Mouse axin and axin2/conductin proteins are functionally equivalent in vivo.

Axin is a central component of the canonical Wnt signal transduction machinery, serving as a scaffold for the β-catenin destruction complex (23, 31, 36, 45, 52). Axin has specific binding sites for many proteins involved in Wnt signal transduction, including β-catenin, glycogen synthase kinase 3 (GSK3), CKI, adenomatous polyposis coli (APC), Dvl, LRP, and protein phosphatase 2A (Fig. ​(Fig.1A)1A) (23, 31). Its key function in this pathway is to bring together β-catenin and the protein kinases CKI and GSK3, thus promoting the phosphorylation and consequent destruction of β-catenin. In the presence of a Wnt signal, this function is overcome, allowing β-catenin to accumulate and enter the nucleus (9, 13). The mechanism by which a Wnt signal leads to the inactivation of the Axin complex is not entirely clear, but it is thought to involve the binding of Axin to the Wnt coreceptor LRP as well as to Dvl (7, 34, 42, 44, 45). This results in the dephosphorylation of Axin, leading to a decrease in its affinity for β-catenin, and in a decrease in the level of Axin (20, 47, 48). Axin also enters the nucleus and appears to play a role in the nuclear-cytoplasmic shuttling of β-catenin (8, 46). FIG.1. Targeted replacement of the Axin gene with myc-tagged Axin2 cDNA or myc-tagged Axin cDNA. (A) Schematic diagram of Axin and Axin2 proteins and binding partners. Percent similarities between the conserved RGS and DIX domains and the GSK3 and β-catenin ... The major product of the Axin gene is a protein of 832 or 868 amino acids (depending on alternative splicing) containing two highly conserved domains (52): the RGS domain, which encompasses the binding site for APC (16, 18), and the DIX domain, a region of homology with Dvl proteins that is implicated in the binding of Axin to Dvl as well as in homodimerization (10, 17, 22, 24, 29, 40). (The open reading frame of Axin cDNA potentially encodes proteins of 956 and 992 amino acids, but proteins of this length have not been detected, and the major protein begins at codon Met-125 of the original sequence.) Mutant embryos lacking Axin die at embryonic day 9.5 (E9.5) with abnormalities including truncation of the forebrain, neural tube defects, and embryonic axis duplications (14, 35). Axin is expressed ubiquitously during embryogenesis, and the presence of axis duplications in its absence is thought to be a consequence of the abnormal accumulation of β-catenin, mimicking a Wnt signal, in the early embryo. Axin2 (also known as Conductin) is 44% identical to Axin and shares the RGS and DIX domains (Fig. ​(Fig.1A)1A) (5, 39, 49) as well as the binding sites for β-catenin, GSK3, Diversin, and Smad3 (Fig. ​(Fig.1A)1A) (5, 11, 39, 49). While Axin2 has been studied less extensively than Axin, most data suggest that they are similar in function. Thus, when overexpressed in cultured cells, both proteins reduce the levels of β-catenin and the expression of Wnt target genes, and when expressed in frog embryos, both inhibit the development of dorsal structures (5, 19, 37, 49, 52). However, they are not fully redundant in vivo, as Axin2 is clearly unable to compensate for the absence of Axin in Axin-null embryos (52). Furthermore, humans with heterozygous germ line AXIN2 mutations have familial tooth agenesis (25), while mice homozygous for loss of Axin2 have skull abnormalities (51), indicating that Axin does not fully substitute for the lack of Axin2. In addition, deletions or mutations in AXIN or AXIN2 have been observed in a number of types of tumors, including colorectal cancer and hepatocellular carcinoma, indicating that each gene is a tumor suppressor and that neither gene can replace the other for this function (30, 38, 43). One reason for the lack of full redundancy may be the different expression patterns of Axin and Axin2. While Axin mRNA is ubiquitous (52), Axin2 is expressed in tissue- and developmental-stage-specific patterns, due (at least in part) to its transcriptional upregulation by canonical Wnt signaling (4, 21, 27). Thus, Axin2 potentially constitutes a negative-feedback mechanism regulating the response to Wnt signals (21, 27, 32, 50). In the mouse embryo, the inability of Axin2 to compensate for the lack of Axin might be explained simply by the fact that Axin2 is not expressed in every cell. However, the two proteins might also differ significantly in function, a possibility supported by their different subcellular localizations in at least some cell types (3). If the functional differences were subtle, they might not be detected using assays that involve overexpression; since the normal level of Axin is extremely low (26), the abnormally high levels of expression in these assays could mask a functional difference. To address the question of functional redundancy in a more physiological way, we generated knockin mice in which the Axin gene was replaced with an Axin2 cDNA sequence. This gene replacement strategy is similar to those used successfully to compare the functions of other paralogous gene pairs (6, 12, 15, 33, 41). The main advantage of this approach over in vitro assays or other in vivo functional assays (e.g., transgenic rescue) is that targeting of a cDNA to the appropriate genetic locus results in a normal pattern and level of expression. In animals homozygous for the resulting allele, AxinAx2, there will be no Axin, but Axin2 will be expressed ubiquitously from the Axin locus at a physiological level, as well as in its normal tissue-specific pattern from the unmodified Axin2 locus. Thus, the total level of Axin plus Axin2 expression in any cell type should be unchanged from that of the wild type. The ability of these mice to develop and survive normally thus tests the capacity of Axin2 to replace Axin in vivo.