Snai1-deficient mice die early in development before the differentiation of mesodermal lineages (Carver et al., 2001; Peinado et al., 2007). Hence, we generated mice in which Snai1 could be inactivated in selected tissues by Cre/loxp-mediated recombination (Fig. 2, A–C). Fibroblasts isolated from a Snai1^+/fl mouse were treated with an adenoviral Cre recombinase construct (adeno-Cre) or a control adenovirus (β-galactosidase [β-gal]), and recombination at the Snai1 locus was verified by PCR. As shown in Fig. 2 D, although adeno–β-gal–infected fibroblasts yield P1/P2 amplicons corresponding to both the wild-type and loxp alleles of Snai1, adeno-Cre–infected fibroblasts yielded a single amplicon corresponding to the wild-type Snai1 allele with P1 and P2, as well as a P3/P4 amplicon representing the Snai1^- allele. Fibroblasts isolated from Snai1^fl/fl mice and infected with adeno-Cre display a 95% reduction in Snail1 mRNA, whereas Snail1 protein expression is undetectable by Western blotting or immunocytochemistry (Fig. 2, F and G).

In addition to its well-defined role in promoting EMT, Snail1 can regulate cell cycle progression and sensitivity to proapoptotic stresses (Vega et al., 2004; Barrallo-Gimeno and Nieto, 2005; Escriva et al., 2008). Snail1-deficient fibroblasts proliferate, however, at normal rates, with no observed changes in apoptosis under serum-free conditions (Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200810113/DC1). Furthermore, though Snail1 can promote a motile phenotype in epithelial cells (Barrallo-Gimeno and Nieto, 2005; Peinado et al., 2007), Snail1-deleted fibroblasts migrate at rates comparable to wild-type fibroblasts in a two-dimensional wound assay (Fig. S1, C and D). Likewise, whereas increased fibronectin synthesis and matrix assembly are characteristic features of EMT programs (Barrallo-Gimeno and Nieto, 2005; Peinado et al., 2007), Snail1-deficient fibroblasts deposit a fibronectin matrix at rates comparable to control fibroblasts (Fig. S1 E). Consequently, insights into Snail1 function were alternatively sought by interrogating the gene expression patterns of Snail1-deleted fibroblasts. Recent studies have demonstrated that cell behavior in vitro more closely recapitulates that observed in vivo when cells are cultured within a 3D ECM (Hotary et al., 2003; Yamada and Cukierman, 2007; Zhou et al., 2008). Hence, Snail1 wild-type and deficient cells were suspended in type I collagen matrices, the dominant matrix component of interstitial tissues (Grinnell, 2003; Sabeh et al., 2004), and subjected to transcriptional profiling. Using cutoffs of P ≤ 0.005 and a minimum fold change of 1.5, Snail1 deficiency in fibroblasts exerts a global effect on transcription, with >1,000 significant changes in gene expression detected (Fig. 3 A and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200810113/DC1). Gene ontology (GO) analysis further demonstrates that Snail1 governs multiple processes critical to fibroblast motile behavior, including adhesion, migration, and proteolysis (Fig. 3 B). Snail1 deletion did not trigger a mesenchymal-to-epithelial transdifferentiation process, as assessed by transcriptional analysis, which suggests that Snail1 is required for the induction, but not maintenance, of the mesenchymal phenotype during development.

To assess the consequences of Snail1 loss on 3D ECM invasion, a critical component of fibroblast wound and tumor responses (Martin, 1997; Grinnell, 2003; Bhowmick et al., 2004; Sabeh et al., 2004; Orimo et al., 2005), cells were embedded within a 3D bed of type I collagen. Snail1-deleted, but not Snail1-rescued, fibroblasts displayed a significant defect in their ability to negotiate the type I collagen barrier (Fig. 4, A and B; and Fig. S2, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200810113/DC1). Focusing on candidate genes implicated previously in 3D cell motility and invasion (Yamaguchi and Condeelis, 2007; Olson and Sahai, 2008; Sakurai-Yageta et al., 2008), probe sets corresponding to transcripts for cortactin (Cttn), enabled homologue (Enah), ezrin, moesin, rhoA, ROCK1, myosin light chain kinase, tropomyosin, and membrane type-1 matrix metalloproteinase (MT1-MMP; Mmp14) are significantly altered in Snail1-deficient fibroblasts (Fig. 3 C). Consistent with the altered patterns of gene expression revealed by the microarray data and confirmed by quantitative PCR (Fig. S2C), Snail1-deficient fibroblasts exhibit a significant reduction in the cortactin-rich membrane protrusions that mark invadopodia, the actin-rich, cellular processes that focus proteolytic activity at sites of cell–ECM contact (i.e., 83.3 ± 8.3% cortactin-positive processes in wild-type fibroblasts vs. 31.2 ± 13.2% in Snail1-deleted fibroblasts; P < 0.01; Fig. 4 C; Gimona et al., 2008).

As cortactin-rich invadopodia play a critical role in recruiting MT1-MMP, a membrane-anchored collagenase critical for cell invasion, to zones of pericellular proteolysis (Artym et al., 2006; Clark et al., 2007; Li et al., 2008), wild-type and Snail1-deficient fibroblasts were cultured with fibrillar gels of Alexa 594–labeled type I collagen, and collagenolysis was monitored (Sabeh et al., 2004). Whereas wild-type fibroblasts generate collagenolytic zones that are associated with adhesive sites enriched for actin spikes and cortactin, Snail1-deficient fibroblasts exhibit a significantly diminished ability to degrade collagen or mobilize invadopodia-like structures (Fig. 4, D and E). In accordance with these collagenolytic defects, invadopodial clusters of MT1-MMP and cortactin localized at the fibroblast–collagen interface are reduced by ~80% in Snail1-deficient cells (Fig. 4, F and G). Reconstitution of Snail1-deficient fibroblasts with full-length human Snail1 normalizes expression of cortactin and MT1-MMP (Fig. S2 D). Furthermore, consistent with GO enrichment scores that did not detect changes in cell cycle or apoptosis regulation, wild-type or Snail1-deleted fibroblasts embedded within 3D collagen gels proliferate at indistinguishable rates (7.8 ± 3.2% Ki67-positive for Snail1 wild-type cells vs. 8.1 ± 1.4% Ki67-positive for Snail1-null cells; n = 3) and display similar low levels of apoptosis (Snail1 wild-type, 1.6 ± 0.8%; Snail1-null, 1.4 ± 1.4%; assessed by TUNEL; n = 3).

Though Snail1-deficient cells display defects in the pericellular proteolysis and invasion of homogeneous collagenous barriers in vitro, connective tissue barriers in vivo are more complex, multimolecular composites of ECM macromolecules (Grinnell, 2003; Hotary et al., 2003; Yamada and Cukierman, 2007; Zhou et al., 2008). As such, wild-type and Snail1-deleted fibroblasts were cultured atop the chorioallantoic membrane (CAM) of live chick embryos (Sabeh et al., 2004), a tissue characterized by a type IV collagen-rich basement membrane and an underlying interstitium containing both type I and type III collagens (the stroma also contains blood vessels circumscribed by type IV collagen-positive basement membranes; Fig. 5 A). Although wild-type fibroblasts efficiently breach the CAM basement membrane and invade into the underlying stroma, Snail1-deficient fibroblasts exhibit a complete defect in invasion and fail to penetrate the CAM surface (Fig. 5, B and C), a phenotype identical to that described previously for MT1-MMP–null fibroblasts (Sabeh et al., 2004). In vivo, fibroblasts can initiate neovascularization during wound healing (Martin, 1997), but Snail1-deficient fibroblasts also demonstrate a significantly attenuated ability to induce neovessel formation (Fig. 5, B and D). Neither proliferative nor apoptotic indices of the fibroblasts are affected in the CAM model (Fig. 5, E and F). Collectively, the data identify Snail1 as a master regulator of activated fibroblast function in vivo by controlling tissue-invasive as well as proangiogenic functions.

Snail1 exerts global effects on epithelial cell gene expression by binding consensus sequences within the promoter regions of target genes while recruiting histone deacetylases, arginine methyltransferase, and DNA methyltransferases to chromatin remodeling complexes (Peinado et al., 2007; Herranz et al., 2008; Hou et al., 2008). Despite the remarkable range of Snail1's impact on epithelial cell fate determination, a functional role for Snail1 in terminally differentiated mesenchymal cells has not been explored previously. Unexpectedly, under 3D culture conditions, GO analyses revealed that major shifts had occurred in fibroblast behavior in the absence of Snail1 expression, with changes concentrated in functional programs tightly linked to cell adhesion, migration, proteolysis, and morphogenesis. Among Snail1-regulated targets, cortactin has been found to regulate MT1-MMP–dependent proteolysis, an activity critical for mesenchymal cell trafficking through ECM barriers (Chun et al., 2004; Sabeh et al., 2004; Filippov et al., 2005; Artym et al., 2006; Hotary et al., 2006; Clark et al., 2007). As such, the defects in cortactin and MT1-MMP expression and function observed in Snail1-deficient fibroblasts, in tandem with predicted changes in accessory molecules such as rhoA, ROCK, myosin light chain kinase, and tropomyosin, correlated with a marked loss in collagenolytic potential as well tissue-invasive activity in vitro and in vivo. Snail1-deleted fibroblasts were also unable to initiate an angiogenic response, a result likely consistent with the ability of MT1-MMP to induce angiogenesis by generating bioactive collagen fragments, regulating VEGF expression, or mediating semaphorin 4D shedding (Sounni et al., 2004; Weathington et al., 2006; Basile et al., 2007).