Daniel J. Sussman

Social Interaction and Sensorimotor Gating Abnormalities in Mice Lacking Dvl1

Mice completely deficient for Dvl1, one of three mouse homologs of the Drosophila segment polarity gene Dishevelled, were created by gene targeting. Dvl1-deficient mice are viable, fertile, and structurally normal. Surprisingly, these mice exhibited reduced social interaction, including differences in whisker trimming, deficits in nest-building, less huddling contact during home cage sleeping, and subordinate responses in a social dominance test. Sensorimotor gating was abnormal, as measured by deficits in prepulse inhibition of acoustic and tactile startle. Thus, Dvl1 mutants may provide a model for aspects of several human psychiatric disorders. These results are consistent with an interpretation that common genetic mechanisms underlie abnormal social behavior and sensorimotor gating deficits and implicate Dvl1 in processes underlying complex behaviors.

Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development

Dendritic arborization is required for proper neuronal connectivity. Rho GTPases have been implicated in the regulation of dendrite development. However, the signaling pathways that impinge on these molecular switches remain poorly understood. Here we show that Wnt7b, which is expressed in the mouse hippocampus, increases dendritic branching in cultured hippocampal neurons. This effect is mimicked by the expression of Dishevelled (Dvl) and is blocked by Sfrp1, a secreted Wnt antagonist. Consistent with these findings, hippocampal neurons from mice lacking Dvl1 show reduced dendritic arborization. Activation of the canonical Wnt-Gsk3β pathway does not affect dendritic development. In contrast, Wnt7b and Dvl activate Rac and JNK in hippocampal neurons. Dominant-negative Rac, dominant-negative JNK or inhibition of JNK blocks Dvl-mediated dendritic growth. These findings demonstrate a new function for the non-canonical Wnt pathway in dendrite development and identify Dvl as a regulator of Rho GTPases and JNK during dendritic morphogenesis.

Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1.

Wnt proteins transduce their signals through dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3β (GSK), leading to the accumulation of cytosolic β‐catenin and activation of TCF/LEF‐1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF‐1, we investigated the roles of Axin and Frat1 in Wnt‐mediated activation of LEF‐1 in mammalian cells. We found that Dvl interacts with Axin and with Frat1, both of which interact with GSK. Similarly, the Frat1 homolog GBP binds Xenopus Dishevelled in an interaction that requires GSK. We also found that Dvl, Axin and GSK can form a ternary complex bridged by Axin, and that Frat1 can be recruited into this complex probably by Dvl. The observation that the Dvl‐binding domain of either Frat1 or Axin was able to inhibit Wnt‐1‐induced LEF‐1 activation suggests that the interactions between Dvl and Axin and between Dvl and Frat may be important for this signaling pathway. Furthermore, Wnt‐1 appeared to promote the disintegration of the Frat1–Dvl–GSK–Axin complex, resulting in the dissociation of GSK from Axin. Thus, formation of the quaternary complex may be an important step in Wnt signaling, by which Dvl recruits Frat1, leading to Frat1‐mediated dissociation of GSK from Axin.

Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure

The murine dishevelled 2 (Dvl2) gene is an ortholog of the Drosophila segment polarity gene Dishevelled, a member of the highly conserved Wingless/Wnt developmental pathway. Dvl2-deficient mice were produced to determine the role of Dvl2 in mammalian development. Mice containing null mutations in Dvl2 present with 50% lethality in both inbred 129S6 and in a hybrid 129S6-NIH Black Swiss background because of severe cardiovascular outflow tract defects, including double outlet right ventricle, transposition of the great arteries and persistent truncus arteriosis. The majority of the surviving Dvl2-/- mice were female, suggesting that penetrance was influenced by sex. Expression of Pitx2 and plexin A2 was attenuated in Dvl2 null mutants, suggesting a defect in cardiac neural crest development during outflow tract formation. In addition, ∼90% of Dvl2-/- mice have vertebral and rib malformations that affect the proximal as well as the distal parts of the ribs. These skeletal abnormalities were more pronounced in mice deficient for both Dvl1 and Dvl2. Somite differentiation markers used to analyze Dvl2-/- and Dvl1-/-;Dvl2-/- mutant embryos revealed mildly aberrant expression of Uncx4.1, delta 1 and myogenin, suggesting defects in somite segmentation. Finally, 2-3% of Dvl2-/- embryos displayed thoracic spina bifida, while virtually all Dvl1/2 double mutant embryos displayed craniorachishisis, a completely open neural tube from the midbrain to the tail. Thus, Dvl2 is essential for normal cardiac morphogenesis, somite segmentation and neural tube closure, and there is functional redundancy between Dvl1 and Dvl2 in some phenotypes.

Dishevelled Proteins Lead to Two Signaling Pathways REGULATION OF LEF-1 AND c-Jun N-TERMINAL KINASE IN MAMMALIAN CELLS

Dishevelled (Dsh/Dvl) proteins are known to mediate Wnt signaling by up-regulating β-catenin levels and stimulating T cell factor (TCF)/LEF-1-dependent transcription. We have identified a new Dvl-mediated signaling pathway in that mouse Dvl proteins, when expressed in COS-7 cells, stimulate c-Jun-dependent transcription activity and the kinase activity of the c-Jun N-terminal kinase (JNK). The DEP domain of Dvl1 is essential for JNK activation. By contrast, all three conserved domains of Dvl, including DIX, PDZ, and DEP, are required for up-regulation of β-catenin and for stimulation of LEF-1-mediated transcription in mammalian cells. Thus, Dvl can lead to two different signaling pathways. Furthermore, the small G proteins of Cdc42 or Rac1, which are involved in JNK activation by many stimuli, do not appear to play a major role in Dvl-mediated JNK activation, because the dominant negative mutants of Cdc42 and Rac1 could not inhibit Dvl-induced JNK activation. This suggests that Dvl may activate JNK via novel pathways.

Murine dishevelled 3 functions in redundant pathways with dishevelled 1 and 2 in normal cardiac outflow tract, cochlea, and neural tube development.

Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3 −/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3 −/− and LtapLp/+ mutants, Dvl3 +/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.

Wnt glycoproteins regulate the expression of FoxN1, the gene defective in nude mice

T cell development and selection require the fully mature and diverse epithelial microenvironment of the thymus. Acquisition of these characteristics is dependent on expression of the forkhead (also known as winged-helix) transcription factor FoxN1, as a lack of functional FoxN1 results in aberrant epithelial morphogenesis and an inability to attract lymphoid precursors to the thymus primordium. However, the transcriptional control of Foxn1 expression has not been elucidated. Here we report that secreted Wnt glycoproteins, expressed by thymic epithelial cells and thymocytes, regulate epithelial Foxn1 expression in both autocrine and paracrine fashions. Wnt molecules therefore provide regulatory signals critical for thymic function.

Endogenous Protein Kinase CK2 Participates in Wnt Signaling in Mammary Epithelial Cells

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors, transformed cell lines, and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves, as has been seen in a number of human breast cancers, or through mutation of intermediates in the Wnt pathway, such as adenomatous polyposis coli or β-catenin, as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development, but overexpression of certain Wnts, such as Wnt-1, leads to proliferation and transformation of cells. We report that upon stable transfection ofWnt-1 into the mouse mammary epithelial cell line C57MG, morphological changes and increased proliferation are accompanied by increased levels of CK2, as well as of β-catenin. CK2 and β-catenin co-precipitate with the Dvl proteins, which are Wnt signaling intermediates. A major phosphoprotein of the size of β-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated β-catenin, Dvl-2, and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells, abrogates phosphorylation of β-catenin, and reduces β-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells.

Conservation of dishevelled structure and function between flies and mice: isolation and characterization of Dvl2

The segment polarity gene dishevelled (dsh) of Drosophila is required for pattern formation of the embryonic segments and the adult imaginal discs. dsh encodes the earliest-acting and most specific known component of the signal transduction pathway of Wingless, an extracellular signal homologous to Wnt1 in mice. We have previously described the isolation and characterization of the Dvl1 mouse dsh homolog. We report here the isolation of a second mouse dsh homolog, Dvl2, which maps to chromosome 11. The Dvl2 amino acid sequence is equally related to the dsh sequence as is that of Dvl1, but Dvl2 is most similar to the Xenopus homolog Xdsh. However, unlike the other vertebrate dsh homologs. Like the other genes, Dvl2 is ubiquitously expressed throughout most of embryogenesis and is expressed in many adult organs. We have developed an assay for dsh function in fly embryos, and show that Dvl2 can partially rescue the segmentation defects of embryos devoid of dsh. Thus, Dvl2 encodes a mammalian homolog of dsh which can transduce the Wingless signal.

Isolation and characterization of mouse Dishevelled-3

The Drosophila dishevelled (dsh) segment polarity gene is required to establish cell fates specified by wingless/Wnt signal transduction during development. We have previously reported the cloning and characterization of a mouse homolog of dishevelled, Dvl1. Utilizing RT‐PCR with degenerate primers, we isolated another member of the mouse Dishevelled (Dvl) gene family, Dvl3. The Dvl3 gene maps to mouse chromosome 16. The predicted amino acid sequence shares 64 and 62% identity to Dvl1 and Dvl2, respectively. The region of highest conservation between all three Dvl coding regions, at 97% identity, is noted at the PDZ domain (also termed the DHR domain or GLGF motif), a motif of 60 amino acids present in all dishevelled encoded proteins and first described in the Drosophila discs large (dlg) tumor suppressor gene. In adult mice, Dvl3 expression is widespread with highest levels exhibited in brain, ovary, and heart. In embryos, Dvl3 is expressed in every tissue between 7.5 and 9.5 days postcoitum, and by 10.5 days postcoitum highest expression was seen in the dorsal root ganglia, somites, limb buds, branchial arches, heart, gut, and throughout the developing central nervous system.