Fig. 4

Ectopic expression of foxo3b impairs primitive hematopoiesis mimicking that of eaf1 knockdown. (A) Erythropoiesis was disrupted by injection of foxo3b mRNA. At 52 hpf, O-dianisidine staining showed lower hemoglobin levels at the ventral yolk in embryos injected with foxo3b mRNA (b) as compared with control embryos injected with the same dosage of GFP mRNA (a). At 48 hpf, expression of hbae3 (d) and hbbe1 (f) was reduced in embryos injected with foxo3b mRNA as compared with control embryos injected with GFP mRNA (c and e, respectively); at 24 hpf, expression of alas in the posterior blood island was reduced in embryos injected with foxo3b mRNA (h) as compared with embryos injected with GFP mRNA (g). (B) Semi-quantitative RT-PCR confirmed that the reduction of expression of alas2 in embryos injected with foxo3b mRNA as compared with embryos injected with GFP mRNA, but expression of mesoderm marker myoD was not altered (a); the expression of injected foxo3b mRNA in 26 hpf embryos was confirmed by Western blot using anti-Myc antibody (b). (C) Expression of myeloid markers l-plastin (b) and mpo (d) was reduced in embryos injected with foxo3b mRNA as compared with embryos injected with GFP mRNA (a and c) at 48 hpf. (D) Definitive hematopoiesis was not affected by ectopic expression of foxo3b. No obvious alternations of definitive hematopoiesis markers runx1 (a and b) and c-myb (c and d) were detected in embryos with ectopic expression of foxo3b. (E) The lymphoid markers rag1 and rag2 were not altered after ectopic expression of foxo3b at 4.5 dpf (b versus a; d versus c). foxo3b mRNA, 900 pg/embryo.