Moriwaki J et al. (2000), Isolation of Xenopus frizzled-10A and frizzled-...

XB-ART-9904

Biochem Biophys Res Commun 2000 Nov 19;2782:377-84. doi: 10.1006/bbrc.2000.3808.

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Isolation of Xenopus frizzled-10A and frizzled-10B genomic clones and their expression in adult tissues and embryos.

Moriwaki J , Kajita E , Kirikoshi H , Koike J , Sagara N , Yasuhiko Y , Saitoh T , Hirai M , Katoh M , Shiokawa K .

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Frizzled genes, encoding WNT receptors, play key roles in cell fate determination. Here, we isolated two Xenopus frizzled genes (Xfz10A and Xfz10B), probably reflecting pseudotetraploidy in Xenopus. Xfz10A (586 amino acids) and Xfz10B (580 amino acids) both encoded by a single exon, consisted of the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal Ser/Thr-X-Val motif. Xfz10A and Xfz10B were 97.0% identical at the amino acid level, and Xfz10B was 100% identical to previously reported Xfz9, yet Xfz10A was 85.3% and 62.4% identical to FZD10 and FZD9, respectively. Xfz10 mRNA appeared as 3.4 kb in adult tissues and embryos. RT-PCR analyses revealed the expression of more Xfz10A mRNA in stomach, kidney, eye, skeletal muscle, and skin, and more Xfz10B mRNA in heart and ovary, but in embryos, two mRNAs were equally expressed from the blastula stage with their peak expression at the late gastrula stage. The main site of Xfz10 mRNA expression was neural fold at the neurula stage and the dorsal region of midbrain, hindbrain, and spinal cord at the tadpole stage. These results suggest that Xfz10 has important roles in neural tissue formation.

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Species referenced: Xenopus
Genes referenced: fzd10 fzd9 mhc2-dmb

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	FIG. 1. Nucleotide sequence alignment of Xfz10A and Xfz10B. Nucleotide sequences of Xfz10A and Xfz10B are aligned, and the nucleotide numbers are indicated at the right. In-frame stop codons in the 59-UTR of Xfz10A and Xfz10B are indicated in green, translation initiation codon in red, and stop codon in blue. Seven regions corresponding to transmembrane domains are indicated in light blue and numbered consecutively. RT-PCR primers and probes for Northern blot analysis and whole-mount in situ hybridization are indicated. Sequences confirmed to occur in the cDNAs reverse-transcribed from Xenopus liver total RNA are indicated in light yellow.
	FIG. 2. (a) Deduced amino acid sequences of Xenopus laevis Xfz10A, Xfz10B, human FZD10, and FZD9. Amino acids are numbered at the right. Transmembrane domains (open box), conserved cysteine residues in the N-terminal extracellular region (cross), potential N-glycosylation sites in the N-terminal extracellular region (dot), conserved cysteine residues in the second and third extracellular region (sharp), and the Ser/Thr-X-Val motif in the C-terminus (dark box) are indicated. (b) Sequences confirmed to be present in the cDNAs reverse-transcribed from Xenopus liver total RNA. The coding region is depicted as an open box and the noncoding region as a solid bar. RT-PCR products (10A-01 and 10A-02 for Xfz10A, and 10B-01 and 10B-02 for Xfz10B) confirmed to be present are indicated by solid bars.
	FIG. 3. Expression of Xfz10 in adult Xenopus tissues. (a) Northern blot analysis on Xfz10. Northern blot filter containing 20 mg of total RNA per lane was hybridized with the XF10S probe, corresponding to a part of the 59-UTR and the coding region of Xfz10A (the nucleotide position 207–872 of Xfz10A) (Fig. 1). A single band of 3.4 kb in size was detected. 18S and 28S rRNAs on gel were stained before transfer to the membrane and indicated to show the loading equivalency. (b) Multiplex RT-PCR analysis. Xfz10A cDNA fragment (684 bp) and b-actin cDNA fragment (291 bp) were detected by multiplex RT-PCR with XF10A-05, XF10A-06, XBAC-01, and XBAC-02 as primers (A). Xfz10B cDNA fragment (473 bp) and b-actin cDNA fragment (291 bp) were detected by multiplex RT-PCR with XF10B-05, XF10B-06, XBAC-01, and XBAC-02 as primers (B).
	FIG. 4. Expression of Xfz10 in developing Xenopus embryos. (a) Northern blot analysis. (b) Multiplex RT-PCR analysis. For explanations, see the legend to Fig. 3.
	FIG. 5. Whole-mount mRNA in situ hybridization analyses on Xfz10. The XF10AIS probe (antisense probe) corresponded to the 39-UTR of Xfz10A, whereas the XF10BIS probe (antisense probe) corresponded to the 39-UTR of Xfz10B. Xenopus embryos were hybridized with the XF10AIS probe (a, b, c, d, e, and g), or the XF10BIS probe (f ). Xenopus embryos were at stage 18 (a, b), stage 22 (c), or stage 35 (d to g). When sense probes were used no signal was obtained (data omitted). nf, neural fold; nt, neural tube; mb, midbrain; hb, hindbrain; tt, tail tip; dmb, dorsal midbrain.