Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3892-3897, April 1996 Developmental Biology Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture RONIT LAHAV*, CATHERINE ZILLER, ELISABETH DUPIN, AND NICOLE M. LE DOUARIN Institut d'Embryologie Cellulaire et Moleculaire du Centre National de la Recherche Scientifique et du College de France, 49bis Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France Contributed by Nicole M. Le Douarin, December 28, 1995 ABSTRACT Mutations in the endothelin 3 (EDN3) gene receptor-B (EDNRB) and its ligand endothelin 3 (EDN3), severely affect the development of neural crest-derived mela- respectively (11, 12). Mutations in the EDN3 and EDNRB nocytes. In this paper, we report the action ofEDN3 on neural genes affect two types of NC derivatives: melanocytes and crest cells in vitro. The presence of EDN3 leads to a large enteric ganglia. It has been proposed, on the basis of coelomic increase in the number of cells, the majority of which even- skin transplantation experiments, that both s/and Is mutations tually differentiate into melanocytes that aggregate to form a affect melanocyte precursors in a cell autonomous manner (13, reproducible pigmentation pattern. Quantitative analysis of 14), whereas the defect in the enteric innervation of Is mice has the effect of different culture conditions revealed that EDN3 clearly been attributed (by aggregation chimera experiments) initially promotes neural crest cell proliferation. This phase of to the environment of the gut in which the NC cells differen- expansion, which can be prolonged for a few weeks if the cells tiate (15, 16). are replated regularly, is followed by both a decrease in cell We have decided to further investigate this problem byusing proliferation and the onset of melanocytic differentiation. avian embryos both in vivo and in vitro. For this purpose, the Therefore, EDN3 is a potent mitogen for early neural crest cell EDNRB gene of the quail has been cloned and used to precursors that can give rise to melanocytes. determine its spatiotemporal pattern of expression in migrat- ing crest cells and in NC derivatives (V. Nataf, L. Lecoin, A. The neural crest (NC) is a transient structure of the vertebrate Eichmann, and N.M.L.D., unpublished data); moreover, be- embryo whose cells migrate to different target sites and cause endothelins exhibit a high level of zoological conserva- differentiate into a large variety of cell types, including mela- tion (12), we looked for the effect of human-rat EDN3 on nocytes (1-3). In vitro clonal analysis of the potentialities ofthe primary cultures of quail NC cells by using a protocol that was migrating NC cell population demonstrated that it is composed similar to the one used for SCF. We found that during the first of a mixture of pluripotent and of more or less restricted few days ofcultivation, EDN3 had a profound effect on NC cell progenitors (4, 5). In vivo experiments using the quail-chicken proliferation while at the same time temporarily inhibiting chimera system have allowed us to establish a fate map of NC their differentiation. During a second phase, cell proliferation derivatives and to demonstrate that the differentiation of NC diminished significantly and differentiation was strongly cells largely depends on the type of environmental influence(s) skewed toward the melanocytic pathway. that they receive during their migration and/or within their homing site (for review, see ref. 6). The identity of the MATERIALS AND METHODS environmental factors that influence the emergence of the final differentiated phenotypes and the patterning of diverse NC Cultures. Quail NC cells were obtained from neural NC derivatives is a critical problem for our understanding of tubes cultured for 48 hr, as described (10). About 1 x 104 cells NC ontogeny. were replated in a small volume (30 ,l) of culture medium One approach to identify such factors is to analyze loci that containing Dulbecco's modified Eagle's medium (DMEM)/ affect the development of NC derivatives in mice. Such is the 10% fetal calf serum (FCS), on collagen-coated 8-well Lab- case for the stem cell factor (SCF) the steel gene product and Tek tissue culture dishes and left overnight to attach. The next its receptor c-kit encoded by the dominant white spotting (W) day, the medium was changed to either medium containing gene which play a role in NC-derived melanocyte development DMEM/10% FCS/2% chick embryonic extract (CEE) or to (for review, see ref. 7). We have previously investigated the DMEM supplemented with a defined serum substitution [Ul- role of the SCF/c-kit system in the differentiation of quail NC troser G (IBF, Villeneuve-la-Garenne, France) containing cells in culture and in vivo. We found that the NC cells that growth factors, adhesion factors, hormones, vitamins, miner- migrate along the dorso-lateral pathway to the skin, start to als, and bovine serum albumin] with orwithout END3 (human, express the c-kit receptor as they are engaged in the melano- rat; Sigma). Additional cultures using Ultroser G, in the cytic differentiation pathway (8) and when they also express presence or absence of EDN3, were supplemented with 100 ng the premelanocytic marker MelEM (melanocyte earlymarker) of avian recombinant SCF per ml (10). Analysis of the in- (9). The results obtained in culture are in agreement with these corporation of BrdUrd (Amersham) was carried out for 24 hr. findings. Chicken SCF added to cultured NC cells has a The cultures that were incubated with enriched medium were discrete effect on cell proliferation and an essential action in too dense for quantitative analysis; therefore, they were enhancing the survival and terminal differentiation of pre- treated with trypsin, resuspended, counted, and then replated melanoblasts, rather than affecting their engagement into the sparsely in collagen-coated Lab-Tek wells (1-3 x 104 cells/ melanocytic differentiation pathway (10). well) in order to quantify the number of BrdUrd-labeled cells, Recently, the gene products of other mutant genes associ- premelanocytes [identified by the MelEM monoclonal anti- ated with spotting and megacolon in piebald-lethal (s') and body (mAb); ref. 9] and melanocytes. The cultures were fixed lethal spotted (Is) mice were identified as the endothelin Abbreviations: EDN3, endothelin 3; NC, neural crest; SCF, stem cell factor; MelEM, melanocyte early marker; EDNRB, endothelin recep- The publication costs of this article were defrayed in part by page charge tor-B; FCS, fetal calf serum; CEE, chicken embryonic extract; mAb, payment. This article must therefore be hereby marked "advertisement" in monoclonal antibody. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3892 Downloaded by guest on September 24, 2021 Developmental Biology: Lahav et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3893 and analyzed by indirect immunofluorescence as described the incorporation of BrdUrd by cells in cultures incubated with (10). Neurons and adrenergic cells were detected using anti- EDN3 decreased but remained significantly higher than in bodies to neurofilament protein (Sigma) and tyrosine hydrox- controls until day 12 (except for those cultures incubated with ylase (5), respectively. Glial cells were identified by the avian low concentrations of EDN3) (Fig. 2B). Schwann cell myelin protein mAb, which is a constitutive The effect of EDN3 on the appearance of premelanocytes marker for the glial lineage in the avian embryo (17, 18). was revealed by the MelEM mAb that labels quail NC-derived The experiments done with SCF were performed with premelanocytes (9). In the first 3 days of culture, EDN3 cultures of sparsely plated NC cells (about 6 x 103 cells/0.5 ml) inhibited the appearance of MelEM-labeled cells in a dose- (10) in medium containing DMEM and Ultroser G supple- dependent manner (Fig. 2C). Starting with day 12, however, ment as described. when the characteristic pattern shown in Fig. 1 F and H is well The analyses of total cell number, BrdUrd-positive cells, established, the percentage of MelEM-positive cells gradually MelEM-labeled cells, and pigment-containing cells were done and significantly increased in the presence of EDN3. By day 16, at 1, 3, 5, 9, 12, and 16 days of culture. Each analysis reported the proportion of MelEM-labeled cells (incubated with 100 in Figs. 2 and 3 contained values from experiments repeated nM EDN3) reached 90%, while only 5% of the cells in control four times. Each experiment contained duplicate wells for each cultures were labeled (Fig. 2C). Therefore, the unpigmented concentration. Mean values of total number of cells, standard cells in these cultures were, in fact, mostly premelanocytes. deviations, and significance were calculated using the Stu- The same effect of EDN3 on melanogenesis was also dent's t test (n = 4-6). The percentage of cells was calculated revealed by evaluating the proportion ofpigmented cells grown by the ratio of A/B, where A = number of stained cells and B for different periods of times in culture. The inhibitory phase = total number of nuclei stained by bisbenzimide (Hoechst- was again visible during the first 3 days (Fig. 2D); this was Roussel). For each well, 6-12 fields were counted and included followed by a sharp increase in the proportion of melanocytes, at least 100 counted cells. Statistical analysis of the data was and by day 16, a 67-fold increase in the number of melanocytes performed using the X2 test. The experiments with END3 and was observed in those cultures incubated with 100 nM EDN3 SCF (Fig. 4) in sparse cultures were analyzed on day 5 of when compared with controls. culture as described above. The presence of EDN3 (100 nM), however, did not prevent Replating Protocol. Cells that were cultured in Lab-Tek the appearance of other cell types. After 5 days in culture, very wells in medium supplemented with FCS and CEE plus 100 few neurons and adrenergic cells were detected in either the nM EDN3 were trypsinized and counted after either 5 or 16 EDN3-treated or control cultures (<10 cells/well). During the days. Cells (1 x 105) were then replated in 35-mm culture period between 12 and 16 days, cells expressing the early avian dishes in the same medium. This procedure was repeated when glial specific marker Schwann cell myelin protein (17, 18) were the cells reached confluence. observed in both controls and EDN3-treated cultures (-20-60 cells/well). When NC cells were grown for 3 or 6 days in the presence of 100 nM EDN3 and then switched to a RESULTS medium deprived of EDN3, the expression of Schwann cell Effect of EDN3 Added to FCS- and CEE-Containing Me- myelin protein-labeled glial cells increased greatly, demon- dium. We first analyzed the effect of EDN3 (concentrations strating that, in cultures treated with EDN3, glial precursors ranging from 100 pM to 100 nM) on quail NC cells cultured in were present but their differentiation was largely impaired. DMEM/10% FCS/2% CEE for 16 days. The effect of high In summary, EDN3 in a FCS- and CEE-supplemented concentrations of EDN3 (10 and 100 nM) was spectacular medium enhanced the proliferation of NC cells while inhibit- when compared with controls (Fig. 1 B, D, F, and G). The first ing the onset of melanogenesis. With time, cell proliferation apparent effect, visible after only a few hours of culture, was diminished, melanogenesis began, MelEM-positive cells be- a change in cell morphology; the cells became elongated and came apparent, and finally melanocytes emerged. The number acquired long processes and/or a spindle-like shape (Fig. 1 A of these cells was highly increased in EDN3-treated cultures and B). A large increase in cell number and an inhibition of when compared to the controls. pigment cell emergence was then apparent from day 1 to day Effect of EDN3 Added to a Defined Medium. The effects of 5 of culture (Fig. 1 C and D). From day 9, cultures containing EDN3 were also examined in serum- and CEE-free cultures 100 nM EDN3 became progressively pigmented, and at day 16 using DMEM supplemented with the defined serum substitute the cultures contained a large number of fully differentiated Ultroser G. During the first days in culture, high concentra- melanocytes; moreover, the pigment cells aggregated to form tions of EDN3 (10 and 100 nM) significantly enhanced BrdUrd a highly reproducible pattern as seen in Fig. 1 F and H, with incorporation by NC cells (Fig. 3B), but no significant increase a continuous network of closely aggregated pigmented cells in total cell number was observed when compared with around mostly unpigmented cells. In control cultures, the total controls (Fig. 3A). A subsequent decline in total cell number number of cells and that of melanocytes were much more (Fig. 3A) was accompanied by the inhibition of melanogenic modest, and the melanocytes were randomly distributed on the differentiation (Fig. 3 C and D). substrate (Fig. 1 E and G). Therefore, in both conditions EDN3 initially increased NC To quantify these effects, the cultured cells were resus- cell proliferation and inhibited melanocyte differentiation. In pended and replated at low density for analysis. The total the defined medium, however, the effect on cell survival and number of cells grown in the presence of EDN3 increased differentiation was severely compromised, suggesting a re- rapidly in a concentration-dependent manner. Between day 1 quirement for additional factor(s) present in the CEE and/or and day 5, a 70-fold increase in cell number was found in FCS and acting in combination with EDN3 to permit pro- cultures containing 100 nM EDN3 (Fig. 2A). From day 5 to the longed survival followed by enhanced differentiation of mela- end of the culture period, the total number of cells in these nocytes. cultures remained stable. In control cultures, an -4-fold Is SCF Acting in Cooperation with EDN3 During Melano- increase in total cell number was observed between days 1 and genic Differentiation in Culture? A possible candidate for the 16. additional factor (found in CEE and/or FCS) was SCF because Analysis of BrdUrd-labeled cells showed that the rate of it was previously shown to favor the survival of mouse and crest cell division increased significantly in the presence of avian NC precursors and to promote melanocyte differentia- EDN3 in a dose-dependent manner. During the first day, 92% tion (10, 19). We therefore added SCF (100 ng/ml) to NC cells of the cells had incorporated BrdUrd (100 nM EDN3) com- cultured in serum- and CEE-free media. Cells were cultured pared to 37% of cells in control cultures. As time progressed, from days 1 to 16 in the absence or presence of EDN3 (at Downloaded by guest on September 24, 2021 3894 Developmental Biology: Lahav et al. Proc. Natl. Acad. Sci. USA 93 (1996)

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0 LU. o dl d3 d5 .Dd9 d 12 d 16 dl d3 d ..-I....l d5 d9 12 d16 dl d3 d5 d9 d12 d16 FIG. 2. Quantification of EDN3-induced changes at different time points. Cultures in enriched medium with different EDN3 concentrations were analyzed during a culture period of 16 days. (A) Total cell numbers. (B) Cell proliferation was determined by the proportion of BrdUrd incorporation. (C) Proportion of premelanocytes labeled by the MelEM mAb. (D) Proportion of melanocytes. Significant differences are indicated by the presence of a star at the top of the column. erated with EDN3 to enhance NC cell survival and mela- cells can be propagated and expanded in this order of mag- nocyte differentiation. nitude. The growth of NC cells in the presence of EDN3 is The NC Cell Proliferation Phase Can Be Protracted by accompanied by the appearance of a striking spatial pattern of Subculturing the Cells. The question as to whether the stabi- pigmentation. So far, models to explain pigmentation patterns lization in cell number, observed after 5 days ofculture in FCS- (clonal growth, chemotaxis, gradients, thresholds, and reaction and CEE-supplemented medium in the presence of 100 nM diffusion) are based on in vivo observations in animals where EDN3 (Fig. 2A), could be due to contact inhibition was these patterns appear after the mechanisms leading to their addressed. Cells were cultured for 5 days and then replated in establishment have been in operation for a considerable period 35-mm dishes in the same medium. These cells resumed of time. The culture system described here produces an growth immediately and could be replated approximately opportunity to analyze the mechanisms leading to a highly every 3 days for 3 weeks. During this time, the total cell number reproducible pattern involving pigment cells at the time it is increased about eight times at each replating step. For 2 weeks being established. the proliferating cells did not appear pigmented. Melanocytes One can rule out SCF as the factor acting together with were observed during the third week as the rate ofproliferation EDN3 during avian melanocyte ontogeny. This conclusion is decreased. From weeks 3 to 6, the increase in cell number was supported by the expression pattern of an avian homologue of -2-fold per week (data not shown). In fact, during the whole EDNRB that was recently cloned in our laboratory and was culture period, cell proliferation and viability depended on the shown to be expressed by the NC cells as soon as they become presence of EDN3 because both diminished drastically after individualized in the neural primordium and during the early removal of the factor from the culture medium. This reveals stages of neural crest cell emigration (at embryonic day 2) (V. that, when contact inhibition is avoided by frequent replating, Nataf, L. Lecoin, A. Eichmann, and N.M.L.D., unpublished the high rate of cell proliferation induced by EDN3 continues data). In contrast, the SCF receptor c-kit does not appear until for a while and then slows down spontaneously. The reduction later when the NC cells have migrated to the skin at the stage in cell growth is concomitant with the onset of melanogenesis. when MelEM expression starts and the cells become commit- Therefore, NC cells cannot respond indefinitely to EDN3 by ted to the melanocytic differentiation pathway (i.e., from dividing. Their response switches to cell differentiation ac- embryonic day 4 onward) (8). cording to a program whose timing only, can be modulated by Consistent with the pattern of expression in vivo, the in vitro monitoring cell density. study reported here has demonstrated that EDN3 is active early in NC cell development, before these cells express the DISCUSSION melanocytic marker MelEM. Likewise, a comparative in vivo study of melanocyte precursors in a mutant mouse (s'), using Our experiments have shown that EDN3 acts in a synergistic a lineage specific marker for melanoblasts, tyrosinase-related manner with factor(s) found in CEE plus FCS-containing protein 2, has shown that tyrosinase-related protein-2-positive medium to strongly stimulate proliferation, survival, and mel- cells do not appear in the affected areas of s1 compared with anogenic expansion ofavian NC cells. To our knowledge, there wild type. These data support the notion that EDNRB acts were no previous reports of culture conditions in which NC early in melanocyte development by affecting NC cells before Downloaded by guest on September 24, 2021 3896 Developmental Biology: Lahav et al. Proc. Natl. Acad. Sci. USA 93 (1996)

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%i-- I d ..-_i... · -L:j 0 _L*m d l d9 d16 dl d9 d 16 FIG. 3. Quantification of EDN3-induced changes in defined medium. (A) Total cell number. (B) Percentage of BrdUrd-incorporating cells. (C) Percentage of MelEM-labeled cells. (D) Percentage of melanocytes. The cultures were analyzed at different time points, with different EDN3 concentrations during a culture period of 16 days. Significant differences are indicated by the presence of a star at the top of the column. they express this marker. It was therefore concluded that, in studies are needed to determine whether their differentiation the mouse, EDNRB acts upstream to c-kit (20). The in vivo could, as has now been shown for the melanogenic precursors, expression pattern of c-kit and EDNRB genes in chickens as be enhanced in combination with other specific environmental well as our results support this conclusion and point to the stimuli. This could account for the megacolon phenotype intervention of EDN3 and SCF at two successive steps of resulting from EDN3 and EDNRB gene mutation. melanocyte ontogeny. These results are in agreement with studies carried out in the Another striking effect of EDN3 was the change in cell mouse suggesting that, when the supply ofEDN3 is reduced (as morphology that appeared in all culture conditions tested so in Is mutants) (14) or when the response to EDN3 is abolished far. These changes are obviously connected with the reorga- by a defect in the EDNRB (as in piebald mutant) (13), the nization of cytoskeletal and/or adhesion molecules. Such melanoblast population is depleted, resulting in its failure to changes may influence motility and invasiveness. This would occupy certain skin areas, therebygenerating the white spotted correlate well with the expression of EDNRB during early coat color. EDN3 function might be crucial at the trunk level stages of NC emigration. Indeed, a role for EDNRB in where murine melanoblast number is small when compared promoting motility and invasiveness was suggested in human with the head and tail regions; this is consistent with the fact melanoma cells (21). that the areas which are depigmented in sl and Is mice are The results presented here show that EDN3 triggers a large generally located in the trunk (20). expansion of early NC-derived melanogenic precursors in the avian system. In contrast, the appearance of neuronal and glial We thank Yann Rantier, Frangoise Viala, and Helene San- precursors was not enhanced in the presence ofEDN3. Further Clemente for their help in preparing this manuscript and Corinne .::.. Control m ..J SCF 4 0C 5 A * EDN3 B ,.' EDN3 + SCF o 4J X 4 >I 3 0 x E v- r 3 E q- 0.5 0 L.U 2 >'0( o 2 0 1 E-0- E Z -o -0 CU 1 E * * i z 0 ··I··. 0 4- 0 L o i LL.L= 1 L FIG. 4. EDN3 inhibits the effects of SCF on NC cell survival (A), number of MelEM-labeled cells (B), and number of melanocytes (C) after 5 days. Downloaded by guest on September 24, 2021 Developmental Biology: Lahav et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3897 Glavieux for technical assistance. The Institut d'Embryologie Cellu- 9. Nataf, V., Mercier, P., Ziller, C. & Le Douarin, N. (1993) Exp. laire et Moleculaire is supported by grants from the Centre National Cell Res. 207, 171-182. de la Recherche Scientifique, the Institut National de la Sante et de 10. Lahav, R., Lecoin, L., Ziller, C., Nataf, V., Carnahan, J. F., la Recherche Medicale (Grant 920806), the Ministere de la Recherche Martin, F. H. & Le Douarin, N. M. (1994) Differentiation 58, et de la Technologie (Grant 509112S), and the following private 133-139. foundations: Ligue Nationale Francaise contre le Cancer, Association 11. Hosoda, K., Hammer, R. E., Richardson, J. A., Baynash, A. G., pour la Recherche contre le Cancer, Fondation pour la Recherche Cheung, J. C., Giaid, A. & Yanagisawa, M. (1994) Cell 79, Medicale Francaise, and Association Francaise contre les Myopathies. 1267-1276. R.L. is supported by a fellowship of the Soci6te de Secours des Amis 12. Baynash, A. G., Hosoda, K., Giaid, A., Richardson, J. A., Emoto, des Sciences. N., Hammer, R. E. & Yanagisawa, M. (1994) Cell 79, 1277-1285. 13. Mayer, T. C. (1977) Dev. Biol. 56, 255-262. 1. Le Douarin, N. M. (1982) The Neural Crest (Cambridge Univ. 14. Mayer, T. C. & Maltby, E. (1964) Dev. Biol. 9, 269-286. Press, Cambridge). 15. Kapur, R. P., Yost, C. & Palmiter, R. D. (1993) Development 2. Couly, G., Coltey, P. & Le Douarin, N. M. (1993) Development (Cambridge, U.K) 117, 993-999. (Cambridge, U.K) 117, 409-429. 16. Rothman, T. P., Goldowitz, D. & Gershon, M. D. (1993) Dev. 3. Le Douarin, N. M., Ziller, C. & Couly, G. F. (1993)Dev. Biol. 159, Biol. 159, 559-573. 24-49. 17. Dulac, C. & Le Douarin, N. M. (1991) Proc. Natl. Acad. Sci. USA 4. Baroffio, A., Dupin, E. & Le Douarin, N. M. (1988) Proc. Natl. 88, 6348-6362. Acad. Sci. USA 85, 5325-5329. 18. Cameron-Curry, P., Dulac, C. & Le Douarin, N. M. (1993) Eur. 5. Baroffio, A., Dupin, E. & Le Douarin, N. M. (1991)Development J. Neurosci. 5, 594-604. (Cambridge, U.K) 112, 301-305. 19. Morrison-Graham, K. & Weston, J. A. (1993) Dev. Biol. 159, 6. Le Douarin, N. M. & Ziller, C. (1993) Curr. Opin. Cell Biol. 5, 346-352. 1036-1043. 20. Pavan, W. J. & Tilghman, S. M. (1994) Proc. Natl. Acad. Sci. USA 7. Williams, D. E., De Vries, P., Namen, A. E., Widmer, M. B. & 93, 7159-7163. Lyman, S. D. (1992) Dev. Biol. 151, 368-379. 21. Yohn, J. J., Smith, C., Stevens, T., Hoffman, T., Morelli, T. A., 8. Lecoin, L., Lahav, R., Martin, F. H., Teillet, M.-A. & Le Douarin, Hurt, J. G., Yanagishawa, D. L., Kane, M. & Zamora, M. R. N. M. (1995) Dev. Dyn. 203, 106-118. (1995) Biochem. Biophys. Res. Commun. 201, 449-457. 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