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First published online November 10, 2005
doi: 10.1242/10.1242/dev.02141

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The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature

¹ Stahlman Cardiovascular Laboratories, Department of Medicine and Program for Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
² MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
³ Department of Cell and Developmental Biology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA

View larger version (95K): [in a new window]   Fig. 1. Wt1 and cytokeratins as markers for mesothelial cells in the heart and gut. (A,B) Expression of Wt1 and cytokeratin mark epicardial mesothelium. (A) Merged image of Wt1 (green) and cytokeratin (pCK, red) colocalization in mesothelial cells (arrows) over the heart (h) and the adjacent liver (l) at E11.5. Boxed area in A is shown in B-B'' to delineate colocalization. (C-G) Wt1 and cytokeratin also mark serosal mesothelium. (C) Expression of Wt1 is first observed in the urogenital ridge at E9.5 (arrowheads) but is absent from the mesentery (arrows) and gut tube. (D) At E10.5, Wt1 staining extends over the mesentery (arrows) but does not cover the entire gut. (E) Cytokeratin staining is absent from the gut surface, but residual expression is seen in splanchnic mesoderm of the mesentery (arrows). Note cytokeratin staining in somatic mesoderm (asterisk) of the body wall. (F,G) At E11.5, Wt1 (F, arrows) and cytokeratin (G, arrows) staining is evident on the entire surface of the mesentery and gut tube. Note that only cytokeratin is expressed in the endodermal linings of the gut (e). lua, left umbilical artery; m, mesentery; nd, nephric duct; rua, right umbilical artery; sm, somatic lateral mesoderm. Scale bars: 20 µm in B-B''; and 100 µm for A,C-G.

View larger version (118K): [in a new window]   Fig. 2. Blood vessel formation in mesentery and gut. (A) Pecam is expressed in the endodermally associated plexus in the gut tube and mesentery (A, arrowheads) at E11.5. No surface staining is observed. (B) At E13.5, Pecam-positive cells are seen on the surface of the gut (arrows), connecting to the plexus via endothelial bridges (arrowhead). (C) At this time, no vascular smooth muscle cells are detected on the gut surface by anti-SMA staining; however, visceral muscle (vism) is detected. (D,E) In later stages, smooth muscle is associated with developing endothelial tubes in the mesentery and gut tube. e, endoderm; lua, left umbilical artery. Scale bars: 100 µm in A; 50 µm in B-E.

View larger version (64K): [in a new window]   Fig. 3. Molecular genetic analysis of serosal mesothelial development. (A-D) Whole-mount analysis of lacZ expression marks the advance of the serosal mesothelium. (A) At E11.5, lacZ staining is prominent at the root of mesentery and intestine (small arrows), but is absent from the herniated gut (large arrows). Note the staining in nephric tubules (black arrowheads) and the urogenital ridge (white arrowhead). (B) At E12.5, lacZ-positive cells are seen covering more distal portions of the gut (arrows). (C) By E14.5, the intestine and mesentery (m) are positive for lacZ staining. (D) At E18.5, lacZ staining is strong over the entire gut tube and in the mesentery. (E-E'') Immunohistochemistry for ß-Gal (E, red) and Wt1 (E', green) reveal that, at E12.5, most of the serosal mesothelial cells express ß-Gal; ß-Gal staining colocalizes with Wt1 staining (arrows; E''). e, endoderm; in, intestine; st, septum transversum. Scale bars: 500 µm in A-C; 250 µm in D; 50 µm in E-E''.

View larger version (72K): [in a new window]   Fig. 4. Molecular genetic and vital dye lineage marking of serosal EMT. (A-F) Localization of Wt1 (green) and ß-Gal (red) proteins during gut development. Wt1 expression is largely confined to the serosal mesothelium in the embryo (A-C) and newborn (NB0; D-F). The anti-ß-Gal antibody marks cells in the subserosal space of the developing gut tube (arrows in B,C,E,F). Both Wt1 and ß-Gal are expressed in most cells of the serosal mesothelium (C,F, arrowheads). (G-K) CCSFE (green) was used to vitally label surface cells of gut explants from E12.5 Wt1-Cre; Rosa26R embryos. (G) At day 0 of culture, surface cells were positive for CCFSE (area framed by arrowheads), but the subjacent mesenchyme is negative. (H,I) After two days of culture, CCFSE-positive cells (arrowheads) at the gut surface co-label with Wt1. CCFSE-marked cells in the subserosal space are not positive for anti-Wt1 antibody (arrows). Also, many mesenchymal cells label with both anti-ß-Gal antibody and CCFSE (arrows, J,K). ß-Gal-positive cells unmarked by CCFSE are also seen in the mesenchyme (open arrowheads). Note a small number of CCFSE-labeled cells without the ß-Gal marker (filled arrowheads). Scale bars: 20 µm.

View larger version (51K): [in a new window]   Fig. 5. Progeny of the serosal mesothelium contribute to blood vessels of the mesentery and gut. (A-D) Whole-mount lacZ staining characterizes Wt1-expressing cells and their descendents during postnatal stages in the gut (A-C) and heart (D). (A,B) After birth, major vessels in the mesentery and on the gut surface (black arrowheads) contain lacZ-positive cells. Veins are very lightly stained in whole mounts (B, white arrowheads). Note the blue staining outside of the vessel (B, white arrows). (C) At high magnification, lacZ-stained samples show the characteristic `corkscrew' deposition of smooth muscle cells around a sample artery (arrowheads). (D) In the newborn (NB0) heart, coronary vessels contain lacZ-positive cells (arrowheads). in, intestine; m, mesentery. Scale bars: 1 mm in A; 500 µm in B,D; 200 µm in C.

View larger version (116K): [in a new window]   Fig. 6. Progeny of the serosal mesothelium differentiate into vascular smooth muscle cells. (A-H) Immunohistochemistry for ß-Gal and vessel markers in the adult gut. In co-labeling studies for ß-Gal and SMA (A), or ß-Gal and Pecam (E), the anti-ß-Gal antibody marks blood vessels (asterisks) and the serosal mesothelium (arrowheads; A,E), but not the visceral smooth muscle (vism) of the intestine (A). High magnification (white box in A) of anti-SMA and anti-ß-Gal staining reveals colocalization of ß-Gal and SMA (arrowheads, B-D). Note the additional ß-Gal-marked cells interspersed (arrows) between circular (cvm) and longitudinal visceral smooth muscle layers (lvm). High magnification (white box in E) of anti-Pecam and anti-ß-Gal staining shows virtually no overlap of expression (arrowheads, F-H). Note that ß-Gal-marked cells are arranged perpendicular to the endothelial Pecam-positive cells. Scale bars: in A, 50 µm for A,E; in B, 30 µm for B-D,F-H.

View larger version (76K): [in a new window]   Fig. 7. ß-Gal colocalizes with SMA, but not Pecam. Confocal images of immunohistochemistry in the newborn (NB0) gut (A-F) and the (adult) heart (G-L) of ß-Gal with SMA (A-C,G-I), and with Pecam (D-F,J-L). Significant co-labeling is seen with anti-SMA and anti-ß-Gal in vascular smooth muscle of the mesentery (A-C), and of the heart (G-I). By contrast, anti-Pecam and anti-ß-Gal antibodies reveal essentially no overlap in endothelial cells of the mesentery (D-F), and the heart (J-L).

View larger version (32K): [in a new window]   Fig. 8. The serosal mesothelium gives rise to mural cells of gut vasculature. (Left) At E9.5, the embryonic gut is not covered by a mesothelium. The gut consists only of endoderm (e) and splanchnic mesoderm (splm). A vascular plexus (red) runs between endoderm and splanchnic mesoderm, and within the mesentery. (Center) At E10.5, the Wt1/Cytokeratin-positive serosal mesothelium (green) starts to cover the mesentery and, subsequently, by E11.5, coats the entire gut. (Right) Serosal mesothelial cells undergo EMT into the subserosal space, where these mesenchymal cells discontinue Wt1 expression (open green triangles). (Box) Progeny of the serosal mesothelial cells differentiate into smooth muscle cells surrounding the blood vessels (red) that form in the subserosal space. A subset of these progeny differentiates into, as yet unidentified, non-vessel cells (triangle with question mark). da, dorsal aorta.