Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
The complex sequence of inductive events responsible for the generation of the neural crest at the border between the neural plate and the epidermis, triggers a genetic cascade involving several families of transcription factors. Two members of the Snail family, Snail and Slug, have both been implicated in this cascade. In chick and Xenopus, loss- and gain-of-function experiments have provided evidence that Slug plays a key role in neural crest development. However, in contrast to the chick, Snail rather than Slug is expressed in the premigratory neural crest in the mouse and, in Xenopus, Snail precedes Slug expression in this population. Thus, in order to study the function of Snail in neural crest development in Xenopus, we have carried out conditional gain- and loss-of-function experiments using different Snail constructs fused to a glucocorticoid receptor element. We show that Snail is able to induce the expression of Slug and all other neural crest markers tested (Zic5, FoxD3, Twist and Ets1) at the time of specification. This activation is observed in whole embryos and in animal caps, in the absence of neural plate and mesodermal markers. We show that Snail is required for neural crest specification and migration and that it works as a transcriptional repressor. These functions have been previously attributed to SLUG: However, Slug alone is unable to induce other neural crest markers in animal cap assays, and we show that Snail and Slug can be functionally equivalent when tested in overexpression studies. This suggests that, in Xenopus embryos, at least some of the functions previously attributed to Slug can be carried out by SNAIL: This is additionally supported by rescue experiments in embryos injected with dominant-negative constructs that indicate that Snail lies upstream of Slug in the genetic cascade leading to neural crest formation and that it plays a key role in crest development.
Fig. 2. Expression pattern of Snail and Slug analysed by whole-mount in situ hybridisation. (A) Dorsal view of a stage 9 embryo. An, animal pole; Veg, vegetal pole. Notice the expression in the dorsal marginal zone. (B) Dorsal view of a stage 11.5 embryo. b, dorsal blastopore lip. Snail is expressed in the prospective neural crest (arrowheads) and in a continuous band at the anterior border of the neural plate (arrow). (C) Same embryo as in B (stage 11.5) but hybridised for Slug expression. No expression is seen in the ectoderm (white arrowheads). (D) Dorsovegetal view of the embryo shown in B. Arrow indicates Snail staining in the anteriorectoderm. This is also visible around the blastopore lip in the marginal zone, apart from the dorsal region where Snail has been switched off. (E) Dorsal view of a stage 12.5 embryo. Expression is visible in the mesoderm near the blastopore and in the prospective neural crest (arrowhead). An, animal pole; Veg, vegetal pole. (F) Earliest Slug expression. Dorsal view of a stage 12.5 embryo. Expression in the prospective neural crest (arrowheads). An, animal pole; Veg, vegetal pole. (G) Anterior view of a stage 17 embryo. Expression in the superficial (white arrowheads) and in the deep (black arrowheads) layers of the ectoderm and in the anterior neural fold (a, arrow). (H,I) Schematic representation of Snail and Slug expression at stages 11 and 12.5. Anterior is upwards and posterior is downwards.
Fig. 3. Snail participates in the early specification of the neural crest. One blastomere of a two-cell stage embryo was injected with 700 pg of Xsnail-GR mRNA, treated with dexamethasone at stage 12.5, fixed at stage 19, and the expression of several genes analysed. The arrowheads indicate the injected side that contained FLDx (see Materials and Methods). Anterior is towards the left. (A-D) Neural crest markers. Notice the expansion of the markers on the injected side. (A) Snail expression. (B) Slug expression. (C) Twist expression. (D) FoxD3 expression. (E) Expression of the neural plate marker Sox2, is reduced on the injected side. The broken line indicates the dorsal midline and the brackets indicate the width of the neural plate. (F) Expression of the epidermal marker Cytokeratin 81A (dorsal view), is almost completely inhibited on the injected side. (G,H) Lateral views of the same embryo where the inhibition of Cytokeratin expression is better assessed. G corresponds to the injected side.
Fig. 4. The expansion of the neural crest territory induced by Snail or Slug does not require cell proliferation. One blastomere of a two-cell stage embryo was injected with 700 pg of Xsnail-GR mRNA (A,B) or Xslug-GR (C,D), treated with dexamethasone and HUA at stage 12.5, fixed at stage 19 and the expression of the neural crest markers Snail (A,C) and Slug (B,D) analysed. The injected side, which can be recognised by the blue FLDx staining, is indicated by the arrowhead. Note the expansion in the expression of the neural crest markers on the injected side. (E) HUA treated and (F) control embryos stained for histone H3 to verify the blockade in cell proliferation induced by the treatment. The inset shows a higher magnification of the embryos. Note the staining in absence of HUA treatment, but the lack of staining after HUA treatment.
Fig. 6. Inhibition of Snail activity blocks the expression of neural crest markers. One blastomere of a 2-cell stage embryo was injected with 700 pg of the different dominant negative constructs, treated with dexamethasone at stage 12.5, fixed at stage 19, and the expression of the neural crest markers analysed. The injected side is indicated by an arrowhead. (A-D) XsnailZnFGR: dominant-negative of the Snail zinc fingers. Note that the dominant negative construct inhibited the expression of all the neural crest markers analysed. (E,F) Rescue of XsnailZnFGR by XsnailGR: both mRNAs were injected in equivalent amounts and analysed as previously described. Note the normal expression of the neural crest markers in the injected side. (G,H) SnailN-GR: dominant-negative using the Snail N-terminal domain. Note that the dominant-negative constructs inhibited the expression of all the neural crest markers analysed. (I,J) Rescue of XsnailNGR by XsnailGR: both mRNAs were injected in equivalent amounts and analysed as previously described. Note the normal expression of the neural crest markers in the injected side.
Fig. 7. Snail lies upstream of Slug in the cascade leading to neural crest development. One blastomere of a two-cell stage embryo was co-injected with the different dominant-negative constructs and the wild type, treated with dexamethasone at stage 12.5, fixed at stage 19 and the expression of the neural crest markers analysed. The injected side is indicated by an arrowhead. (A,B) XsnailZnFGR rescued by XslugGR: the effect of the zinc fingers dominant-negative Snail construct was rescued by co-expression of Slug. Note the normal expression of neural crest markers in the injected side. (C,D) Effect of injecting XslugNGR dominant-negative construct. Note the inhibition in the expression of the markers in the injected side. (E,F) Co-injection of XslugNGR and XsnailGR: note that the effect of the dominant-negative Slug construct can not be rescued by co-expression of Snail.
Fig. 8. Snail functions as a transcriptional repressor. One blastomere of a two-cell stage embryo was injected with 700 pg of a Snail repressor construct (A-D) or the Snail activator construct (E-H), treated with dexamethasone at stage 12.5, fixed at stage 19, and the expression of neural crest markers analysed. Arrowhead, injected side. Note that the Snail repressor construct (XsnailZnF-GR-EnR) produced an expansion of the neural crest markers on the injected side (A-D), while the Snail activator lead to an inhibition in the expression of the markers (E-H).
Fig. 9. Snail controls neural crest migration. One blastomere of a two-cell stage embryo was injected with 700 pg of SnailGR (A,D), its dominant negative (B,E) or SlugGR (C,F) treated with dexamethasone at stage 16, fixed between stages 22 and 23, and the expression of the neural crest markers Slug analysed. (A-C) Injected side (arrowhead). (D-F) Uninjected side of the embryos shown in A-C. The leading edge of migration is indicated with a broken line. Note that SnailGR (A,D) and SlugGR (C,F) produces a stronger migration in the injected side; while the injection of ZnFXsnailGR (B,E) leads to an inhibition in the migration of the crest cells.
Fig. 10. Functional equivalence of the Snail genes assayed in Xenopus embryos. One blastomere of a two-cell stage embryo was injected with 500 pg of mRNA encoding for different members of the Snail gene family, treated with dexamethasone at stage 12.5, fixed at stage 25, and the expression of the neural crest marker Slug analysed. Arrowhead indicates injected side. (A,B) Xenopus genes: Xsnail-GR (A) or Xslug-GR (B). (C,D) Chick genes: Snail-GR (C) or Slug-GR (D). Note that in all the injected sides of the embryos a more vigorous and larger population of migratory crest cells (asterisks).
