Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis (2018)
Figure 6

Figure 6. Spatiotemporal expression of Kitl-tdTomato at pre-liver sites of hematopoiesis

A. Confocal whole-mount immunofluorescence (WM-IF) analysis of Kitl-tdTomato expression in the E8.5 (6-7sp) conceptus (maximum intensity 3D projection from a 500μm-thick Z-stack). Kitl-tdTomato expression (red) is seen in the YS, allantois (a; from which the umbilical artery derives), and para-aortic splanchnopleura (PAS) of Kitl-tdTomato::23GFP transgenic concepti (the embryo proper, from which the head has been removed, is outlined with a purple dashed line). The 23GFP transgene (green) labels the YS blood islands (bi; yellow dashed line); this GFP reporter is transcribed from a heterologous hsp68 promoter under the spatiotemporal control of the Runx1 hematopoietic +23 enhancer, resulting in GFP expression in all primitive erythroid(-fated) cells, hemogenic endothelium, and emerging HSPCs[2,74]. Endothelial cells (CD31+) are in white. Arrowheads indicate the paired aortae (pa). Purple boxed area is magnified in (B); yellow boxed area is magnified in (C). Scale bar: 100μm. N=2 embryos analyzed.

B. Single 2.5μm-thick Z slice from the region highlighted in the purple box in (A). Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells in one of the paired aortae (pa). Scale bar: 25μm.

C. Single 2.5μm-thick Z slice from the region highlighted in the yellow box in (A), showing Kitl-tdTomato expression in the E8.5 YS BI. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells; the arrow indicates a Kitl-tdTomato+ perivascular cell. Scale bar: 25μm.

D. Single 2.5μm-thick Z slice of 10.5 (35sp) YS (WM-IF) showing Kitl-tdTomato (red), Kit (white) and CD31 (green) expression. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells. N=3 embryos analyzed. Scale bar: 50μm.

E. Confocal WM-IF analysis of Kitl-tdTomato expression (red) in a E10.5 (36sp) AGM region. Top panels (3D view) show a maximum intensity 3D projection from a 400μm-thick Z stack. Bottom panels (slice view) show a single 2.5μm-thick Z slice of the dorsal aorta, from the boxed region in the top panels. CD31 (vasculature; green); Kit (hematopoietic progenitors; white). Scale bars: 100μm (top); 25μm (bottom). fl: fetal liver; va: vitelline artery plexus; da: dorsal aorta; fp: floor plate; nt: neural tube. N=6 embryos analyzed.

F. Confocal immunofluorescence analysis of cryosections through the AGM region of E10.5 (34-36sp) Kitl-tdTomato transgenic embryos. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells (top), Kitl-tdTomato+ PDGFR-β+ (middle) or Kitl-tdTomato+ α-SMA+ (bottom) perivascular smooth muscle cells. Scale bars: 25μm or 50μm (PDGFR-β panel). N>6 embryos analyzed.

G. Confocal immunofluorescence analysis of Kitl-tdTomato expression in the E10.5 FL. A cryosection through a E10.5 (35-36sp) Kitl-tdTomato transgenic FL is shown. Arrowheads indicate Kitl-tdTomato+ CD31+ Kit- endothelial cells. Asterisks indicate Kitl-tdTomato+ CD31- Kit- mesenchymal cells or hepatoblasts. Note proximity of Kitl-tdTomato+ cells and Kit+ hematopoietic cells. Scale bars: 50μm (left panel), 25μm (magnified inset). N>5 embryos analyzed.