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Primary cilia as signaling organelles in development and disease

  • Author(s): Dowdle, William Eric
  • Advisor(s): Reiter, Jeremy F
  • et al.
Abstract

In this dissertation, I describe the regulation of signal transduction by primary cilia, microtubule-based organelles present on nearly every vertebrate cell, during embryonic development and in the pathogenesis of disease. Using a combination of molecular biology, biochemistry, cell biology, and mouse genetics, I have investigated the role of primary cilia in canonical Wnt signaling and the role of B9-domain containing proteins in the formation and function of cilia during mouse development and in the etiology of Meckel-Gruber syndrome.

I show that the primary cilium restricts the activity of the canonical Wnt pathway in mouse embryos, primary fibroblasts, and embryonic stem cells. Non-ciliated cells activate transcription in response to Wnt stimulation, but do so more robustly than ciliated cells. Loss of Kif3a, but not Ift88 and Ofd1 genes, causes constitutive phosphorylation of Dishevelled (Dvl) in a casein kinase I (CKI) activity dependent manner. These results suggest that Kif3a restrains canonical Wnt signaling both by restricting the CKI-dependent phosphorylation of Dvl and through a separate cilia dependent mechanism. More generally, these findings reveal that, in contrast to its role in promoting Hedgehog (Hh) signaling, the cilium restrains canonical Wnt signaling.

In addition, I describe the role of B9-proteins, found in nearly every ciliated organism, in the formation and function of cilia. Mice lacking B9d1 displayed phenotypes identical to those caused by Meckel syndrome (MKS), a severe inherited human disorder, and neural tube patterning defects concomitant with compromised ciliogenesis, ciliary protein localization and Hedgehog (Hh) signal transduction. With coimmunoprecipitation and mass spectrometric analysis, we found that all three B9-proteins encoded in the genome interact physically with one another and with additional proteins implicated in MKS. In collaboration, we screened MKS patients for mutations in B9D1 and B9D2 and identified a homozygous B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. I also observed that B9d1 is not required for the formation of all cilia in tissues, but is required for the localization of ciliary membrane proteins to the cilia that remain in its absence. Cumulatively, these data suggest that primary cilia are important in the regulation of multiple signaling pathways including Wnt and Hh and in the development and homeostasis of multiple organ systems in vertebrates.

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