Semba K et al. (1996), cDNA cloning and its pronephros-specific expres...

XB-ART-17579

Gene 1996 Oct 10;1751-2:167-72. doi: 10.1016/0378-1119(96)00143-6.

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cDNA cloning and its pronephros-specific expression of the Wilms' tumor suppressor gene, WT1, from Xenopus laevis.

Semba K , Saito-Ueno R , Takayama G , Kondo M .

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The Wilms' tumor suppressor gene, WT1, plays a crucial role during early urogenital development in mammals. To elucidate the function of WT1 in other vertebrates, we isolated the Xenopus WT1 homolog (XeWT1) from a testis cDNA library. Comparison of the XeWT1 protein with other WT1 proteins revealed that the zinc finger domain of XeWT1 is identical to that of the human WT1 except for the first two amino acid residues. An alternative splice II site, located between the third and the fourth zinc finger is also conserved. In the transcriptional regulatory region, however, three domains, a glycine stretch, a proline stretch and one alternative splice site which are present in the mammalian WT1, are not conserved in amphibians. The XeWT1 gene is expressed in testis and kidney, and whole mount in situ hybridization revealed that the onset of the WT1 gene expression coincides with the early stages of pronephros development in Xenopus. These findings implicate the involvement of WT1 protein during urogenital development in amphibians as well as in mammals.

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Species referenced: Xenopus laevis
Genes referenced: kit scn1a wt1

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	Fig. 1. Primary structure of Xenopus WT1. (A) Nucleotide and predicted aa sequence of Xenopus WT1. Nucleotide numbers are indicated on the sequence, and aa are numbered at the right. An in-frame stop codon in the 5'-non-coding region is underlined. Oligonucleotides NI: 5'-CGCGGATCCGTACGGTCGGCATC- (AGCT)GA(AG)AC-3', N2: 5'-CGCGGATCCAGATATTTTAAGCT- (AGCT)TC(AGTC)CA-Y, Ch 5'-CGCCTCGAGCTGATGCAT- (AG)TT(AG)TG(AG)TG-3' and C2: 5'-CGCCTCGAGGGTCATGTT( CT)CT(CT)TG(AG)TG-3' were used for RT-PCR amplification of the Xenopus WT1 sequence. PCR reactions were performed using cDNA prepared from 0.05pg of poly(A)+RNA with the following conditions: one cycle of 95°C for 5 min, 45°C for 2 min, 72°C for 3 min and 95°C for 2 rain, 35 cycles of 45°C for 1 min, 72°C for 1.5 rain and 95°C for 0.5 min, and one cycle of 45°C for 2 rain, 72°C for 10 min and 5°C for 5 min. One tenth of the PCR reaction mixture was used for the second round of PCR under the same conditions as described above. The oligonucleotide, Y-CA (CY) AC (AGCT) GG (AGCT) GA (AG) AA (AG) CC (AGCT)-3' was used as a probe for Southern blot hybridization. Hybridization and washing conditions were as described (KOster et al., 1988). The positive fragment was cloned and sequenced to confirm the presence of a WTl-like sequence. Using this DNA as a probe, Xenopus WT1 cDNA No. 65 was isolated from X. laevis testis cDNA library (constructed with TimeSaver cDNA synthesis kit, Pharmacia Biotech and Lambda ZAP II/EcoRI/Gigapack Cloning Kits, Stratagene). Sequencing of both strands of the cDNA clone was carried out using a deletion kit for kilo-sequencing (Takara Biochemicals) and an automated DNA sequencer (Applied Biosystems). Sequence was analyzed by the Genetyx-Mac program (Software Development Co., Ltd.). The nt sequence reported in this paper has been submitted to the DDBJ, EMBL and GenBank nt sequence databases with accession No. D82051. (B) +KTS and -KTS variants. Comparison of Xenopus WT1 cDNA No. 65 (-KTS form) and cDNA No. 37 (+ KTS form). Asterisks indicate identical aa and dashes indicate absence of a residue at each position.
	Fig. 2. Amino acid alignment of vertebrate WT1 proteins. Predicted aa sequences of human (Gessler et al., 1990), mouse (Buckler et al., 1991), rat (Sharma et al., 1992), chick (Kent et al., 1995), alligator (Kent et al, 1995) and zebrafish (Kent et al., 1995) WT1 are compared. Identical aa are indicated by dots and absent residues by dashes. Aa residues labeled with asterisks are described in the text.
	Fig. 3. Expression of the Xenopus WT1 gene in adult frog tissues. Poly(A) + RNA was isolated from organs of adult frogs using ISOGEN (Nippongene) and Oligotex-dT30 (Takara Biomedicals). 3pg of poly(A)+RNAs were electrophoresed in 1% agarose gel containing 2.2 M formaldehyde and then subjected to blot hybridization as described. A 1.5-kb NotI fragment of clone No. 65 was labeled with 3/p using Megaprime DNA labeling systems (Amersham). Arrow indicates WT1 transcript. Bars indicate mobility of molecular weight markers, 9.49, 7.46, 4.40, 2.37, 1.35 kb (RNA Ladder, Gibco BRL). The autoradiogram was exposed for 5 days.
	Fig. 4. Spatial and temporal distribution of Xenopus WT1 transcript during embryogenesis. (A) Results from a tailbud embryo, stages 30, 26, 25 and 23 (from top to bottom): pronephros anlage was stained in purple. (B) Tailbud, stage 30: both sides of pronephros were stained. (C) Sense probe did not detect any specific regions. WT1 sense and antisense probes were generated by in vitro transcription in the presence of rUTP-digoxigenin (Boehringer Mannheim) using T3 and T7 RNA polymerases (Promega). The length of both probes was 1.5 kb. Hybridization was performed according to the previously described method (Harland, 1991), with the following modifications. Wild type embryos were bleached with 6% hydrogen peroxide after rehydration and then hybridized at 60°C in a buffer containing 50% formamide, 5 × SSC, 1 mg/ml torula RNA, 100 mg/ml heparin, 1 xDenhardt's, 0.1% Tween 20, 0.1% CHAPS and 10mM EDTA. After hybridization, embryos were washed at 60°C with 0.2 × SSC, at 60°C with 0.2 × SSC, and then at RT with maleic acid buffer (100 mM maleic acid, 150 mM NaC1, pH 7.5). Alkaline phosphate-coupled anti- DIG antibody and BM Purple (Boehringer Mannheim) were used for detection of the probes. Stained embryos were postfixed in MEMPFA and cleared with benzyl benzoate/benzyl alcohol (2:1) and photographed.