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Dev Biol
2003 Jul 01;2591:19-33. doi: 10.1016/s0012-1606(03)00161-1.
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Sox10 regulates the development of neural crest-derived melanocytes in Xenopus.
Aoki Y
,
Saint-Germain N
,
Gyda M
,
Magner-Fink E
,
Lee YH
,
Credidio C
,
Saint-Jeannet JP
.
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The transcription factors of the Sox family play important roles in diverse developmental processes. A number of genetic studies have established that Sox10 is a major regulator of neural crest formation. Here, we report the cloning and functional analysis of the Xenopus Sox10 gene. Sox10 mRNA accumulates during gastrulation at the lateral edges of the neural plate, in the neural crest-forming region. In this tissue, Sox10 expression is regulated by Wnt signaling and colocalizes with two major regulators of neural crest formation, Slug and Sox9. While initially expressed in neural crest cells from all axial levels, at the tailbud stage, Sox10 is downregulated in the cranial neural crest and persists mostly in neural crest cells from the trunk region. Overexpression of Sox10 causes a dramatic expansion of the Slug expression domain. We show that the C-terminal portion of Sox10 is sufficient to mediate this activity. Later during embryogenesis, Sox10-injected embryos show a massive increase in pigment cells (Trp-2-expressing cells). The responsiveness of the embryo to Sox10 overexpression by expansion of the Slug expression domain and ectopic production of Trp-2-positive cells and differentiated melanocytes is lost during gastrulation, as revealed by a hormone-inducible Sox10 construct. These results suggest that Sox10 is involved in the specification of neural crest progenitors fated to form the pigment cell lineage.
Fig. 2. Developmental expression of Sox10 by whole-mount in situ hybridization. (A) Sox10 expression at the neurula stage (stage 14) is detected at the lateral edges of the neural plate. Slug (B) and Sox9 (C) expression at a similar stage are shown for comparison. (A–C) Dorsal views, anterior to top. (D) Transverse section of a stage 14 embryo. Sox10 expression is restricted to the neural crest-forming region. (E–H) Stage 16 embryo, dorsal (E, G) or lateral (F, H) views, anterior to top. As the neural tube closes, Sox10 (E, F) remains strongly expressed in the neural crest at all axial levels. Sox9 expression (G, H) is shown for comparison. (I) At stage 22, Sox10 is detected in migrating cranial neural crest cells (red arrow) as well as in neural crest at the dorsal midline in the trunk region (green arrow). An additional domain of expression includes the otic placode (yellow arrow). Dorsal view, anterior to top. (J–L) In stage 25 embryos, Sox10 is detected in the streams of cranial neural crest that populate the branchial arches (red arrows), in the dorsal aspect of the neural tube (green arrow) and in the otic placode (yellow arrow). Lateral (J) and dorsal (K) views, anterior to left. (L) Transverse section of a stage 25 embryo, highlights Sox10 expression in neural crest progenitors at the dorsal midline. (M-P) At stage 32, Sox10 expression is downregulated in the branchial arches but persists in the otic vesicle (yellow arrow), at the dorsal midline (green arrow), in the forebrain, and in discrete domains adjacent to the hindbrain. Lateral (M) and dorsal (P) views, anterior to left. Higher magnification view of Sox10 expression in the head (N). Sox9 expression in the head of an embryo at a similar stage is shown for comparison (O). In a lateral view of an embryo at stage 35 (Q), Sox10 is detected in the forebrain, the otic vesicle (yellow arrow), a number of cranial ganglia and in differentiating melanoblasts. (R) Transverse section at the level of the hindbrain illustrates Sox10 expression in the otic vesicle (yellow arrow) and adjacent VII/IX cranial ganglion (purple arrow).
Fig. 3. Regulation of Sox10 expression in the neural crest-forming region. (A) Regulation of Sox10 and Slug expression by Wnt signaling. Injection of Xwnt-1 plasmid (100 pg) expands Slug (79%, n = 53) and Sox10 (82%, n = 50) expression domains. Conversely, overexpression of GSK3 beta (1 ng), known to block Wnt signaling, prevents Slug (81%, n = 42) and Sox10 (65%, n = 51) expression on the injected side. (B) Sox9 and Slug are required for Sox10 expression. Sox9-AS injection (10 ng) blocks Slug (89%, n = 35) and Sox10 (96%, n = 54) expression. Similarly, a dominant negative Slug (delta Slug, 1 ng) blocks Slug (61%, n = 51) and Sox10 (56%, n = 50) expression. (C) Sox9 and Slug overexpression upregulate Sox10 expression. Sox9 (1 ng) overexpression leads to a limited expansion of Slug (83%, n = 58) and Sox10 (96%, n = 79) expression domains. Overexpression of Slug (0.1 ng) results in a robust expansion of Slug (90%, n = 30) and Sox10 (98%, n = 44). In all panels, early neurula stage embryos are viewed from the dorsal side, anterior to top. RNA encoding the lineage tracer beta-galactosidase was coinjected to identify the injected side (red staining), the right side in all panels.
Fig. 4. The C-terminal domain of Sox10 is sufficient to induce neural crest progenitors. (A) Embryos were injected in one blastomere at the two-cell stage with mRNA encoding either wild type Sox10 (Sox10) or various Sox10 deletion constructs (Delta-Sox10, Sox10-delta-C, or Nsox10) and analyzed for Slug expression at stage 17. Sox10 injection (1 ng) expands Slug expression domain (white arrow). A transverse section through a Sox10-injected embryo illustrates Slug expansion on the injected side (black arrow). Delta-Sox10-injected embryos (1 ng) display a dramatic expansion of the Slug expression domain. By contrast, injection of Sox10-delta-C (2 ng) resulted in a moderate inhibition of Slug expression, whereas NSox10-injected embryos (1 ng) were unaffected. RNA encoding the lineage tracer beta-galactosidase was coinjected to identify the injected side (red staining), the right side in all panels. (B) Schematic representation of wild type Sox10 and various Sox10 deletion constructs. The HMG box is depicted in blue. Quantification of the in situ hybridization results. (1), expansion of Slug expression domain; (2), reduction of Slug expression domain; (3), Slug expression domain is unaffected. N, number of cases analyzed.
Fig. 5. Sox10 is sufficient to induce pigment cell lineage as revealed by Trp-2 expression. (A) Developmental expression of Xenopus Trp-2 by Northern hybridization. The stages are according to Nieuwkoop and Farber (1967). (B) By whole-mount in situ hybridization at stage 27/28, Trp-2 is detected in the developing retina and in individual cells scattered at the dorsal midline at a level posterior to the hindbrain. Dorsal view, anterior to the left. (C) An embryo injected at the two-cell stage with 1 ng of Sox10 mRNA and analyzed at stage 30 displays massive ectopic expression of Trp-2 (left panel), while the uninjected side is unperturbed (right panel). Lateral view, anterior is to the right. (D) RT-PCR analysis of animal explants derived from embryos injected at the two-cell stage with 1 ng of Sox9 or Sox10 mRNA. Only Sox10 overexpression induces strong expression of Trp-2 at stage 33. EF1-alpha is used as a loading control. (E) In situ hybridization of explants overexpressing Sox9, Sox10, or Slug. Induction of Trp-2 is exclusively detected in Sox10-injected animal explants at equivalent stage 32. Trp2 expression in an uninjected embryo (control) at stage 32 is shown (left panel).
Fig. 6. Sox10 expands Slug and induces ectopic Trp2-expressing cells and melanocytes at the gastrula stage. (A) Ectopic Slug expression in embryos injected with Sox10-GR at the two-cell stage and treated with dexamethasone at stage 6 or stage 10 (left panels). Slug expression on the uninjected side is shown for comparison (right panels). (B) Ectopic Trp-2 expression in embryos injected with Sox10-GR at the two-cell stage and treated with dexamethasone at stage 10 (upper left panel). Trp-2 expression is unaffected in the neural crest lineage of embryos treated with dexamethasone at stage 13 (lower left panel). When targeted in the head region, Sox10-GR blocks Trp-2 expression in the pigmented epithelium of the retina (arrow, left panel). Trp-2 expression on the uninjected side is presented for comparison (right panels). Lateral view, anterior is to the left (left panels) or to the right (right panels). RNA encoding the lineage tracer beta-galactosidase was coinjected to identify the injected side (red staining). (C) Quantification of the in situ hybridization results. The number of cases analyzed for each time point (+Dex) is indicated in parentheses. (D) Western blot analysis. Detection of Sox10-GR protein in extracts from Sox10-GR-injected embryos (+Sox10-GR) collected at different stages after injection at the two-cell stage. The fusion protein is expressed at similar levels at all stages (st 10–22). This Sox10 antibody does not allow detection of endogenous Sox10 protein (first lane). Tubulin is presented as a loading control. (E) Development of ectopic melanocytes in Sox10-GR-injected embryos. Embryos injected with Sox10-GR at the two-cell stage and treated with dexamathasone at stage 10 (upper panel, +Dex st 10) displays ectopic formation of pigment cells in the head (white arrow) and the trunk (black arrows) regions. The pattern of pigment cells is unaffected in sibling embryos treated with dexamethasone at stage 16 (lower panel, +Dex st 16). (F) Induction of differentiated melanocytes in animal explants at equivalent stage 40. Animal explants isolated at the blastula stage from embryo injected with 2 ng of Sox10-GR mRNA at the two-cell stage were treated with dexamethasone at stage 9 (+Dex st. 9) or at stage 16 (+Dex st. 16). Induction of melanocytes is only observed in animal explants treated by dexamethasone at stage 9.
