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First published online 16 June 2004
doi: 10.1242/dev.01193

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Cell-autonomous and cell non-autonomous signaling through endothelin receptor B during melanocyte development

¹ Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-4472, USA
² Cell and Developmental Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
³ Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (69K): [in a new window]   Fig. 1. Lack of melanocyte differentiation in EdnrblacZ/EdnrblacZ neural crest cells in culture. Primary cultures from embryos of the indicated genotypes were established and kept in the presence of EDN3 for 2 weeks before fixation. X-gal staining was then performed and the cultures were photographed in bright field to show melanin and ß-gal staining. (A) EdnrblacZ/+ cultures show melanin-positive cells, many of which co-express ß-gal (arrow) although some lack clear ß-gal staining (arrowhead). (B) EdnrblacZ/EdnrblacZ cultures lack melanin-positive cells, although ß-gal-positive cells are present (arrow).

View larger version (110K): [in a new window]   Fig. 2. Expression of MITF and KIT does not depend on Ednrb, whereas that of tyrosinase does. Neural crest cultures were established from embryos of the indicated genotypes and kept in the absence of EDN3 for 2 days (A-D) or in the presence of EDN3 for 2 weeks (E,F). They were then subjected to double indirect immunofluorescent labeling using MITF- and ß-gal-specific antibodies (A,B), ß-gal-specific antibody alone (C,D), or tyrosinase- and ß-gal-specific antibodies (E,F). (A,B) Merged images show that regardless of the Ednrb genotype, MITF/ß-gal double-positive cells were generated. (C,D) Regardless of the Ednrb genotype, cells double-positive for cytoplasmic ß-gal (marking Ednrb expression) and nuclear ß-gal (marking Kit expression) were generated (arrows). Note that in cultures from KitlacZ/+ embryos, ß-gal staining is confined to the nucleus (Hou et al., 2000) (data not shown), and in cultures from EdnrblacZ/+ embryos, to the cytoplasm. (E,F) Merged images show Tyr/ß-gal double-positive cells along with ß-gal single-positive cells in EdnrblacZ/+ cultures (E) but only ß-gal single-positive cells in EdnrblacZ/EdnrblacZ cultures (H). Scale bar: 25 µm for A,B,E,F; 20 µm for C,D.

View larger version (46K): [in a new window]   Fig. 3. Ednrb wild-type neural tubes (NTs) induce tyrosinase expression in Ednrb-deficient cultures. (Top) Tissue recombination experiments. In a first step, E9.5 NT explants were isolated from (EdnrblacZ/+ x EdnrblacZ/+) matings as well as from [(Mitf mi–ew/Mitf mi–ew; Ednrb+/+) x (Mitf mi–ew/Mitf mi–ew; Ednrb+/+)] or [(KitlSl/+; Ednrb+/+) x (KitlSl/+; Ednrb+/+)] matings. The NTs were placed separately into individual dishes and the corresponding embryos were genotyped. On the following day, the NTs from identified EdnrblacZ/EdnrblacZ cultures were removed and replaced with NTs from the respective Ednrb+/+ cultures. As a control, NTs from EdnrblacZ/EdnrblacZ cultures were given to other EdnrblacZ/EdnrblacZ cultures whose own NTs had been removed. The cultures were kept for 2 weeks and then stained with ß-gal and tyrosinase antibodies as described in the legend for Fig. 2 and the Materials and methods section. (Bottom panel) (A) EdnrblacZ/EdnrblacZ culture control-reconstituted with EdnrblacZ/EdnrblacZ NT. (B) EdnrblacZ/EdnrblacZ culture reconstituted with (Mitf mi–ew/Mitf mi–ew; Ednrb+/+) NT. (C) EdnrblacZ/EdnrblacZ culture reconstituted with (KitlSl/KitlSl; Ednrb+/+) NT. EdnrblacZ/EdnrblacZ cultures always generated ß-gal-positive cells. However, only (Mitf mi–ew/Mitf mi–ew; Ednrb+/+) NTs (B), but not EdnrblacZ/EdnrblacZ NTs (A) or (KitlSl/KitlSl; Ednrb+/+) NTs (C), led to the generation of tyrosinase-positive cells. Neither (Mitf mi–ew/Mitf mi–ew; Ednrb+/+) nor (KitlSl/KitlSl; Ednrb+/+) cultures carry the lacZ marker, nor are they capable of generating tyrosinase-positive cells on their own (Hou et al., 2000) (this paper), indicating that Tyr/ß-gal double-positive cells in B are derived from the EdnrblacZ/EdnrblacZ culture. Scale bar: 25 µm.

View larger version (54K): [in a new window]   Fig. 4. KITL rescues melanoblast differentiation in EdnrblacZ/EdnrblacZ neural crest cells. Primary cultures from embryos of the indicated genotypes were established and kept in the presence of KITL for 2 weeks before fixation. The cultures were then double-labeled for ß-gal and tyrosinase as described in Fig. 2. Note the presence of Tyr/ß-gal double-positive cells in KITL-treated cultures, regardless of whether they were derived from EdnrblacZ/+ (A) or EdnrblacZ/EdnrblacZ embryos (B). Scale bar: 25 µm.

View larger version (49K): [in a new window]   Fig. 5. TPA but not EDN1 rescues pigmentation in KITL-treated, tyrosinase-positive Ednrb-deficient melanoblasts. (A-C) Cultures from embryos with the indicated genotypes were kept in the presence of KITL for 6 days, and then in the presence of KITL and TPA (A,C) or KITL alone (B) for another 8 days. Mature melanocytes are present in TPA-treated EdnrblacZ/+ and EdnrblacZ/EdnrblacZ cultures (A,C) but absent in melanocytes in EdnrblacZ/EdnrblacZ cultures when TPA was omitted (B). (D,E) Cultures of the indicated genotypes were treated with KITL and EDN1 for 14 days. Pigmented cells are not present in EdnrblacZ/EdnrblacZ cultures (E).

View larger version (22K): [in a new window]   Fig. 6. The effect of EDNRB signaling on melanocyte development both cell-autonomously and cell non-autonomously. Both melanoblasts and non-melanogenic NT or neural crest cells express Ednrb. As the addition of Ednrb+/+ NTs can induce tyrosinase expression in Ednrb-deficient melanoblasts, we postulate that in response to EDN3, these Ednrb+/+ NTs or their neural crest derivatives produce one or several factors that indirectly help melanoblasts to differentiate. As these rescuing Ednrb+/+ NTs need not be capable of generating melanocytes themselves but must be capable of providing KITL, the helper cell type(s) can be non-melanocytic but must provide KITL as a major helper factor. It is likely that KITL acts directly on melanoblasts even though an indirect action involving yet other cell types cannot be excluded. Nevertheless, even though the cell non-autonomous action of Ednrb can rescue Ednrb mutant cells to the tyrosinase-positive stage, it is insufficient to induce pigmentation. Pigmentation can be seen, however, if the rescued cells are given TPA which induces PKC as does EDNRB signaling. Thus, terminal differentiation to mature, pigmented melanocytes probably depends on an additional, cell-autonomous EDNRB signaling step. Note that the above scenario does not preclude the possibility that in wild-type melanoblasts, Ednrb can also act cell-autonomously throughout development.