Transcriptional repression by Snail family members contributes to many biological and cellular processes, including Xenopus neural crest development, by affecting cellular differentiation, survival, and migration (Carl et al. 1999
; LaBonne and Bronner-Fraser 2000
; Aybar et al. 2003
; Tribulo et al. 2004
; Zhang et al. 2006
). An N-terminal SNAG domain is both necessary and sufficient for Snail/Slug-mediated repression, but the precise mechanisms regulating repression in different tissues and organisms remain unclear. Here, we show that Ajuba family LIM proteins, Ajuba, LIMD1, and WTIP, specifically function as Snail/Slug corepressors in mammalian cells as well as in vivo
to regulate Xenopus neural crest development. In support of this conclusion; 1) Ajuba LIM proteins interact predominately with the SNAG domain of Snail/Slug in cells and accumulate in the nucleus in a SNAG-dependent manner. 2) Ajuba LIM proteins contribute to Snail-dependent repression of E-cadherin transcription in cells. 3) Ajuba LIM proteins are present on endogenous promoters of Snail-regulated genes (e.g. E-cadherin), but only in the presence of Snail. 4) Expression of Ajuba LIM proteins in Xenopus embryos mimics expression of Snail/Slug in that they both enhance neural crest development, and this effect of Ajuba LIM proteins requires Slug expression. 6) Only isoforms of LIMD1 capable of interacting with Snail cause increased neural crest in Xenopus. 7) Depletion of Ajuba LIM proteins in Xenopus embryos phenocopies depletion of Slug with both resulting in a block or inhibition of neural crest development. 8) The effect of Ajuba LIM proteins upon Xenopus neural crest development occurs via mechanisms similar to those regulated by Snail/Slug. Upon loss of either, there is a compensatory increase in Sox2 (neural) expression in border territories and a decrease in cell survival, with no change in proliferation. 9) The block in Xenopus neural crest development upon depletion of Ajuba LIM proteins was not rescued by concurrent Slug expression but can be rescued by the dominant repressor protein, EngR.SlZnF.
Although Ajuba LIM proteins play a number of roles in cells that could potentially affect neural crest development, our evidence strongly supports a major role as Snail/Slug corepressors. Throughout, gain- and loss-of function experiments of Ajuba LIM proteins consistently phenocopy Snail and Slug. It is possible that Ajuba LIM proteins directly or indirectly (through affecting a non-Snail/Slug-mediated neural crest pathway) affect the expression of Snail or Slug, and this could have resulted in similar phenotypes. Mapping experiments, however, revealed a direct correlation between the ability of LIMD1 to interact with Snail and its capacity to enhance neural crest development when overexpressed. Moreover, if influencing expression of Snail/Slug is a role for Ajuba LIM proteins, we would expect expression of Snail or Slug to rescue the loss of Ajuba LIM proteins expression in Xenopus. They do not rescue that loss, but the EngR.SlZnF fusion protein, which eliminates a dependence on SNAG interactions, does. While we cannot rule out the possibility that other functions of Ajuba LIM proteins may also contribute to neural crest development, these results demonstrate that the Snail/Slug corepressor function of Ajuba LIM proteins is likely their primary role in this process.
We propose a model in which Ajuba LIM proteins bind nuclear Snail on specific gene promoters through an interaction of the LIM region (LIM1 and/or 2) with the SNAG domain of Snail. LIM domains do not directly bind DNA, but LIM proteins have been shown to regulate gene expression through direct LIM domain interaction with known transcription factors (Zhao et al. 1999
; Sharp et al. 2004
; Srichai et al. 2004
; Guo et al. 2006
). In most cases, the LIM region directs the interaction with the DNA binding protein. Accumulated data on Ajuba function have shown that the LIM and preLIM regions often interact with distinct targets to bring together proteins that contribute to common cellular functions (Marie et al. 2003
; Pratt et al. 2005
). We hypothesize that the preLIM regions are important for corepressor activity, perhaps through interactions with repressor complex proteins (see ). This model is based, in part, on evidence that overexpression of the LIM region, which mediates the Snail interaction, blocks or inhibits association of full-length Ajuba and Snail in cells, inhibits Snail-dependent repression of E-cadherin transcription in transient assays, and inhibits neural crest development in vivo
(unpublished data). It will be critical to define the specific role(s) of the preLIM region in Snail/Slug-mediated repression.
All three Ajuba LIM proteins interact with Snail family members, suggesting that they serve redundant functions. The potential for compensation between Ajuba LIM proteins is apparent in mice as we do not observe overt developmental pathologies in Ajuba−/−
mice ((Marie et al. 2003
; Feng et al. 2007
) and unpublished data). In Xenopus, however, we observe a dose-dependent response to depletion of either XLIMD1 or XWTIP. While the precise reason why depletion of only one family member gives a significant phenotype in Xenopus neural crest is not known, it may be that this system is more sensitive to reduction in total amount of Ajuba LIM proteins. Indeed, we observe a greater effect on neural crest development when both XLIMD1 and XWTIP are depleted. In addition, Slug overexpression rescued loss of XLIMD1 alone, but not the loss of both proteins ( and unpublished data). The discrepancy between the murine and Xenopus neural crest phenotypes following loss of Ajuba LIM proteins is most likely explained by the fact that neither Snail nor Slug is required for early neural crest development in mice (Murray and Gridley 2006
The precise hierarchy of transcription factors controlling Xenopus neural crest development is complicated by multiple feedback loops and cross talk. Ajuba LIM proteins, as corepressors to both Snail and Slug, likely impact at multiple points during this process. Through a temporal analysis of neural crest markers, we found that Ajuba LIM proteins are required early in neural crest development. Loss of either XLIMD1 or XWTIP results in disruption of Pax3 and Snail staining as early as stage 12 (supplemental Table 1
). In developing neural crest, Snail expression has been observed as early as stage 11−12, preceding Slug expression (Linker et al. 2000
), suggesting that this early function of Ajuba LIM proteins may be due to its interaction with Snail instead of Slug. However, we also observed that the Slug depletion caused a similar loss of early neural crest markers (supplemental Table 1
). This suggests that Slug may function earlier than previously appreciated and may contribute in a feedback loop to maintain expression of Snail, as well as other neural crest markers. Further work will be necessary to fully understand the temporal relationships between Ajuba LIM proteins, Snail, Slug, and other factors that regulate neural crest development, as well as to distinguish between the Snail and Slug corepressor functions of the Ajuba LIM proteins during neural crest development.
We describe a nuclear role for Ajuba LIM proteins as corepressors of Snail that contribute to E-cadherin repression. Ajuba LIM proteins are also components of epithelial cell-cell junctions (Marie et al. 2003
; Srichai et al. 2004
). Ajuba influences the formation and/or stability of adherens junctions, possibly by coupling the E-cadherin adhesive complex to the actin cytoskeleton (Marie et al. 2003
). Because of distinct roles for separate cellular pools, Ajuba may play an integral role in communicating between cell surface adhesive complexes and the nucleus to provide precise regulation of epithelial dynamics. While we do not have evidence that Ajuba LIM proteins alone initiate EMT, we observe Ajuba accumulation in the nucleus at Snail-regulated promoters in the presence of Snail. Ajuba LIM proteins, as Snail co-repressors, could then contribute to a feed forward loop to maintain the mesenchymal phenotype. By contributing to E-cadherin repression, Ajuba may also indirectly allow more Ajuba to be available for entry into the nucleus. Consistent with this, Ajuba is released from adherens junctions upon expression of Snail and subsequent transcriptional downregulation of E-cadherin (Jamora et al. 2005
). We have also observed the release of Ajuba from epithelial junctions in HaCaT epithelial cells upon treatment with TGF-β, which induces EMT in these cells (unpublished data).
Whether Ajuba LIM proteins also play a role in mesenchymal to epithelial transitions (MET) is an interesting possibility. MET occurs physiologically during mammalian nephrogenesis (Kanwar et al. 2004
) and pathologically as metastatic cells reform epithelial-like tumors at metastatic sites (Thiery and Sleeman 2006
). Recently, Snail was shown to be capable of repressing its own transcription (Peiro et al. 2006
), perhaps providing a mechanism to initiate MET. Ajuba, as a Snail corepressor, may enhance Snail-mediated repression of Snail transcription. As Snail expression decreases, Ajuba would exit the nucleus and E-cadherin transcription would resume. Ajuba could then be recruited to newly forming adherens junctions and contribute to epithelia formation.