Abstract
Mutations in human prominin 1 (PROM1), encoding a transmembrane glycoprotein localized mainly to plasma membrane protrusions, have been reported to cause retinitis pigmentosa, macular degeneration, and cone-rod dystrophy. Although the structural role of PROM1 in outer-segment (OS) morphogenesis has been demonstrated in Prom1-knockout mouse, the mechanisms underlying these complex disease phenotypes remain unclear. Here, we utilized a zebrafish model to further investigate PROM1's role in the retina. The Prom1 orthologs in zebrafish include prom1a and prom1b, and our results showed that prom1b, rather than prom1a, plays an important role in zebrafish photoreceptors. Loss of prom1b disrupted OS morphogenesis, with rods and cones exhibiting differences in impairment: cones degenerated at an early age, whereas rods remained viable but with an abnormal OS, even at 9 months postfertilization. Immunofluorescence experiments with WT zebrafish revealed that Prph2, an ortholog of the human transmembrane protein peripherin 2 and also associated with OS formation, is localized to the edge of OS and is more highly expressed in the cone OS than in the rod OS. Moreover, we found that Prom1b deletion causes mislocalization of Prph2 and disrupts its oligomerization. We conclude that the variation in Prph2 levels between cones and rods was one of the reasons for the different PROM1 mutation-induced phenotypes of these retinal structures. These findings expand our understanding of the phenotypes caused by PROM1 mutations and provide critical insights into its function.
Highlights
Mutations in human prominin 1 (PROM1), encoding a transmembrane glycoprotein localized mainly to plasma membrane protrusions, have been reported to cause retinitis pigmentosa, macular degeneration, and cone–rod dystrophy
Human PROM1 gene mutations lead to extremely variable retinal clinical phenotypes, including cone photoreceptor– or macula-dominated disorders such as macular degeneration (MD), autosomal dominant Stargardtlike macular dystrophy [7], autosomal dominant bull’s-eye macular dystrophy [7], autosomal dominant cone–rod dystrophy (CRD) [7, 8], autosomal recessive CRD (8 –15), and the rod photoreceptor– dominated disorder autosomal recessive retinitis pigmentosa (RP) (6, 16 –20)
We detected Prom1b protein expression by Western blotting, and the result confirmed that no Prom1b protein existed in the mutant zebrafish, which further demonstrated the successful construction of the prom1bϪ/Ϫ zebrafish line (Fig. 1G)
Summary
We constructed prom1a- and prom1b-knockout zebrafish lines by the transcription activator–like effector nuclease (TALEN) technology. At 3 dpf, the normal stacked disk morphology of photoreceptor OSs could be clearly observed in WT zebrafish; by contrast, there were few, or occasionally several, quite small whorl-like disk structures detected in prom1bϪ/Ϫ zebrafish at the same age (Fig. 6A). Prom1b deletion in zebrafish exerted different effects on the disk formation of rod and cone OSs, with the pattern of disks being similar to that seen in Prph2ϩ/Ϫ mice, all of which led us to consider whether there was a relationship between prom1b and prph. To examine the cellular localization of Prph, we labeled photoreceptors with specific antibodies (Prph, Rhodopsin, and Opn1lw) in retinal sections from WT and prom1bϪ/Ϫ zebrafish at 2 mpf (rods and a few red cones were still present in the retina at this period). Based on the fact that photoreceptor number was reduced at an early age, the decreased level of Prph likely resulted from a consequence of the OS phenotypes following Prom1b deletion
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