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. 2017 Aug 31;12(8):e0183837.
doi: 10.1371/journal.pone.0183837. eCollection 2017.

Mouse models of human ocular disease for translational research

Mark P Krebs  1 , Gayle B Collin  1 , Wanda L Hicks  1 , Minzhong Yu  2   3 , Jeremy R Charette  1 , Lan Ying Shi  1 , Jieping Wang  1 , Jürgen K Naggert  1 , Neal S Peachey  2   3   4 , Patsy M Nishina  1
Affiliations

Mouse models of human ocular disease for translational research

Mark P Krebs et al. PLoS One. .

Abstract

Mouse models provide a valuable tool for exploring pathogenic mechanisms underlying inherited human disease. Here, we describe seven mouse models identified through the Translational Vision Research Models (TVRM) program, each carrying a new allele of a gene previously linked to retinal developmental and/or degenerative disease. The mutations include four alleles of three genes linked to human nonsyndromic ocular diseases (Aipl1tvrm119, Aipl1tvrm127, Rpgrip1tvrm111, RhoTvrm334) and three alleles of genes associated with human syndromic diseases that exhibit ocular phentoypes (Alms1tvrm102, Clcn2nmf289, Fkrptvrm53). Phenotypic characterization of each model is provided in the context of existing literature, in some cases refining our current understanding of specific disease attributes. These murine models, on fixed genetic backgrounds, are available for distribution upon request and may be useful for understanding the function of the gene in the retina, the pathological mechanisms induced by its disruption, and for testing experimental approaches to treat the corresponding human ocular diseases.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Rapid photoreceptor degeneration in Aipl1 mutants.
(A) Fundus photographs of B6J control and homozygous Aipl1tvrm119 (tvrm119) or Aipl1tvrm127 (tvrm127) mice at one and three months (mo) of age. (B) Retinal sections stained with hematoxylin and eosin (H&E) obtained from B6J control, tvrm119, and tvrm127 mice at P8 (n = 3, 4 and 4, respectively), P12 (n = 3, 2 and 3, respectively) and one month (n = 3, all strains) of age visualized by light microscopy. An allele test of Aipl1tvrm119/Aipl1tvrm127 compound heterozygous (tvrm119/tvrm127) mice showed rapid loss of photoreceptors at one month of age (n = 8). GC, ganglion cell; INL, inner nuclear layer; ONL, outer nuclear layer. Bar, 25 μm.
Fig 2
Fig 2. IHC, western analysis and ERG of homozygous Aipl1 mutants.
(A) Immunostaining of Aipl1tvrm119 (n = 2) and Aipl1tvrm127 (n = 3) and B6J control (n = 3) retinas at P12. Lower panel: mislocalization of rhodopsin (green) to the ONL was observed in both mutants. Upper panel: anti-cone arrestin (red) staining shows uniform cells bodies on the scleral side of the photoreceptor ONL while cell bodies were found scattered throughout the ONL in Aipl1tvrm119 mutants. Nuclear staining with DAPI (blue) shows a more pronounced photoreceptor degeneration in Aipl1tvrm127 mutants. Bar, 50 μm. (B) Western analysis at P10–12 with ROM1 antibody, an OS marker, shows similar relative expression in homozygous Aipl1tvrm119 (n = 2) and B6J control (n = 4) mice, but reduced expression in homozygous Aipl1tvrm127 mice (n = 4). Analysis with PDE6α antibody shows loss of PDE6α in Aipl1tvrm119 and Aipl1tvrm127 mutant mice. β-actin was probed as a loading control. (C) Dark- and light-adapted ERG analysis of P18 Aipl1tvrm119 (n = 2) and Aipl1tvrm127 (n = 6) mutants and B6J control (n = 2) mice.
Fig 3
Fig 3. mRNA isoforms in targeted and chemically induced Rpgrip1 murine models.
(A) Wild-type (WT) mice produce a full-length Rpgrip1 transcript, isoform 1 (NM_023879.3), and a short transcript, isoform 2 (NM_001168515.1) that contains an extension of exon 13 leading to an early termination. (B) The targeted insertion of a large cassette in Rpgrip1tm1Tili mice between exons 14 and 15 leads to undetectable full-length Rpgrip1 but does not affect the shorter Rpgrip1 isoform. (C). Rpgrip1nmf247 splice-donor mutation produces multiple isoforms that lead to premature termination of both WT Rpgrip1 isoforms. Aberrant splicing into intron 6 generates a 96 bp insertion, but is predicted to terminate at the mutation site because it generates a stop codon (top). Skipping of exon 7 (middle) or exons 7 and 8 (bottom) results in a frame-shift that is predicted to lead to a premature termination in exon 9. (D) Rpgrip1tvrm111 splice-donor mutation in intron 8 produces multiple transcripts that lead to premature termination or in-frame deletion of WT Rpgrip1 isoforms. In one transcription product detected by cDNA sequencing, aberrant splicing to a cryptic site 214 bp downstream in intron 8 (top) is predicted to yield isoforms that encode an additional eight amino acid residues and result in premature termination. In a second product, skipping of exons 7 and 8 results in a frame-shift that is predicted to lead to a premature termination in exon 9 (middle). In a third product, skipping of exon 8 leads to an in-frame deletion of eight residues (bottom). This splicing event is predicted to occur in both full-length and short Rpgrip1 isoforms. Exons are colored boxes while intron sequence is represented by lines. Exons and introns are not to scale. Red arrows denote location of mutations.
Fig 4
Fig 4. Progressive photoreceptor degeneration in Rpgrip1 mutants.
(A) Fundus images of homozygous Rpgriptvrm111 mice (tvrm111) at 1 and 6 months of age. (B) Retinal sections stained with H&E obtained from B6J at 1 month of age (n = 3) and Rpgrip1tvrm111 at 1 (n = 3) and 3 (n = 6) months of age, or compound heterozygous Rpgrip1tvrm111/Rpgrip1nmf247 (tvrm111/nmf247) mice at 1 month (n = 3) of age, and visualized by light microscopy. Retinal layers are labeled as in Fig 1B. Bar, 25 μm. (C) Dark- and light-adapted ERG responses in Rpgrip1nmf247 homozygotes at 1 (n = 6) and 6 (n = 5) months of age and in B6J (n = 4) controls at 1 month of age. (D) Dark- and light-adapted ERG response amplitudes at varying illuminance determined from the samples tested as in C. Values indicate mean ± SEM.
Fig 5
Fig 5. Ultrastructural analysis of OS alterations in Rpgrip1tvrm111 mice.
Transmission electron micrographs at P14 show disorganization of photoreceptor OS discs in tvrm111 (n = 3) compared to control (n = 3) mice. As can be seen at higher magnification, some discs have a vertical arrangement within the OS (right, asterisk) similar to the Rpgriptm1Tili mutant [34]. Bar, left panel, 2 μm; right, 1 μm.
Fig 6
Fig 6. Clinical and histological alterations in heterozygous RhoTvrm334 mice indicate a rapid, early onset degeneration.
(A) Fundus photographs at P21 indicate atypical RPE characteristic of the grainy retinal appearance. (B) Retinal sections stained with H&E obtained from WT littermate control and heterozygous RhoTvrm334 (Tvrm334) mice at P14 (n = 4, both genotypes) and P21 (n = 5 and 3, respectively) as visualized by light microscopy. Retinal layers are labeled as in Fig 1B. Bar, 25 μm. (C) ERGs recorded at P21 from WT littermate control (black; n = 4) and heterozygous RhoTvrm334 (red; n = 4) mice. (D) Immunostaining of heterozygous Tvrm334 mutants and WT littermate controls with anti-rhodopsin (green) at P21 (n = 3, both genotypes). RPE, retinal pigment epithelium. ONL, outer nuclear layer; OS, outer segment. Bar, 20 μm.
Fig 7
Fig 7. Alms1tvrm102 recapitulates clinical phenotypes observed in patients with Alström syndrome.
(A) Obesity is an early clinical feature of homozygous Alms1tvrm102 (tvrm102) mice (n = 6). Control mice (n = 10) include WT and heterozygous littermates. Graph shows that male mutant mice begin weight gain between 8–12 weeks of age. Values indicate mean ± standard deviation; *, p<0.0001. (B) Fundus images of WT littermate control and homozygous Alms1tvrm102 mice at 15 months of age. Arrowheads, small bright spots. (C) Brightfield microscopy of H&E stained retinal sections showing a slow progression of photoreceptor loss in Alms1tvrm102 mutants at 5 (n = 4) and 18 months (n = 3) of age, and WT and heterozygous controls at 5–6 months (n = 3) and 18 months (n = 3) of age. Retinal layers are labeled as in Fig 1B. (D) Immunostaining with rhodopsin (red) in homozygous Alms1tvrm102 mice at 5–6 (n = 3) and 18–20 (n = 3) months of age. Arrowheads show mislocalized rhodopsin in photoreceptor somata in the ONL. Controls at the same ages are as in C (n = 3). Bars, 30 μm.
Fig 8
Fig 8. Gradual photoreceptor loss in homozygous Clcn2nmf289 mutants.
(A) Fundus imaging of homozygous Clcn2nmf289 (nmf289) mice at 3 months of age indicates vascular atrophy compared to WT littermate controls. (B) Brightfield images of H&E-stained retinal sections of WT or heterozygous littermate controls at P14 (n = 5) and 1 month (n = 3) of age, and homozygous Clcn2nmf289 at P14 (n = 3) and at 1 month (n = 3) of age. Compound heterozygous Clcn2nmf289/Clcn2nmf240 (nmf289/nmf240) mice at P14 (n = 6) show similar ONL loss. Retinal layers are labeled as in Fig 1B. Bar, 20 μm. (C) Dark-adapted ERG series obtained from WT littermate control (left; n = 3) and homozygous and Clcn2nmf289 (right; n = 5) mice at 1 month of age (left to right). D. Immunostaining with anti-ezrin antibody shows elongated apical microvilli in homozygous Clcn2nmf289 mice (bottom; n = 3) compared to WT littermates (top; n = 3) at P14. Bar, 10 μm.
Fig 9
Fig 9. Clcn2nmf289 mice develop leukoencephalopathy and azoospermia.
(A) Sagittal section of a control BALB/cByJ brain at 3–6 months of age stained with H&E (n = 4). (B) Similarly stained section of homozygous Clcn2nmf289 brain (mutant littermate; n = 5). Arrows depict marked vacuolization in the white matter tracts of the corpus callosum (a), brainstem (b) and cerebellum (c) of mutant mice. (C) Higher power image of the cerebellum of BALB/cByJ mice. (D) The cerebellum of homozygous Clcn2nmf289 mice exhibits vacuolization. (E) Toluidine blue-stained sections show normal spermatogenesis in BALB/cByJ testes (n = 4). (F) Atrophic seminiferous tubules were observed in testes of mutant littermates at 3–6 months of age (n = 4). Bars, (A-B) 2 mm, (C-D) 500 μm, (E-F) 100 μm.
Fig 10
Fig 10. Aberrant clinical, histological and functional effects of the homozygous Fkrptvrm53 allele.
(A) Fundus of B6J control mice at 6 months and Fkrptvrm53 (tvrm53) mutants at 1 and 6 months of age. (B) Fundus images of B6J control (top; n = 4) and Fkrptvrm53 (bottom; n = 3) mice at 1 month of age, digitally processed to highlight vascular dysmorphology in the mutant strain. (C) Brightfield image of H&E stained-B6J control (n = 3) and Fkrptvrm53 (n = 3) retina at one month of age. Arrowheads depict vascular abnormalities in the vitreous. Bar, 100 μm. (D) Dark-adapted (left) and light-adapted (right) ERGs obtained from a representative B6J control (n = 2) and Fkrptvrm53 (n = 3) mouse at 7 weeks of age. (E) Spinal muscle showing normal distribution of nuclei at the periphery of muscle fibers in B6J mice (left; n = 3) and mislocalized nuclei in Fkrptvrm53 mice (right; n = 4) at 12 months of age. Bar, 50 μm. (F) Quantitation of central nuclei in B6J (n = 3) and Fkrptvrm53 mice (n = 4). Mean value and standard deviation is indicated.
Fig 11
Fig 11. Marker analysis of homozygous Fkrptvrm53 mice at 1–2 months of age.
(A) Localization of FKRP in B6J control retina (n = 3). FKRP (red) colocalizes with β-dystrophin to synaptic processes in the OPL. (B) Co-staining of FKRP (red) with CTBP2 (green) shows FKRP surrounds the synaptic ribbons. (C) IHC of rhodopsin (red) and pan-laminin (green) in B6J control (n = 6) and homozygous Fkrptvrm53 mice (n = 7). (D) IHC of pan-laminin in ocular cryosections of samples as in C. (E) IHC of collagen IV (COL IV, red) and β-dystroglycan (β-DYS, green) in cryosections (n = 5 and 6 for B6J and mutant mice, respectively). (F) Glial fibrillary acidic protein (GFAP) staining of retinal cryosections indicated Müller cell activation in homozygous Fkrptvrm53 mice (n = 4) compared to B6J controls (n = 3). Retinal layers are labeled as in Fig 1B. Asterisks, abnormal vessels in (D, E). Bars, (A) 20 μm, (B) 5 μm, (C-D) 50 μm. (E-F) 100 μm.
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