To generate mice in which Axin is replaced by Axin2, we inserted a mouse Axin2 cDNA in place of exon 2 of the Axin
gene (Fig. ). The resulting AxinAx2
allele cannot encode Axin but should express Myc-tagged Axin2 from the Axin
locus. As a control, we generated a second allele, AxinAx
, in which Axin cDNA was inserted into the Axin
locus. Although Axin
normally encodes two isoforms that differ by the presence or absence of 36 amino acids (52
), a cDNA can encode only a single isoform, and we used form 1 Axin, which lacks the 36 amino acids. There is only one known isoform of Axin2.
Correctly targeted ES cell lines were obtained (Fig. ) and used to generate germ line chimeric mice. Heterozygotes for both alleles appeared normal and were mated to produce homozygotes, which were identified by Southern blotting and PCR (Fig. ). AxinAx2/Ax2 and AxinAx/Ax homozygotes were found in the expected proportions and also appeared normal. Both sexes were fertile and had an apparently normal life span. No premature deaths, obvious behavioral defects, or overt tumors were observed among approximately 50 AxinAx2 homozygotes over a period of 18 months. Histological analysis on a number of organs (liver, lungs, kidney, small and large intestines, stomach, heart, and spleen), including several where Wnt signaling is known to be important for development, revealed no abnormalities (data not shown). Of course, we cannot rule out the possibility that mice expressing only Axin2 have subtle defects yet to be detected.
As a more stringent test, we crossed AxinAx2
mice to those carrying the AxinTg1
-null allele (52
), thus generating AxinAx2/Tg1
compound heterozygotes, in which the level of Axin2 should be only half that in AxinAx2/Ax2
homozygotes. If Axin2 were less active than Axin at performing their shared functions, the AxinAx2/Tg1
compound heterozygotes might reveal a phenotypic defect not seen in AxinAx2/Ax2
homozygotes. However, these mice were indistinguishable from the AxinAx2/Ax2
The only minor defect we detected, both in AxinAx2/Ax2 and AxinAx/Ax homozygotes, was a reduction of 11 to 14% in birth weight compared to that of wild-type littermates. AxinAx2/Ax2 mice were 11% smaller than wild-type mice at birth (P = 0.06), 12% smaller at 14 days (P = 0.06), and 12% smaller at 28 days (P = 0.03). AxinAx/Ax mice were 14% smaller than wild-type mice at birth (P = 0.001), 12% smaller at 14 days (P = 0.003), and 11% smaller at 28 days (P = 0.06). However, by 6 weeks of age, the difference was only 5 to 6% and was not statistically significant (P = 0.55 for AxinAx2/Ax2 mice and P = 0.32 for AxinAx/Ax mice). As the AxinAx2 and AxinAx alleles had the same effect, this does not reflect a functional difference between Axin and Axin2 but rather a property of both knockin alleles. One possibility was that these alleles might express too little or too much Axin/Axin2. To compare the amounts of protein encoded by the wild-type and AxinAx alleles, we performed immunoblotting with AxinAx/+ heterozygous embryos and an anti-Axin antiserum that detects both Myc-tagged and endogenous Axin. Myc-tagged Axin is larger than endogenous Axin, and the two bands are easily resolved (Fig. ). This analysis showed that the AxinAx allele encodes Myc-Axin at the same level as endogenous Axin, ruling out this explanation for the low birth weight. An alternative possibility is that the Myc tag, present in both AxinAx and AxinAx2 alleles, causes this transient defect.
FIG. 2. Expression of Axin2 and Axin in AxinAX2 and AxinAX mice. (A) Protein lysates from wild-type (WT) and AxinAx/+ heterozygous embryos were probed with anti-Axin antibodies. The heterozygotes expressed equal amounts of Myc-tagged Axin from the Axin (more ...)
Although our targeting strategy precluded the expression of any functional Axin from the AxinAx2 locus (since the initiation codon and exon 2, which encodes the essential RGS domain, were deleted), we wanted to test this at the protein level. We therefore performed immunoblotting with anti-myc and anti-Axin antisera on extracts of AxinAx2 embryos (Fig. ). This confirmed that the AxinAx2/Ax2 homozygotes expressed Myc-tagged Axin2 and lacked endogenous Axin.
Our results show that Axin2, when expressed under the control of Axin
regulatory sequences, can carry out all the essential functions of Axin during development as well as in adult life. If Axin
are functionally identical, why have the two genes been conserved during evolution? One explanation is that the total level of Axin-like protein (Axin plus Axin2) needs to be elevated in certain cells; Axin
provides a basal level in all cells (52
), while Axin2
, which is induced by Wnt/β-catenin signaling, is regulated to provide elevated levels where needed (4
). Additional insight into this question derives from the analysis of a null-mutant allele (lacZ
insertion) of Axin2
). Mice lacking Axin2
are viable and fertile but display skull malformations resembling craniosynostosis in humans, due to the premature fusion of cranial sutures (51
). Thus, in the presence of a normal complement of Axin
is dispensable for most developmental processes, although it is important for postnatal skull development. To further examine the relationship between Axin
, mice with various combinations of AxinTg1
- and Axin2lacZ
-null alleles were examined (B. Jerchow and W. Birchmeier, personal communication). Double homozygotes lacking both Axin
died much earlier (by E6.5) than those lacking only Axin
, indicating that endogenous Axin2
can partially compensate for the absence of Axin
in early embryogenesis. Furthermore, while mice with only one wild-type Axin
) are normal (52
), the further removal of Axin2
) resulted in severe brain and craniofacial abnormalities at birth. Thus, one Axin
allele is sufficient in the presence of Axin2
but not in its absence. Overall, these findings argue that when Axin is absent or reduced, Axin2 can partially compensate for its developmental functions. Axin2 does not fully compensate because it is not ubiquitously expressed. However, in the AxinAx2
allele, where the pattern of Axin2 expression is altered to resemble that of Axin, it can replace the functions of Axin.
The functional equivalence of the Axin and Axin2 proteins during mouse development implies that any amino acids that are not conserved between the two proteins are not required for their function. Similarly, any interactions with other proteins that are not shared by Axin and Axin2 are unlikely to be important. Axin interacts directly with at least 17 other proteins (Fig. ) (31
), but only a small subset of these has been tested for interaction with Axin2. We suggest that one way to evaluate the importance of these multiple protein-protein interactions for the functions of Axin would be to test if they interact similarly with Axin2; those that fail are unlikely to be required for the developmental functions of Axin.
Our results also call into question the significance of the differences in subcellular locations observed for Axin and Axin2. Anderson et al. (3
) previously reported that in normal colon epithelial cells, Axin was found in several locations—diffusely in the nucleus, along cell membranes, and often in the cytoplasm—while Axin2 was uniformly expressed in the nucleus. In adenomatous polyps, Axin was strongly cytoplasmic while Axin2 remained nuclear. Given our results, it is unlikely that these differences in localization reflect important differences in the functions of the two proteins. While other studies have shown previously that Axin shuttles between the nucleus and the cytoplasm (8
), it is very unlikely that all of the functions of Axin and Axin2 can be carried out in the nucleus. Therefore, the apparent nuclear localization of Axin2 probably does not reflect its major site of action. Perhaps a low level of the protein in the cytoplasm or at the cell surface, below the level of detection by immunostaining, is sufficient to carry out its essential cytoplasmic functions. The equivalence of Axin and Axin2 proteins, despite their different subcellular localizations, is reminiscent of the finding that APC1 and APC2 are functionally redundant in the fly, although these proteins also display different intracellular locations (1
The insertion of form 1 Axin cDNA into the Axin locus (in the AxinAx allele), while primarily intended as a control for the AxinAx2 allele, provided some useful information. First, it showed that the long isoform of Axin, form 2, is not required for normal development. On the other hand, the transient growth defects observed in both AxinAx and AxinAx2 homozygotes might be due to the lack of form 2 Axin or to the absence of the upstream Axin coding sequences. Second, the normal development of AxinAx/Ax homozygotes suggests that this targeting strategy can be used to efficiently generate new mutant alleles of Axin, in which specific domains or amino acids are altered. This approach is currently being used to examine the importance in vivo of several conserved sequences believed to play important roles in the functions of Axin.