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???
We have investigated the molecular interactions underlying neural crest formation in Xenopus. Using chordin overexpression to antagonize endogenous BMP signaling in whole embryos and explants, we demonstrate that such inhibition alone is insufficient to account for neural crest induction in vivo. We find, however, that chordin-induced neural plate tissue can be induced to adopt neural crest fates by members of the FGF and Wnt families, growth factors that have previously been shown to posteriorize induced neural tissue. Overexpression of a dominant negative XWnt-8 inhibits the expression of neural crest markers, demonstrating the necessity for a Wnt signal during neural crest induction in vivo. The requirement for Wnt signaling during neural crest induction is shown to be direct, whereas FGF-mediated neural crest induction may be mediated by Wnt signals. Overexpression of the zinc finger transcription factor Slug, one of the earliest markers of neural crest formation, is insufficient for neural crest induction. Slug-expressing ectoderm will generate neural crest in the presence of Wnt or FGF-like signals, however, bypassing the need for BMP inhibition in this process. A two-step model for neural crest induction is proposed.
Fig. 1. Xslug expression is altered in response to changes in BMP activity. Whole-mount in situ hybridization showing Xslug expression in stage 17 control embryos (A) and in embryos injected in one cell at the 2-cell stage with 50 pg (B), 200 pg (C) or 1 ng (D) BMP4 mRNA. With increasing BMP concentration Xslug expression domains become deflected toward the midline (B), fuse (C) and are lost in ventralized embryos lacking a discernible neural plate (D). (E,F) Xslug expression in embryos injected in one cell at the 2-cell stage with 200 pg of chordin mRNA. Xslug expression is expanded laterally (E) and/or ventrally (F) on the injected side.
Fig. 2. Chordin induces low levels of neural crest gene expression in ectodermal explants. RT-PCR analysis of ectodermal explants from embryos injected with a range of concentrations (10 pg-2 ng) of chordin mRNA. Representative doses are shown. Explants were isolated at late stage 9 and harvested at stage 17. Control explants are from uninjected embryos. Whole embryos and chordin-expressing explants conjugated with dlmz serve as positive controls. All samples were analyzed with primers specific for the neural crest markers Xsna and Xslug, the pan-neural marker NCAM, the mesodermal marker muscle actin and epidermal keratin. EF1! serves as a loading control.
Fig. 3. Chordin-expressing ectoderm can be lateralized to neural crest fates by members of the FGF and Wnt families. (A) Schematic of the assay. Conjugates were made by combining explants from control (C) or chordin (CHD)-injected embryos with explants expressing XeFGF or XWnt-8 at late stage 9. Explants were isolated at stage 17 for analysis by in situ hybridization or RT-PCR. (B) In situ hybridization showing Xslug expression in conjugates. Xslug expression is found in CHD/XWnt-8 and CHD/XeFGF conjugates but not in conjugates of control ectoderm with CHD, XeFGF or XWnt-8. (C) Section through a CHD/Wnt conjugate slug expression is found throughout the conjugate except for a crenelated epithelial-like outer layer. (D) In situ hybridization showing expression of the neural plate marker Xsox2 in stage 17 embryos and in conjugates. Xsox2 expression is found throughout C/CHD conjugates, but is diminished in CHD/XWnt-8 conjugates. (E) RT-PCR analysis of gene expression in conjugates. Controls and primers are the same as in Fig. 1. (F) Co- expression of a dominant inhibitory Wnt (dnWnt) inhibits the induction of neural crest markers in CHD/FGF conjugates.
Fig. 4. Xslug expression is altered in response to changes in Wnt activity. In situ hybridization showing Xslug (A-C) and Xsox2 (D-F) expression in control embryos (A,D) or embryos injected in one cell at the 2-cell stage with 200 pg XWnt-8 mRNA (B,E) or 1 ng of dominant negative XWnt-8 mRNA (D,F). Arrowhead denotes the injected side of embryos in C and F.
Fig. 5. Effects of !-catenin overexpression in whole embryos and conjugates. (A) in situ hybridization showing expression of Xslug and Xsox-2 in embryos injected in one cell at the 2-cell stage with 100 pg !-catenin. Expanded expression of the neural crest marker and diminished expression of the neural plate marker is seen on the injected side (white arrowheads). (B) In situ hybridization of conjugates which tBR and !-catenin were each expressed in only one half of each conjugate, or were coexpressed in both halves of each conjugate. Xslug expression is found only in conjugates in which tBR and !-catenin are coexpresed in the same cells.
Fig. 6. The effects of Wnt signaling on neural crest induction are direct. (A) Section through the midbrain region of a stage 18 embryo coinjected in one cell at stage 2 with !-catenin and !-galactosidase mRNAs. Embryos were stained for !- galactosidase activity (light blue) and processed for in situ hybridization using a Xslug probe (purple). Ectopic Xslug expression is restricted to cells expressing !-galactosidase. (B) Section through the rostral trunk region of a similarly treated embryo. !-galactosidase expression is seen exclusively in cells of ectodermal lineage. !-catenin overexpression induced ectopic Xslug expression lateral to endogenous expression domains, at the expense of epidermis. (C) Section through the caudal hindbrain region of a stage 16 embryo coinjected with dnWNT and !-galactosidase mRNAs. Expression of the injected mRNAs is exclusively ectodermal and mesodermal derivatives appear normal. (D) Closeup of a region of residual Xslug expression. Inhibition of Xslug expression was noted only in or directly adjacent to cells which expressed high levels of !-galactosidase activity.
Fig. 7. Effects of Slug overexpression in whole embryos. In situ hybridization showing expression of Xtwi (A,B) and Xslug (C-E) in control embryos (A, both embryos; C, rightembryo) and embryos injected in one cell at the 2-cell stage with 0.5 ng Slug mRNA. Expanded expression of neural crest markers is seen on the injected side. Increased numbers of melanocytes are found when such embryos are allowed to develop until stage 41. (F) Leftembryo is injected, rightembryo is a sibling control. (G) Top two embryos are injected, bottom two are sibling controls.
Fig. 8. Neural crest markers, but not neural markers, are induced in Slug/Wnt conjugates. (A-D) In situ hybridization showing expression of Xslug in conjugates of control or Slug-expressing explants with explants expressing Xwnt-8 or BMP4. Expression of Xslug is seen only in Slug-expressing explants exposed to a Wnt signal. (E-H) Xsox-2 expression is not seen in Slug-expressing conjugates.
Fig. 9. Neural crest induction in Slug-expressing conjugates is BMP- sensitive. RT-PCR analysis of gene expression in Slug-expressing conjugates in the presence and absence of increased BMP levels. BMP is co-expressed in either the Slug-expressing or Wnt- expressing halves of the conjugate. Xtwi is a neural crest marker. Other primers and controls are the same as in Fig. 1.
