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Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

Veth KN, Willer JR, Collery RF, Gray MP, Willer GB, Wagner DS, Mullins MC, Udvadia AJ, Smith RS, John SW, Gregg RG, Link BA - PLoS Genet. (2011)

Bottom Line: Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes.This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis.Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

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Expression of the gap43:GFP transgene in lrp2 mutants.A–L Expression of the gap43:GFP transgene in flat-mounted retinas from 2-month bugeye heterozygotes (wild-type) (A–E) and homozygous mutants (F–J). Shown are 5 representative samples for each condition. Images capture a single plane of the nerve fiber layer using equal gain settings on a confocal microscope. Scale bar  = 200 µm. Similar analysis on 6-month bugeye heterozygotes (wild-type) (K–O) and homozygous mutants (P–T). Note the stronger activation and unique wandering/circling phenotype in mutant axons.
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pgen-1001310-g008: Expression of the gap43:GFP transgene in lrp2 mutants.A–L Expression of the gap43:GFP transgene in flat-mounted retinas from 2-month bugeye heterozygotes (wild-type) (A–E) and homozygous mutants (F–J). Shown are 5 representative samples for each condition. Images capture a single plane of the nerve fiber layer using equal gain settings on a confocal microscope. Scale bar  = 200 µm. Similar analysis on 6-month bugeye heterozygotes (wild-type) (K–O) and homozygous mutants (P–T). Note the stronger activation and unique wandering/circling phenotype in mutant axons.

Mentions: Because the ultrastructural signature of degenerating axons following a crush injury is relatively short-lived in the optic nerve tract of teleost fish as compared to mammals [54], we utilized a genetic tool to label damaged and regenerating axons over a longer period of time [55]. We crossed Tg(3.6Frgap43:GFP)mil1 transgenic fish with lrp2 homozygous mutants and then used the resulting progeny to backcross with non-transgenic lrp2 mutant fish. This breeding scheme resulted in families with equal proportions of lrp2 heterozygous and homozygous mutant fish carrying single insertions of the 3.6Frgap43:GFP transgene. This transgene contains 3.6 kb of regulatory sequence (5′ flanking region and first intron) from theTakifugu rubripes gap43 locus driving GFP. Importantly, in these transgenic fish, GFP is expressed in axons following injury [55]. For our analysis, we compared large-eyed lrp2 homozygous mutant fish (>0.07 E:B ratio) to normal-eyed heterozygous siblings (Figure 8K–8T). In all large-eyed mutant fish we observed strong activation of GFP in a sub-set of retinal ganglion cells. In the majority of mutant retinas examined (6 of 6 at 6 months, Figure 8P–8T; and 10 of 12 at 12 months, data not shown), there was a characteristic axon ‘wandering’ and ‘circling’ around the optic nerve head. This axon phenotype, where GFP-positive axons approached the optic nerve head in a disorganized and circuitous fashion, was never observed in retinas from age matched lrp2 heterozygotes (Figure 8K–8O) or from 12-month wild-type fish that carried the 3.6Frgap43:GFP transgene (data not shown). The transgene was activated with variability at 2 months in both wild-type or lrp2 mutant fish (Figure 8A–8J), but the wandering axon phenotype was only rarely observed in mutants at this early timepoint. Weak expression of the transgene was noted in the nerve fiber layer of non-mutant retinas, consistent with the ongoing neurogenesis of zebrafish. In addition, older wild-type fish occasionally showed stronger GFP-positive axons, suggesting sporadic age-related degeneration. In wild-type eyes, all of the low-GFP expressing axons, as well as the occasional high-GFP expressing axons, exited the eye directly without wandering or circling the optic nerve head like those of mutants. To address whether the chronic stress conditions of lrp2 mutants differ from acute injury, we performed optic nerve crushes on adult gap43:GFP fish. At 6 days post-crush there was significant up-regulation of GFP across the retina (Figure S4). By 5 weeks post-crush, when axons had regrown [56], there was only an occasional wandering axon. Most samples following nerve crush, however, showed accurate and direct axon targeting through the optic nerve head. By 11 weeks post-crush, there was significant reduction in transgene activation and no axons showed wandering or circling at the optic nerve head like age-matched lrp2 mutants. This comparison highlights the differences between the chronic stresses caused by the lrp2 mutation versus the acute, crush injury model, in which the genetic model results in changes at the optic nerve head that are not evident in the post-nerve head crush paradigm.


Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

Veth KN, Willer JR, Collery RF, Gray MP, Willer GB, Wagner DS, Mullins MC, Udvadia AJ, Smith RS, John SW, Gregg RG, Link BA - PLoS Genet. (2011)

Expression of the gap43:GFP transgene in lrp2 mutants.A–L Expression of the gap43:GFP transgene in flat-mounted retinas from 2-month bugeye heterozygotes (wild-type) (A–E) and homozygous mutants (F–J). Shown are 5 representative samples for each condition. Images capture a single plane of the nerve fiber layer using equal gain settings on a confocal microscope. Scale bar  = 200 µm. Similar analysis on 6-month bugeye heterozygotes (wild-type) (K–O) and homozygous mutants (P–T). Note the stronger activation and unique wandering/circling phenotype in mutant axons.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040661&req=5

pgen-1001310-g008: Expression of the gap43:GFP transgene in lrp2 mutants.A–L Expression of the gap43:GFP transgene in flat-mounted retinas from 2-month bugeye heterozygotes (wild-type) (A–E) and homozygous mutants (F–J). Shown are 5 representative samples for each condition. Images capture a single plane of the nerve fiber layer using equal gain settings on a confocal microscope. Scale bar  = 200 µm. Similar analysis on 6-month bugeye heterozygotes (wild-type) (K–O) and homozygous mutants (P–T). Note the stronger activation and unique wandering/circling phenotype in mutant axons.
Mentions: Because the ultrastructural signature of degenerating axons following a crush injury is relatively short-lived in the optic nerve tract of teleost fish as compared to mammals [54], we utilized a genetic tool to label damaged and regenerating axons over a longer period of time [55]. We crossed Tg(3.6Frgap43:GFP)mil1 transgenic fish with lrp2 homozygous mutants and then used the resulting progeny to backcross with non-transgenic lrp2 mutant fish. This breeding scheme resulted in families with equal proportions of lrp2 heterozygous and homozygous mutant fish carrying single insertions of the 3.6Frgap43:GFP transgene. This transgene contains 3.6 kb of regulatory sequence (5′ flanking region and first intron) from theTakifugu rubripes gap43 locus driving GFP. Importantly, in these transgenic fish, GFP is expressed in axons following injury [55]. For our analysis, we compared large-eyed lrp2 homozygous mutant fish (>0.07 E:B ratio) to normal-eyed heterozygous siblings (Figure 8K–8T). In all large-eyed mutant fish we observed strong activation of GFP in a sub-set of retinal ganglion cells. In the majority of mutant retinas examined (6 of 6 at 6 months, Figure 8P–8T; and 10 of 12 at 12 months, data not shown), there was a characteristic axon ‘wandering’ and ‘circling’ around the optic nerve head. This axon phenotype, where GFP-positive axons approached the optic nerve head in a disorganized and circuitous fashion, was never observed in retinas from age matched lrp2 heterozygotes (Figure 8K–8O) or from 12-month wild-type fish that carried the 3.6Frgap43:GFP transgene (data not shown). The transgene was activated with variability at 2 months in both wild-type or lrp2 mutant fish (Figure 8A–8J), but the wandering axon phenotype was only rarely observed in mutants at this early timepoint. Weak expression of the transgene was noted in the nerve fiber layer of non-mutant retinas, consistent with the ongoing neurogenesis of zebrafish. In addition, older wild-type fish occasionally showed stronger GFP-positive axons, suggesting sporadic age-related degeneration. In wild-type eyes, all of the low-GFP expressing axons, as well as the occasional high-GFP expressing axons, exited the eye directly without wandering or circling the optic nerve head like those of mutants. To address whether the chronic stress conditions of lrp2 mutants differ from acute injury, we performed optic nerve crushes on adult gap43:GFP fish. At 6 days post-crush there was significant up-regulation of GFP across the retina (Figure S4). By 5 weeks post-crush, when axons had regrown [56], there was only an occasional wandering axon. Most samples following nerve crush, however, showed accurate and direct axon targeting through the optic nerve head. By 11 weeks post-crush, there was significant reduction in transgene activation and no axons showed wandering or circling at the optic nerve head like age-matched lrp2 mutants. This comparison highlights the differences between the chronic stresses caused by the lrp2 mutation versus the acute, crush injury model, in which the genetic model results in changes at the optic nerve head that are not evident in the post-nerve head crush paradigm.

Bottom Line: Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes.This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis.Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

Show MeSH
Related in: MedlinePlus