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

Abstract

Axin is a central component of the canonical Wnt signal transduction machinery, serving as a scaffold for the beta-catenin destruction complex. The related protein Axin2/Conductin, although less extensively studied, is thought to perform similar functions. Loss of Axin causes early embryonic lethality, while Axin2-null mice are viable but have craniofacial defects. Mutations in either gene contribute to cancer in humans. The lack of redundancy between Axin and Axin2 could be due to their different modes of expression: while Axin is expressed ubiquitously, Axin2 is expressed in tissue- and developmental-stage-specific patterns, and its transcription is induced by canonical Wnt signaling. Alternatively, the two proteins might have partially different functions, a hypothesis supported by the observation that they differ in their subcellular localizations in colon epithelial cells. To test the functional equivalence of Axin and Axin2 in vivo, we generated knockin mice in which the Axin gene was replaced with Myc-tagged Axin or Axin2 cDNA. Mice homozygous for the resulting alleles, Axin(Ax) or Axin(Ax2), express no endogenous Axin but express either Myc-Axin or Myc-Axin2 under the control of the Axin locus. Both Axin(Ax/Ax) and Axin(Ax2/Ax2) homozygotes are apparently normal and fertile, demonstrating that the Axin and Axin2 proteins are functionally equivalent.

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 (β-cat) binding regions of Axin and Axin2 are indicated, as are those of other less-conserved regions. The solid lines at the top indicate the regions of Axin involved in binding to the indicated proteins (31). Of these, only APC, GSK3, β-catenin, Diversin, and Smad3 are known to bind to Axin2. aa, amino acids; PP2A, protein phosphatase 2A. (B) Schematic diagram of the Axin genomic locus, targeting constructs, and targeted alleles. Exons 1 and 2 (EX1 and EX2) are depicted as grey boxes, and intron sequences are depicted as solid lines. The positions of the restriction enzyme sites and the probe PB are indicated. Small harpoons (⇁ and ↼) show the PCR primers AXL1 (a), MTAXR1 (b), and MTCONR1 (c). (C) Southern blot analysis of G418-resistant colonies after electroporation of ES cells with targeting constructs. Probing with probe PB following digestion of DNA with Hpa1 detected a band of 9.8 kb for the Axin^AX2 allele, a band of 10.5 kb for the Axin^AX allele, and a band of 8.8 kb for the wild-type allele. (D) Identification of homozygous, heterozygous, and wild-type (WT) Axin^AX2 and Axin^AX mice by PCR.

FIG. 2.

Expression of Axin2 and Axin in Axin^AX2 and Axin^AX mice. (A) Protein lysates from wild-type (WT) and Axin^Ax/+ heterozygous embryos were probed with anti-Axin antibodies. The heterozygotes expressed equal amounts of Myc-tagged Axin from the Axin^AX allele and wild-type Axin from the endogenous allele, showing that the Axin^AX allele is expressed at a normal level. (B, C, and D) Protein lysates from wild-type, Axin^AX/AX, and Axin^AX2/AX2 homozygous embryos were probed with anti-Myc antibody (B) or with two different anti-Axin antisera (C and D). In wild-type embryos, the anti-Axin antibodies detected endogenous Axin (∼110 kDa), while the anti-Myc antibody detected only background bands. In Axin^AX/AX embryos, both the anti-Axin and the anti-Myc antibodies detected Myc-tagged Axin (∼130 kDa), while endogenous Axin was absent. In Axin^AX2/AX2 embryos, the anti-Axin antibodies detected only background bands also seen in wild-type embryos, while the anti-Myc antibody detected Myc-tagged Axin2. Anti-Axin antiserum FF, a gift from François Fagotto, was raised against amino acids 406 to 685 of mouse Axin, while anti-Axin antiserum DV, a gift from David Virshup, was raised against full-length mouse Axin. Molecular size markers (in kilodaltons) are noted at the left of blots.