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The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Luhmann UF, Carvalho LS, Holthaus SM, Cowing JA, Greenaway S, Chu CJ, Herrmann P, Smith AJ, Munro PM, Potter P, Bainbridge JW, Ali RR - Hum. Mol. Genet. (2014)

Bottom Line: Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines.By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified.This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and u.luhmann@ucl.ac.uk.

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Assessment of OLM integrity and associated Müller glia activation in the superior and inferior retina of different Crb1rd8/rd8 mouse lines. Corresponding confocal bright field images of the superior (A–E) and inferior (F–J) OLM (white arrow heads) in retinas of animals from respective lines. Scale bar: 20 µm. (K–O) Corresponding images of the inferior retina obtained by immunohistochemistry for glial fibrillary acidic protein (GFAP, white, activated Müller glia and astrocytes), the tight junction protein ZO-1 (green) and 4′,6-diamidino-2-phenylindole (DAPI, blue, nuclei) on sagittal retinal cryo-sections. Scale bar: 50 µm. (Ki–Oi) Magnified views of the inferior disrupted OLM. Scale bar: 25 µm. Red arrow heads indicate predominant disruption of the OLM in areas of the inferior retina where photoreceptor disruption in the ONL is associated with localized Müller cell activation (white, GFAP+ processes of Müller glia cells). (P–U) Assessment of integrity of the OLM by TEM. Representative TEM images with electron dense adhesions plaques (white arrow heads) and Müller glia processes (red arrows) from the superior and inferior central retina of Crb1rd8/rd8/J mice, C57BL/6 Crb1rd8/rd8 (line 1) and wild-type mice. Scale bar: 5 µm. (V) Quantification of number of electron dense AJ normalized to the assessed distance of OLM and expressed as number of densities per 10 µm. Different colours and shapes represent the origin of individual wild-type or Crb1rd8/rd8 mice (black square: C57BL/6J controls, yellow circle: C57BL/6J Crb1rd8/rd8 (1), green square: C57BL/6J Crb1rd8/rd8 (2) and open diamond: Crb1rd8/rd8/J). Overall, the number of AJ per 10 µm shows a similar range of variable reduction in the superior and inferior retina of Crb1rd8/rd8 mice compared with wild-type mice, although this feature was not consistent in all Crb1 deficient mice.
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DDU424F4: Assessment of OLM integrity and associated Müller glia activation in the superior and inferior retina of different Crb1rd8/rd8 mouse lines. Corresponding confocal bright field images of the superior (A–E) and inferior (F–J) OLM (white arrow heads) in retinas of animals from respective lines. Scale bar: 20 µm. (K–O) Corresponding images of the inferior retina obtained by immunohistochemistry for glial fibrillary acidic protein (GFAP, white, activated Müller glia and astrocytes), the tight junction protein ZO-1 (green) and 4′,6-diamidino-2-phenylindole (DAPI, blue, nuclei) on sagittal retinal cryo-sections. Scale bar: 50 µm. (Ki–Oi) Magnified views of the inferior disrupted OLM. Scale bar: 25 µm. Red arrow heads indicate predominant disruption of the OLM in areas of the inferior retina where photoreceptor disruption in the ONL is associated with localized Müller cell activation (white, GFAP+ processes of Müller glia cells). (P–U) Assessment of integrity of the OLM by TEM. Representative TEM images with electron dense adhesions plaques (white arrow heads) and Müller glia processes (red arrows) from the superior and inferior central retina of Crb1rd8/rd8/J mice, C57BL/6 Crb1rd8/rd8 (line 1) and wild-type mice. Scale bar: 5 µm. (V) Quantification of number of electron dense AJ normalized to the assessed distance of OLM and expressed as number of densities per 10 µm. Different colours and shapes represent the origin of individual wild-type or Crb1rd8/rd8 mice (black square: C57BL/6J controls, yellow circle: C57BL/6J Crb1rd8/rd8 (1), green square: C57BL/6J Crb1rd8/rd8 (2) and open diamond: Crb1rd8/rd8/J). Overall, the number of AJ per 10 µm shows a similar range of variable reduction in the superior and inferior retina of Crb1rd8/rd8 mice compared with wild-type mice, although this feature was not consistent in all Crb1 deficient mice.

Mentions: To better understand how the loss of the evenly distributed CRB1 protein at the OLM might lead to a preferential degeneration in the inferior retina and to see whether all Crb1rd8/rd8 homozygous mice are similarly affected, we evaluated the integrity of the OLM in the three differentially affected Crb1rd8/rd8 lines (Fig. 4). Confocal evaluation on semithin sections across the whole retina suggested that the OLM of C57BL/6 Crb1rd8/rd8 (1) mice is macroscopically similar to that of wild-type mice (white arrow heads; Fig. 4D versus E and I versus J). Also the tight junction protein ZO-1 is evenly distributed along the whole OLM in all Crb1rd8/rd8 and wild-type mice (Fig. 4K–O and Ki–Oi). ZO-1 labelling only became disrupted in the inferior retina if photoreceptor columns were disorganized (red arrow heads, Fig. 4F–J), photoreceptor nuclei dropped out of the ONL (red arrow heads, Fig. 4F–J) and localized Müller glia became activated (Fig. 4K–M). All these features were barely detectable in C57BL/6 Crb1rd8/rd8 (1) mice (Fig. 4N and Ni, red arrowhead) and difficult to distinguish from observations in wild-type mice (Fig. 4E and J and O and Oi).Figure 4.


The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Luhmann UF, Carvalho LS, Holthaus SM, Cowing JA, Greenaway S, Chu CJ, Herrmann P, Smith AJ, Munro PM, Potter P, Bainbridge JW, Ali RR - Hum. Mol. Genet. (2014)

Assessment of OLM integrity and associated Müller glia activation in the superior and inferior retina of different Crb1rd8/rd8 mouse lines. Corresponding confocal bright field images of the superior (A–E) and inferior (F–J) OLM (white arrow heads) in retinas of animals from respective lines. Scale bar: 20 µm. (K–O) Corresponding images of the inferior retina obtained by immunohistochemistry for glial fibrillary acidic protein (GFAP, white, activated Müller glia and astrocytes), the tight junction protein ZO-1 (green) and 4′,6-diamidino-2-phenylindole (DAPI, blue, nuclei) on sagittal retinal cryo-sections. Scale bar: 50 µm. (Ki–Oi) Magnified views of the inferior disrupted OLM. Scale bar: 25 µm. Red arrow heads indicate predominant disruption of the OLM in areas of the inferior retina where photoreceptor disruption in the ONL is associated with localized Müller cell activation (white, GFAP+ processes of Müller glia cells). (P–U) Assessment of integrity of the OLM by TEM. Representative TEM images with electron dense adhesions plaques (white arrow heads) and Müller glia processes (red arrows) from the superior and inferior central retina of Crb1rd8/rd8/J mice, C57BL/6 Crb1rd8/rd8 (line 1) and wild-type mice. Scale bar: 5 µm. (V) Quantification of number of electron dense AJ normalized to the assessed distance of OLM and expressed as number of densities per 10 µm. Different colours and shapes represent the origin of individual wild-type or Crb1rd8/rd8 mice (black square: C57BL/6J controls, yellow circle: C57BL/6J Crb1rd8/rd8 (1), green square: C57BL/6J Crb1rd8/rd8 (2) and open diamond: Crb1rd8/rd8/J). Overall, the number of AJ per 10 µm shows a similar range of variable reduction in the superior and inferior retina of Crb1rd8/rd8 mice compared with wild-type mice, although this feature was not consistent in all Crb1 deficient mice.
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DDU424F4: Assessment of OLM integrity and associated Müller glia activation in the superior and inferior retina of different Crb1rd8/rd8 mouse lines. Corresponding confocal bright field images of the superior (A–E) and inferior (F–J) OLM (white arrow heads) in retinas of animals from respective lines. Scale bar: 20 µm. (K–O) Corresponding images of the inferior retina obtained by immunohistochemistry for glial fibrillary acidic protein (GFAP, white, activated Müller glia and astrocytes), the tight junction protein ZO-1 (green) and 4′,6-diamidino-2-phenylindole (DAPI, blue, nuclei) on sagittal retinal cryo-sections. Scale bar: 50 µm. (Ki–Oi) Magnified views of the inferior disrupted OLM. Scale bar: 25 µm. Red arrow heads indicate predominant disruption of the OLM in areas of the inferior retina where photoreceptor disruption in the ONL is associated with localized Müller cell activation (white, GFAP+ processes of Müller glia cells). (P–U) Assessment of integrity of the OLM by TEM. Representative TEM images with electron dense adhesions plaques (white arrow heads) and Müller glia processes (red arrows) from the superior and inferior central retina of Crb1rd8/rd8/J mice, C57BL/6 Crb1rd8/rd8 (line 1) and wild-type mice. Scale bar: 5 µm. (V) Quantification of number of electron dense AJ normalized to the assessed distance of OLM and expressed as number of densities per 10 µm. Different colours and shapes represent the origin of individual wild-type or Crb1rd8/rd8 mice (black square: C57BL/6J controls, yellow circle: C57BL/6J Crb1rd8/rd8 (1), green square: C57BL/6J Crb1rd8/rd8 (2) and open diamond: Crb1rd8/rd8/J). Overall, the number of AJ per 10 µm shows a similar range of variable reduction in the superior and inferior retina of Crb1rd8/rd8 mice compared with wild-type mice, although this feature was not consistent in all Crb1 deficient mice.
Mentions: To better understand how the loss of the evenly distributed CRB1 protein at the OLM might lead to a preferential degeneration in the inferior retina and to see whether all Crb1rd8/rd8 homozygous mice are similarly affected, we evaluated the integrity of the OLM in the three differentially affected Crb1rd8/rd8 lines (Fig. 4). Confocal evaluation on semithin sections across the whole retina suggested that the OLM of C57BL/6 Crb1rd8/rd8 (1) mice is macroscopically similar to that of wild-type mice (white arrow heads; Fig. 4D versus E and I versus J). Also the tight junction protein ZO-1 is evenly distributed along the whole OLM in all Crb1rd8/rd8 and wild-type mice (Fig. 4K–O and Ki–Oi). ZO-1 labelling only became disrupted in the inferior retina if photoreceptor columns were disorganized (red arrow heads, Fig. 4F–J), photoreceptor nuclei dropped out of the ONL (red arrow heads, Fig. 4F–J) and localized Müller glia became activated (Fig. 4K–M). All these features were barely detectable in C57BL/6 Crb1rd8/rd8 (1) mice (Fig. 4N and Ni, red arrowhead) and difficult to distinguish from observations in wild-type mice (Fig. 4E and J and O and Oi).Figure 4.

Bottom Line: Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines.By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified.This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and u.luhmann@ucl.ac.uk.

Show MeSH
Related in: MedlinePlus