Retinal vascular patterning in <i>Gpr116</i>^-/- mice.

<p>A. Vascular network in P4 retinas. Dashed line indicates the limits of the retina (the picture shown is representative of at least 5 mice for each genotype). B. Quantification of the retinal vascular outgrowth at P4 (n = 5 for WT, n = 12 for heterozygotes and n = 6 for knockout). C. Vascular patterning in P7 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. Isolectin (red), CD31 (green) and Erg (grey) were used to visualize endothelium, and NG2 (green) and ASMA (red) to detect mural cells (the images shown are representative of 3 mice for each genotype). D. Vascular patterning in P7 retinas from <i>Gpr116</i> ECKO and littermates controls. Isolectin (red) is used to visualize endothelium, and NG2 (green) and smooth muscle actin α (ASMA, blue) to detect mural cells (the images show are representative of 2 mice per genotype). E. Isolectin (red) and FITC-dextran (green) distribution in P21 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. CD31 (green) is used to stain the endothelium, and nuclei are stained with Hoechst (blue) (the images shown are representative of 3 mice per genotype). F. Monolayers formed by isolated endothelial cells from <i>Gpr116</i> WT, heterozygous and knockout brain. Endothelial cells (CD31) and nuclei (Hoechst) are indicated in green and blue, respectively (the pictures shown are representative of 3 mice for each genotype)</p

Vascular expression and genetic ablation of the <i>Gpr116</i> gene in mouse.

<p>A. <i>Gpr116</i> mRNA expression in the published organ-specific EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref045" target="_blank">45</a>]. B. <i>Gpr116</i> mRNA expression assessed by qRT-PCR in EC from 3-weeks-old and 3-months-old ROSA^mT/mG x Tie2-Cre mice. Results are normalized by brain EC expression. Error bars represent SD. (n = 3 mice per genotype). C. <i>Gpr116</i> mRNA expression in the published brain-specific vascular and EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref046" target="_blank">46</a>]. D. Schematic representation of the area targeted by homologous recombination in the <i>Gpr116</i> locus. Dotted lines indicate the regions of homology in between the Gpr116 locus and the cassette. The dark grey arrow indicates the position of WT primers: both are located in the untranslated region of exon 21, but the area recognized by the forward primer is lost in the mutant allele. The light grey arrow represents the knockout primer, specific for the cassette. Critical Gpr116 domains (SEA, IgG, GAIN and transmembraine, TM) are indicated above the corresponding encoding exons. E. Example of genotyping PCR products on genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. WT primers amplify a 325-bp fragment in the 3´UTR exon 21 of <i>Gpr116</i> gene representing the wild type allele. The 401 bp band is specific for the mutant allele. F. Example of genotyping PCR products using genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. LacZ primers amplify a 210 bp fragment in LacZ gene present in the insert replacing exon 4 to 21. G. <i>Gpr116</i> exon 17–18 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). H. <i>Gpr116</i> exon 2–3 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). I. mRNA detection by RNAscope in brain cortical capillary vessels from <i>Gpr116</i> WT (top row), knockout (middle row) and ROSA^mTmG X Tie2-Cre mice (lower row) at 3 weeks. On the left column, note that only the probe signal (red) and the nuclear staining (blue) are visible. On the right column, an endothelial staining (green) is merged to the probe and the nuclear signal: a CD31 antibody staining is on the two upper rows, while Tie-2 Cre mediated GFP is on the lower row. (n = 1 mouse per genotype).</p

Normalized pathological angiogenesis in <i>Gpr116</i>^-/- retinas.

<p>A. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (the images shown are representative of 5 mice per genotype). B. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype). C. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (n = 5 mice at least per genotype). D. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (n≥7 mice at least per genotype). E. Confocal images of post-OIR tufts (blue arrows) in <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype)</p

Blood brain barrier breakdown in <i>Gpr116</i>^-/- mice.

<p>A. Whole brain images taken after 1kDa cadaverine perfusion (left) and associated quantification of extravasated cadaverine (right) in aged <i>Gpr116</i> WT, heterozygous and knockout mice (n≥5 mice for each genotype). B. Whole brain images taken 70 kDa tetramethylrhodamine dextran perfusion (left) and quantification of extravasated tracer (right) in <i>Gpr116</i> WT and heterozygous and <i>Gpr116</i> ECKO mice (n = 3 for wild type and ECKO, n = 2 for <i>PDGF-B</i>^{<i>ret/ret</i>}, n = 1 for uninjected control). C. Confocal images of cerebral cortex from aged <i>Gpr116</i> WT, heterozygous and knockout mice. Astrocytes (GFAP) appear in green, endothelial cells (CD31) in red (the images are representative of 4 mice per genotype) and associated quantification of perivascular associated astrocytes in aged <i>Gpr116</i> WT, heterozygous and knockout mice (n = 4 mice for each genotype, 2 sections at least quantified per genotype). D. Whole brain fluorescence images taken after Alexa 555-cadaverine circulation (upper) and quantification of extravasated cadaverine (lower) in 1.5-month-old <i>Gpr116</i> knockout (n = 3 mice per genotype). E. Whole brain fluorescent images taken after cadaverine circulation (upper) and associated quantification of extravasated cadaverine (lower) in 2-months-old <i>Gpr116</i> AEC KO (n = 6 mice per genotype). F. Whole brain fluorescent images taken after cadaverine circulation (upper) and quantification of extravasated cadaverine (lower) in 2-months-old <i>Gpr116</i> ECKO (n = 7 mice per genotype)</p

Massive accumulation phenotype in lungs of aged <i>Gpr116</i>^-/- mice.

<p>A. Bright field image of the inflated lung from <i>Gpr116</i> WT, heterozygous and knockout littermates. B. Weights of whole lungs over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). C. Bright field images of heart from <i>Gpr116</i> WT, heterozygous and knockout littermates. D. Weights of the heart (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). E. Bright field images of the spleen from <i>Gpr116</i> WT, heterozygous and knockout littermates. F. Weights of the spleen (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). G. BALF collected from <i>Gpr116</i> WT, heterozygous and knockout littermates (The picture shown is representative of 3 mice for each genotype). H. Quantification of saturated phosphatydilcholine in BALF by ELISA (n = 3 mice per genotype). I. Quantification of protein content in BALF by BCA assay (n = 3 mice per genotype). J. Surfactant proteins detection in BALF by western blot. Molecular weights are indicated on the right. (n = 2 mice per genotype). K. Bright field images of the lung, after hematoxylin and eosin staining. The black arrowheads indicate alveolar macrophages (the image is representative of 4 mice for each genotype). L. Electron microscopy view of <i>Gpr116</i> wildtype and knockout lungs (n = 2 mice for each genotype). M. Confocal images of lung sections stained with ADRP (white) and nuclear stain (Hoechst, blue). Note that a red autofluorescent signal appears in knockout lungs. (the image shown is representative of 2 mice for each genotype). N. Confocal images of lung sections stained with nuclear marker Hoechst (blue) to show autofluorescent cells accumulated in the alveolar space, either in the green or red channel (the image is representative of 3 mice for each genotype). O. Autofluorescence emission spectrum of macrophages in the old knockout lung, upon 405 nm excitation (the image is representative of 2 mice). P. Detection of autofluorescent cells from <i>Gpr116</i> knockout lung by FACS (n = 2 mice per genotype)</p