EvoprintHD of Axn

BIOLOGICAL OVERVIEW

The vertebrate Axin protein, the product of the mouse fused gene, binds to beta-catenin to inhibit Wnt signaling. A homolog of vertebrate Axin, Drosophila Axin (here termed Axin in conformity to FlyBase usage) has been identified. Axin knockout produces phenotypes that are similar to overexpression of the Drosophila Wnt gene wingless (wg). Overexpression of Axin produces phenotypes similar to loss of wg. Axin overexpression can modify phenotypes elicited by wg and another Drosophila Wnt gene, Wnt oncogene analog 2. Using immunoprecipitation of endogenous Axin embryonic protein, it has been shown that Axin interacts with Armadillo and Zeste-white 3. The loss-of-function and overexpression phenotypes show that Axin, like its mammalian counterpart, acts as a negative regulator of wg/Wnt signaling (Willert, 1999b). Together, Axin, APC and Shaggy/Zeste-white 3 form a protein complex termed the "ß-catenin destruction complex."

Two approaches have been used to clone Axin. Hamada (1999) performed a yeast two-hybrid screen of a Drosophila embryo cDNA library using the Armadillo repeat domain of Armadillo as target and identified Axin as an Arm-interacting protein. Willert (1999b) identified Axn by searching the EST database with the protein sequence of mouse Axin. An EST with significant homology to the DIX (a domain similar between Axin and Dishevelled) domain of Axin was identified and used to isolate the full-length clone from an embryonic Drosophila cDNA library (Willert, 1999b).

Loss-of-function mutant phenotypes can be mimicked by the injection of double stranded RNA. This method, referred to as RNA interference (RNAi), was initially successfully used in C. elegans and more recently in Drosophila. RNAi was used to disrupt the function of Axn during patterning of the embryonic cuticle, a process that requires Wg signaling. The wild-type embryo secretes a cuticle consisting of a repeated pattern of denticle belts with intervening naked regions. Loss of wg leads to a cuticle covered with denticles -- one lacking any naked areas. Overexpression of wg in the embryo leads to loss of all denticle structures, i.e. a naked cuticle. Disruption of AXN expression by RNAi leads to a similar naked cuticle, suggesting that Axn functions to down-regulate Wg signaling. Virtually all injected embryos had extra naked cuticle, ranging from partially to nearly completely naked. Control injection of either sense or antisense single stranded AXN mRNA does not cause any phenotypic changes. As expected, wg RNAi produces a partial wg-like cuticle. Thus, mimicking loss of Axn function by RNAi leads to phenotypes similar to overexpressing wg, consistent with the model that Axn is a negative regulator of Wg signaling (Willert, 1999b).

A P-element insertion near the beginning of the Axn gene has been shown to disrupt expression of the gene to produce a loss-of-function allele of Axn. Embryos lacking zygotic Axn are still wild type and only upon removal of the maternally contributed Axn gene product is a naked cuticle revealed. Thus, the RNAi experiments successfully disrupt the maternally contributed Axn gene product and produce a phenotype identical to that of a loss-of-function mutation in the gene (Willert, 1999b).

To address further whether Axn regulates Wg signaling, the UAS-Gal4 system was used to overexpress Axn in various tissues. The Daughterless (Da)-Gal4 driver expresses early during embryogenesis; when combined with UAS-wg, it produces a completely naked cuticle. Overexpression of Axn using the Da-Gal4 driver produces a loss of wg-like phenotype, a cuticle covered with denticles, consistent with overexpression of Axn blocking Wg signaling in the embryonic epidermis. To extend this study Axn was misexpressed in the wing using the 69B-Gal4 driver. wg is expressed in a narrow stripe along the presumptive wing margin where it is required for proneural achaete-scute complex gene expression and for the formation of margin bristles. Loss of Wg signaling along the wing margin, as in the case of dsh loss-of-function clones, leads to loss of these margin bristles and notches along the wing. Overexpression of Axn in the wing also produces this wing notching effect, a result consistent with Axn interfering with the Wg signaling pathway at the wing margin (Willert, 1999b).

Deletion of the RGS domain of mouse Axin produces a dominant negative Axin protein, as assayed by its ability to induce a secondary axis when overexpressed in Xenopus embryos (Itoh, 1998 and Zeng, 1997). An analogous mutation was constructed in Axn that deletes the entire RGS domain (DaxinDeltaRGS; deletion of amino acids 50 to 172). Surprisingly, when overexpressed, this Axn mutant produces the same phenotypes in the embryo and in the wing as does wild-type Axn overexpression. Thus, the RGS domain is dispensable in overexpression assays for Axn. Thus, the RGS domain, which interacts with the APC protein (Behrens, 1998 and Hamada, 1999), is dispensable for overexpressed Axinís ability to block Wnt signaling. The discrepancy between the Xenopus and fly data with respect to the RGS deleted Axin may be explained in a number of ways. In analogy to APC, Axin overexpression in various organisms may produce distinct phenotypes. APC overexpression in mouse cell culture is known to down regulate Wnt signaling, while overexpression of APC in Xenopus embryos leads to axis duplication, a hallmark phenotype of genes that activate Wnt signaling. As another exception to the model of APC being a negative factor, the C. elegans APC homolog Apr-1 has been shown to positively regulate Wnt signaling. The reasons for the inconsistent phenotypes for APC loss- and gain-of-function are not yet understood (Willert, 1999b and references).

In support of the findings presented by Willert (1999b), Hart (1998) has shown that overexpression of human Axin lacking the APC binding domain (i.e. the RGS domain) promotes Axinís ability to downregulate beta-catenin levels. Although both mutant forms of mouse Axin (Zeng, 1997) and Axn primarily delete the RGS domain, there are subtle differences in the deletions: DaxinDeltaRGS deletes the entire RGS domain (amino acids 50-172 in Axn corresponding to amino acids 89-216 in mouse Axin) while mouse AxinDeltaRGS deletes part of the RGS domain and some N-terminally flanking sequence (amino acids 124-227 in Axin corresponding to amino acids 87-183 in Axn). In sum, it remains to be established whether the interaction between Axin and APC is critical for Axin to inhibit Wnt signaling. A genetic analysis of the Axn gene and identification of mutations that disrupt this interaction will likely provide insights into this question (Willert, 1999b).

The above data clearly demonstrate that Axn loss-of-function or overexpression produces phenotypes similar to wg overexpression or loss-of-function, respectively. However, it fails to demonstrate that Axn can modify the effects of Wnt signaling. To examine whether ectopic Axn expression can interfere with the effects of Wnt signaling, wg was misexpressed in the eye and DWnt-2 was misexpressed in the ovary. When overexpressed in the eye, wg produces a glassy eye that is greatly reduced in size compared to a wild-type eye. Misexpression of Axn in the eye produces a weakly rough eye. When Axn and wg are both overexpressed, the morphology of the eye is intermediate to that of a wild-type eye and that of a wg eye, indicating that Axn can interfere with the Wg signal (Willert, 1999b).

In order to address whether Axin inhibits only Wg, or whether it can function as a more general inhibitor of Wnt signals, an examination was carried out to see whether Axn could interfere with a different Wnt signal. Four Wnt genes have been identified in Drosophila (see The World Wide Web Wnt Window ), but mutations have been described only for two of them: wg and DWnt-2. Loss of DWnt-2 produces a muscle migration defect in the male gonads, resulting in male sterility, and a lack of the characteristic pigment cells that migrate over the male testis. Ovaries are normally not surrounded by pigment cells, but misexpression of DWnt-2 in females can induce ectopic male-specific pigment cells. This dominant phenotype of DWnt-2 misexpression was used to address whether Daxin can block ectopic pigment cell formation. Overexpression of Axn strongly reduces the frequency of DWnt-2-mediated pigment cell formation in ovaries, demonstrating that Axn can block the DWnt-2 signal in addition to the Wg signal. Overexpression of a dominant-negative Pangolin/dTCF lacking the amino-terminal Arm binding domain (DN-dTCF) also blocks the ectopic DWnt-2 induced pigment cells in the ovary (Willert, 1999b).

GENE STRUCTURE

Sequence analysis has identified two Axn species, termed A1 and A2. These forms of Axn are revealed by amino acid differences between two independent Axin isolates: Willert (1999b) and Hamada (1999). Form A2 (Willert, 1999b) includes the six amino acids SRSGSS while Form A1 of Hamada, 1999, does not.

PROTEIN STRUCTURE

Amino Acids - 739

Structural Domains

The Axin cDNA contains two alternative polyadenylation sites and an open reading frame encoding a 739 amino acid protein (form A1). Several clones (3 out of 5) contain an 18 basepair (bp) insert (form A2). This insert in Drosophila is in a similar, albeit not identical, region where in mouse, Axin contains a 108 bp insert to give rise to two different forms (form 1 and 2). The 18 nucleotides in the fly gene code for six amino acids, four of which are serine residues, directly amino-terminal to the DIX domain. No difference in the activities of the two isoforms of either mouse Axin or Drosophila Axn have been identified. The recently published sequence of Drosophila Axin (Hamada, 1999) is identical to form A2, but lacks two serine residues at position 644 and 645. The differences in sequences, including forms A1 and A2, may reflect naturally occurring polymorphisms in the Axn gene. Overall, the identity between mouse and Axn is 21% , although several domains are more highly conserved. The Axn protein is more distantly related (17%) to Conductin (Behrens, 1998), suggesting that it is the vertebrate Axin ortholog. Like the vertebrate Axin proteins, Axn contains an RGS domain near the amino terminus. These domains are found in a family of proteins that regulate G-proteins. Although no such G-protein signaling function has been attributed to the mouse Axin RGS domain (Mao, 1998), the RGS has been shown to be required for the interaction with the APC protein (Behren, 1998). Deletion of this domain in mouse Axin produces a dominant negative Axin protein, as assayed by axis duplication activity in Xenopus embryos (Itoh, 1998; Zeng, 1997). In contrast, overexpression of wild-type mouse Axin inhibits axis duplication, suggesting that interaction with APC is crucial for Axinís ability to block the Wnt signaling pathway. The region between the RGS and DIX domains, where both GSK-3beta and beta-catenin binding sites are located in Axin and Conductin, contains stretches of lower similarity. Despite this low similarity, Axn does in fact interact with both Arm and Zw3. Hamada (1999) mapped the Arm binding domain in Axn to amino acids 459-538 by precipitating in vitro translated Arm with GST-Axn fusion proteins, a region which contains significant homology with Axin and Conductin. The Axn protein contains four potential nuclear localization signals, but the mouse Axin protein, that also contains a nuclear localization signal, has been localized predominantly to the cytoplasm (Behrens, 1998 and Fagotto, 1999) (Willert, 1999b).

date revised: 6 October 99

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