Cdx4 Is a Direct Target of the Canonical Wnt Pathway

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Developmental Biology 289 (2006) 55 – 63 www.elsevier.com/locate/ydbio

Cdx4 is a direct target of the canonical Wnt pathway

Nicolas Pilon a,1, Karen Oh a, Jean-Rene´ Sylvestre a, Nathalie Bouchard a, Joanne Savory d, David Lohnes a,b,c,d,*

a Institut de Recherches Cliniques de Montreál, 110 Avenue des Pins ouest, Montreál, Que´bec, Canada H2W 1R7 b Department of Molecular Biology, Universite´ de Montreál, Montreál, Que´bec, Canada c Division of Experimental Medicine, McGill University, Montreál, Que´bec, Canada d Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Canada K1H 8M5

Received for publication 19 April 2005, revised 3 October 2005, accepted 4 October 2005

Abstract

There is considerable evidence that the Cdx gene products impact on vertebral patterning by direct regulation of Hox gene expression. Data from a number of vertebrate model systems also suggest that Cdx1, Cdx2 and Cdx4 are targets of caudalizing signals such as RA, Wnt and FGF. These observations have lead to the hypothesis that Cdx members serve to relay information from signaling pathways involved in posterior patterning to the Hox genes. Regulation of Cdx1 expression by RA and Wnt in the mouse has been well characterized; however, the means by which Cdx2 and Cdx4 are regulated is less well understood. In the present study, we present data suggesting that Cdx4 is a direct target of the canonical Wnt pathway. We found that Cdx4 responds to exogenous Wnt3a in mouse embryos ex vivo, and conversely, that its expression is down-regulated in Wnt3avt/vt embryos and in embryos cultured in the presence of Wnt inhibitors. We also found that the Cdx4 promoter responds to Wnt signaling in P19 embryocarcinoma cells and have identified several putative LEF/TCF response elements mediating this effect. Consistent with these data, chromatin immunoprecipitation assays from either embryocarcinoma cells or from the tail bud of embryos revealed that LEF1 and h-catenin co-localize with the Cdx4 promoter. Taken together, these results suggest that Cdx4, like Cdx1, is a direct Wnt target. D 2005 Elsevier Inc. All rights reserved.

Keywords: Cdx; Wnt; LEF/TCF; Transcription; Vertebral patterning

Introduction critical regulators of axial patterning (Ikeya and Takada, 2001; Marshall et al., 1996; Yamaguchi et al., 1994; Chen et al., 1998; The somites arise from periodic segmentation of the paraxial Christ and Ordahl, 1995; Favier et al., 1996; Horan et al., 1995). mesoderm. Somites subsequently differentiate into dermatome, In the mouse, the 39 Hox genes are distributed in four myotome and sclerotome, the latter being the anlage of the clusters, Hoxa to d, which are believed to have derived from vertebrae and the ribs. Many vertebrae exhibit specific morpho- duplication of an ancestral complex related to the Drosophila logical characteristics according to their position along the melanogaster HOM-C (Duboule, 1998; Duboule and Dolle´, antero-posterior (AP) axis and must therefore be subject to 1989; Ferrier and Holland, 2001). In the mouse, Hox patterning. A number of signaling pathways, including retinoic transcripts are first detected at embryonic day 7.5 (E7.5) in acid (RA), Wnt/wingless (Wnt), and fibroblast growth factor the primitive streak with expression subsequently reaching a (FGF), are implicated in vertebral patterning, ultimately predetermined rostral limit in the neuroectoderm and paraxial impacting on Hox gene expression, the products of which are mesoderm (Deschamps and Wijgerde, 1993; Deschamps et al., 1999; Oosterveen et al., 2003; Roelen et al., 2002). The time of onset and rostral limit of expression are generally related to the * Corresponding author. Department of Cellular and Molecular Medicine, chromosomal location of a given Hox gene within a cluster, University of Ottawa, 451 Smyth Road, Canada K1H 8M5. Fax: +1 613 with 3V members of a given cluster expressed earlier and 562 5434. V E-mail address: [email protected] (D. Lohnes). reaching a more rostral limit than 5 members. This co-linear 1 Present address: De´partement de biome´decine ve´te´rinaire, Faculte´de pattern of expression results in staggered domains of Hox me´decine ve´te´rinaire, Universite´ de Montre´al, St-Hyacinthe, Que´bec, Canada. expression along the AP axis, which is believed to comprise a

‘‘Hox code’’ (Burke et al., 1995; Gaunt, 1994; Kessel and LEF1 and TCF1 are the most likely effectors of the Wnt Gruss, 1991). The results from grafting experiments in chick signal in the primitive streak and tail bud of the mouse embryo embryos have demonstrated that AP patterning and Hox (Galceran et al., 1999), and LEF1, TCF1 and a number of Wnt expression are established before overt somite segmentation, ligands, including Wnt3a, are co-expressed with the Cdx suggesting that the molecular cues impacting on Hox genes, genes in the caudal embryo (Beck et al., 1995; Gamer and and ultimately vertebral patterning, operate shortly after Wright, 1993; Meyer and Gruss, 1993; Oosterwegel et al., gastrulation (Christ and Ordahl, 1995; Christ et al., 1974; 1993; Takada et al., 1994). Consistent with these observations, Kieny et al., 1972; Nowicki and Burke, 2000). Wnt3a null mutants and the Wnt3a hypomorph vestigial tail Considerable work has been directed to an understanding of (Wnt3avt/vt) exhibit cervical vertebral defects which are the molecular regulation of vertebral Hox gene expression associated with reduced Cdx1 expression and posteriorized (Barna et al., 2002; Bel-Vialar et al., 2002; Charite´ et al., 1998; Hox expression (Ikeya and Takada, 2001; Prinos et al., 2001). Di Rocco et al., 2001; Gould et al., 1997; Isaacs et al., 1998; More posterior vertebral defects are also observed in Wnt3a Marshall et al., 1996). In the mouse, members of the vertebrate null mutants, suggesting that Wnt3a either directly regulates Cdx family, Cdx1, Cdx2 and Cdx4 have emerged as important the expression of more 5VHox genes or impacts on expression players in this process (Beck et al., 1995; Gamer and Wright, of another mediator of Hox expression; Cdx4 is a candidate 1993; Meyer and Gruss, 1993). for such an intermediary. The Cdx genes encode homeodomain transcription factors We now provide data suggesting that Cdx4 is a direct Wnt and are related to the Drosophila gene caudal. In the mouse target gene. We found that Cdx4 expression is induced by embryo proper, the Cdx genes are sequentially activated exogenous Wnt3a treatment ex vivo and that Cdx4 expression beginning at late streak stage (E7.5) and subsequently exhibit is attenuated in the tail bud of Wnt3avt/vt embryos or in nested domains of expression in both ectodermal and mesoder- embryos cultured in the presence of a Wnt antagonist. mal lineages of the caudal embryo. Numerous studies have Moreover, data from transfection analysis, electrophoretic demonstrated that Cdx members play key roles in vertebral mobility shift assays and chromatin immunoprecipitation patterning, at least in part by direct regulation of Hox expression. studies strongly suggest that this regulation is mediated by Loss-of-function studies in the mouse have shown that Cdx1À/À, LEF/TCF response elements in the Cdx4 proximal promoter. Cdx2+/À and Cdx1À/ÀCdx2+/À compound mutants display vertebral defects which correlate with a posterior shift in the Materials and methods rostral limit of mesodermal expression of a number of Hox genes (Chawengsaksophak et al., 1997; Subramanian et al., 1995; van Mice and in situ hybridization analysis den Akker et al., 2002). Cdx binding sites have been identified in vt/vt the promoter regions of a number of Hox genes (Beck et al., Vestigial tail (Wnt3a ) mice were obtained from Jackson Laboratory (Bar 1995; Subramanian et al., 1995), several of which have been Harbor, Maine, USA) and maintained on a C57BL/6 background. CD1 mice were used as wild-type controls; no difference in the expression of relevant demonstrated to regulate spatial expression in vivo (Charite´et genes between these wild type backgrounds has been noted. For in situ al., 1998). Gain- and loss-of-function studies in both chick and hybridization analysis, animals were mated overnight, and noon of the day of Xenopus models also support a role for Cdx proteins in vertebral detection of a vaginal plug was considered as E0.5. In situ hybridization was patterning via regulation Hox gene expression (Bel-Vialar et al., performed as previously described (Allan et al., 2001). Embryos to be 2002; Isaacs et al., 1998). compared were processed in parallel to control for variation in signal intensity and stage-matched according to somite count. Studies performed using diverse vertebrate model systems Embryo culture was performed as described previously (Prinos et al., 2001). have led to the hypothesis that Cdx members might serve, at least Briefly, embryos were dissected out in PBS containing 10% fetal bovine serum in part, to convey the caudalizing signals of RA, Wnt, and FGF and stored briefly in Dulbecco’s modified Eagle’s medium (DMEM) buffered pathways to the Hox genes (Lohnes, 2003). In this regard, we with HEPES. Embryos were cultured in DMEM/rat serum (50:50) pre- - and others have demonstrated that Cdx1 is directly regulated by equilibrated with 5% O2/5%CO2 in N2 at 37 C in the presence of supernatant from Wnt3a-expressing cells (Shibamoto et al., 1998) or control supernatant for RA and Wnt signaling (Houle et al., 2000, 2003; Lickert et al., 3 h prior to in situ hybridization analysis. Alternatively, embryos were cultured 2000; Ikeya and Takada, 2001; Prinos et al., 2001). FGF and/or for 8 h in the absence or presence of soluble Frizzled-Related Protein-2 (sFRP- Wnt pathways have also been suggested to impact on the 2, 0.5–5 Ag/ml, Cedarlane Laboratories) prior to in situ hybridization. expression of Cdx2 and/or Cdx4 (Shimizu et al., 2005; Pownall et al., 1996), although the precise mechanisms conveying these Isolation of Cdx4 genomic sequences and plasmid derivation effects is less well understood. To this end, we investigated a potential role for canonical Wnt signaling in affecting expression Three BACs harbouring Cdx4 were obtained by hybridization with a probe derived from the Cdx4 cDNA (Gamer and Wright, 1993) using a mouse BAC of Cdx4. library (Roswell Park Cancer Institute, Buffalo, USA). One of these was Wnt signaling is involved in numerous developmental employed as a template for PCR amplification of Cdx4 5Vflanking sequences processes (Moon et al., 2002; Wodarz and Nusse, 1998). In using the oligonucleotides: 5V-CTCGAGGGTGATGTGTCCACAGTTAC- the canonical pathway, Wnt occupation of the Frizzled receptor CAC-3V (forward); and 5V-CTCGAGGGTGACTTGGATAGAAGAAGTTGC- leads to stabilization of cytoplasmic h-catenin which then CC-3V(reverse); Xho I sites, used for subsequent cloning, are underlined. The anticipated 3967-bp product was subcloned into the pGEM-T vector translocates to the nucleus and associates with members of the (Promega), sequenced and used to generate a luciferase-based reporter LEF/TCF family of transcription factors (LEF1, TCF1, TCF3 construct (denoted 4 kb hereafter) using the Xho I site of pXP2 (Nordeen, and TCF4) resulting in transcription of target genes. 1988). The Cdx4 1.1-kb luciferase reporter plasmid, which consists of 1112 bp N. Pilon et al. / Developmental Biology 289 (2006) 55–63 57 of 5Vflanking sequences, was generated by Sma I digestion and religation of the were performed as described (Beland et al., 2004). DNAwas phenol–chloroform 4-kb vector. extracted and ethanol precipitated prior to PCR amplification. The 5V sequences were analyzed for potential regulatory elements using For re-ChIP analysis, an aliquot of anti-LEF1 immunoprecipitated MatInspector (Genomatix). Point mutations were introduced into three putative chromatin was liberated from A/G agarose beads by the addition of 100 Al10 LEF/TCF response elements (LRE) of the 1.1-kb luciferase reporter using the mM DTT. Samples were then diluted 1/40 in dilution buffer (1% tritonX-100, 2 Quick Change site-directed mutagenesis kit (Stratagene) according to mM EDTA, 150 mM NaCl, 20 mM Tris–HCl pH 8.1), incubated overnight manufacturer’s instructions. Upper strand sequences of the oligonucleotides with 5 Ag of rabbit anti-h-catenin antibody (Upstate 06–734) and processed as used for site-directed mutagenesis were LRE1 5V-CCCTTGACGTCAGCC- above. CAG CCGGATCCAACCCTCCTATGGCTCAC-3V; LRE2 5V-CAAATATC- For both cell and embryo samples, PCR was performed using primers TATCTGTCC AGAGAGATATCGCCCTCTCCTTGTGTGTGAG-3V; LRE3 flanking LRE2, LRE3 or exon 3 of Cdx4; the latter was used as a negative 5V-CCTGTCCTTTAGG GCACTGGAATTCAAGAAGACAGACGGAAAC- control. 30 or 36 amplification cycles (96- for 35 s, 60- for 30 s and 72- for 30 s) 3V(point mutations are denoted in bold). were used for cell and embryo samples, respectively. Products were resolved on a Hemagglutinin (HA) epitope-tagged LEF1, provided by L. Attisano, was 1.5% agarose gel, transferred to a Hybond N membrane (Amersham) and subcloned into pSG5 (Stratagene), while a Flag-tagged b-catenin expression hybridized with end-labeled internal probes specific to the predicted amplifica- vector was provided by D. Rimm. tion product.

Cell culture and transfection analysis Results

P19 embryocarcinoma cells were propagated as described previously Cdx4 expression is regulated by Wnt3a ex vivo (Prinos et al., 2001). For transfection analysis, approximately 400,000 cells were seeded into 6-well plates and transfected the same day using the calcium To investigate potential regulation of Cdx4 by Wnt phosphate precipitation method. Transfections consisted of 0.5 Ag of reporter vector, a LacZ expression vector (used for normalization of transfection signaling, E8.5 wild-type embryos were cultured in the efficiency) and equal amounts of LEF-1 and b-catenin expression vectors or presence of control media or media from Wnt3a producing empty vector (where required) to a total of 1 Ag of DNA per transfection. The cells and expression monitored by in situ hybridization. As following day, medium was replenished, and culture continued for 24 h. Cell shown in Fig. 1, Cdx4 expression was marginally, but h lysates were generated and assessed for luciferase and -galactosicase activity consistently, induced in embryos cultured in Wnt3a-condi- as described previously (Beland et al., 2004). tionned medium (Fig. 1B) relative to controls (Fig. 1A). Cdx4 F9 embryocarcinoma and Cos7 cells were cultured as previously described vt/vt (Beland et al., 2004). Transfections were performed as described above except expression was also assessed in Wnt3a embryos (Greco et that 100-mm plates were used with 25 Ag of relevant expression vector per al., 1996). Although expression was not detectably altered at plate. E8.5 (compare Figs. 1C and D), the rostral domain of expression was modestly, but reproducibly, reduced in the tail Electromobility shift assay (EMSA) bud of E9.5 Wnt3avt/vt embryos (Fig. 1F, compare to control in Fig. 1E). This outcome was not likely due to a general effect on Three micrograms of nuclear extracts from mock- or HA-LEF1-transfected mesoderm in the Wnt3avt/vt background, as expression of both Cos7 cells was used in each reaction as previously described (Houle et al., 2000). The upper strands of each wild-type double-stranded probe used were Cyp26A1 and Brachyury (T)(Abu-Abed et al., 1998; Wil- LRE1 5V-TGACGTCAGCCCAGCCTTTGGTAACCCTCCTATGGCTCAC-3V; kinson et al., 1990) was unaffected (Figs. 1H and J) relative to LRE2 5V-CTATCTGTCCAGAGAAACAAAGCCCTCTCCTTGTGT-3V; LRE3 wild-type controls (Figs. 1G and I). In this regard, although T is 5V-CCTTTAGGGCACTGACTTTGAAGAAGACAGACGGAAA-3V (putative also a Wnt3a target (Yamaguchi et al., 1999b), T transcripts are LREs sequences are underlined). The oligonucleotides comprising mutated stabilized at later stages in the tail bud of Wnt3avt/vt embryos LRE sequences were as described for site-directed mutagenesis. Antibody supershift assays were performed as described (Houle et al., 2000) using 1 Ag (Aulehla et al., 2003). of mouse monoclonal anti-HA antibody (Covance, Berkeley, California, USA). Cdx4 expression is attenuated by sFRP Chromatin immunoprecipitation (ChIP) analysis Alterations in expression of Cdx4 mediated by exogenous vt/vt Chromatin immunoprecipitation assays from either mock or HA-LEF1 Wnt3a or in the Wnt3a background were modest but transfected F9 embryocarcinoma cells were performed as previously described reproducible. In the case of Wnt3avt/vt, this minor effect could (Beland et al., 2004) using mouse monoclonal anti-HA antibody (Covance, be due to the relatively weak nature of this allele and/or Berkeley, California, USA). ChIP assays from embryos were performed using material from seventy five E9.5 embryos, separated into head or tail bud regions compensation by other Wnts, while induction by exogenous and fixed and dehydrated as described for in situ hybridization (Allan et al., Wnt3a may be masked by high Wnt activity in the caudal 2001). After rehydration, head and tail bud regions were cross-linked with 1% embryo. To further investigate this relationship, we assessed formaldehyde in PBS for 15 min at room temperature. Cross-linking was stopped the impact of the Wnt antagonist sFRP-2 (Kawano and Kypta, by adding glycine to a final concentration of 0.125 M. Samples were then 2003)onCdx4 expression. Embryos cultured ex vivo for 8 h in centrifuged, and pellets were washed with ice-cold PBS. Pellets were resuspended in 1 ml of RIPA buffer (10 mM Tris pH8.0, 140 mM NaCl, 1 mM the presence of sFRP-2 showed a marked reduction in EDTA, 1% TX-100, 0.1% SDS and 0.1% deoxycholate) containing protease expression of Cdx4 (Figs. 2B and C, compare to control in inhibitors and tissues disrupted by passage through a 26-G needle. After a 1- panel A). This was unlikely to be secondary to general effects h incubation, chromatin extracts were sonicated with 8 12-s bursts using a Sonic on the primary axis, as expression of the caudal marker Dismembrator M100 sonicator (Fisher Scientific) to an average DNA size of Cyp26A1 was not attenuated under identical conditions (Figs. 400–800 bp, pre-cleared with protein A/G agarose beads for 1 h followed by an overnight incubation with 5 Ag of goat anti-LEF1 antibody (Santa Cruz sc-8509). 2E and F, compare to control in panel D). These results are The following day, samples were incubated with protein A/G agarose for 4 h. consistent with a role for Wnt signaling in regulating Cdx4 Post-immunoprecipitation washes, chromatin elution and cross-link reversion expression. 58 N. Pilon et al. / Developmental Biology 289 (2006) 55–63

manner, suggesting that elements mediating this response are restricted to the proximal sequences. Analysis of this proximal interval using the MatInspector software revealed three putative LEF/TCF response elements located 41 bp (LRE1), 447 bp (LRE2) and 797 bp (LRE3) upstream of the transcription start site (Fig. 4A). These putative LREs exhibited perfect conservation to the core of the consensus motif (Fig. 4B; Clevers and van De, 1997). These motifs were assessed for binding ability by EMSA, which revealed that LEF1 could associate with both LRE2 and LRE3 in a manner similar to the binding observed to the Cdx1 LRE1 (Fig. 4C lanes 3, 11 and 26; Prinos et al., 2001). LEF1, however, did not exhibit detectable binding to the Cdx4 LRE1 under identical conditions (Fig. 4C, lane 19). These observations are in accordance with the respective homology of these motifs to the LRE consensus sequence, especially as regards conservation at position 7 (Fig. 4B; Clevers and van De, 1997). The specificity of LEF1 binding was further substantiated by supershift of the HA-LEF1/DNA complexes with an anti-HA antibody (Fig. 4C, lanes 4 and 12) and by lack of LEF1 association with mutated LRE2 and LRE3 probes (Fig. 4C, lanes 7 and 15). Consistent with the above data, mutation of LRE2 and LRE3 alone or in combination attenuated transactivation by LEF1-h-catenin in transfection assays in P19 cells (Fig. 4D). It is notable, however, that although LEF1 did not exhibit detectable binding to the LRE1 under our EMSA conditions, this motif did contribute to LEF1-h-catenin mediated transactivation (Fig. 4D), suggesting that it is a functional element. It is possible that LEF1 associated only weakly with these sequences in EMSA, or that chromatin-dependent events alter its affinity for LEF1 binding in vivo.

LEF1 and b-catenin co-localize on the endogenous Cdx4 promoter Fig. 1. Cdx4 expression is regulated by Wnt3a. Whole mount in situ hybridization of Cdx4 in stage-matched wild-type embryos following ex vivo culture in presence of control supernatant (A) or supernatant from Wnt3a- To further investigate Cdx4 as a potential Wnt target, we secreting cells (B). (C–F) Cdx4 expression was stage specifically down- performed chromatin immunoprecipitation (ChIP) analysis regulated in Wnt3avt/vt embryos. Note that expression was not altered at E8.5 in vt/vt using F9 embryocarcinoma cells as well as E9.5 embryos. As Wnt3a embryos (D) relative to wild-type controls (C), but the rostral limit of shown in Fig. 5A, in transfected F9 cells, HA-LEF1 was expression was reproducibly reduced in E9.5 Wnt3avt/vt embryos (F) compared to controls (E). By contrast, expression of the tail bud markers Cyp26A1 (G and associated with the Cdx4 promoter on intervals spanning either H) and T (I and J) was comparable between E9.5 Wnt3avt/vt embryos (H and J) LRE2 or LRE3 but not on more downstream (exon 3) sequences. and wild-type controls (G and I). Importantly, an identical outcome was observed for endogenous LEF1 in the tail bud, but not the head, of E9.5 embryos (Fig. 5B); this finding is consistent with high levels of LEF1 in the caudal Cdx4 is a direct target of LEF1-b-catenin embryo at this stage (Galceran et al., 1999; Oosterwegel et al., 1993). Moreover, LEF1 is likely active on the Cdx4 promoter in PCR was used to isolate 3967 bp of Cdx4 5V-flanking vivo, as re-ChIP analysis revealed that h-Catenin co-localized sequences including 3806 bp 5V of the transcription start site with LEF1 on the LRE2 interval specifically in the embryonic (Gamer and Wright, 1993). These sequences were used to tail bud (Fig. 5C). Taken altogether, these data strongly suggest generate luciferase reporter constructs using either the full- that Cdx4 is a direct target of the canonical Wnt pathway. length promoter fragment (denoted as the 4-kb Cdx4 reporter) or with the proximal 1112 bp (1.1-kb Cdx4 reporter). To Discussion determine whether Cdx4 is a potential direct target of the canonical Wnt pathway, these reporters were co-transfected We present evidence suggesting that Cdx4 is a direct Wnt together with LEF1 and h-catenin expression vectors in P19 target and is regulated by multiple LEF/TCF response elements embryocarcinoma cells. As shown in Fig. 3, both constructs in the proximal Cdx4 promoter. These LREs are necessary to responded to LEF1+h-catenin in a similar, dose-dependent mediate transactivation of the Cdx4 promoter by LEF1 and h- N. Pilon et al. / Developmental Biology 289 (2006) 55–63 59

Fig. 2. sFRP attenuates Cdx4 expression ex vivo. Cdx4 expression was assessed by whole mount in situ hybridization of wild type E9.5 embryos following ex vivo culture in the presence of recombinant mouse sFRP-2. (A–C) Cdx4 expression was down regulated in the presence of sFRP while Cyp26A1 (D–F) was unaffected. catenin in tissue culture. The attenuation of Cdx4 expression in with previous studies. For example, RA treatment, or Wnt3avt/vt embryos at E9.5 (or by the Wnt inhibitor sFRP-2) disruption of Cyp26A1 (which leads to high caudal RA together with ChIP data further supports the existence of this levels) results in down regulation of both Wnt3a and Cdx4 relationship in vivo. (Abu-Abed et al., 2003; Iulianella et al., 1999; Sakai et al., 2001); our present results suggest that this outcome reflects Cdx4 is a direct target of Wnt signaling Wnt-dependent regulation of Cdx4. Moreover, Wnt3avt/vt offspring exhibit defects affecting the lower thoracic, lumbar The present study suggests that Cdx4, like Cdx1,isa and sacral vertebrae (Ikeya and Takada, 2001). While similar direct Wnt target gene, an observation that is in accordance defects are not observed in Cdx1 null or Cdx2 heterozygote offspring, Cdx4 is expressed in the tail bud during the temporal window when the somites giving rise to these vertebrae are generated (Gamer and Wright, 1993; Tam et al., 2000). This observation, together with our present findings, is consistent with a reduction in Cdx4 expression in Wnt3avt/vt embryos leading to these homeosis. In this regard, although a definitive role for Cdx4 in vertebral patterning awaits analysis of the cognate null mutant, ectopic expression of Cdx4 has been shown to impact on Hoxb8 expression in the mouse (Charite´ et al., 1998). In addition, gain- and loss-of-function studies of zebrafish, Xenopus as well as chick also supports a role for Cdx4 in regulating Hox gene expression and axial patterning (Bel- Vialar et al., 2002; Davidson et al., 2003; Isaacs et al., Fig. 3. The Cdx4 proximal promoter sequences respond to canonical Wnt 1998). signaling in vitro. P19 embryocarcinoma cells were transfected with reporter In situ hybridization analysis suggested that Cdx4 expres- vt/vt constructs alone or with various amounts (0.05 to 0.25 Ag each) of LEF1 and b- sion was affected only at later stages (E9.5) in Wnt catenin expression vectors. Reporter vectors consisted of 4 or 1.1 kb of Cdx4 5V offspring. Is it difficult, however, to conclude that this genomic sequences that had been subcloned in the pXP2 luciferase expression observation is indicative of stage-specific effects, as residual vector. 48 h post-transfection, cells were harvested, and cell lysates were assessed for luciferase activity. Results are presented as fold induction mediated Wnt3a activity in the vestigial tail hypomorph may suffice for by LEF1 and h-catenin relative to values observed using the reporter vector early stages of Cdx4 expression. Alternatively, other Wnts alone. may compensate for a reduction in Wnt3a signaling during 60 N. Pilon et al. / Developmental Biology 289 (2006) 55–63

Fig. 4. Identification and characterization of LEF/TCF response elements in the proximal Cdx4 promoter. (A) Schematic representation and position relative to the transcription start site of the 3 putative LREs in the Cdx4 proximal promoter. (B) Sequence comparisons of the putative Cdx4 LREs relative to the consensus sequence. Mismatches are denoted by lowercase lettering. Abbreviations: Y, C or T; W (weak) A or T. (C) Electrophoretic mobility shift assay of the Cdx4 LREs. Double-stranded end-labeled oligonucleotides harboring sequences corresponding to LRE3 wild-type (lanes 1–4) or mutated sequences (lanes 5–8), LRE2 wild- type (lanes 9–12) or mutated sequences (lanes 13–16) and LRE1 wild-type (lanes 17–20) or mutated sequences (lanes 21–24) were compared to the LRE1 from the Cdx1 promoter as a positive control (lanes 25–27). Lanes 1, 5, 9, 13, 17 and 21 contained probe with no protein, lanes 2, 6, 10, 14, 18, 22 and 25 were incubated with nuclear extracts from mock-transfected Cos7 cells and lanes 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 26 and 27 were prepared from Cos7 cells that had been transfected with HA-LEF1. Binding specificity was assessed by adding a mouse monoclonal anti-HA antibody to the DNA–protein binding reaction (lanes 4, 8, 12, 16, 20, 24 and 27). LEF1 binding and anti-HA supershift (aHA SS) are indicated by arrows. All reactions were performed in parallel under identical conditions. (D) Impact of LRE mutation on LEF1-h-catenin induction of the 1.1-kb reporter. P19 embryocarcinoma cells were transfected with wild-type or mutated Cdx4 reporter constructs alone or with a LEF1 and b-catenin expression vectors. Results are expressed as fold induction compared to the relevant reporter vector alone. Note that concomitant mutation of all 3 LREs was necessary to totally abolish response. m, mutant. this window. Wnt3, Wnt5b and Wnt8 all exhibit overlapping pathways that are likely to regulate each gene. Cdx1, for expression patterns with Cdx4 in the primitive streak and tail example, is subject to synergistic regulation by retinoid and bud (Bouillet et al., 1996; Liu et al., 1999; Takada et al., Wnt pathways whereby loss of the Wnt signal leads to a 1994; Yamaguchi et al., 1999a) and are potential candidates profound reduction in the acuity of response to RA (Prinos et for such a role. In this regard, it is interesting to note that, al., 2001). unlike Cdx4, expression of the Wnt target gene Cdx1 is attenuated in Wntvt/vt embryos at E8.5 (Prinos et al., 2001; Cdx genes as integrators of AP patterning and axial elongation Ikeya and Takada, 2001). This may reflect variable sensitivity of Cdx1 and Cdx4 promoters to the Wnt signal per se or may Data from a number of studies suggest that the vertebrate be related to the differential interaction between combinatorial Cdx genes are direct targets of caudalizing signals, including N. Pilon et al. / Developmental Biology 289 (2006) 55–63 61

phak et al., 1997, 2004). A role for Cdx4 in this process is supported by work in Xenopus, where expression of a Cdx4 repressor (XcadEn-R) leads to an arrest of axial elongation (Isaacs et al., 1998). In addition zebrafish embryos homozygous for an autosomal recessive mutation in the Cdx4 ortholog ZFcad3 also exhibit severe tail defects (Davidson et al., 2003). Prior work (Lickert et al., 2000; Ikeya and Takada, 2001; Prinos et al., 2001) and the present data strongly suggest that both Cdx1 and Cdx4 are targets of canonical Wnt signaling. Given the axial truncation phenotype exhibited by both Wnt3a null mutants (Takada et al., 1994) and a role for Cdx1 and Cdx4 in this process (van den Akker et al., 2002), it is tempting to speculate that these Cdx members mediate the effects of Wnt3a signaling on axial elongation. However, in contrast to our findings, others have reported no detectable loss of expression of Cdx4 in Wnt3a null mutants (Ikeya and Takada, 2001). While our data strongly suggest that Cdx4 is a direct Wnt target gene, the lack of an appreciable loss of expression in Wnt3a null mutants suggests that there is not a simple linear relationship between Wnt3a, Cdx4 and axial elongation. However, this does not preclude combinatorial effects of loss of Wnt3a function on expression of both Cdx1 and Cdx4. Moreover, it is conceivable that other Wnts expressed in the caudal embryo may also impact on expression of these Cdx members. Consistent with this, morpholino knockdown of zebrafish Cdx1a and Cdx4 phenocopies the effects of Wnt3a/Wnt8 loss of function, leading to the complete loss of tail structures (Shimizu et al., 2005). Additional studies h Fig. 5. LEF1 and -catenin co-localize on the endogenous Cdx4 promoter. (A, are required to identify the complement of Wnt members B) Chromatin immunoprecipitation analysis of endogenous Cdx4 proximal promoter sequences in F9 embryocarcinoma cells (A) and in E9.5 embryos (B, impactingonexpressionofCdx4andtomorefully C). (A, B) Primers flanking LRE2, LRE3 or exon 3 of Cdx4 (as a negative understand the role of this transcription factor in the Wnt control) were used to amplify anti-LEF1 immunoprecipitated DNA from mock signaling pathway. In this regard, preliminary transgenic transfected (A, upper panels) or HA-LEF1 transfected (A, lower panels) F9 reporter analysis using the 4-kb genomic sequences isolated cells (A) or from E9.5 embryo heads (B, upper panels) and tail buds (B, lower for this study failed to reproducibly recapitulate the pattern panels). PCR products were resolved by agarose gel electrophoresis and assessed by Southern blot analysis using an internal oligonucleotide probe of expression of Cdx4 (data not shown), suggesting that specific to the predicted amplification products. Lanes 1 to 4 (input) and 5 to factors in addition to Wnt signaling are also required to 8 (immunoprecipitate) represent serial dilutions of DNA used for PCR govern Cdx4 expression. amplification. Lane 9 is a negative control in which DNA was not included. (C) Lanes 1 and 3 are PCR products from chromatin inputs prior to initial Acknowledgments immunoprecipitation with anti-LEF1. Lanes 2 and 4 are PCR products specific for the LRE2 or exon 3 from the head or tail bud, as indicated, generated from chromatin immunoprecipitated with anti-LEF1 and subsequently re-immuno- We thank Drs. C. Wright, B. Herrmann, M. Petkovich, L. precipitated with anti-h-catenin. Note the PCR product specific for LRE2 that Attisano and D. Rimm for their kind gifts of cDNA was generated only from the tail bud (lane 2, lower panel). plasmids and James Holcroft for his assistance with ChIP analysis. This work was supported by funding from the RA, Wnt and FGF. This has prompted the hypothesis that CIHR (Canada). N.P. is supported by a fellowship from Cdx genes serve to relay these signals to programs involved CIHR (Canada) and D.L. by the FRSQ (Que´bec). in axial patterning, such as regulation of Hox gene expression (Lohnes, 2003). The present data extend this hypothesis and References suggest that multiple Cdx members are targets of the canonical Wnt pathway. Abu-Abed, S.S., Beckett, B.R., Chiba, H., Chithalen, J.V., Jones, G., Metzger, In addition to vertebral patterning, Cdx gene products are D., Chambon, P., Petkovich, M., 1998. Mouse P450RAI (CYP26) also involved in axial elongation. For example Cdx2+/À expression and retinoic acid-inducible retinoic acid metabolism in F9 cells À/À +/À are regulated by retinoic acid receptor gamma and retinoid X receptor alpha. heterozygotes, Cdx1 Cdx2 compound mutants and J. Biol. Chem. 273, 2409–2415. chimeric Cdx2 null mutants all exhibit foreshortening of Abu-Abed, S., Dolle´, P., Metzger, D., Wood, C., MacLean, G., Chambon, P., the AP axis (van den Akker et al., 2002; Chawengsakso- Petkovich, M., 2003. Developing with lethal RA levels: genetic ablation of 62 N. Pilon et al. / Developmental Biology 289 (2006) 55–63

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