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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-3116 |
| Date | 15 May 2009 |
| Creators | Sukumaran, Sujita |
| Contributors | Perkins, Brian D. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | electronic, application/pdf, born digital |
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