Abstract
Purpose :
The Wnt/β-catenin pathway controls postnatal bone development and prenatal development of retinal blood vessels. In humans, loss of function mutations of the Wnt co-receptor, Lrp5, cause reduced bone mineral density and familial exudative vitreoretinopathy (FEVR). Although mouse Lrp5 knockout models exist, rats are preferred for orthopedic studies because their size makes them more amenable to modeling bone repair and surgical treatments. We developed an Lrp5 knockout rat model using CRISPR/Cas9 technology to investigate the effects of loss of function on bone mass and determine whether a FEVR-like phenotype is present.
Methods :
CRISPR/Cas9 was used to inactivate the Lrp5 gene in Sprague-Dawley rats. Femurs from 6 month homozygous knockout (KO) and wildtype (WT) littermate animals were collected and bone parameters were assessed using micro-CT analysis. Retinas of P8 and 3 month rats were fixed in PFA and the retinal vasculature was stained with Alexa Fluor 594-conjugated isolectin B4. Images of the retinal vasculature were analyzed using AngioTool software.
Results :
Guides targeting exon 2 of Lrp5 generated an 18 bp deletion causing early termination of the Lrp5 gene resulting in reduced bone mineral density, thinner bones and low bone quality due to decreased trabecular bone. Development of the retinal vasculature was delayed at P8, with truncated vessels ending in glomeruloid tufts compared to WT rats. Three month KO rats had sparse retinal vasculature with extensive yellow-green fluorescent exudates and significant reduction in vessel branch points: 42.3/mm2 compared to 86.3/mm2 in WT animals. WT vessels covered 37.7% of the retinal area compared to 25.4% in KO. Median vessel length was 1.75 mm in WT rats and 0.63 mm in KO rats. Heterozygous rats had normal retinal vessels. Rats with a homozygous 1 bp deletion in the Lrp5 gene had reduced bone mass and disorganized, tortuous retinal vessels with abundant exudates.
Conclusions :
A novel Lrp5 knockout rat model was produced in which bone development is more similar to humans than mice. The animals have reduced bone mass and their retinas express a FEVR-like phenotype, suggesting that this model will be useful for studying Lrp5 mutations in humans who exhibit both skeletal and retinal vascular abnormalities.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.