Figure 1.

Lymphatic Endothelial Cells Elongate, Reorient by 90°, and Migrate Collectively to Vessel Lumen to Initiate Valve Leaflet Formation

(A and B) Whole-mount immunofluorescence of E16.5 (A) and E17.5 (B) wild-type mesenteric vessels for Prox1 (green) and PECAM-1 (red). Note the elongation of Prox1^high cells at E16.5 (arrowheads in A) and their reorientation by 90° at E17.5 (arrowheads in B).

(C–F′) Labeling of individual lymphatic endothelial cells with a membrane targeted GFP in Prox1-CreER^T2;R26-mTmG mesentery. At all stages analyzed (E16.5–E17.5), Prox1^high valve forming cells show elongated shape (arrowheads) compared to cells on the vessel wall (arrows). Note polarized membrane protrusions in reorienting cells (open arrowhead in F and F′).

(G and H) Visualization of a “ring-shaped” valve in E17.5 mesenteric lymphatic vessel of Prox1-CreER^T2;R26-mTmG reporter mouse (G). The boxed area shows a valve that was analyzed by serial sectioning for light microscopy and 3D reconstruction (H, shown at two different angles). Arrow in (H) shows the direction of flow. Blue color highlights valve endothelial cells forming a disc and gray represents the vessel wall.

(I and J) Semi-thin section stained with 1% toluidine blue showing a cross section of a valve “disc” in E17.5 mesentery. Boxed area in (I) is magnified in (J). Endothelial cells are present in multiple layers (arrowheads in J).

(K–M′) Transmission electron microscopy of developing (E17.5; K, L, and L′) and mature (P6; M and M ′) valves in mesenteric lymphatic vessels. Boxed area in (K) is magnified in (L), and the areas in (L) and (M) are magnified in (L′) and (M′), respectively. Note discontinuous cell-cell junctions (arrowheads in L and L′) and large intercellular gaps (asterisks in L and L′) at E17.5, compared to continuous overlapping cell-cell junctions in mature valves (arrowhead in M and M′). Extracellular matrix core of the valve leaflet is highlighted in red in (M) and (M′).

Scale bars represent 40 μm (A–F), 100 μm (G and H), 10 μm (I), 5 μm (J and K), and 1 μm (L–M′).

See also Figure S1 and Movie S1.