Figure 1.
Immunofluorescence and FACS analysis reveals cells that co-express stromal and endothelial markers.
A–A’’. Wholemount immunofluorescence reveals that PECAM positive vessels (A, red) are present within the Foxd1 positive renal stroma (A’, green) on the merged image (A’’). B. IF in the cortex of an E18.5 kidney shows that developing endothelial cells stained with PECAM (blue) are present in the stroma stained with Foxd1 (green) but not in nephron precursors stained with Six2 (red). B’–B’’’. Higher power views from “B” reveal expression of the stromal marker Foxd1 in the nucleus (green, arrowhead) and PECAM in the cytoplasm (blue) of the same cell. C–C’’. High power image of IF at E18.5 shows co-expression of the stromal marker Foxd1 (green) and the endothelial marker Erg (red) in nuclei of some renal cells (arrowhead). D. Flow sorted kidneys from E18.5 Foxd1EGFPcre mouse shows co-expression of GFP (Foxd1-positive stromal marker, X axis) and Flk1 (Y axis) in a subset of cells (P8). Scale bars A = 50 µm, B and C = 10 µm.
Figure 2.
Renal stromal cells can differentiate into cells with endothelial like properties.
A–E. Tubulogenesis assay: A. Foxd1EGFPcre positive sorted cells appear rounded when cultured at day zero (arrowhead). B. HUVECs (positive controls) develop the endothelial-like tubules after two days in culture. C. After 7 days of growth, the Flk1-positive populations remain rounded demonstrating that they had lost the ability to form endothelial-like tubules. D–E: After 7 days of growth, Foxd1-positive cells readily formed endothelial like tubes (D) and expressed PECAM by IF (E). F. Acetylated LDL assay: Under hypoxic conditions, cultured FACsorted Foxd1-positive renal stroma cells endocytosed Ac-LDL (green), which is a function specific to endothelial cells (blue = DAPI). Scale bars A–E = 50 µm, F = 5 µm.
Figure 3.
Stromal derivatives form endothelium that is present in peritubular capillaries.
A. IF in E18.5 Foxd1creCAG mouse section reveals RFP stromal derivatives (red) that co-label with Erg (green) in a peritubular capillary (arrowhead); in the glomerulus (G), the stromal-derived cells form the mesangium (red) but do not express Erg (green). B. Flow sorted kidneys from E18.5 Foxd1creCAG reporter mice show co-expression of RFP (Foxd1-stromal derivative, X axis) and Flk1 (Y axis) in a subset of cells (P5). C-C’’. IF in a P30 Foxd1creCAG kidney section reveals a peripheral peritubular capillary with co-expression of RFP stromal derivatives (red) and PECAM (green) (arrowhead). D. IF from a P30 Foxd1creCAG kidney section shows a glomerulus (G) and major vessel (V), that express RFP stromal derivatives in the mesangium and vascular smooth muscle (red), respectively, but that do not co-express RFP and PECAM (green) in endothelium in those tissues. E. Flow sorted kidneys from P30 Foxd1creCAG reporter mice show that many RFP stromal derived cells (X axis) co-express PECAM (Y axis) as shown in P5. Scale bars A = 30 µm, C = 20 µm, D = 50 µm.
Figure 4.
The majority of embryonic stromally derived endothelium does not express pericyte markers.
Embryonic tissues FACS sorted using a back-gating strategy. This involved identifying the Foxd1creCAG/Endothelial cells (box surrounding cells in B, C and D) and gating these against the pericyte specific markers CD73 and CD44 (B’, C’ and D’). A. Representative single color control for RFP-positive cells, the red dots represent the RFP positive cells. B. Representative FACS plot for control cells that are negative for CD73 and CD44, the red dots represent control cells that are negative for pericyte markers. C. E13.5 Foxd1creCAG kidney cells stained with PECAM showing that 0.2% of the cells are Foxd1 derived endothelium (box). C’. Cells from “C” were then stained for pericyte markers (CD73 and CD44) and back gated against the pericyte markers, 75.3% of the cells that are endothelial-positive were pericyte negative. D. E15.5 kidney sample showing that 0.2% of the cells are Foxd1 derived endothelium (box). D’. Cells from “D” were then back gated against pericyte markers (CD73 and CD44), 84.0% of the cells that were endothelial positive were pericyte negative. E. E15.5 lung sample showing that 0.4% of the cells are Foxd1 derived endothelium (box). E’. Cells from “E” were then back gated against pericyte markers (CD73 and CD44), 83.4% of the cells that are endothelial positive are pericyte negative.
Figure 5.
The adult lung contains Foxd1-derived endothelium.
Representative FACS plot of adult lung showing a population of stromally derived endothelium (box, P5).
Figure 6.
Foxd1-positive derivatives express markers of endothelium in the lung at E15.5.
A. FACS sorting of a Foxd1creCAG E15.5 lung shows co-expression of RFP (Foxd1-expressing cells- X axis) and Flk1 (Y axis) in a number of cells (panel S2). B. IF of an E15.5 Foxd1creCAG lung reveals co-expression of RFP-positive Foxd1 derivatives (red) and the endothelial marker Flk1 (green) (box). B’–B’’’. High power images of panel B (box) confirms co-expression of RFP positive Foxd1 derivatives (red, nucleus) and Flk1 (green, cell membrane) in some pulmonary cells C. IF of Foxd1creCAG E15.5 lung reveals co-expression of RFP-positive Foxd1 derivative (red) and nuclear endothelial marker Erg (green) (box). C’–C’’’. High power images of panel C (box) shows co-expression of the RFP-positive Foxd1 derivatives (red) and Erg (green) in nuclei of some pulmonary cells (yellow). Scale bar = 50 µm.
Figure 7.
Foxd1 positive lung cells differentiate into endothelial-like tubules and take up Ac-LDL.
A–C. Tubulogenesis assay: A. Foxd1EGFPcre/GFP positive sorted cells are rounded when cultured at day zero. B. After 7 days of growth, the PECAM positive populations remain rounded having lost the ability to form endothelial-like tubules. C. After 7 days of growth, Foxd1EGFPcre/GFP positive cells readily formed endothelial-like tubular networks. D. Acetylated LDL assay: Under hypoxic conditions, Foxd1EGFPcre/GFP cells have endocytosed Ac-LDL (green) (blue = DAPI). Scale bars A–C = 50 µm, D = 5 µm.