Investigating Unc119 as a Novel Ciliopathy Candidate

Abstract

Cilia are microtubule-based structures that project from nearly every cell in the vertebrate body. While cilia in different contexts can have either sensory or motile functions, all cilia rely upon a core set of genes. When these genes are mutated either singly or in various combinations, a pleiotropic set of cellular phenotypes leading to disease (ciliopathies) can result. However up to 70% of ciliopathy patients don't have a clear genetic pathogenesis, which highlights the importance of identifying unrecognized ciliary genes that may be disease-causing. This thesis touches on three candidate gene families: the unc119 gene family of trafficking factors, an unc119 binding protein (unc119BP), and members of the fox transcription factor gene family. The main focus is on the Unc119 proteins, which are required for ciliary trafficking in a process called lipidated protein intraflagellar targeting (LIFT) or through vesicle transport. However, although unc119 has been studied in a variety of contexts, either organismal constraints or genetic redundancies has largely restricted their study in ciliary contexts. One possible solution for this is to use the zebrafish where the unc119 genes have not yet been well described. This thesis focused on studying the zebrafish unc119 genes from an evolutionary and developmental perspective. My work has shown in a condensed species tree that the presence of unc119 genes correlates with the presence of cilia across eukaryotes and that phylogenetic evidence suggests there are three subgroups of UNC119 proteins. Zebrafish contain all three of these subgroups: two vertebrate-specific UNC119As, one vertebrate-specific UNC119B, and one UNC119. Expression analyses show that each of the zebrafish unc119 genes are maternally-expressed and have overlapping but distinct expression in ciliated tissues, such as the eye, pronephric duct, and spinal cord. Knock-down of the most promising ciliary unc119 paralogue results in phenotypes consistent with a loss of ciliary trafficking – truncated photoreceptors in the eye and motile cilia within the Kupffer’s vesicle (KV), mild hydrocephaly, and tail curvature. Furthermore, the KV cilia are non-functional since they aren’t able to produce a fluid flow, which ultimately results in left-right axis abnormalities. In contrast, construction of combinatorial unc119 mutants doesn’t reveal any obvious embryonic morphological phenotypes, although there is evidence of late-onset photoreceptor degeneration that may be due to aberrant synaptic vesicle trafficking. Despite the lack of phenotypes in the genetic zebrafish model, the expression patterns of this gene family and their strong conservation indicates that this gene family is still fulfilling an essential role in the developing zebrafish. Altogether, the work done in this thesis illustrates the many complications that can be associated with modeling disease, and taking those into consideration, provides future avenues for studying the unc119 genes in the ciliary context.