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Fibulin-3 knockout mice demonstrate corneal dysfunction but maintain normal retinal integrity

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Abstract

Fibulin-3 (F3) is an extracellular matrix glycoprotein found in basement membranes across the body. An autosomal dominant R345W mutation in F3 causes a macular dystrophy resembling dry age-related macular degeneration (AMD), whereas genetic removal of wild-type (WT) F3 protects mice from sub-retinal pigment epithelium (RPE) deposit formation. These observations suggest that F3 is a protein which can regulate pathogenic sub-RPE deposit formation in the eye. Yet the precise role of WT F3 within the eye is still largely unknown. We found that F3 is expressed throughout the mouse eye (cornea, trabecular meshwork (TM) ring, neural retina, RPE/choroid, and optic nerve). We next performed a thorough structural and functional characterization of each of these tissues in WT and homozygous (F3−/−) knockout mice. The corneal stroma in F3−/− mice progressively thins beginning at 2 months, and the development of corneal opacity and vascularization starts at 9 months, which worsens with age. However, in all other tissues (TM, neural retina, RPE, and optic nerve), gross structural anatomy and functionality were similar across WT and F3−/− mice when evaluated using SD-OCT, histological analyses, electron microscopy, scotopic electroretinogram, optokinetic response, and axonal anterograde transport. The lack of noticeable retinal abnormalities in F3−/− mice was confirmed in a human patient with biallelic loss-of-function mutations in F3. These data suggest that (i) F3 is important for maintaining the structural integrity of the cornea, (ii) absence of F3 does not affect the structure or function of any other ocular tissue in which it is expressed, and (iii) targeted silencing of F3 in the retina and/or RPE will likely be well-tolerated, serving as a safe therapeutic strategy for reducing sub-RPE deposit formation in disease.

Key messages

• Fibulins are expressed throughout the body at varying levels.

• Fibulin-3 has a tissue-specific pattern of expression within the eye.

• Lack of fibulin-3 leads to structural deformities in the cornea.

• The retina and RPE remain structurally and functionally healthy in the absence of fibulin-3 in both mice and humans.

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Acknowledgments

The authors would like to personally thank Amber Wilkerson, the UTSW Histo-Pathology Core, and the UTSW Electron Microscopy for their assistance with data acquisition and sample processing. The authors would like to also thank Dr. Zainah Alsagoff, MD (Southland Hospital, Invercargill, New Zealand) for performing the ocular exam.

Funding

VQC was supported by a funding from the UT Southwestern Summer Medical Student Research Program. JDH is supported by an endowment from the Roger and Dorothy Hirl Research Fund, a vision research grant from the Karl Kirchgessner Foundation, a BrightFocus Foundation Macular Degeneration Research Grant (M2016200), an NEI R01 grant (EY027785), and a Career Development Award from Research to Prevent Blindness (RPB). Additional support was provided by an NEI Visual Science Core grant (P30 EY030413) and an unrestricted grant from RPB (both to the UT Southwestern Department of Ophthalmology). GZ is supported by an NEI R01 grant (EY026177). WMP is supported by an NEI R01 grant (EY013322).

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Authors and Affiliations

  1. Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, USA

    Steffi Daniel, Marian Renwick, Viet Q. Chau, Shyamtanu Datta, W. Matthew Petroll & John D. Hulleman

  2. Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA, USA

    Shyamtanu Datta

  3. Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX, USA

    Prabhavathi Maddineni & Gulab Zode

  4. Department of Women’s and Children’s Health, Dunedin School of Medicine, University of Otago, Dunedin, 9016, New Zealand

    Emma M. Wade & Stephen P. Robertson

  5. Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, USA

    John D. Hulleman

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  1. Steffi Daniel
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Supplemental Figure 1:

Confirmation of genotypes and F3 knockout. (a) Genotyping products for WT, F3+/-, and F3-/- animals. (b) Primer coverage for Sybr green and TaqMan probes. (c) Confirmation of F3 knockdown using Sybr green and Taqman probes. (PDF 690 kb)

Supplemental Figure 2:

Limbal stem cells are present and proliferating in the absence of F3. (a) Representative images of WT and F3-/- corneas stained for K15, Ki67, and DAPI. Both WT and F3-/- show evidence of limbal stem cell (K15 positive) as well as proliferating cells (Ki67 positive) in 8 mo mice. Dashed lines represent limbal limit (n=4) (scale bar = 100 μm). (PDF 2615 kb)

Supplemental Figure 3:

Additional histologic analyses and IOP measurements. (a) Histological comparison of ciliary body (CB) and TM between WT and F3-/- mice is unremarkable. (b) F3-/- mice exhibit lower IOP than WT mice, but these readings are likely an underestimation of the actual IOP measurements due to thinner corneas in F3-/- mice at 6 mo. (c) Representative images of immunostaining shows localization of F3 in the retina in WT as well as absence of F3 staining in F3-/- (scale bar = 100 μm). (PDF 2184 kb)

Supplemental Figure 4:

Absence of F3 does not lead to astrogliosis. (a) Representative images of immunostaining shows GFAP labeled astrocytes in WT and F3-/- retinas (scale bar = 100 μm). (b) Graph representing intensities (AU) shows no differences between WT and F3-/- in 8 mo mice (n=4). (c) Representative images of immunostaining shows Iba-1 labeled microglia in WT and F3-/- (inner and outer) retinas (scale bar = 100 μm). (d) Graph representing cell density (cells/mm2) shows no differences between WT and F3-/- in 8 mo mice (n=4). (PDF 1864 kb)

Supplemental Figure 5:

Normal nerve fiber, ganglion cell, and inner plexiform thickness in a patient with biallelic loss-of-function mutations in F3. (a, b) Normal combined thickness of the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL) as determined by OCT suggests no indications of inner retinal thinning or glaucoma. (PDF 1306 kb)

Supplemental Figure 6:

Normal structure and tissue thickness surrounding the optic nerve head in a patient with biallelic loss-of-function mutations in F3. (a, b) Normal retinal nerve fiber layer (RNFL) structure and thickness surrounding the optic nerve head suggests a healthy optic disk with no evidence of cupping or dysfunction indicative of glaucoma. (PDF 2137 kb)

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Daniel, S., Renwick, M., Chau, V.Q. et al. Fibulin-3 knockout mice demonstrate corneal dysfunction but maintain normal retinal integrity. J Mol Med 98, 1639–1656 (2020). https://doi.org/10.1007/s00109-020-01974-z

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