Mills KR et al. (1999), Elucidating the origins of the vascular system:...

XB-ART-13029

Dev Biol 1999 May 15;2092:352-68. doi: 10.1006/dbio.1999.9245.

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Elucidating the origins of the vascular system: a fate map of the vascular endothelial and red blood cell lineages in Xenopus laevis.

Mills KR , Kruep D , Saha MS .

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Required to supply nutrients and oxygen to the growing embryo, the vascular system is the first functional organ system to develop during vertebrate embryogenesis. Although there has been substantial progress in identifying the genetic cascade regulating vascular development, the initial stages of vasculogenesis, namely, the origin of vascular endothelial cells within the early embryo, remain unclear. To address this issue we constructed a fate map for specific vascular structures, including the aortic arches, endocardium, dorsal aorta, cardinal veins, and lateral abdominal veins, as well as for the red blood cells at the 16-cell stage and the 32-cell stage of Xenopus laevis. Using genetic markers to identify these cell types, our results suggest that vascular endothelial cells can arise from virtually every blastomere of the 16-cell-stage and the 32-cell-stage embryo, with different blastomeres preferentially, though not exclusively, giving rise to specific vascular structures. Similarly, but more surprisingly, every blastomere in the 16-cell-stage embryo and all but those in the most animal tier of the 32-cell-stage embryo serve as progenitors for red blood cells. Taken together, our results suggest that during normal development, both dorsal and ventral blastomeres contribute significantly to the vascular endothelial and red blood cell lineages.

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Species referenced: Xenopus laevis
Genes referenced: aplnr hbg1 ret sst.1

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	FIG. 2. Identification of vascular structures and ventral blood islands. Whole-mount in situ hybridization was performed on hatching- stage embryos (stage 33/34) using (A) X-msr to identify vascular tissue and (B) Beta-globin to identify the ventral blood islands. (C) Schematic diagram indicating the location of structures examined in this study. aa, aortic arches; acv, anterior cardinal vein; da, dorsal aorta; endo, endocardium; lav, lateral abdominal vein; pcv; posterior cardinal vein; sa, somitic arteries; tb, tailbud; vbi, ventral blood islands.
	FIG. 3. Colocalization of X-msr signal and RLDx. Following in situ hybridization with an X-msr probe, embryos were embedded in wax, transversely sectioned, and then analyzed for the presence of both X-msr and RLDx. Black arrowheads (A, D, and G) indicate X-msr signal in anterior cardinal veins, endocardium, and posterior cardinal veins, respectively; white arrowheads (B, E, and H) indicate the same location when viewed under fluorescence. Black arrows (C, F, and I) show overlay of X-msr signal with fluorescence in the identical location. (A, B, and C) Examples of X-msr staining in the anterior cardinal veins colocalizing with RLDx from blastomere D2.2. (A) X-msr signal in anterior cardinal vein, (B) fluorescence in the same section, and (C) an overlay of both images showing colocalization. (D, E, and F) Examples of X-msr staining in the endocardium colocalizing with RLDx from blastomere D1.1. (D) X-msr signal, (E) fluorescence in the same section, and (F) an overlay of both images. Insets represent an enlargement of the upper left region of the endocardium showing X-msr signal (C), fluorescence (D), and overlay (E). Arrowheads in the insets point to two cells showing both X-msr signal and fluorescence; directly to the right of the asterisk is depicted a cell that shows X-msr signal but does not show fluorescence colocalization. (G, H, and I) Examples of X-msr signal in the posterior cardinal vein colocalizing with RLDx signal from blastomere V2.2. (G) X-msr signal, (H) fluorescence in the same section, and (I) an overlay of both images. For A, dorsal is toward the top of the page. Magnification for A is 250x, D is 400x, G and H is 200x, and I is 180x. Note that the magnification of G and H is slightly greater than that of I. (J) Schematic indicating planes of section: I (A, B, C), II (D, E, F), and III (G, H, I). Diagram adapted from Nieuwkoop and Faber (1967). acv, anterior cardinal vein; endo, endocardium; fg, foregut; mb, midbrain; myo, myocardium; nc, notochord; ret, retina; som, somite; pcv, posterior cardinal vein.
	FIG. 4. Colocalization of X-msr or B-globin and RLDx. Following in situ hybridization, embryos were embedded in wax and transversely sectioned and then analyzed for the presence of either X-msr or B-globin and RLDx. Black arrowheads in A, D, and G indicate in situ hybridization signal for lateral abdominal veins, dorsal aorta, and ventral blood islands; white arrowheads in B, E, and H indicate the same location viewed under fluorescence. Arrows in C, F, and I show overlay of in situ hybridization signal with fluorescence in same region. (A, B, and C) Examples of X-msr staining in the lateral abdominal veins colocalizing with RLDx from blastomere V2.2. (A) X-msr signal, (B) fluorescence in the same section, and (C) an overlay of both images showing colocalization. (D, E, and F) Examples of X-msr signal colocalizing with RLDx from blastomere V2.1. (D) X-msr staining in the dorsal aorta, which lies ventral to the notochord and hypochord, (E) fluorescence in the same section, and (F) an overlay of both images. (G, H, and I) Examples of B-globin signal in the ventral blood islands colocalizing with RLDx from blastomere D2.1. (G) B-Globin signal, (H) fluorescence in the same section, and (I) an overlay of both images. Dorsal is toward the top of the page. Magnification for A and G is 200x, D is 250x. (J) Schematic indicating planes of section: I (A, B, C), II (D, E, F), and III (G, H, I). Diagram adapted from Nieuwkoop and Faber (1967). da, dorsal aorta; hg, hindgut; lav, lateral abdominal vein; nc, notochord; som, somite; vbi, ventral blood island.

	Examples of X-msr staining in the endocardium colocalizing with RLDx from blastomere.
	Whole-mount in situ hybridization was performed on hatching- stage embryos (stage 33/34) using X-msr to identify vascular tissue.
	FIG. 1.Identification of injected blastomeres. Blastomeres were designated according to Moody (1987a,b)
	FIG. 5.Schematic summary of fate map for (A) the 16-cell- and (B) the 32-cell-stage embryos. The embryos schematically depict theprogeny of each of the blastomeres of the 16- and 32-cell-stage embryos. The percentage of injected embryos for a given blastomere thatcontributed to a specific tissue is indicated as follows: Structures denoted in large bold red letters indicate that 75–100% of the injectedblastomeres contributed to that structure; structures shown in large blue letters denote that 50 –74% of the injected embryos contributeprogeny to that structure; structures shown in small black letters indicate that 25– 49% of the injected blastomeres contributed progenyto the structure.366Mills, Kruep, and SahaCopyright © 1999 by Academic Press. All rights of reproduction in any form reserved.