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Overview of the role of -catenin in specification of the dorsal-ventral axis of Xenopus

Randall T. Moon
Howard Hughes Medical Institute and Department of Pharmacology
University of Washington School of Medicine
Seattle, WA 98195-7370

The issue of how a fertilized egg develops dorsal-ventral polarity during the cleavage stages has attracted many investigators, as solution of this question would lead to understanding the hierarchy of signals leading to formation of the gastrula organizer. It is clear that in Xenopus embryos the multi-functional protein -catenin is required for formation of the endogenous gastrula organizer, based on elegant antisense oligo-knockout experiments of Janet Heasman and Chris Wylie. Four important issues regarding the role of -catenin in axis specification are considered below:

Is endogenous -catenin expressed in a manner consistent with a role in axis specification? This is an important question, as it is irrelevant that ectopic -catenin induces an axis if endogenous -catenin does not show greater levels, or activity, on the future dorsal side of the embryo. Similarly, the loss-of-function experiments are hard to interpret when you don't know where the protein even functions.

This question has now been resolved by confocal microscopy of the early embryo. Larabell et al. in J. of Cell Biology show that -catenin accumulates opposite the sperm entry point by the end of the first cell cycle. It continues to accumulate in dorsal (i.e., opposite the sperm entry point) but not ventral cytoplasm through the early cleavage stages. By the 16- to 32- cell stages it accumulates in dorsal but not ventral nuclei. Remarkably, the pattern of dorsal accumulation of -catenin closely parallels the ability of transplanted dorsal cells to induce an axis when implanted into host embryos (reviewed by Larabell et al., 1997). Thus, -catenin is the first signaling molecule to show a dorso-ventral polarity in the early embryo. Combined with the loss-of-function data from Heasman et al., it is now clear that when fertilization elicits a cortical rotation, and displacement of material and organelles to the future dorsal side (Rowning et al., 1997, Proc. Natl. Acad. Sci. USA 94: 1224-1229), this leads to a dorso-ventral asymmetry in -catenin, which is required for axis formation.

The next question is "what establishes this asymmetry in -catenin?" This is partially answered by Yost, Torres et al. in Genes and Development. The results show that a serine/threonine kinase, glycogen synthase kinase-3 (Xgsk-3), directly phosphorylates -catenin in vitro at an amino terminal site. Deletion or mutation of this site greatly reduces phosphorylation of -catenin in embryos, and inhibition of endogenous Xgsk-3 with a dominant negative protein also reduces phosphorylation of -catenin. The experiments show further that phosphorylation of -catenin by Xgsk-3 targets it for rapid degradation in the embryo. When this phosphorylation by Xgsk-3 is inhibited in the embryo, -catenin accumulates in both the cytoplasm and in the nucleus. The nuclear -catenin likely functions in conjunction with HMG-box architectural transcription factors to modulate expression of specific genes (Molenaar et al., Cell, August, 1996).

Thus, Yost, Torres et al. propose that after fertilization of Xenopus eggs, dorsal-ventral differences in Xgsk-3 activity may arise, due to presently unknown mechanisms, though Rowning et al. (1997) suggest how this could be linked to cortical rotation. These differences in kinase activity lead to dorsal-ventral differences in -catenin phosphorylation and thus steady-state levels. Greater dorsal -catenin then participates in establishing dorsal mesoderm, which leads to formation of the gastrula organizer.

The third question is "what lies downstream of -catenin?" Restated, we know that injection of Wnts (McMahon and Moon, 1989, Cell 58: 1075-1084), or -catenin (work of Gumbiner and colleagues), on the ventral side of an embryo, induces a large number of genes and an ectopic axis. Therefore, any or all of these genes might normally be regulated by -catenin in the unperturbed embryo. However, are these genes a direct target of -catenin, or is this highly indirect, perhaps working without -catenin ever entering a nucleus?

The issue of whether any dorsally expressed genes are the direct target of -catenin has just been answered. Brannon et al. in Genes and Development (in press) show that the HMG Box factor XTcf-3 directly binds the siamois promoter. In the absence of -catenin, XTcf-3 inhibits gene expression. However, on the dorsal side of the embryo, -catenin binds the XTcf-3, and now activates the gene. This is notable because siamois is a homeobox gene likely playing a major role in specifying formation of Spemann's Organizer. So, a dorso-ventral difference in -catenin forms within an hour or two of fertilization, directly regulating a key homeobox gene in the blastula, thus contributing to forming Spemann's Organizer on the dorsal side of the gastrula.

The next issue is "where in the cell does -catenin function?" From the discussion above, you would probably say that since -catenin accumulates in dorsal nuclei, and directly interacts with a transcription factor, -catenin probably works in the nucleus. However, keep in mind that -catenin is found at the plasma membrane in association with cadherins, at the plasma membrane in association with the tumor suppressor promoter APC and microtubles, in the cytoplasm, and in the nucleus. So, it cannot be presumed that it must work only in the nucleus to "signal" a Wnt response.

Recently, Klymkowsky and colleagues (Merriam et al.. 1997. Develop. Biol.185: 67-81) found that ectopic membrane-tethered plakoglobin (related to -catenin) was active in axis induction. They speculated that plakoglobin, and -catenin, work by keeping a repressor of dorsal cell fate out of the nucleus, and speculate this repressor is XTcf-3 (Molenaar et al., 1996). Miller and Moon (J. Cell Biol., 1997, in press) performed similar experiments with ectopic membrane tethered -catenin, and likewise found it to be active in axis induction. However, they came to a very different conclusion and interpretation. They find that ectopic membrane-tethered -catenin, and even ectopic wild-type plakoglobin, bind to endogenous APC. Since APC normally plays a role in degrading -catenin, this results in the artefactual stabilization and accumulation of -catenin. As -catenin levels rise, it accumulates in nuclei. So, if this nuclear -catenin were responsible for the axis inducing activity of membrane-tethered -catenin, you might expect that co-expression of cadherin (which would titrate the free -catenin to the membrane) would block the axis inducing activity of membrane tethered -catenin. On the other hand, if -catenin functions to keep a repressor out of the nucleus, then cadherin probably should not block its axis inducing activity. The results show that cadherin does block the axis inducing activity of membrane-tethered -catenin. A final answer of where -catenin functions in the cell during Wnt signaling is under investigation in several labs, in a genetic background where -catenin is not present.

Return to Initiating the Embryonic Body Plan: Dorsalization of the Xenopus Embryo.

Digging Deeper:

A current model of Wnt signaling has recently been posted on the Moon laboratory Web site.

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Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Thursday, June 25, 1998