FIGURE 2.

In Cspg4-Cre line, tamoxifen treatment results in Cre-induction across a sparse population of contractile vascular cells. (a) In the oil injected mice, no “leaky” activation of CreERT2 was detected. Scale bar 100 μm. (b) Tamoxifen ip induced TdTomato expression in sparsely distributed vascular mural cells. Scale bar 100 μm. (c) High resolution image from the area highlighted in (b). When TdTomato brightness was strongly amplified, weak TdTomato signal was detected around soma in the ganglion cell layer. Scale bar 20 μm. (d) In the reporter mouse, weak TdTomato signal was found in the absence of Cre. Please note that the TdTomato signal was amplified in comparison with settings for (a–b). Scale bar 100 μm. (e–g) High magnification of an area from (d) taken at the inner limiting membrane, where red TdTomato signal was colocalized with Muller cell marker, glutamine synthetase (GS, white). Scale bar 20 μm. (h) Phenotypic characterization of sparely labeled by TdTomato mural cells along the vascular tree. Cells expressing SMA are marked. Asterisks mark fine processing along branching vessels. Scale bar 25 μm. (i–n) Structural assessment of the artery branching point. Scale bar 10 μm. (i) A projection through the sphincter cell. (j) The branching complex labeled for endothelial cells (CD31, white), smooth muscle actin (SMA, blue), sphincter (TdTomato, red), and basement membrane (Iso, green). (k) Sphincter cell (red ring) has very low SMA level in comparison with surrounding SMA-positive cells (blue labeling, arrowheads). (l) The branching point is strongly insulated by especially thick basement membrane (green). (m) Two endothelial cells are located at the base of the branch (CD31, white) with additional endothelial cells surrounding the entrance into the branch (pink arrow). All endothelial cells described here were also well insulated with the basement membrane (l).