Developmental platelet endothelial cell adhesion molecule expression suggests multiple roles for a vascular adhesion molecule

Abstract

Platelet endothelial cell adhesion molecule (PECAM) is used extensively as a murine vascular marker. PECAM interactions have been implicated in both vasculogenesis and angiogenesis. To better understand the role of PECAM in mammalian development, PECAM expression was investigated during differentiation of murine embryonic stem (ES) cells and in early mouse embryos. Undifferentiated ES cells express PECAM, and as in vitro differentiation proceeds previously unidentified PECAM-positive cells that are distinct from vascular endothelial cells appear. PECAM expression is gradually restricted to endothelial cells and some hematopoietic cells of differentiated blood islands. In embryos, the preimplantation blastocyst contains PECAM-positive cells. PECAM expression is next documented in the postimplantation embryonic yolk sac, where clumps of mesodermal cells express PECAM before the development of mature blood islands. The patterns of PECAM expression suggest that undifferentiated cells, a prevascular cell type, and vascular endothelial cells express this marker during murine development. PECAM expression in blastocysts and by ES cells suggests that PECAM may function outside the vascular/hematopoietic lineage.

Figure 1.

Immunofluorescence of ES cell clumps with an anti-PECAM antibody. ES cells were incubated with the anti-PECAM antibody Mec 13.3 (A and C) or an isotype-matched anti-IgE FcR control antibody (B and D), followed by detection with a B-phycoerythrin-conjugated secondary antibody. Fluorescent (A, B) and phase-contrast (C, D) images are shown. Scale bar, 30 μm.

Figure 2.

Immunofluorescence of dissociated ES cells. ES cell cultures were treated with collagenase and then incubated with the following antibodies or sets of antibodies: (A, D), anti-PECAM antiserum and mouse IgM control; (B, E), mouse anti-SSEA-1, (C, F), anti-PECAM antiserum and mouse anti-SSEA-1. Secondary antibodies were goat anti-rabbit FITC (green) and donkey anti-mouse B-phycoerythrin (red). A–C: FITC fluorescence; D–F: B-phycoerythrin fluorescence. Scale bar, 60 μm.

Figure 3.

PECAM RNA analysis of ES cell differentiation. Total RNA was reverse transcribed using random primers and then amplified using either PECAM (A) or β-actin (B) primers. Lane 1, ES cells; lanes 2–8, days 1–7 of differentiation; lane 9, 3T3 fibroblasts. The predicted sizes of the PECAM and β-actin products are 215 and 147 bp, respectively. The positions of molecular weight standards are indicated to the right of each gel.

Figure 4.

PECAM immunofluorescence of ES cell differentiation. Dispase-treated ES cells were plated onto tissue culture dishes on day 0. Representative plates were fixed every other day. Wells were stained with an anti-PECAM antibody and a B-phycoerythrin-conjugated secondary antibody. A, D: day 2; B, E: day 4; C, F: day 6; G, J: day 8; H, K: day 10; I, J: day 12. A–C, G–I: fluorescence photomicrograph; D–F, J–L: the corresponding phase contrast photomicrograph. Arrowheads in H point to PECAM strain at cell borders. Scale bar, 30 μm.

Figure 5.

Double immunofluorescence for PECAM and CD34 in ES cells and during ES cell differentiation. A, F: undifferentiated ES cells, fixed 3 days after passage. B–E, G–J: Dispase-treated ES cells were cultured in Petri dishes for 3 days, then plated onto tissue culture dishes. Representative plates were fixed every day, beginning with day 5. Wells were stained with biotin-conjugated Mec 13.3 (A–C, E) and an anti-CD34 rat monoclonal antibody (F–I). Control plates had either the anti-PECAM primary antibody (D) or the anti-CD34 antibody primary antibody (J) omitted. Detection was with streptavidin-conjugated B-phycoerythrin and donkey anti-rat FITC. A, F, undifferentiated ES cells; B, G, day 6; C–E, H–J, day 10. A–E, PECAM-specific B-phycoerythrin fluorescence; F–J, CD34-specific FITC fluorescence of the same wells. In B and G, the arrow shows cells that are CD34-negative and PECAM-positive and the arrowhead indicates cells that are double positive.

Figure 6.

A time course of ES colony recovery and PECAM immunostaining. ES cells were treated with Dispase and cultured in differentiation medium. Each day 2 sets of dishes were either fixed and stained for PECAM or treated with collagenase and replated for ES colony formation. A: ES colonies were counted 4 days after plating either 200,000 or 500,000 cells treated with collagenase and their number calculated as a percent of maximum colony number. B: Parallel cultures were visually examined and the relative amount of PECAM staining in three patterns was assessed: balls, tight clumps of cells with a dense staining pattern, similar to Figure 1A ▶ (ES-like); clumps, small to medium aggregations of cells with distinct cell border staining, similar to Figure 4, A–C ▶ ; vessels, cells arranged with continuous staining at cell-cell borders, similar to Figure 4, G–I ▶ . The area covered by each pattern of staining was estimated as follows: ±, trace; +, 2–5%; ++, 5–10%; +++, 10–30%; ++++, >30%.

Figure 7.

PECAM RNA analysis of mouse blastocysts. Poly A⁺ RNA (blastocysts) or total RNA (cell lines) was reverse transcribed using random primers, then amplified using either β-actin (lanes 1–3) or PECAM (lanes 4–6) primers. Lanes 1, 4: blastocysts; lanes 2, 5: Py-4–1 endothelial cells; lanes 3, 6 : 3T3 fibroblasts. The predicted sizes of the PECAM and β-actin products are 215 and 147 bp, respectively. The positions of molecular weight standards are indicated in the right margin.

Figure 8.

PECAM immunofluorescence of mouse blastocysts. Fixed blastocysts were stained with a control antibody directed against IgE FcR (A, D) or two different anti-PECAM antibodies, Mec13.3 (B, E) or EA-3 (C, F), followed by detection with a goat anti-rat B-phycoerythrin secondary antibody. A–C: fluorescent photomicrographs; D–F: the corresponding phase-contrast micrographs. Scale bar, 25 μm.

Figure 9.

Whole-mount PECAM antibody staining of postimplantation embryos. E 7.5 embryos (mid-streak to early head fold stage) were subjected to whole-mount antibody staining with Mec 13.3 rat anti-mouse PECAM antibody (A). Embryos were then embedded in paraffin and sectioned (B–D). B: cross-section through the yolk sac and allantois of a mid-streak embryo. PECAM staining is seen at the cell borders of a clump of mesodermal cells that, by position, will form a blood island. Additional PECAM reactivity is seen in a small clump closer to the embryonic/extraembryonic border (arrowhead) and in a subset of cells within the allantois (arrows) that are likely to be endothelial precursors. C: another clump of PECAM⁺ yolk sac mesodermal cells from the same embryo. D: a clump of mesodermal yolk sac cells from a slightly older embryo, with a mixture of PECAM⁺ and PECAM-negative cells (arrow).