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Localization of complement factor H gene expression and protein distribution in the mouse outer retina
Zeljka Smit-McBride1, Sharon L Oltjen1, Roxana A Radu2
1Department of Ophthalmology, University of California, Davis, CA.
Purpose:
To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.
Methods:
Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.
Results:
Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.
Conclusions:
Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.
Insights
Complement factor H (Cfh) mRNA is found in mouse RPE, but the protein is unexpectedly located in photoreceptors. This suggests Cfh is secreted and present at low levels in RPE cells.
Area of Science:
- Ophthalmology
- Immunology
- Molecular Biology
Background:
- Complement factor H (Cfh) plays a crucial role in regulating the complement system.
- Understanding Cfh localization in the retina is vital for comprehending its function in ocular health and disease.
Purpose of the Study:
- To precisely determine the localization of complement factor H (Cfh) mRNA and its corresponding protein within the mouse outer retina.
Main Methods:
- Quantitative real-time PCR (qPCR) to analyze Cfh and Cfh-related (Cfhr) transcript expression in the RPE/choroid.
- In situ hybridization (ISH) using the RNAscope 2.0 FFPE assay for Cfh mRNA localization in the outer retina.
- Immunohistochemistry (IHC) and western blotting to confirm Cfh protein localization and antibody specificity.
Main Results:
- qPCR detected Cfh and Cfhr2 transcripts in the RPE/choroid; other Cfhr transcripts were not found.
- ISH revealed abundant Cfh mRNA specifically in the retinal pigment epithelium (RPE) across tested mouse strains.
- Unexpectedly, IHC localized Cfh protein to photoreceptor cells, not RPE cells, with validated antibody specificity.
Conclusions:
- Only Cfh and Cfhr2 genes are expressed in the mouse outer retina.
- Cfh mRNA is present in the RPE, but the protein is not detected there, suggesting low steady-state levels due to secretion.
- The study highlights a novel localization of Cfh protein in photoreceptors, distinct from its mRNA in RPE.
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