Cell signalling pathways triggered by Wnt proteins control gene expression and cell behaviour, especially during development. Work on frogs reveals another specific role for these proteins, and the signalling involved.
Living organisms tend to be highly economical, using the same proteins or cell-to-cell signalling processes in different contexts and for different purposes. Take, for example, the family of proteins known as NFATs. These are transcription factors — they regulate gene expression — and they have traditionally been implicated in controlling genes involved in immunity. The events leading up to such regulation include an increase in the levels of calcium ions in a cell; the NFAT transcription factors then move into the nucleus and form a complex with relevant regions of DNA (and, in some cases, with other transcription factors1). Several different signalling pathways are known to activate NFAT by changing calcium levels, and to this list can now be added one of the pathways that is triggered by Wnt proteins, as Saneyoshi and colleagues2 describe on page 295 of this issue. Rather than being involved in immunity, however, these events are key to early vertebrate development.
Members of the Wnt protein family are secreted from cells and picked up by receptor proteins on the surface of cells, triggering intracellular signalling pathways. These pathways regulate cell proliferation, death, fate and behaviour in contexts ranging from early embryonic development to colorectal cancer. The best-understood Wnt pathway is the 'canonical' one: here, Wnt-induced signalling suppresses degradation of the β-catenin protein, enabling it to accumulate in the nucleus3. Nuclear β-catenin then binds to particular transcription factors (not the NFATs) and thereby modulates gene expression.
Over the past few years it has become apparent that certain Wnt proteins and their receptors (proteins of the Frizzled family) can also activate 'non-canonical' pathways. Although the details of the canonical Wnt/Frizzled pathway are firmly supported by genetic, biochemical and cell-biological experiments in many species, the non-canonical pathways are less well understood. Nonetheless, it is known that a non-canonical 'planar cell polarity' pathway is used in fruitflies to orientate cells4.
There also seems to be more than one Wnt/Frizzled pathway at work in vertebrate embryos. Early studies of the South African clawed frog, Xenopus laevis, established that the overexpression of some Wnt proteins stabilizes β-catenin and results in twinned embryos, complete with two heads. Yet overexpression of other Wnt proteins perturbs cell movements during gastrulation3 — a crucial stage of development that leads to formation of the three main tissue layers: endoderm, mesoderm and ectoderm. These different effects hinted that there is more than one Wnt pathway in vertebrates.
The idea was substantiated by further work in zebrafish and Xenopus embryos, which suggested that the Wnts that perturb cell movements activate a non-canonical Wnt/Frizzled pathway3 involving increases in intracellular Ca2+ levels and the consequent activation of the enzymes protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)5. But questions remain, including the relationship between the non-canonical pathways in fruitflies compared with vertebrates, whether the Wnt/Ca2+ pathway works in any context other than early embryos, and whether it modulates gene expression or only gastrulation movements.
Saneyoshi et al.2 enter this arena of uncertainty by proposing that if signalling from certain Wnt proteins leads to rises in intracellular Ca2+ levels, it may also activate calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, leading to the removal of phosphate groups from NFAT and hence its accumulation in the nucleus. And indeed, they find that Wnt5A and Frizzled-2 — the same Wnt–receptor combination reported to lead to intracellular Ca2+ increases in zebrafish and to activate CaMKII and PKC in Xenopus — strongly induces the movement of NFAT into the nucleus in cells of Xenopus embryos.
The authors also observe that overexpression of a constitutively active NFAT perturbs the cell movements that occur during gastrulation in Xenopus, and encourages cells to take on characteristics typical of the belly (ventral) rather than the dorsal part of the embryo. Finally, this hyperactive NFAT antagonizes the duplication of the dorsal axis that can be induced by the canonical Wnt pathway. Similar effects have been seen3 with overexpression of Wnt5A. So Saneyoshi et al.'s findings support the existence of the Wnt/Ca2+ pathway in Xenopus, and identify NFAT as a likely downstream target of this pathway.
What, then, is the most plausible role of normal Wnt/Ca2+ signalling and NFAT in early frog embryos? Saneyoshi et al.'s data lend much weight to the previous suggestion5 that this non-canonical pathway helps to determine a ventral fate for cells and opposes the canonical pathway, which promotes dorsal cell fates in early embryos (Fig. 1). In support of this, Saneyoshi et al. show that expressing a presumed inhibitor of NFAT in frog embryos results in a new dorsal axis, as would be predicted if the NFAT had been promoting ventral cell fates.
So the new data are a tantalizing hint that Wnt/Ca2+ signalling promotes ventral cell fates by activating NFAT. But there are questions to be answered before we can be sure. First, does NFAT actually activate gene transcription in response to a Wnt/Ca2+ signal? Saneyoshi et al. show that, in the presence of one of its cooperative partners — the transcription factor AP-1 — a constitutively active Xenopus NFAT activates transcription of a 'reporter' gene inserted into Xenopus embryos. But it is not yet certain whether normal NFAT would do so in response to Wnt/Ca2+ signalling, or whether Wnt5A leads to the transcription of NFAT-responsive genes, as would be predicted. There is evidence that Wnt5A regulates AP-1-dependent transcription as well as Ca2+-mediated signalling6. So the Wnt/Ca2+ gene targets may encompass those regulated by AP-1 alone, or those regulated by complexes of AP-1 and NFAT.
Second, Saneyoshi et al.'s data predict that more NFAT proteins will be found in cell nuclei on the future ventral side of Xenopus embryos than on the future dorsal side. If that is the case, does this nuclear accumulation depend on Wnt signals? The activity of CaMKII is increased on the future ventral side in a Wnt-dependent manner5, but it remains to be seen whether the nuclear accumulation of NFAT is likewise increased. Third, we need to test the proposed role of NFAT by knocking out its function, using genetic or molecular means.
Finally, it will be interesting to see whether the Wnt5A-driven movement of NFAT to the nucleus is unique to early vertebrate development, where all characterization of the Wnt/Ca2+ pathway has been conducted to date. Unpublished work from Chris Hughes (Univ. California, Irvine) suggests the contrary: that this Wnt protein also induces NFAT to accumulate in the nuclei of the immune system's T cells. Whatever the answers, it is clear that it takes many Wnt pathways to make a frog — and, by extension, any vertebrate.
Macian, F., Lopez-Rodriguez, C. & Rao, A. Oncogene 20, 2476–2489 (2001).
Saneyoshi, T., Kume, S., Amasaki, Y. & Mikoshiba, K. Nature 417, 295–299 (2002).
Kuehl, M., Sheldahl., L. C., Park, M., Miller, J. R. & Moon, R. T. Trends Genet. 16, 279–283 (2000).
Axelrod, J. D. & McNeill, H. Sci. World J. (in the press).
Kuehl, M., Sheldahl, L. C., Malbon, C. C. & Moon, R. T. J. Biol. Chem. 275, 12701–12711 (2000).
Yamanaka, H. et al. EMBO Rep. 3, 1–7 (2002).
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