Iraha F et al. (2002), Common and distinct signals specify the distrib...

XB-ART-6344

Dev Growth Differ 2002 Oct 01;445:395-407. doi: 10.1046/j.1440-169x.2002.00653.x.

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Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos.

Iraha F , Saito Y , Yoshida K , Kawakami M , Izutsu Y , Daar IO , Maéno M .

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In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie-2, a Xenopus homolog of the tie-2 receptor tyrosine kinase, was examined. Whole-mount in situ hybridization analysis revealed that Xtie-2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie-2-positive cells are predominantly present just outside the boundary of alpha-globin-positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie-2 and alpha-globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a "dorsal determinant" from the vegetal cytoplasm at the 1-cell stage. Second, expression of both Xtie-2 and alpha-globin were markedly induced in ectodermal explants (animal caps) from embryos co-injected with activin and bone morphogenetic protein (BMP)-4 RNA. Furthermore, both Xtie-2 and alpha-globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP-4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP-4-dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 fgf2 hba1 kdr tek

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	Fig. 1. Whole-mount in situ hybridization analysis of Xtie-2 expression in embryos. Lateral views of (A) stage 24 and (B) stage 27 embryos showing positive signals in the prospective endocardiac region (h) and in the surrounding regions of the ventral blood islands (v) in stage 27 embryos. (C) In stage 32 embryos, Xtie-2 is prominently expressed in the endocardium (h) and in the head mesenchyme around the dorsal region of the optic capsule (e) and in the mesenchyme overlying the auditory region (a). The expression is also evident in the branchial region (b), in the cardinal veins (c) and in the peripheral region of ventral blood islands (v). (D) Stage 32 embryos hybridized with Xtie-2 sense probe as a control experiment. Comparison of (E) Xtie-2 and (F) -globin expression in the ventral mesoderm of a stage 32 embryo, showing that the expression patterns of these two genes are mutually exclusive in the ventral blood islands. All the figures are shown at the same magnification. Bar in (A), 0.5 mm.
	Fig. 2. Whole-mount in situ hybridization analysis of Xtie-2, flk-1 and -globin expression in ventralized or dorsalized embryos. Expression of (A,D) Xtie-2, (B,E) flk-1 and (C,E) -globin is shown in the ventralized embryos (A,B,C) and in the dorsalized embryos (D,E,F). Expression of an anterior marker, XCG7, is also shown as a control experiment (G, dorsalized). In ventralized embryos, all the three markers were preferentially detected in a large area at the opposite end from the position of the yolk plug. In contrast, in dorsalized embryos, Xtie-2 and flk-1 expressions were detected in the surrounding region of the -globin-positive area (closed arrowheads in D and E). (E) flk-1 expression was also observed in the head mesenchyme, the cardiac region (arrow) and the cardinal vein (open arrowheads). (G) Expression of XCG7, a cement gland marker, shows the most anterior portion of the embryo. Embryos in (A,B,C) are the sibling embryos at stage 32 and the yolk plug is located at the bottom end of these figures. Embryos in (D,E,F,G) are the sibling embryos at stage 32. The posterior side of the embryos is positioned downward. All the figures are shown at the same magnification. Bars in (A) and (H), 0.5 mm. Comparison of sections through the ventralized embryos that were stained by (H) Xtie-2 and (I) -globin probes. Messages of both were detected in the same region of the mesodermal layer.
	Fig. 3. Induction of Xtie-2 and -globin messages in animal cap explants. Embryos injected with activin RNA and different doses of bone morphogenetic protein (BMP)-4 RNA at the 2-cell stage were allowed to develop until the blastula stage and animal caps were dissected. Explants were further cultured until the sibling control embryos reached stage 32. (A–D) Wholemount in situ hybridization analysis, showing the expression of (A,B) Xtie-2 and (C,D) -globin in the animal cap explants that had received injection of 1 ng XBMP-4 and 0.05 ng activin RNA (B,D) or in the animal cap explants without injection (A,C). All the figures are shown at the same magnification. Bar in (A), 0.5 mm. (E) Semi-quantitative analysis of Xtie-2 and -globin expression by reverse transcription (RT)–polymerase chain reaction (PCR). Total RNA from 10 to 15 explants of each group were used for the RT–PCR analysis as described in the Materials and Methods. Amplified products were run on 5% polyacrylamide gels and dried gels were exposed to X-ray film. The densitometric analysis was performed as described in Materials and Methods. Note that both Xtie-2 and -globin were maximally expressed in explants that had been injected with 0.5 ng XBMP-4 and 0.05 ng activin RNA. Blue box, Xtie-2; pink box, -globin.
	Fig. 4. Expression of Xtie-2 is regulated by bone morphogenetic protein (BMP)-4 signaling. Embryos injected with RNA at the 4-cell stage were allowed to develop until the early gastrula stage (stage 10+), and either the dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) was excised. Explants were further cultured until the sibling embryos reached stage 32. (A–H; J–Q) Whole-mount in situ hybridization analysis. Expression of (A–D) Xtie-2 and (E–H) -globin was examined in BMP-4-injected DMZ explants (B,F) or tBR-injected VMZ explants (D,H). Uninjected DMZ explants (A,E) and VMZ explants (C,G) are also represented. Note that both Xtie-2 and -globin were strongly induced in the DMZ explants injected with BMP-4 RNA, and that these markers disappeared in the VMZ explants injected with tBR RNA. All the figures are shown at the same magnification. Bars in (A) and (J), 0.5 mm. (I) Semi-quantitative analysis of Xtie-2 and - globin expression by reverse transcription (RT)–polymerase chain reaction (PCR). The assay was done as described for Figure 3. The result was consistent with in situ hybridization analysis. (J–Q) Expression of (J–M) Xtie-2 and (N–Q) -globin in the DMZ explants that were injected with (J,N) 0 ng, (K,O) 0.08 ng, (L,P) 0.4 ng and (M,Q) 2 ng BMP-4 RNA and were cultured until stage 32. The maximum induction of Xtie-2 and –globin occurs in the explants receiving the highest dose of BMP-4 RNA. Blue box, Xtie-2; pink box, -globin.
	Fig. 5. Distinct expression of Xtie-2 and –globin in ventral marginal zone (VMZ) explants. The VMZ was excised from stage 10+ embryos and cultured until the sibling embryos reached stage 32. Whole-mount in situ hybridization analysis shows the expression of (A,B) Xtie-2 and (C,D) –globin. Arrowheads in (C) show the point of the closed yolk plug. (B,D) Some explants were sectioned to examine the exact location of the stained area. (E–M) Lineage-tracing experiment. (E) represents the experimental strategy. Two blastomeres (A4, B4, C4 or D4) of the 32-cell stage embryo were injected with -galactosidase RNA. The VMZ region was excised at the gastrula stage and cultured until stage 32. The explants were then fixed and X-gal staining (blue) was performed. Typical results after injection of -galactosidase RNA into (F,G) A4, (H,I) B4, (J,K) C4 and (L,M) D4 are shown. (G,I,K,M) are sections of (F,H,J,L), respectively. Bar in (C), 0.5 mm; bars in (D) and (M), 0.2 mm.
	Fig. 6. Fibroblast growth factor (FGF) signal is involved in the distribution pattern of vascular and blood cells in the ventral marginal zone (VMZ) explant. Various amounts of bFGF or XFD (dominant negative FGF receptor) mRNA were injected into C4 blastomeres of the 32-cell stage embryos that contain blood and vascular precursor cells and the VMZ were excised at stage 10+ for subsequent culture until stage 32. Whole-mount in situ hybridization was performed to show the expression of Xtie-2 and -globin in these explants. FGF expanded Xtie-2-positive area in the explants (A–C), whereas it inhibited the expression of - globin in the explants (D–F). In contrast, XFD diminished the Xtie- 2-positive area (G–I) and enlarged the -globin-positive area (J–L). (A,D,G,J), controls; (B,E) 0.5 ng bFGF; (C,F) 1.0 ng bFGF; (H,K) 0.05 ng XFD; (I,L) 0.1 ng XFD. Bars in (F) and (L), 0.5 mm.