di;H‘kw~ f in fion of’ Melanophores iw Corn 1f 21res

and Xm fhophores

Melanophore differentiation was strongly promoted by the epidermis and this finding corroborates previous observations which have indicated that the epidermis regulates the development of melanophores in the em-

Diff‘erent iatitw

337

bryo (DeLanney, 1941; Lehman, 1953; Landesman and Dalton, 1964; Hoperskaya and Golubeva, 1982; Keller ef trl., 1982). DOPA incubation revealed that even peripheral, apparently undifferentiated cells in the outgrowths of the tube-crest and epidermis-crest explants displayed phenol oxidase activity. However, since these peripheral melanophores did not turn visibly black, it seems that “melanizing stimulus” was lacking. the appropriate Thus, there is reason to believe that even though early migrating neural crest cells may initially be directed by the neural tube or dorsal epidermis towards a melanogenie developmental bias (Derby and Newgreen, 1982; Matsuda, 1983), they require continuous environmental stimulation through close association with these tissues to complete their differentiation. In amphibians, it has been proposed that pigment cells may occur in mosaic forms, showing ultrastructural characteristics of both melanophores and xanthophores (Bagnara et trl., 1979). However, cells that simultaneously contained both visible amounts of melanin and showed discernible pteridine fluorescence were never observed in our coculturcs. This does not rule out the possibility, however, that such “dual phenotype cells” (Loring et ul., 19sa) might arise under different experimental conditions or later in development. The role of different embryonic tissues in promoting neural crest cell differentiation has been stressed in several previous reports. For instance, melanocyte differentiation is stimulated in avian neural crest cell cultures by the neural tube, the removal of which from cultures within 18 hr after explantation causes a marked decline in overt differentiation (Glimelius and Weston, 1981b; Derby and Newgreen, 1982). Likewise, the establishment of crest-derived adrenergic structures seems to be affected by the neural tube (Cohen, 1972; Norr, 1973; Teillet et ctl., 1978). Semitic and notochordal mesenchymal tissues, which constitute the immediate embryonic microenvironment of neural crest cells giving rise to sympathetic nerve traits, have been found to enhance adrenergic differentiation considerably (Cohen, 1972; Teillet cjf ~1.. 1978; Ziller et nl., 1979; Smith and Fauquet, 1984). Ch row u toph ore Ex-pressio?1 ox Condif imed Microcu rriers There was a good temporal correlation between the discovery of expressed chromatophores on the condi-

FIG. 20. Ruthenium red-stained, subepidermal ECM from a region similar to that indicated in Fig. 19. SEM. x5000. FIG. 21. Neural crest cells migrating along the dorsolateral pathway in a position corresponding to that indicated in Fig. 19. The cells contact the ECM fibrils which apparently are used as substrate for their locomotion. X19,800. FIG. 22. Isolated premigratory neural crest cells, immediately after explantation, deposited on an ECM-covered microcarrier. Some cells were also placed beside the carrier-directly on the plastic surface of the culture dish (arrows). x200. FIG. 23. SEM micrograph showing a neural crest cell which was explanted and deposited on a conditioned microcarrier. The cell has become enmeshed by fibrils of the ECM substrate adsorbed on the carrier. X5500. ECM = extracellular matrix, EP = epidermis, MC = microcarrier; NC r neural crest. NT z neural tube.

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tioned carriers and the external appearance of melanophores as well as the possibility to identify xanthophores by alkali-induced fluorescence in control, companion embryos. This, in turn, indicates that the normal developmental time schedule of chromatophore differentiation could be simulated in our bioassay. The potential usefulness of our microcarrier technique is highlighted by these results and parallels the earlier findings that the extracellular matrix might be a timing factor for developmental processes in the embryo (Lofberg ef al., 1985). The importance of cell-ECM contact for the promotion of chromatophore differentiation was clearly demonstrated in the cases where isolated neural crest cells were placed beside the conditioned microcarriers and failed to differentiate. This result, taken together with the finding that some of the neural crest cells which had migrated away from their initial position on the ECM substrate also showed chromatophore expression implies that differentiation could not have been triggered by diffusible components from the explanted ECM, but only by a short-lasting contact with it. Our present results, showing that close range interactions between crest cells and ECM are crucial for chromatophore differentiation, suggest that substratemediated regulation of chromatophore expression might be the working mechanism in the embryo, also. In the subepidermal space the extracellular matrix is associated with the neural tube and dorsal epidermis and close interactions with these tissues are found to promote chromatophore differentiation as well. When explanted, both the neural tube and epidermis might provide a similar extracellular matrix which would serve as a mediator for the promotion of this phenotypic expression. Apparently, the competence of isolated premigratory neural crest cells to respond to ECM and develop into melanophores and xanthophores was not lost within the first 24 hr of culture. Two possible explanations for this observation may be discussed: At explantation, premigratory neural crest cells could be developmentally labile and susceptible to environmental stimuli for a certain time even in culture. Alternatively, already in the premigratory position the cells could be determined toward the chromatophore differentiation pathway, this commitment persisting in culture but phenotypic expression occurring only when the appropriate stimulus is offered. In a number of neural crest cell explants cultured on

ECM microcarriers, no fluorescent cells could be disclosed. This finding might be explained, however, by Epperlein and Lofberg’s (1984) observations of the appearance of early expressed groups of xanthophores in a premigratory position within the neural crest, though at later developmental stages than those at which crest cells were isolated here. Thus, when discrete clusters of neural crest cells were dissected out from the early premigratory crest cord, these clusters might have been devoid of cells competent to develop into xanthophores, possibly explaining why, in such cultures, this chromatophore type did not emerge. This interpretation is based on the possibility that determined but unexpressed chromatophores might be present in the early premigratory crest before the time of explantation. The importance of extracellular matrix in affecting various differentiation processes is well documented in numerous developing systems (Hay, 1981; Sanes, 1983; Kemp and Hinchliffe, 1984; Trelstad, 1984). Close association of neural crest cells with somite-conditioned substrates has been shown to promote adrenergic differentiation and to suppress melanogenesis proportionally (Loring ef al., 1982). Melanocyte differentiation has been found to be induced by deoxycholate-resistant, structural constituents of the extracellular matrix of the quail embryo (Derby, 1982). In that case, dermal mesenchyme matrix was obtained from explants or dispersed monolayers, the cells of which had been removed with the aid of the detergent deoxycholate. Melanogenesis has also been reported to have been prevented and the production of the S 100 protein, characteristic of glial cells, to have been enhanced in response to developmental cues provided by components at or near the surface of neuronal cells of chick ganglia (Holton and Weston, 1982). Moreover, from a large number of culture experiments it has been proposed that neural crest cell differentiation is regulated by interactions between the cells and nondiffusible components of the pericellular matrix (Newsome, 1976; Bee and Thorogood, 1980; Hall, 1982; Smith and Thorogood, 1983; Loring ef uh, 1982). In accordance with these data, our results suggest that in the embryo the information for the establishment of melanophore and xanthophore traits might be provided by the subepidermal extracellular matrix. The present data also indicate that although progenitors of melanophores and xanthophores in the neural crest might have attained competence for terminal specification at earlier developmental stages than those studied here,

FIGS. 24 AND 26. Phase-contrast micrographs of 5-day-old cultures of neural crest cells grown on ECM-covered microcarriers (MC), hefore DOPA (Fig. 24) and alkali treatment (Fig. 26). Fig. 24, X950; Fig. 26, X1200. FIGS. 25 AND 27. DOPA- (Fig. 25) and AIPF-positive cells (Fig. 27) expressed in the same cultures as shown in Figs. 24 and 26, respectively. Neural crest cells exhibiting phenol oxidase activity (arrows) and AIPF-positive xanthophores are restricted to those cells that remain on the ECM substrate attached to the carriers. Figs. 25 and 27, X1550.

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premigratory crest cells require stimuli from the surrounding tissue environment along their migratory routes in order to express these chromatophore phenotypes. However, even if the ability of the neural tube, the dorsal epidermis and the subepidermal ECM to promote neural crest cell differentiation has been clearly demonstrated, it remains to be established whether the inductive information provided by the extracellular matrix is of an instructive or a permissive character (Saxen, 1977; Slack, 1984). If the inductive signals are of an instructive nature, it would be of great interest to know how restricted the potency of neural crest cells is at these developmental stages. However, our results, together with those of Epperlein and Lofberg (1984), indicate that the interactions leading to chromatophore differentiation could be permissive, implying that environmental factors that are normally encountered by the neural crest cells, i.e., extracellular matrices, would selectively promote the expression of already committed chromatophore phenotypes. Thus, stimulating signals should be necessary for the final chromatophore differentiation but should not influence the developmental pathway selected by the crest cells. We thank Vibeke Nilsson, Charlotte Fallstrom, Lars-Erik Jonsson, and Henrik Olson for technical assistance. The study was supported by the Swedish Natural Science Research Council (Grant B-Bu 3810-100). REFERENCES BAGNARA, J. T., MATSIJMOTO, J., FERRIS, W., FROST, S. K., TURNER, W. A., TCHEN, T. T., and TAYLOR, J. D. (1979). Common origin of pigment cells. Science (Wuah,ington, D. C.) 203, 410-415. BARALD, K. F. (1983). Monoclonal antibodies to chick ciliary ganglion isolate a neural crest subpopulation by fluorescence activated cell sorting. Sot. Neurosci. S(l), Abstr. 103.2. BEE, J., and THOROGOOD,P. (1980). The role of tissue interactions in the skeletogenic differentiation of avian neural crest cells. Dev. Biol. 78,47-62. BORDZILOVSKAYA, N. P., and DETTLAFF, T. A. (1979). Table of stages of the normal development of axolotl embryos and the prognostication of timing of successive developmental stages at various temperatures. Axolotl Neu&tt.7,2-22 (Dept. of Biology, Indiana Univ., Bloomington). COCHARD, P., and COLTEY, P. (1983). Cholinergic traits in the neural crest: Acetylcholinesterase in crest cells of the chick embryo. Dev. BioL 98,221-238. COHEN, A. M. (1972). Factors directing the expression of sympathetic nerve traits in cells of neural crest origin. J. Exp. 2001. 179,167-182. COHEN, A. M., and KONIGSBERG, I. R. (1975). A clonal approach to the problem of neural crest cell differentiation. Dev. Bid. 46,262-280. DELANNEY, L. E. (1941). The role of the ectoderm in pigment production, studied by transplantation and hybridization. J. Exp. Zool. 87,323341. DERBY, M. A. (1982). Environmental factors affecting neural crest differentiation by crest cells exposed to cell-free (deoxycholate-extracted) dermal mesenchyme matrix. Cell Tissue Res. 225.379-386. DERBY, M. A., and NEWGREEN, D. F. (1982). Differentiation of avian neural crest cells in vitro: Absence of a developmental bias toward melanogenesis. Cell Tissue Res. 225, 365-378.

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