Endothelin signalling in the development of neural crest-derived ...

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Endothelin signalling in the development of neural crest-derived melanocytes Karin Opdecamp, Lidia Kos, Heinz Arnheiter, and William J. Pavan

Abstract: In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest-derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early Dct+ cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+ melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-α-methylleucyl-D-1methoxycarbonyltryptophanyl-D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+ melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation. Key words: neural crest, melanocyte, endothelin, differentiation. Résumé : Chez l’homme comme chez la souris, des mutations du gène codant le récepteur B des endothélines ou du gène codant l’un de ces ligands, l’endothéline 3, mènent à des défauts de développement de deux lignages cellulaires issus de la crête neurale, à savoir les mélanocytes et les neurones entériques. Les différentes étapes du développement mélanocytaire susceptibles d’être affectées par les endothélines ont été étudiées dans un système murin de cultures primaires de crêtes neurales. Lorsque de telles cultures sont maintenues en présence de sérum de veau foetal, nous observons l’apparition de cellules exprimant le marqueur mélanoblastique précoce Dct (Dct+) indépendamment de la présence d’un ester de phorbol (tetradecanoyl phorbol acetate, le TPA) ou d’endothélines. Ces cellules ne sont cependant capables de proliférer et de se pigmenter qu’après addition de TPA ou d’endothélines. Nous montrons également que le TPA et l’endothéline 2 présentent une même efficacité à générer des cellules Dct+ et pigmentées, mais celle-ci est cependant moindre que celle apportée par les endothélines 1 et 3. Un antagoniste spécifique du récepteur B des endothélines, le BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-α-methylleucyl-D-1methoxycarbonyltryptophanyl-D-norleucine), inhibe l’effet prolifératif des trois endothélines, ce qui suggère que le même récepteur est utilisé lors de la stimulation par ces molécules. La présence de ce récepteur a été détectée dans les cellules Dct+ (ainsi que dans des cellules Dct–). Nos résultats montrent donc que les endothélines sont de puissants stimulateurs de la prolifération et de la différenciation mélanocytaire. Mots clés : crête neurale, mélanocyte, endothéline, différenciation.

Introduction Neural crest cells are multipotent cells initially localized on the dorsal side of the folding neural tube. Shortly before or just after closure of the tube, the cells detach from the

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neuroepithelium, start to migrate along characteristic pathways, and ultimately differentiate into a variety of cell types typical for the axial level from where they originate. The cranial and hindbrain crest, for instance, but not the vagal, trunk, and sacral crest give rise to bone and cartilage cells.

Received September 4, 1998. Revised October 8, 1998. Accepted October 20, 1998. Abbreviations: BQ-788, N-cis-2,6-dimethylpiperidinocarbonyl-L-α-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine; Dct, DOPAchrome tautomerase; Edn1, Edn2, and Edn3, endothelin-1, endothelin-2, and endothelin-3; Ednra, endothelin A receptor, Ednrb, endothelin B receptor; Edns, endothelins; FCS, fetal calf serum; Kit, tyrosine kinase receptor; Mgf, mast cell growth factor; TPA, tetradecanoyl phorbol acetate. K. Opdecamp1 and H. Arnheiter.2 Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 5D06, 36 Convent Drive, MSC-4160 Bethesda, MD 20892, U.S.A. L. Kos3 and W. Pavan. Laboratory of Genetic Disease Research, National Center for Human Genome Research, National Institutes of Health, Bethesda, MD 20892, U.S.A. 1

Present address: Université Libre de Bruxelles, Laboratoire d’Embryologie Moléculaire, Rue des Chevaux, 67, B-1640 Rhode-StGenèse, Belgique. 2 Author to whom all correspondence should be addressed (e-mail: [email protected]). 3 Present address: Florida International University, Department of Biological Sciences, University Park, Miami, FL 33199, U.S.A. Biochem. Cell Biol. 76: 1093–1099 (1998)

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Fig. 1. Eleven-day-old murine neural crest cell cultures (unstained) showing different numbers of pigmented cells depending on culture conditions. (A) Control condition consisting of F10 medium supplemented with 10% FCS and 20 nM cholera toxin. No pigmented cells are seen. (B) Medium supplemented with 16 nM TPA. (C) Medium supplemented with 1 nM Edn1 instead of TPA. (D) Medium supplemented with 1 nM Edn2 instead of TPA. (E) Medium supplemented with 1 nM Edn3 instead of TPA.

The different regions also generate distinct subsets of peripheral nervous system neurons, but other cell types, such as glia and pigment cells, are common to all axial levels (reviewed in Le Douarin 1982; Bronner-Fraser 1995). These observations pose intriguing questions about the mechanisms that guide the development of the neural crest and the differentiation of its derivatives (Anderson 1997). The most straightforward way to identify such mechanisms is the molecular analysis of neural crest mutations. Among these, mutations associated with pigment abnormalities have long been favored among geneticists since they are easily visible and since pigment cells per se are not essential for an organism’s viability. In fact, in the mouse alone, over 80 different loci have been described that either affect the generation of pigment cells or the quality of their pigment. About a quarter of these loci have now been analyzed molecularly (Mouse Genome Database 1998), and it has become apparent that pigment cell development is controlled by a plethora of transcription factors and signalling systems, in addition to proteins involved in melanin synthesis and processing. Some of the mutations show that besides determining coat color patterns, melanocytes are also critical in the development and function of the inner ear (Steel and Barkway 1989; Tachibana et al. 1992). Furthermore, many of these loci not only affect pigment cells but also other neural crest-derivatives such as neurons and glial cells, and, not unexpectedly, unrelated cell types derived from other tissues. The analysis of pigment loci is thus important far beyond the narrow goal of understanding pigmentation. An interesting set of murine pigment loci are the piebald and lethal spotting loci. Mutations at either of these loci produce similar phenotypes characterized by varying degrees of pigment loss and aganglionic megacolon owing to lack of neural crest-derived enteric ganglion cells in the hindgut (Mouse Genome Database 1998). The gene affected in piebald mice encodes the endothelin B receptor (Ednrb), a G-coupled heptahelical receptor (Hosoda et al. 1994), whose human homolog, EDNRB, is mutated in approximately 5% of cases with Hirschsprung disease (reviewed in Chakravarti 1996). Ednrb mutations have also been identified at the spotting-lethal locus in the rat (Ceccherini et al.

1995; Gariepy et al. 1996; Shin et al. 1997) and in horses with the lethal white foal syndrome (Santschi et al. 1998). The murine lethal spotting locus encodes the Ednrb ligand, endothelin-3 (Edn3) (Baynash et al. 1994). Edn3 is a member of a group of three related peptides, Edn1, Edn2, and Edn3, each encoded by a separate gene. The peptides are produced as inactive preproendothelins that undergo successive proteolytic cleavages to generate the 22 amino acid active endothelins (Edns). The three oligopeptides bind Ednrb with similar affinity. There is at least one second endothelin receptor, Ednra, which is encoded by a different gene and shares approximately 60% sequence similarity with Ednrb. Bovine Ednra, expressed from a cDNA, binds Edn1 and Edn2 with similar affinity and Edn3 with a 1000– 2000 fold lower affinity (Inoue et al. 1989; Miller et al. 1993; Opgenorth 1995). Additional Ednrb receptor subtypes have been described in Xenopus (ETC, Karne et al. 1993) and quail (Ednrb2, Lecoin et al. 1998). The earliest cells of the melanocyte lineage that are affected in mutant embryos are the melanoblasts. Operationally, neural crest-derived melanoblasts can be defined as cells that express the tyrosine kinase receptor Kit, the melanogenic enzyme DOPAchrome tautomerase (Dct), also known as tyrosinase-related protein-2 (Trp2), and the transcription factor Mitf (Hodgkinson et al. 1993; Opdecamp et al. 1997; Nakayama et al. 1998). In the mouse, melanoblasts first appear at E10.5 adjacent to the anterior cardinal vein and later can be seen along the anterior-posterior axis with the highest density in the head and tail regions (Pavan and Tilghman 1994). In mice homozygous for Ednrbs-l (piebaldlethal), in which the Ednrb gene is deleted (Hosoda et al. 1994), melanoblasts do not appear before E11.5, and then are found around the head region and hind limb bud region but remain sparse. While this suggests that expression of functional Ednrb is important for the generation of melanoblasts, it also says that this receptor is not an absolute requirement in the process (Pavan and Tilghman 1994). Similarly, mutations at Edn3 also cause a reduction, and not elimination, of melanoblasts (Yoshida et al. 1996) and allow for some pigmented patches to occur (Baynash et al. 1994). In previous studies, the developmental expression patterns of Ednrb and Edn3 have been analyzed mostly in avian em© 1998 NRC Canada

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bryos. These studies showed that Edn3 is expressed in the environment through which neural crest cells migrate while Ednrb is expressed by the neural crest cells themselves (Nataf et al. 1996, 1998). Avian cells giving rise to melanocytes, however, express not Ednrb but a closely related receptor, Ednrb2 (Lecoin et al. 1998). In contrast, in the mouse, little information is available as to the expression pattern of Ednrb and Edn3, and the existence of an Ednrb2 homolog has not so far been reported. In both quail and mouse, however, the effect of Edn3 on in vitro neural crest development has been studied in some detail. In the quail, Edn3 markedly increases the proliferation of pluripotent neural crest cells and eventually stimulates dramatic increases in the number of melanocytes (Lahav et al. 1996; Stone et al. 1997). In primary mouse neural crest cell cultures, Edn3 increases the number of Kit and Dct+ melanoblasts and acts synergistically with the melanoblast survival factor, mast cell growth factor (Mgf) (Reid et al. 1996; Opdecamp et al. 1997). In fact, as will be demonstrated here, Edn3 treatment alone allows these cells to differentiate into melanocytes and thus overcomes the need for treatment of the cultures with phorbol esters to stimulate melanogenesis. These observations are consistent with the genetic data of Ednrb mutations and the previous demonstration that melanoblasts have binding sites for radiolabeled Edn1, which binds both Ednra and Ednrb (Reid et al. 1996). However, a direct demonstration of Ednrb expression in mouse melanoblasts is still missing, and it has not so far been shown that Edn3 indeed signals through Ednrb. In this study, we characterize the in vitro response of cells of the melanocyte lineage to Edn1, Edn2, and Edn3. We also show that each of the three Edns are able to increase the number of melanoblasts, though to different extents, and that they signal through Ednrb and not Ednra, thus providing a rational for the observation that Ednra does not complement Ednrb/Edn3 mutants.

Materials and methods Mice and cell lines

1095 whose middle was marked by touching with the heated back end of a Pasteur pipette. This procedure allowed for the drop to stay inside the marked ring until the cells were attached and the dish could be filled with medium. The ring marked a surface approximately corresponding to that covered by a 4-day-old culture. Greater than 50% of the cells survived replating, yielding cultures whose cell density was similar to that of cultures that have not been replated.

Culture conditions, media, and growth factors Neural crest cells were cultured on 3.5-cm Falcon Primaria dishes coated with 10 µg/mL of fibronectin (GIBCO BRL) diluted in PBS. The culture medium was F10 medium supplemented with 10% fetal calf serum and 20 nM cholera toxin (GIBCO BRL). Where indicated, control medium was further supplemented with 16 nM tetradecanoyl phorbol acetate (TPA), or, instead, Edn1, Edn2, or Edn3, each at 1 nM (SIGMA). For dose–response curves, Edns were added at the concentrations indicated. BQ-788 (N-cis-2,6dimethylpiperidinocarbonyl-L-α-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine; Research Biochemicals International), a competitive inhibitor of Ednrb (Ishikawa et al. 1994), was used at a concentration of 10–8 to 10–6 M.

In situ hybridizations In situ hybridizations of neural crest cultures were performed as previously described (Opdecamp et al. 1997). The template for the Dct riboprobes has been described (Steel et al. 1992) and corresponds to a 1200-bp mouse Dct cDNA. The template for mouse Ednrb was pWP40, which was isolated by RT PCR and corresponds to position 555–1514 of the mouse Ednrb sequence (Genbank accession No. U32329). Its 32P-labeled insert was first tested by Northern assays. It was found to hybridize strongly with a 4.4-kb message of mRNA from E7.5, 11.5, 15.5, and 17.5 wildtype embryos and from adult heart, brain, lung, and testis or melan c cells. From both templates, sense and antisense riboprobes were generated.

Antibodies and immunostaining Dct positivity was scored either by in situ hybridization as described (Opdecamp et al. 1997) or by immunocytochemistry using a rabbit anti-Dct antibody provided by Dr. V. Hearing.

BrdU incorporation and immunostaining

Wild-type embryos were either obtained from C57BL/6 females or superovulated FVB/N females mated with the respective males. Noon of the day a plug was found was defined as embryonic day 0.5 day (E0.5). Melan-c cells were obtained from Dr. Vincent Hearing.

For bromodeoxyuridine labeling, cultures were kept for 2 days and exposed in the morning of the 3rd day to 10 mM BrdU for 60 min. They were immediately fixed in 4% paraformaldehyde for 20 min, and processed for BrdU immunostaining according to the manufacturer’s protocol (Boehringer Mannheim). Subsequent in situ hybridization with the Dct riboprobe was as described above.

Neural crest cell preparation

Results

Neural tube explants were obtained from E9.5 embryos of a C57BL/6/C3H mixed background (15–30 pairs of somites). Embryos were removed from the uterus and placed in a 3.5-cm Petri dish containing equal volumes of Dulbecco’s minimal essential medium and PBS. Using fine forceps under a dissecting microscope, embryos were cleaned of membranes and soft tissues, and a portion of the neural tube corresponding to the 10 posterior-most somites was cut and digested on ice for 3 min in 1% trypsin in PBS. The trypsin was neutralized by transferring the neural tube to a new 3.5-cm Petri dish containing Dulbecco’s minimal essential medium supplemented with 10% fetal calf serum (FCS). Using fine forceps, the neural tube was cleaned from the surrounding somites transferred to the culture dishes, usually one tube per dish. However, for dose–response and growth curves, the neural tubes were pooled and cultured for 1 day. Then, the cells were trypsinized and aliquotted into drops whose number corresponded to the number of embryos from which the pooled tubes were originally harvested. The drops were spotted onto Primaria dishes

Endothelins promote the generation of pigmented cells in culture Neural crest cell cultures were prepared form E9.5 embryos as previously described (Reid et al. 1996; Opdecamp et al. 1997). As shown in Fig. 1A, when such cultures were kept in the absence of TPA, no pigmented cells developed even after prolonged periods of time. However, in the presence of the phorbol ester TPA, pigmented cells developed starting around day 10 of culture (Fig. 1B). Since TPA activates protein kinase C (Oka et al. 1996) as do endothelins (Imokawa et al. 1996), it was possible that addition of endothelins would substitute for the requirement for TPA in generating pigmented cells. Indeed, as shown in Figs. 1C–1E, all three endothelins, added at 1 nM (approximately 10-fold above their Kd for Ednrb), promoted the formation of pigmented © 1998 NRC Canada

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cells. Inclusion of Edn2 (Fig. 1D) resulted in similar numbers of pigmented cells as TPA alone, while inclusion of Edn1 or Edn3 (Figs. 1C and 1E) led to greatly increased melanocyte numbers. The results indicated that either of the Edns can replace TPA for the generation of pigmented cells but that they differ in their potency even though previous studies showed that their affinities for Ednrb are similar. Endothelins increase the number of melanoblasts in culture and signal through Ednrb The above results suggest that Edns may be effective in promoting melanocyte differentiation of an otherwise similar population of precursor cells, or they may increase the number of these precursor cells. Thus, we tested whether addition of these factors would differentially affect the numbers of melanoblasts. Either one of the endothelins, TPA, or control medium was added to individual cultures from day 0 through day 5, i.e., during a period where no pigmented cells can be detected. The cultures were then fixed and processed for in situ hybridization using a riboprobe for the melanoblast marker Dct. This marker is expressed as early as day 1 after neural tube explantation and persists even after the cells have reached their terminal differentiation (Opdecamp et al. 1997). In cultures kept in the absence of both FCS and TPA, Dct+ cells were not observed (not shown). However, as shown in Fig. 2 (open circles), when FCS but not TPA was added (Fig. 2, CON), Dct+ cells could be detected, although there were never more than 30 cells/culture. In the presence of TPA, the number of melanoblasts increased, ranging from 30 to 160. When Edn1 or Edn3 was added, this number was further increased while addition of Edn2 was less effective. Even though there is a considerable variability between cultures (most likely owing to different numbers of melanoblast precursor cells present in the neural tube at times of harvest), it appears that Edns increase the number of pigmented cells as shown in Fig. 1 by increasing the number of Dct+ melanoblasts, and that Edn1 and Edn3 do so with much greater efficiency than Edn2. This effect was not simply the result of a general increase in cell proliferation and survival. Edn1, for instance, increased the number of melanoblasts up to 20-fold over that of control, but the size of the culture and the number of cells per culture changed little (not shown). An increase in the number of melanoblasts by Edns could result from signaling either through the Ednrb or the Ednra receptor; except for Edn3, which binds only Ednrb with high affinity, the other endothelins bind the two receptors with similar affinity. However, in vivo, an intact Ednra gene does not compensate for the loss of Ednrb. It was likely, therefore, that signaling by endothelins in melanoblasts occurs through Ednrb. To formally test this possibility, we made use of a selective antagonist of Ednrb, the pseudotripeptide BQ788 (Ishikawa et al. 1994). In different cell systems, this antagonist has a 1000-fold higher inhibitory concentration (IC50) for Ednra compared to Ednrb (Ishikawa et al. 1994; Opgenorth 1995). As shown in Fig. 2 (filled circles), BQ788 (10–6 M) does not have agonistic effects nor is it toxic: cultures receiving control medium with or without BQ-788 showed similar numbers of melanoblasts, and the numbers obtained in TPA-treated cultures also remained unchanged

Biochem. Cell Biol. Vol. 76, 1998 Fig. 2. Edns increase the number of Dct+ melanoblasts and signal through Ednrb. Cultures were exposed to different conditions from day 0–5 and then scored for the number of Dct+ cells. Each circle represents one culture, which is the equivalent of the cells derived from the neural tube of a single embryo. s, cultures maintained in control medium (10% FCS, 20 nM cholera toxin) or control medium supplemented with either 16 nM TPA, 1 nM Edn1, 1 nM Edn2, or 1 nM Edn3. Both Edn1 and Edn3 generate high numbers of Dct+ cells when compared with TPA or Edn2. d, same conditions as described above but supplemented in addition with 1 µM BQ-788, a selective antagonist of Ednrb. BQ-788 reduces the number of cells generated in cultures supplemented with Edns but not in cultures supplemented with TPA or left without supplement. CON, control.

when BQ-788 was added. However, BQ-788 reduced the number of Dct+ cells in cultures treated with Edn3, but only when used at doses higher than 10–7 M (not shown). As shown in Fig. 2, BQ-788 (10–6 M) efficiently inhibited all three endothelins. These results suggest that Edns signal through the Ednrb receptor and that the Ednra signaling pathway, if at all available to melanoblasts, may only play a minor role. Ednrb is expressed in Dct+ melanoblasts To confirm that the Ednrb receptor is indeed expressed in cultured mouse melanoblasts, we performed a double in situ hybridization assay, using a digoxigenin-labeled Ednrbspecific riboprobe and an 35S-labeled Dct riboprobe on 2-dayold neural crest cell cultures. The results are shown in Fig. 3. Among the labeled cells less than 9% were Dct singlelabeled, 55% Ednrb single-labeled, and 36% Ednrb/Dct double-labeled. These results indicate that the majority of Dct+ melanoblasts in culture co-expressed the Ednrb gene, and that Dct- cells, which may belong to different cell lineages, also express Ednrb. © 1998 NRC Canada

Opdecamp et al. Fig. 3. Expression of Ednrb in neural crest cell cultures. Cultures were kept in control conditions for 2 days and then processed for double in situ hybridization using a digoxigeninlabeled Ednrb-specific riboprobe (dark cells) and an 35S-labeled Dct-specific ribroprobe (grains). Note double-labeled cells (arrows) as well as Ednrb single-labeled cells (large arrowheads). A small percentage of cells was Dct single-labeled (not shown).

Dose–response curves of Edns The above variabilities in numbers of melanoblasts from culture to culture precluded the establishment of dose–response curves for the different endothelins. To overcome the problem of variability, comparable numbers of cells were plated in individual dishes in the following way. Neural tubes derived from individual embryos were first pooled together in a single dish. They were then cultured for 1 day in control conditions (without TPA or endothelins but with FCS), which, as shown above, generates some Dct+ cells. The cultures were then trypsinized and replated in as many dishes as embryos were pooled initially. The replating was done such that the cell density per surface area corresponded to that of the original cultures. One hour later, after the cells had attached, they were refed with fresh medium supplemented with one of the endothelins at different concentrations. Each dish was then kept for 4 days, fixed, and scored for the number of melanoblasts, again using Dct immunolabeling as a marker. The results are shown in Fig. 4. Edn1 and Edn3 generated more than 700 melanoblasts/culture at saturation (10–50 nM) while Edn2 was less effective across all doses tested, generating never more than 700 Dct+ cells. It is worth noting that in these replated cultures, the complete absence of TPA or Edns led to approximately 100 Dct+ cells/culture, that is, about three times as many as would be expected from cultures that were not replated. When similar numbers of cells were seeded at lower densities, lower numbers of Dct+ cells were obtained. Thus, it appeared that trypsinization and remixing as well as plating density were crucial additional parameters affecting melanoblast proliferation. We also noted that a reduction of the concentration of FCS below 10% led to progressive death of the culture, regardless of the addition of Edns or trypsinization and replating.

1097 Fig. 4. Dose–response curves showing the number of Dct+ cells generated in 5-day-old neural crest cultures as a function of the concentration of Edn1 (e), Edn2 (u), or Edn3 (s). Curves were obtained by pooling tubes derived from different embryos, maintaining them for 1 day in control conditions and then trypsinizing and replating the cells. Edn1 and Edn3 generate similar numbers of cells while Edn2 is less effective across all doses tested. Note that for each Edn, the number of melanoblasts generated in these replated cultures is higher than those obtained in primary cultures (see Fig. 2).

Effect of Edn3 on melanoblast proliferation and survival While the above experiments showed an effect of Edns after 4–5 days of exposure, they did not provide insights into the early time course of melanoblast development in culture. To test what the fate of the initially generated Dct+ cells were in presence or absence of Edn3, we again established individual cultures derived from pooled neural tubes. Pooled neural tubes were cultured in the absence of added factors for 1 day, split, and replated into 10 dishes. Five dishes were kept in control medium and five were supplemented with 1 nM of Edn3. One control and one Edn3-supplemented culture were fixed each day and the number of Dct+ cells was determined immunocytochemically. As shown in Fig. 5, the 2-day control and Edn3-supplemented culture initially showed a similar number of Dct+ cells. During the following days, in the presence of Edn3, the number of Dct+ cells increased with an average doubling time of 32 h. In the absence of Edn3, however, the number of Dct+ cells gradually decreased. These results were confirmed by a DNA synthesis assay in which cells cultured for 2 days were subjected to a 1-h BrdU pulse and then double-labeled for BrdU and Dct. In this assay, 7.5% of the Edn3-treated Dct+ cells (n = 8) and 6% of the TPA treated Dct+ cells (n = 6) were BrdU-labeled © 1998 NRC Canada

1098 Fig. 5. Growth curves showing the number of Dct+ cells generated in neural crest cell cultures as a function of time. Cultures were obtained by pooling neural tubes derived from different embryos and replated cultures were established as for Fig. 4. Note that both conditions initially generated a similar number of Dct+ cells. e, control conditions (FCS, no TPA); u, 1 nM Edn3.

compared to none of the Dct+ cells kept in the absence of Edn3. These results confirmed that addition of Edn3 is not required initially for the cells to reach the Dct+ stage. They also showed that in the absence of Edn3, Dct+ cells do not proliferate and are not maintained, either because they downregulate the expression of this marker or because they do not survive. In the presence of Edn3, however, these cells proliferate and survive.

Discussion We have used primary neural crest cell cultures to define the role of the three known endothelin oligopeptides in melanocyte development. Interestingly, melanoblasts, defined as neural crest-derived cells expressing the lineage marker gene Dct, were generated initially regardless of the culture condition, provided FCS was present. In control conditions, however, these initial Dct+ melanoblasts did not proliferate and were soon lost. Thus, additional factors are required for the melanoblast population to expand and finally differentiate into melanocytes. Crucial among these factors are undefined components present in FCS and growth factors such as Mgf and, as demonstrated here, Edns. However, none of these factors alone is sufficient. All three Edns tested were capable of replacing TPA for the generation of pigmented cells. They effectively increased the number of Dct+ cells, though to different extents. Edn3 affected the early proliferation and survival of the initially generated Dct+ cells, suggesting that Edns increase the number of pigmented cells not simply by stimulating terminal differentiation of an otherwise similar population of melanoblasts but also by increasing the number of melanoblasts. An unexpected finding was the observation that neural crest cells, trypsinized and replated after a short initial cul-

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ture period, generated a greater number of Dct+ cells in control conditions and an even greater number after addition of Edns. In particular, Edn2, which had little activity on primary cultures, had a greater activity on these secondary cultures, indicating that Edn2 was not simply a peptide of generally low activity but was active under different culture conditions. The observations suggest that cell-to-cell contact is an important parameter influencing the generation, proliferation, and survival of melanoblasts. Membrane-bound Mgf, perhaps present in the mixed neural crest cell cultures, is a candidate for mediating this effect. Alternatively, local concentrations of secreted factors could be higher in these cultures. In an analysis of chimeric mice composed of homozygous Ednrbs-l and wild-type cells, it was found that given optimal ratios between mutant and wild-type cells, mutant neural crest cells were perfectly able to colonize the distal large intestine (which they fail to colonize in nonchimeric mutant mice). This observation suggested that the mutant cells had been helped in their development by wild-type cells capable of responding to Edns, or, in other words, that the Ednrb mutation does not act strictly in a cellautonomous fashion (Kapur et al. 1995). A comparatively higher activity of Edns in replated wild-type cultures would be consistent with an indirect effect on melanoblasts mediated by the action of Edns on other cells that express Ednrb. In fact, we have identified in neural crest cell cultures, Ednrb-expressing cells that do not co-express the melanoblast marker Dct, consistent with an earlier report in avian neural crest cell cultures (Nataf et al. 1996) and with in situ hybridization studies on mouse embryos (Kos et al., unpublished). Edns can signal through both the Ednrb and Ednra receptor. Whole-mount in situ hybridizations show that both receptors are expressed in embryos, although in different regions. Ednrb expression extends along the anterior–posterior axis and Ednra expression is prominent in the head region but below the level of detectability in the tail region (Reid et al. 1996). We, therefore, tested the effect of the Ednrb-selective antagonist, BQ-788, for its effect on the Edn-mediated increase in the number of melanoblasts. The results suggest that in culture, all three Edns signal through Ednrb which we found to be expressed in melanoblasts, consistent with the fact that Ednrb null mutants are not complemented by wildtype Ednra. It is worthwhile to compare this observation with in vivo findings in mice with Edn3 mutations. Such mice are severely deficient in melanocytes, indicating that Edn1 or Edn2 cannot complement the loss of Edn3. Thus, even though all Edns have activity in culture, only Edn3 is relevant in vivo, suggesting that Edn1 or Edn2 are not available for rescuing Edn3-deprived melanoblasts during development. In conclusion, our results provide evidence that Edns, particularly Edn1 and Edn3, are crucial factors that promote the proliferation and survival of early melanoblasts in culture.

Acknowledgements We thank Drs. Lynn Hudson and M. Dubois-Dalcq and members of the Pavan and Arnheiter laboratories for helpful comments and Dr. Vincent Hearing for antibodies against Dct. © 1998 NRC Canada

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