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Abstract
The neural crest is a transient population of precursor cells that arises at the border between the neural plate and prospective epidermis in vertebrate embryos. The earliest known response to neural-crest-inducing signals is the expression of the zinc-finger transcription factors slug and snail. Although it is widely believed that these transcription factors play an essential role in neural crest development, relatively little is understood about their mechanism of action during this process. We have previously shown that overexpression of XSlug leads to expanded expression of neural crest markers and an excess of at least one neural crest derivative, melanocytes. In order to further investigate XSlug function, we overexpressed mutant constructs in which the DNA-binding domain was fused to either the activation domain from Gal4 or the repressor domain from Drosophila Engrailed. The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. In contrast, overexpression of either the activation domain fusion or the DNA-binding domain alone was found to inhibit XSlug function. Using a hormone-inducible inhibitory mutant, we show that inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration, demonstrating for the first time that this transcriptional repressor is required during multiple stages of neural crest development.
FIG. 1. XSlug functions as a transcriptional repressor. (A) Diagram of the constructs used for overexpression experiments. The zinc-finger region of XSlug was fused in fram e to either the activation domain of Gal4 (G4A-Slug) or the repressor domain from Drosophila Engrailed (EnR-Slug). (B) Whole-mount in situ hybridization of embryos injected in one cell with mRNA encoding either G4A-Slug (B, D, F) or EnR-Slug (C, E, G). Embryos were examined at midneurula stages (stage 17-18) for the expression of XSlug (B, C), XTwist (D, E), or XSox-2 (F, G). Overexpression of the activation domain fusion caused a loss of neural crest marker expression and an increase in neural plate marker expression on the injected side (black arrowheads), while overexpression of the repressor fusion had the opposite effects.
FIG. 2. Inhibition of XSlug activity blocks the expression of early neural crest markers. Whole-mount in situ hybridization of embryos injected in one cell with a truncation mutant encoding the DNA-binding domain of XSlug (delta-Slug; A, C) or coinjected with full-length XSlug (B, D). Embryos were examined at midneurula stages (stage 178) for the expression of the neural crest markers XSlug (A, B) and XTwist (C, D). Overexpression of the truncation mutant inhibited the expression of early neural crest markers on the injected side (black arrowheads). These effects could be rescued by coexpression of the full-length protein.
FIG. 3. Inhibition of XSlug function prevents neural crest formation. Donor embryos expressing GFP alone or coexpressing GFP and delta-Slug were cultured to midneurula stages (stage 16-17). Cranial neural folds were explanted from donor embryos displaying strong GFP expression in this region (A) and grafted into the same location on stage-matched unlabeled host embryos (B). Host embryos were cultured through late neurula stages and migration of neural crest cells was followed using fluorescence microscopy. Grafts from donor embryos injected with GFP alone produced copious numbers of neural crest cells that migrated in well-characterized mandibular (M), hyoid (H), anterior branchial (AB), and posterior branchial (PB) arch stream s (C). In contrast, grafts from embryos coinjected with GFP and delta-Slug failed to produce migratory neural crest cells (D).
FIG. 4. Inducible inhibition of XSlug function. The zinc-finger region of XSlug was fused in frame to the hormone-binding domain of the glucocorticoid receptor (GR-delta-Slug; A). Embryos injected in one cell with mRNA encoding this fusion were cultured in the absence of dexamethasone (B, D) or treated with dexamethasone from stage 11.5 onward (C, E). Embryos were examined by in situ hybridization at midneurula stages (stage 17-18) for expression of XSlug (B, C) or XTwist (D, E). In embryos treated with dexamethasone, the expression of neural crest markers was lost or diminished on the injected side (black arrowheads). In the absence of hormone, the expression of neural crest markers was unchanged.
FIG. 5. A later role for XSlug in neural crest migration. Embryos injected in one cell with mRNA encoding the GR-delta-slug were treated with dexamethasone from stage 17/18 onward (A) or left untreated (F). Embryos were examined by in situ hybridization for expression of XSlug (A) or XTwist (D) at late neurula stages, when cranial neural crest cells are migrating. In embryos treated with hormone, cells expressing neural crest markers on the injected side of the embryo (black arrowheads) remained as a mass of cells adjacent to the neural tube. Normal migration patterns were observed on the uninjected side of hormone-treated em bryos and in injected embryos reared in the absence of hormone.
