Academic journal article Genetics

The Zebrafish Pob Gene Encodes a Novel Protein Required for Survival of Red Cone Photoreceptor Cells

Academic journal article Genetics

The Zebrafish Pob Gene Encodes a Novel Protein Required for Survival of Red Cone Photoreceptor Cells

Article excerpt

ABSTRACT

The zebrafish mutant, partial optokinetic response b (pob), was isolated using an N-ethyl N-nitrosourea (ENU)-based screening strategy designed to identify larvae with defective optokinetic responses in red but not white light. Previous studies showed that red-light blindness in pob is due to the specific loss of long-wavelength photoreceptor cells via an apoptotic mechanism. Here, we used positional cloning to identify the mutated pob gene. We find that pob encodes a highly conserved 30-kDa protein of unknown function. To demonstrate that the correct gene was isolated, we used the Tol2 transposon system to generate transgenic animals and rescue the mutant phenotype. The Pob protein contains putative transmembrane regions and protein-sorting signals. It is localized to the inner segment and synapse in photoreceptor cells, and when expressed in COS-7 cells it localizes to intracellular compartments. We also show that the degeneration of red cone photoreceptors in the mutants occurs independently of light. On the basis of our findings, we propose that Pob is not involved in phototransduction but rather plays an essential role in protein sorting and/or trafficking.

RDD and cone photoreceptors are highly polarized cells designed to transmit visual information with amazing fidelity. Proper function of these compartmentalized cells requires a large number of molecules that are specific to photoreceptors or that have specialized functions within photoreceptors. The most apical part, the outer segment, is composed of stacks of membranous disks containing the phototransduction machinery, 80% of which is the opsin protein. The photoreceptor synapse is a ribbon type with structural and functional characteristics designed for rapid, continual transmission of visual information over several orders of magnitude of light illumination (BOWLING 1987). While much is known about the molecules involved in photoreceptor function, relatively little is known about the mechanisms and regulation of the proper sorting and targeting of these molecules.

The nonessential aspect of vision has made it possible to identify mutations in a large number of genes that are critical for the proper function and/or viability of photoreceptor cells. Over 80 genes that are essential for normal visual function have been identified in humans (PACIONE et al. 2003; see http^www.sph.uth.tmc.edu/ Retnet/). In addition, genetic screens in Drosophila have uncovered many genes necessary for invertebrate visual function and development (PAK 1995). These genetic strategies have identified proteins (1) unique to photoreceptors, such as phototransduction components; (2) unique to photoreceptors, but not easily identified using other experimental strategies; and (3) essential to photoreceptor function with widespread patterns of expression. The latter two categories include premRNA splicing factors, molecules involved in fatty acid biosynthesis, tissue inhibitor metalloproteinases, inosine monophosphate dehydrogenase, and other important cellular components (PACIONE et al. 2003).

Defects that cause subtle visual phenotypes are not likely to be identified in humans since identification of mutations requires clinical observation by an ophthalmologist and extensive genetic and molecular analysis. Furthermore, recessive mutations that are rare may also be missed because large inbred pedigrees within most human populations are uncommon. A more suitable genetic model would benefit from a relatively short generation time, rapid development of the visual system, large brood size, and ease in identifying visual function defects. While Drosophila sufficiently meets these criteria, the photoreceptors of invertebrates are very different from vertebrates (YARFITZ and HURLEY 1994). In contrast, the zebrafish provides a vertebrate model that has the qualities needed for identifying genes necessary for visual function and has been used in recent years to isolate genes essential for this process (Li and BOWLING 1997; KAY et al 2001; BROCKERHOFF et al 2003; TAYLOR étal. …

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