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Abstract
The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anteriorhead development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.
Fig 1. Induction of ubiquitous Xwnt-8 expression in transgenic Xenopus embryos. (a) An illustration of the DNA construct containing Xwnt-8 cDNA and GFP cDNA under the transcriptional control of the Xhsp70 promoter, which was used to generate transgenic embryos. Embryos that expressed GFP or were developing abnormally were removed from the experiment before induction (see Table 1). Normally developing embryos with no GFP expression were heat treated during (b–e) early (stage 13) and (f–i) late (stage 20) neurulation to induce transient gene expression. These embryos were fixed at stages (b–e) 29 and (f–i) 36. The same embryos were photographed under (b,d,f,h) visible light and (c,e,g,i) UV light. (d,e,h,i) Transgenic embryos with induced Xwnt-8 expression were (e,i) recognised by detecting GFP under UV light (note bluish green GFP colour, specifically in eye, fin and axial tissue) and separated from (b,c,f,g) non-transgenic embryos (note (c,g) only yellowish autofluorescence in yolky tissue). Note that development of the cement gland (the darkly pigmented organ at most anterior position) and anterior development were inhibited by transient ubiquitous Xwnt-8 expression in early neurula ((d); compare with (b)). Transient ubiquitous expression of Xwnt-8 at the end of neurulation did not visibly effect anterior development ((f); compare with (h)). Embryos are oriented with anterior to the left.
Fig 2. Induction of GFP expression in transgenic Xenopus embryos. Transgenic embryos were generated with a DNA construct containing Xwnt-8 cDNA and GFP cDNA under the transcriptional control of the Xhsp70 promoter (see Figure 1a). Normally developing embryos with no GFP expression were heat treated during early neurulation (stage 13) to induce transient gene expression. The embryos were fixed after neurulation (stage 21) and divided into GFP-expressing and non-expressing groups as detected under UV light. After sectioning, GFP expression in internal tissues was confirmed by detection with an anti-GFP antibody (see Materials and methods). Note (a) GFP protein expression in a section from a GFP-expressing embryo and (b) compare with the absence of GFP protein expression in a non-expressing embryo. Sections are oriented with dorsal to the top.
Fig 3. Induction of ubiquitous Xfz7 expression in transgenic Xenopus embryos. A DNA construct containing Xfz7 cDNA and GFP cDNA under the transcriptional control of the Xhsp70 promoter was used to generate transgenic Xenopus embryos. Embryos that expressed GFP or were developing abnormally were removed from the experiment before induction. Normally developing embryos with no GFP expression were heat treated during (a) early neurulation (stage 13), (b) late neurulation (stage 20) and (c) shortly before the tailbud stage (stage 25) to induce gene expression. These embryos were fixed at approximately stage (a,b) 34 or (c) 36. Gained GFP expression (data not shown) distinguishes transgenic embryos with induced Xfz7 expression (Exp) from non-transgenic embryos (Con). Ubiquitous Xfz7 expression affected the length of the dorsal axis, but different parts of the dorsal axis were affected by Xfz7 over-expression at different stages of development. (a) During early neurulation, transient Xfz7 over-expression affected the development of dorsal axial structures at the level of the hindbrain (arrowhead). In the illustrated experiment, 53% of a total of 17 embryos with GFP expression had a strong anterior trunk phenotype, whereas 92% of 109 embryos without any detectable GFP expression were normal. (b) During late neurulation, however, transient Xfz7 over-expression affected dorsal axial structures at the level of the posterior trunk (arrowhead). In the illustrated experiment, 33% of a total of 18 embryos with GFP expression had a strong posterior trunk phenotype, whereas 95% of 126 embryos without any detectable GFP expression were normal. (c) Transient Xfz7 over-expression at a stage just before the tailbud formation affects the developing tail (arrowhead). In the illustrated experiment, 71% of a total of 14 embryos with GFP expression had a strong tail phenotype, whereas 84% of 72 embryos without any detectable GFP expression were normal. Embryos are oriented with anterior to the left.
