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Fig. 1. Expression of peroxidase 2 (POX2) in normal and exogastrula embryos (A–C). Whole-mount in situ hybridization analysis at the neurula stage (stage 13 and stage 15) (A and B) and early tailbud stage (stage 19) (C), showing that expression of POX2 starts in the two separated portions of the anterior ventral region (arrowheads in 1A and 1B). (D–I) Expression of POX2 (D, G), otx2 (E), nkx2.5 (F) and α-globin (H) in exogastrula embryos. The exogastula embryos were made as described in the Materials and methods section, and these embryos were cultured until stage 20 (D, E, F) or stage 30 (G, H). The expression of α-globin in a control stage 30 embryo is shown in I. The POX2 message was localized in the anterior mesoderm where otx-2 and nkx2.5 were also located.
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Distribution of anterior ventral (AV)-derived cells and dorsal marginal zone (DMZ)-derived cells in the embryos at the tailbud stage. (A) Beta-galactosidase (beta-gal) RNA (300 pg/embryo) was injected into the DMZ at the 4-cell stage. AV region at stage 13–14 or DMZ at stage 10+ was excised from the beta-gal-injected embryos and transplanted into stage 13–14 embryos. Host embryos were fixed at the tailbud stage (stage 32), stained with red-gal and subjected to whole-mount in situ hybridization analysis. A tailbud embryo (B, C) grafted with AV region (asterisk in B) and a tailbud embryo (D, E) grafted with DMZ region (asterisk in D), showing red-gal-positive cells (red) and peroxidase 2 (POX2)-positive cells (blue). Existence of double- positive cells (purple cells in C and E) in the lateral trunk region of the embryo revealed that a group of cells derived from the transplanted area migrated in the posterior direction and differentiated into POX2-positive leukocytes.
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Fig. 3. Distribution of anterior ventral (AV)- and ventral blood island (VBI)-derived cells in the mesenchyme and peripheral blood at the early tadpole stage. The AV regions of Xenopus borealis embryos at stage 13–14 were excised and orthotopically transplanted into Xenopus laevis embryos. As a control experiment, the VBI regions of borealis embryos at stage 20–21 were transplanted. The host embryos were allowed to develop until appropriate stages and were fixed for histological examination to detect borealis (donor) cells. (A–F) Quinacrine-stained sections of AV-grafted animals (A–C) and VBI-grafted animals (D–F) at stage 46. A’–F’ are high magnification views of A–F. Borealis nuclei have brightly stained spots after staining with quinacrine (arrowheads in A’–F’). Significant numbers of borealis cells were observed in the mesenchyme near the epidermis (A, A’), the spinal cord (C, C’) and in the somite (B, B’) of AV-grafted animals. In contrast, many borealis cells were observed in the heart (D, D’), blood vessels (E, E’) and mesenchyme (F, F’) of VBI-grafted animals.
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Fig. 4. Expression of peroxidase 2 (POX2) in dorsal marginal zone (DMZ) and ventral marginal zone (VMZ) explants. (A–I) Whole-mount in situ hybridization analysis showing the expression of POX2 (B, C, H, I) and α-globin (E, F) in DMZ (B, E, H) and VMZ (C, F, I) explants. Explants were harvested at stage 30 (B, C, E, F) or at stage 37/38 (H, I). Control whole embryos (w. e) are also shown (A, D, G). (J) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. Explants and the control whole embryos (w. e) were harvested at stage 30 or stage 37/38 and processed for the RT-PCR analysis to show the expression of POX2 and EF1α. The results demonstrated that both the DMZ and VMZ explants contain leukocyte precursor cells, but only the cells in DMZ explants differentiate at the early stage (stage 30).
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Fig.5. Induction of peroxidase 2 (POX2) expression in ventral marginal zone (VMZ) explants. Prospective VMZ at the 4-cell stage injected with mRNA coding for the BMP signal inhibitor and/or the wnt signal inhibitors. VMZ explants excised at the early gastrula stage were cultured until stage 28–30 and processed for whole-mount in situ hybridization analysis. Expression of POX2 (A–F), nkx2.5 (G), otx2 (H), α-globin (I) and LuRP (J, K) was examined in cultured VMZ explants that had been previously injected with β-gal (300 pg) (A), tBR (1000 pg) (B), tBR (1000 pg) + dkk1 (50 pg) (C), dkk1 (50 pg) (D, G, H, I, K), frzb-1 (800 pg) (E), or cerberus (600 pg) (F) mRNA. POX2 was strongly induced by dkk1 mRNA but was not induced by tBR mRNA. The VMZ explants injected with dkk1 mRNA did not express other anterior tissue markers, such as otx2 or nkx2.5. Instead, they expressed a ventral mesoderm marker, such as α- globin. Another leukocyte marker, LURP-1, was also strongly induced by dkk1 mRNA injection (50 pg) (J, K). L shows the expression of LURP-1 in stage 30 embryos.
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Fig.6. Dose-dependent induc- tion of peroxidase 2 (POX2) and nkx2.5 in ventral marginal zone (VMZ) explants after injection of dkk1 mRNA. Prospective VMZ at the 4-cell stage injected with dkk1 mRNA. VMZ explants excised at the early gastrula stage were cultured until stage 28 and processed for whole-mount in situ hybridization analysis. Expression of POX2 (A–D) and nkx2.5 (E-H) was examined in cultured VMZ explants that had been injected with 300 pg β-gal (A, E), 50 pg dkk1 (B, F), 200 pg dkk1 (C, G) or 600 pg dkk1 (D, H) mRNA. Expression of POX2 was induced at a low dose of dkk1 RNA, whereas nkx2.5 was induced at a high dose of dkk1 RNA.
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Induction of peroxidase 2 (POX2) in animal cap explants. The animal cap was excised from a mid-blastula embryo, and cells were dissociated in a Ca2+-free medium for 20 min and then reaggregated in the medium containing activin, as described previously (Ariizumi et al. 2003). Aggregates were further cultured until stage 33 and fixed to process for reverse transcription-polymerase chain reaction (RT-PCR) and whole- mount in situ hybridization analyses. (A) Expression of POX2, tnIc (a heart marker), transferrin (a liver marker) and EF1α (loading control) was examined by RT-PCR analysis. POX2 expression was strongly induced in the explants treated with 100 ng/mL activin. The explants also expressed tnIc but did not express transferrin. (B–E) Expression of POX2 and tnIc was examined by whole-mount in situ hybridization analysis.
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Fig. 8. Induction and distribution of myeloid cells derived from the anterior ventral (AV) region of the neurula embryo. At the early neurula stage wnt inhibitors, such as dkk1, frzb-1 and crescent, were expressed in the prechordal plate and anterior endoderm (diagonal area). A high dose of dkk1 induces the heart mesoderm, while a low dose of dkk1 induces myeloid cells. The differentiated leukocytes start to migrate toward the posterior area at the tailbud stage. At the swimming tadpole stage, ventral blood island (VBI)- derived leukocytes appear, in addition to AV-derived leukocytes, and the former become a major population at the feeding tadpole stage.
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