Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract, Cochlea, and Neural Tube Development

and Neural Tube Development. PLoS Genet 4(11): e1000259. doi:10.1371/journal.pgen.1000259 Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract, Cochlea, and Neural Tube Development S. Leah Etheridge 0 1 Saugata Ray 0 1 Shuangding Li 0 1 Natasha S. Hamblet 0 1 Nardos Lijam 0 1 Michael Tsang 0 1 Joy Greer 0 1 Natalie Kardos 0 1 Jianbo Wang 0 1 Daniel J. Sussman 0 1 Ping Chen 0 1 Anthony 0 1 David R. Beier, Harvard Medical School, United States of America 0 a Current address: Burnham Institute for Medical Research, La Jolla, California, United States of America b Current address: Department of Medicine, Eastern Virginia Medical School, Norfolk, Virginia, United States of America c Current address: Department of Cell Biology, School of Medicine, University of Alabama , Birmingham, Alabama , United States of America 1 1 Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Emory University School of Medicine, Atlanta, Georgia, United States of America, 3 Columbus State Community College , Columbus , Ohio, United States of America, 4 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine , Pittsburgh , Pennsylvania, United States of America , 5 New Horizons Diagnostics, Columbia , Maryland, United States of America, 6 Department of Pediatrics and Institute for Human Genetics, University of California San Francisco School of Medicine , San Francisco, California , United States of America Dishevelled (Dvl) proteins are important signaling components of both the canonical b-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. Dvl32/2 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 Dvl32/2 and LtapLp/+ mutants, Dvl3+/2;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. - Funding: This work was supported by grants from the March of Dimes to AWB., the National Institutes of Health to AWB. (HD43173), PC (DC007423), and the UCSD Neuroscience Microscopy Core (NS47101), as well as a postdoctoral fellowship from the American Heart Association to SLE. Competing Interests: The authors have declared that no competing interests exist. Normal mammalian development is the result of complex signaling networks that regulate and coordinate cell behavior. Wnt signaling controls a broad spectrum of cell fate decisions during embryogenesis and is critical for cell to cell communication in mammalian development. Through the activation of specific target genes, the canonical Wnt pathway tightly regulates cell proliferation, differentiation, adhesion and survival, and controls embryonic patterning [1,2]. A non-canonical Wnt planar cell polarity (PCP) pathway, parallel to that first discovered in flies, has been described in mammals, where it regulates cell polarity and convergent extension (CE) movements. In these coordinated cell movements, cells migrate medially and intercalate, producing an elongation and narrowing of the tissue along the anterior-posterior axis [37]. Dishevelled (Dvl) proteins, of which three have been identified in humans and mice [813] are highly conserved components of both the canonical Wnt and PCP signaling cascades. They function as essential scaffold proteins that interact with diverse proteins, including kinases, phosphatases and adaptor proteins [14,15]. In the canonical Wnt pathway, Dvl transduces the signal activated by Wnt binding to membrane-bound Frizzled (Fz) receptors and low-density lipoprotein-related receptor protein (LRP) 5/6 co-receptors, causing the stabilization and cytosolic accumulation of the critical mediator, b-catenin. Following the Multi-gene families, comprising a set of very similar genes with shared nucleotide sequences, are common in mammals. Individual family members may be expressed in different places and perform separate functions. Alternatively, the genes may have redundant functions, but distinct dosage requirements. Mammals share three Dishevelled (Dvl) family members and while the roles of Dvl1 and Dvl2 have been described previously, the functions of Dvl3 have remained elusive. Here, we show that the lack of Dvl3 in mice affects the formation of the heart, neural tube, and inner ear. We further show that the defects in these tissues are much more severe when the mice are deficient in more than one Dvl family member, indicating redundant functions for these genes. Congenital heart disease affects approximately 75 in every 1,000 live human births, and approximately 30% of these diseases are due to disruptions in the outflow tract, the region affected in mice lacking Dvl genes. Neural tube defects, similar to those observed in the Dvl mutants, are also common in humans. The animal models described here provide useful tools to elucidate the genetic mechanisms that underlie these abnormalities and may provide novel ways of treating these disorders in the future. translocation of b-catenin to the nucleus, it then binds with members of the T-cell factor (TCF)/lymphocyte enhancer factor (Lef) family of transcription factors to regulate the expression of target genes. Dvl is also one of the core components of the PCP signaling pathway, in addition to Fz, Van Gogh/Strabismus (Vang/Stbm), Flamingo/Starry night (Fmi/Stan), Diego (Dgo) and Prickle (Pk). The specific, highly con (...truncated)