February 1, 2003;
Expression of scFv antibodies in Xenopus embryos to disrupt protein function: implications for large-scale evaluation of the embryonic proteome.
We evaluated the use of single-chain antibody (scFv) expression as a tool to disrupt the function of specific proteins in embryos of the frog, Xenopus laevis. The expression of scFvs that recognize the bone
morphogenetic protein receptor (ALK3
) or the fibroblast
growth factor receptor1 (FGFR1
) as endoplasmic reticulum
-anchored proteins caused distinct developmental defects that were virtually indistinguishable from the defects caused by expression of the dominant negative forms of each receptor. These results demonstrate that scFvs from phage-display libraries can be readily fashioned into effective and specific inhibitors of signaling pathways in developing embryos. In addition, as several effective scFvs against a specific target can be isolated rapidly, this approach represents a valuable new tool for large-scale functional analysis of the embryonic proteome.
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FIG. 2. Intracellular expression of the anti-ALK3 scFv D1 blocks ALK3 function in developing frog embryos as effectively as DN-ALK3. mRNA encoding the anti-ALK3 scFvs D1, E4, F2, DN-ALK3, the anti-FGFR1 scFv A2, the anti-Erb-B2 scFv F5, or the anti-cytochrome B scFv were injected into the two caudal cells (the so-called entralcells) of four-cell stage frog embryos. When the embryos reached the tadpole stage they were scored morphologically for trunk duplications characteristic of disrupting ALK3 receptor function. A: Expression of the anti-ALK3 scFv-D1 caused embryos to develop with trunk duplications. The data were compiled from three separate experiments and the number of injected embryos is indicated. B: Secondary trunks resulting from anti-ALK3 scFv-D1 expression are similar if not identical to those observed in embryos expressing the DN-ALK3. The trunk-duplications are indicated by the arrow. In addition to trunk duplications, a small number of the embryos expressing anti-ALK3 scFv-D1 or DN-ALK3 developed with enlarged heads, a phenotype also caused by disrupting ALK3 function (data not shown). C: Coexpression of wild-type ALK3 with anti-ALK3 scFv-D1 rescues the trunk duplications caused by anti-ALK3 scFv-D1 alone. D: Secondary trunks caused by anti-ALK3 scFv-D1 or the DN-ALK3 contained ectopic muscle (detected using whole-mount immunocytochemistry and the muscle-specific antibody 12/101) (Kintner and Brockes, 1984). The large arrowhead indicates the ectopic muscle. E: Anti-ALK3 scFv-D1 or the DN-ALK3 both directed the formation of secondary trunks that contained ectopic neural tubes (detected using whole-mount immunocytochemistry and the NCAM-specific 6F11 antibody). The large arrowhead indicates the ectopic neural tube that forms in the secondary trunks due to inhibition of ALK3 function. F: Expression of anti-ALK3 scFv-D1 or the DN-ALK3 caused ectopic expression of the myogenic factor myf-5. Stage 10.5 embryos expressing either anti-ALK3 scFv-D1 or the DN-ALK3 were analyzed for myf-5 expression using whole-mount in situ hybridization. i: Uninjected controls. ii: Embryos expressing DN-ALK3. iii: Embryos expressing Anti-ALK3 scFv-D1. iv: Embryos expressing anti-FGFR1 scFv-A2. Anti-ALK3 scFv-D1 and the DN-ALK3 caused ectopic myf-5 expression (marked by the arrow), while anti-FGFR1 scFv-A2 did not. The asterisk marks the early blastopore lip and Spemann organizer. Reproduced with permission of the Publisher, John Wiley & Sons.
FIG. 3. Intracellular expression of Anti-FGFR1 scFvs blocks FGFR1 function in frog embryos to cause posterior defects. mRNA encoding either the anti-FGFR1 scFvs A2, C3, A7, A23, DNFGFR1, the noninhibitory FGFR1 HAV anti-ALK3 scFv-D1, anti-Erb-B2 scFv-F5, or the anti-cytochrome B scFv were injected into two anterior cells (the so-called dorsal cells) of four-cell stage frog embryos. A: When embryos reached the tadpole stage they were scored morphologically for defects in posterior development characteristic of disrupting FGFR1 function. The data in this graph were compiled from three separate experiments and the number of injected embryos is indicated. B: Expression of the anti-FGFR1 scFv-A2 caused embryos to develop with significantly reduced trunks and tails identical to the phenotypes caused by DN-FGFR1. C: Coexpression of wild-type FGFR1 with anti-FGFR1 scFv-A2 rescues the posterior defects caused by anti-FGFR1 scFv-A2 alone. D: Embryos expressing anti-FGFR1 scFv-A2 or the DN-FGFR1 developed with reductions in embryonic muscle (indicated with the arrow) compared to the normal amounts of muscle present in controls. Muscle was detected using whole-mount immunocyto- chemistry and the muscle-specific antibody 12/101 (Kintner and Brockes, 1984). E: Expression of anti-FGFR1 scFv-A2 or the DN-FGFR1 disrupted xbra expression. The CD1 cells of 16-cell embryos were injected with mRNAs encoding anti-FGFR1 scFv-A2, DN-FGFR1, or anti-ALK3 scFv-D1. At Stage 10 the embryos were analyzed for xbra expression using whole-mount in situ hybridization. i: Uninjected controls. ii: Embryos expressing DN-FGFR1. iii: Embryos expressing Anti-FGFR1 scFv-A2. iv: Embryos expressing Anti-ALK3 scFv-D1. Anti-FGFR1 scFv-A2 and the DN-FGFR1 caused disrupted xbra expression (the region marked by the arrows) while anti-ALK3 scFv-D1 did not. Reproduced with permission of the Publisher, John Wiley & Sons.