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Vasculogenesis and hematopoiesis are closely linked in developing vertebrates. Recently, the existence of a common progenitor of these two tissues, the hemangioblast, has been demonstrated in different organisms. In Xenopus early vascular and hematopoietic cells differentiate in a region called the anterior ventral blood island (aVBI). Differentiating cells from this region migrate out to form embryonic blood and part of the vascular structures of the early frog embryo. A number of members of the ETS family of transcription factors are expressed in endothelial cells and some of them play important roles at various stages of vascular development. The loss of ER71 function in mice led to a complete loss of blood and vascular structures. Similarly, knock down of the zebrafish homolog of er71, etsrp, greatly affected development of vascular structures and myeloid cells. We have identified the Xenopus ortholog of er71 and could show that er71 function in Xenopus is required for vasculogenesis, but not for the development of hematopoietic cells.
Figure 3. Spatial analyses of er71 expression. A: Whole-mount in situ hybridization of wild type embryos of different stages. er71 is expressed in progenitor cells of the vascular system. a,b: er71 expression at NF stage 17–18. er71 is expressed in the anterior portion of the embryo in the vicinity of the cement gland. c–j: er71 expression between NF stage 20 and 37. k–m: Sections of a NF stage-34 embryo show expression of er71 in endothelial structures in the retinal veins (k,l), intersomitic veins (l), branchial arches and duct of cuvier (m), and anterioraorta and vitelline veins (n). aa, anterioraorta; ap, anal porus; (ba, branchial arches; dc, duct of cuvier; h, heart anlage; isv, intersomitic veins; mnc, migrating neural crest; pcv, posterior cardinal vein; pp, posterior precursors; rv, retinal vein; vp, ventral precursors; vbi, ventralblood islands; vv, vitelline veins.
Figure 4. er71 is required for vascular development. A: Microinjection of X. laevis-specific er71 antisense morpholino (er71-MO-xl) leads to a reduction in vascular development. At NF stage 37, knock-down of er71 function leads to a severe reduction in the expression of vascular marker genes flk1, Ami, and ve-cadherin in the vitelline veins, in the intersomitic veins, in the posterior cardinal vein, and in the vascular structures of the head. To trace the fate of the microinjected morpholinos, synthetic mRNA for β-galactosidase was co-injected. In control morpholino-injected embryos, expression of marker genes appears normal. B: er71-MO-xl specifically reduces translation of er71-mRNA in a coupled transcription/translation assay. ER71 protein expressed from a pCS2+ vector that contains 5′-UTR and the N-terminus of er71 is reduced upon addition of er71-MO-xl but not by the application of a X. tropicalis-specific antisense morpholino (er71-MO-xt). C: Sequences of X. laevis- and X. tropicalis-specific antisense morpholinos, aligned to the initial AUG of er71-mRNA. isv, intersomitic veins; pcv, posterior cardinal vein; vv, vitelline veins.
Figure 5. er71 is not required for hematopoietic gene expression. Microinjection of X. laevis-specific er71 antisense morpholino (er71-MO-xl) had no effect on the expression of the myelogenic genes runx1, lmo, and mpo or the erythrogenic genes SCL and globin. The arrows mark the position of the posterior cardinal vein or the ventralblood islands. The injection was traced by the co-injection of synthetic mRNA encoding β-galactosidase.
Figure 6. er71 overexpression induces endothelial gene expression. Injected (a–h) and non-injected (i–l) sides of NF stage-37 embryos are shown. Microinjection of er71-mRNA in concentrations up to 250 pg/embryo led to a dramatic increase in the expression of the endothelial genes Ami and msr. Whereas on the non-injected side, the development of the vasculature occurs normally, the analysis of the injected side reveals massive ectopic spots of Ami and msr expression, which also led to large protrusions of the embryos. Dashed red lines indicate the level of the section. Arrows point to vitelline veins and posterior cardinal veins on the non-injected side and the respective area on injected side. The injection was traced by the co-injection of synthetic mRNA encoding β-galactosidase. pc, pigmented cells; pcv, posterior cardinal vein; vv, vitelline veins.
Figure 7. Overexpression of er71 in animal caps had a dramatic effect on the morphology of the caps. Increasing amounts of synthetic er71 mRNA were injected into fertilized eggs. Animal caps excised from those embryos or whole embryos were incubated until they reached NF stage 36. At higher concentrations, er71 overexpression led to gastrulation defects in most embryos. In animal cap assays, er71 overexpression caused the cells to lose contact with each other.
cdf (complement factor D (adipsin)) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 37, lateral view, anteriorleft, dorsal up.
