Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, we isolated and characterized a novel mouse frizzled gene Fzd5. Fzd5 mRNA was expressed in the yolk sac, eye and lung bud at 9.5 days post coitum. Fzd5 specifically synergized with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, we have generated Fzd5 knockout mice. While the heterozygotes were viable, fertile and appeared normal, the homozygous embryos died in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days post coitum, prior to any morphological changes, endothelial cell proliferation was markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days post coitum, large vitelline vessels were poorly developed, and the capillary plexus was disorganized. At this stage, vasculogenesis in the placenta was also defective, although that in the embryo proper was normal. Because Wnt5a and Wnt10b co-localized with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac.

Wnt proteins form one of the major families of secreted ligands that play key roles in developmental signaling (Nusse and Varmus, 1992; Miller and Moon, 1996; Cadigans and Nusse, 1997). Wnts are expressed in various tissues in the developing mouse embryo and the most common sites of expression are the limbs and central nervous system (Gavin et al., 1990; Parr et al., 1993). The expression patterns of certain Wnt genes overlap, suggesting a degree of functional redundancy among them (McMahon and Bradley, 1990; McMahon et al., 1992). Null mutations have been generated for a number of Wnt genes and in most cases, the homozygous mutants are embryonic lethal, demonstrating their essential roles in development. For example, Wntl knockout mice have defects in the cerebellum often with a complete loss of the midbrain structures (McMahon and Bradley, 1990; Thomas and Capecchi, 1990); Wnt2 mutant mice have defects in placentation (Monkley et al., 1996); Wnt3 knockout mice do not form a primitive streak, mesoderm or node (Liu et al., 1999); Wnt4 mutant mice have abnormal kidney development (Stark et al., 1994), with mutant females exhibiting defects in the reproductive organs (Vainio et al., 1999); and Wnt5a-deficient mice show truncated limbs and AP axis, and reduced numbers of proliferating cells (Yamaguchi et al., 1999).

The frizzled gene was initially identified as a Drosophila tissue polarity gene. It belongs to a family of genes conserved from C. elegans to mammals (Chan et al., 1992; Zhao et al., 1995; Sawa et al., 1996; Wang et al., 1996). The frizzled family of proteins contain a conserved extracellular cysteine-rich domain at the N terminus that mediates the binding of the Wnt ligands (Bhanot et al., 1996; Yang-Snyder et al., 1996). They also contain seven transmembrane domains and an intracellular C terminus that frequently harbors a PDZ-binding motif (Vinson et al., 1989; Wang et al., 1996). Consistent with being Wnt receptors, several mammalian frizzled homologs have been shown to bind Drosophila Wnt (wingless) at the cell surface and stabilize cytoplasmic β-catenin (Bhanot et al., 1996).

In Xenopus embryos, misexpression of some Wnts causes ectopic axes (Miller and Moon, 1996), while disruption or depletion of other components of the Wnt pathway can inhibit the dorsal axis formation (Heasman et al., 1994; Pierce and Kimelman, 1995; Dominguez et al., 1995; Zeng et al., 1997; Molenaar et al., 1996). This axis-promoting activity has provided a valuable in vivo assay system for studying the Wnt pathway (Cadigan and Nusse, 1997). In particular, determining synergistic interactions between specific Wnts and frizzleds by co-injection into Xenopus embryos, has been a useful method with which to identify which Wnt ligand interacts with which frizzled protein. For example, rat frizzled 1 (Fzd1) synergistically induces Wnt-responsive genes with Xenopus Xwnt-8 (Yang-Snyder et al., 1996), and human frizzled 5 (FZD5) can induce secondary axes when co-expressed with Xwnt5a, but neither can induce secondary axes alone (He et al., 1997).

These results suggest that the frizzled proteins function as receptors for Wnt ligands. The diversity of the Wnt and frizzled genes expressed in distinct spatiotemporal patterns during development, suggest that the regulation of Wnt signaling is likely to be complex. Some of the important issues that remain to be resolved include identifying which Wnts interact with particular frizzled receptors and determining the biological function of these signaling events. Here we present evidence that the mouse homolog of human FZD5 functions as a receptor for Wnt5a, Wnt10b and Wnt2, and that the homozygous Fzd5 knockout mice are embryonically lethal, owing to defects in the yolk sac vasculogenesis.

Cloning of Fzd5

Total RNA isolated from newborn mouse guts was reverse transcribed with oligo-dT. The resulting cDNA was PCR amplified with degenerate frizzled motif primers (forward, 5'-TAY CCD GAR MGV CCH AT-3′; reverse, 5′-RGC WGC YAR RAA CCA-3′) for 35 cycles of (1.0 minute at 94°C; 2.5 minutes at 50°C; and 1.5 minutes at 72°C).

Gene mapping of Fzd5

We used panels of DNA samples from interspecific backcross mice that have been characterized for over 1100 genetic markers throughout the genome at a resolution of 50 to 85 centi-Morgans (Seldin et al., 1998). Genomic DNA from F1 animals of the following cross; C3H/HeJ-gld and (C3H/HeJ-g/d x Mus spretus) were digested with various restriction endonucleases and hybridized with a mouse Fzd5 genomic probe to identify restriction fragment length variants (RFLVs) to allow a haplotype analysis.

In situ hybridization

Whole mounts or sections of embryonic tissues were hybridized with digoxigenin-11-UTP (Roche Diagnostics, Germany) -labeled probes as previously described (Saga et al., 1996). A 0.5 kb cDNA Fzd5 fragment containing a 250 bp 5′ noncoding region was used for the Fzd5 probe. Mouse Wnt2, Wnt10b and Wnt5a cDNA probe fragments were PCR amplified with the following primer pairs: Wnt2, 5'-GGT TAA TAT GAA CGT CCC TCT CGG TG-3′ and 5'-AGG TCA TGT AGG CGT CGC CCA GTC GG-3′; Wnt10b, 5'-GGA GGG CAG CGC CAG AGT TCC-3′ and 5'-AGG CTG CCA CAG CCA TCC AAG AGG-3′; and Wnt5a, 5'-GAA TTG GTG GTG TGA ATG AAC TGG GG-3′ and 5'-GCG AAG GAG AAA AAC GTG GCC AAA G-3′. The probes for 4311 and pl1 were obtained from J. Rossant and D. Linzer, respectively.

Xenopus microinjection constructs

The Fzd5, Wnt2, Wnt10b and Wnt5a cDNAs were isolated by PCR amplification with the following primer pairs: Fzd5, 5'-ACG ATG GCT CGA CCC GAC CCG TGT G-3′ and 5'-CTT CAA CTT CCA GCA CTG TCC CTG G-3′; Wnt2, 5'-GGT TAA TAT GAA CGT CCC TCT CGG TG-3′ and 5'-AGG TCA TGT AGG CGT CGC CCA GTC GG-3′; Wnt10b, 5'-GAC ATG CTG GAG GAG CCC CGG TCT C- 3′ and 5'-CTT CAT TTA CAC ACA TTG ACC CAC TCT GTG-3′; and Wnt5a, 5'-GCC ATG AAG AAG CCC ATT GGA ATA TTA-3′ and 5'-CTA TTT GCA CAC GAA CTG ATC CAC AAT C-3′. For Xenopus microinjection constructs, the PCR products were cloned into pCS2+ (Turner and Weintraub, 1994). Human WNT5A was obtained from R. T. Moon.

RNA synthesis and Xenopus embryo injections

Expression plasmids for Fzd5 and Wnts in pCS2 + were linearized, and capped synthetic mRNA was transcribed using an SP6 MEGA script kit (Ambion), with a cap analog 7mGpppG:GTP ratio of 5:1. Xenopus embryos were microinjected in one ventral blastomere at the four-cell stage with the following RNA samples; either 1 ng Fzd5, 5-10 pg of each Wnt, or both. Embryos were scored for secondary axis formation at the tail bud stage.

Construction of Fzd5 targeting vector

A mouse Fzd5 genomic clone was isolated from a 129/Sv genomic λ phage library (Stratagene) using a PCR-amplified Fzd5 cDNA fragment as a probe. From this λ clone, a 10 kb NotI fragment containing the single Fzd5 exon, and a 1.0 kb NotI-SacI fragment (downstream of the exon) were isolated and subcloned in pBluescript II SK (Stratagene). A PGKneobpA cassette (Neo in Fig. 4A; Soriano et al., 1991) was then inserted into the NotI site of Fzd5.

Transfection of ES cells and selection of targeted clones

The targeting vector DNA was linearized with XmnI and electroporated into embryonic stem (ES) cell line RW4 (Genome Systems), which was cultured on neomycin-resistant (neor) mouse embryonic fibroblasts (Oshima et al., 1995). G418 (Geneticin, Sigma)-resistant colonies were screened for homologous recombinants by PCR using primers complementary to Neo (PGK- F5, 5'-GCG CAT GCT CCA GAC TG-3′) and to Fzd5 downstream from the 3 SacI (Mfz5-R3, 5 -GAT GCG GAA GAG TGA CAC GA- 3′) (see Fig. 4A). Samples were amplified for 40 cycles of (0.5 minutes at 94°C; 0.5 minutes at 52°C; and 2 minutes at 72°C) with homologous recombinants generating a 1.2 kb product (see Fig. 4B). Candidates of homologous recombinants were expanded and verified by a Southern analysis. The probe used was a 2.0 kb fragment downstream from the 3′ SacI site (see Fig. 4A) which hybridized to 3.0 kb and 4.6 kb bands for the wild-type and knockout alleles, respectively (see Fig. 4C).

Generation and genotyping of mutant mice

Chimeric mice were generated by injection of the ES cells into C57BL/6 N (B6) blastocysts, followed by transfers to MCH foster mothers (CLEA, Japan), and backcrossed to B6 mice. Genotypes were determined by PCR and/or Southern analysis of the tail DNA samples. The wild-type allele was identified using two primers: Mfz5-eN3, upstream of the NotI site in the exon (5 -GAG GCA AAG GGA AGA AGG AGA-3′) and Mfz5-R3 (described above). PCRs were performed under the same condition as above, except that annealing was at 60°C, and a 1.3 kb fragment was amplified.

Histology, immunohistochemistry and BrdU labeling

After removal from the decidua, embryos were staged and genotyped as described above using DNA extracted from the yolk sac or from the embryo proper. For paraffin sectioning, samples were fixed in 4% paraformaldehyde and embedded according to the standard procedures.

Immunohistochemistry was performed as previously described (Nishikawa et al., 1998). Samples were stained with anti-PECAM1 antibody (Pharmingen) or anti-Flk1 (AVAS12) (Kataoka et al., 1997). The antibody reactions on the paraffin wax-embedded sections were visualized using the Vectastain ABC kit (Vector).

BrdU incorporations were performed for 1 hour for embryos, as previously described (Oshima et al., 1996).

Cloning of the gene for the mouse Wnt receptor frizzled 5 (Fzd5)

To isolate mouse frizzled homologues degenerate oligonucleotide primers were designed based on the conserved amino acids between the mammalian and Drosophila proteins (Bhanot et al., 1996; Wang et al., 1996), and sequences were amplified from a mouse intestinal cDNA pool. Among the isolated sequences, we identified ‘clone 6′, which was very similar to human frizzled 5, FZD5 (Wang et al., 1996). Chromosomal mapping of clone 6 indicated that it is on mouse chromosome 1, cosegregating with the γ crystallin complex (Cryg (mapping data available at http://www.informatics.jax.org, under the Accession Number J:52605). Because these data matched with those of a partial fragment of Fzd5 whose sequence has not been published (Wang et al., 1996), we concluded that ‘clone 6′ was Fzd5. We then isolated the entire ORF of Fzd5 by 5'- and 3'-RACE. The deduced amino acid sequence of Fzd5 showed a 577-residue protein (Fig. 1) with an expected molecular weight of 63.5 kD. Fzd5 contained a signal sequence, ten highly conserved cysteines, seven hydrophobic domains and a potential PDZ-binding site; all motifs characteristic of frizzled proteins. At the amino acid level, Fzd5 showed similarities to human FZD5 (identity, 84%); Xenopus laevis frizzled 8 protein, Xfz8 (67%); zebrafish frizzled homolog, zfzA (66%), mouse Fzd8 (66%); and Drosophila, Fz2 (57%); whereas the originally identified Drosophila frizzled Fz1 was more divergent (identity, only 41%).

Fig. 1.

Comparison of the deduced amino acid sequences of Fzd5 homologs from mouse Fzd5, human FZD5 and Drosophila Fz2 cDNA sequences. Signal sequences are boxed, ten highly conserved cysteines of the putative extracellular ligand binding domain are indicated by asterisks, seven transmembrane regions are underlined and the putative PDZ-binding sites are indicated by an arrowhead. The cDNA and amino acid sequences of Fzd5 have been deposited with GenBank (Accession number, AF272146).

Fig. 1.

Comparison of the deduced amino acid sequences of Fzd5 homologs from mouse Fzd5, human FZD5 and Drosophila Fz2 cDNA sequences. Signal sequences are boxed, ten highly conserved cysteines of the putative extracellular ligand binding domain are indicated by asterisks, seven transmembrane regions are underlined and the putative PDZ-binding sites are indicated by an arrowhead. The cDNA and amino acid sequences of Fzd5 have been deposited with GenBank (Accession number, AF272146).

The transcripts of Fzd5 were detected in various adult mouse tissues by RT-PCR. Expression was especially abundant in the heart and kidney (data not shown), which is consistent with the results of RNase protection assays (Wang et al., 1996). To determine the spatiotemporal expression of Fzd5 in development, we performed an in situ hybridization analysis of mouse embryos (Fig. 2 and data not shown). As described recently (Borello et al., 1999), Fzd5 transcripts were first observed in the ventral part of telencepharon at 8.5 days post coitum (dpc), and its expression was restricted to the eye and lung bud after 9.5 dpc (data not shown), as well as in the yolk sac (Fig. 2A). Although the embryonic expression pattern of Fzd5 was not identical to that of any known Wnt genes, its pattern of expression in the yolk sac was very similar to that of Wnt5a and Wnt10b (Fig. 2B,D, respectively). However, we did not detect the transcripts of Wnt2 (Fig. 2C) or Wnt2b (data not shown) in the yolk sac at 9.5 dpc. The coincident yolk sac expression patterns suggest that Wnt5a and Wnt10b are likely candidates for physiological ligands of Fzd5 (see below). At this stage (9.5 dpc), strong expression was also found in the placenta (Fig. 2E), especially in the labyrinthine trophoblasts (see below).

Fig. 2.

Expression of Fzd5 and Wnt mRNAs in the yolk sac, placenta and intestine. (A-D) Yolk sacs at 10.25 dpc stained in situ with the probes for Fzd5 (A), Wnt5a (B), Wnt2 (C) and Wnt10b (D). Note that expression of Fzd5 mRNA in vascular endothelial cells coincide with Wnt5a and Wnt10b (arrowheads), whereas that of Wnt2does not. (E) Placenta at 10.5 dpc stained with the Fzd5probe. Note the expression in the labyrinthine layer. (F) Adult small intestine stained with the Fzd5 probe. Scale bars: 100 μm in A-D,F.

Fig. 2.

Expression of Fzd5 and Wnt mRNAs in the yolk sac, placenta and intestine. (A-D) Yolk sacs at 10.25 dpc stained in situ with the probes for Fzd5 (A), Wnt5a (B), Wnt2 (C) and Wnt10b (D). Note that expression of Fzd5 mRNA in vascular endothelial cells coincide with Wnt5a and Wnt10b (arrowheads), whereas that of Wnt2does not. (E) Placenta at 10.5 dpc stained with the Fzd5probe. Note the expression in the labyrinthine layer. (F) Adult small intestine stained with the Fzd5 probe. Scale bars: 100 μm in A-D,F.

In the adult small intestine, the source of the cDNA clone, the Fzd5 transcripts were observed in the epithelial cells (Fig. 2F). This expression was stronger in the crypt than in the villus. In the colon, Fzd5 was expressed also in the epithelial cells, especially in goblet cells (data not shown).

Synergistic effects of Fzd5 and Wnts in the secondary axis induction

To analyze whether Fzd5 functions as a component of Wnt signaling pathway, we first performed ectopic expression experiments in Xenopus embryos. Injection of Fzd5 mRNA into all blastomeres at the four-cell stage resulted in bent embryos with shortened or absent trunks but with intact heads and tails (Fig. 3A), a phenotype similar to that observed with Xfz3 (Shi et al., 1998) and Xfz7 (Sumanas et al., 2000) overexpression, respectively. This result suggests that Fzd5 is interacting with endogenous ligands and/or interfering with the signaling through endogenous receptors, causing suppression of the axial trunk development.

Fig. 3.

Functional interaction of Fzd5and Wnts in Xenopus embryos. (A) Radial injection of Fzd5 mRNA (250 pg/blastomere) at the fourcell stage results in bent embryos lacking the axial trunk structures. (B) Fzd5 synergizes with Wnt2, Wnt5a and Wnt10b. Each test RNA (1 ng of Fzd5 and/or 5-10 pg of each Wnt) was micro-injected into one of the ventral blastomeres at the four-cell stage. The injected embryos were scored for the secondary axis formation at the tail bud stage. The histogram summarizes the results from two to three separate injection experiments for each RNA sample. Partial secondary axes without head structures are indicated in gray, while those with head structures, such as eyes and cement glands are shown in black.

Fig. 3.

Functional interaction of Fzd5and Wnts in Xenopus embryos. (A) Radial injection of Fzd5 mRNA (250 pg/blastomere) at the fourcell stage results in bent embryos lacking the axial trunk structures. (B) Fzd5 synergizes with Wnt2, Wnt5a and Wnt10b. Each test RNA (1 ng of Fzd5 and/or 5-10 pg of each Wnt) was micro-injected into one of the ventral blastomeres at the four-cell stage. The injected embryos were scored for the secondary axis formation at the tail bud stage. The histogram summarizes the results from two to three separate injection experiments for each RNA sample. Partial secondary axes without head structures are indicated in gray, while those with head structures, such as eyes and cement glands are shown in black.

To determine the likely ligands for Fzd5, we then tested various candidate Wnts for synergy with Fzd5 using the Xenopus secondary axis assay. Injection of the Fzd5 mRNA alone into a single ventral blastomere resulted in a low frequency of axis duplication, less than 20% (n = 102) (Fig. 3B). In similar ventral injections, we also tested activity of some candidate ligands: mouse Wnt2, Wnt2b, Wnt4, Wnt5a and Wnt10b, which are expressed in the yolk sac (Austin et al., 1997; Fig. 2, and data not shown). Consistent with a previous report (Landesman et al., 1997), only Wnt2 mRNA gave a high frequency of secondary axes in 72% (n = 105) of the embryos (Fig. 3B). All of the Wnt2-induced secondary axes were partial without head structures.

To investigate functional interactions between Fzd5 and the candidate ligands, we co-injected Fzd5 with each of the Wnts and assayed for synergism in the secondary axis induction. Coinjection of Fzd5 mRNA with Wnt2b or Wnt4 mRNA gave no additional effects to the injections of the Fzd5 mRNA alone (Fig. 3B). In contrast, Fzd5 clearly synergized with Wnt2, Wnt5a and Wnt10b, giving a robust induction of the secondary axis in each case. The combination of Fzd5 and Wnt2 resulted in a high frequency of secondary axes with head structures such as eyes and cement glands in 60% (n = 92) of embryos, while Wnt2 alone never gave heads (Fig. 3B). Co-injections of Fzd5 mRNA with mouse Wnt5a or Wnt10b mRNA induced secondary axes in 62% (n = 72) and 92% (n = 94) of embryos, respectively. Co-injection of Fzd5mRNA with Xenopus Wnt5a mRNA also resulted in secondary axes in 63% (n = 100) of the embryos (data not shown). Taken together, these results indicate that Fzd5 functions as a Wnt receptor, and that Fzd5 can interact with Wnt2, Wnt10b and Wnt5a, but not with Wnt2b or Wnt4. Because Wnt5a and Wnt10b are expressed in the yolk sac and Wnt2 is not, the physiological ligands for Fzd5 in mouse yolk sac at 9.5 dpc are likely to be Wnt5a and Wnt10b (see below).

Generation of Fzd5 knockout mice

To determine the in vivo functions of Fzd5, we constructed null knockout mice using standard gene targeting strategy (Fig. 4). While the heterozygous mice were viable, fertile and appeared normal, their intercrosses did not yield any homozygous mutant pups. We then chronologically examined the concepti of the heterozygous intercrosses. As shown in Table 1, the homozygous embryos of 9.5 dpc and 10.25 dpc were morphologically indistinguishable from their wild-type or heterozygous littermates. At 10.75 dpc, however, the homozygotes contained little blood flow in the yolk sac with an anemic appearance (compare Fig. 5B,D with 5A,C), although the heart was still beating. Interestingly, some free embryonic erythrocytes with nuclei were found sitting inside the yolk sac cavity (data not shown), suggesting internal bleeding from the vitelline vessels. In about 10% of the homozygous embryos, internal bleeding was found in the head and/or heart as well, with an underdeveloped branchial arch and nasal process. Some homozygous embryos also exhibited pericardial edema (data not shown; see below).

Table 1.

Genotypes of offspring from Fzd5 heterozygotes intercrosses

Genotypes of offspring from Fzd5 heterozygotes intercrosses
Genotypes of offspring from Fzd5 heterozygotes intercrosses
Fig. 4.

Construction of Fzd5 knockout mice. (A) Targeting strategy. (Top) Wild-type allele; (middle) targeting vector pMfz5; (bottom) knockout allele. The black boxes show the single exon of Fzd5, whereas the white boxes indicate the selection cassettes for the neomycin resistance (Neo) and the diphtheria toxin a-subunit (DT) genes with their transcriptional orientations shown by arrows underneath. The unbroken horizontal lines represent the noncoding sequences. The positions of the PCR primers (arrowheads) and the Southern hybridization probe (horizontal bar) are also shown. The sizes of the NotI fragments detected by Southern hybridization are shown for the wild-type (3.0 kb) and knockout alleles (4.6 kb), respectively. Crossed lines show regions of homologous recombination whereas broken lines indicate the same genomic loci. (B) PCR detection of homologous recombinant ES cell clones. The arrow on the right shows the position of the 1.2 kb PCR product on agarose gel electrophoresis. (C) Verification of homologous recombination in ES cell clones by Southern hybridization of the genomic DNA preparations. Arrows on the right indicate the positions for the NotI fragments for the 3.0 kb wild-type (WT) and 4.6 kb knockout (KO) alleles. (D) RT-PCR analysis of Fzd5 mRNA in 10.5 dpc embryos. Samples in lanes RT + contained reverse transcriptase while RT- did not.

Fig. 4.

Construction of Fzd5 knockout mice. (A) Targeting strategy. (Top) Wild-type allele; (middle) targeting vector pMfz5; (bottom) knockout allele. The black boxes show the single exon of Fzd5, whereas the white boxes indicate the selection cassettes for the neomycin resistance (Neo) and the diphtheria toxin a-subunit (DT) genes with their transcriptional orientations shown by arrows underneath. The unbroken horizontal lines represent the noncoding sequences. The positions of the PCR primers (arrowheads) and the Southern hybridization probe (horizontal bar) are also shown. The sizes of the NotI fragments detected by Southern hybridization are shown for the wild-type (3.0 kb) and knockout alleles (4.6 kb), respectively. Crossed lines show regions of homologous recombination whereas broken lines indicate the same genomic loci. (B) PCR detection of homologous recombinant ES cell clones. The arrow on the right shows the position of the 1.2 kb PCR product on agarose gel electrophoresis. (C) Verification of homologous recombination in ES cell clones by Southern hybridization of the genomic DNA preparations. Arrows on the right indicate the positions for the NotI fragments for the 3.0 kb wild-type (WT) and 4.6 kb knockout (KO) alleles. (D) RT-PCR analysis of Fzd5 mRNA in 10.5 dpc embryos. Samples in lanes RT + contained reverse transcriptase while RT- did not.

Fig. 5.

Morphology of wild-type (+/+) and Fzd5 homozygous mutant (-/-) embryos at 10.75 dpc. (A,B) Embryos isolated together with their yolk sacs. (C,D) Yolk sacs at a higher magnification of the embryos shown in A,B, respectively. Note that the vitelline vessels visible in the wild-type (+/+) embryo (A,C) are missing in the homozygous mutant (-/-) yolk sac (B,D).

Fig. 5.

Morphology of wild-type (+/+) and Fzd5 homozygous mutant (-/-) embryos at 10.75 dpc. (A,B) Embryos isolated together with their yolk sacs. (C,D) Yolk sacs at a higher magnification of the embryos shown in A,B, respectively. Note that the vitelline vessels visible in the wild-type (+/+) embryo (A,C) are missing in the homozygous mutant (-/-) yolk sac (B,D).

Yolk sac defects in the homozygous Fzd5 mutant mice

To visualize the yolk sac vitelline vessels we immunostained embryos at 10.75 dpc with an anti-PECAM antibody (Fig. 6). In the homozygous mutant embryos, large vitelline vessels were poorly developed, and the capillary plexus was disorganized compared with those of the wild-type or heterozygous littermates (Fig. 6A). In flat-mount micrographs, the small vessels of the homozygous yolk sacs were not reorganized or remodeled into a well-connected network (compare Fig. 6C,E with Fig. 6B,D, respectively). The homozygous yolk sacs contained fewer blood cells inside the vasculature, compared with the heterozygous yolk sacs (compare Fig. 6D with 6E). In homozygous mutants, the endothelial cells that lined inside the yolk sac were poorly developed and tended to peel from the mesodermal layer, which had reduced blood cell numbers (compare Fig. 6F with 6G). These degenerating endothelial cells were better visualized in the DAB/nickel stained samples, while the sinusoidal spaces in the mutant developed to a similar diameter as that found in wild-type yolk sacs (compare Fig. 6I with 6H).

Fig. 6.

Yolk sac angiogenesis in Fzd5 homozygous mutant (-/-) embryos at 10.75 dpc as determined by anti-PECAM1 antibody. (A) Whole-mount in situ staining of heterozygous (+/-) and homozygous (-/-) mutant yolk sacs. Arrowheads indicate the areas shown in B and C. (B,C) Higher magnification photographs of the samples shown in A. Note that the large vessels of the homozygous (-/-) yolk sac are narrower in diameter with less branching (C, arrowhead), compared with those of the heterozygotes (B, arrowheads). (D,E) Flat-mount micrographs of the same samples in A-C showing the fine vascular networks. Note that the small vessels of the homozygous (-/-) yolk sacs are poorly reorganized or remodeled into a well-connected network (arrowheads). Note also the less number of the blood cells found inside the vasculature (E, arrow), compared with the heterozygous (+/-) yolk sac (D, arrows). (F,G) Histological sections of the yolk sacs. Note in the homozygous (-/-) yolk sac that the endothelial cells are detached from the mesodermal layers (G, arrow) and the blood vessels contain few blood cells. (H,I) Nickel staining of the endothelial cells of the yolk sacs. Note detached and degenerating endothelial cells in the homozygous (-/-) yolk sacs (I). Scale bars: 100 μ m in B-I.

Fig. 6.

Yolk sac angiogenesis in Fzd5 homozygous mutant (-/-) embryos at 10.75 dpc as determined by anti-PECAM1 antibody. (A) Whole-mount in situ staining of heterozygous (+/-) and homozygous (-/-) mutant yolk sacs. Arrowheads indicate the areas shown in B and C. (B,C) Higher magnification photographs of the samples shown in A. Note that the large vessels of the homozygous (-/-) yolk sac are narrower in diameter with less branching (C, arrowhead), compared with those of the heterozygotes (B, arrowheads). (D,E) Flat-mount micrographs of the same samples in A-C showing the fine vascular networks. Note that the small vessels of the homozygous (-/-) yolk sacs are poorly reorganized or remodeled into a well-connected network (arrowheads). Note also the less number of the blood cells found inside the vasculature (E, arrow), compared with the heterozygous (+/-) yolk sac (D, arrows). (F,G) Histological sections of the yolk sacs. Note in the homozygous (-/-) yolk sac that the endothelial cells are detached from the mesodermal layers (G, arrow) and the blood vessels contain few blood cells. (H,I) Nickel staining of the endothelial cells of the yolk sacs. Note detached and degenerating endothelial cells in the homozygous (-/-) yolk sacs (I). Scale bars: 100 μ m in B-I.

To obtain a further insight into the mechanism of the endothelial cell degeneration, we examined the growth rates of the yolk sac cells by BrdU incorporation (Fig. 7). At 9.25 dpc, the BrdU labeling index in the homozygous mutant yolk sac endothelial cells was not different from that of the wild-type yolk sac. However at 10.25 dpc, prior to the degeneration of the endothelial cells, the labeling index for the homozygous mutant yolk sacs was markedly decreased when compared with that of the wild-type yolk sacs. There was no evidence of excessive apoptosis in the homozygous yolk sac at these stages; therefore, it appears that the endothelial cells in the mutants were not proliferating. These results indicate that Fzd5 plays a key role in maintaining the growth of the yolk sac vascular structure.

Fig. 7.

BrdU incorporation of yolk sac endothelial cells at 9.25 and 10.25 dpc. Seventeen independent sections that contained endothelial cells from each mouse were photographed. Two mice were sampled for each stage and genotype (Fzd5 homozygotes and wild-type littermates). The BrdU labeled proliferating endothelial cells were scored in unit areas (diameter 100 μ m).

Fig. 7.

BrdU incorporation of yolk sac endothelial cells at 9.25 and 10.25 dpc. Seventeen independent sections that contained endothelial cells from each mouse were photographed. Two mice were sampled for each stage and genotype (Fzd5 homozygotes and wild-type littermates). The BrdU labeled proliferating endothelial cells were scored in unit areas (diameter 100 μ m).

Placental defects in the homozygous Fzd5 mice

Next, we analyzed the embryonic and placental vasculature by staining with an anti-Flk antibody. At 10.75 dpc, the plexus of the wild-type embryo proper developed into a highly branched and intricate vascular network (Fig. 8A). The homozygous embryo (Fig. 8B) was indistinguishable from the wild type, although the extent of vascular remodeling showed some fluctuations among individual embryos.

Fig. 8.

Angiogenesis in embryo proper and placenta at 10.75 dpc. (A,B) The Fzd5-homozygous and wild-type embryos proper stained with anti-Flk antibody. Note the normal vascular pattern in the homozygote (B), indistinguishable from the wild-type littermate control (A). (C,D) The placental vasculature visualized by anti-Flk immunostaining. Note that the embryo-derived blood vessels penetrate deeper in the wild-type placenta (C), whereas the vasculature remains in the superficial layer of the placenta in the homozygote (D). (E,F) Sections of the placenta shown in C,D, respectively (Hemotoxylin and Eosin staining). Note in the labyrinthine layer of the wild-type placenta, the extensive intermingling of the maternal (arrowheads, containing small enucleated erythrocytes) and embryonic blood vessels (arrows, identified by large nucleated erythrocytes inside) (E). Note that the blood vessels of the homozygous Fzd5 placenta do not penetrate into the labyrinthine layer (F). Scale bars: 100 μm in E,F.

Fig. 8.

Angiogenesis in embryo proper and placenta at 10.75 dpc. (A,B) The Fzd5-homozygous and wild-type embryos proper stained with anti-Flk antibody. Note the normal vascular pattern in the homozygote (B), indistinguishable from the wild-type littermate control (A). (C,D) The placental vasculature visualized by anti-Flk immunostaining. Note that the embryo-derived blood vessels penetrate deeper in the wild-type placenta (C), whereas the vasculature remains in the superficial layer of the placenta in the homozygote (D). (E,F) Sections of the placenta shown in C,D, respectively (Hemotoxylin and Eosin staining). Note in the labyrinthine layer of the wild-type placenta, the extensive intermingling of the maternal (arrowheads, containing small enucleated erythrocytes) and embryonic blood vessels (arrows, identified by large nucleated erythrocytes inside) (E). Note that the blood vessels of the homozygous Fzd5 placenta do not penetrate into the labyrinthine layer (F). Scale bars: 100 μm in E,F.

In contrast, in the placenta there was a clear difference between the wild-type and the Fzd5-homozygous knockout mice. Although, the embryonic blood vessels of the wild-type placenta penetrated deeper into the labyrinthine layer (Fig. 8C), those of the Fzd5 homozygotes remained in the superficial layer (Fig. 8D). Histological studies demonstrated that the embryonic blood vessels in the mutant placenta were totally separated from the maternal blood flow (compare Fig. 8F with 8E). Placentas were then stained with probes for Pl1 (mouse placental lactogen 1) and 4311, marker genes for giant trophoblastic cells (Colosi et al., 1987) and spongiotrophoblasts (Lescisin et al., 1988), respectively. The mutant placenta contained similar numbers of the trophoblastic cells compared with those found in the wild-type placenta (data not shown), suggesting that the poor growth of the embryonic placental blood vessels was primarily responsible for the defective placental development, as in the mutant yolk sac. Together with the previous studies, our results indicate that Fzd5 plays an important role in extra-embryonic angiogenesis.

Mouse Fzd5is expressed in embryo, yolk sac and placenta

To study the role of endogenous frizzled proteins in mammalian development, we have identified several novel mouse frizzled homologs. In this report, we have described the isolation and characterization of the Fzd5 gene, whose product carries seven predicted transmembrane domains, a cysteine- rich extracellular domain and other characteristics of frizzled proteins (Fig. 1). Fzd5 showed a high amino acid sequence similarity to human FZD5. In addition to the embryonic tissues such as telencephalon, optic vesicles and lung buds (Borello et al., 1999), Fzd5 was strongly expressed in the yolk sac after 9.5 dpc. Because some Wnt genes are also expressed in the yolk sac, these expression patterns suggest that the Wnt signaling plays an important role in the yolk sac development. In stages as late as 10.5 dpc, Fzd5 was also expressed in the placental blood vessels of embryonic origin (see below).

Fzd5 functionally interacts with Wnt2, Wnt5a and Wnt10b in Xenopus embryos

The human FZD5 protein interacts with Wnt5a and induces a secondary axis in Xenopus embryo (He et al., 1997). We used this Xenopus second axis assay to identify the potential Wnt ligands of Fzd5. In our experiments, co-injection of Fzd5 mRNA with either Wnt5a, Wnt10b or Wnt2 mRNA synergistically induced the formation of secondary axes. In contrast, Wnt2b and Wnt4 did not functionally interact with Fzd5 to induce second axes. These results indicate that Fzd5 can function as a Wnt receptor, and can interact with Wnt5a, Wnt10b and Wnt2 as ligands. In addition to these five Wnts, more than ten Wnt genes have been reported. It is therefore possible that additional Wnts may also have ligand activities for the Fzd5 receptor.

Fzd5-deficient mice are embryonically lethal and have defects in yolk sac and placental vasculogenesis

The phenotype of the Fzd5 homozygous mutant embryo is similar to but distinct from that of such Wnt gene knockout mice as Wnt5a (Yamaguchi et al., 1999) or Wnt2 (Monkley et al., 1996). The lethal stage of the Fzd5 homozygous mutant embryo is earlier than that in the Wnt5a- or Wnt2-deficient embryos. Moreover, yolk sac defects are not observed in these Wnt mutants (Wnt10b mutant mice have not been reported so far). Because Wnt5a and Wnt10b are expressed in the early yolk sac but Wnt2 is not, collectively, these results suggest that Wnt5a and Wnt10b are likely physiological ligands functioning through Fzd5 in the yolk sac. On the other hand, the Wnt2-deficient embryos show placental defects such as decreased numbers of the fetal capillaries beginning 14.5 dpc, suggesting that Wnt2 may play a crucial role as a ligand for Fzd5 during later placental development (Monkley et al., 1996).

In Fzd5 mutant yolk sac, the primary capillary plexus was formed and large vessels appeared normal before 10.25 dpc. Thereafter, however, these vessels started to degenerate, and many endothelial cells peeled off the expanding yolk sac. These results suggest that Fzd5-dependent signals support endothelial cell growth rather than their initial differentiation. Consistent with this interpretation, our BrdU labeling experiments showed a marked decrease in proliferating endothelial cells in the mutants by 10.25 dpc, 12 hours before the morphological defects became apparent.

Our studies also demonstrate that the knockout of the Fzd5 gene caused a separation of the embryonic placental blood vessels from the maternal blood, which may also have resulted from defective vascularization. When the genes essential for organizing a functional placenta are mutated, embryonic lethality often results at characteristic developmental stages (Ihle, 2000). For example, null mutations of p38a (Adams et al., 2000) or integrin av (Bader et al., 1998) causes a striking reduction in the labyrinthine layer and a lack of intermingling between the embryonic and the maternal blood vessels. Because yolk sac vasculogenesis takes place earlier than placental formation, and is not affected in the p38a or integrin av mutant, the yolk sac defects in the Fzd5 mutant are unlikely to be due to the secondary effects of a defective placenta. These results suggest that Wnt signaling via Fzd5 is important in vasculogenesis of extra-embryonic tissues around 10.5 dpc.

In contrast, defects in the vasculature were not apparent in the embryo proper, although some embryos showed growth retardation, cardiac edema, and/or internal breeding. These phenotypes could be caused by the increased cardiac load due to the yolk sac vascular defects (Copp, 1995).

Fzd5 mediates signaling for endothelial cell growth

In addition to frizzled transmembrane receptors, several secreted Wnt-binding proteins have recently been identified that are structurally related to the frizzled extracellular domain (Hoang et al., 1996; Leyns et al., 1997; Rattner et al., 1997; Melkonyan et al., 1997). Some of these secreted Wnt-binding proteins have been shown to suppress Wnt signaling. For example, one such secreted frizzled antagonist, FrzA, inhibits the secondary axis induction by human Wnt2, and binds in vitro to wingless (Xu et al., 1998). Interestingly, bovine FrzA is expressed in non-proliferating and differentiated endothelial cells (Duplaa et al., 1999). The mouse ortholog of FrzA, known as Sfrp1, is expressed in the visceral yolk sac and the expression is confined to the inner lining of the endothelial cell layer at 9.5 dpc (Jaspard et al., 2000). Thereafter, Sfrp1 expression decreases and disappears by 12.5 dpc, which should allow the Wnt ligands to bind Fzd5 receptors, resulting in endothelial growth. In the Fzd5 homozygous mutant embryos, however, the free Wnt ligands would not be able to signal through Fzd5 (Fig. 9). These results suggest that the Wnt signaling via frizzled receptors plays a key role in the endothelial cell growth, and that Frz5 activity may be regulated by secreted Wnt-receptor antagonists.

Fig. 9.

Schematic diagram of endothelial cell growth regulation by Wnt signaling through frizzled and soluble frizzled antagonist Sfrp1. (A) In the primary capillary plexus stage at 9.5 dpc, Sfrp1 binds the ligands, Wnt5a, Wnt10b and/or other Wnts, and therefore the unliganded receptor Fzd5 cannot be activated. (A) Along with remodeling and expansion of the vascular network, Sfrp1 expression is decreased and Fzd5-dependent signaling is activated, which induces growth of the endothelial cells and completion of the mature network.

Fig. 9.

Schematic diagram of endothelial cell growth regulation by Wnt signaling through frizzled and soluble frizzled antagonist Sfrp1. (A) In the primary capillary plexus stage at 9.5 dpc, Sfrp1 binds the ligands, Wnt5a, Wnt10b and/or other Wnts, and therefore the unliganded receptor Fzd5 cannot be activated. (A) Along with remodeling and expansion of the vascular network, Sfrp1 expression is decreased and Fzd5-dependent signaling is activated, which induces growth of the endothelial cells and completion of the mature network.

In conclusion, we have isolated a novel Wnt receptor Fzd5 that plays a key role in the blood vessel formation. Moreover, the results of Xenopus embryo injection assays for a secondary embryo axis suggest that Wnt2, Wnt5a and Wnt10b are possible ligands for this receptor. The co-localization of Wnt5a and Wnt 10b with Fzd5 in the developing yolk sac further suggests that Wnt5a and Wnt10b are two physiological ligands for Fzd5-dependent angiogenesis in the yolk sac, whereas Wnt2 is a likely ligand for vascularization in the embryonic placenta.

We thank J. Inuzuka and N. Matsuda for excellent technical assistance, Satomi Nishikawa and M. Hirashima for comments on immunohistochemistry, and K. Nakada for discussions for Xenopus microinjection constructs. A. M. Z. is grateful to Julia Mason for technical assistance. This work was supported by the Joint Research Fund between Banyu Pharmaceutical Co. and The University of Tokyo; Grants from the Japanese Government (MESSC) and Organization for Pharmaceutical Safety and Research (OPSR), Japan; and Grants from the National Institutes of Health (HG00734 to M. F. S.); and the Wellcome Trust, UK (A. M. Z.).

Accepted 4 October; published on WWW 27 November 2000

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