Smith SJ et al. (2002), XPOX2-peroxidase expression and the XLURP-1 pro...

XB-ART-6622

Mech Dev 2002 Sep 01;1171-2:173-86. doi: 10.1016/s0925-4773(02)00200-9.

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XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus.

Smith SJ , Kotecha S , Towers N , Latinkic BV , Mohun TJ .

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Phagocytic myeloid cells provide the principle line of immune defence during early embryogenesis in lower vertebrates. They may also have important functions during normal embryo morphogenesis, not least through the phagocytic clearance of cell corpses arising from apoptosis. We have identified two cDNAs that provide sensitive molecular markers of embryonic leukocytes in the early Xenopus embryo. These encode a peroxidase (XPOX2) and a Ly-6/uPAR-related protein (XLURP-1). We show that myeloid progenitors can first be detected at an antero-ventral site in early tailbud stage embryos (a region previously termed the anterior ventral blood island) and transiently express the haematopoetic transcription factors SCL and AML. Phagocytes migrate from this site along consistent routes and proliferate, becoming widely distributed throughout the tadpole long before the circulatory system is established. This migration can be followed in living embryos using a 5 kb portion of the XLURP-1 promoter to drive expression of EGFP specifically in the myeloid cells. Interestingly, whilst much of this migration occurs by movement of individual cells between embryonic germ layers, the rostral-most myeloid cells apparently migrate in an anterior direction along the ventral midline within the mesodermal layer itself. The transient presence of such cells as a strip bisecting the cardiac mesoderm immediately prior to heart tube formation suggests that embryonic myeloid cells may play a role in early cardiac morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: lcp1 mpo myl2 myl4 nkx2-5 plaur runx1 slurp1l tal1 tbx2

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	Fig. 2. Whole-mount in situ hybridisation analysis of Xenopus POX2 (A–H,L), LURP-1 (I,J) and L-plastin (K) expression. Right-lateral views of stage 19 (A), 24 (C) and 27 (E) embryos showing XPOX2-expressing cells. (B,D,F) Ventral views of the same embryos depicted in (A,C,E). (G) Higher magnification image of the stage 27 embryo depicted in (F) to illustrate the streams of XPOX2-expressing cells that emanate from a focal point (red arrow) located at the ventral midline within the heart-forming region. (H) Right-lateral view of a stage 30 embryo. (I,J) Left-lateral views of embryos at stages 24 and 36, respectively, showing cells that express XLURP-1. (K,L) Right-lateral view of the posterior trunk of a stage 35 tadpole after sequential, double whole-mount in situ hybridisation staining to reveal first (K), Xenopus L-plastin (pale blue) and second (L), POX2 (magenta) expression. The co-localisation of the two chromogenic reagents results in a dark blue colour (L). A, anterior; P, posterior.
	Fig. 3. Transverse sections (10 μm) through Xenopus tailbud stage embryos after whole-mount in situ hybridisation for POX2 (A–J) or LURP-1 (K,L). (A) Section through the centre of the XPOX2 expression domain of a stage 20 embryo, showing the location of the high magnification detail (B). A stage 25 embryo (C) shows the relative location of five ventral-half view images (D–H) that are representative sections marking progressively posterior slices. Sections are numbered (top right of each panel) commencing from the posterior limit of the cement gland. Red arrows mark the ventral-most XPOX2-expressing mesodermal cells within the heart-forming region. (I,J) Sections of stage 27 embryos showing a ventral-half view of the posterior trunk (I) and a dorsal-half view at the level of the eye (J). Ventral-half view of a stage 28 embryo (K) gives the location of a detail (L), which depicts a cell that expresses XLURP-1 located between mesodermal and endodermal layers that was fixed while it apparently phagocytosed cell material from the endoderm (L, black arrow). Additionally, this phagocyte formed such tight contacts with two mesodermal cells that they were prised away from the bulk of the mesoderm as the germ layers became separated during the sectioning procedure. All sections have been counterstained to reveal cell nuclei. Red boxes mark the location of detail images. Ect/Mes, expressing cells located between ectodermal and mesodermal germ layers; Mes/End, expressing cells located between mesodermal and endodermal layers; CG, cement gland; Bla, remnant of the blastocoel cavity; RP, roof plate; NT, neural tube; E, eye.
	Fig. 4. XPOX2 expression within embryo explants. Left-lateral views of representative posterior-dorsal (A) and anterior-ventral (B) embryo dissections fixed at stage 21 and assayed for XPOX2 expression by in situ hybridisation. (C) Ventral view of the same fragment shown in (B). Arrows indicate the posterior limit of the XPOX2-expressing cells. Left-lateral views of posterior-dorsal (D) and anterior-ventral (E) dissections that were cultured till stage 33 and assayed as before. (F) Control stage 33 embryo with XPOX2-positive cells present in posterior and dorsal locations (arrows). The position of the myocardium (Mc) of the forming heart was also assayed by MLC2 expression (Chambers et al., 1994) in this experiment (E,F). Pigmented-wildtype embryos were used for this experiment. CG, cement gland; A, anterior; P, posterior.
	Fig. 7. XPOX2 expression adjacent to the heart-forming region. Left-lateral views of Xenopus stage 20 (A), 24 (B) and 26 (D) embryos after double whole-mount in situ hybridisation for Nkx2-5 (pale blue) and POX2 (magenta). Higher magnification ventral views (C,E) of the heart field of the same embryos depicted in (B) and (D), respectively. Red arrow indicates expressing cells at the focal point located posterior of the cement gland (CG) on the ventral midline. (F) High magnification ventral view of the myocardial plate (Mc.P) of a stage 30 embryo after double whole-mount in situ hybridisation for MLC1av (pale blue) and POX2 (magenta). The stage 20 embryo (A) is a pigmented-wildtype example while the rest are albino embryos. A, anterior; P, posterior.
	Fig. 8. Transverse sections through the heart-forming region of Xenopus embryos after double whole-mount in situ hybridisation for the two markers of cardiogenesis (in pale blue), Nkx2-5 (A–F) and MLC1av (G–K), in combination with POX2 (magenta). (A–C) Three representative sections marking progressively posterior slices through the same stage 24 embryo depicted in Fig. 7(B,C). (D–F) Three progressively posterior sections through the stage 26 embryo depicted in Fig. 7(D,E). Red arrows mark the ventral-most XPOX2-expressing mesodermal cells within the heart field. (G) Transverse section through the myocardial plate of a stage 30 embryo. (H–J) Three progressively posterior sections through the myocardium of a stage 32 embryo. (K) Transverse section through the looping heart tube of a stage 35 embryo. Sections (10 μm) are numbered (top right of each panel) commencing from the posterior limit of the cement gland (A–C and D–F) or the anterior limit of the myocardium (G–K). Ect, ectoderm; Mes, mesoderm; End, endoderm; Mc, myocardium; Ec, endocardium.
	Fig. 9. XPOX2 expression in the anterior VBI. Double whole-mount in situ hybridisation for the haematopoietic transcription factor, SCL (pale blue), in combination with POX2 (magenta). Embryos were photographed successively after the colour reactions for the SCL (A–D) and POX2 (E–H) probes were developed. High magnification ventral views of the site of myeloid cell development at stages 20 (A,E), 22 (B,F) and 24 (C,G). (D,H) High magnification ventral views of the trunk of a stage 28 embryo showing the posterior VBI. LS, lateral SCL expression; PS, posterior SCL expression; pVBI, posterior ventral blood island; A, anterior; P, posterior; red arrow, narrow rostral POX2 expression within heart field.
	Fig. 11. XPOX2 expression in progeny cells of the dorsal-vegetal blastomeres. (A) Diagram illustrating the injection site of the cell-lineage marker, biotin-dextran (pale blue), into an individual dorsal-vegetal blastomere of an eight-cell stage Xenopus embryo. The animal and vegetal poles, and the dorsal-ventral axis, are indicated. The embryos were allowed to develop until stages 22–23, whereupon they were fixed and assayed for XPOX2 mRNA expression, and stained to reveal the daughter cells of the injected blastomere. In 27 correctly targeted embryos, when viewed from the ventral side (Lane and Smith, 1999), we observed two subtly different juxtapositions of myeloid gene expression and the posterior boundary of the dorsal-vegetal progeny cells, which are shown (B–D,E–G). Nevertheless, with both observed configurations, the myeloid cells are wholly derived from the dorsal-vegetal blastomeres. The converse cell-fate mapping experiment of ventral-vegetal blastomere cell-lineage injection never resulted in ventrally derived mesoderm present within the domain of myeloid cells at stage 22 (data not shown). (B,E) Ventral views of the site of myeloid cell development for such representative tailbud embryos showing the progeny cells of the dorsal-vegetal blastomere (pale blue) and XPOX2 expression (magenta). The stage 22 embryo (B) has left-sided lineage staining while the stage 23 embryo (E) has right-sided lineage staining. Black lines indicate the positions of the transverse sections illustrated (C,D,F,G). Red arrows (C,F,G) on the sections mark mesodermal regions of coincident cell-lineage staining and XPOX2 expression, which appears a dark blue colour. Sections (10 μm) are numbered (top right of each panel) commencing from the anterior limit of the XPOX2 expression domain. Pigmented-wildtype embryos were used for this experiment. A, anterior; P, posterior; Ect, ectoderm; Mes, mesoderm; End, endoderm.
	mpo (myeloperoxidase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, ventral view, anterior right.
	slurp1l (secreted LY6/PLAUR domain containing 1-like) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.