In Vitro Cell. Dev. Biol. 28A:348-354, May 1992 9 1992 Tissue Culture Association 0883-8364/92 $01.50+0.00
DIFFERENTIATION OF REPTILIAN NEURAL CREST CELLS I N V I T R O LING HOU ~ v TAKUJI TAKEUCHI1
Biological Institute, Faculty of Science, Tohoku University, Aoba-yama, Sendai 980, Japan (Received 25 September 1991; accepted 20 November 1991)
SUMMARY
An attempt was made to culture neural crest cells of the turtle embryo in vitro. Trunk neural tubes from the St. 9 / 1 0 embryos were explanted in culture dishes. The developmental potency of the turtle neural crest cells in vitro was shown to be essentially similar to that of avian neural crest cells, although they seem to be more sensitive to melanocyte-stimulating hormone (MSH) stimulation. We describe conditions under which explanted neural tube gives rise to neural crest cells that differentiate into neuronal cells and melanocytes. The potency of melanocyte differentiation was found to vary according to the concentration of fetal bovine serum (FBS, from 5 to 20%). Melanization of neural crest cells cultured in the medium containing FBS and a-MSH was more extensive than those cultured with FBS alone, combinations of FBS and chick embryo extract, or turtle embryo extract. These culture conditions seem to be useful for the study of the developmental potency of the neural crest cells as well as for investigating local environmental factors.
Key words: neural crest cells; differentiation; cell culture; melanocytes; c~-melanocyte-stimulating hormone; reptiles. nocytes are located in various extracutaneous tissues of reptiles (Durker, 1985; Hou and Takeuchi, 1991). Mechanisms that produce the difference in the distribution of pigment cells between cold-blooded and warm-blooded animals are not yet known. In an attempt to elucidate this problem we have focused on the interaction of neural crest cells with their surrounding tissues of reptile embryos. So far there are few reports concerning the behavior and fate of neural crest cells of developing reptile embryos in vitro. Therefore, we first attempted to obtain neural crest cells of turtle embryos in vitro. The soft-shelled turtle (Trionyx sinensisjaponicas) was chosen because the normal developmental stage of the embryo and differentiation of extracutaneous melanocytes have been described by us (Hou, 1984, 1986, 1987a, b; Hou and Takeuchi, 1991). In this paper, we have found conditions under which explanted neural tubes give rise to neural crest cells that differentiate into neuronal cells and melanocytes in the presence of fetal bovine serum (FBS), and chick embryo extract (CEE), or turtle embryo extract (TEE). A pronounced effect of melanocyte-stimulating hormone (MSH) on melanogenic differentiation of the turtle neural crest cells was also noted.
INTRODUCTION
One of the basic questions in developmental biology is how differentiated cells arise from common precursor cells. The neural crest cell is an excellent material for the study of cell differentiation because it gives rise to diverse cellular phenotypes during early stages of vertebrate embryogenesis. These cell types include the pigment cells (melanocytes, iridophores, xanthophores, and erythrophores), neurons, support cells of the peripheral nervous systems, adrenomedullary cells, and most of the craniofacial cartilage, skeletal, and connective tissues (Hall, 1988; Le Douarin, 1982). How this diversity of cell types arises from common precursors in neural crest has been investigated extensively (for review, see Bagnara, 1987; Ciment, 1990; Weston, 1986). In particular, culture of vertebrate neural crest cells has made it possible to test their developmental potency under controlled conditions as previously described by many authors (amphibia: Fukuzawa and Bagnara, 1989; Perris et al., 1988, bird: Campbell, 1989; Derby, 1982; Loring et al., 1982; Rogers et al., 1990; Sieber-Blum, 1989, mammal: Agamy and Hornby, 1988; ho and Takeuchi, 1984; MorrisonGraham et al., 1990). It has been suggested that neural crest cells are muhipotent and that environmental cues can influence the differential fate of crest eells such as differentiation of pigment cells. On the other hand, differences occur in the distribution of pigment cells among different classes of vertebrates. In mammals, neural crest-derived melanocytes are usually confined to choroid, epidermis, and hair. In cold-blooded animals, three types of pigment cells occur not only in skin but also at various locations of the body. In fish and amphibia, melanophores, iridophores, and xanthophores are observed in extraeutaneous tissues, and abundant mela-
MATERIALS AND METHODS
Incubation of eggs. Fertilized eggs of the wild-type, soft-shelled turtle were collected at oviposition from females nesting in Hattori-Nakamura Nursery of Shizuoka, Japan, during the nesting season. The method of incubation for eggs of this species was previously described (Hou, 1984, 1986). Briefly,the eggs were incubated at 33 ~ C, with about 90% relative humidity in an incubator. The assignment of each embryo to a developmental stage was made by isolating it from the egg. Isolation of neural tube. Neural tubes were isolated from file turtle embryos at stages 9/10 (Hou, 1984). These stages were chosen because crest-cell migration has not yet started at the posterior levels (unpublished observation). Before the isolation of neural tubes, embryos were removed
1 To whom correspondence should be addressed. 348
349
DIFFERENTIATION OF CREST CELLS
MSH is known to promote melanocyte differentiation as described previously (Takeuchi, 1985). Identification of melanocytes. Melanocytes were identified by their melanization in vitro. The number of melanocytes and the total number of cells were determined by counting cells in a hemacytometer after dispersing the cells with 0.05% trypsin in CMF-phosphate buffered saline. RESULTS
FIG. 1. Photograph of a 11-somite (stage 9) turtle embryo showing the section between arrowheads from which the neural tube is isolated for a neural crest culture. •
from eggs and washed in 60-mm dishes containing Tyrode solution. Trunks of those at five to six last somite levels were dissected with tungsten needles (Fig. 1). The obtained tissues were treated with 1% trysin (trysin 1:250: Difco, Detroit, MI) in Tyrode solution at 4 ~ C for 25 min, then transferred to culture medium (the same medium that was later used for the culture of neural tubes). Then neural tubes were separated from the surrounding tissues by gentle pipeuing using a Pasteur pipette. The isolated neural tubes were checked for purity by visual inspection. Culture of neural tube. Isolated neural tubes were placed in 35-mm tissue culture dishes (3001, Falcon) containing 2 ml Eagle's minimum essential medium (MEM, Nissui 1, Tokyo) supplemented with 0.3% L-glutamine and various concentrations of FBS (no. 29101896 and 9050183, Flow, Irvine, Scotland), and/or CEE, TEE, a-MSH. They were then incubated in a humidified atmosphere of 5% CO2:95% air, at 32 ~ C, the normal temperature for the hatching of this species (Hou, 1984, 1987b), for 14 to 15 days. Half of the medium was replaced with fresh medium every 3rd day. Chick embryoextract. CEE was prepared from the trunks of 10-day-old chick embryos. After being washed in Tyrode solution, the trunks were homogenized and then mixed with an equal volume of Tyrode solution. The mixture was freeze-thawed 3 times, centrifuged twice at 3 000 rpm for 15 min, and the supernatant thus obtained was used as CEE. Turtle embryo extract. Soft-shelled TEE was prepared from the trunks of 20-to 22-day-old embryos, following the process as described in CEE experimental procedures. Treatment with MSH. Neural tube cultures were treated with 10-s M a-MSH (Sigma, St. Louis, MO) in the medium containing 15 or 20% FBS.
Culture of neural tube explant. We first attempted to find suitable conditions for maintaining crest cell growth. In our study, Eagle's MEM supplemented with FBS supported optimal growth and survival. Without FBS, most of the neural tubes failed to adhere to the culture dish. With 5, 15, 20% FBS all the neural tubes attached to the culture dish, although with higher concentrations of 15 and 20% FBS, a higher number of crest cells emigrated from the neural tubes. The cells also survived for a longer period of time in these conditions. In the neural tube explants from embryos of stage 9, a rapid increase in the number of cells in the culture was observed for 1 to 4 days. In the avian embryo, it was shown that isolation of neural tubes on a plastic dish prevented adhesion of explants, resulting in formation of cell clusters which after subeuhuring on a tissue culture dish produced homogeneous colonies of melanocytes (Glimelius and Weston, 1981; Loring et al., 1981; Yamamoto et al., 1987). The turtle neural tubes cultured for 1 to 4 days on plastic dishes, however, did not produce clusters of neural crest cells but sometimes formed small clusterhke aggregates on tubes in the medium containing FBS and TEE or CEE. After the transfer of these aggregates to other culture dishes, no significant indication of melanocyte differentiation was found. Effects of removal of the neural tube. When neural tubes were removed on Day 2 of culture and half the medium was replaced with fresh medium containing 5, 15, and 20% FBS, most cells were lysed. In some cases, however, cells were relatively intact, with the medium containing 20% FBS. This result suggests that the presence of diffusible factors produced by the neural tube is important for survival of neural crest cells in culture, as previously described in rat neural crest cell culture (Smith-Thomas and Fawcett, 1989). Addition of 8% CEE (prepared from 10 days embryos) to the culture medium reduced neither the incidence of cell lysis nor the proliferation of the neural crest cells. Morphologic appearance of cells migrated from explanted neural tube. Twenty-four to forty-eight hours after explantation, the cultured neural tubes were surrounded by numerous cells. Two clearly distinct cell populations were observed at this time; one contained cells that exhibit a stellar morphology with spaces between them, the other, flat cells. The former was similar to those found in avian and mammalian neural crest culture (Cohen and Konigsberg, 1975; ho and Takeuchi, 1984; Loring et al., 1981). We therefore assume that these cells are neural crest cells. The latter population contained cells that displayed an epithelioid morphology and formed a confluent monolayer attached to the ventral part of the neural tube (Fig. 2 B). These cells are similar to the neural tubederived cells observed in the ventral neural tube explant culture of the rat embryo (Smith-Thomas and Fawcett, 1989). This indicates that these cells are neural tube derivatives. After 4 to 5 days in culture, a complex network of axons and a neuronlike structure was observed in the culture. Most of the axons seemed to be connected to the neural tube explants (Fig. 2 C). In
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FIG. 2. Isolated neural tube explant after (,4) 0 h, (B) 48 h, (C) 7-day and 12-day of cuhure in MEM containing 20% FBS (A,B,C), 20% FBS and 1% TEE (D), 14-day of cuhure in 20% FBS and 4% CEE (E,F). A, a neural tube isolated from the last somite region of embryo; B, neural crest outgrowth from a neural tube cultured for 48 h. Two cell types were observed in the culture. C, crest cells in MEM with 20% FBS. The typical axonlike processes are observed. Most of the axons seemed connected to the neural tube explant. D, neural crest cells cultured in MEM with 20% FBS and 1% TEE. The numerous gangllonlike structures (g) and they connected with the axonlike processes (arrowhead) are found. E,F, dendritic melanocytes (arrowheads) were located in epithelial sheet. Phase contrast (A-E), bright-field (F). X50.
particular, when neural crest cells were cultured in the medium containing FBS supplemented with TEE or CEE, the ganglionlike structures and axonlike processes were observed. The axonlike processes connected with these structures were also found (Fig. 2 D). In addition to axons and neuronal cells, we observed melanocytes recognizable by their morphology and the presence of melanin granules (a marker of melanocyte). The melanocytes were located in association with epithelial sheet (Fig. 2 E,F) and observed as
isolated cells at the edge of the outgrowth. The intensity of melanocyte differentiation was found to vary according to the concentration of the serum; it was higher in the medium containing 20% FBS (Table 1). It has been reported that for avian neural crest cells the combination of FBS and CEE at various concentrations promotes growth and differentiation of particular derivatives of the neural crest such as melanocytes (Glimelius and Weston, 1981; Loring et al., 1981). We also observed improvement in growth or in the
351
DIFFERENTIATION OF CREST CELLS TABLE 1 DIFFERENTIATION POTENCY OF NEURAL CREST CELLS INTO MELANOCYTES IN THE CULTURE
FBS
CEE
TEE"
Exp, 1
Exp. 2
Exp. 3
Total
days
Number ofb Melanocytes per Culture
5% 15% 20% 15% 15% 20% 15% 20%
---4% 8% 4% ---
------4% 4%
0/1 0/2 2/3 1/3 2/2 3/3 1/2 2/2
1/2 1/3 4/4 2/3 3/3 NT 2/3 2/2
1/3 2/4 1/3 NT NT NT NT NT
2/6 3/7 8/10 3/6 5/5 3/3 3/5 4/4
9-10 9-10 9-10 9-10 9-10 9-10 9-10 9-10
0-2 0-5 0-21 0-11 2-73 11-38 0-10 3-37
Culture Condition
No. of Explants with Melanocytes/Total Number of Explants
Time of Pigment Appearance,
~ CEE from lO-day; TEE from 20-day; NT = not tested. bNumber of melanizedcells was scored by phase contrast and bright field microscopy 14 day after the explant culture.
potency of melanocyte differentiation when extract of chick or turtle embryos was added in combination with FBS (Table 1), although melanization in neural crest cells was much lower than in the case of avian (data not shown). Effect of o~-MSH on the melanogenic differentiation. MSH is also known to induce the differentiation of neural crest cells into melanocytes in various organisms. It is considered that this ot-MSH action is mediated by cyclic AMP (cAMP), the second messenger (for a review, see Takeuchi and Yamamoto, 1988). To investigate whether the promotion of melanogenic differentiation of the turtle neural crest cells is also induced by ot-MSH, the cultures were treated with the hormone. When neural crest cells were directly inoculated in the medium containing MSH, fully melanized cells first appeared in all the explants after culturing for 4 to 5 days (Table 2), whereas only a few melanocytes appeared in some control cultures even after 9 to 10 days. Treated with 10 -s M ot-MSH, melanocytes were first observed at 4 to 5 days and by Day 7 after plating, numerous melanocytes appeared (Fig. 3 A,B), whereas no melanocyte was seen in control cultures even after 7 to 8 days (Fig. 3 C,D). It was also shown that MSH not only accelerated melanocytes differentiation, but also promoted melanization of neural crest cell population in a dose-dependent manner (Fig. 4). In these a-MSH-treated cultures, about 25% of neural crest cells underwent melanization, whereas only a few melanocytes were found when cultured in the medium containing 20% FBS after 14 days plating (Fig. 5 A,B). They also stimulated neural crest-derived melanocytes to become more dendritic and more pigmented than the control cultures (Fig. 5 C,D). These melanocytes were often distributed at the edge of the outgrowth and located in association with epithelial sheets. In some
cases, crest cells did not migrate out of the dorsal surface of the neural tube, although they differentiated into melanoeytes by MSH stimulation. Such melanized cells did not exhibit dendritic character (data not shown).
DISCUSSION
Reptilian cells grow well in media designed for warm-blooded animal cells (Fergnson et al., 1983; Goldstein, 1981), but reptilian cells require incubation temperatures below that for warm-blooded animal cells. Reptilian cell lines established at wide incubation temperatures have been described (Clark et al., 1970). In our study, the incubation temperature was selected at 32 ~ C because this normal temperature has been used for hatching this species (Hou, 1984, 1987b). We found that this temperature was adequate for our purpose. Eagle's MEM, which has usually been used in cultures of reptilian cells (Ferguson et al., 1983; Goldstein, 1981), was found to be a suitable medium to support the turtle neural crest cell growth. The addition of FBS was necessary, and at 15 or 20% yielded better outgrowth of cells from the explanted neural tubes. Without FBS the tube did not adhere to the culture dish. Although there is no reason to doubt that melanoeytes of reptiles are derived from neural crest cells, we first provided experimental evidence for the origin of melanocytes with cultured neural crest cells in reptiles. In the present study, culture of trunk neural tube gives rise to the appearance of a population of small stellate cells. Their characteristic morphology seems to indicate that these cells are neural crest cells (Cohen and Konigsberg, 1975; Ito and Takeuehi, 1984; Loring et al., 1981). This assumption was verified by
TABLE 2 EFFECTS OF ot-MSH ON DIFFERENTIATIONNEURAL CREST CELLS INTO MELANOCYTES IN CULTURE
FBS
a-MSH
Exp. I
Exp. 2
Exp. 3
Total
days
Number of" Melanocytes per Culture, Mean + SE
15% 20%
10-s M 10-s M
2/2 2/2
3/3 3/3
2/2 3/3
7/7 8/8
4-5 4-5
349 + 21.7 361.6 + 16.2
Culture Condition
No. of Explants with Melanocytes/Total Number of Explants
a Number of melanizedcells was counted as described in Materialsand Methods.
Time of Pigment Appearance,
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FIG. 3. Acceleration of neural crest cells melanization by a-MSH. Neural crest cells were directly treated with a-MSH. Photographs were taken 7 days after plating. A,B, ot-MSH treated culture in MEM containing 20% FBS and 10-s M a-MSH. Numerous melanocytes have already differentiated. C,D, control culture in MEM containing 20% FBS. No melanocyte is seen. Phase contrast (A,C), bright-field (B,D). •
using HNK-1 antibody, which specifically recognizes neural crest cells (unpublished observation). In contrast to quail neural crest, differentiation of the turtle crest cells into melanocytes has been found less in number in explant culture, even after a prolonged culture of 2 wk. The poverty of a development bias toward melano-
FIG. 4. Effect of ot-MSH on melanization of crest cells in culture. Proportions of melanized cells after 14 days in culture were counted. Each point is the average of four to six individual neural tube culture.
genesis in the turtle crest cell culture contrasts with Xenopus and quail crest cell cultures in which melanocytes differentiate readily. In quail crest cell cultures, melanocyte differentiation is favored by the presence of CEE and certain batches of FBS, both of which were present in our culture medium. On the other hand, mouse and rat neural crest cells under these culture conditions fully differentiated melanocytes never appeared, even after treatment with c~-MSH (ho and Takeuchi, 1984; Smith-Thomas and Fawcett, 1989). The poverty of differentiation ability toward melanogenesis in the turtle and mammalian crest cell cultures suggests that the melanocyte differentiation in vitro requires an environmental stimulus, such as hormonal factors, extracellular matrix (ECM), or other melanogenic factors. As one approach to this problem, we investigated the effects of a-MSH on melanogenic differentiation of the turtle neural crest cells. In fish, Chen et al. (1973) reported that differentiation of melanoblasts into melanocytes was promoted by MSH treatment. In amphibia, it has been shown that MSH accelerated melanophore development in vitro and in vivo (Wahn et al., 1976) and that MSH played a role in chromatoblast determination of early development (Bagnara, 1987). In quail, MSH was also shown to promote the melanogenic differentiation in neural crest cells in vitro (Satoh and Ide, 1987). Furthermore, Ito and Takeuchi (1984) reported that finally differentiated melanocytes have only been detected in mouse neural crest cell cultures under the presence of MSH. Our result
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DIFFERENTIATION OF CREST CELLS
FIG. 5. Differentiation of neural crest cells into melanocytes in MEM with 20% FBS alone and combinations of l0 -s M a-MSH in culture for 14 days. A,B, culture in the media containing 20% FBS; few melanocytes are observed. C,D, a-MSH treated culture, in MEM with 20% FBS; intense melanocytes are shown. X50.
suggests that MSH stimulates melanogenic differentiation of the turtle neural crest cells in culture. It is also suggested that a morphologically undifferentiated population of the crest cells already possess a-MSH receptors. In a-MSH-treated culture, a population (about 25%) of neural crest cells undergo melanization. It is likely that differences in surface receptors result in the heterogeneity in responsiveness to MSH. As to the heterogeneity in the neural crest population, two possibilities can be suggested, a) The turtle neural crest cells at this stage consist of heterogeneous cell populations. Cells that expressed MSH receptors, thus committing into melanocyte differentiation pathway, are only a proportion of the crest cell population; and b) the neural crest cells at this stage are still underway to produce MSH receptors. About 75% of the cells are not yet competent to MSH induction. Our result was taken as further evidence that the developing neural crest is heterogeneous and that an environmental stimulus is required for final differentiation of neural crest cells into melanocytes in the embryos of turtle. In fact, the pigment promoting factor (PPF) from mouse epidermis and skin tissue (particularly ECM) has been reported to promote melanocyte differentiation (Agamy and Hornby, 1988; Morrison-Graham et al., 1990). It is further suggested that an environmental signal controls differentiation of melanocytes from neural crest cells in the mouse and that their interaction may occur after the migration of neural crest cells for cartilage development (Hall, 1987). On the other hand, differentiation of pigment cells may occur at the beginning of
migration of crest cells in amphibian (Epperlein and Liifberg, 1984). The different timing of the interaction of neural crest cells and surrounding tissues among different species of vertebrate groups probably indicate that neural crest cells are determined at different stages or that environmental factor(s) that induce neural crest cell differentiation exert at different stages. It is unknown whether a PPF-like factor plays a role in the melanocyte differentiation in reptiles in vivo. It is possible, however, that the final differentiation of neural crest-derived melanoblasts in the integument or in extracutaneous tissues is promoted by particular tissues that elaborate stimulatory factor(s) (Hou and Takeuchi, 1991). It is feasible that these tissues exert peptide factors similar to MSH. ACKNOWLEDGEMENTS This work was supported in part by a Grant-in Aid from the Ministry of Education, Science and Culture, Japan (to T. Takeuchi). We are grateful to Hattofi-Nakamura Nursery of Shizuoka for providing us with the eggs used in this research. REFERENCES Agamy, E.; Hornby, J. E. Pigment promoting factor for mouse neural crest cells. Pigment Cell Res. 1:272; 1988. Bagnara, J. T. The neural crest as a source of stem ceil. In: Maderson,
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P. F. A., ed. Developmental and evolutionary aspects of the neural crest. London: J. Wiley & Sons; 1987:57-87. Campbell, M. Melanogenesis of avian neural crest cell in vitro is influenced by external cues in the periorbital mesenchyme. Development 66:717-726; 1989. Chen, S.; Tchen, T. T.; Taylor, J. D. The role of c-AMP in hormone (MSH or ACTH)-induced melanocyte development in organ cultures of cauda fines of xanthic goldfish. In: Riley, V., ed. Pigment cell. Basel: Karger; 1973:1-5. Ciment, G. The melanocyte/Sehwann cell progenitor: a bipotent intermediate in the neural crest lineage. Comments Dev. Neurobiol. 1:207223; 1990. Clark, H. F.; Cohen, M. M.; Karzon, D. T. Characterization of reptilian cell lines established at incubation temperatures of 230-36 ~ C. Proc. Soc. Exp. Biol. Med. 33:1039-1047; 1970, Cohen, A. M.; Konigsberg, I. R. A clonal approach to the problem of neural crest determination. Dev. Biol. 46:262-280: 1975. Derby, M. A. Environmental factors affecting neural crest differentiation: melanocyte differentiation by crest cell exposed to cell-free (deoxycholate-extracted) dermal mesenchyme matrix. Cell Tissue Res. 225:379-383; 1982. Durker, H. R. The neural crest. In: Bagnara, J. T.; Klaus, S.; Paul, E., et al., eds. Tokyo: University of Tokyo Press; 1985:255-267. Epperlein, H. H.; Li~tberg, J. Xanthophores in chromatophore groups of the premigratory crest initiate the pigment pattern of the axolotl larva. Wilhelm Roux's Arch. Dev. Biol. 193:357-369; 1984. Ferguson, M. W. J.; Honig, L. S.; Bringnas, P., et al, Alligator mandibular development during long-term organ culture. In Vitro 19:385-393; 1983. Fukuzawa, T.; Baguara. J. T. Control of melanoblasts differentiation in amphibia by a-melanocyte stimulating hormone, a serum melanization factor, and a melanization inhibiting factor. Pigment Cell Res. 2:171-181; 1989. Glimelius, B.; Weston, J. A. Analysis of developmentally homogeneous neural crest cell populations in vitro. 1Ii. Role of culture environment in cluster formation and differentiation. Cell Differ. 10:5767; 1981. Goldstein, S. Aging in vitro. Growth of cultured cells from the galapagos tortoise. Exp. Cell. Res. 83:297-302; 1981. Hall, B. K. Tissue interaction in the development and evolution of the vertebrate head. In: Madenson, P. F. A., ed. Development and evolutionary aspects of the neural crest. New York:John Wiley & Sons; 1987:215-259. Hall, B. K. The neural crest. London: Oxford University Press; 1988. Hou, L. Acquirement and use of the turtle embryos for experimental purposes. J. Zool. 4:39-40; 1986. Hou, L. Studies on the embryonic development of the turtle, Trionyx sinensis. Natl. Sci. J. Hunan Normal Univ. 2:59-64; 1984.
Hou, L. Cytogenesis of the primordial germ cells in the embryos of the turtle. Acta Herpertol Sin. 6:5-10; 1987a. Hou, L. Effect of incubation temperature on sexual differentiation of two turtles. J. Hunan Sei. Technol. Univ. 3:71-76; 1987b. Hou, L.; Takeuehi, T. Differentiation of extracutaneous melanocytes in embryos of the turtle (Trionyx sinensisjaponicus). Pigment Cell Res. 4:158-162; 1991. Ito, K.; Takeuchi, T. The differentiation in vitro of the neural crest cells of the mouse embryo. J. Embryol. Exp. Morphol. 84:49-62; 1984. Le Douarin, N. The neural crest. England: Cambridge University Press; 1982. Loring, J.; Glimelius, B.; Ericson, C., et al. Analysis of developmentally homogeneous neural crest cell population in vitro. I. Formation, morphology, and differentiation behavior. Dev. Biol. 82:86-94; 1981. Loring, J.; Glimelius, B.; Weston, J. A. Extracellular matrix materials influence quail neural crest cell differentiation in vitro. Dev. Biol. 90:165-174; 1982. Morrison-Graham, K.; West-Johnsrud, L.; Weston, J. A. Extracellular matrix from normal but not Steel mutant mice enhances melanogenesis in cultured mouse neural crest cells. Dev. Biol. 139:299-307; 1990. Perris, R.; Von Boxberg, Y.; LiStberg, J. Local embryonic matrices determine region-specific phenotypes in neural crest cells. Science 241:86-89; 1988. Rogers, S. L.; Bernard, L.; Weston, J. A. Substratum effects on cell dispersal, morphology, and differentiation in culture of avian neural crest cells. Dev. Biol. 141:173-182; 1990. Satoh, M.; lde, H. Melanocyte stimulating hormone affects melanogenic differentiation of quail neural crest cells in vitro. Dev. Biol. 119:579-586; 1987. Sieber-Blum, M. Commitment of neural crest cells to the sensory neuron lineage. Science 243:1608-1610; 1989. Smith-Thomas, L. C.; Fawcett, J. W. Expression of schwann cell markers by mammalian neural crest cells in vitro. Development 105:251262; 1989. Takeuchi, T. Gene controlling intercellular communication during melanocyte differentiation in the mouse. Zool. Sci. (Tokyo) 2:823-831; 1985. Takeuchi, T.; Yamamoto, H. Genetic regulation of melanocyte differentiation. Pigment Cell Res. 1:32-37; 1988. Wahn, H. L.; Taylor, D.; Tchen, T. T. Acceleration of amphibian embryonic melanophore development by melanophore-stimulating hormone, N6, O2-dlbutyryl 9 . 3,5 , ,-monophosphate and theophadenosine ylline. Dev. Biol. 49:470-478; 1976. Weston, J. A., Phenotypic diversification in neural crest-derived cell: the time and stability of commitment during early development. Curr. Top. Dev. Biol. 20:195-210; 1986. Yamamoto, H.; ho, K.; lshiguro, S., et al. Gene controlling a differentiation step in the quail melanocyte. Dev. Genet. 8:179-185; 1987.
DIFFERENTIATION OF REPTILIAN NEURAL CREST CELLS I N V I T R O LING HOU ~ v TAKUJI TAKEUCHI1
Biological Institute, Faculty of Science, Tohoku University, Aoba-yama, Sendai 980, Japan (Received 25 September 1991; accepted 20 November 1991)
SUMMARY
An attempt was made to culture neural crest cells of the turtle embryo in vitro. Trunk neural tubes from the St. 9 / 1 0 embryos were explanted in culture dishes. The developmental potency of the turtle neural crest cells in vitro was shown to be essentially similar to that of avian neural crest cells, although they seem to be more sensitive to melanocyte-stimulating hormone (MSH) stimulation. We describe conditions under which explanted neural tube gives rise to neural crest cells that differentiate into neuronal cells and melanocytes. The potency of melanocyte differentiation was found to vary according to the concentration of fetal bovine serum (FBS, from 5 to 20%). Melanization of neural crest cells cultured in the medium containing FBS and a-MSH was more extensive than those cultured with FBS alone, combinations of FBS and chick embryo extract, or turtle embryo extract. These culture conditions seem to be useful for the study of the developmental potency of the neural crest cells as well as for investigating local environmental factors.
Key words: neural crest cells; differentiation; cell culture; melanocytes; c~-melanocyte-stimulating hormone; reptiles. nocytes are located in various extracutaneous tissues of reptiles (Durker, 1985; Hou and Takeuchi, 1991). Mechanisms that produce the difference in the distribution of pigment cells between cold-blooded and warm-blooded animals are not yet known. In an attempt to elucidate this problem we have focused on the interaction of neural crest cells with their surrounding tissues of reptile embryos. So far there are few reports concerning the behavior and fate of neural crest cells of developing reptile embryos in vitro. Therefore, we first attempted to obtain neural crest cells of turtle embryos in vitro. The soft-shelled turtle (Trionyx sinensisjaponicas) was chosen because the normal developmental stage of the embryo and differentiation of extracutaneous melanocytes have been described by us (Hou, 1984, 1986, 1987a, b; Hou and Takeuchi, 1991). In this paper, we have found conditions under which explanted neural tubes give rise to neural crest cells that differentiate into neuronal cells and melanocytes in the presence of fetal bovine serum (FBS), and chick embryo extract (CEE), or turtle embryo extract (TEE). A pronounced effect of melanocyte-stimulating hormone (MSH) on melanogenic differentiation of the turtle neural crest cells was also noted.
INTRODUCTION
One of the basic questions in developmental biology is how differentiated cells arise from common precursor cells. The neural crest cell is an excellent material for the study of cell differentiation because it gives rise to diverse cellular phenotypes during early stages of vertebrate embryogenesis. These cell types include the pigment cells (melanocytes, iridophores, xanthophores, and erythrophores), neurons, support cells of the peripheral nervous systems, adrenomedullary cells, and most of the craniofacial cartilage, skeletal, and connective tissues (Hall, 1988; Le Douarin, 1982). How this diversity of cell types arises from common precursors in neural crest has been investigated extensively (for review, see Bagnara, 1987; Ciment, 1990; Weston, 1986). In particular, culture of vertebrate neural crest cells has made it possible to test their developmental potency under controlled conditions as previously described by many authors (amphibia: Fukuzawa and Bagnara, 1989; Perris et al., 1988, bird: Campbell, 1989; Derby, 1982; Loring et al., 1982; Rogers et al., 1990; Sieber-Blum, 1989, mammal: Agamy and Hornby, 1988; ho and Takeuchi, 1984; MorrisonGraham et al., 1990). It has been suggested that neural crest cells are muhipotent and that environmental cues can influence the differential fate of crest eells such as differentiation of pigment cells. On the other hand, differences occur in the distribution of pigment cells among different classes of vertebrates. In mammals, neural crest-derived melanocytes are usually confined to choroid, epidermis, and hair. In cold-blooded animals, three types of pigment cells occur not only in skin but also at various locations of the body. In fish and amphibia, melanophores, iridophores, and xanthophores are observed in extraeutaneous tissues, and abundant mela-
MATERIALS AND METHODS
Incubation of eggs. Fertilized eggs of the wild-type, soft-shelled turtle were collected at oviposition from females nesting in Hattori-Nakamura Nursery of Shizuoka, Japan, during the nesting season. The method of incubation for eggs of this species was previously described (Hou, 1984, 1986). Briefly,the eggs were incubated at 33 ~ C, with about 90% relative humidity in an incubator. The assignment of each embryo to a developmental stage was made by isolating it from the egg. Isolation of neural tube. Neural tubes were isolated from file turtle embryos at stages 9/10 (Hou, 1984). These stages were chosen because crest-cell migration has not yet started at the posterior levels (unpublished observation). Before the isolation of neural tubes, embryos were removed
1 To whom correspondence should be addressed. 348
349
DIFFERENTIATION OF CREST CELLS
MSH is known to promote melanocyte differentiation as described previously (Takeuchi, 1985). Identification of melanocytes. Melanocytes were identified by their melanization in vitro. The number of melanocytes and the total number of cells were determined by counting cells in a hemacytometer after dispersing the cells with 0.05% trypsin in CMF-phosphate buffered saline. RESULTS
FIG. 1. Photograph of a 11-somite (stage 9) turtle embryo showing the section between arrowheads from which the neural tube is isolated for a neural crest culture. •
from eggs and washed in 60-mm dishes containing Tyrode solution. Trunks of those at five to six last somite levels were dissected with tungsten needles (Fig. 1). The obtained tissues were treated with 1% trysin (trysin 1:250: Difco, Detroit, MI) in Tyrode solution at 4 ~ C for 25 min, then transferred to culture medium (the same medium that was later used for the culture of neural tubes). Then neural tubes were separated from the surrounding tissues by gentle pipeuing using a Pasteur pipette. The isolated neural tubes were checked for purity by visual inspection. Culture of neural tube. Isolated neural tubes were placed in 35-mm tissue culture dishes (3001, Falcon) containing 2 ml Eagle's minimum essential medium (MEM, Nissui 1, Tokyo) supplemented with 0.3% L-glutamine and various concentrations of FBS (no. 29101896 and 9050183, Flow, Irvine, Scotland), and/or CEE, TEE, a-MSH. They were then incubated in a humidified atmosphere of 5% CO2:95% air, at 32 ~ C, the normal temperature for the hatching of this species (Hou, 1984, 1987b), for 14 to 15 days. Half of the medium was replaced with fresh medium every 3rd day. Chick embryoextract. CEE was prepared from the trunks of 10-day-old chick embryos. After being washed in Tyrode solution, the trunks were homogenized and then mixed with an equal volume of Tyrode solution. The mixture was freeze-thawed 3 times, centrifuged twice at 3 000 rpm for 15 min, and the supernatant thus obtained was used as CEE. Turtle embryo extract. Soft-shelled TEE was prepared from the trunks of 20-to 22-day-old embryos, following the process as described in CEE experimental procedures. Treatment with MSH. Neural tube cultures were treated with 10-s M a-MSH (Sigma, St. Louis, MO) in the medium containing 15 or 20% FBS.
Culture of neural tube explant. We first attempted to find suitable conditions for maintaining crest cell growth. In our study, Eagle's MEM supplemented with FBS supported optimal growth and survival. Without FBS, most of the neural tubes failed to adhere to the culture dish. With 5, 15, 20% FBS all the neural tubes attached to the culture dish, although with higher concentrations of 15 and 20% FBS, a higher number of crest cells emigrated from the neural tubes. The cells also survived for a longer period of time in these conditions. In the neural tube explants from embryos of stage 9, a rapid increase in the number of cells in the culture was observed for 1 to 4 days. In the avian embryo, it was shown that isolation of neural tubes on a plastic dish prevented adhesion of explants, resulting in formation of cell clusters which after subeuhuring on a tissue culture dish produced homogeneous colonies of melanocytes (Glimelius and Weston, 1981; Loring et al., 1981; Yamamoto et al., 1987). The turtle neural tubes cultured for 1 to 4 days on plastic dishes, however, did not produce clusters of neural crest cells but sometimes formed small clusterhke aggregates on tubes in the medium containing FBS and TEE or CEE. After the transfer of these aggregates to other culture dishes, no significant indication of melanocyte differentiation was found. Effects of removal of the neural tube. When neural tubes were removed on Day 2 of culture and half the medium was replaced with fresh medium containing 5, 15, and 20% FBS, most cells were lysed. In some cases, however, cells were relatively intact, with the medium containing 20% FBS. This result suggests that the presence of diffusible factors produced by the neural tube is important for survival of neural crest cells in culture, as previously described in rat neural crest cell culture (Smith-Thomas and Fawcett, 1989). Addition of 8% CEE (prepared from 10 days embryos) to the culture medium reduced neither the incidence of cell lysis nor the proliferation of the neural crest cells. Morphologic appearance of cells migrated from explanted neural tube. Twenty-four to forty-eight hours after explantation, the cultured neural tubes were surrounded by numerous cells. Two clearly distinct cell populations were observed at this time; one contained cells that exhibit a stellar morphology with spaces between them, the other, flat cells. The former was similar to those found in avian and mammalian neural crest culture (Cohen and Konigsberg, 1975; ho and Takeuchi, 1984; Loring et al., 1981). We therefore assume that these cells are neural crest cells. The latter population contained cells that displayed an epithelioid morphology and formed a confluent monolayer attached to the ventral part of the neural tube (Fig. 2 B). These cells are similar to the neural tubederived cells observed in the ventral neural tube explant culture of the rat embryo (Smith-Thomas and Fawcett, 1989). This indicates that these cells are neural tube derivatives. After 4 to 5 days in culture, a complex network of axons and a neuronlike structure was observed in the culture. Most of the axons seemed to be connected to the neural tube explants (Fig. 2 C). In
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FIG. 2. Isolated neural tube explant after (,4) 0 h, (B) 48 h, (C) 7-day and 12-day of cuhure in MEM containing 20% FBS (A,B,C), 20% FBS and 1% TEE (D), 14-day of cuhure in 20% FBS and 4% CEE (E,F). A, a neural tube isolated from the last somite region of embryo; B, neural crest outgrowth from a neural tube cultured for 48 h. Two cell types were observed in the culture. C, crest cells in MEM with 20% FBS. The typical axonlike processes are observed. Most of the axons seemed connected to the neural tube explant. D, neural crest cells cultured in MEM with 20% FBS and 1% TEE. The numerous gangllonlike structures (g) and they connected with the axonlike processes (arrowhead) are found. E,F, dendritic melanocytes (arrowheads) were located in epithelial sheet. Phase contrast (A-E), bright-field (F). X50.
particular, when neural crest cells were cultured in the medium containing FBS supplemented with TEE or CEE, the ganglionlike structures and axonlike processes were observed. The axonlike processes connected with these structures were also found (Fig. 2 D). In addition to axons and neuronal cells, we observed melanocytes recognizable by their morphology and the presence of melanin granules (a marker of melanocyte). The melanocytes were located in association with epithelial sheet (Fig. 2 E,F) and observed as
isolated cells at the edge of the outgrowth. The intensity of melanocyte differentiation was found to vary according to the concentration of the serum; it was higher in the medium containing 20% FBS (Table 1). It has been reported that for avian neural crest cells the combination of FBS and CEE at various concentrations promotes growth and differentiation of particular derivatives of the neural crest such as melanocytes (Glimelius and Weston, 1981; Loring et al., 1981). We also observed improvement in growth or in the
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DIFFERENTIATION OF CREST CELLS TABLE 1 DIFFERENTIATION POTENCY OF NEURAL CREST CELLS INTO MELANOCYTES IN THE CULTURE
FBS
CEE
TEE"
Exp, 1
Exp. 2
Exp. 3
Total
days
Number ofb Melanocytes per Culture
5% 15% 20% 15% 15% 20% 15% 20%
---4% 8% 4% ---
------4% 4%
0/1 0/2 2/3 1/3 2/2 3/3 1/2 2/2
1/2 1/3 4/4 2/3 3/3 NT 2/3 2/2
1/3 2/4 1/3 NT NT NT NT NT
2/6 3/7 8/10 3/6 5/5 3/3 3/5 4/4
9-10 9-10 9-10 9-10 9-10 9-10 9-10 9-10
0-2 0-5 0-21 0-11 2-73 11-38 0-10 3-37
Culture Condition
No. of Explants with Melanocytes/Total Number of Explants
Time of Pigment Appearance,
~ CEE from lO-day; TEE from 20-day; NT = not tested. bNumber of melanizedcells was scored by phase contrast and bright field microscopy 14 day after the explant culture.
potency of melanocyte differentiation when extract of chick or turtle embryos was added in combination with FBS (Table 1), although melanization in neural crest cells was much lower than in the case of avian (data not shown). Effect of o~-MSH on the melanogenic differentiation. MSH is also known to induce the differentiation of neural crest cells into melanocytes in various organisms. It is considered that this ot-MSH action is mediated by cyclic AMP (cAMP), the second messenger (for a review, see Takeuchi and Yamamoto, 1988). To investigate whether the promotion of melanogenic differentiation of the turtle neural crest cells is also induced by ot-MSH, the cultures were treated with the hormone. When neural crest cells were directly inoculated in the medium containing MSH, fully melanized cells first appeared in all the explants after culturing for 4 to 5 days (Table 2), whereas only a few melanocytes appeared in some control cultures even after 9 to 10 days. Treated with 10 -s M ot-MSH, melanocytes were first observed at 4 to 5 days and by Day 7 after plating, numerous melanocytes appeared (Fig. 3 A,B), whereas no melanocyte was seen in control cultures even after 7 to 8 days (Fig. 3 C,D). It was also shown that MSH not only accelerated melanocytes differentiation, but also promoted melanization of neural crest cell population in a dose-dependent manner (Fig. 4). In these a-MSH-treated cultures, about 25% of neural crest cells underwent melanization, whereas only a few melanocytes were found when cultured in the medium containing 20% FBS after 14 days plating (Fig. 5 A,B). They also stimulated neural crest-derived melanocytes to become more dendritic and more pigmented than the control cultures (Fig. 5 C,D). These melanocytes were often distributed at the edge of the outgrowth and located in association with epithelial sheets. In some
cases, crest cells did not migrate out of the dorsal surface of the neural tube, although they differentiated into melanoeytes by MSH stimulation. Such melanized cells did not exhibit dendritic character (data not shown).
DISCUSSION
Reptilian cells grow well in media designed for warm-blooded animal cells (Fergnson et al., 1983; Goldstein, 1981), but reptilian cells require incubation temperatures below that for warm-blooded animal cells. Reptilian cell lines established at wide incubation temperatures have been described (Clark et al., 1970). In our study, the incubation temperature was selected at 32 ~ C because this normal temperature has been used for hatching this species (Hou, 1984, 1987b). We found that this temperature was adequate for our purpose. Eagle's MEM, which has usually been used in cultures of reptilian cells (Ferguson et al., 1983; Goldstein, 1981), was found to be a suitable medium to support the turtle neural crest cell growth. The addition of FBS was necessary, and at 15 or 20% yielded better outgrowth of cells from the explanted neural tubes. Without FBS the tube did not adhere to the culture dish. Although there is no reason to doubt that melanoeytes of reptiles are derived from neural crest cells, we first provided experimental evidence for the origin of melanocytes with cultured neural crest cells in reptiles. In the present study, culture of trunk neural tube gives rise to the appearance of a population of small stellate cells. Their characteristic morphology seems to indicate that these cells are neural crest cells (Cohen and Konigsberg, 1975; Ito and Takeuehi, 1984; Loring et al., 1981). This assumption was verified by
TABLE 2 EFFECTS OF ot-MSH ON DIFFERENTIATIONNEURAL CREST CELLS INTO MELANOCYTES IN CULTURE
FBS
a-MSH
Exp. I
Exp. 2
Exp. 3
Total
days
Number of" Melanocytes per Culture, Mean + SE
15% 20%
10-s M 10-s M
2/2 2/2
3/3 3/3
2/2 3/3
7/7 8/8
4-5 4-5
349 + 21.7 361.6 + 16.2
Culture Condition
No. of Explants with Melanocytes/Total Number of Explants
a Number of melanizedcells was counted as described in Materialsand Methods.
Time of Pigment Appearance,
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FIG. 3. Acceleration of neural crest cells melanization by a-MSH. Neural crest cells were directly treated with a-MSH. Photographs were taken 7 days after plating. A,B, ot-MSH treated culture in MEM containing 20% FBS and 10-s M a-MSH. Numerous melanocytes have already differentiated. C,D, control culture in MEM containing 20% FBS. No melanocyte is seen. Phase contrast (A,C), bright-field (B,D). •
using HNK-1 antibody, which specifically recognizes neural crest cells (unpublished observation). In contrast to quail neural crest, differentiation of the turtle crest cells into melanocytes has been found less in number in explant culture, even after a prolonged culture of 2 wk. The poverty of a development bias toward melano-
FIG. 4. Effect of ot-MSH on melanization of crest cells in culture. Proportions of melanized cells after 14 days in culture were counted. Each point is the average of four to six individual neural tube culture.
genesis in the turtle crest cell culture contrasts with Xenopus and quail crest cell cultures in which melanocytes differentiate readily. In quail crest cell cultures, melanocyte differentiation is favored by the presence of CEE and certain batches of FBS, both of which were present in our culture medium. On the other hand, mouse and rat neural crest cells under these culture conditions fully differentiated melanocytes never appeared, even after treatment with c~-MSH (ho and Takeuchi, 1984; Smith-Thomas and Fawcett, 1989). The poverty of differentiation ability toward melanogenesis in the turtle and mammalian crest cell cultures suggests that the melanocyte differentiation in vitro requires an environmental stimulus, such as hormonal factors, extracellular matrix (ECM), or other melanogenic factors. As one approach to this problem, we investigated the effects of a-MSH on melanogenic differentiation of the turtle neural crest cells. In fish, Chen et al. (1973) reported that differentiation of melanoblasts into melanocytes was promoted by MSH treatment. In amphibia, it has been shown that MSH accelerated melanophore development in vitro and in vivo (Wahn et al., 1976) and that MSH played a role in chromatoblast determination of early development (Bagnara, 1987). In quail, MSH was also shown to promote the melanogenic differentiation in neural crest cells in vitro (Satoh and Ide, 1987). Furthermore, Ito and Takeuchi (1984) reported that finally differentiated melanocytes have only been detected in mouse neural crest cell cultures under the presence of MSH. Our result
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DIFFERENTIATION OF CREST CELLS
FIG. 5. Differentiation of neural crest cells into melanocytes in MEM with 20% FBS alone and combinations of l0 -s M a-MSH in culture for 14 days. A,B, culture in the media containing 20% FBS; few melanocytes are observed. C,D, a-MSH treated culture, in MEM with 20% FBS; intense melanocytes are shown. X50.
suggests that MSH stimulates melanogenic differentiation of the turtle neural crest cells in culture. It is also suggested that a morphologically undifferentiated population of the crest cells already possess a-MSH receptors. In a-MSH-treated culture, a population (about 25%) of neural crest cells undergo melanization. It is likely that differences in surface receptors result in the heterogeneity in responsiveness to MSH. As to the heterogeneity in the neural crest population, two possibilities can be suggested, a) The turtle neural crest cells at this stage consist of heterogeneous cell populations. Cells that expressed MSH receptors, thus committing into melanocyte differentiation pathway, are only a proportion of the crest cell population; and b) the neural crest cells at this stage are still underway to produce MSH receptors. About 75% of the cells are not yet competent to MSH induction. Our result was taken as further evidence that the developing neural crest is heterogeneous and that an environmental stimulus is required for final differentiation of neural crest cells into melanocytes in the embryos of turtle. In fact, the pigment promoting factor (PPF) from mouse epidermis and skin tissue (particularly ECM) has been reported to promote melanocyte differentiation (Agamy and Hornby, 1988; Morrison-Graham et al., 1990). It is further suggested that an environmental signal controls differentiation of melanocytes from neural crest cells in the mouse and that their interaction may occur after the migration of neural crest cells for cartilage development (Hall, 1987). On the other hand, differentiation of pigment cells may occur at the beginning of
migration of crest cells in amphibian (Epperlein and Liifberg, 1984). The different timing of the interaction of neural crest cells and surrounding tissues among different species of vertebrate groups probably indicate that neural crest cells are determined at different stages or that environmental factor(s) that induce neural crest cell differentiation exert at different stages. It is unknown whether a PPF-like factor plays a role in the melanocyte differentiation in reptiles in vivo. It is possible, however, that the final differentiation of neural crest-derived melanoblasts in the integument or in extracutaneous tissues is promoted by particular tissues that elaborate stimulatory factor(s) (Hou and Takeuchi, 1991). It is feasible that these tissues exert peptide factors similar to MSH. ACKNOWLEDGEMENTS This work was supported in part by a Grant-in Aid from the Ministry of Education, Science and Culture, Japan (to T. Takeuchi). We are grateful to Hattofi-Nakamura Nursery of Shizuoka for providing us with the eggs used in this research. REFERENCES Agamy, E.; Hornby, J. E. Pigment promoting factor for mouse neural crest cells. Pigment Cell Res. 1:272; 1988. Bagnara, J. T. The neural crest as a source of stem ceil. In: Maderson,
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