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???displayArticle.abstract??? Hes3 belongs to the Hes basic helix-loop-helix family of transcriptional repressors that play central roles in maintaining progenitor cells and regulating binary cell fate decisions in the embryo. During Xenopus laevis development, hes3 is expressed in the embryonic ectoderm in a horseshoe shape domain at the edge of the developing neural pate. Hes3 mis-expression at early neurula stage blocks neural crest (snai2, sox8, sox9 and sox10) and cranial placode (six1 and dmrta1) gene expression, and promotes neural plate (sox2 and sox3) fate. At tailbud stage, these embryos exhibited a massive up-regulation of both sox8 and sox10 expression, associated with an increase in genes important for melanocytes differentiation (mitf and dct). Using a hormone inducible construct we show that Hes3 does not induce a pigment cell differentiation program de novo, rather it maintains progenitor cells in an undifferentiated state, and as Hes3 expression subsides overtime these cells adopt a pigment cell fate. We demonstrate that mechanistically Hes3 mediates its activity through inhibition of Wnt/β-catenin signaling, a molecular pathway critical for neural crest specification and pigment cell lineage differentiation. We propose that Hes3 at the edge of the neural plate spatially restricts the response to mesoderm-derived Wnt ligands, thereby contributing to the establishment of sharp boundaries of gene expression at the neural plate border.
Fig. 1. Sequence comparison of Hes proteins across species. (A) The amino acid sequences of human (Hs), mouse (Mm), zebrafish (Dr), Xenopus tropicalis (Xt), Xenopus laevis (Xl) Hes3 genes were aligned using ClustalX2. Hes3 amino acid conservation among species is highlighted in different shades of grey. The b-HLH is underlined in blue and the Groucho-binding domain is underlined in red. (B) Phylogenetic tree of Hes proteins from Xenopus laevis (Xl), human (Hs) mouse (Mm), chicken (Gg) and zebrafish (Dr).
Fig. 2. Developmental expression of Hes3. (A-N) Developmental expression of hes3 visualized by ISH at the gastrula (A-D), neurula (E-J) and tailbud (K-N) stages. (A-I, K) Dorsal views, anterior to top. (J, L) Anterior views, dorsal to top. (M) Posterior view, dorsal to top. (N) Lateral view, dorsal top, anterior to left. (O-R) Serial (anterior to posterior) transverse sections of a neurula stage embryo show the antero-lateral expression domain of hes3 in the neural plate. Dorsal to top. (S) Higher magnification view of panel (R). (T-U) Longitudinal sections highlight the anterior expression domain of hes3. Anterior to left, dorsal to top. (A-U) In each image, the embryonic stage (NF) is indicated in the lower left corner. (V-ZZ) single (V-W, Y-Z) and double (X, ZZ) ISH of neurula stage (NF stage 15) embryos for the genes indicated in the lower left corner of each image. There is no overlap between hes3 and the NC (snai2) or the PPR (dmrta1) genes. (V-X) Dorsal views, anterior to top. (X-ZZ) Anterior views, dorsal to top.
Fig. 3. Hes3 represses NPB fates. (A) Unilateral injection of hes3GR mRNA (500 pg) at the 2-cell stage followed by Dex treatment at the gastrula stage (NF stage 10.5) blocked snai2, sox10, foxd3 and dmtra1 expression, while the expression domain of pax3 and zic1 was expanded. In these embryos, the neural plate genes sox2 and sox3 were also expanded (red line). (B) Injection of hes3GR mRNA in the absence of Dex had not effect on snai2 and sox10 expression. (A-B) Dorsal views, anterior to top. Injected side is to the right. The frequency of the phenotypes shown (%) and the number of embryos analyzed (n) are indicated in the upper right corner. (C) qRT-PCR analyses indicate that Hes3GR blocks NC induction (snai2 and sox10) by Wnt8 and Noggin in animal cap explants, and promote neural plate fate (sox2). (D) Similarly, Hes3GR blocks PPR gene induction (six1 and dmtra1) by Noggin in animal cap explants. Values are normalized to ef1α and presented as mean  ±  s.e.m. Statistically significant changes are indicated; (*) p < 0.05 and (**) p < 0.01 (Student's t-test), from three independent samples.
Fig. 4. Hes3 mis-expression induces ectopic pigment cells differentiation. (A) Unilateral injection of hes3GR mRNA (500 pg) at the 2-cell stage followed by Dex treatment at the gastrula stage (NF stage 10.5) induced ectopic sox10 (NF stages 25 and 31) and dct (NF stage 35/36) expression. Sibling embryos at the tadpole stage (NF stage 41) showed supernumerary pigment cells in the head region (arrows). In all panels, lateral views are shown, dorsal to top. (B) At the tailbud stage, hes3GR-injected embryos show a decrease in neural (sox2, sox3 and pax6) and placode (dmrta1 and runx1) gene expression. Anterior views, dorsal to top. Injected side is to the right. (A-B) The frequency of the phenotypes shown (%) and the number of embryos analyzed (n) are indicated in the upper right corner. (C) In animal cap explants, Hes3 is sufficient to activate sox10 and mitf expression (22 h in culture), while dct expression was unchanged over the same period of time. (D) Hes3 is also sufficient to induce pax3 (NPB specifier), snai2 (NC specifier) and sox2 (neural) genes after 22 h in culture. Values are normalized to ef1α and presented as mean± s.e.m. Statistically significant changes are indicated; (*) p < 0.05 and (**) p < 0.01 (Student's t-test), from three independent samples.
Fig. 5. Hes3 mis-expression differentially affects soxE gene expression. (A) sox10 and sox8 follow a similar expression pattern upon Hes3 mis-expression in the embryo. Their expression is lost at the neurula stage (NF stage 17), but progressively reactivated as development proceeds, NF stage 17–20 for sox8 and NF stage 20–23 for sox10. (B) Under the same conditions, sox9 expression is also lost at the neurula stage (NF stage 17), however its expression is not reactivated at the tailbud stage (NF stage 23). (A-B) Dorsal views, anterior to top. Injected side is to the right. (B) Stage 23 lateral views, dorsal to top, anterior to right (control side), anterior to left (injected side).
Fig. 6. Hes3 does not induce de novo a pigment cell differentiation program. Unilateral injection of hes3GR mRNA (500 pg) at the 2-cell stage followed by Dex treatment at the gastrula stage (NF stage 10.5) induced ectopic sox10 expression, and repressed sox9 expression at the tailbud stage. By contrast Dex treatment at the neurula stage (NF stage 17) had no impact on sox10 or sox9 expression. Lateral views, dorsal to top, anterior to right (control side) or anterior to left (injected side). The frequency of the phenotypes shown (%) and the number of embryos analyzed (n) are indicated in the upper right corner.
Fig. 7. Hes3 interferes with Wnt/β-catenin signaling. (A) Hes3GR blocks snai2 and sox10 induction by Wnt8 and Ctnnb1. Dorsal views, anterior to top. Injected side is to the right. (B) wnt8 (200 pg mRNA) expression activates a TOP-FLASH reporter (10 pg DNA) construct in animal cap explants, this activity is inhibited by hes3GR mRNA coinjection (1 ng). (C) Ventral injection of wnt8 mRNA (50 pg) in the marginal zone of 4-cell stage embryos induces secondary axis formation. Coinjection of hes3GR mRNA (500 pg) completely blocked axis duplication by Wnt8. Dorsal views, anterior to left. (A, C) The frequency of the phenotypes shown (%) and the number of embryos analyzed (n) are indicated in the upper right corner.
Fig. 8. Model for the establishment of the NP/NC boundary by Hes3. Hes3 expression at the lateral margin of the NP restricts the response to Wnt8 to establish the NP/NC boundary. The different embryonic territories on this transverse section of a stage 15 embryo (Fig. 2S) are outlined. NC, neural crest; No, notochord; NP, neural plate; PM, paraxial mesoderm.
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