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. Author manuscript; available in PMC: 2004 Nov 8.
Published in final edited form as: Genes Dev. 2001 Jun 1;15(11):1427–1434. doi: 10.1101/gad.194301

A mutation in the Gsk3–binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon

Carl-Philipp Heisenberg 1,5, Corinne Houart 1,6, Masaya Takeuchi 1,6, Gerd-Jörg Rauch 2, Neville Young 3, Pedro Coutinho 4, Ichiro Masai 1, Luca Caneparo 1, Miguel L Concha 1, Robert Geisler 2, Trevor C Dale 3, Stephen W Wilson 1,7, Derek L Stemple 4
PMCID: PMC312705  NIHMSID: NIHMS7463  PMID: 11390362

Abstract

Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. Here we show that mbl−/− embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl−/− embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3β can restore eye and telencephalic fates to mbl−/− embryos. Our data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories.

Keywords: Axin, Wnt, forebrain, zebrafish

Although considerable progress has been made in elucidating the genetic pathways that regulate dorsoventral patterning of the vertebrate CNS (Lee and Jessell 1999; Jessell 2000), it remains uncertain how early anterior-posterior (AP) pattern is established (Lumsden and Krumlauf 1996). Studies performed mainly in amphibia have suggested various models of AP patterning in which early neural tissue has anterior character upon which posterior character is imposed by signals originating in more caudal regions of the embryo (Gamse and Sive 2000). Among the signals that are suggested to influence regional identity along the AP axis are Fgfs, retinoic acid, Nodals, and Wnt proteins (Gamse and Sive 2000; Wilson and Rubenstein 2000).

Misexpression studies have suggested that suppression of Wnt signaling is required for induction of the vertebrate head (Niehrs 1999). Direct genetic evidence supporting this conclusion has come from the recent demonstration that headless (hdl) mutant zebrafish embryos carry a mutation that abolishes the repressor function of Tcf3, a transcriptional modulator of Wnt signaling (Kim et al. 2000). Although the evidence implicating the Wnt pathway in head development is substantial, it remains to be determined where, when, and how Wnt signaling must be suppressed to promote head formation.

Levels of Wnt signaling can be regulated negatively through the activity of a variety of extracellular proteins that include members of the Cerberus, Dickkopf, and Sfrp families (Niehrs 1999). Additionally, several intracellular components of the Wnt signaling pathway can modulate the degree to which Wnt target genes are activated or repressed (Brown and Moon 1998; Wodarz and Nusse 1998). For instance, the protein kinase Gsk3 phosphorylates β-catenin, targeting it for degradation (Aberle et al. 1997; Ikeda et al. 1998; Kishida et al. 1999), thereby limiting the amount of β-catenin available to activate Wnt target genes. Gsk3 functions in multicomponent complexes, one of which appears to include adenomatous polyposis coli, Axin, and additional proteins (Kim et al. 1999; Barker and Clevers 2000). Within this complex, Axin is believed to function as a scaffolding protein that enables Gsk3 to efficiently colocalize with, and thereby phosphorylate β-catenin (Ikeda et al. 1998; Kishida et al. 1999). The eventual nuclear readout of Wnt signals therefore can be modulated in many ways and at several different positions in the Wnt signal transduction pathway.

In this study, we show that alterations in Wnt signaling underlie one of the most dramatic mutations known to affect AP patterning of the brain. masterblind (mbl) mutant embryos exhibit major fate switches within the prospective forebrain and here we show that the mbl phenotype is a result of a mutation in Axin1 that leads to overactivation of Wnt signaling.

Results and Discussion

mbl mutant embryos show a fate transformation of telencephalon and eyes to diencephalon

Zebrafish homozygous for the mbltm213 mutation (mbl−/−) possess a head but exhibit major alterations in fate allocation within the forebrain such that eyes and telencephalon are reduced or absent while the diencephalic epiphysis is expanded anteriorly (Heisenberg et al. 1996; Masai et al. 1997) (Fig. 1A–F). The expressivity of the mbl−/−phenotype is temperature-dependent and small eyes are present in 20% of mutants reared at 22°C (Fig. 1C,D) while they are absent when rearing is at 30°C (Fig. 1E,F). This suggests that the mbl allele may generate a protein that has temperature-dependent activity.

Figure 1.

Figure 1

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mbl−/− embryos exhibit fate transformations within the forebrain. (A,C,E) Lateral views of living wild-type (A) and mbl−/−embryos raised at 22°C (C) and 30°C (E). (B,D,F) Lateral views of forebrains of wild-type (B) and mbl−/− embryos raised at 22°C (D) and 30°C (F) stained with an α-acetylated tubulin antibody. Eyes (arrowheads) and telencephalon (asterisks) are reduced in the embryo raised at 22°C, whereas they are absent in the embryo raised at 30°C indicating that the mbl mutation is temperature-sensitive. (G–R) Differences in gene expression between neural plates of mbl−/− (mbl) and wild-type embryos. Genes analyzed a re indicated at bottom right. Axial mesendodermal tissue is labeled additionally with a flh probe in (G,H) and gsc and flh probes in (Q,R). For deltaB (M,N), both diencephalic expression (arrow) and expression in prospective trigeminal neurons (asterisk) is rostrally expanded in the mbl−/−embryo. Gene expression characteristic of telencephalic and eye field fates (G–L) are reduced or absent in mbl−/− embryos while gene expression characteristic of more caudal diencephalic fates (M–P) is expanded.

The fate changes within the forebrain of mbl−/− embryos are reflected in alterations to the expression of genes within the anterior neural plate from gastrulation stages onwards. In mbl−/− embryos, genes expressed in the prospective telencephalon (anf, emx1; Fig. 1G–J) and eyes (rx3; Fig. 1K,L) are reduced or absent by bud stage, whereas genes expressed within the mid/caudal diencephalon (fkd3, deltaB; Fig. 1M–P) are expanded anteriorly. In contrast, the expression domains of genes within the prospective midbrain (pax2.1) are only mildly altered or show no obvious changes in mbl−/− embryos (Fig. 1Q,R). These results indicate that anterior forebrain territory adopts posterior forebrain character by late gastrulation in mbl−/− embryos.

mbl−/− embryos carry a mutation in the Gks3 binding domain of Axin1

To understand the role of Mbl in early forebrain patterning, we identified the mutation likely to be responsible for the mbl−/− phenotype. Mapping of the mbl mutation showed it to be adjacent to the centromeric region of linkage group 3, close to the Wnt scaffolding protein encoding gene, axin1 (Shimizu et al. 2000) (Fig. 2A). Mice lacking Axin function have axis duplications (Jacobs-Cohen et al. 1984), supporting the conclusion that Axin is a negative regulator of Wnt signaling (Zeng et al. 1997). As suppression of Wnt signaling can promote anterior fates (Niehrs 1999), we tested axin1 as a candidate for harboring the mbl mutation. Sequencing of the mbl axin1 allele revealed a leucine to glutamine (L→Q) amino-acid exchange at position 399 within the Gsk3 binding domain (Ikeda et al. 1998) of Axin1 (Fig. 2B) raising the possibility that altered Axin1 function indeed could be responsible for the mbl−/− phenotype.

Figure 2.

Figure 2

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mbl−/− embryos harbor a mutation in the axin1 gene. (A) Mapping of mbl on linkage group 3 close to axin1 on the corresponding region of a radiation hybrid map. (B) Schematic representation of the Axin1 protein showing the amino-acid exchange (L399→Q) and underlying point mutation (T→A) within the predicted Gsk3 binding site in the mbl Axin1 allele. (C–F) Localization of axin1 transcripts in lateral (D,E) and animal pole (C,F) views of wild-type embryos at the 1 6 cell stage (C), sphere stage (D), 80% epiboly (E) and bud stage (F). axin1 transcripts are maternally provided and ubiquitously expressed in wild-type embryos. (G–L) Phenotypes of wild-type (G,J), mbl−/− (H,K), and mbl−/− embryos injected with axin1 RNA (I,L) stained for the expression of flh in the epiphysial region of the diencephalon (arrow,G) at bud stage (G–I) or stained with α-acetylated tubulin antibody at pharyngula stage (J–L). Injection of axin1 RNA at the one-cell-stage rescues the mbl−/− mutant phenotype determined by the pattern of flh expression at bud stage and the presence of telencephalon (asterisks) and eyes (arrowheads) at pharyngula stage.

To determine if axin1 is expressed at appropriate times and places to regulate early forebrain patterning, we analysed axin1 RNA distribution. axin1 is maternally provided and ubiquitously expressed throughout early development with slightly higher expression anteriorly at the end of gastrulation (Fig. 2C–F). The expression of axin1 is not noticeably changed in mbl−/− mutant embryos (data not shown). Axin1 therefore is likely to be present throughout the embryo at stages when we believe Mbl function is required.

To test if altered Axin1 function is indeed responsible for the mbl−/− phenotype, we provided mbl−/− embryos with RNA encoding wild-type Axin1. Widespread expression of wild-type axin1 RNA in mbl−/− embryos efficiently rescues early neural plate patterning and restores eyes and telencephalic structures at later stages (Fig. 2G–L; Table 1). In contrast, injection of equivalent (or higher) levels of mbl axin1 RNA into mbl−/− embryos did not rescue the mbl−/− phenotype (Table 1; data not shown). Therefore, the mutation within Axin1 is likely to be responsible for the fate transformations within the anterior neural plate of mbl−/− embryos.

Table 1.

Wild-type Axin1 rescues eye1 development in mbl−/− embryos

Injected RNA Genotype Normal (n) Absent (n) Small (n) Total (n)
GFP (control) mblxmbl 78 27 0 105
axin1(L→Q) mblxmbl 87 29 0 116
axin1 mblxmbl 77 0 20 97
gsk3β mblxmbl 73 15 8 96
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1

Size of the eyes was scored at pharyngula stage. 1 0 pg/embryo of GFP, axin1 and axin1 (L→Q) RNA, and 200 pg/embryo of gsk3β RNA were injected.

Axin1/Gsk3 interactions are disrupted by the mbl mutation

The L→Q amino-acid change in Axin1/Mbl occurs within the Gsk3 binding domain (e.g., Ikeda et al. 1998) raising the possibility that Axin1/Gsk3 interactions may be disrupted in mbl−/− embryos. Mutations at the same or nearby sites in human Axin have been identified in cells from colon cancers and in vitro assays have shown that such mutations can reduce binding of Axin to Gsk3 (Webster et al. 2000). We therefore introduced the L→Q amino-acid change at an equivalent position in a murine Axin construct (Fig. 3A) and tested if this affects binding to Gsk3. Gsk3 is efficiently immunoprecipitated by the wild-type Axin construct but not by the Axin construct incorporating the mbl mutation (Fig. 3B). Therefore, the L→Q amino-acid exchange in mbl Axin1 is likely to impair Axin1 binding to Gsk3 in mbl−/− embryos.

Figure 3.

Figure 3

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The mbl mutation a bolishes binding of Axin to Gsk3. (A) Comparison of the amino-acid sequence around the L→Q mutation site within the Gsk3 binding domain of Mbl/Axin1 between zebrafish, mouse, and human shows a high degree of conservation. (B) Assessment of binding of Gsk3 β to a wild-type Axin construct (FlgΔN-Axin) and to an Axin construct incorporating the L→Q amino-acid exchange (FlgΔN-Axin L–Q). Wild-type and/or mutant (L–Q) FlgΔN-Axin and HA-Gsk3β were immunoprecipitated and immunoblotted with α-Gsk-3β and α-Flg antibodies. The L–Q mutation prevents both endogenous Gsk-3-β and HA-Gsk-3β association with the Axin protein. The ΔN variant of Axin (amino acids 351–965) was used in preference to full length Axin because of its greater solubility in vitro (Smalley et al. 1999). (C) Assessment of TCF-dependent transcription in the presence of Flg-ΔNAxin and Flg-ΔNAxin L–Q. Expression of Flg-ΔNAxin L–Q activates TCF-dependent transcription indicating that it can act as a dominant active protein while wild-type Flg-ΔNAxin has little or no effect compared to control (pcDNA). The dominant effect of Flg-ΔNAxin L–Q is comparable to the previously described L–P mutation at the same position (lane4; Smalley et al. 1999). Expression of transfected Axin and endogenous Gsk3β (control) is shown by immunoblot analysis. Lane 5 shows transcription following transfection with a constitutively active form of beta;-catenin. Counts are luciferase reporter assay readouts from TOP-FLASH plasmid (TCF-binding motif) and FOP-FLASH plasmid (mutant motif as control) that have been adjusted for transfection efficiency as described previously (Smalley et al. 1999).

Mutations in the Gsk3 binding site of murine Axin can lead to mutant forms of Axin functioning in a dominant negative way, most likely by interfering with wild-type Axin function (Smalley et al. 1999). Therefore, we examined if Axin constructs incorporating the L→Q amino-acid exchange show any dominant negative effects. Tcf-dependent transcription is increased in the presence of Axin1 constructs incorporating the L→Q amino-acid exchange while it shows little or no change in the presence of wild-type Axin1 constructs (Fig. 3C). This suggests that the Axin construct incorporating the mbl amino-acid change can interfere with wild-type Axin1 function in vitro. Despite this, embryos heterozygous for the mbl mutation have no obvious phenotype indicating that a single mbl allele does not lead to significant disruption of the function of wild-type Axin1 or related proteins in vivo. To test if high levels of the mbl allele of Axin1 could function in a dominant negative manner in vivo, we injected high concentrations of both wild-type and mbl axin1. Unfortunately, this led to the disruption of several processes (dorsoventral patterning, cell movements, and AP patterning) during gastrulation making interpretation of phenotypes problematic. These experiments did not allow us to unequivocally determine if the mbl allele of Axin1 does indeed have weak dominant-negative activity in vivo.

Excess Gsk3β can partially rescue the mbl phenotype

Association with Axin is believed to enable Gsk3 to efficiently phosphorylate β-catenin (Ikeda et al. 1998; Kishida et al. 1999), and so compromised Gsk3 activity in mbl−/− embryos may contribute to the mbl−/− phenotype. Indeed, wild-type zebrafish embryos treated with lithium, an inhibitor of Gsk3 (Klein and Melton 1996), have mbl-like phenotypes in which telencephalon and eyes are reduced or absent and trigeminal neurons are ectopically expanded (Macdonald et al. 1994). To test if reduced Gsk3 activity in mbl−/− mutants can be overcome by an excess of Gsk3, we overexpressed gsk3β RNA (Tsai et al. 2000) in mbl−/− embryos. About 30% of mbl−/− embryos overexpressing gsk3β had small eyes and this was also reflected by restoration of neural-plate patterning at earlier stages (Fig. 4A–F, Table 1). This data suggests that increasing Gsk3β activity can partially alleviate the requirement for Axin1 and supports the hypothesis that reduced Gsk3 activity contributes to the mbl−/− phenotype. It also indicates that Gsk3β still can have activity in the absence of wild-type Axin1, possibly through association with other Axin family proteins.

Figure 4.

Figure 4

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Mbl/Axin1 functions both in anterior neural plate patterning and axis development. (A–F) Dorsal views of neural plates and brains of wild-type (A,D), mbl−/− (B,E), and mbl−/− embryos injected with gsk3 β RNA (C,F) stained for the expression of flh in the epiphysial region of the diencephalon (arrow, A) at bud stage (A–C) or stained with an α-acetylated tubulin antibody at pharyngula stage (D–F). The embryos injected with gsk3β RNA show partial rescue of thembl−/− phenotype as determined by the pattern of flh expression at bud stage and the presence of telencephalon (asterisk) and small eyes (arrowhead) at pharyngula stage. (G–H) Dorsal views of neural plates of wild-type (G), mbl−/− (H), and mbl−/− embryos in which wild-type cells expressing axin1 RNA were transplanted into the anterior neural plate at 70% epiboly stage (I) showing expression of fkd3 (characteristic of mid/caudal diencephalon identity in wild-type) at bud stage. Ectopic rostral fkd3 expression is absent in the transplanted cells and in some of the mbl−/− host cells adjacent to the transplanted cells. (J–L) Dorsal (J,K) and animal pole (L) views of a wild-type embryo and embryos injected with α-axin1 morpholino antisense oligonucleotides (K,L) stained for the expression of flh (a marker of organizer tissue, arrowheads) at shield stage. (J) An uninjected wild-type control embryo showing a single expression domain of flh within the dorsal organizer region. In (K), the injected embryo shows expansion of flh expression on the dorsal side of the embryo. In (L), the injected embryo has multiple sites of flh expression indicative of widespread dorsalization. (M–O) Animal pole views of bud-stage control embryo (M) or embryos injected with increasing (left to right) doses of wnt8 RNA (N,O). In addition to variably causing dorsalization (data not shown), pax2.1/noi expression (normally restricted to the presumptive midbrain), spreads into the anteriormost regions of the neural plate of injected embryos. This expansion is unlikely to be simply a result of dorsalization of the ectoderm, as inhibition of BMP signaling also dorsalizes ectoderm but does not lead to anterior expansion of pax2.1 expression (e.g., Barth et al. 1999).

The mbl mutation has no major consequences upon organizer/germ-ring formation or fates

Taken together, the experiments described above suggest that compromised Axin1 function in mbl−/− embryos results in reduced Gsk3 activity with the consequence that less β–catenin is targeted for degradation and Wnt pathway target genes are overactivated. Activation of the Wnt pathway promotes organizer fates (Larabell et al. 1997) and the zebrafish organizer can indirectly influence anterior neural fates (Fekany et al. 1999; Saude et al. 2000; Shimizu et al. 2000; Sirotkin et al. 2000). This raises the possibility that the CNS phenotype of mbl−/−embryos may be due to incorrect organizer function. However, unlike mutations such as bozozok (Koos and Ho 1999; Fekany-Lee et al. 1999, 2000) and squint (Feldman et al. 1998), which primarily affect organizer tissues, we observed apparently normal expression of gsc, flh, and other markers of organizer-derived mesendodermal fates in mbl−/− embryos (Fig. 1H; data not shown). This suggests that there is no major disruption to organizer function or fates in mbl−/− embryos.

Wnt8 is a candidate posteriorizing factor that can inhibit anterior neural fates and in bozozok and tcf3/hdl mutant embryos that lack forebrain structures, there is increased and ectopic expression of wnt8 in germ-ring cells during gastrulation (Fekany-Lee et al. 2000; Kim et al. 2000). However, in clutches of embryos from heterozygous mbl−/− fish, we were unable to detect any obvious differences in wnt8 expression between mbl−/− embryos and their siblings (data not shown). Therefore, mbl−/−embryos do not show the same early alterations in gene expression that occur in embryos carrying other mutations that are known to directly or indirectly affect Wnt signaling activity.

Axin1/Mbl function is required during gastrulation for forebrain patterning

The apparent absence of major early patterning defects in mbl−/− embryos suggests that the mbl mutation may affect primarily Wnt-dependent patterning events after the onset of gastrulation. To address when Mbl/Axin1 may be required, we performed two sets of experiments. First, we transplanted wild-type ectodermal cells (taken from random positions in donor embryos) overexpressing axin1 into the anterior neural plate of mbl−/− mutants at 70% epiboly stage. axin1 expressing wild-type cells within the anterior neural plate partially rescue the mbl−/− mutant phenotype as determined by the expression of fkd3 at bud stage and the presence of eyes at later stages (n = 9) (Fig. 4G–I; data not shown). Therefore, restoration of Axin1 function to neural plate cells during gastrulation is sufficient to restore anterior forebrain character in mbl−/− embryos. As a second approach, we made use of the temperature-dependent expressivity of the mbl−/− phenotype. To assess when Mbl function is required to promote eye development, we shifted mbl−/−embryos from 28°C to 22°C at the onset and at the end of gastrulation. Five percent of embryos shifted to the lower temperature at the onset of gastrulation had small eyes whereas embryos shifted at bud stage lacked eyes (Table 2). Together these studies suggest that suppression of Wnt activity during gastrulation stages influences the regional subdivision of the neural plate into telencephalic, diencephalic, and optic territories.

Table 2.

mbl is a temperature-sensitive mutation affecting eye1 development

Temp (°C) Genotype Normal (n) Absent (n) Small (n) Total (n)
28 mblxmbl 126 38 0 164
22 mblxmbl 118 32 13 163
28→222 mblxmbl 148 42 11 201
28→223 mblxmbl 155 48 0 203
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1

Size of the eyes was scored at pharyngula stage;

2

embryos were shifted from 28°C to 22°C at shield stage;

3

embryos were shifted from 28°C to 22°C at bud stage.

Axin1 also functions in early dorsoventral patterning

Mice lacking Axin function have multiple axes (Zeng et al. 1997) whereas mbl−/− fish embryos exhibit normal early dorsoventral patterning of mesodermal structures. This difference could be from the presence of maternally provided wild-type Axin1 in mbl−/− embryos. To assess if this may be the case, we injected antisense morpholino oligonucleotides (Nasevicius and Ekker 2000) against axin1 into wild-type embryos (n > 200) and assessed effects upon the development of axial structures, using the organizer/notochord marker, flh (Fig. 4J). About 20% of injected embryos exhibited either marked dorsal expansion of the domain of flh expression (Fig. 4K) or ectopic expression of flh around the germ ring (Fig. 4L). About 50% of surviving injected embryos showed expansion of axial tissue at bud stage (data not shown). To confirm that the morpholino injections led to a reduction in Axin1 function, we performed Western blot analysis on wild-type and axin1-morpholino injected embryos. Weak anti-Axin immunoreactivity was detected in extracts of morpholino-injected embryos but levels were considerably reduced compared to wild-type (data not shown). We also confirmed phenotypes with two other axin morpholinos (data not shown). These results indicate that morpholino-based reduction in Axin1 function at early developmental stages can lead to the expansion, or ectopic induction, of axial structures. Therefore, it is likely that the function of Axin is conserved between mouse and fish, but that the mbl allele reveals a novel requirement for the gene that has not been amenable to study in mouse because of the severity of the early phenotype.

Wnt signaling establishes regional fates along the anterior-posterior axis of the prospective brain

Our study reveals a novel role for Axin1 in the regional subdivision of prospective forebrain territories. We propose that Wnt signaling must be suppressed to allow the development of telencephalic and optic fates and that if this fails to occur, prospective forebrain cells adopt a more caudal, diencephalic identity. It is intriguing that headless/tcf3 mutant zebrafish embryos, in which the canonical Wnt pathway is also likely to be overactivated, show expansion of midbrain (or midbrain/hindbrain boundary) fates at the expense of forebrain fates (Kim et al. 2000). Similarly, graded overexpression of Wnt8 can lead to expansion of midbrain-specific gene expression into prospective forebrain territories (Fig. 4M–O). These observations raise the possibility that thresholds of Wnt activity may specify different posterior to anterior fates within the neural plate in a manner analogous to that proposed for graded Bmp activity in the allocation of fates along the dorsoventral axis of the neural plate (Barth et al. 1999; Nguyen et al. 2000).

Materials and methods

In-situ hybridization and antibody labeling

Antibody staining and in-situ hybridization with digoxygenin-incorporated antisense RNA probes was done as described (Heisenberg et al. 1996). Fish embryo raising and staging was done as described (Westerfield 1995).

Linkage analysis, mapping, and cloning

mbltm213 was mapped in F2 offspring of a TÜ × WIK reference cross as described (Rauch et al. 1997), using Simple Sequence Length Polymorphism markers (Knapik et al. 1996) on pools of 48 mutants and 48 siblings. Linkages from the pools were confirmed and refined by genotyping single embryos. To identify mutations, RNA from Tue wild-type and homozygous mbltm213 embryos at bud stage was reverse-transcribed and then amplified by PCR with pfu polymerase for sequence analysis.

RNA and morpholino oligonucleotide injections

The wild-type axin1 injection construct (pCS2SNaxin1) was obtained from Dr. Hibi, Osaka University, and the wild-type wnt8 injection construct from D r. R. Moon, Washington University. The mutated mbl axin1 injection construct was assembled from two PCR fragments for which internal oligonucleotide primers were designed to introduce the L399→Q amino-acid exchange using the wild-type pCS2SNaxin1 injection construct as template and subsequently cloned into the pCS2SN vector. RNA was injected into one-cell-stage embryos as described previously (Barth and Wilson 1995). Antisense axin1 morpholino oligonucleotides (sequences available on request) were injected at the one-cell stage or into individual blastomeres at early blastula stages. Injection of 2.5 ng morpholino antisense oligonucleotides/embryo led to reproducible phenotypes in the injected embryos. Results were comparable for both classes of injection and results shown in the paper are from injections at the one-cell stage. Western blot analysis (data not shown) used an affinity purified anti-Axin1 polyclonal antibody that revealed a band of expected size. Control morpholino oligonucleotides (Gene Tools) and morpholino antisense oligonucleotides against various other genes (lfng, sqt, cyc) were injected to determine the specificity of the obtained phenotypes. After phenotypic evaluation, in some cases, single embryos were genotyped taking advantage of a BpmI restriction polymorphism induced by the axin1/mbl point mutation. Genomic DNA from single embryos was amplified a round the mutation site and the amplified fragment subsequently digested using BpmI restriction enzyme.

Immunoprecipitation

Full-length murine Axin constructs were subcloned from a Myc-tagged Axin Form 1 cDNA (supplied by F. Constantini) into the CMV expression vector pcDNA3.1+ (Invitrogen). The N-terminal deletion construct was created using restriction enzymes (Smalley et al. 1999). The ΔNAxin L–Q mutation was created using the QuickChange Site-directed Mutagenesis Kit (Stratagene). The HA-tagged GSK-3β was made from pTM3β-tag (supplied by J. Woodgett) and cloned into pcDNA3.1+. 293 cells were transfected using FuGENE 6 (Boehringer Mannheim) according to the manufacturer’s instructions and lysed 48 h later with a buffer containing 10mM Tris pH 7.5, 1 mM EDTA, NaCl 50 mM, 0.6 mM PMSF (Sigma Cat. No. P-7626), protease inhibitor cocktail (Sigma Cat. No. P8340), and 100 nM Okadaic Acid (Sigma Cat. No. O-1506). The cell lysates were precleared with 50 μL protein G sepharose beads (Amersham Pharmacia Biotech), previously washed in lysis buffer, for 30 min on a rotating wheel at 4°C. Aliquots containing 750 μg protein were made up to a volume of 500 μL and incubated, again on the rotating wheel at 4°C, with 2 μg antibody and 50 μL protein G sepharose beads for 4 h. The beads then were pelleted and washed three times with lysis buffer and used for Western analysis. Antibodies used were mouse α-Flag M 2 monoclonal antibody (Sigma), mouse α-HA monoclonal antibody 12CA5, and mouse α-GSK-3β (Transducin Labs).

Luciferase essay

293 cells were seeded at 7.5 × 104 cells/well in 6-well dishes (NUNC), 4 8 h before an experiment. Each well was transfected with 0.5 μg of one of the luciferase reporters TOPFLASH or FOPFLASH (Korinek et al. 1997), 0.15 μg of CMV-hTCF4 (Korinek et al. 1997), 2 5 ng of CMV-lacZ as an internal control and 0.3 ng of the test-construct (CMV-driven Flg-tagged ΔNAxin1). The CMV promoter-containing plasmid pcDNA3.1+ (Invitrogen) was used to equalize the total amount of CMV-driven plasmid per well. Carrier DNA (salmon sperm) was added to a total of 2 μg. Transfection was done in triplicate using FuGENE 6 (Boehringer Mannheim) according to the manufacturers instructions. Luciferase and β-galactosidase activity were measured 48 h later. For the luciferase assays, cells were harvested into PBS and pelleted with a low-speed spin. Cell pellets then were lysed with reporter lysis buffer and processed to generate cell supernatant and pellet fractions according to manufacturer’s recommendations (Promega Luciferase Assay system). Both fractions were kept for Western analysis. Luciferase activity in 20 μL supernatant fraction was measured in an AutoLumat LB953 (Berthold). For the β-galactosidase activity measurements, 20 μL of the supernatant fractions were used in a spectrometric kinetic assay based on the substrate CPRG (Boehringer Mannheim). Luciferase results were calculated after correcting for β-galactosidase activity. For Western analysis, the triplicate samples containing TOPFLASH were combined and the supernatant and pellets run separately on SDS-PAGE gels after normalizing protein loading for β-galactosidase expression. Unless otherwise stated, only data from supernatant fractions are presented adjacent to the luciferase TCF-transcription assay data. Antibodies used were mouse α-GSK-3β antibody (Transducin Labs) and mouse α-Flag M 2 monoclonal antibody (Sigma). Secondary antibodies used were against the appropriate species and HRP conjugated for visualization with enhanced chemiluminescence Western Blotting detection reagents (Amersham Pharmacia Biotech). Immunoblots shown in the figures are of the supernatant fraction unless otherwise stated.

Cell transplantations

Cell transplantations between wild-type and mbl−/− mutant embryos at midgastrulation stages were performed as previously described (Houart et a l. 1998).

Acknowledgments

We thank many colleagues who provided reagents that enabled us to test axin1 and several other genes as candidates for the mbl mutation. In particular, we are indebted to Masahiko Hibi, Ken Irvine, Antonio Jacinto, Yun-Jin Jiang, Julian Lewis, and Tom Vogt for help and advice. We thank Ajay Chitnis and Dana Zivkovic for providing information prior to publication. This study was supported primarily by grants from the EMBO and EC to C.P.H., Wellcome Trust and EC to S.W.W., from the MRC to D.S., from Naito to M.T., from the DHGP to G.J.R. and R.G., and from the CRC/ICR to T.D. P.C. was supported by a PhD studentship from Fundação para a Ciência e Tecnologia, Programa PRAXIS XXI. S.W.W. is a Wellcome Trust Senior Research Fellow.

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