1 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
2 Department of Immunology, University Hospital, PO Box 85500, 3508 GA Utrecht, The Netherlands
Present address: Westburg b.v., PO Box 214, 3830 AE Leusden, The Netherlands
Present address: Laboratory of Plant Cell Biology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
*Author for correspondence: dana{at}niob.knaw.nl
Accepted July 25, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: masterblind, zebrafish, Wnt, GSK3ß, lithium
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The AP patterning of the neural plate also appears to be tightly regulated by basal activity of Wnt pathway components that act to destabilise ß-catenin. In the absence of Wnt signal, glycogen synthase kinase-3ß (Gsk3ß), Axin, the adenomatous poliposis coli (APC) protein and phosphoprotein phosphatase 2A (PP2A) form a complex that binds and phosphorylates ß-catenin thereby stimulating its degradation by the ubiquitin-proteosome system (Aberle et al., 1997; Ikeda et al., 1998; Bienz and Clevers, 2000). In the presence of Wnts that trigger the canonical pathway, inhibition of Gsk3ß activity allows the cytoplasmic accumulation of stabilised ß-catenin. This unphosphorylated ß-catenin then translocates into the nucleus where it modulates target gene transcription by interacting with members of the TCF/LEF1 family of transcription factors (Behrens et al., 1996; Molenaar et al., 1996).
In model organisms as well as in mammalian cells in culture, Gsk3ß plays a key role in cell fate decisions by negatively regulating ß-catenin (Welsh et al., 1996; Larabell et al., 1997). Lithium has been shown to inhibit Gsk3ß, thereby mimicking Wnt signalling (Klein and Melton, 1996; Stambolic et al., 1996; Hedgepeth et al., 1997). Indeed, treatment of frog and fish embryos with lithium after the mid blastula transition (MBT) leads to posteriorization of the CNS that is strikingly similar to that induced by ectopic expression of Wnts (Yamaguchi and Shinagawa, 1989; Fredieu et al., 1997; Kelly et al., 1995). Consistent with the role of Wnt antagonist in anteriorization of the neural plate is targeted overexpression of gsk3ß mRNA in Xenopus embryos that induces ectopic neural tissue of anterior character (Itoh et al., 1995; Pierce and Kimelman, 1996). Another negative regulator of the Wnt pathway is Axin that promotes Gsk3ß-dependent phosphorylation of ß-catenin thereby stimulating its degradation. Mutations in axin, such as those encoded by the fused locus in mouse and loss of function of Drosophila axin, phenocopy overexpression of Wnt/wg (Zeng et al., 1997; Hamada et al., 1999). The phenotype of fused homozygous mutants is characterised by axial duplications that are reminiscent of induction of the secondary axes in frogs by ectopic overexpression of Wnts (McMahon and Moon, 1989). Indeed, in the Xenopus axis-induction assay, axin inhibits induction of a secondary axis by negatively regulating Wnts (Zeng et al., 1997; Fukui et al., 2000). fused mutants also have neuroectodermal abnormalities which supports a role of axin in AP patterning of the CNS (Perry et al., 1995). Recently, genetic evidence has been presented to show that components of the Wnt pathway downstream of axin play a role in head induction. Severe head defects in the zebrafish headless (hdl) mutant are due to a loss of function of tcf3 (Pelegri et al., 1998) that represses Wnt target genes (Kim et al., 2000).
masterblind (mbl) is a zebrafish recessive zygotic mutation from the Tübingen screen (Haffter et al., 1996). mbl is characterised by the absence or reduction in size of the telencephalon complemented by the anteriorward expansion of the epiphysis (Heisenberg et al., 1996; Masai et al., 1997). The phenotype of mbl suggests that the locus is required for normal development of the eyes and the telencephalon, the same structures that are negatively affected by ectopic Wnt signalling.
We present evidence that the function of mbl in normal development is to antagonise Wnt signalling and in doing so to establish and maintain the most anterior CNS. At the molecular and cellular levels, the posteriorised phenotype of mbl phenocopies the inhibition of Gsk3ß in zebrafish embryos. By manipulating in embryos the dose of Gsk3ß we show that the mbl mutant phenotype arises as a consequence of ectopic Wnt signalling. The defects in mbl were rescued by overexpression of gsk3ß or axin1 mRNA implicating that an ectopic Wnt signal underlies its phenotype. We detected in mbl mutants a point mutation in the minimal gsk3ß binding domain of axin1 (Heisenberg et al., 2001) that when injected into wild-type embryos had a weak dominant negative activity. Our findings provide new insights into the mechanisms that generate AP polarity of the neural plate and extend evidence for the crucial role of Wnt-pathway components such as mbl/axin therein.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
mbl (tm13) heterozygous fish were generated from mbl outcrosses in Tüwt background (a kind gift from Pascal Haffter, Tübingen) and subsequently in HLwt background.
Lithium treatments
Lithium (0.3 M LiCl) treatments were carried out on a shaking platform at 27°C in egg water (Westerfield, 1995) on sphere stage embryos (4hpf) (post-MBT treatment or lithium treatment). Treated embryos were subsequently washed 3 times in egg water and used in different experiments.
"Head" measurements (µm) of lithium-treated and wild-type embryos were carried out using an ocular micrometer in a dissecting microscope. Rostrocaudal distance was determined from the most anterior part of the head to the otic vesicles while dorsoventral distance was determined from the dorsal aspect of the yolk to the top of the head at the mid-hindbrain boundary.
Microinjection of synthetic mRNAs
One or 0.5 nl with identical end concentration of mRNA in water was injected into one or two-cell stage embryos with needles of approx. 5 µm diameter using a pneumatic picopump microinjector (WPI). Capped synthetic mRNAs were prepared using the SP6 mMessage mMachine kit (Ambion). The following constructs were used to prepare RNA for injection: full length (fl Xgsk3ß), dominant negative (dn Xgsk3ß), and frame shift Xenopus gsk3ß (fs Xgsk3ß) in pCS2+ linearized with NotI (kind gift from Dr Kimelman) (Pierce and Kimelman, 1995); zebrafish gsk3ß linearized with NotI and zebrafish axin1 linearized with AscI (kind gift from Dr Hirano). In controls, equivalent quantities of GFP mRNA were injected and its expression assessed in living embryos.
DNA microinjection
Supercoiled plasmid DNA (1.4 nl of 40 µg/ml) for full length (fl Xgsk3ß) or dominant negative (dn Xgsk3ß) driven by the CMV promoter (kind gift from Dr Kimelman) (Pierce and Kimelman, 1995) was injected as previously described (Joore et al., 1996).
Whole-mount in situ hybridisation
Whole-mount in situ hybridisations were carried out as previously described (Joore et al., 1994).
Antisense DIG (Boehringer) labelled riboprobes were synthesised as described elsewhere: otx2 (Li et al., 1994), shh (Krauss et al., 1993), Krox20 (also known as egr2) (Oxtoby and Jowett, 1993), fgf8 (Reifers et al., 1998), emx1 (Morita et al., 1995), flh (Talbot et al., 1995), pax6 (Krauss et al., 1991), wnt1 (Molven et al., 1991).
Embryo genotyping
To verify whether microinjections of mRNAs for gsk3ß or axin1 rescued the mbl phenotype we genotyped injected and control embryos. In PCRs we tested primer pairs for markers that are closely linked to the mbl locus, z15747, z24851 and z25683 on LG 3 (G.-J. Rauch and R. Geisler, pers. comm.). DNA samples of individual embryos were used for genotyping. Each 20 µl reaction contained 5 µl template DNA. The marker that showed the highest linkage to mbl was z24851 on 60.3 cM (purchased from MWG AG Biotech) (http://zebrafish.mgh.harvard.edu/cgi-bin/ssr_map/view_lg.cgi and http://wwwmap.tuebingen.mpg.de) and Fig.6.
|
bp 6-734: gacagagtgcagggacactatgagc and aagactttgccgttgcctgacaccg
bp 498-1248: cgtatcccggcagatcaaacccg and cgcctctcgttccctcaacaccc
bp 947-1816: acgtgaactctggctacgcgctggc and tggtgttgggaccgacgctcattcc
bp 1511-2076: gccattctccgaagtcccgctcg and ccccagacgctccaccgaactgg
bp 1842-2645: ggcagcacgctatccaagcgaccg and tggctctccagcacgtccacgttcg
PCR reactions were performed using Amplitaq Gold (Perkin Elmer). Conditions were: 20 seconds 94°C, 20 seconds 67°C and 30 seconds 72°C.
PCR fragments were subsequently subcloned into pGEMT and sequenced in the presence of 1.3 M Betaine and 1.3% DMSO. The mutation found in mbl was confirmed by direct sequencing of the PCR product.
To make an mbl expression construct from which we made L399Q mutant axin1 mRNA, a NsiI-NarI fragment from wild-type axin1 was replaced by a NsiI-NarI fragment from mbl containing the mutation.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine the sensitivity period for eye loss, embryos were exposed to lithium at different times after MBT. The most sensitive interval was from late blastula to early gastrulation, 4 to 6 hpf, when lithium induced loss of eyes or small eyes in almost 100% of embryos. During 7 to 8 hpf, susceptibility to lithium declined and progressively more embryos with small and wild-type eyes were observed (data not shown) (Stachel et al., 1993; Yamaguchi and Shinagawa, 1989).
The loss of anterior brain could be predicted at the tailbud stage by aberrant expression of neural plate regional marker genes. wnt1, a marker for the presumptive mid/hindbrain boundary, was expanded anteriorly in the apparently narrowed neural plate (Fig. 1A,B), while the expression of the midline marker shh was weaker in the anterior neural plate (Fig. 1C). Interestingly, the modification of wnt1 expression in treated embryos was suggestive of abnormal convergent extension (CE) movements in the anterior neural plate (Fig. 1B). At 24 hpf the mild lithium phenotype was characterised by the loss of brain rostral to the D2/3 diencephalic boundary including the eyes, as revealed by loss of eye-specific pax6 expression (Fig. 1D,E) and by an anterior shift of otx2 (Fig. 1F). The severe phenotype was characterised by the loss of brain anterior to, and often including, the mid/hindbrain boundary as shown by loss of Pax6 (Fig. 1D) and Wnt1 expression (Fig. 1G). The hindbrain was unaffected as shown by expression of Krox20 (Fig. 1H).
|
To investigate whether the loss of brain induced by lithium was due to apoptosis, we carried out the TUNEL assay on lithium-treated tailbud and 24 hpf embryos. Since there was no difference in labelling between lithium-treated and wild-type embryos we conclude that apoptosis does not underlie the lithium phenotype (data not shown).
The data show that the loss of anterior brain caused by lithium-mediated ectopic activation of the Wnt pathway is the consequence of posteriorization of the neuroectoderm during early- to midgastrula.
Overexpression of dn Xgsk3ß mRNA in wild-type embryos phenocopies mbl and lithium treatment
Lithium treatment inhibits Gsk3ß activity and phenocopies the mbl defects. To test whether inhibition of Gsk3ß activity is sufficient to phenocopy mbl, we overexpressed dn Xgsk3ß mRNA (Pierce and Kimelman, 1996) in wild-type embryos with the expectation of inducing loss of eyes. Indeed, overexpression of dn Xgsk3ß mRNA caused loss of eyes or reduction in their size in approx. 40% of injected embryos (n=154) (Fig. 2A,E). Although we titrated the mRNA concentrations to be injected and chose the concentration that induced the highest number of embryos with the eyeless phenotype associated with the lowest number of gastrulation phenotypes, there was still high mortality of injected embryos (Fig. 2A). It is likely that this mortality was caused by the disturbance of the Wnt pathway during axis formation (Harland and Gerhart, 1997; Moon and Kimelman, 1998). Consistent with this is the observation that a number of dn Xgsk3ß mRNA-injected embryos displayed axial abnormalities at the tailbud stage as indicated by truncated and bifurcated expression of flh, the marker for the notochord anlage (not shown). To circumvent interference of the "early" Wnt pathway by dn Xgsk3ß mRNA, we overexpressed the plasmid DNA containing dn Xgsk3ß driven by the CMV promoter (1.4 nl of 40 µg/ml). In three independent experiments 13.5% (14/102) of embryos had loss of eyes and partial loss of forebrain. Since the efficiency with which dn Xgsk3ß DNA overexpression induced the eyeless phenotype was very low, most likely due to mosaicism, we concluded that in our experimental design overexpression of dn Xgsk3ß mRNA was to be preferred. The specificity of the observed effects of dn Xgsk3ß mRNA on eye development was confirmed by injecting the inactive fs Xgsk3ß mRNA (Pierce and Kimmelman, 1996), which caused a mutant eye phenotype in less than 5% of embryos (Fig. 2C). To study gain of function of gsk3ß we overexpressed full length fl Xgsk3ß mRNA and observed an eye/brain phenotype in approx. 10% of embryos (Fig. 2B) that was characterised by the deletion of the ventral forebrain and accompanying partial eye-fusion (Fig. 2F,G). Together these data suggest that the inhibition as well as ectopic activation of gsk3ß signalling affects eye development.
|
In zebrafish, fgf8 (acerebellar) is expressed at the MHB, in the dorsal diencephalon, retinae and optic stalks and in the facial ectoderm (FEC) (Fig. 2K) (Reifers et al., 1998). Overexpression of dn Xgsk3ß mRNA that induced small eyes in the majority of these embryos did not affect the fgf8 pattern (16/24) (Fig. 2L, left panel). In the minority of embryos with small eyes the optic stalk labelling was absent (7/24) (Fig. 2L, right panel), while exceptionally (1/24), fgf8 was expressed only at the MHB (not shown). In most of the embryos that lost their eyes upon dn Xgsk3ß mRNA overexpression, fgf8 was expressed at the MHB only (26/40) or not at all (14/40) (not shown). The data indicate that experimental activation of the Wnt pathway in zebrafish embryos through dn gsk3ß overexpression can differentially affect telencephalon and eyes.
Common molecular mechanisms underlie mbl and lithium-generated phenotypes
To investigate whether a common mechanism underlies gsk3ß inhibition and the mbl phenotype, we treated mbl mutants with a sub-effective dose of lithium. We reasoned that, owing to the dosage effect, mbl heterozygotes would be more sensitive to partial gsk3ß inhibition than wild types. We used 5-10 minute exposure to lithium (Fig. 3). A 6 minute treatment applied to the offspring of mbl+/ outcrossed to HLwt, results in 50% eyeless embryos and 50% wild-type embryos (Fig. 3A). This same treatment induced less than 2% of embryos affected in wildtypes (Fig. 3B), demonstrating an enhanced sensitivity of mbl heterozygotes to ectopic activation of the Wnt pathway. Interestingly, the same treatment results in 25% small eyes, 25% eyeless and 50% wild types when the offspring of mbl+/ outcrossed to Tüwt is used (not shown). Therefore the HLwt background is more sensitive to gsk3ß inhibition, possibly because of genetic differences in modifier genes that interact with the gsk3ß signalling.
|
Overexpression of Xgsk3ß rescues eyes of mbl embryos
To test the hypothesis that wild-type Mbl protein antagonizes the Wnt pathway we investigated the effects of injections of dn Xgsk3ß or fl Xgsk3ß mRNAs into progeny of in-crosses of mbl heterozygous fish. Overexpression of dn Xgsk3ß mRNA induced loss or reduction of eyes in approx. 80% of embryos (Fig. 4A). When effects of dn Xgsk3ß on wild-type embryos (Fig. 2A) as well as the increased susceptibility of heterozygous mbl embryos to lithium (Fig. 3A) are considered then 80% of embryos with a mutant phenotype would be predicted. To investigate whether the overexpression of fl Xgsk3ß mRNA could rescue eyes in mbl/ embryos, we injected this mRNA into the progeny of in-crosses of mbl heterozygotes. Because of the variable penetrance of the eye phenotype in mbl embryos that ranges from eyelessness to small eyes (Heisenberg et al., 1996), we present analysis of only eyeless progeny (Fig. 4B,D). Upon injection of fl Xgsk3ß mRNA in embryos from eyeless clutches a reduction in eyeless embryos from 25% to 5% was accompanied by a corresponding increase in embryos with small eyes from 0% to 20% (Fig. 4B,E). These data strongly suggest that a simultaneous increase in the number of embryos with small eyes and a decrease of eyeless embryos reflects eye-rescue of homozygous eyeless mbl/ embryos by fl Xgsk3ß mRNA.
|
|
To study whether the wild-type brain has been restored in fl Xgsk3ß- rescued mbl/ embryos with small eyes, we analysed expression of flh, emx1 and fgf8. The zebrafish homeobox gene floating head (flh) that is essential in notochord development (Talbot et al., 1995) is required for progression of neurogenesis in the epiphysis. In mbl/ embryos epiphysal neurons are generated throughout the dorsal forebrain within the ectopic flh expression domain that delineates the front of the neural plate as early as the tailbud stage (Fig. 4G) (Masai et al., 1997). Overexpression of fl Xgsk3ß in mbl embryos resulted in a marked downregulation of this ectopic flh-domain at the anterior neural plate (Fig. 4H) that now resembled the wild-type expression (Fig. 4F) and most likely predicted eye rescue.
The telencephalic domain of eyeless and small eyes mbl embryos was shifted ventrally and anteriorly as revealed by an expanded emx1 domain (Fig. 5B,C). These data suggest that telencephalic domain in mbl embryos is in molecular terms not lost, but transformed, with, as a consequence, the lack of differentiated telencephalon. The data show that overexpression of fl Xgsk3ß may rescue eyes, without fully restoring the wild-type emx1 domain (Fig. 5D). To further analyse the effect of rescue on brain regionalisation, mbl embryos were labelled with fgf8. In uninjected mbl embryos, the loss of eyes was accompanied by the disappearance of fgf8 expression in the optic stalks and retinae and weakening or disappearance of the expression in the dorsal diencephalon, whereas expression at the mid/hindbrain boundary and in facial ectoderm was present (Fig. 5F). fgf8 expression in rescued embryos with small eyes was restored to various degrees (Fig. 5G,H). In addition to the wild-type pattern (left eye in Fig. 5H), eye rescue could be established without restoration of fgf8 in retinae (left eye in Fig. 5G), suggesting that Xgsk3ß is sufficient to fully restore eyes in mbl/ embryos.
|
The data demonstrate that the mbl locus corresponds to axin1 and that the recovered point mutation results in an altered axin1 function that leads to activation of ß-catenin /TCF.
In summary, mutation in minimal GID of axin1/mbl as well as a fine-tuned post-MBT treatment with lithium or injection of dn gsk3ß mRNA all induce loss of eyes in zebrafish embryos, while overexpression of gsk3ß or axin1 rescues eyes of mbl embryos. The data are consistent with ectopic activation of ß-catenin/TCF signalling in mbl embryos.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interestingly, the block on gsk3ß activity and a consequent activation of the Wnt pathway by the post-MBT lithium treatment, in the most severe cases, phenocopies zebrafish hdl (headless) mutants (Kim et al., 2000). The common molecular mechanisms underlying this phenotypic resemblance is evident since in hdl the loss of function of Tcf3 (Pelegri et al., 1998), a transcriptional repressor in the canonical Wnt pathway results in derepression of Wnt targets (Kim et al., 2000), while lithium treatment inhibits gsk3ß and apparently activates an overlapping set of targets (Fredieu et al., 1997) (this work). The phenotypes of mbl and hdl as well as of lithium-treated and dn gsk3ß-injected embryos are presumably due to activation of Wnt target genes, which are repressed in the anterior CNS in normal development.
Mutation in mbl and inhibition of gsk3ß induce loss of eyes but have distinct effects on brain regionalisation
The posteriorising effect of an ectopic Wnt signal results in loss of eyes (Heisenberg et al., 1996; Masai et al., 1997; Kelly et al., 1995; Fredieu et al., 1997; Stachel et al., 1993; Kim et al., 2000) (this work). Vertebrate eye formation is initiated by protrusion of optic vesicles from anterior neural tissue. The optic vesicle develops into the optic cup while inducing lens in the opposing ectoderm (Grainger, 1992). As a consequence of ectopic Wnt signal neither the optic vesicle nor the lens can form in mbl (Heisenberg et al., 1996). Our experiments show that this anterolateral region of the early neural plate that gives rise to optic vesicles is transformed in mbl embryos and now ectopically expresses flh. Accordingly, in mbl embryos rescued with gsk3ß the wild-type flh pattern is restored. The optic vesicle presumably does not form because anterior neural tissue is transfated to posterior and has lost its morphogenetic properties. Interestingly, it has been suggested that overexpression of gsk3ß in Xenopus interferes with signalling that controls morphogenesis of optic vesicles (Itoh et al., 1995). During early gastrulation this most anterior presumptive retina/forebrain field represents the source of the lens inducing planar signal (Grainger, 1992). Since in mbl embryos this region is posteriorised, the lens-inducing signals are likely not generated at all and as a consequence the lens fails to develop.
Although eye loss is common to mbl, hdl mutants, and experimental inhibition of gsk3ß, the brainpattern is differentially affected. Although previous observations that in mbl embryos the telencephalon is reduced or missing are still valid (Heisenberg et al., 1996), we find that emx1 expression, which in wild type delineates the most dorsal telencephalon, is in mbl not reduced or lost, but rather is expanded and shifted anteroventrally. It appears that the emx1 domain is anteriorly displaced from its dorsal position as indicated by the diencephalic marker flh [compare Fig. 5B,C and Fig. 7B (Masai et al., 1997)]. The consequence appears to be the lack of differentiated telencephalon that in order to differentiate may require signals from the most anterior ventral CNS, which has been transformed in mbl. In contrast to mbl, overexpression of dn gsk3ß either does not affect emx1 expression at all, or in the most severe cases its domain is deleted together with all of the fore- and midbrain. However in 1 out of 26 embryos overexpressing dn gsk3ß we observed the expanded emx1 domain that is typical for mbl mutants. Moreover, our data show that overexpression of fl Xgsk3ß in mbl mutants may rescue eyes, without restoring telencephalon, as evidenced by an aberrant emx1 domain in these embryos that remains, as in mutant embryos, ventrally expanded. An exciting hypothesis is that eye and telencephalon would require different levels of Wnt inhibition for their development. To assess whether this hypothesis is valid we are currently investigating the localisation, abundance and phosphorylation status of ß-catenin in mbl embryos as well as in embryos that received different experimental treatments.
The mbl locus corresponds to axin1
Our data suggest that interference with gsk3ß signalling in the embryo results in different phenotypes as a consequence of quantitative differences in phosphorylation of ß-catenin. Indeed, we show that the mbl locus signals in a dose-dependent fashion since a sub-threshold lithium treatment of mbl heterozygote embryos, which only marginally affected wild-type embryos, resulted in eyeless phenotype in heterozygotes. Conversely when gsk3ß was overexpressed in homozygous mbl it was capable of rescuing eyes but not their wild-type size. We deduced that mbl contributes to Gsk3ß-mediated phosphorylation of ß-catenin. These experiments place the mbl gene in the canonical Wnt pathway in parallel or upstream of the complex consisting of APC, Gsk3ß, PP2A and ß-catenin (Bienz and Clevers, 2000). Identification of axin1 as the mbl locus (this work) (Heisenberg et al., 2001) was therefore in agreement with the role proposed for mbl. axin was identified as the gene mutated in the fused mice that as homozygotes have axial duplications (Perry et al., 1995). Moreover, when tested in the Xenopus axis-induction assay, axin inhibits induction of a secondary axis by negatively regulating Wnt (Zeng et al., 1997; Fukui et al., 2000). In a multimeric complex, Axin simultaneously binds to APC, Gsk3ß, PP2A and ß-catenin to promote Gsk3ß-mediated phosphorylation of ß-catenin and its subsequent degradation (Bienz and Clevers, 2000; Ratcliffe et al., 2000). In this fashion axin prevents interaction of stabilised unphosphorylated ß-catenin with TCF transcription factors and subsequent regulation of Wnt target genes (Roose et al., 1999; Bienz and Clevers, 2000). mbl is a point mutation in axin1 resulting in a leucine to glutamine substitution at position 399 that leads to its loss of function. Leu 399 is conserved in a 25 aa sequence in Axin, which represents the minimal Gsk3ß interaction domain (GID) that directly binds to Gsk3ß (Hedgepeth et al., 1999b). The analogous mutation L525M in murine Axin1 present as L396M in human colorectal cancers also interfered with gsk3ß binding and resulted in low level activation of TCF-dependent transcription (Webster et al., 2000). Yet another L521P point mutation, also in GID of murine axin1, transformed it into a transcriptional activator (Smalley et al., 1999). Our data are in agreement with these findings, since we demonstrate that overexpression of axin1 L399Q in wild-type embryos induces 30% phenotype most likely through its dose-dependent dominant negative activity. In contrast to the mbl phenotype, overexpression of axin1 L399Q in wild-type embryos induces a deletion of the telencephalic region in a majority of embryos (80%), whereas only 20% becomes eyeless. The molecular basis for the dominant negative activity of axin1 that is mutated in GID domain (Hinoi et al., 2000; Smalley et al., 1999; Webster et al., 2000) may be based on its interference with the function of wild-type axin1. In this scenario, mutant axin1 would compete with wild-type axin1 for components that form the multimeric complex with it, such as APC, PP2A and ß-catenin. In this manner, mutant axin1 would impair the function of wild-type axin1,which is to antagonise the Wnt signal. If true, those regions of the embryo containing lower levels of axin1 mRNA would, upon overexpression of mutant axin1, be more sensitive to its dominant negative activity. Strikingly, zebrafish telencephalon contains less axin1 mRNA than the midbrain (Fig. 6E,F). It is therefore tempting to speculate that the telencephalon would be the most sensitive target to phenotypic alteration by dominant negative activity of overexpressed mutant axin1. Our experiments appear to support this possibility.
With regard to the mbl phenotype, it is interesting to note that fused homozygous mice display strong neuroectodermal abnormalities (Perry et al., 1995). Expression of axin1 in the anterior midbrain of Xenopus embryos (Hedgepeth et al., 1999a) its anterior overlap at the mid/forebrain boundary with tcf4 and its dorsal overlap with wnt1 and wnt3A (Konig et al., 2000) a), as well as its strong expression in the midbrain of the zebrafish embryo (Fig. 6E,F) further support its role in regionalisation of the anterior brain.
Why does overexpression of gsk3ß only rarely result in full phenotypic rescue of mbl?
Overexpression of axin1 more efficiently and more completely rescues mbl than gsk3ß overexpression (Table I), although in both cases rescue is expected to arise as a consequence of ß-catenin phosphorylation and, as a result, counteraction of the ectopic Wnt signal. It has been shown that ß-catenin is not a good substrate for Gsk3ß in vitro, but is efficiently phosphorylated in the presence of Axin1 (Ikeda et al., 1998). Since the point mutation in GID of mbl/axin1 abolishes binding of gsk3ß to Axin1 (Heisenberg et al., 2001), this is likely to be the limiting factor for its enzymatic activity. We speculate that overexpression of gsk3ß can partially overcome this limitation and to an extent rescue the mbl phenotype because it may very efficiently interact with maternal axin1. For instance, it has been shown in Xenopus egg extracts that the inhibitory effect of GBP and dsh on ß-catenin degradation is reversed by high concentrations of gsk3ß due to titration of GBP and dsh on axin (Salic et al., 2000). The same work suggests that the interaction between axin- gsk3ß is highly dynamic since dn gsk3ß blocks degradation of ß-catenin in extracts. If so, then overexpression of gsk3ß in mbl embryos would shift the binding equilibrium towards gsk3ß axin, and, as a consequence, degradation of ß-catenin. The reason for the incomplete phenotypic rescue of mbl may be the limiting concentrations of maternal axin1. Alternatively, it is possible that the weak dominant negative activity of L399Q mutant axin1 cannot be overruled by overexpression of gsk3ß.
Does a gradient of Wnt signalling pattern the neural ectoderm?
Comparison of loss-of-function hdl/tcf3, lithium treatment, overexpression of dn gsk3ß and loss of Gsk3ß binding activity of mbl/axin1 reveals that although they all activate an ectopic Wnt signal their resulting phenotypes differ. This may be due to the type of Wnt target genes and/or the extent to which they are ectopically activated. Expression patterns of Wnts and their regulators suggest that there is an AP gradient of activation/repression of Wnt targets in the anterior neural plate.
For instance, in the most severe phenotypic category of hdl/tcf3, dn gsk3ß overexpression and severe lithium phenotype, Wnt target genes that during normal development are activated at the MHB and/or posterior to it, are in mutant and treated embryos derepressed and ectopically expressed anterior to the MHB (present work) (Kim et al., 2000). During normal development repression of these genes in the most anterior domain secures head induction. The most anterior neuroectoderm is characterised not only by the highest expression of tcf3, the absence of Wnt ligands and high levels of axin1/mbl in the midbrain caudally to it, but is also exposed to secreted Wnt antagonists such as dkk1 (Hashimoto et al., 2000) and perhaps others as well. These are only a few molecules among those that play a role in establishment of a gradient of Wnt signal. At the present moment the challenge is to identify expression and function of other potential players that generate neuroectodermal polarity, both known ones such as APC and PP2A and novel ones. Moreover, it should be possible, using specific antibodies, to carry out spatiotemporal analysis of stabilised unphosphorylated ß-catenin and thus investigate whether there exists an AP gradient of Wnt signalling.
Since phosphorylation of ß-catenin represents an integrating point of numerous input signals within the Wnt pathway we propose that there may be a gradient of constitutive inhibition (phosphorylation of ß-catenin) with its apex at the anterior pole of the embryo. This gradient acts upon the posteriorising signal, which is the unphosphorylated ß-catenin with the apex at the posterior pole.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aberle, H., Bauer, A., Stappert, J., Kispert, A. and Kemler, R. (1997). ß-catenin is a target for the ubiquitin-proteosome pathway. EMBO J. 16, 3797-3804.
Beddington, R. S. and Robertson, E. J. (1998). Anterior patterning in mouse. Trends Genet. 14, 277-284.[Medline]
Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Bierchmeier, W. (1996). Functional interaction of ß-catenin with the transcription factor LEF-1. Nature 382, 638-642.[Medline]
Berridge, M. J., Downes, C. P. and Hanley, M. R. (1989). Neural and developmental actions of lithium: a unifying hypothesis. Cell 59, 411-419.[Medline]
Bienz, M. and Clevers, H. (2000). Linking colorectal cancer to Wnt signaling. Cell 103, 311-320.[Medline]
Christian, J. L. and Moon, R. T. (1993). Interaction between Xwnt-8 and Spemann organizer signalling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus laevis. Genes Dev. 7, 13-28.[Abstract]
Fekany-Lee, K., Gonzalez, E., Miller-Bertoglio, V. and Solnica-Krezel, L. (2000). The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of Bmp2/4 and Wnt pathways. Development 127, 2333-2345.
Fredieu, J. R., Cui, J., Maier, D., Danilchik, V. and Christian, J. L. (1997). Xwnt-8 and lithium can act upon either dorsal mesodermal or neuroectodermal cells to cause a loss of forebrain in Xenopus embryos. Dev. Biol. 186, 100-114.[Medline]
Fukui, A., Kishida, S., Kikuchi, A. and Asashima, M. (2000). Effects of rat Axin domains on axis formation in Xenopus embryos. Dev. Growth Differ. 42, 489-498.[Medline]
Gathpande, S. K., Mulherkar, L. and Modak, S. P. (1993). Lithium chloride and trypan blue induce abnormal morphogenesis by suppressing cell population growth. Dev. Growth Differ. 35, 409-420.
Geisler, R., Rauch, G. J., Baier, H., van Bebber, F., Brobeta, L., Dekens, M. P., Finger, K., Fricke, C., Gates, M. A., Geiger, H., Geiger-Rudolph, S., Gilmour, D., Glaser, S., Gnugge, L., Habeck, H., Hingst, K., Holley, S., Keenan, J., Kirn, A., Knaut, H., Lashkari, D., Maderspacher, F., Martyn, U., Neuhauss, S., Haffter, P. et al. (1999). A radiation hybrid map of the zebrafish genome. Nature Genet. 23, 86-89.[Medline]
Glinka, A., Wu, W., Delius, H., Monaghan, P. A., Blumenstock, C. and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391, 357-362.[Medline]
Grainger, R. M. (1992). Embryonic lens induction: shedding light on vertebrate tissue determinatioin. Trends Genet. 8, 349-355.[Medline]
Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P., Kelsh, R. N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C. and Nusslein-Volhard, C. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1-36.
Hall, T. S. (1942). The model of action of lithium salts in amphibian development. J. Exp. Zool. 89, 1-33.
Hamada, F., Tomoyasu, Y., Takatsu, Y., Nakamura, M., Nagai, S., Suzuki, A., Fujita, F., Shibuya, H., Toyoshima, K., Ueno, N. and Akiyama, T. (1999). Negative regulation of Wingless signalling by D-Axin, a Drosophila homolog of axin. Science 283, 1739-1742.
Harland, R. and Gerhart, J. (1997). Formation and function of Spemanns organizer. Annu. Rev. Cell Dev. Biol. 13, 611-667.[Medline]
Hashimoto, H., Itoh, M., Yamanaka, Y., Yamashita, S., Shimizu, T., Solnica-Krezel, L., Hibi, M. and Hirano, T. (2000). Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation. Dev. Biol. 217, 138-152.[Medline]
Hedgepeth, C. M., Conrad, L. J., Zhang, J., Huang, H.-C., Lee, V. M. Y. and Klein, P. S. (1997). Activation of the Wnt signaling pathway: A molecular mechanism for lithium action. Dev. Biol. 185, 82-91.[Medline]
Hedgepeth, C. M., Deardorff, M. and Klein, P. S. (1999a). Xenopus axin interacts with glycogen synthase kinase-3 beta and is expressed in the anterior midbrain. Mech. Dev. 80, 147-151.[Medline]
Hedgepeth, C. M., Deardorff, M. A., Rankin, K. and Klein, P. S. (1999b). Regulation of glycogen synthase kinase 3ß and downstream Wnt signaling by axin. Mol. Cell. Biol. 19, 7147-7157.
Heisenberg, C.-P., Brand, M., Jiang, Y. J., Warga, R. M., Beuchle, D., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, D. A., Kelsh, R. N., Mullins, M. C., Odenthal, J. and Nusslein-Volhard, C. (1996). Genes involved in forebrain development in the zebrafish, Danio rerio. Development 123, 191-203.
Heisenberg, C.-P. et al. (2001). A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon. Genes Dev. 15, 1427-1434.
Hinoi, T., Yamamoto, H., Kishida, M., Takada, S., Kishida, S. and Kikuchi, A. (2000). Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3ß-dependent phosporylation of ß-catenin and down-regulates ß-catenin. J. Biol. Chem. 275, 34399-34406.
Ho, R. and Kane, D. A. (1990). Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors. Nature 348, 728-730.[Medline]
Hoppler, S., Brown, J. and Moon, R. T. (1996). Expression of a dominant negative Wnt blocks induction of myoD in Xenopus embryos. Genes Dev. 10, 2805-2817.[Abstract]
Houart, C., Westerfield, M. and Wilson S. W. (1998). A small population of anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391, 788-792.[Medline]
Hsieh, J.-C., Kodjabachian, L., Rebbert, M. L., Rattner, A., Smallwood, P. M., Samos, C. H., Nusse, R., Dawid, I. B. and Nathans, J. (1999). A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature 398, 431-436.[Medline]
Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S. and Kikuchi, A. (1998). Axin, a negative regulator of the wnt signaling pathway, forms complex with GSK-3ß and ß catenin and promotes GSK-3ß-dependent phosphorylation of ß catenin. EMBO J. 17, 1371-1384.
Itoh, K., Tang, T. L., Neel, B. G. and Sokol, S. Y. (1995). Specific modulation of ectodermal cell fates in Xenopus embryo by glycogen synthase kinase. Development 121, 3979-3988.
Joore, J., van der Lans, G. B., Lanser, P. H., Vervaart, J. M., Zivkovic, D., Speksnijder, J. E. and Kruijer, W. (1994). Effects of retinoic acid on the expression of retinoic acid receptors during zebrafish embryogenesis. Mech. Dev. 46, 147-150.
Kazanskaya, O., Glinka, A. and Niehrs, C. (2000). The role of Xenopus dickkopf-1 in prechordal plate specification and neural patterning. Development 127, 4981-4992.
Kelly, G. M., Greenstein, P., Erezyilmaz, D. F. and Moon, R. (1995). Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways. Development 121, 1787-1799.
Kim, C.-E., Oda, T., Motoyuki, I., Jiang, D., Artinger, S. B., Chandrasekharappa, S. C., Driever, W. and Chitnis, A. B. (2000). Repressor activity of Headless/Tcf3 is essential for vertebrate head formation. Nature 407, 913-916.[Medline]
Klein, P. S. and Melton, D. A. (1996). A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA 93, 8455-8459.
Konig, A., Gradl, D., Kuhl, M. and Wedlich, D. (2000). The HMG-box transcription factor XTcf-4 demarcates the forebrain-midbrain boundary. Mech. Dev. 93, 211-214.[Medline]
Krauss, S., Johansen, T., Korzh, V., Moens, U., Ericson, J. and Fjose, A. (1991). Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube. EMBO J. 10, 3609-3619.[Abstract]
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444.[Medline]
Larabell, C. A., Torres, M., Rowning, B. A., Yost, C., Miller, J. R., Wu, M., Kimelman, D. and Moon, R. T. (1997). Establishment of dorso-ventral axis in Xenopus embryos is prestaged by asymmetries in ß catenin that are modulated by the Wnt signaling pathway. J. Cell Biol. 136, 1123-1136.
Leyns, L., Bouwmeester, T., Kim, S. H., Piccolo, S. and De Robertis E. M. (1997). Frzb-1 is a secreted antagonist of Wnt signalling expressed in the Spemann organizer. Cell 88, 747-756.[Medline]
Li, Y., Allende, M. L., Finkelstein, R. and Weinberg, E. S. (1994). Expression of two zebrafish orthodenticle-related genes in the embryonic brain. Mech. Dev. 48, 229-244.[Medline]
Macdonald, R., Xu, Q., Barth, K. A., Mikkola, I., Holder, N., Fjose, A., Krauss, S. and Wilson, S. W. (1994). Regulatory gene gene expression boundaries demarcate site of neuronal differentiation in the embryonic zebrafish forebrain. Neuron 13, 1039-1053.[Medline]
Masai, I., Heisenberg, C.-P., Anukampa Barth, K., Macdonald, R., Adamek, S. and Wilson, S. (1997). Floating head and masterblind regulate neuronal patterning in the roof of the forebrain. Neuron 18, 43-57.[Medline]
McMahon, A. P. and Moon, R. T. (1989). Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads duplication of the embryonic axis. Cell 58, 1075-1084.[Medline]
Molven, A., Njolstad, P. R. and Fjose, A. (1991). Genomic structure and restricted neural expression of the zebrafish wnt-1 (int-1) gene. EMBO J. 10, 799-807.[Abstract]
Moolenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Korinek, V., Roose, J., Destree, O. and Clevers, H. (1996). XTcf-3 transcription factor mediates ß-catenin-induced axis formation in Xenopus embryos. Cell 86, 391-399.[Medline]
Moon, R. T. and Kimelman, D. (1998). From cortical rotaion to organizer gene expression: toward a molecular explanation of axis specification in Xenopus. BioEssays 20, 536-545.[Medline]
Morita, T., Nitta, H., Kiyama, Y., Mori, H. and Mishina, M. (1995). Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain. Neurosci. Lett. 198, 131-134.[Medline]
Niehrs, C. (1999). Head in the Wnt- the moleculair nature of Spemanns head organizer. Trends Genet. 15, 314-319.[Medline]
Nieuwkoop, P. D., Boterenbrood, E. C., Kremer, A., Bloesma, F. S. N., Hoessels, E. L. M. J., Meyer, G. and Verheyen, F. J. (1952). Activation and organization of the central nervous system in amphibians. J. Exp. Zool. 120, 1-108.
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21, 1087-1095.[Abstract]
Pelegri, F. and Maischein, H. M. (1998). Function of zebrafish beta catenin and TCF-3 in dorsoventral patterning. Mech. Dev. 77, 63-74.[Medline]
Perry, W. L. I., Vasicek, T. J., Lee, J. J., Rossi, J. M., Zeng, L., Zhang, T., Tilghman, S. M. and Costantini, F. (1995). Phenotypic and moleculair analysis of a transgenic insertional allele of mouse Fused locus. Genetics 141, 321-332.
Piccolo, S., Agius, E., Leyns, L., Phattacharyya, S., Grunz, H., Bouwmeester, T. and De Robertis, E. M. (1999). The head inducer cerberus is a multifunctional antagonis of Nodal, BMP and Wnt signals. Nature 397, 707-710.[Medline]
Pierce, S. B. and Kimelman, D. (1995). Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3. Development 121, 755-765.
Pierce, S. B. and Kimelman, D. (1996). Overexpression of Xgsk-3 disrupts anterior ectodermal patterning in Xenopus. Dev. Biol. 175, 256-264.[Medline]
Ratcliffe, M. J., Itoh, K. and Sokol, S. Y. (2000). A positive role for the PP2A catalytic subunit in Wnt signal transduction. J. Biol. Chem. 275, 35680-35683.
Reifers, F., Böhli, H., Walsh, E., Crossley, P., Stainier, D. and Brand, M. (1998). Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenisis. Development 125, 2381-2395.
Roose, J., Huls, G., van Beest, M., Moerer, P., van der Horn, K., Goldsschmeding, R., Logtenber, T. and Clevers, H. (1999). Synergy between tumor supressor APC and the ß catenin-Tcf4 target Tcf1. Science 285, 1923-1926.
Salic, A., Lee, E., Mayer, L. and Kirschner, M. W. (2000). Control of ß-catenin stability: reconstitution of the cytoplasmic steps of the Wnt pathway in Xenopus egg extracts. Mol. Cell 5, 523-532.[Medline]
Shimizu, T., Yamanaka, Y., Ryu, S.-L., Hashimoto, H., Yabe, T., Hirata, T., Bae, Y.-K., Hibi, M. and Hirano, T. (2000). Cooperative roles of Bozozok/Dharma and Nodal-related proteins in the formation of the dorsal organizer in zebrafish. Mech. Dev. 91, 293-303.[Medline]
Sirotkin, H. I., Dougan, S., Schier, A. F. and Talbot, W. S. (2000). bozozok and squint act in parallel to apecify dorsal mesoderm and anterior neuroectoderm in zebrafish. Development 127, 2583-2592.
Smalley, M. J., Sara, E., Paterson, H., Naylor, S., Cook, D., Jayatilake, H., Fryer, L. G., Hutchinson, L., Fry, M. J. and Dale, T. C. (1999). Interaction of Axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription. EMBO J. 18, 2823-2835.
Stachel, S., Grunwald, D. J. and Myers, P. Z. (1993). Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development 117, 1261-1274.
Stambolic, V., Ruel, L. and Woodgett, J. R. (1996). Lithium inhibits glycogen synthase kinase-3 and mimics wingless signalling in intact cells. Curr. Biol. 6, 1664-1668.[Medline]
Talbot, W., Trevarrow, B., Halpern, M., Melby, A., Farr, G., Postlethwait, J., Jowett, T., Kimmel, C. and Kimelman, D. (1995). A homeobox gene essential for zebrafish notochord development. Nature 378, 150-157.[Medline]
Webster, M. T., Rozycka, M., Sara, E., Davis, E., Smalley, M., Young, N., Dale, T. C. and Wooster, R. (2000). Sequence variants of the axin gene in breast, colon, and other cancers: an analysis of mutations that interfere with gsk3ß binding. Genes Chrom. Cancer 28, 443-453.[Medline]
Welsh, G. I., Wilson, C. and Proud, C. G. (1996). GSK-3: a SHAGGY frog story. Trends Cell Biol. 6, 274-279.
Westerfield, M. (1995). The Zebrafish Book. Eugene: University of Oregon Press.
Woo, K. and Fraser, S. (1995). Order and coherence in the fate map of the zebrafish nervous system. Development 121, 2595-2609.
Yamada, T. (1994). Caudalization by the amphibian organizer; brachyury, convergent extension and retinoic acid. Development 120, 3051-3062.
Yamaguchi, Y. and Shinagawa, A. (1989). Marked alteration at midblastula transition in the effect of lithium on formation of the larval body pattern of Xenopus laevis. Dev. Growth Differ. 31, 531-541.
Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S. and Aizawa, S. (1997). Emx1 and emx2 functions in development of dorsal telencephalon. Development 124, 101-111.
Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., Lee, J. J., Tilghman, S. M., Gumbiner, B. M. and Costantini, F. (1997). The mouse fused locus encodes axin, an inhibitor of the wnt signaling pathway that regulates embryonic axis formation Cell 90, 181-192.[Medline]