Analysis of photoreceptor outer segment morphogenesis in zebrafish ift57, ift88 and ift172 intraflagellar transport mutants

Vertebrate photoreceptors are polarized cells that consist of a specialized sensory structure termed the outer segment required for phototransduction and an inner segment that contains the cellular organelles. Proteins synthesized in the inner segment are transported to the outer segment via a connecting cilium by a process called Intraflagellar Transport (IFT). The IFT mechanism refers to the movement of a multisubunit complex along the flagellar axoneme, and mutations in some IFT components cause retinal degeneration. To better understand the role of IFT in early photoreceptor development, we studied zebrafish with mutations in genes encoding three specific subunits of the IFT particle: IFT57, IFT88 and IFT172. These mutants exhibit photoreceptor defects by five days post fertilization (dpf); however, it is not known whether outer segment formation initiates at earlier time points and then degeneration occurs or if outer segments never form at all. To understand this, we performed transmission electron microscopy to study the ultrastructure of photoreceptors at 60, 72 and 96 hours post fertilization (hpf). At 60 hpf, developing outer segments were seen in IFT57 mutant and wild type embryos, however, disorganized membranous structures were observed in IFT88 and IFT172 mutants. At 72 hpf, the number of outer segments in the IFT57 mutants was reduced by 88% when compared to wild type, indicating a defect in initiation of outer segment formation. By 96 hpf we see a reduction in both outer segment length and number in IFT57 mutants. In comparison, the IFT88 and IFT172 mutants do not grow outer segments at any time point. To complement our ultrastructural analysis, we performed immunohistochemistry to understand cell morphology and protein trafficking in these mutants. Zpr1, a marker for cone morphology, showed the presence of normal cones initially that began to degenerate at later time points. Immunohistochemistry with rhodopsin, a phototransduction protein that localizes to the outer segment, revealed that rhodopsin was mislocalized in all the three mutants by 96 hpf. Connecting cilia labeled with acetylated tubulin were highly reduced in IFT57 mutants whereas none were observed in IFT88 and IFT172 mutants. Together these data indicate that IFT57 is required for maintenance and growth of outer segments whereas IFT88 and IFT172 are required for initiating outer segment formation.