Asahi H et al. (2002), Expression of FTZ-F1alpha in transgenic Xenopus...

XB-ART-6004

Dev Growth Differ 2002 Dec 01;446:509-16. doi: 10.1046/j.1440-169x.2002.00663.x.

Show Gene links Show Anatomy links

Expression of FTZ-F1alpha in transgenic Xenopus embryos and oocytes.

Asahi H , Takase M , Yuge M , Matsui K , Mori M , Fujita T , Nakamura M .

???displayArticle.abstract???
Fushi tarazu transcription factor-1 (FTZ-F1) was originally found as a regulator of fushi tarazu gene expression in Drosophila. The frog homologue (FTZ-F1alpha) and the 3.5 kb 5'-flanking region of the FTZ-F1alpha gene have been cloned, and it has been shown by reverse transcription-polymerase chain reaction that FTZ-F1alpha expression begins in embryos at stage 11 and becomes stronger after that. By in situ hybridization analysis, the FTZ-F1alpha mRNA was also found in immature frog oocytes. In this study, immunohistology revealed that the product of FTZ-F1alpha was localized in the cytoplasm of the immature oocyte. To analyze the promoter activity of the Rana rugosa FTZ-F1alpha gene, transgenic Xenopus were produced carrying the fusion construct, consisting of truncated 5'-flanking regions (3.0, 1.8 and 0.3 kb) of the FTZ-F1alpha gene and the green fluorescent protein (GFP) open reading frame. The 0.3 kb 5'-flanking region could drive GFP expression in Xenopus embryos at stage 20 and in immature oocytes in the ovary 2 months after metamorphosis. Gel mobility shift assay was used to test whether proteins in extracts from Xenopus embryos and ovaries bound to the 0.3 kb DNA. The extract from embryos at stage 11 formed one retarded band. The extract from ovaries formed a different retarded band. The results, taken together, indicate that production of transgenic Xenopus is very useful for the analysis of the promoter activity of genes in amphibians. The results also suggest that at least two proteins (one in the embryo and the other in the ovary of 2-month-old postmetamorphosing Xenopus) bind the 0.3 kb 5'-flanking region of the FTZ-F1alpha gene. These proteins may be involved in the regulation of FTZ-F1alpha gene expression in amphibians.

???displayArticle.pubmedLink??? 12492509
???displayArticle.link??? Dev Growth Differ

???displayArticle.antibodies??? Nr5a2 Ab1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Proximal 5-flanking region of the FTZ-F1 promoter. Schematic diagram of the linearized pFTZ-F1-EGFP-1 plasmid. The 3.45 kb 5-flanking region of the Rana rugosa FTZ-F1 gene is shown as a solid bar and the green fluorescent protein (GFP) coding region as an open box. The GFP non-coding region is shown as a thin bar. The number on the restriction enzyme indicates nucleotide positions of the 5-upstream region of the FTZ-F1 gene.
	Fig. 2. DNA sequence of the 0.3 kb 5-flanking region of the FTZ-F1 gene. Putative transcription factor-binding sites are underlined. Exon I of FTZ-F1 is boxed. Nucleotides in a genomic clone are numbered from the first nucleotide of the transcript from the FTZ-F1 gene which is +1. The deduced amino acid sequence is shown below the nucleotide sequence.
	Fig. 3. Expression of pFTZ-F1-EGFP-1 plasmids in transgenic Xenopus embryos. The pFTZ-F1(–3030/+6)-EGFP-1 and pFTZF1(– 306/+6)-EGFP-1 plasmids were injected into Xenopus eggs. Green fluorescent protein (GFP)-specific fluorescence was observed under fluorescent illumination in the whole embryo at stage 20 (a) and in the dorsal body of the embryo at stage 31 (c) after injection of the pFTZ-F1(–3030/+6)-EGFP-1 plasmid, and in the whole embryo at stage 20 (b) after injection of the pFTZ-F1(–306/+6)-EGFP-1 plasmid.
	Fig. 4. Expression of FTZ-F1 and green fluorescent protein (GFP) in oocytes of frog ovaries. (a,b) In situ hybridization analysis. The FTZ-F1 mRNA in oocytes of the frog Rana rugosa was hybridized with digoxigenin (DIG)-labeled antisense (a), or sense (b) RNA probe. Bar, 50 μm. (c,d) Immunohistological analysis of oocytes expressing FTZ-F1 protein in the ovary of the frog R. rugosa. Sections of the R. rugosa ovary were stained with anti-FTZ-F1 IgG (c), or non-immune rabbit serum (d) followed by an incubation with fluorescein isothiocyanate (FITC)-labeled antirabbit IgG. Bar, 50 μm. (e–j) Immunohistological analysis of oocytes expressing green flourescent protein (GFP) in the ovary of transgenic Xenopus. Localization of GFP antibody binding to the ovary of transgenic (e,g–j), or nontransgenic (f) Xenopus 2 months after metamorphosis was determined. Transgenic Xenopus were produced by injection of the 3.0 (g,h) and 0.3 (i,j) kb 5-flanking regions of the FTZ-F1 gene. Sections of Xenopus ovaries were photographed under fluorescent illumination (e,f), and stained with the GFP antibody (g,i), or non-immune rabbit serum (h,j), followed by an incubation with avidin–biotin complex to detect immunoreactive products. Bar, 50 μm. (k,l) Immunohistological analysis of transgenic Xenopus liver. GFP antibody binding to liver of transgenic (k), and non-transgenic (l) Xenopus 2 months after metamorphosis were determined. The liver was taken from the same non-transgenic or transgenic Xenopus as used for the analysis of (e–h), and stained with the GFP antibody, followed by an incubation with avidin–biotin complex to visualize immunoreactive products. Bar, 50 μm.
	Fig. 5. A gel shift assay with extracts from Xenopus embryos at stage 11 and ovaries 2 months after metamorphosis. This assay was performed with the extract from embryos at stage 11 (right panel) and ovaries 2 months after metamorphosis (left panel). The figures were produced using Adobe Photoshop. Ovary: 1, control; 2, extract; 3, extract + 10 competitors. Embryo: 1, control; 2, extract; 3, extract + 10 competitors; 4, extract + 20 competitors.