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. 2022 Dec 1;11:e73105. doi: 10.7554/eLife.73105

Figure 1. Scl38a5 expression is enriched in retinal blood vessels and down-regulated in the retinas and retinal blood vessels of Wnt signaling deficient Lrp5−/− and Ndpy/− mice.

(A–B) mRNA levels of Slc38a5 were measured by RT-PCR in Lrp5−/− (A) and Ndpy/− (B) retinas compared with their respective wild type (WT) controls during development at postnatal day 5 (P5), P8, P12, and P17. (C) Retinal blood vessels stained with isolectin B4 (red, outlined by the white dashed lines) were isolated by laser-captured microdissection (LCM) from retinal cross-sections. Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, blue) for illustration purpose only. LCM retinal samples were stained with only isolectin B4 without DAPI. Slc38a5 mRNA levels in LCM isolated retinal blood vessels were compared with the whole retinal levels using RT-qPCR. Scale bars: 50 µm. (D) Immunohistochemistry of retinal cross sections from WT eyes shows colocalization of SLC38A5 antibody (green) in retinal blood vessels stained with isolectin (red), counterstained with nuclear staining DAPI (blue). (E) Slc38a5 mRNA levels in LCM-isolated Lrp5−/− and Ndpy/− retinal blood vessels were quantified with RT-qPCR and compared with their respective WT controls. (F–G) Protein levels of SLC38A5 (52 kDa) in P17 Lrp5−/− and Ndpy/− retinas and their WT controls were quantified with Western blot (F) and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 37 kDa) levels (G). RGC: retinal ganglion cells, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer. Data are expressed as mean ± SEM. n=3–4 per group. *p≤0.05, **p≤0.01, ****p≤0.0001.

Figure 1—source data 1. Raw data for Figure 1.

Figure 1.

Figure 1—figure supplement 1. Distinct expression of Slc38a5 in vascular endothelium in mouse and human retina with single-cell transcriptomics.

Figure 1—figure supplement 1.

(A) Dot plot of Slc38a5 and endothelial cell marker Pecam1 gene expression (scaled) for different retinal cell types in P14 C57BL/6 J mouse retinas. Slc38a5 was distinctly expressed in vascular endothelium cluster. Data source: Study - P14 C57BL/6 J mouse retinas (https://singlecell.broadinstitute.org/single_cell/study/SCP301) (Macosko et al., 2015). (B). Dot plot of SLC38A5 and endothelial cell marker PECAM1 gene expression (scaled) for different retinal cell types at human fovea and peripheral retina. Slc38a5 was highly expressed in endothelium cluster. Data source: Study - Cell atlas of the human fovea and peripheral retina (https://singlecell.broadinstitute.org/single_cell/study/SCP839) (Yan et al., 2020). Scaling is relative to each gene’s expression across all cells of a cluster. Gene expression is scaled from 0 to 1 (0.5=the mean across all cells in the cluster file referenced for dot plotting). % expressing is the percent of cells that have one or more transcripts for the gene of interest.