Retinal expression of Wnt-pathway mediated genes in low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice

Abstract

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5(-/-)) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5(-/-) mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5(-/-) and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5(-/-) retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5(-/-) retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5(-/-) retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

Figure 1. Delayed development of the superficial retinal vasculature and persistent hyaloid vessels in Lrp5 null mice.

(A) Left: retinal whole mounts stained with isolectin B₄-594 from WT and Lrp5 null mice at post-natal day (P) 8. Right: quantification of vascularized retinal area in WT and Lrp5 null mice at P8. n = 5–10 per group, ***p<0.001. (B) Retinal cross sections of WT mice and Lrp5 null mice at P8 stained for endothelial cells with isolectin B₄-594 (red) and cell nuclei (DAPI, blue). Arrows indicate persistent hyaloid vessels in Lrp5 null retina. Scale bars: 500 µm.

Figure 2. Abnormal vascular development in the inner and outer retina and forebrain of Lrp5 null mice.

(A) Retinal whole mounts of WT and Lrp5 null mice stained for endothelial cells with isolectin B₄-594 at P12 and 17. Enlarged images highlight the abnormal vessel growth in the Lrp5 null retina. (B) Retinal cross sections from P30 WT and Lrp5 null mice stained for endothelial cells with isolectin B₄-594 (red) and cell nuclei with DAPI (blue). Arrows indicate deep layers of retinal vasculature which is present in WT retina but absent in Lrp5 null retina. (C) Cross sections of the forebrain from P8 WT and Lrp5 null mice with endothelial cells stained with isolectin B₄-594. Scale bar: 100 µm.

Figure 3. Expression levels of Lrp5, Norrin, Frizzled4 and Dvl mRNA during retinal development in wild type mice.

Quantification of mRNA (A) Lrp5, (B) Norrin, (C) Frizzled4, (D) Dvl1, (E) Dvl2, and (F) Dlv3 was performed with RT-qPCR during normal retinal development from P1-P17. Expression levels were normalized against house keeping gene Cyclophilin A. Trend lines were fitted with polynomial, linear or power regression.

Figure 4. Regulation of tight junction, membrane transport, angiogenic, and cell adhesion genes in the Lrp5 null retina.

Heat maps illustrate the results of a gene array run from whole retinal total mRNA. The most regulated families of genes were (A) claudin family genes, (B) membrane transport genes, (C) angiogenic regulatory genes, and (D) cell adhesion/cell-cell junction genes. Each sample is represented by a block: either wild-type (WT) samples 1 through 3, and Lrp5 null samples 1–3. Relative down-regulation of expression in Lrp5 null retina compared to WT retina is represented by green, while relative up-regulation is in red. No relative regulation is black (See scale on Figure).

Figure 5. Confirmation of gene expression differentially regulated in Lrp5 null retina with RT-qPCR.

Quantification of mRNA (A) Cln5 and Slc38a5, (B) Gja1, Msfd2, Sox18, and vWF, and (C) Plvap and EMP1 in WT and Lrp5 null retina with RT-qPCR at P8. Expression levels were normalized against housekeeping gene Cyclophilin A. *p<0.05, **p<0.01.

Figure 6. Wnt ligands and receptor regulated in P8 Lrp5 null retina.

Wnt ligand Norrin, Wnt5a, Wnt7b, and Wnt10b mRNA expression in WT and Lrp5^−/− whole retinas were quantified with RT-qPCR and normalized to Cyclophilin A expression. *p<0.05, **p<0.01.