In the Drosophila, a single copy of the phosphoprotein Dishevelled (Dsh) is found. In the genomes of higher organism (including mammals), three genes encoding isoforms of Dishevelled (Dvl1, Dvl2, and Dvl3) are present. In the fly, Dsh functions in the Wnt-sensitive stabilization of intracellular beta-catenin and activation of the Lef/Tcf-sensitive transcriptional response known as the Wnt "canonical" pathway. In the current work we explore the expression of Dishevelleds in mammalian cells and provide an estimate of the relative cellular abundance of each Dvl. In mouse F9 cells, all three Dvls are expressed. Dvl2 constitutes more than 95% of the total pool, the sum of Dvl1 and Dvl3 constituting the remainder. Similarly, Dvl2 constitutes more than 80% of the Dvl1-3 pool in mouse P19 and human HEK 293 cells. siRNA-induced knock-down of individual Dvls was performed using Wnt3a-sensitive canonical pathway in F9 cells as the read-out. Activation of the canonical signaling pathway by Wnt3a was dependent upon the presence of Dvl1, Dvl2, and Dvl3, but to a variable extent. Wnt3a-sensitive canonical transcription was suppressible, by knock-down of Dvl1, Dvl2, or Dvl3. Conversely, the overexpression of any one of the three Dvls individually was found to be capable of promoting Lef/Tcf-sensitive transcriptional activation, in the absence of Wnt3a, i.e., overexpression of Dvl1, Dvl2, or Dvl3 is Wnt3a-mimetic. Graded suppression of individual Dvl isoforms by siRNA was employed to test if the three Dvls could be distinguished from one another with regard to mediation of the canonical pathway. Canonical signaling was most sensitive to changes in the abundance of either Dvl3 or Dvl1. Changes in expression of Dvl2, the most abundant of the three isoforms, resulted in the least effect on canonical signaling. Dvl-based complexes were isolated by pull-downs from whole-cell extracts with isoform-specific antibodies and found to include all three Dvl isoforms. Rescue experiments were conducted in which depletion of either Dvl3 or Dvl1 suppresses Wnt3a activation of the canonical pathway and the ability of a Dvl isoform to rescue the response evaluated. Rescue of Wnt3a-stimulated transcriptional activation in these siRNA-treated cells occurred only by the expression of the very same Dvl isoform depleted by the siRNA. Thus, Dvls appear to function cooperatively as well as uniquely with respect to mediation of Wnt3a-stimulated canonical signaling. The least abundant (Dvl1, 3) plays the most obvious role, whereas the most abundant (Dvl2) plays the least obvious role, suggesting that individual Dvl isoforms in mammals may operate as a network with some features in common and others rather unique.

Retinoic acid is a well known morphogen involved in many aspects of mammalian development (1). Our understanding of the mechanism(s) by which retinoic acid functions is quite detailed at the level of nuclear receptors controlling gene expression for this morphogen (2, 3). Retinoids like retinoic acid act via the retinoic acid receptor and retinoid receptor proteins, which modulate gene expression through binding to retinoic acid-responsive elements of target genes. Another class of morphogens, Wnts, is a class of vertebrate genes encoding secreted signaling proteins that appear to modulate diverse processes in developing vertebrate embryos and some adult tissues (4 -7). The actions of Wnts are thought to be mediated by the function of members of the frizzled gene family of prospective heptihelical receptors (8 -13). In the absence of a Wnt signal, active glycogen synthase kinase 3 (zesty white 3/shaggy in Drosophila) phosphorylates ␤-catenin at an amino-terminal site (14), targeting it for ubiquitination and degradation through a proteasome pathway that also involves axin and the product of the adenomatous polyposis coli or APC gene (15-24). Signaling by Wnt-1 via Frizzled homologues activates the function of Disheveled, which represses the activity of glycogen synthase kinase 3 (6, 25), promoting the elevation of intracellular ␤-catenin levels and accumulation of ␤-catenin in nuclei (14, 26). Nuclear ␤-catenin interacts with members of the T-cell factorlymphoid enhancer-binding factor (Tcf-Lef) 1 classes of architectural high mobility group box transcription factors (27-30) to regulate the expression of genes involved in vertebrate development (27,(31)(32)(33)(34)(35).Recently, we demonstrated that the activation of the Frizzled-1 receptor expressed in mouse F9 teratocarcinoma cells like retinoic acid promotes the formation of primitive endoderm (36). The Frizzled-1 receptor signals via heterotrimeric G-proteins to the ␤-catenin-Tcf-Lef-sensitive transcriptional pathway (37). Studies with the native rat Frizzled-1 receptor (36) as well as with a novel chimeric receptor employing the cytoplasmic domains of Frizzled-1 with the transmembrane and exofacial regions of the ␤ 2 -adrenergic receptor (37) demonstrate the stabilization and accumulation of ␤-catenin, the activation of Tcf-Lef-sensitive transcription, and the formation of primitive endoderm. Retinoic acid stimulates the formation of primitive endoderm in mammalian stem cells (38,39). Cross-regulation between retinoid signaling and the ␤-catenin-Tcf-Lef pathways has been reported in MCF-7 breast cancer cells in culture (40). In these cells, retinoic acid acts in opposition to the ␤-cateninTcf-Lef pathway. In the current work, we investigate a novel hypothesis that the ␤-catenin/Lef-Tcf pathway participates in the ability of retinoic acid to promote the formation of primitive endoderm much like it does in Wnt-Frizzled-1 signaling. The results demonstrate a new role for ␤-catenin-Tcf-Lef signaling in enabling retinoic acid to promote the formation of primitiv...

The heterotrimeric G-protein G 13 mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this G␣ 13 -mediated pathway, whereas that between G␣ 13 and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active G␣ 13 was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated G␣ 13 . Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of G␣ 13 . Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated G␣ 13 -stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from G␣ 13 to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm.