Unc119 proteins form a structurally distinct family composed of proteins that are strikingly conserved among all metazoans. Intriguingly, this small family is implicated in diverse activities, including initiating the cellular immune response, promoting neural development in C. elegans, and coordinating signal transduction in mice. Despite these seemingly distinct roles, orthologues appear to be functionally interchangeable, suggesting that a common biochemical mechanism underlies each of these cellular processes and that redundancies may arise where paralogues exist.
A compelling unifying mechanism is intraflagellar transport (IFT), which is a protein trafficking system initially described in cilia. Cilia are microtubule-based protrusions from the cell body and are critically important for vertebrate organ function, body patterning and signalling pathways. IFT factors are additionally required for cellular processes unrelated to cilia, like those that involve Unc119 proteins. Since Unc119 proteins appear to be required for all cellular processes that rely upon IFT and have been shown to be required for trafficking, I propose that they are novel IFT factors. I hypothesize that the highly conserved Unc119 family proteins are previously unrecognized ciliary factors that promote proper cellular trafficking via the IFT machinery. To test this hypothesis, I am studying the cellular and phenotypic consequences of a loss of unc119 genes using zebrafish.
Zebrafish have four paralogues of unc119, none of which have previously been studied in great detail in terms of cilia function. To begin, I wanted to determine whether any of the paralogues have a potential role in cilia by performing in situ hybridization. I found that each paralogue has a distinct expression pattern, although there is a large degree of overlap particularly in ciliated organs. To assay for ciliary phenotypes, I have been using CRISPR-Cas9 technology to create deletion mutations in each of the zebrafish paralogues. My preliminary results using morpholinos to target one of the paralogues (unc119b) have revealed that there are ciliary phenotypes (hydrocephaly, kidney cysts, l-r asymmetry defects), but these are milder and less penetrant than expected, which is consistent with redundancy between paralogues. Given the conserved nature of these genes and the overlap in expression between paralogues, it’s possible that there is functional redundancy, which we will be testing using rescue assays. Thus far, we have shown that at least one of the paralogues is involved in cilia but it is likely that the other paralogues act similarly molecularly to affect cilia function.