The Netrin-related domain of Sfrp1 interacts with Wnt ligands and antagonizes their activity in the anterior neural plate
© Lopez-Rios et al.. 2008
Received: 28 February 2008
Accepted: 20 August 2008
Published: 20 August 2008
Secreted frizzled related proteins (SFRPs) are multifunctional modulators of Wnt and BMP (Bone Morphogenetic Protein) signalling necessary for the development of most organs and the homeostasis of different adult tissues. SFRPs fold in two independent domains: the cysteine rich domain (SfrpCRD) related to the extracellular portion of Frizzled (Fz, Wnt receptors) and the Netrin module (SfrpNTR) defined by homologies with molecules such as Netrin-1, inhibitors of metalloproteinases and complement proteins. Due to its structural relationship with Fz, it is believed that SfrpCRD interferes with Wnt signalling by binding and sequestering the ligand. In contrast, the functional relevance of the SfrpNTR has been barely addressed.
Here, we combine biochemical studies, mutational analysis and functional assays in cell culture and medaka-fish embryos to show that the Sfrp1NTR mimics the function of the entire molecule, binds to Wnt8 and antagonizes Wnt canonical signalling. This activity requires intact tertiary structure and is shared by the distantly related Netrin-1NTR. In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated β-catenin transcriptional activity.
On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.
Secreted frizzled related protein
Bone morphogenetic protein:
Secreted frizzled related proteins (SFRPs) compose a family of soluble factors widely involved in the control of embryonic development and the homeostasis of adult tissues. Members of this family were independently isolated using a variety of approaches and immediately proposed as Wnt signalling inhibitors because of their ability to interfere with Wnt-induced embryonic axis duplication and forebrain development in Xenopus [1, 2]. Many studies have thereafter confirmed that addition of SFRPs can block Wnt-mediated signalling activation in different experimental paradigms showing possible binding preferences between SFRP and Wnt pairs (reviewed in ). Whether SFRP-mediated interference with Wnt signalling activation is the result of a single biochemical interaction between Wnt and SFRPs or instead reflects multiple binding mechanisms among SFRP, Wnt and their Frizzled (Fz) receptors is, however, a still unresolved issue.
Indeed, SFRP molecules fold in two independent domains: an amino-terminal cysteine-rich domain (CRD) and a carboxy-terminal Netrin-related motif (NTR) [4, 5]. The SfrpCRD contains ten cysteines with a pattern of five disulfide bridges identical to that of the extracellular CRD of Fz [6, 7]. Due to this structural relationship, it is generally assumed that Sfrp-mediated Wnt signalling inhibition results from the interaction between the ligand and SfrpCRD, which has been actually shown to immunoprecipitate with Wnt1 and Wnt2 [8, 9]. However, SfrpCRD can also form homo- and heterodimers with the CRD domain of Fz receptors [8, 10], suggesting potential alternative mechanisms of action.
The carboxy-terminal SfrpNTR is separated from the SfrpCRD by a linker region and is characterized by the presence of several conserved blocks of hydrophobic residues and a pattern of six conserved cysteines. NTR domains with similar features are found in a wide range of otherwise unrelated proteins, including Netrin-1, tissue inhibitors of metallo-proteinases (TIMPs), complement proteins and type I procollagen C-proteinase enhancer proteins (PCOLCEs) . Despite an initial suggestion that the SfrpNTR may interact with Wnt ligands , the participation of this domain in SFRP function has not been addressed.
Here, we have combined biochemical studies, mutational analysis and functional assays in cell culture and medaka-fish embryos to test the functional relevance of the SfrpNTR in Wnt signalling modulation. We show that the Sfrp1NTR mimics the function of the full-length Sfrp1, binds to Wnt ligands and prevents Wnt canonical signalling activation, effects shared by distantly related NTR domains such as that of Netrin-1. In contrast, Sfrp1CRD fails to interact with Wnt but binds to Fz receptors, possibly explaining the potential that the CRD has to inhibit Wnt signalling. We thus conclude that SFRPs modulate Wnt signalling by interacting with both Wnt ligands and Fz receptors but through different domains of the molecule and propose possible models of SFRP function that may reconcile data available in the literature.
Sfrp1NTR mimics the effect of the full-length protein in the anterior neural plate
Anteriorised phenotypes induced by over-expression of different Sfrp variants
Percentage of embryos
showing an enlarged forebrain
Sfrp1 (200 ng/μl; n = 70)
Sfrp1 CRD (100 ng/μl; n = 162)
Sfrp1 CRD-2(100 ng/μl; n = 86)
Sfrp1 NTR (120 ng/μl; n = 158)
Sfrp1 NTR-C177S;C180S (120 ng/μl; n = 48)
Sfrp2 (200 ng/μl; n = 62)
Sfrp2 NTR (120 ng/μl; n = 47)
Sfrp3 (200 ng/μl; n = 51)
4 (42; n = 40)†
Sfrp3 NTR (120 ng/μl; n = 38)
3 (27; n = 56)†
Sfrp3 CRD (100 ng/μl; n = 36)
0 (0; n = 75)†
Netrin-1 NTR (120 ng/μl; n = 61)
Netrin-1 NTR-C471S;C475S (120 ng/μl; n = 40)
Quite surprisingly, over-expression of Sfrp1CRD, the domain postulated to mediate SFRP-Wnt interactions, did not result in comparable phenotypes (Table 1). Instead, Sfrp1 CRD mRNA injected embryos presented a small but appreciable reduction of the forebrain (Figure 2c), which was associated with a diminished expression of prosencephalic markers (Figure 2g,k,o). Forebrain reduction was more evident at earlier stages of differentiation even with lower doses of mRNA (data not shown), supporting that the Sfrp1CRD gain-of-function phenotype did not reflect lower levels of peptide expression. Accordingly, Western blot analysis of embryos injected with haemagglutinin (HA)-tagged versions of the peptides indicated that Sfrp1 and Sfrp1 NTR mRNA were efficiently translated at comparable levels while the Sfrp1 CRD mRNA was produced in a larger amount, which existed in a monomeric and possibly a dimeric form (Figure 3).
Together, these data suggested that the molecular events induced by the two domains of Sfrp1 were probably different in nature. The Sfrp1 CRD -induced phenotype was difficult to explain according to the generally accepted view that this domain binds Wnt ligands and antagonizes their activity. In contrast, the strong anteriorisation observed after Sfrp1 and Sfrp1 NTR over-expression could be easily explained as the result of an early and generalized antagonism of the canonical Wnt pathway, since inhibition of this pathway induces similar anteriorised and dorsalised phenotypes in both fish and Xenopus embryos [1, 2, 13].
To investigate this possibility, we next assayed whether injection of Sfrp1 and Sfrp1 NTR could alleviate the phenotypes caused by Wnt8-mediated activation of canonical Wnt signalling. As previously shown in other species [14, 15], Wnt8 over-expression in medaka fish embryos led to a strong reduction of the forebrain associated with loss of the rx3-positive optic vesicles (Table 2; compare Figure 5a,e with Figure 5b,f). These anterior defects were similar to those observed after Sfrp1 CRD injections (Figures 2c,k and 5c,g; Table 2) but opposite to those induced by Sfrp1 or Sfrp1 NTR over-expression (Figures 2b,d,j,l and 5d,h; Table 2). Upon co-injection, Wnt8 and Sfrp1 mRNAs appeared to counteract each other's activity, resulting in mildly anteriorised embryos (Figure 5i,l; Table 2) that, however, still presented partial posterior truncations or axis duplications (Figure 5i,l). This suggests that, in the concentration range tested, Wnt8 cannot completely counteract Sfrp1-induced axial defects. In agreement with our previous observations, Sfrp1 NTR mRNA abrogated the Wn8-induced phenotype, restoring almost completely the size of the rx3 expression domain (Figure 5k,n; Table 2; compare to control embryos in Figure 5a,b). In contrast, Sfrp1 CRD , rather than counteracting, accentuated the reduction of the forebrain induced by Wnt8 (Figure 5j,m).
Antagonistic interaction between Sfrp variants and Wnt8/Wnt5
Wnt8 (50 ng/μl)
Wnt5 (50 ng/μl)
embryos showing a
embryos showing a
None (Wnt8/Wnt5 alone)
Sfrp1 (200 ng/μl)
Sfrp1 CRD (100 ng/μl)
Sfrp1 NTR (120 ng/μl)
Sfrp3 (200 ng/μl)
Sfrp3 CRD (120 ng/μl)
Sfrp3 NTR (100 ng/μl)
Altogether, these results challenged the view that the CRD domain of the Sfrp1 protein plays an important role in Wnt antagonism. To exclude the possibility that inadequate folding or destabilization of the Sfrp1CRD construct could mislead this interpretation, we designed an additional construct encoding the CRD and the entire linker region (Sfrp1CRD2; Figure 1) to ensure proper folding of the Sfrp1 CRD domain . Over-expression of this new construct, Sfrp1 CRD2, caused phenotypes similar to those observed upon Sfrp1 CRD injection (Additional file 1). As an alternative explanation, the behaviour of the Sfrp1CRD could reflect a peculiarity of this specific member of the SFRP family. Therefore, the CRD domain of Sfrp3 (Sfrp3CRD; Figure 1), the family member that diverges the most from Sfrp1 , was also analyzed. Interestingly, over-expression of Sfrp3 CRD had no morphologically evident effects on embryonic development, even at high concentrations (Additional file 1; Table 1) and, in contrast to Sfrp1 CRD , failed to enhance Wnt8-induced phenotype (Additional file 1; Table 2).
Sfrp1NTR effects are shared by distantly related NTRs and require intact tertiary structure
We next asked whether the tertiary structure of Sfrp1NTR was important for its function. The NTR motif is, in general, poorly conserved and mainly defined by the presence of six conserved cysteine residues that form three disulfide bonds [5, 11]. Mutations of the first two of these residues (Cys177 and Cys180) are predicted to disrupt two disulfide bonds, thus destabilizing the tertiary structure of the NTR domain. Indeed, over-expression of such a mutated construct (Sfrp1NTR-C177S;C180S; Figure 1) did not alter medaka embryonic development (Figure 7h; Table 1), indicating that intact tertiary structure of the NTR motif is required for Sfrp1 activity. Notably, analogous mutations of the first two conserved cysteines of the Netrin-1NTR (Netrin-1NTR-C471S;C475S; Figure 1) also interfered with, but surprisingly not totally abolished, the anteriorising activity of this domain (Figure 7j; Table 1).
Altogether, these data strongly support that the NTR domain has a relevant role in mediating SFRP function and that this role is conserved also in distantly related domains.
Sfrp1NTR and Sfrp1CRD bind to Wnt8 and Frizzled, respectively, antagonizing canonical signalling
In agreement with our finding that NTR domains of SFRPs are functionally relevant to Wnt signalling modulation, in vitro studies of the interaction between Sfrp1 and Wingless have mapped the relevant SFRP binding site to the carboxyl terminus of the protein . To assess whether a similar biochemical interaction between Wnt8 and Sfrp1NTR could explain our over-expression experiments in medaka fish embryos, we challenged Wnt8 interaction with the two Sfrp1 domains.
To further test the functionality of this interaction in β-catenin-mediated Wnt signalling and to compare it with that of other NTR domains, we performed TCF-luciferase reporter-based assays in embryonic retinal cells, where β-catenin-mediated transcriptional activity is physiologically low . We thus transfected retina cells with Fz5, a Wnt β-catenin associated receptor expressed in the anterior neural plate  to ensure Wnt8-mediated signalling activation . Fz5 alone or in combination with Sfrp1, Sfrp1CRD or Sfrp1NTR did not modify basal β-catenin activity (Additional file 3). Instead, co-transfection or addition of Sfrp1, Sfrp1NTR or Netrin-1NTR conditioned media strongly inhibited reporter activity induced by Wnt8 and Fz5 over-expression (Figure 8b; Additional file 3). Equivalent amounts of Sfrp3 or Sfrp3NTR were less effective (Figure 8b), in good agreement with what is observed in medaka fish embryos (Figure 7). In apparent contrast with immunoprecipitation experiments, co-transfection of Sfrp1 CRD also resulted in a significant decrease in reporter activity (Figure 8b). Notably, co-transfection with Sizzled or Sizzled CRD , a SFRP family member that does not appear to interfere with Wnt signalling , had a weaker activity (Additional file 3).
Sfrp1 has been shown to form complexes with Fz6  and Fz2 , while crystallographic studies have shown that Fz8CRD and Sfrp3CRD can form dimers . It was possible, therefore, that Sfrp1CRD-mediated inhibition of β-catenin transcriptional activity could result from Sfrp1CRD binding to the Fz5 receptor, thus preventing signal activation as previously proposed . To test this possibility, we performed co-immunoprecipitation studies using cell lysates from HEK 293T cells transfected with Fz5-HA, Sfrp1-myc or its derivatives or co-transfected with Fz2-HA, as a positive control , and Sfrp1-myc or its derivatives. As shown in Figure 8c, both Sfrp1 and Sfrp1CRD, but not Sfrp1NTR, interacted with Fz5-HA, supporting the possibility that Sfrp1CRD could impede Fz5 activation in TCF-luciferase reporter-based assays by competing with Wnt8 for binding to the Fz receptor. A similar interaction was also observed between Fz2-HA and Sfrp1CRD-myc as well as with the entire protein (Additional file 2), confirming and extending previous studies .
Wnt signalling contributes to the regional specification of the anterior neural plate. Acquisition of diencephalic, eye and telecencephalic identities, however, require a differential contribution from canonical and non-canonical Wnt pathways, which are regulated by different Wnt antagonists, including Sfrp1 . Accordingly, Mo-based knock-down of Sfrp1, a Wnt antagonist broadly expressed in the anterior neural plate, strongly reduces the eye field size, concomitantly expanding the telencephalic but not the diencephalic or mesencephalic territories in the medaka fish . Conversely, Sfrp1 over-expression leads to expansion of the forebrain associated with posterior truncations and axial duplications . Taking advantage of these activities, we have shown here that the NTR domain of Sfrp1 mimics the function of the full-length protein, binds to Wnt8 and antagonizes Wnt-canonical signalling. This activity requires an intact tertiary structure and is shared by the distantly related Netrin-1NTR. In contrast, the Sfrp1CRD does not mirror the effects of Sfrp1 over-expression but interacts in vitro with Fz receptors and antagonizes Wnt8-mediated β-catenin transcriptional activity, indicating that Wnt signalling modulation may involve multiple and differential interactions among Wnt, Fz and SFRPs.
These are somewhat surprising observations because it is generally accepted that Wnt-SFRP interaction takes place through the CRD domain due to its high degree of conservation with the extracellular portion of the Fz receptors [8, 9]. Several studies in fact have provided convincing evidence that, when used in large amounts compared to Wnt protein concentration, SFRPs or their respective SfrpCRD can efficiently block Wnt signalling in different contexts, such as in Xenopus axis formation [1, 9], neural tube , somites  and heart formation , although a certain specificity among SFRPs has been observed. Furthermore, studies using cell lysates from co-transfected cell lines have shown physical interactions between Wnt1 or Wnt2 and Sfrp3CRD [8, 9].
In contrast with this view, we have provided evidence in favour of the relevance of the NTR domain in SFRP-Wnt interaction. Although our data suggest that SfrpCRD more likely interacts with Fz receptors, there are several possibilities worth considering as to why we may have failed to observe a clear interaction between Sfrp1CRD and Wnt. In the simpler scenario, the difference we have observed between the Sfrp1NTR and Sfrp1CRD domains' abilities to mimic the effect of the entire molecule could have been related to a differential translation efficiency of their respective mRNA within the embryos. However, this possibility seems quite unlikely because western blot analysis of embryo lysates injected with equimolar amounts of tagged molecules indicated that the different peptides were produced with similar efficiency and, if any, the Sfrp1CRD was expressed at higher levels. Similarly, Sfrp1CRD-myc was retrieved at consistently higher levels in the culture medium from transfected cell lines  and in primary cultures from retinal cells (unpublished observations). Furthermore, the reduction of the eye field observed after Sfrp1 CRD injections was observed even with low mRNA doses.
A second possibility may relate to the stoichiometry of the SfrpCRD-Wnt interaction. It has been proposed that a dimer of the CRD Fz8 domain binds Wnt8  and dimerisation of the receptor may increase efficiency of signal transduction . If Sfrp1CRD dimers form and bind Wnt8 more efficiently, it is possible that we may have missed this interaction since we noticed that we mostly immunoprecipitate the monomeric form (Figure 8a(iv)). This possibility, however, does not explain why in the reverse inmunoprecipitations (Additional file 2) the Wnt8-Sfrp1CRD immunocomplex was not observed. Similarly, it does not explain why Sfrp1CRD cannot counteract Wnt1/5/8 function in vivo, where both monomers and possible dimers seem to be present in similar amounts (Figure 3).
As a third possibility, failure of the SfrpCRD to antagonize Wnt signalling may reflect specificity of binding. Although we have shown that SfrpCRD failed to interact with Wnt8 and did not counteract the effect of Wnt1, Wnt5 and Wnt8 overexpression, we cannot exclude that Sfrp1 might show selectivity of binding through the two domains with Wnts other than those we have tested.
In agreement with our view of the importance of the SfrpNTR domain in Wnt activity, several studies have provided indirect evidence in favour of the relevance of this domain. In Drosophila, the CRD motif of Dfz or Dfz2 is dispensable for Wg signal transduction and Frizzled proteins lacking the CRD can fully rescue the simultaneous loss of different Fz receptors or partially rescue the canonical signalling in fz/fz2 double mutants . Furthermore, a carrier function for the CRD has been suggested in studies where the CRD domain of the Drosophila fz receptor has been substituted with the structurally distinct Wnt-binding domain or with wingless itself . A recent study, aimed at demonstrating the interaction between Norrin and Fz4, failed to reveal a positive interaction between the CRD domain of all human SFRP family members and Xwnt8, which instead interacts with the CRD domain of Fz4, 5, 7 and 8 (see Figure 2 in ). Furthermore, in vitro analysis of the interaction between Sfrp1 and Wingless mapped the relevant SFRP binding site to the carboxyl terminus of the protein . Our biochemical and functional data are in line with this set of data, strongly supporting the proposal that the NTR domain has a relevant role in mediating Sfrp function. This role is conserved also in distantly related domains. Indeed, the NTR of Sfrp1, 2, and 5 shares a quite similar pattern of cysteine spacing, related to that of Netrin-1. Conformational similarities are, therefore, likely to explain why over-expression of Sfrp1NTR, Sfrp2NTR and Netrin-1NTR results in all cases in forebrain expansion and effective inhibition of Wnt8-induced β-catenin activation. In contrast, Sfrp3NTRand Sfrp4NTR display a different cysteine spacing and, thus, a distinct pattern of disulphide bonds , supporting that variations in the NTR structural features could underlie the differences in activities observed among the distinct subgroups of the family [5, 16], as we have observed with Sfrp3NTR.
The crystallographic resolution of the structure of the mouse Sfrp3 and Fz8 CRD domains revealed the potential for the different CRDs to homo- or heterodimerise . This potential has also been demonstrated in biochemical studies where SFRPs and Fzs and/or their CRDs have been shown to form homo- and/or hetero-complexes [8, 24, 31]. In line with these data, we have demonstrated a physical interaction between Sfrp1CRD and Fz5 and Fz2. This binding may very well justify the potential of the Sfrp1CRD to antagonize, albeit with lower efficiency, Wnt8-induced β-catenin activation, as we have observed in our experimental conditions mimicking the physiological extracellular interactions among Fz, Wnt and SFRPs. This interaction also provides a mechanism, based on functional inactivation of the receptor, to explain why, in many studies, addition of high levels of the CRD alone is sufficient to prevent Wnt signalling activation. The reason why, in our studies, Sfrp1 CRD over-expression in medaka fish embryos seems to synergize rather than prevent the effect of Wnt8 over-expression (Figure 2) is, however, unclear. As a tempting speculation, Sfrp1CRD may have higher affinity for Fz receptors that, like Fz2 , are involved in mediating non-canonical signalling, which, in turn, has been shown to antagonize the Wnt canonical pathway during eye field specification . Alternatively, in the embryo, Sfrp1CRD may interfere with other cell signalling pathways, as demonstrated for the CRD of Sizzled, a related family member that binds and inhibits Tolloid/BMP1, metalloproteases that normally degrade the BMP inhibitor chordin, thereby promoting BMP signalling [23, 37].
Genetic manipulations selectively eliminating one or the other domain of SFRPs may provide further insights and help resolve the accuracy of these models. Additional studies characterizing the functionally relevant interactions among SfrpNTR-Wnt or SfrpCRD-Fz pairs are also undoubtedly needed. Interaction with additional components of the Wnt signalling cascade also needs to be addressed. Particularly relevant might be the contributions of proteoglycans, which are known to bind Wnts  and may additionally interact with the SfrpNTR (PE, unpublished observations). An accurate establishment of SFRP mode of action is indeed particularly important given the growing interest in these molecules raised by the observations that their expression is altered in different type of cancers, bone pathologies, retinal degenerations and hypophosphatemic diseases, pointing to their potential value as therapeutic targets.
Materials and methods
Whole-mount in situ hybridisation
Whole-mount in situ hybridizations were performed in medaka embryos using digoxigenin- and fluorescein-labelled riboprobes. A minimum of 40 embryos were hybridized for each marker and condition. All embryos shown correspond to Iwamatsu stage 19–20 .
olSfrp1, mWnt8a, zWnt5 and zWnt1 expression constructs have been described [13, 36, 41, 42]. zSizzled was a kind gift of Dr Hibi and xSizzled of Dr E De Robertis. Medaka Sfrp2 full length clone corresponds to the expressed sequence tag MF01SSA080C03, kindly provided by Dr. Takeda. zSfrp3 and olNetrin-1 where cloned by RT-PCR using specific primers. Full length, truncated and chimerical coding sequences of Sfrp1, Sfrp2, Sfrp3 and Netrin-1 where cloned by PCR into pCS2+. All chimerical constructs where designed so that the signal peptide of the corresponding protein was fused in frame with the linker region that precedes the NTR domain, ensuring proper secretion of the corresponding peptide (Figure 1). Cysteine to serine mutations were introduced into the NTR of both Sfrp1 and Netrin-1 by PCR. Given the structural similarity between serine and cysteine, this substitution is expected to disrupt di-sulphide bridge formation without altering the secondary structure of the peptide. Carboxy-terminal 3xHA tagged constructs of Sfrp1, Sfrp1CRD and Sfrp1NTR were generated with linker oligos. All constructs were fully sequenced to ensure in-frame fusions.
mRNA and morpholino injections
pCS2 plasmids were linearised and transcribed in vitro using the SP6 Message mMachine kit (Ambion, Austin, TX, USA). The synthesized mRNA was purified and injected into two-cell stage embryos at different concentrations (titration curve: 50–300 ng/μl) and the severity of the induced phenotypes was dose dependent in all the cases. Injection solutions included 30 ng/ml of hGFP mRNA as a lineage tracer. Selected working concentrations correspond to equimolecular amounts of the different Sfrp mRNAs (full length, truncated and chimerical) to obtain equivalent protein levels (Tables 1 and 2). Mo studies were performed as previously described  using the following tested Mo (Gene Tools, LLC, Philomath, OR, USA) designed against olSfrp1: 5'-CTGTGTTT GTAGGAACCTCGACTGG-3'. Mo were injected at the final concentration of 0.3 mM into one blastomere of embryos at the two-cell stage. For co-injection experiments, 60 ng of Sfrp1 or 30 ng of Sfrp1 CRD or 35 ng of Sfrp1 NTR mRNAs were used. At least three independent experiments were conducted for each marker and condition.
Protein expression and immunoprecipitations
To determine the efficiency of translation of the Sfrp1 and its derivatives, triply-HA tagged constructs were generated (see above) and their respective mRNAs were injected into medaka embryos in equimolecular amounts (Sfrp1-3HA, 200 ng/μl; Sfrp1 CRD -3HA, 100 ng/μl; and Sfrp1 NTR -3HA, 120 ng/μl) together with GFP mRNA as a tracer. For each construct, 30 embryos were treated with lysis buffer (150 mM NaCl; 1% NP40; 50 mM Tris pH 8; 10 μg/ml aprotinin; 10 μg/ml leupeptin and 1 mM phenylmethanesulphonylfluoride (PMSF). Lysates were precipitated with a polyclonal anti-HA (Sigma-Aldrich, St Louis, MI, USA) and Protein G-Sepharose for enrichment. The protein complex present in each of the pellets was re-suspended in 2 × SDS sample buffer containing 1 M urea. The proteins were resolved by SDS-PAGE blotted and the membranes probed with a monoclonal anti-HA (Sigma-Aldrich). Proteins from total cell extracts were subjected to SDS-PAGE, blotted and the membranes probed with an anti-GFP antibody (Molecular Probes, Invitrogen, Carlsbad CA, USA) and a secondary anti-rabbit-POD antibody.
Sub-confluent HEK 293T cells were transiently and separately transfected with constructs encoding chick Wnt8c-HA, chick Sfrp1-myc or Sfrp1CRD-myc or Sfrp1NTR-myc in 2% fetal calf serum. After 2 days, the conditioned media were collected and clarified by centrifugation. The amount of protein present in the conditioned media was evaluated by western blot and similar amounts of peptides derived from each Sfrp1-myc present in the conditioned media were mixed with conditioned medium from Wnt8-HA or mock transfected for 2 hours. Sample volumes were adjusted to 600 μl with lysis buffer (as above). Proteins from conditioned media were precipitated with 3 μg of an anti-HA polyclonal antibody (Sigma-Aldrich) and Protein G-Sepharose. After four washes with lysis buffer, the protein complex was subjected to SDS-PAGE, blotted and the membranes probed with a monoclonal anti-myc antibody (9E10) and a secondary anti-mouse-POD antibody. Signal was detected with the Advanced ECL Western blotting detection Kit analysis (GE Healthcare Life Sciences, Pollards Wood, Buckinghamshire, UK). Reverse inmunoprecipitation experiments were performed using similar incubations of conditioned media. Proteins were precipitated with a polyclonal anti-myc antibody (SIGMA). The immunocomplexes were subjected to SDS-PAGE, blotted and the membranes probed with a monoclonal anti-myc antibody (9E10) and a secondary anti-mouse-POD antibody.
For Fz2 and Fz5 immunoprecipitations, HEK 293T cells were transiently transfected with mouse Fz2-HA, chick-Sfrp1-myc or Sfrp1 CRD-myc or Sfrp1 NTR-myc or cotransfected with mouse Fz5-HA and chick-Sfrp1-myc or Sfrp1 CRD-myc or Sfrp1 NTR-myc expression constructs. After 2 days, cells were scraped in lysis buffer (as above). Immunoprecipitations were performed as previously described .
Dissociated cells from embryonic day (E)5 central retinas were prepared as described , seeded in 24-well plates and transfected 3 hours later using the FuGENE HD Transfection Reagent (Roche, Nutley, NJ, USA). In each case the 700 ng/well of total DNA contained 200 ng of a plasmid containing a 4xLef-1 responsive luciferase reporter and 50 ng of pRL-TK (Promega, Madison, WI, USA) together with variable amounts of the effector plasmids or the empty vector. After 24 hours, luciferase activities were determined using a dual-luciferase assay system (Promega). The LEF-1 reporter luciferase activity was normalized with that of the Renilla luciferase to account for transfection efficiency. Data were statistically evaluated using the SPSS v15.0 software (SPSS Inc., Chicago, Illinois, USA) applying a one-way ANOVA test plus post hoc test (Dunnet test).
Live embryos were visualized at room temperature under a Leica stereomicroscope equipped with a PLANAPO objective. Embryos processed for in situ hybridization were fixed with 4% paraformaldehyde (PFA) and equilibrated in 80% glycerol. After removal of the yolk, embryos were mounted and visualized under a Leica microscope. In all cases, images were captured with a Leica digital camera controlled by the Leica software.
We are grateful to Drs F Cavodeassi, EM De Robertis, JL Gomez-Skarmetha, CP Heisenberg, M Hibi, and H Takeda for providing us with Wnt1, Xsizzled, Wnt8, Wnt5, zSizzled and Sfrp2 plasmids, respectively. We are also in debt to Drs E Cisneros, JR Martinez-Morales, G Nusspaumer, S Rodríguez de Córdoba, and R Zeller for critical reading of the manuscript and Dr K Heath for editorial assistance. This work was supported by grants from the Spanish MEC (BFU2004-01585 and BFU2007-61774), the Fundación la Caixa (BM04-77-0), the Fundación Mutual Madrileña (2006-0916), and Comunidad Autonoma de Madrid (CAM, P-SAL-0190-2006) to PB.
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