Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm

Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.     

GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation

GATA-6 is expressed in presumptive cardiac mesoderm before gastrulation, but its role in heart development has been unclear. Here we show that Xenopus and zebrafish embryos, injected with antisense morpholino oligonucleotides designed specifically to knock-down translation of GATA-6 protein, are severely compromised for heart development. Injected embryos express greatly reduced levels of contractile machinery genes and, at the same stage, of regulatory genes such as bone morphogenetic protein-4 (BMP-4) and the Nkx2 family. In contrast, initial BMP and Nkx2 expression is normal, suggesting a maintenance role for GATA-6. Endoderm is critical for heart formation in several vertebrates including Xenopus, and separate perturbation of GATA-6 expression in the deep anterior endoderm and in the overlying heart mesoderm shows that GATA-6 is required in both for cardiogenesis. The GATA-6 requirement in cardiac mesoderm was confirmed in zebrafish, an organism in which endoderm is thought not to be necessary for heart formation. We therefore conclude that proper maturation of cardiac mesoderm requires GATA-6, which functions to maintain BMP-4 and Nkx2 expression.     

Contribution of ventral and dorsal mesoderm to primitive and definitive erythropoiesis in the salamander Hynobius retardatus

Previously, we found that the conversion of hemoglobins (Hbs) from the larval to the adult type occurred within a single erythroid cell population in a salamander, Hynobius retardatus ("Hb switching" model), whereas the transition involves replacement of red-blood-cell (RBC) populations ("RBC replacement" model) in many amphibians (M. Yamaguchi, H. Takahashi, and M. Wakahara, 2000, Dev. Gene Evol. 210, 180-189). To further characterize the Hb transition, developmental changes in the erythropoietic sites have been intensively analyzed using larval- and adult-specific globin antibodies and globin and GATA-3 RNA probes. Cells of the ventral blood island (VBI) and the dorsolateral plate (DLP) in embryos differentiate in situ to erythroid cells that contain larval globin mRNA, suggesting that both the VBI and the DLP contribute to "primitive" erythropoiesis. In contrast, the expression pattern of the GATA-3 gene suggests that cells of the DLP may contribute to "definitive" hematopoiesis. In order to determine whether it is possible to define a definitive erythropoiesis in H. retardatus or not, further experiments were done: (1) when metamorphosing larvae were treated with phenylhydrazine to induce anemia and then bled at the postmetamorphic stage after recovery from the anemia, a precocious Hb transition was observed in these animals; (2) an RBC population expressing only adult Hb was confirmed by subtracting the number of RBCs expressing larval Hb from the total number of RBCs during metamorphosis. All these results support the existence of a definitive erythroid cell population that contributes only adult RBCs in this species.