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Primary cilia are signaling organelles that project from the apical surface of almost all vertebrate cells. The signaling capabilities of primary cilia stem from their unique membrane protein composition: even though the ciliary membrane is continuous with the plasma membrane, certain membrane proteins localize specifically to the ciliary membrane. This dissertation describes how the ciliary membrane protein composition is maintained, and how the complex responsible for ciliary membrane composition is localized. Using a combination of molecular biology, cell biology, and biochemistry, I have investigated the role of TMEM231 in the formation and function of the transition zone in cilia during development and in disease, and the role of INTU in localizing TMEM231 during ciliogenesis. I show that TMEM231 is a component of the Meckel Syndrome (MKS) complex, and is responsible for organizing the MKS complex at the transition zone. In the absence of Tmem231, mouse embryos display phenotypes similar to those found in MKS, which is a genetic disorder characterized by polydactyly, cystic kidneys, and exencephaly. Using co-immunoprecipitation, I confirmed that TMEM231 physically interacts with other known MKS complex components, including B9D1 and MKS1. In addition, I show that TMEM231 functions with the MKS complex, which is required for ciliary membrane proteins to localize properly. In collaboration with human geneticists, we identified compound heterozygous mutations in TMEM231 in Oro-facio-digital syndrome type III (OFD3) patients, as well as homozygous mutations in TMEM231 in MKS patients. I discovered that these mutations are hypomorphic, as they are unable to restore full ciliary membrane protein localization in a rescue assay. These findings confirm that TMEM231 is a functional component of the MKS complex, and suggest that the mutations identified in OFD3 and MKS patients underlie the disease phenotypes. In addition, I investigated the role of INTU in cilia function more...

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 homozygo more...