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The expression pattern of endothelin 3 in the avian embryo
Mechanisms of Development 73 (1998) 217–220
Gene expression pattern
The expression pattern of endothelin 3 in the avian embryo V. Nataf a,*, A. Amemiya b, M. Yanagisawa b, N.M. Le Douarin a a
Institut d’Embryologie Cellulaire et Mole´culaire du Centre National de la Recherche Scientifique et du Colle`ge de France, 49 bis Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France b Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas, TX, USA Received 29 December 1997; revised version received 2 March 1998; accepted 4 March 1998
Abstract We investigated the expression pattern of the endothelin 3 gene, of which the mutation, as well as mutation of its receptor (endothelin-B receptor), affects the development of two neural crest derivatives: enteric nervous system and melanocytes. After previous work showing that these neural crest derived cells express endothelin-B receptor or its subtype endothelin-B2 receptor in the avian embryo, we have demonstrated that endothelin 3 is expressed by the environment of enteric nervous system and melanocyte precursors. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Endothelin 3; Avian; Embryo; Expression pattern; Enteric nervous system; Melanocytes
Vertebrate neural crest (NC) development is tightly controlled by the environmental cues encountered by NC cells during their migration and at their target sites. Some of the factors able to influence NC cell fate have been identified (reviewed in Le Douarin et al., 1994; Anderson, 1997). Both the knock-out of endothelin (EDN) genes or their receptors (EDNRs), and analysis of spontaneous mouse mutants, clearly indicate that these molecules are involved in NC development. In particular, EDN3 and its receptor EDNRB interact in the development of melanocytes and enteric nervous system, which were severely affected in these mutants (Baynash et al., 1994; Hosoda et al., 1994). It was therefore of interest to analyse the spatiotemporal patterns of expression of the genes encoding both EDNRB and EDN3, in order to assess whether these molecules act directly or indirectly on NC cell development. We previously had analysed the spatiotemporal expression pattern of EDNRB in the avian embryo (Nataf et al., 1996), a model in which NC development has been particularly well documented. EDNRB is expressed in the peripheral nervous system, including its enteric component, but is not expressed by melanocytes and migrating melanoblasts. However, a novel subtype of EDNRB (designated EDNRB2), found to be specific to the melanocytic lineage * Corresponding author. Tel: +33 1 45141515; fax: +33 1 48734377.
0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (98 )0 0048-3
and to also bind EDN3, was recently cloned by Lecoin et al. (1998) in the quail. In the present work we have investigated where and when EDN3 is expressed in the chick embryo by using a radioactively labelled EDN3 riboprobe. In situ hybridisation experiments on sections of chick embryos from the 12-somite stage (ss) up to 10 days of incubation (E10) were performed. At all the stages studied, we found that neither NC cells nor NC derivatives express EDN3 except for the NCderived mesectoderm of the head and hypobranchial region (Couly et al., 1993). In contrast, the EDN3 message was detected in the mesenchymal environment where NC cells migrate. The earliest stage at which a signal was clearly detected was 17ss. Fig. 1A–D shows EDN3 transcripts in the splanchnopleure at the level of the pharynx and even more posteriorly in the trunk. In contrast, the endoderm was negative. The splanchnopleure later yields the intestinal mesenchyme in which enteric neural precursors, expressing EDNRB (Nataf et al., 1996), migrate in a rostrocaudal colonisation (Le Douarin and Teillet, 1973). Thus EDN3 is expressed very early in the (future) environment of the precursors of the enteric nervous system. EDN3 transcripts are not detected elsewhere in the embryo at this stage. At 17 and 21 ss, we did not find expression of EDN3 in the environment of NC cells migrating dorsoventrally. These had, however, been found (Nataf et al., 1996) to express EDNRB at
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V. Nataf et al. / Mechanisms of Development 73 (1998) 217–220
Fig. 1. Transverse sections of 17 somite-stage (ss) (A–D), E3 (E,F,I,J) and E10 (G,H) chick embryos hybridised to EDN3. The section in (A) is at the level of the pharynx. (C) the section is of the same embryo as in (A) but at a more caudal level. (A,C,E,G,I) bright field; (B,D,F,H,J) corresponding dark field. sp, splanchnopleure; end, endoderm; ec, ectoderm; ep, epidermis; fb, feather bud; ba, branchial arch. Bar, 50 mm.
these stages. It is important to note that the derivatives of the NC which migrate in the dorsoventral pathway and yield the dorsal root and sympathetic ganglia are not affected in
either lethal spotted nor in piebald lethal mice, spontaneous mutants of EDN3 and EDNRB respectively (Baynash et al., 1994; Hosoda et al., 1994). From E3 onward, the ectoderm
V. Nataf et al. / Mechanisms of Development 73 (1998) 217–220
219
Fig. 2. Transverse sections of E4 (A,B,E–H) and E6 (C,D) chick embryos hybridised to EDN3. (A,C,E,G), bright field; (B,D,F,H), corresponding dark field. g, gizzard; ca, caeca; my, myotome; lb, limb bud. Bar = 50 mm.
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V. Nataf et al. / Mechanisms of Development 73 (1998) 217–220
expresses EDN3 (Fig. 1E,F). At E3 and E4, melanoblasts are still in the subectodermal mesenchyme (Teillet, 1971). It is clear that neural crest cells are directly under the influence of EDN3 when they are in the epidermis, where EDN3 expression is maintained at least to E10 (Fig. 1G,H), the last time-point investigated. It is interesting to note that melanoblasts proliferate abundantly before differentiating within the feather germs, where they start to produce melanin at E9 (Teillet, 1971). At E3 and E4, EDN3 is also expressed in the branchial arches (Fig. 1I,J). At these stages, the branchial arches are filled with rhombencephalic NC cells from which mesectodermal derivatives will later derive. This expression of EDN3 in the rhombencephalic NC is transient, since NCderived mesectodermal tissues such as cephalic dermis, Meckel and hyoid cartilages are negative (not shown). This domain of expression was unexpected because EDNRB is not expressed in the branchial arches of the avian embryo (Nataf et al., 1996). Moreover, on the basis of studies carried out in the mouse (Kurihara et al., 1994; Clouthier et al., 1998; Yanagisawa et al., 1998), it has been shown that EDN1/EDNRA act in the development of the NC mesectodermal derivatives. At E4 and E6, EDN3 transcripts are present along the whole digestive tract (Fig. 2A–D) where the signal is found only in the mesenchymal compartment. At E6, EDN3 expression is found throughout the mesenchyme of the gizzard wall, but only in its periphery, where myenteric ganglia are located (not shown). Expression of EDN3 was also observed in the developing myotome at E4 and E6 (Fig. 2E,F). The antero-proximal part of the limb is strongly positive in both the ectoderm and the mesenchyme (Fig. 2G,H). Gonads are positive, while kidneys, heart and liver are negative at all the stages studied (not shown). In conclusion, in birds, EDN3 is expressed in the environment of two NC-derived populations expressing either EDNRB or its subtype EDNRB2: the enteric nervous system and the melanocytic lineages. These data are in agreement with the phenotypes of the mice carrying null mutations of both EDNRB and EDN3 (Baynash et al., 1994; Hosoda et al., 1994). Antisense and sense RNA probes were prepared from the chicken EDN3 full-length cDNA (unpublished sequence, 2.3 kb) by in vitro transcription using T3 and T7 RNA polymerase. In situ hybridisation is described in more detail in Eichmann et al. (1993).
Acknowledgements The authors thank D. Champeval for technical assistance, and F. Viala and S. Gournet for helping with photography
and illustrations. This work was supported by the Centre National de la Recherche Scientifique. M.Y. is an Investigator and A.A. is an Associate of the Howard Hughes Medical Institute.
References Anderson, D.J., 1997. Cellular and molecular biology of neural crest cell lineage determination. Trends Genet. 13, 276–280. Baynash, A.G., Hosoda, K., Giaid, A., Richardson, J.A., Emoto, N., Hammer, R.E., Yanagisawa, M., 1994. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79, 1277–1285. Couly, G.F., Coltey, P.M., Le Douarin, N.M., 1993. The triple origin of the skull in higher vertebrates – a study in quail-chick chimeras. Development 117, 409–429. Clouthier, D.E., Hosoda, K., Richardson, J.A., Williams, S.C., Yanagisawa, H., Kuwaki, T., Kumada, M., Hammer, R.E., Yanagisawa, M., 1998. Cranial and cardiac neural crest defects in endothelin-A receptordeficient mice. Development 125, 813–824. Eichmann, A., Marcelle, C., Bre´ant, C., Le Douarin, N.M., 1993. Two molecules related to the VEGF receptor are expressed in early endothelial cells during avian embryonic development. Mech. Dev. 42, 33– 48. Hosoda, K., Hammer, R.E., Richardson, J.A., Greenstein Baynash, A., Cheung, J.C., Giaid, A., Yanagisawa, M., 1994. Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 79, 1267– 1276. Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagai, R., Oda, H., Kuwaki, T., Cao, W.H., Kamada, N., Jishage, K., Ouchi, Y., Azuma, S., Toyoda, Y., Ishikawa, T., Kumada, M., Yazaki, Y., 1994. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368, 703–710. Lecoin, L., Sakurai, T., Ngo, M.T., Abe, Y., Yanagisawa, M., Le Douarin, N.M., 1998. Cloning and characterization of a novel endothelin receptor subtype in the avian class. Proc. Natl. Acad. Sci. USA, in press. Le Douarin, N.M., Teillet, M.A., 1973. The migration of neural crest cells to the wall of the digestive tract in avian embryo. J. Embryol. Exp. Morphol. 30, 31–48. Le Douarin, N.M., Dupin, E., Ziller, C., 1994. Genetic and epigenetic control in neural crest development. Curr. Opin. Genet. Dev. 4, 685– 695. Nataf, V., Lecoin, L., Eichmann, A., Le Douarin, N.M., 1996. EndothelinB receptor is expressed by neural crest cells in the avian embryo. Proc. Natl. Acad. Sci. USA 93, 9645–9650. Teillet, M.A. (1971). Recherches sur le mode de migration et la diffe´renciation des me´lanoblastes cutane´s chez l’embryon d’oiseau: Etude expe´rimentale par la me´thode des greffes he´te´rospe´cifiques entre embryons de Caille et de Poulet. Ann. Embryol. Morphogen. 95–109. Yanagisawa, H., Yanagisawa, M., Kapur, R.P., Richardson, J.A., Williams, S.C., Clouthier, D.E., de Wit, D., Emoto, N., Hammer, R.E., 1998. Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme1 gene. Development 125, 825–836.
Gene expression pattern
The expression pattern of endothelin 3 in the avian embryo V. Nataf a,*, A. Amemiya b, M. Yanagisawa b, N.M. Le Douarin a a
Institut d’Embryologie Cellulaire et Mole´culaire du Centre National de la Recherche Scientifique et du Colle`ge de France, 49 bis Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France b Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas, TX, USA Received 29 December 1997; revised version received 2 March 1998; accepted 4 March 1998
Abstract We investigated the expression pattern of the endothelin 3 gene, of which the mutation, as well as mutation of its receptor (endothelin-B receptor), affects the development of two neural crest derivatives: enteric nervous system and melanocytes. After previous work showing that these neural crest derived cells express endothelin-B receptor or its subtype endothelin-B2 receptor in the avian embryo, we have demonstrated that endothelin 3 is expressed by the environment of enteric nervous system and melanocyte precursors. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Endothelin 3; Avian; Embryo; Expression pattern; Enteric nervous system; Melanocytes
Vertebrate neural crest (NC) development is tightly controlled by the environmental cues encountered by NC cells during their migration and at their target sites. Some of the factors able to influence NC cell fate have been identified (reviewed in Le Douarin et al., 1994; Anderson, 1997). Both the knock-out of endothelin (EDN) genes or their receptors (EDNRs), and analysis of spontaneous mouse mutants, clearly indicate that these molecules are involved in NC development. In particular, EDN3 and its receptor EDNRB interact in the development of melanocytes and enteric nervous system, which were severely affected in these mutants (Baynash et al., 1994; Hosoda et al., 1994). It was therefore of interest to analyse the spatiotemporal patterns of expression of the genes encoding both EDNRB and EDN3, in order to assess whether these molecules act directly or indirectly on NC cell development. We previously had analysed the spatiotemporal expression pattern of EDNRB in the avian embryo (Nataf et al., 1996), a model in which NC development has been particularly well documented. EDNRB is expressed in the peripheral nervous system, including its enteric component, but is not expressed by melanocytes and migrating melanoblasts. However, a novel subtype of EDNRB (designated EDNRB2), found to be specific to the melanocytic lineage * Corresponding author. Tel: +33 1 45141515; fax: +33 1 48734377.
0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (98 )0 0048-3
and to also bind EDN3, was recently cloned by Lecoin et al. (1998) in the quail. In the present work we have investigated where and when EDN3 is expressed in the chick embryo by using a radioactively labelled EDN3 riboprobe. In situ hybridisation experiments on sections of chick embryos from the 12-somite stage (ss) up to 10 days of incubation (E10) were performed. At all the stages studied, we found that neither NC cells nor NC derivatives express EDN3 except for the NCderived mesectoderm of the head and hypobranchial region (Couly et al., 1993). In contrast, the EDN3 message was detected in the mesenchymal environment where NC cells migrate. The earliest stage at which a signal was clearly detected was 17ss. Fig. 1A–D shows EDN3 transcripts in the splanchnopleure at the level of the pharynx and even more posteriorly in the trunk. In contrast, the endoderm was negative. The splanchnopleure later yields the intestinal mesenchyme in which enteric neural precursors, expressing EDNRB (Nataf et al., 1996), migrate in a rostrocaudal colonisation (Le Douarin and Teillet, 1973). Thus EDN3 is expressed very early in the (future) environment of the precursors of the enteric nervous system. EDN3 transcripts are not detected elsewhere in the embryo at this stage. At 17 and 21 ss, we did not find expression of EDN3 in the environment of NC cells migrating dorsoventrally. These had, however, been found (Nataf et al., 1996) to express EDNRB at
218
V. Nataf et al. / Mechanisms of Development 73 (1998) 217–220
Fig. 1. Transverse sections of 17 somite-stage (ss) (A–D), E3 (E,F,I,J) and E10 (G,H) chick embryos hybridised to EDN3. The section in (A) is at the level of the pharynx. (C) the section is of the same embryo as in (A) but at a more caudal level. (A,C,E,G,I) bright field; (B,D,F,H,J) corresponding dark field. sp, splanchnopleure; end, endoderm; ec, ectoderm; ep, epidermis; fb, feather bud; ba, branchial arch. Bar, 50 mm.
these stages. It is important to note that the derivatives of the NC which migrate in the dorsoventral pathway and yield the dorsal root and sympathetic ganglia are not affected in
either lethal spotted nor in piebald lethal mice, spontaneous mutants of EDN3 and EDNRB respectively (Baynash et al., 1994; Hosoda et al., 1994). From E3 onward, the ectoderm
V. Nataf et al. / Mechanisms of Development 73 (1998) 217–220
219
Fig. 2. Transverse sections of E4 (A,B,E–H) and E6 (C,D) chick embryos hybridised to EDN3. (A,C,E,G), bright field; (B,D,F,H), corresponding dark field. g, gizzard; ca, caeca; my, myotome; lb, limb bud. Bar = 50 mm.
220
V. Nataf et al. / Mechanisms of Development 73 (1998) 217–220
expresses EDN3 (Fig. 1E,F). At E3 and E4, melanoblasts are still in the subectodermal mesenchyme (Teillet, 1971). It is clear that neural crest cells are directly under the influence of EDN3 when they are in the epidermis, where EDN3 expression is maintained at least to E10 (Fig. 1G,H), the last time-point investigated. It is interesting to note that melanoblasts proliferate abundantly before differentiating within the feather germs, where they start to produce melanin at E9 (Teillet, 1971). At E3 and E4, EDN3 is also expressed in the branchial arches (Fig. 1I,J). At these stages, the branchial arches are filled with rhombencephalic NC cells from which mesectodermal derivatives will later derive. This expression of EDN3 in the rhombencephalic NC is transient, since NCderived mesectodermal tissues such as cephalic dermis, Meckel and hyoid cartilages are negative (not shown). This domain of expression was unexpected because EDNRB is not expressed in the branchial arches of the avian embryo (Nataf et al., 1996). Moreover, on the basis of studies carried out in the mouse (Kurihara et al., 1994; Clouthier et al., 1998; Yanagisawa et al., 1998), it has been shown that EDN1/EDNRA act in the development of the NC mesectodermal derivatives. At E4 and E6, EDN3 transcripts are present along the whole digestive tract (Fig. 2A–D) where the signal is found only in the mesenchymal compartment. At E6, EDN3 expression is found throughout the mesenchyme of the gizzard wall, but only in its periphery, where myenteric ganglia are located (not shown). Expression of EDN3 was also observed in the developing myotome at E4 and E6 (Fig. 2E,F). The antero-proximal part of the limb is strongly positive in both the ectoderm and the mesenchyme (Fig. 2G,H). Gonads are positive, while kidneys, heart and liver are negative at all the stages studied (not shown). In conclusion, in birds, EDN3 is expressed in the environment of two NC-derived populations expressing either EDNRB or its subtype EDNRB2: the enteric nervous system and the melanocytic lineages. These data are in agreement with the phenotypes of the mice carrying null mutations of both EDNRB and EDN3 (Baynash et al., 1994; Hosoda et al., 1994). Antisense and sense RNA probes were prepared from the chicken EDN3 full-length cDNA (unpublished sequence, 2.3 kb) by in vitro transcription using T3 and T7 RNA polymerase. In situ hybridisation is described in more detail in Eichmann et al. (1993).
Acknowledgements The authors thank D. Champeval for technical assistance, and F. Viala and S. Gournet for helping with photography
and illustrations. This work was supported by the Centre National de la Recherche Scientifique. M.Y. is an Investigator and A.A. is an Associate of the Howard Hughes Medical Institute.
References Anderson, D.J., 1997. Cellular and molecular biology of neural crest cell lineage determination. Trends Genet. 13, 276–280. Baynash, A.G., Hosoda, K., Giaid, A., Richardson, J.A., Emoto, N., Hammer, R.E., Yanagisawa, M., 1994. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79, 1277–1285. Couly, G.F., Coltey, P.M., Le Douarin, N.M., 1993. The triple origin of the skull in higher vertebrates – a study in quail-chick chimeras. Development 117, 409–429. Clouthier, D.E., Hosoda, K., Richardson, J.A., Williams, S.C., Yanagisawa, H., Kuwaki, T., Kumada, M., Hammer, R.E., Yanagisawa, M., 1998. Cranial and cardiac neural crest defects in endothelin-A receptordeficient mice. Development 125, 813–824. Eichmann, A., Marcelle, C., Bre´ant, C., Le Douarin, N.M., 1993. Two molecules related to the VEGF receptor are expressed in early endothelial cells during avian embryonic development. Mech. Dev. 42, 33– 48. Hosoda, K., Hammer, R.E., Richardson, J.A., Greenstein Baynash, A., Cheung, J.C., Giaid, A., Yanagisawa, M., 1994. Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 79, 1267– 1276. Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagai, R., Oda, H., Kuwaki, T., Cao, W.H., Kamada, N., Jishage, K., Ouchi, Y., Azuma, S., Toyoda, Y., Ishikawa, T., Kumada, M., Yazaki, Y., 1994. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368, 703–710. Lecoin, L., Sakurai, T., Ngo, M.T., Abe, Y., Yanagisawa, M., Le Douarin, N.M., 1998. Cloning and characterization of a novel endothelin receptor subtype in the avian class. Proc. Natl. Acad. Sci. USA, in press. Le Douarin, N.M., Teillet, M.A., 1973. The migration of neural crest cells to the wall of the digestive tract in avian embryo. J. Embryol. Exp. Morphol. 30, 31–48. Le Douarin, N.M., Dupin, E., Ziller, C., 1994. Genetic and epigenetic control in neural crest development. Curr. Opin. Genet. Dev. 4, 685– 695. Nataf, V., Lecoin, L., Eichmann, A., Le Douarin, N.M., 1996. EndothelinB receptor is expressed by neural crest cells in the avian embryo. Proc. Natl. Acad. Sci. USA 93, 9645–9650. Teillet, M.A. (1971). Recherches sur le mode de migration et la diffe´renciation des me´lanoblastes cutane´s chez l’embryon d’oiseau: Etude expe´rimentale par la me´thode des greffes he´te´rospe´cifiques entre embryons de Caille et de Poulet. Ann. Embryol. Morphogen. 95–109. Yanagisawa, H., Yanagisawa, M., Kapur, R.P., Richardson, J.A., Williams, S.C., Clouthier, D.E., de Wit, D., Emoto, N., Hammer, R.E., 1998. Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme1 gene. Development 125, 825–836.