Fig 1.
Three-dimensional analysis of c-Kit+ cells in the embryonic head.
(A) 3D confocal image of c-Kit (green) and CD31 (magenta) expression in the mouse head region at E10.5 (35 sp). The whole-head image was acquired using tile scanning (20 tiles). (B) Cartographic distribution of c-Kit+ cells in the head. The c-Kit+ cells observed in the left half of the head are plotted. The head region for counting c-Kit+ cells is indicated by the broken line (above the first pharyngeal arch). PA1: first pharyngeal arch. (C) The number of c-Kit+ cells in the head vasculature at different times of development. E9.5 (n = 2, 25 and 26 sp), E10.5 (n = 4, 35 and 36 sp) and E11.5 (n = 4, 45–47 sp) were analyzed.
Fig 2.
Spatio-temporal quantitation of c-Kit+ cells in the head arteries.
(A) Illustrative example showing arteries (red) and veins (blue) in the E10.5 (35 sp) head. Scale bar: 300 μm. (B) Number of c-Kit+ cells in the dorsal aorta (n = 3) and the head vasculature (n = 4) at E10.5 (35–37 sp). (C) Cartographic distribution of c-Kit+ cells in the head arteries. The c-Kit+ cells observed in the left half of the head are plotted (red circles). The grey region represents arteries. See S3 Fig for the complete mapping of the c-Kit+ cells in the head vasculature. ICA: internal carotid artery. (D) Number of c-Kit+ cells in the head arteries at different times of development. E9.5 (n = 2, 25 and 26 sp), E10.5 (n = 4, 35 and 36 sp) and E11.5 (n = 4, 45–47 sp) were analyzed. (E) The number of hematopoietic clusters with more than 10 cells at E10.5 (35–37 sp).
Fig 3.
Morphological analysis of c-Kit+ cells in the head vasculature.
(A) Examples of spherical, quasi-spherical and hemispherical cells localized adjacent to the endothelial layer. (B) c-Kit+ cells in the E10.5 (36 sp) dorsal aorta. Blue, orange and white arrows indicate hemispherical, quasi-spherical and spherical cells, respectively. (C) c-Kit+ cells in the E10.5 (36 sp) head artery. (D) Frequency of spherical, quasi-spherical and hemispherical c-Kit+ cells in the dorsal aorta (n = 179), head arteries (n = 131) and head veins (n = 194). Seven embryos (E10.5, 35–37 sp) were analyzed. Scale bars: 10 μm in A; 50 μm in B and C.
Fig 4.
Absence of Runx1-GFP+ endothelial cells in the head vasculature.
Whole-mount immunostaining of the E10.5 (35 sp) Runx1-GFP transgenic mice for GFP (green) and CD31 (magenta) expression. (A) Dorsal aorta. Note that Runx1-GFP is expressed in the flat endothelial cells (arrow) and the hemispherical cells (arrowhead). VA: vitelline artery. Scale bars: 100 μm in the left panel; 50 μm in the right panel. (B) Head. Runx1-GFP was detected in the round cells (blue arrows), but not in the endothelial cells. Scale bars: 300 μm in the left panel; 100 μm in the right panel.
Fig 5.
Runx1-GFP and CD45 expression in head c-Kit+ cells.
(A-C) Whole-mount immunostaining of E10.5 Runx1-GFP transgenic mice for GFP (green), c-Kit (red) and CD31 (blue) expression. (A) Sagittal image of the dorsal aorta (35 sp). The c-Kit+ circulating cells (arrowheads) were negative for Runx1-GFP. (B and C) Runx1-GFP expression in c-Kit+ cells. (C) Quantitation of GFP+ cells from two embryos (35 sp). The frequency of Runx1-GFP+ cells in the head artery c-Kit+ cells is similar to that of circulating blood c-Kit+ cells. (D and E) Whole-mount immunostaining of E10.5 mouse embryos for c-Kit (green), CD31 (blue) and CD45 (magenta) expression. (D) Representative confocal images of the dorsal aorta and head region (36 sp). The arrowheads indicate c-Kit+CD45+ cells. (E) Quantitation of CD45+ cells from three embryos (36 and 37 sp). The frequency of CD45+ cells in the head artery c-Kit+ cells is similar to that of circulating blood c-Kit+ cells. Scale bars: 50 μm.
Fig 6.
Majority of head artery c-Kit+ cells originate from other organs.
(A) Experimental design for tracing the progenies of yolk sac hemogenic endothelium. Runx1SACre/+:: ROSA26YFP/+ embryos were labeled at E7.5 and analyzed at E10.5. Note that many YFP+ endothelial cells (arrowheads) were observed in the yolk sac but not in the head. Scale bar: 50 μm. (B) Representative confocal image of an YFP+ c-Kit+ cell (arrowhead) in the head. Scale bar: 20 μm. (C) Quantitation of YFP+ c-Kit+ cells from three embryos (36–38 sp).