| |
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. |
