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FZD4S, a Splicing Variant of Frizzled-4, Encodes a Soluble-Type Positive Regulator of the WNT Signaling Pathway
Biochemical and Biophysical Research Communications 282, 750 –756 (2001) doi:10.1006/bbrc.2001.4634, available online at http://www.idealibrary.com on
FZD4S, a Splicing Variant of Frizzled-4, Encodes a Soluble-Type Positive Regulator of the WNT Signaling Pathway Norihiko Sagara,* ,† Hiroyuki Kirikoshi,* Harumi Terasaki,‡ Yukuto Yasuhiko,‡ Gotaro Toda,† Koichiro Shiokawa,‡ and Masaru Katoh* ,1 *Genetics and Cell Biology Section, Genetics Division, National Cancer Center Research Institute, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; †Division of Gastroenterology and Hepatology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo 105-0003, Japan; and ‡Department of Developmental Biology, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
Received March 6, 2001
Frizzled-1 (FZD1)–FZD10 are seven-transmembranetype WNT receptors, and SFRP1–SFRP5 are solubletype WNT antagonists. These molecules are encoded by mutually distinct genes. We have previously isolated and characterized the 7.7-kb FZD4 mRNA, encoding a seven-transmembrane receptor with the extracellular cysteine-rich domain (CRD). Here, we have cloned and characterized FZD4S, a splicing variant of the FZD4 gene. FZD4S, corresponding to the 10.0-kb FZD4 mRNA, consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD, and was expressed in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage did not induce axis duplication by itself, but augmented the axis duplication potential of coinjected Xwnt-8 mRNA. These results indicate that the FZD4 gene gives rise to solubletype FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing, and strongly suggest that FZD4S plays a role as a positive regulator of the WNT signaling pathway. © 2001 Academic Press Key Words: Frizzled; WNT receptor; alternative splicing; Xenopus laevis.
The WNT signaling pathway plays a critical role in embryonic development and tumor development (1, 2). The WNT signal is transduced to the -catenin–TCF pathway (3), the Jun-N-terminal kinase pathway (4),
or the Ca 2⫹-releasing pathway (5) through seventransmembrane-receptors Frizzled-1 (FZD1)–FZD10 (6 –2). FZDs are seven-transmembrane-receptors with the frizzled-type cysteine-rich domain (CRD). CRD is the WNT-binding domain, which also exists in secretedtype Frizzled-related proteins SFRP1–SFRP5 (13–15). FZDs function as WNT receptors, whereas SFRPs function as WNT antagonists. Although FZDs and SFRPs are mutually homologous in CRD, these two groups of proteins are encoded by distinct genes. Thus, SFRP is not a splicing variant of FZD. We have previously reported molecular cloning of the FZD4 gene, encoding a seven-transmembrane receptor (11). The open reading frame (ORF) of the 7.7-kb FZD4 mRNA is split within the CRD by intron 1. Here, we have cloned and characterized the FZD4S cDNA, which consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD due to the appearance of a new stop codon within the retained intron 1. FZD4S was expressed as the 10.0-kb FZD4 mRNA in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage augmented the axis duplication potential of the coinjected Xenopus wnt-8 (Xwnt-8) mRNA. This is the first report on cloning and characterization of a soluble-type isoform produced by alternative splicing of a member of the FZD gene family. MATERIALS AND METHODS
The nucleotide sequence data of human FZD4S will appear in the DDBJ/EMBL/GenBank Data Bases with the Accession No. AB054881. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-3-3541-2685. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
cDNA library screening. Human fetal lung cDNA library in gt10 (Clontech Laboratories) was screened with the FZGC4 probe (nucleotide position 3342– 4114 of the FZD4 gene) as described previously (16). cDNA inserts were excised with EcoRI digestion, and subcloned
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FIG. 1. Structure of FZD4S cDNA and amino-acid sequence of FZD4S. (A) Schematic presentation of the FZD4S cDNA. The coding region and UTRs are depicted as an open box and bold bars, respectively. Four overlapping FZD4S cDNAs (HF4-5, HF4-6, HF4-7, and HF4-9) are shown by solid bars. (B) Amino-acid sequence of FZD4S. Amino acids are numbered on the right. The signal peptide (bold over-line), CRD (open box), and the unique sequence of FZD4S isoform (over-line) are shown. The conserved amino acids in the N-terminal part of CRD (asterisk), and N-glycosylation site (sharp) are also shown. FZD4S is a soluble-type polypeptide with the N-terminal part of CRD. (C) Kyte and Doolittle hydrophobicity analysis on FZD4S. SP: signal peptide.
into the pUC118 vector (Takara) for nucleotide sequence analyses with ABI310 Sequencer (PE Applied Biosystems). cDNA-PCR. cDNA was synthesized from 40 ng of poly(A) ⫹ RNAs of human fetal kidney (Clontech Laboratories) with the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech). The subsequent PCR with P4-101 and P4-102 primers were performed by using the KOD Plus DNA polymerase (Toyobo) as described previously (16). Nucleotide sequences of PCR primers are as follows: PF4-101 (sense), 5⬘-ATCCCACACAGTCGCGCG-3⬘; PF4-102 (antisense), 5⬘CCAATGGGGATGTTGATCTTC-3⬘. Northern blot analyses. MTN Northern blot filters (Clontech Laboratories) were hybridized with a [␣- 32P] dCTP-labeled probe at 68°C for 1 h in QuikHyb solution (Stratagene). After washing, filters were exposed to the Imaging plate (Fuji) for the image analysis in the Storm system (Molecular Dynamics) as described previously (16). The HF4A probe (nucleotide position 79 – 470 of the FZD4 gene) was derived from exon 1, the HF4B probe (nucleotide position 821–1202 of the FZD4 gene) was derived from intron 1, and the HF4S probe (nucleotide position 6788 –7185 of the FZD4 gene) was derived from exon 2. In vitro synthesis of the FZD4S mRNA. PCR with P4-103 and P4-104 primers were performed to amplify the ORF of the FZD4S from the HF4-9 cDNA. Nucleotide sequences of PCR primers are as follows: PF4-103 (sense), 5⬘-GTGGATCCTGGGGGTGTCTGCCAGAG-3⬘; PF4-104 (antisense), 5⬘-GTGAATTCTTTCCCAGGCCAGATGAC-3⬘. BamHI linker and XhoI linker were added to the 5⬘-end of PF4-103 and PF4-104 primers, respectively. The FZD4S-ORF cDNA fragment (nucleotide position 242–794 of the FZD4 gene) was digested with BamHI and XhoI, and subcloned into the pCS2⫹ vector kindly provided by Dr. Dave Turner (Fred Hutchinson Cancer Research Center). After nucleotide sequence analysis, a FZD4SORF/pCS2⫹ plasmid without misincorporation during PCR was selected for linearization with PvuII digestion. By using the linearized
FZD4S-ORF/pCS2⫹ plasmid as a template, the capped FZD4S mRNA was synthesized with the MEGAscript SP6 Kit (Ambion) in the presence of RNA cap structure analog (New England Biolabs). Xenopus axis duplication assay. Twelve hours after injection of gonatropin (Teikoku Zouki) into female Xenopus laevis, eggs were manually ovulated and artificially fertilized (17). Fertilized eggs were dejellied by treatment with 4.5% cysteine hydrochloride. After washing, embryos were transferred to culture dishes containing the 0.1⫻ Steinberg’s solution. Synthetic RNA was injected into the ventral marginal zone or the dorsal marginal zone of Xenopus embryos at the 4-cell stage as described previously (17). Phenotypes of Xenopus embryos were examined 24 h after mRNA injection.
RESULTS Isolation of FZD4S cDNAs We have previously isolated eight positive clones from a human fetal lung cDNA library with the FZGC4 probe (11). Two FZD4 cDNA clones, corresponding to the 7.7-kb FZD4 mRNA, consisted of exon 1 and exon 2 of the FZD4 gene (11). However, nucleotide sequence analyses of three cDNA clones, such as HF4-5, HF4-6, and HF4-7, revealed that these clones consisted of intron 1 and exon 2 of the FZD4 gene. We then performed cDNA-PCR with P4-101 and P4-102 primers to isolate the variant FZD4 cDNA which contains intron 1. We isolated here a 2846-bp cDNA fragment, designated HF4-9, from poly(A) ⫹ RNAs of fetal kidney by cDNAPCR with reverse transcriptase. Since such a cDNA
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FIG. 2. Expression profile of FZD4S. (A–D) Northern blot analyses. Multiple tissue Northern filters for adult tissues (A), fetal tissues, (B, D), and cancer cell lines (C) (Clontech Laboratories) containing 2 g of poly(A) ⫹ RNA on each lane were hybridized with the [␣- 32P]dCTP-labeled HF4B probe (A–C), or HF4A probe (D). The HF4B probe hybridized to a single band of 10.0 kb, whereas the HF4A probe hybridized to a major band of 7.7 kb and a minor band of 10.0 kb. The 10.0-kb FZD4 mRNA (arrow) was expressed moderately in fetal kidney, but only faintly in adult heart and fetal lung. (E) RNA dot blot analysis. RNA Master Blot filter (Clontech Laboratories) was hybridized with the HF4B probe (left). Sources of poly(A) ⫹ RNA on each spot is shown (right). Hybridization signal to FZD4S, corresponding to the 10.0-kb FZD4 mRNA, was relatively strong in adult heart (c1) and lung (f 2), and in fetal kidney (g3) and lung (g7).
was not obtained by cDNA-PCR without reverse transcriptase (data not shown), we assumed that theHF4-9 cDNA was derived from a variant FZD4 mRNA rather than from a contaminated genome DNA. Sequence analysis revealed that the HF4-9 cDNA consisted of exon 1, intron 1 and exon 2 of the FZD4 gene.
The total nucleotide sequence of the FZD4S cDNA was determined by combining the nucleotide sequences of the following cDNAs: HF4-5 (nucleotide position 506 –3355), HF4-6 (nucleotide position 1669 – 4242), HF4-7 (nucleotide position 2070 – 4474), and HF4-9 (nucleotide position 1–2847) (FIG. 1A). The FZD4S
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FIG. 3. Alternative splicing of the FZD4 gene. (Top) Structure of the FZD4 gene. Two exons are shown by boxes, and one intron is shown by a bold bar. (Middle) The FZD4 probes used for Northern blot analyses are shown by bold bars. The size(s) of the FZD4 mRNA detected by each probe are also shown. (Bottom) The structure of the 10.0-kb FZD4 mRNA (soluble-type isoform) and the 7.7-kb FZD4 mRNA (seven-transmembrane-type isoform). The coding region is indicated by the closed box, and UTRs by the open boxes. The 10.0-kb FZD4 mRNA consists of exon 1, intron 1, and exon 2, whereas the 7.7-kb FZD4 mRNA consists of exon 1, and exon 2. Thus, the FZD4 gene gives rise to soluble-type FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing of the retained intron type.
cDNA was found to consist of the 164-bp 5⬘-UTR, the 378-bp ORF, and the 3932-bp 3⬘-UTR (Fig. 1A). Amino-Acid Sequence of FZD4S FZD4S was predicted to encode a polypeptide of 125 amino-acid residues (Fig. 1B). The N-terminal 98 amino-acid residues of FZD4S were identical to those of the seven-transmembrane-receptor FZD4 (11), but the following 27 amino-acid residues were unique. The N-terminal hydrophobic region, corresponding to the signal peptide, was identified in FZD4S by the Kyte and Doolittle hydrophobicity analysis (Fig. 1C). FZD4S contained the N-terminal part of CRD of FZD4, but contained neither the latter half of CRD nor seven transmembrane domains of FZD4 (Fig. 1B). These results indicate that FZD4 is a soluble-type polypeptide with only the N-terminal part of CRD.
bridized only to 10.0-kb RNA. The 10.0-kb FZD4 mRNA was expressed moderately in fetal kidney, and only faintly in adult heart and fetal lung (Figs. 2A and 2B). The 10.0-kb FZD4 mRNA was almost undetectable in human cancer cell lines HL-60, HeLa S3, K-562, MOLT-4, Raji, SW480, A539, and G361 (Fig. 2C). Expression profile of the 10.0-kb FZD4 mRNA among various human tissues was investigated further by using RNA Master Blot filter (Clontech Laboratories). FZD4S, corresponding to the 10.0-kb FZD4 mRNA, was expressed in adult heart and lung, and in fetal kidney and lung (Fig. 2E). Hybridization signals of FZD4S in adult heart, adult lung, and fetal lung were more clearly detected by RNA master blot analysis than by Northern blot analysis, probably due to higher sensitivity of RNA master blot analysis. These results indicated that the 10.0-kb FZD4 mRNA was expressed in adult heart and lung, and in fetal kidney and lung.
Expression Profile of FZD4S The HF4B probe, corresponding to intron 1 of the FZD4 gene, was synthesized by PCR using the HF4-9 cDNA as a template. We expected that this probe would hybridize to the 10.0-kb FZD mRNA with intron 1, but not to the 7.7-kb FZD4 mRNA without intron 1. In fact, northern blot analysis showed that the HF4B probe hy-
FZD4S Is a Splicing Variant of the FZD4 Gene The HF4A probe, the HF4B probe, and the HF4S probe are derived from exon 1, intron 1, and exon 2 of the FZD4 gene, respectively. The HF4A probe hybridized to a major band of 7.7 kb and a minor band of 10.0 kb (Fig. 2D), whereas the HF4B probe hybridized to a
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FIG. 4. Functional analyses of FZD4S in the Xenopus axis duplication assay. Synthetic mRNAs of FZD4S (1 ng), Xwnt-8 (0.05 pg), or -globin (1 ng) were injected into the ventral marginal zone of Xenopus embryos at the 4-cell stage, and the phenotypes were evaluated 24 h after injection. (A) Representative Xenopus embryos injected with -globin alone (upper left), FZD4S alone (upper right), -globin ⫹ Xwnt-8 (lower left), and FZD4S ⫹ Xwnt-8 (lower right) are shown. (B) Incidence of complete axis duplication (closed bar) and partial axis duplication (open bar) in each injection group are shown. Ventral injection of FZD4S did not induce axis duplication by itself (0%). Ventral injection of low-dose Xwnt-8 induced partial axis duplication in 56% of Xenopus embryos, but complete axis duplication only in 8%. Coinjection of FZD4S and low-dose Xwnt-8 induced complete axis duplication with significantly higher frequency (61%) (P ⬍ 0.05). These results indicate that FZD4S augmented the potential of low-dose Xwnt-8 to induce complete axis duplication.
single band of 10.0 kb (Fig. 2B). As shown previously, the HF4S probe hybridized to a major band of 7.7 kb and a minor band of 10.0 kb (11). These results indicate that the 10.0-kb FZD4 mRNA consists of exon 1, intron
1, and exon 2, while the 7.7-kb FZD4 mRNA consists of exon 1, and exon 2 (Fig. 3). Therefore, we concluded that FZD4S was produced from the FZD4 gene due to alternative splicing.
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FZD4S Potentiates Xwnt-8 Signaling in Xenopus laevis Embryos Ectopic activation of the WNT signaling pathway in the ventral region of Xenopus embryos leads to the secondary axis formation, and inhibition of the WNT signaling pathway in the dorsal region leads to loss of the head structure (1, 18). As the dorsal injection of synthetic FZD4S mRNA did not interfere with the head structure formation (data not shown), FZD4S was then injected into the ventral region of Xenopus embryos. Ventral injection 0.5 pg of Xwnt-8 (high-dose Xwnt-8) induced additional head structure with eyes (complete axis duplication) in 50% of embryos, and also additional head like structures without eyes (partial axis duplication) in 15% of embryos (data not shown). Ventral injection of 0.05 pg of Xwnt-8 (low-dose Xwnt-8) induced partial axis duplication in 56% of Xenopus embryos, but complete axis duplication only in 8% (Fig. 4). Ventral injection of FZD4S did not induce axis duplication by itself. Coinjection of FZD4S and low-dose Xwnt-8 induced complete axis duplication in 61%, which was significantly higher than each injection (P ⬍ 0.05) (Fig. 4). These results indicate that FZD4S augmented the potential of low-dose Xwnt-8 to induce complete axis duplication. DISCUSSION The FZD4S cDNA, encoding a soluble-type polypeptide, was isolated and characterized in this report. The FZD4S cDNA isolated in this study consists of exon 1, intron 1, and exon 2 of the FZD4 gene (Fig. 1), while the FZD4 cDNA isolated previously consists of exon 1, and exon 2 of the FZD4 gene (11). The size difference between the 10.0- and 7.7-kb FZD4 mRNAs exactly corresponds to the size of intron 1. Both the HF4A probe (exon 1) and the HF4S probe (exon 2) hybridized to a major 7.7-kb band and a minor 10.0-kb band, whereas the HF4B probe (intron 1) hybridized to a single band of 10.0 kb in size (Fig. 2). These results indicate that the 7.7-kb FZD4 mRNA consists of exon 1, and exon 2, and that the 10.0-kb FZD4 mRNA consists of exon 1, intron 1, and exon 2. Thus, it is reasonable to conclude that the FZD4 gene gives rise to seventransmembrane-type FZD4 as well as soluble-type FZD4S due to alternative splicing of the retained intron type (Fig. 3). Members of the FZD gene family, including FZD1– FZD10, encode seven-transmembrane-type WNT receptors with CRD (10), while members of the SFRP gene family, including SFRP1–SFRP5, encode solubletype WNT antagonists with CRD (15). SFRP is not a splicing variant of any member of the FZD gene family.
Thus, this is the first report on the soluble-type FZD isoform produced by alternative splicing. While the 10.0-kb FZD4 mRNA was detected preferentially in fetal kidney (Fig. 3D), the 7.7-kb FZD4 mRNA is detected preferentially in adult heart, skeletal muscle, ovary, and fetal kidney (11). These results indicate that the 10.0-kb FZD4 mRNA, encoding FZD4S, might be produced preferentially in fetal lung due to a tissue specific splicing mechanism. FZD4S is predicted to be a soluble-type polypeptide with the N-terminal part of CRD (Fig. 1B). We should detect FZD4S protein in the culture medium of mammalian cells transfected with the FZD4S expression vector in the near future. As SFRPs are soluble polypeptides with CRD, which function as WNT antagonists (14), it is reasonable to speculate that FZD4S might function as WNT antagonist, just like SFRP. However, this hypothesis was denied, because injection of synthetic FZD4S mRNA into the dorsal marginal zone of Xenopus embryos did not exerted any effects. Then, the synthetic FZD4S mRNA was injected into the ventral marginal zone of Xenopus embryos at the 4-cell stage; however, ventral injection of FZD4S did not induce axis duplication by itself (0%). We next investigated the effect of FZD4S on Xwnt-8. Ventral injection of low-dose Xwnt-8 induced complete axis duplication at a very low frequency by itself (8%), while coinjection of FZD4S mRNA and low-dose Xwnt-8 mRNA induced complete axis duplication with a significantly higher frequency (61%) (Fig. 4). Based on these results, we concluded that FZD4S functions as a positive regulator of the WNT signaling pathway. Members of the FZD family are classified into the canonical receptors and the non-canonical receptors, and FZD4 is a noncanonical receptor (19). As human FZD4S is highly homologous to Xenopus frizzled-4 (Xfz4) (20) in the N-terminal part of CRD (96% amino-acid identity), FZD4S might interfere with the noncanonical WNT signaling through seven-transmembrane-receptor Xfz4, and might augment the canonical WNT signaling induced by Xwnt-8. Thus, FZD4S, moderately expressed in fetal kidney, might be implicated in kidney morphogenesis through augmentation of the canonical-type WNT signaling as predicted from the Xenopus axis duplication assay. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science, and Culture of Japan (to M.K.). We thank Drs. Dave Turner and Sergei Sokol for kindly providing us with the CS2⫹ plasmid and the Xwnt8-pXT7 plasmid, respectively.
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FZD4S, a Splicing Variant of Frizzled-4, Encodes a Soluble-Type Positive Regulator of the WNT Signaling Pathway Norihiko Sagara,* ,† Hiroyuki Kirikoshi,* Harumi Terasaki,‡ Yukuto Yasuhiko,‡ Gotaro Toda,† Koichiro Shiokawa,‡ and Masaru Katoh* ,1 *Genetics and Cell Biology Section, Genetics Division, National Cancer Center Research Institute, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; †Division of Gastroenterology and Hepatology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo 105-0003, Japan; and ‡Department of Developmental Biology, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
Received March 6, 2001
Frizzled-1 (FZD1)–FZD10 are seven-transmembranetype WNT receptors, and SFRP1–SFRP5 are solubletype WNT antagonists. These molecules are encoded by mutually distinct genes. We have previously isolated and characterized the 7.7-kb FZD4 mRNA, encoding a seven-transmembrane receptor with the extracellular cysteine-rich domain (CRD). Here, we have cloned and characterized FZD4S, a splicing variant of the FZD4 gene. FZD4S, corresponding to the 10.0-kb FZD4 mRNA, consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD, and was expressed in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage did not induce axis duplication by itself, but augmented the axis duplication potential of coinjected Xwnt-8 mRNA. These results indicate that the FZD4 gene gives rise to solubletype FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing, and strongly suggest that FZD4S plays a role as a positive regulator of the WNT signaling pathway. © 2001 Academic Press Key Words: Frizzled; WNT receptor; alternative splicing; Xenopus laevis.
The WNT signaling pathway plays a critical role in embryonic development and tumor development (1, 2). The WNT signal is transduced to the -catenin–TCF pathway (3), the Jun-N-terminal kinase pathway (4),
or the Ca 2⫹-releasing pathway (5) through seventransmembrane-receptors Frizzled-1 (FZD1)–FZD10 (6 –2). FZDs are seven-transmembrane-receptors with the frizzled-type cysteine-rich domain (CRD). CRD is the WNT-binding domain, which also exists in secretedtype Frizzled-related proteins SFRP1–SFRP5 (13–15). FZDs function as WNT receptors, whereas SFRPs function as WNT antagonists. Although FZDs and SFRPs are mutually homologous in CRD, these two groups of proteins are encoded by distinct genes. Thus, SFRP is not a splicing variant of FZD. We have previously reported molecular cloning of the FZD4 gene, encoding a seven-transmembrane receptor (11). The open reading frame (ORF) of the 7.7-kb FZD4 mRNA is split within the CRD by intron 1. Here, we have cloned and characterized the FZD4S cDNA, which consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD due to the appearance of a new stop codon within the retained intron 1. FZD4S was expressed as the 10.0-kb FZD4 mRNA in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage augmented the axis duplication potential of the coinjected Xenopus wnt-8 (Xwnt-8) mRNA. This is the first report on cloning and characterization of a soluble-type isoform produced by alternative splicing of a member of the FZD gene family. MATERIALS AND METHODS
The nucleotide sequence data of human FZD4S will appear in the DDBJ/EMBL/GenBank Data Bases with the Accession No. AB054881. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-3-3541-2685. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
cDNA library screening. Human fetal lung cDNA library in gt10 (Clontech Laboratories) was screened with the FZGC4 probe (nucleotide position 3342– 4114 of the FZD4 gene) as described previously (16). cDNA inserts were excised with EcoRI digestion, and subcloned
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FIG. 1. Structure of FZD4S cDNA and amino-acid sequence of FZD4S. (A) Schematic presentation of the FZD4S cDNA. The coding region and UTRs are depicted as an open box and bold bars, respectively. Four overlapping FZD4S cDNAs (HF4-5, HF4-6, HF4-7, and HF4-9) are shown by solid bars. (B) Amino-acid sequence of FZD4S. Amino acids are numbered on the right. The signal peptide (bold over-line), CRD (open box), and the unique sequence of FZD4S isoform (over-line) are shown. The conserved amino acids in the N-terminal part of CRD (asterisk), and N-glycosylation site (sharp) are also shown. FZD4S is a soluble-type polypeptide with the N-terminal part of CRD. (C) Kyte and Doolittle hydrophobicity analysis on FZD4S. SP: signal peptide.
into the pUC118 vector (Takara) for nucleotide sequence analyses with ABI310 Sequencer (PE Applied Biosystems). cDNA-PCR. cDNA was synthesized from 40 ng of poly(A) ⫹ RNAs of human fetal kidney (Clontech Laboratories) with the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech). The subsequent PCR with P4-101 and P4-102 primers were performed by using the KOD Plus DNA polymerase (Toyobo) as described previously (16). Nucleotide sequences of PCR primers are as follows: PF4-101 (sense), 5⬘-ATCCCACACAGTCGCGCG-3⬘; PF4-102 (antisense), 5⬘CCAATGGGGATGTTGATCTTC-3⬘. Northern blot analyses. MTN Northern blot filters (Clontech Laboratories) were hybridized with a [␣- 32P] dCTP-labeled probe at 68°C for 1 h in QuikHyb solution (Stratagene). After washing, filters were exposed to the Imaging plate (Fuji) for the image analysis in the Storm system (Molecular Dynamics) as described previously (16). The HF4A probe (nucleotide position 79 – 470 of the FZD4 gene) was derived from exon 1, the HF4B probe (nucleotide position 821–1202 of the FZD4 gene) was derived from intron 1, and the HF4S probe (nucleotide position 6788 –7185 of the FZD4 gene) was derived from exon 2. In vitro synthesis of the FZD4S mRNA. PCR with P4-103 and P4-104 primers were performed to amplify the ORF of the FZD4S from the HF4-9 cDNA. Nucleotide sequences of PCR primers are as follows: PF4-103 (sense), 5⬘-GTGGATCCTGGGGGTGTCTGCCAGAG-3⬘; PF4-104 (antisense), 5⬘-GTGAATTCTTTCCCAGGCCAGATGAC-3⬘. BamHI linker and XhoI linker were added to the 5⬘-end of PF4-103 and PF4-104 primers, respectively. The FZD4S-ORF cDNA fragment (nucleotide position 242–794 of the FZD4 gene) was digested with BamHI and XhoI, and subcloned into the pCS2⫹ vector kindly provided by Dr. Dave Turner (Fred Hutchinson Cancer Research Center). After nucleotide sequence analysis, a FZD4SORF/pCS2⫹ plasmid without misincorporation during PCR was selected for linearization with PvuII digestion. By using the linearized
FZD4S-ORF/pCS2⫹ plasmid as a template, the capped FZD4S mRNA was synthesized with the MEGAscript SP6 Kit (Ambion) in the presence of RNA cap structure analog (New England Biolabs). Xenopus axis duplication assay. Twelve hours after injection of gonatropin (Teikoku Zouki) into female Xenopus laevis, eggs were manually ovulated and artificially fertilized (17). Fertilized eggs were dejellied by treatment with 4.5% cysteine hydrochloride. After washing, embryos were transferred to culture dishes containing the 0.1⫻ Steinberg’s solution. Synthetic RNA was injected into the ventral marginal zone or the dorsal marginal zone of Xenopus embryos at the 4-cell stage as described previously (17). Phenotypes of Xenopus embryos were examined 24 h after mRNA injection.
RESULTS Isolation of FZD4S cDNAs We have previously isolated eight positive clones from a human fetal lung cDNA library with the FZGC4 probe (11). Two FZD4 cDNA clones, corresponding to the 7.7-kb FZD4 mRNA, consisted of exon 1 and exon 2 of the FZD4 gene (11). However, nucleotide sequence analyses of three cDNA clones, such as HF4-5, HF4-6, and HF4-7, revealed that these clones consisted of intron 1 and exon 2 of the FZD4 gene. We then performed cDNA-PCR with P4-101 and P4-102 primers to isolate the variant FZD4 cDNA which contains intron 1. We isolated here a 2846-bp cDNA fragment, designated HF4-9, from poly(A) ⫹ RNAs of fetal kidney by cDNAPCR with reverse transcriptase. Since such a cDNA
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FIG. 2. Expression profile of FZD4S. (A–D) Northern blot analyses. Multiple tissue Northern filters for adult tissues (A), fetal tissues, (B, D), and cancer cell lines (C) (Clontech Laboratories) containing 2 g of poly(A) ⫹ RNA on each lane were hybridized with the [␣- 32P]dCTP-labeled HF4B probe (A–C), or HF4A probe (D). The HF4B probe hybridized to a single band of 10.0 kb, whereas the HF4A probe hybridized to a major band of 7.7 kb and a minor band of 10.0 kb. The 10.0-kb FZD4 mRNA (arrow) was expressed moderately in fetal kidney, but only faintly in adult heart and fetal lung. (E) RNA dot blot analysis. RNA Master Blot filter (Clontech Laboratories) was hybridized with the HF4B probe (left). Sources of poly(A) ⫹ RNA on each spot is shown (right). Hybridization signal to FZD4S, corresponding to the 10.0-kb FZD4 mRNA, was relatively strong in adult heart (c1) and lung (f 2), and in fetal kidney (g3) and lung (g7).
was not obtained by cDNA-PCR without reverse transcriptase (data not shown), we assumed that theHF4-9 cDNA was derived from a variant FZD4 mRNA rather than from a contaminated genome DNA. Sequence analysis revealed that the HF4-9 cDNA consisted of exon 1, intron 1 and exon 2 of the FZD4 gene.
The total nucleotide sequence of the FZD4S cDNA was determined by combining the nucleotide sequences of the following cDNAs: HF4-5 (nucleotide position 506 –3355), HF4-6 (nucleotide position 1669 – 4242), HF4-7 (nucleotide position 2070 – 4474), and HF4-9 (nucleotide position 1–2847) (FIG. 1A). The FZD4S
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FIG. 3. Alternative splicing of the FZD4 gene. (Top) Structure of the FZD4 gene. Two exons are shown by boxes, and one intron is shown by a bold bar. (Middle) The FZD4 probes used for Northern blot analyses are shown by bold bars. The size(s) of the FZD4 mRNA detected by each probe are also shown. (Bottom) The structure of the 10.0-kb FZD4 mRNA (soluble-type isoform) and the 7.7-kb FZD4 mRNA (seven-transmembrane-type isoform). The coding region is indicated by the closed box, and UTRs by the open boxes. The 10.0-kb FZD4 mRNA consists of exon 1, intron 1, and exon 2, whereas the 7.7-kb FZD4 mRNA consists of exon 1, and exon 2. Thus, the FZD4 gene gives rise to soluble-type FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing of the retained intron type.
cDNA was found to consist of the 164-bp 5⬘-UTR, the 378-bp ORF, and the 3932-bp 3⬘-UTR (Fig. 1A). Amino-Acid Sequence of FZD4S FZD4S was predicted to encode a polypeptide of 125 amino-acid residues (Fig. 1B). The N-terminal 98 amino-acid residues of FZD4S were identical to those of the seven-transmembrane-receptor FZD4 (11), but the following 27 amino-acid residues were unique. The N-terminal hydrophobic region, corresponding to the signal peptide, was identified in FZD4S by the Kyte and Doolittle hydrophobicity analysis (Fig. 1C). FZD4S contained the N-terminal part of CRD of FZD4, but contained neither the latter half of CRD nor seven transmembrane domains of FZD4 (Fig. 1B). These results indicate that FZD4 is a soluble-type polypeptide with only the N-terminal part of CRD.
bridized only to 10.0-kb RNA. The 10.0-kb FZD4 mRNA was expressed moderately in fetal kidney, and only faintly in adult heart and fetal lung (Figs. 2A and 2B). The 10.0-kb FZD4 mRNA was almost undetectable in human cancer cell lines HL-60, HeLa S3, K-562, MOLT-4, Raji, SW480, A539, and G361 (Fig. 2C). Expression profile of the 10.0-kb FZD4 mRNA among various human tissues was investigated further by using RNA Master Blot filter (Clontech Laboratories). FZD4S, corresponding to the 10.0-kb FZD4 mRNA, was expressed in adult heart and lung, and in fetal kidney and lung (Fig. 2E). Hybridization signals of FZD4S in adult heart, adult lung, and fetal lung were more clearly detected by RNA master blot analysis than by Northern blot analysis, probably due to higher sensitivity of RNA master blot analysis. These results indicated that the 10.0-kb FZD4 mRNA was expressed in adult heart and lung, and in fetal kidney and lung.
Expression Profile of FZD4S The HF4B probe, corresponding to intron 1 of the FZD4 gene, was synthesized by PCR using the HF4-9 cDNA as a template. We expected that this probe would hybridize to the 10.0-kb FZD mRNA with intron 1, but not to the 7.7-kb FZD4 mRNA without intron 1. In fact, northern blot analysis showed that the HF4B probe hy-
FZD4S Is a Splicing Variant of the FZD4 Gene The HF4A probe, the HF4B probe, and the HF4S probe are derived from exon 1, intron 1, and exon 2 of the FZD4 gene, respectively. The HF4A probe hybridized to a major band of 7.7 kb and a minor band of 10.0 kb (Fig. 2D), whereas the HF4B probe hybridized to a
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FIG. 4. Functional analyses of FZD4S in the Xenopus axis duplication assay. Synthetic mRNAs of FZD4S (1 ng), Xwnt-8 (0.05 pg), or -globin (1 ng) were injected into the ventral marginal zone of Xenopus embryos at the 4-cell stage, and the phenotypes were evaluated 24 h after injection. (A) Representative Xenopus embryos injected with -globin alone (upper left), FZD4S alone (upper right), -globin ⫹ Xwnt-8 (lower left), and FZD4S ⫹ Xwnt-8 (lower right) are shown. (B) Incidence of complete axis duplication (closed bar) and partial axis duplication (open bar) in each injection group are shown. Ventral injection of FZD4S did not induce axis duplication by itself (0%). Ventral injection of low-dose Xwnt-8 induced partial axis duplication in 56% of Xenopus embryos, but complete axis duplication only in 8%. Coinjection of FZD4S and low-dose Xwnt-8 induced complete axis duplication with significantly higher frequency (61%) (P ⬍ 0.05). These results indicate that FZD4S augmented the potential of low-dose Xwnt-8 to induce complete axis duplication.
single band of 10.0 kb (Fig. 2B). As shown previously, the HF4S probe hybridized to a major band of 7.7 kb and a minor band of 10.0 kb (11). These results indicate that the 10.0-kb FZD4 mRNA consists of exon 1, intron
1, and exon 2, while the 7.7-kb FZD4 mRNA consists of exon 1, and exon 2 (Fig. 3). Therefore, we concluded that FZD4S was produced from the FZD4 gene due to alternative splicing.
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FZD4S Potentiates Xwnt-8 Signaling in Xenopus laevis Embryos Ectopic activation of the WNT signaling pathway in the ventral region of Xenopus embryos leads to the secondary axis formation, and inhibition of the WNT signaling pathway in the dorsal region leads to loss of the head structure (1, 18). As the dorsal injection of synthetic FZD4S mRNA did not interfere with the head structure formation (data not shown), FZD4S was then injected into the ventral region of Xenopus embryos. Ventral injection 0.5 pg of Xwnt-8 (high-dose Xwnt-8) induced additional head structure with eyes (complete axis duplication) in 50% of embryos, and also additional head like structures without eyes (partial axis duplication) in 15% of embryos (data not shown). Ventral injection of 0.05 pg of Xwnt-8 (low-dose Xwnt-8) induced partial axis duplication in 56% of Xenopus embryos, but complete axis duplication only in 8% (Fig. 4). Ventral injection of FZD4S did not induce axis duplication by itself. Coinjection of FZD4S and low-dose Xwnt-8 induced complete axis duplication in 61%, which was significantly higher than each injection (P ⬍ 0.05) (Fig. 4). These results indicate that FZD4S augmented the potential of low-dose Xwnt-8 to induce complete axis duplication. DISCUSSION The FZD4S cDNA, encoding a soluble-type polypeptide, was isolated and characterized in this report. The FZD4S cDNA isolated in this study consists of exon 1, intron 1, and exon 2 of the FZD4 gene (Fig. 1), while the FZD4 cDNA isolated previously consists of exon 1, and exon 2 of the FZD4 gene (11). The size difference between the 10.0- and 7.7-kb FZD4 mRNAs exactly corresponds to the size of intron 1. Both the HF4A probe (exon 1) and the HF4S probe (exon 2) hybridized to a major 7.7-kb band and a minor 10.0-kb band, whereas the HF4B probe (intron 1) hybridized to a single band of 10.0 kb in size (Fig. 2). These results indicate that the 7.7-kb FZD4 mRNA consists of exon 1, and exon 2, and that the 10.0-kb FZD4 mRNA consists of exon 1, intron 1, and exon 2. Thus, it is reasonable to conclude that the FZD4 gene gives rise to seventransmembrane-type FZD4 as well as soluble-type FZD4S due to alternative splicing of the retained intron type (Fig. 3). Members of the FZD gene family, including FZD1– FZD10, encode seven-transmembrane-type WNT receptors with CRD (10), while members of the SFRP gene family, including SFRP1–SFRP5, encode solubletype WNT antagonists with CRD (15). SFRP is not a splicing variant of any member of the FZD gene family.
Thus, this is the first report on the soluble-type FZD isoform produced by alternative splicing. While the 10.0-kb FZD4 mRNA was detected preferentially in fetal kidney (Fig. 3D), the 7.7-kb FZD4 mRNA is detected preferentially in adult heart, skeletal muscle, ovary, and fetal kidney (11). These results indicate that the 10.0-kb FZD4 mRNA, encoding FZD4S, might be produced preferentially in fetal lung due to a tissue specific splicing mechanism. FZD4S is predicted to be a soluble-type polypeptide with the N-terminal part of CRD (Fig. 1B). We should detect FZD4S protein in the culture medium of mammalian cells transfected with the FZD4S expression vector in the near future. As SFRPs are soluble polypeptides with CRD, which function as WNT antagonists (14), it is reasonable to speculate that FZD4S might function as WNT antagonist, just like SFRP. However, this hypothesis was denied, because injection of synthetic FZD4S mRNA into the dorsal marginal zone of Xenopus embryos did not exerted any effects. Then, the synthetic FZD4S mRNA was injected into the ventral marginal zone of Xenopus embryos at the 4-cell stage; however, ventral injection of FZD4S did not induce axis duplication by itself (0%). We next investigated the effect of FZD4S on Xwnt-8. Ventral injection of low-dose Xwnt-8 induced complete axis duplication at a very low frequency by itself (8%), while coinjection of FZD4S mRNA and low-dose Xwnt-8 mRNA induced complete axis duplication with a significantly higher frequency (61%) (Fig. 4). Based on these results, we concluded that FZD4S functions as a positive regulator of the WNT signaling pathway. Members of the FZD family are classified into the canonical receptors and the non-canonical receptors, and FZD4 is a noncanonical receptor (19). As human FZD4S is highly homologous to Xenopus frizzled-4 (Xfz4) (20) in the N-terminal part of CRD (96% amino-acid identity), FZD4S might interfere with the noncanonical WNT signaling through seven-transmembrane-receptor Xfz4, and might augment the canonical WNT signaling induced by Xwnt-8. Thus, FZD4S, moderately expressed in fetal kidney, might be implicated in kidney morphogenesis through augmentation of the canonical-type WNT signaling as predicted from the Xenopus axis duplication assay. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science, and Culture of Japan (to M.K.). We thank Drs. Dave Turner and Sergei Sokol for kindly providing us with the CS2⫹ plasmid and the Xwnt8-pXT7 plasmid, respectively.
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