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Differences in corneal phenotypes between destrin mutants are due to allelic difference and modified by genetic background.

Kawakami-Schulz SV, Verdoni AM, Sattler SG, Ikeda A, Ikeda S - Mol. Vis. (2012)

Bottom Line: The goal of this study was to determine whether phenotypic differences are due to allelic differences between Dstn(corn1) and Dstn(corn1-2J), or are the result of genetic background effects.Actin accumulation, neovascularization, epithelial proliferation and inflammation in B6.Cg-Dstn(corn1) cornea are significantly reduced when compared to A.BY Dstn(corn1)cornea.Differences in the abnormal phenotypes of Dstn mutants result from allelic differences between Dstn(corn1) and Dstn(corn1-2J) .

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Wisconsin, Madison, WI 53705, USA.

ABSTRACT

Purpose: Mutations in destrin (Dstn) cause corneal abnormalities in mice. A mutation, Dstn(corn1), results in corneal epithelial hyperproliferation, inflammation, and neovascularization in the A.BY background (A.BY Dstn(corn1)). Homozygosity for a point mutation, Dstn(corn1-2J), results in mild thickening of the corneal epithelium but no corneal neovascularization in a C57BL/6 (B6) background (B6 Dstn(corn1-2J)). The goal of this study was to determine whether phenotypic differences are due to allelic differences between Dstn(corn1) and Dstn(corn1-2J), or are the result of genetic background effects.

Methods: We generated two congenic (Cg) mouse lines, B6.Cg-Dstn(corn1) and A.BY.Cg-Dstn(corn1-2J), to compare to the original A.BY Dstn(corn1) and B6 Dstn(corn1-2J) lines. We performed immunohistochemistry to assay F-actin accumulation, neovascularization, proliferation, and inflammation. By western blot analysis we tested the expression of serum response factor (SRF), a known regulator of the Dstn(corn1) phenotype.

Results: The Dstn(corn1) mutation leads to neovascularization, hyperproliferation, and inflammation in the cornea of A.BY Dstn(corn1) as well as B6.Cg-Dstn(corn1) mice. We did not observe significant corneal neovascularization or hyperproliferation in either A.BY.Cg-Dstn(corn1-2J) or B6 Dstn(corn1-2J) mice. Actin accumulation, neovascularization, epithelial proliferation and inflammation in B6.Cg-Dstn(corn1) cornea are significantly reduced when compared to A.BY Dstn(corn1)cornea. SRF changes are consistent in Dstn(corn1) mutants, regardless of genetic background.

Conclusions: Differences in the abnormal phenotypes of Dstn mutants result from allelic differences between Dstn(corn1) and Dstn(corn1-2J) . Moreover, phenotypes of Dstn(corn1) mice are modified by genetic background, suggesting the existence of genetic modifiers. Protein analysis suggests that a genetic modifier affects phenotypic severity functionally downstream from or in a pathway independent from SRF. These data demonstrate that natural genetic variation affects phenotypic severity in Dstn(corn1) mice.

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Corneal neovascularization caused by the Dstncorn1 mutation. A: Immunofluorescence for CD31 highlights blood vessels that infiltrate Dstncorn1 cornea in both the A.BY and B6 background. Significant neovascularization is not observed as a result of the Dstncorn1–2J mutation, making the appearance similar to WT. Bar, 200 μm. B: Quantification of the vascularized area shows that Dstncorn1 cornea have significantly more vasculature compared to Dstncorn1–2J and WT cornea in both genetic backgrounds (p<0.001 for both backgrounds). Note that the resting level of vasculature is higher in B6 than A.BY. C: Genetic background effect on the neovascularization phenotype in the Dstncorn1 cornea is significant at postnatal day 28, and becomes even more significant with age. Sample sizes: A.BY.Cg-Dstncorn1–2J P58 n=5, A.BY Dstncorn1 P58 n=11, A.BY WT P58 n=3, B6 Dstncorn1–2J P58 n=4, B6.Cg-Dstncorn1 P58 n=10, B6 WT P58 n=4, A.BY Dstncorn1 P28 n=11, B6.Cg-Dstncorn1 P28 n=6. Error bars, SEM * denotes statistical significance resulting from t-tests, with omitted bars representing nonsignficance. *p<0.05, **p<0.01, ***p<0.001.
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f3: Corneal neovascularization caused by the Dstncorn1 mutation. A: Immunofluorescence for CD31 highlights blood vessels that infiltrate Dstncorn1 cornea in both the A.BY and B6 background. Significant neovascularization is not observed as a result of the Dstncorn1–2J mutation, making the appearance similar to WT. Bar, 200 μm. B: Quantification of the vascularized area shows that Dstncorn1 cornea have significantly more vasculature compared to Dstncorn1–2J and WT cornea in both genetic backgrounds (p<0.001 for both backgrounds). Note that the resting level of vasculature is higher in B6 than A.BY. C: Genetic background effect on the neovascularization phenotype in the Dstncorn1 cornea is significant at postnatal day 28, and becomes even more significant with age. Sample sizes: A.BY.Cg-Dstncorn1–2J P58 n=5, A.BY Dstncorn1 P58 n=11, A.BY WT P58 n=3, B6 Dstncorn1–2J P58 n=4, B6.Cg-Dstncorn1 P58 n=10, B6 WT P58 n=4, A.BY Dstncorn1 P28 n=11, B6.Cg-Dstncorn1 P28 n=6. Error bars, SEM * denotes statistical significance resulting from t-tests, with omitted bars representing nonsignficance. *p<0.05, **p<0.01, ***p<0.001.

Mentions: To examine the amount of corneal neovascularization, we performed immunofluorescence using an anti-CD31 antibody on whole cornea. Quantification of the vascularized area in A.BY Dstncorn1 to A.BY.Cg-Dstncorn1–2J and B6.Cg-Dstncorn1 to B6 Dstncorn1–2J and WT mice revealed that significant corneal neovascularization occurs only in mice with the Dstncorn1 mutation, in both the A.BY and B6 backgrounds. There is no significant difference in the amount of vasculature in Dstncorn1–2J compared to WT (Figure 3A,B). Neovascularization is significantly reduced in the cornea of B6.Cg-Dstncorn1 mice compared to A.BY Dstncorn1 mice. While this difference is apparent and significant by P28, the difference increases in significance with age (Figure 3C).


Differences in corneal phenotypes between destrin mutants are due to allelic difference and modified by genetic background.

Kawakami-Schulz SV, Verdoni AM, Sattler SG, Ikeda A, Ikeda S - Mol. Vis. (2012)

Corneal neovascularization caused by the Dstncorn1 mutation. A: Immunofluorescence for CD31 highlights blood vessels that infiltrate Dstncorn1 cornea in both the A.BY and B6 background. Significant neovascularization is not observed as a result of the Dstncorn1–2J mutation, making the appearance similar to WT. Bar, 200 μm. B: Quantification of the vascularized area shows that Dstncorn1 cornea have significantly more vasculature compared to Dstncorn1–2J and WT cornea in both genetic backgrounds (p<0.001 for both backgrounds). Note that the resting level of vasculature is higher in B6 than A.BY. C: Genetic background effect on the neovascularization phenotype in the Dstncorn1 cornea is significant at postnatal day 28, and becomes even more significant with age. Sample sizes: A.BY.Cg-Dstncorn1–2J P58 n=5, A.BY Dstncorn1 P58 n=11, A.BY WT P58 n=3, B6 Dstncorn1–2J P58 n=4, B6.Cg-Dstncorn1 P58 n=10, B6 WT P58 n=4, A.BY Dstncorn1 P28 n=11, B6.Cg-Dstncorn1 P28 n=6. Error bars, SEM * denotes statistical significance resulting from t-tests, with omitted bars representing nonsignficance. *p<0.05, **p<0.01, ***p<0.001.
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f3: Corneal neovascularization caused by the Dstncorn1 mutation. A: Immunofluorescence for CD31 highlights blood vessels that infiltrate Dstncorn1 cornea in both the A.BY and B6 background. Significant neovascularization is not observed as a result of the Dstncorn1–2J mutation, making the appearance similar to WT. Bar, 200 μm. B: Quantification of the vascularized area shows that Dstncorn1 cornea have significantly more vasculature compared to Dstncorn1–2J and WT cornea in both genetic backgrounds (p<0.001 for both backgrounds). Note that the resting level of vasculature is higher in B6 than A.BY. C: Genetic background effect on the neovascularization phenotype in the Dstncorn1 cornea is significant at postnatal day 28, and becomes even more significant with age. Sample sizes: A.BY.Cg-Dstncorn1–2J P58 n=5, A.BY Dstncorn1 P58 n=11, A.BY WT P58 n=3, B6 Dstncorn1–2J P58 n=4, B6.Cg-Dstncorn1 P58 n=10, B6 WT P58 n=4, A.BY Dstncorn1 P28 n=11, B6.Cg-Dstncorn1 P28 n=6. Error bars, SEM * denotes statistical significance resulting from t-tests, with omitted bars representing nonsignficance. *p<0.05, **p<0.01, ***p<0.001.
Mentions: To examine the amount of corneal neovascularization, we performed immunofluorescence using an anti-CD31 antibody on whole cornea. Quantification of the vascularized area in A.BY Dstncorn1 to A.BY.Cg-Dstncorn1–2J and B6.Cg-Dstncorn1 to B6 Dstncorn1–2J and WT mice revealed that significant corneal neovascularization occurs only in mice with the Dstncorn1 mutation, in both the A.BY and B6 backgrounds. There is no significant difference in the amount of vasculature in Dstncorn1–2J compared to WT (Figure 3A,B). Neovascularization is significantly reduced in the cornea of B6.Cg-Dstncorn1 mice compared to A.BY Dstncorn1 mice. While this difference is apparent and significant by P28, the difference increases in significance with age (Figure 3C).

Bottom Line: The goal of this study was to determine whether phenotypic differences are due to allelic differences between Dstn(corn1) and Dstn(corn1-2J), or are the result of genetic background effects.Actin accumulation, neovascularization, epithelial proliferation and inflammation in B6.Cg-Dstn(corn1) cornea are significantly reduced when compared to A.BY Dstn(corn1)cornea.Differences in the abnormal phenotypes of Dstn mutants result from allelic differences between Dstn(corn1) and Dstn(corn1-2J) .

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Wisconsin, Madison, WI 53705, USA.

ABSTRACT

Purpose: Mutations in destrin (Dstn) cause corneal abnormalities in mice. A mutation, Dstn(corn1), results in corneal epithelial hyperproliferation, inflammation, and neovascularization in the A.BY background (A.BY Dstn(corn1)). Homozygosity for a point mutation, Dstn(corn1-2J), results in mild thickening of the corneal epithelium but no corneal neovascularization in a C57BL/6 (B6) background (B6 Dstn(corn1-2J)). The goal of this study was to determine whether phenotypic differences are due to allelic differences between Dstn(corn1) and Dstn(corn1-2J), or are the result of genetic background effects.

Methods: We generated two congenic (Cg) mouse lines, B6.Cg-Dstn(corn1) and A.BY.Cg-Dstn(corn1-2J), to compare to the original A.BY Dstn(corn1) and B6 Dstn(corn1-2J) lines. We performed immunohistochemistry to assay F-actin accumulation, neovascularization, proliferation, and inflammation. By western blot analysis we tested the expression of serum response factor (SRF), a known regulator of the Dstn(corn1) phenotype.

Results: The Dstn(corn1) mutation leads to neovascularization, hyperproliferation, and inflammation in the cornea of A.BY Dstn(corn1) as well as B6.Cg-Dstn(corn1) mice. We did not observe significant corneal neovascularization or hyperproliferation in either A.BY.Cg-Dstn(corn1-2J) or B6 Dstn(corn1-2J) mice. Actin accumulation, neovascularization, epithelial proliferation and inflammation in B6.Cg-Dstn(corn1) cornea are significantly reduced when compared to A.BY Dstn(corn1)cornea. SRF changes are consistent in Dstn(corn1) mutants, regardless of genetic background.

Conclusions: Differences in the abnormal phenotypes of Dstn mutants result from allelic differences between Dstn(corn1) and Dstn(corn1-2J) . Moreover, phenotypes of Dstn(corn1) mice are modified by genetic background, suggesting the existence of genetic modifiers. Protein analysis suggests that a genetic modifier affects phenotypic severity functionally downstream from or in a pathway independent from SRF. These data demonstrate that natural genetic variation affects phenotypic severity in Dstn(corn1) mice.

Show MeSH
Related in: MedlinePlus