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Figure 2. eg5 is required for early neural crest specification in Xenopus embryos. A–E: In vitro and in vivo efficiency of eg5 antisense morpholino oligonucleotide (eg5MO). A: Efficiency of eg5 antisense morpholino oligonucleotide. eg5MO injected and control embryos were cultured until stage 14 and homogenized for total protein extraction. Eg5-GFP protein was detected by Western blotting using an anti-green fluorescent protein (GFP) antibody. Each lane was loaded with 100 μg of total protein. eg5MO inhibits translation of eg5 in a dose-dependent manner. eg5MO concentration is expressed in ng/embryo. B–E: Dorsal views of Xenopus laevis embryos under the fluorescence stereo microscope, anterior side is on the left. White arrowheads indicate the injected side. B', D', E': Fluorescence and clear field images of each embryo are shown in merged images. B, B': Embryo injected with mRNA encoding δC-eg5GFP (1 ng/embryo) showing GFP fluorescence on the treated side. C, C': Embryo injected with δC-eg5GFP mRNA (1 ng/embryo) and control antisense morpholino oligonucleotide (CoMO, 20 ng/embryo). D, D', E, E': Embryos injected with δC-eg5GFP mRNA (1 ng/embryo) and eg5MO (C and C', low dose (l), 10 ng/embryo; D and D', high dose (h), 22 ng/embryo. No embryo shows GFP fluorescence at a high dose of eg5MO. F–O: Analysis of eg5MO effects on neural crest early specification. Dorsal views of Xenopus laevis embryos, anterior side is on the left. Black arrowheads indicate the injected side. F, G: eg5MO-injected embryos show inhibition of foxd3 and snail2 neural crest markers, respectively. H, I: The expression of the neural plate marker sox2 is reduced and the epidermal marker xk81a is expanded on the eg5MO-treated side. Brackets indicate the width of the neural plate (H) and the width of the neural plate plus the neural crest domain (I). J: Embryo labeled by double in situ hybridization for sox2 and xk81a genes showing the reduction of prospective neural crest (black brackets) and neural plate (red brackets) domains in the eg5MO-injected side. Green brackets indicate neural crest and neural plate territories together. This result is identical to H and I. K: The neural plate border specification marker pax3 show reduced expression in the eg5MO-injected side. L, M: Coinjection of eg5MO and Xenopus tropicalis eg5 mRNA rescues foxd3 and sox2 expression, respectively. N: Embryo injected with eg5MO and xr11, the Xenopus bcl2 orthologue. The antiapoptotic factor xr11 did not rescue the expression of foxd3 in the neural crest. O: CoMO-injected embryos show normal expression of foxd3.Download figure to PowerPoint |
