Dishevelled Associated Activator of Morphogenesis

Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel formin homology protein Daam1

Wnt signaling via the Frizzled (Fz) receptor controls cell polarity and movement during development, but the molecular nature of Wnt/Fz polarity signal transduction remains poorly defined. In human cells and during Xenopus embryogenesis, Wnt/Fz signaling activates the small GTPase Rho, a key regulator of cytoskeleton architecture. Wnt/Fz activation of Rho requires the cytoplasmic protein Dishevelled (Dvl) and a novel Formin homology protein Daam1. Daam1 binds to both Dvl and Rho, and mediates Wnt-induced Dvl-Rho complex formation. Inhibition or depletion of Daam1 prevents Wnt/Fz activation of Rho and of Xenopus gastrulation, but not of ß-catenin signaling. This study illustrates a molecular pathway from Wnt/Fz signaling to Rho activation in cell polarity signal transduction (Habas, 2001).

Because Dvl2 PDZ domain is required for Fz/Dvl signaling to Rho proteins associated with the PDZ domain, interacting proteins were sought using the yeast two-hybrid technique. The widely expressed human Daam1 protein contains 1078 amino acids, and belongs to the family of Formin homology (FH) proteins that have been implicated in cell polarity from yeast to human. Formin is the product of the limb deformity locus and is required for limb morphogenesis in mice. Daam1 shares 22% to 30% identity with, and thus is distantly related to, several known mammalian FH proteins. Like other FH proteins, Daam1 contains a central proline-rich FH1 domain and a more carboxyl FH2 domain, and represents a novel subfamily that includes a closely related Daam2, Xenopus and zebrafish Daam, and a Drosophila ortholog, dDaam. The Daam subfamily exhibits extensive similarity both within and outside the FH1 and FH2 domains, including the amino and carboxyl terminal regions. Since several FH proteins bind to Rho, Rac, or Cdc42, Daam1 may also bind Rho GTPases (Habas, 2001).

The Daam1 amino terminus binds to Rho-GDP or Rho-GTP, suggesting a role for Daam1 as a scaffolding protein to recruit Rho-GDP (via the amino terminus) and a Rho-GEF (via the C-Daam1 portion), thereby enhancing Rho-GTP formation. The Daam1 amino terminus binds Rho-GTP with apparently higher affinity, raising an intriguing possibility of positive feedback control, a theme common in cell polarization. Polarity establishment relies on signal amplifications that interpret a small difference in a polarity signal field into a polarized cellular response. DFz1 (Frizzled) exhibits a polarized localization that depends on Dsh function, suggesting a positive feedback loop. Rho-GTP binding to the Daam1 amino terminus may stabilize Daam1 in its activated state, or recruit/activate additional Daam1, thereby promoting an amplification of Rho activation. Such a feedback loop would resemble one in pheromone-induced polarity in yeast. The mating pheromone, via its serpentine receptor and the trimeric G protein, recruits and activates a GEF specific for Cdc42. Activated Cdc42, in turn, is required for the GEF localization, thereby leading to further and polarized Cdc42 activation. The possibility that Daam1 may function primarily in such a feedback control cannot be ruled out. In this scenario, Wnt/Fz signaling initiates Rho activation without Daam1, and the activated Rho together with Dvl recruits/activates Daam1 to amplify Rho activation. In any event, Daam1 function is essential for Rho activation triggered by Wnt/Fz signaling (Habas, 2001).

Daam1 is distantly related to several distinct mammalian FH proteins, such as FRL (30% identity), FHOS (27%), mDia1 (28%), and mDia2 (22%), whose functions in GTPase signaling remain to be fully understood. FRL and FHOS bind specifically to Rac in a nucleotide-independent manner, and an activated FHOS is antagonized by Rac and Rac mutants, leading to the suggestion that FRL and FHOS are scaffolding proteins linking Rac to other proteins. Members of the mDia subfamily of FH proteins (see Drosophila Diaphanous) bind to Rho-GTP (and Rho-GDP in some cases), and are proposed to be Rho targets. However, since actin fiber induction by the activated mDia can be blocked by inhibition of Rho in some instances, and the activated mDia can cause RhoA activation, the relationship between mDia and Rho, and between FH proteins and Rho GTPases in general, may be complex and needs further investigation (Habas, 2001).

Vertebrate gastrulation involves polarization and intercalation of dorsal mesodermal cells along the mediolateral axis (convergence), resulting in the elongation of the anterioposterior axis (extension). This morphogenetic process is governed by Wnt-11 PCP signaling. In Xenopus gastrula, endogenous Rho activation is detected mainly in dorsal tissue, and is abolished when Wnt-11/Fz/Xdsh signaling or Daam1 function is inhibited. Conversely, ectopic Wnt-11/Fz/Xdsh signaling or C-Daam1 activates RhoA on the ventral side. Thus, Wnt-11/Fz signaling, via Xdsh and Daam1, is necessary and sufficient for RhoA activation during gastrulation, consistent with the previous finding that interference of Rho function inhibits gastrulation. In an explant assay, inhibition or depletion of Daam1 perturbs morphogenetic movements, whereas C-Daam1 restores the movements even when Wnt-11/Fz or Xdsh is inhibited. Daam1 thus functions downstream of Wnt-11/Fz/Xdsh in governing gastrulation. Finally, inhibition or depletion of Daam1 in the embryo blocks gastrulation and phenocopies the morphogenetic defects caused by inhibition of Wnt-11, Fz, or Xdsh signaling (Habas, 2001).

A molecular pathway for the Wnt/Fz activation of Rho is suggested, which is referred to as the Wnt/Rho pathway to distinguish it molecularly from Wnt/ß-catenin and Wnt/Ca2+ pathways. A Wnt signal activates a Fz receptor, which translocates Dsh to the plasma membrane and promotes Dsh-Daam1-RhoA complex formation and RhoA activation, likely via the recruitment of a Rho-GEF by the Daam1 scaffolding protein. Activated RhoA generates polarized cytoskeleton remodeling via the ROCK kinase, and perhaps also induces changes in gene expression. The zebrafish knypek gene product, a glypican, facilitates Wnt signal reception, whereas LRP5/6, which is the Fz coreceptor for Wnt/ß-catenin signaling, participates in neither PCP signaling nor RhoA activation. Whether and how other PCP gene products function in the Wnt/Rho pathway or in parallel pathways remains to be elucidated (Habas, 2001).

Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. The Formin homology protein Daam1 is important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacts with the FH1 domain of Daam1 and is localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibits stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibits blastopore closure in Xenopus but does not affect convergent extension movements, tissue separation or neural fold closure. These studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation (Sato, 2006).

Role of PKA as a negative regulator of PCP signaling pathway during Xenopus gastrulation movements

Convergent extension (CE) movements in gastrulation are essential for the establishment of the body axis during early vertebrate development. Although the precise molecular mechanisms of CE movements are not clearly understood, noncanonical Wnt pathway is known to be important for the control of CE movements. Evidence is presented that PKA is implicated in noncanonical Wnt pathway. Overexpression and specific depletion of PKA inhibit CE movements. PKA depletion also disrupts cell morphology, protrusive activity, and cortical actin formation in dorsal mesodermal cells. Moreover, PKA activity is negatively regulated by major components of planar cell polarity (PCP) pathway. In line with this, overexpression of PKA can rescue the inhibition of CE movements caused by overexpression of these molecules. This regulation of PKA activity is dependent upon Galphai signaling. As a negative component of PCP signaling, PKA inhibits not only the activation of RhoA and JNK but also the Dsh-Daam1-RhoA complex formation which is essential for the regulation of RhoA activity. Together, this study suggests a molecular pathway from Wnt/Dsh/PKA signaling to Rho activation in PCP signaling (Park, 2006).

DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics

Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 binds to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localizes in dotted pattern at the dorsal side of transfected cells and the protein accumulates in the proximity to the microtubule organizing center. Moreover, ectopic expression of DAAM1 induces a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bind to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 interacts and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, these data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system (Aspenstrom, 2006).

Dishevelled Associated Activator of Morphogenesis: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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