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.
Broders-Bondon F
,
Chesneau A
,
Romero-Oliva F
,
Mazabraud A
,
Mayor R
,
Thiery JP
.
???displayArticle.abstract???
We analyzed the effects of Rho GTPases on XSnail2 expression during neural crest (NC) ontogeny in Xenopus laevis embryos. The ectopic expression of both dominant-negative (N-) and constitutively active (V-) Rho GTPase mutants after RNA or DNA microinjection disrupted the endogenous expression of XSnail2, XFoxD3, and XSnail1. V14RhoA and N17Rac1 were inhibitory, whereas N19RhoA and V12Rac1 increased NC marker gene expression. In reporter assays using a XSnail2 promoter-green fluorescent protein (GFP) construct (alpha700BA-GFP), the ectopic expression of V14RhoA, N17Rac1, or the Rac1 inhibitor NSC 23766 decreased reporter expression in NC-neural plate, whereas N19RhoA or the RhoA inhibitor Y27632 and V12Rac1 enhanced it. Similarly, transgenic embryos expressing Rho GTPase mutants and GFP under control of the alpha700BA promoter displayed variations similar to those observed for ectopic RNA and DNA expression. These results show that Rho GTPases can regulate the expression of XSnail2 during NC ontogeny.
Figure 1. A-E: In situ hybridization with the XSnail2 RNA probe on stage 18N.F. embryos into which 1 ng of mutant RhoGTPase mRNA was microinjected, into one blastomere, at stage 6 N.F. The injected side (left), marked with an asterisk, is compared with the noninjected (right) side; lysine-fixable fluorescein was injected together with mRNA and visualized by blue staining (black arrows). B-E: Embryos were injected with N19RhoA mRNA (B), V14RhoA mRNA (C), N17Rac1 mRNA (D), V12 Rac1 mRNA (E). F: NC territory of embryos scanned with ImageJ 1.36b software. The graph shows the ratio of stained NC areas on the injected side to NC areas on the noninjected side. G-K: In situ hybridization with the XFoxD3 RNA probe on stage 18N.F. embryos into which 140 pg of pCMV DNA constructs encoding RhoGTPase mutants was microinjected into one left dorsal blastomere at stage 3N.F. The injected side (left), marked with an asterisk, is compared with the noninjected (right) side; beta -galactosidase mRNA was co-injected and visualized by Red-Gal staining. H-K: Embryos were injected with pCMV-N19RhoA (H), pCMV-V14RhoA (I), pCMV-N17Rac (J), pCMV-V12 Rac1 (K). L: NC territory of embryos scanned with ImageJ 1.36b software. The graph shows the ratio of stained NC areas in injected sides to NC areas in noninjected sides. M-Q: In situ hybridization with the XSnail1 RNA probe on stage 18N.F. embryos into which 140 pg of pCMV DNA constructs encoding RhoGTPase mutants was microinjected, into one blastomere, at stage 3N.F. The injected side (left), marked with an asterisk, is compared with the noninjected (right) side; beta -galactosidase mRNA was co-injected and visualized by Red-Gal staining. N-Q: Embryos were also co-injected with N19RhoA mRNA (N), V14RhoA mRNA (O), N17Rac1 mRNA (P), V12 Rac1 mRNA (Q). R: NC territory of embryos scanned with ImageJ 1.36b software. The graph shows the ratio of stained NC areas on the injected side to NC areas on the noninjected side. n, total number of scanned embryos; CMV, cytomegalovirus.
Figure 2. Expression of the XSnail2 promoter in neuroectoderm. A: Schematic drawing of the XSnail2 ( alpha 700BA)-GFP DNA construct. B,C: Ectopic DNA expression. B: Stage 3N.F. embryos, into which 100 pg of alpha 700BA-GFP DNA was injected into one left dorsal blastomere, were analyzed at stage 18N.F. C: Transverse section of an embryo expressing alpha 700BA-GFP, showing fluorescent cells in the neuroectoderm and none in the mesoderm or endoderm; the white dotted line delimits the neuroectoderm from the mesoderm. D,E: Stage 18N.F. embryos micro-injected with alpha 700BA-GFP (D) and in situ hybridized with XSnail2 and GFP probes (E): GFP and XSnail2 expressions colocalize in the anterior region of the embryo. F-I: Transgenesis. F: Transgenic embryos expressing alpha 700BA-GFP on both sides or on one side only. G,H: Transverse section of an hemi-transgenic embryo expressing alpha 700BA-GFP, showing fluorescent cells in the neuroectoderm and none in the mesoderm or endoderm; the white-dotted line delimits the neuroectoderm from the mesoderm. I: Stage 23N.F. transgenic embryo expressing GFP in the cephalic arches. GFP, green fluorescent protein.
