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 16 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.

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 gsk3β 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 the mbl^-/- 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).

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 are 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 mbl mutation abolishes 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 Gsk3β 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 (lane 4; 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 β-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).