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The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.
Figure 1 (a) Schematic diagram showing the activator and effector constructs used in this study. Activator constructs contain the yeast transcriptional activator Gal4 under the control of the ubiquitously expressed CMV promoter or the tissue-specific Pax-6 promoter. Effector constructs contain five repeat concatemers of the consensus binding site for Gal4 (UAS), linked to the hsp70 minimal promoter and the gene of interest, green fluorescent protein (GFP) or Xvent-2. (b) F1 progeny from effector lines were tested for the transmission of the transgene and transactivation by Gal4. Embryos were injected into one cell at the two-cell stage with 100 pg of Gal4 mRNA. Two stage 19 sibling F1 embryos from GUASGFP line 2 injected with Gal4 mRNA (arrow). GFP fluorescence is observed in the rightembryo on its right side, and that embryo is deemed to have inherited the UASGFP transgene, which has been transactivated by Gal4. The embryo on the left shows no GFP fluorescence and is assumed not to have inherited the transgene.
Figure 2 GFP is transactivated by Gal4 in temporally and spatially restricted manners on cross-fertilization of founder activator and effector lines. (a–c) Pax-6Gal4 line 2 × UASGFP line 2. (a) GFP fluorescence is restricted to the eye field in a stage 25 F1 embryo. GFP RNA (b) or Gal4 RNA (c) as detected by in situ hybridization is expressed in the anterior neural plate and presumptive hindbrain and spinal cord in stage 19 F1 embryos. (d) Stage 19 F0 embryo transgenic for Pax-6GFP showing the expression of GFP RNA driven by the Pax-6 promoter. (e–g) CMVGal4 line 1× UASGFP line 6. (e) Stage 25 F1 embryo expressing GFP ubiquitously. (f) Sibling stage 25, F1 embryo not expressing GFP. (g) Stage 40, F1 tadpole expressing GFP ubiquitously.
Figure 3 Microcephalic and ventralized phenotypes result from transactivation of Xvent-2 by Gal4 in three independent crosses. (a) CMVGal4 line 1 × UASXvent-2 line 5. (b) Pax-6Gal4 line 1 × UASXvent-2 line 4. (c) Pax-6Gal4 line 1 × UASXvent-2 line 6. (a–c Left) F1 stage 30 embryos showing the characteristic microcephalic or ventralized phenotype (Lower) when compared to an apparently normal sibling (Upper). (Right) PCR genotyping of individual embryos with the indicated phenotype. PCR primer combinations are indicated. −ve, PCR control without DNA. (d and e) Half of F1 progeny from Pax-6Gal4 line 1 × wild type express Gal4 as revealed by in situ hybridization to Gal4 RNA. (d) At stage 10, Gal4 is expressed throughout the marginal zone of the embryo. (e) At stage 19, Gal4 expression is restricted to the eye field in the anterior neural plate.
Figure 4 Analysis of the microcephalic phenotype at stage 3. F1 progeny from CMVGal4 line 1 × UASXvent-2 line 5. (a–f and i) In situ hybridization to the indicated marker genes. Representative sibling F1 embryos that are phenotypically normal (Upper) or microcephalic (Lower) are shown for each marker. (g and j) Transverse sections through the dorsal region of embryos stained for Pax-3 and cardiac actin, respectively, with the section of the microcephalic embryo on the right. (h) MZ15 monoclonal antibody staining of notochordal tissue for normal (Upper) and microcephalic (Lower) F1 embryos.
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