Fig 1.
Loss of Lrp5 causes retinal hypovascularization and neovascularization.
(A) ColIV whole mount IF staining showing retinal vessels of Lrp5-/- and control mice at P9 and P30. Quantification of vascular sprout numbers at P5 shown at right. (B) Adult Lrp5-/- retinas showing persistent hyaloid vessels (black arrows, CD31 IHC staining), aneurysms (open arrow, CD31 IHC staining), neovascular overgrowth (white arrows, fibronectin IF staining) and lack of IPL and OPL vascular development (lower panels, green: FITC-Dextran perfusion). (C) FITC-Dextran perfusion (green) showing retinal vascular leakage (white arrows) in adult Lrp5-/- mice compared to control. Scale bars = 100μm. (D) EM analysis of endothelium of Lrp5-/- and control retinas at P5, P8 and P30. Arrows point to area of endothelial fenestration. Scale bars = 500nm. (E) Total amounts of VEGF protein in retinas of Lrp5-/- and control mice at P5, P8 and P30. Each ELISA was done in duplicate and normalized to total retinal protein amount. n = 9, 5, 12 for controls (at P5, P8, P30) and 9, 8, 8 for Lrp5-/- (at P5, P8, P30). * p<0.05, ** p<0.01. Data are represented as means ± SD. Ctrl, control.
Fig 2.
Conditional knockout of Lrp5 with Tie2-Cre but not VE-Cad-Cre recapitulates retinal vascular defects in Lrp5-/- mice.
(A and B) ColIV IF staining (red) and FITC-Dextran perfusion (green) showing adult retinal vasculature in VE-Cad-Cre;Lrp5fl/- and Tie2-Cre;Lrp5fl/fl CKO mice (4w) compared to Lrp5fl/fl control. Arrows: hyaloid vessels; open arrows: neovascular tufts; arrowheads: neovascular overgrowth. At right: Quantification of vascular branch points in OPL at 4w; ns not significant. (C) TdTomato signals in VE-Cad-Cre;tdTomato (8w) and Tie2-Cre;tdTomato (7w) mice showing predominant endothelial expression of VE-Cad-Cre and Tie2-Cre in the NFL, IPL and OPL of the retina. Note that VE-Cad-Cre is also widely expressed in myeloid cells around the vessels in the NFL (upper left panel, also see D). Arrows point to IB4 (green) positive vascular area with negative VE-Cad-Cre;tdTomato signals in a P5 mouse indicating incomplete VE-Cad-Cre recombination. (D) Myeloid and microglial localization of VE-Cad-Cre;tdTomato and Tie2-Cre;tdTomato signals in P5 (left and mid-left panels) and adult (right and mid-right panels) retinas. Arrows point to myeloid cells and arrowheads point to microglial cell. Green: F4/80 IF staining. Scale bars = 100nm.
Fig 3.
Retinal vascular phenotype in mice with conditional knockout of Lrp5 in myeloid/microglial cells.
(A) Whole mount IF staining with IB4 (blue) or ColIV antibody (right panel, green) showing distribution of LysM-Cre;tdTomato+ (red) myeloid and microglial cells in retinas at P5, P8 and 6w. Most LysM-Cre;tdTomato+ myeloid cells were also F4/80+ (green) (white rectangle in left panel indicates area shown at higher magnification in the upper right corner inset). (B) Localization of LysM-Cre;tdTomato+ myeloid cells in three adult (6w) retinal vascular beds. Green: ColIV. (C) Retinal vasculature of LysM-Cre;Lrp5fl/- CKO mice (12w). Red: IB4. (D) Distribution of CD11b-Cre;tdTomato+ (red) myeloid and microglial cells in retinas at P6 (left and mid panels, green: IB4) and 4w (right panel, cross section). Blue: Hoechst. (E) Localization of CD11b-Cre;tdTomato+ signals (red) in myeloid, microglial, Müller glial and perivascular macrophage cells in adult (4w) retina. Blue: IB4. White rectangle in right panel outlines cell shown at higher magnification in upper right corner inset. (F) Retinal vasculature of CD11b-Cre;Lrp5fl/- CKO mice (4w). Green: FITC-Dextran perfusion. Scale bars = 100nm.
Fig 4.
Flk1-CreBreier is specifically expressed in endothelial cells and conditional knockout of Lrp5 with Flk1-CreBreier recapitulates retinal vascular defects in Lrp5-/- mice.
(A) Specific endothelial location of Flk1-CreBreier;tdTomato signals in developing retinas (P5, upper panels) overlapping with IB4 IF signals (blue). F4/80 IF staining (green) showing that most macrophages were tdTomato negative. In adult retina, Flk1-CreBreier was specifically expressed in all three layers of the retinal vascular beds (lower panels) with no signs of any myeloid cell expression. Penetrance of Flk1-CreBreier expression in ECs could also be incomplete as shown in (D). (B) Specific endothelial location of Flk1-CreBreier;tdTomato signals in adult (4w) bone marrow, cortical bone and skeletal muscle. M: muscle; CB: cortical bone; BM: bone marrow. Blue: Hoechst. (C) Whole mount IF staining of ColIV (red) and IB4 (green) showing retinal vasculature in adult (4w) Flk1-CreBreier;Lrp5fl/- CKO mice with disorganized NFL vessels, vertical vessel branches terminating in ball-like structures in the IPL and lack of OPL vascular bed. Arrow points to persistent hyaloid vessels. Note that regions with vascular abnormalities often had patchy normal-looking areas located in the neighborhood. In the selected OPL image, an area with well-developed vessels is next to another with no vascular development. (D) Whole mount IF staining of IB4 (green) showing that normally developed vascular areas in Flk1-Cre;Lrp5fl/-;tdTomato retinas (8w) included many Flk1-Cre;tdTomato negative vessels (arrows), whereas abnormal vessel structures were all tdTomato positive (open arrows). Scale bars = 100μm.
Fig 5.
Conditionally restoring Lrp5 in endothelial but not myeloid cells rescues retinal vascular defects in Lrp5-/- mice.
(A) FITC-Dextran perfusion showing distorted NFL and IPL vessels and lack of OPL vascular development in Lrp5a214v(n)/- hypomorph mice (lower panels) compared to controls (upper panels) both in retinal whole mount (4w) and cross section (2m) images. (B) VE-Cad-Cre;Lrp5a214v(n)/+ (upper left panels, FITC-Dextran perfusion), VE-Cad-Cre;Lrp5a214v(n)/- (lower left panels, FITC-Dextran perfusion) and Flk1-Cre;Lrp5a214v(n)/- (upper right panels, red: ColIV, green: IB4) mice all developed a normalized three-tier retinal vascular structure, while LysM-Cre;Lrp5a214v(n)/- (lower right panels, red: ColIV) mice displayed similar retinal vascular abnormalities compared to control Lrp5a214v(n)/- mice (A, lower panels). Quantification of vascular branch points in OPL is shown in graph at right; **P<0.01, ns not significant. All mice are between 4 to 5 weeks of age except otherwise labeled. Scale bars = 100nm.
Fig 6.
Endothelium-derived Lrp6 is dispensable for retinal vascular development.
Whole mount IF staining of ColIV (red) and FITC-Dextran perfusion showing (A) Retinal vasculature of Tie2-Cre;Lrp5fl/+;Lrp6fl/+ CKO mice. (B) Retinal vasculature of Tie2-Cre;Lrp5fl/+;Lrp6fl/fl CKO mice. (C) Retinal vasculature of Tie2-Cre;Lrp5fl/fl CKO mice. (D) Retinal vasculature of Tie2-Cre;Lrp5fl/fl;Lrp6fl/+ CKO mice. All mice are 8 weeks of age. Scale bars = 100nm.