Fig. 6. Overexpression of bmp4, scl and gata1 in bls and clo mutants. Plasmid construct with CMV promoter driving (A) GFP, (B) bmp4, (C) scl and (D) gata1 were micro-injected into wild-type, bls and clo embryos. The injected embryos were fixed at 23 hpf and analyzed by scl, gata1 or globin in situ hybridization. (A) Plasmid expression of GFP did not perturb embryo development and did not alter the expression of scl or gata1 when compared with uninjected embryos. Expression of scl can normally be detected in the posterior ICM of bls embryos (arrowhead), and in 5-10 cells of the wedge region in the anterior ICM of clo embryos (arrowhead with asterisk). (B) Wild-type embryos injected with bmp4 were ventralized, with an expanded ICM that expresses scl and gata1. The arrowhead indicates the wedge region of anterior ICM. Ventralized bls embryos lacked scl- or gata1-expressing cells in the anterior ICM, but the number of scl-positive cells in the posterior ICM was slightly increased (arrowhead with asterisk). Ventralized clo embryos did not express scl or gata1. (C) Four scl injected bls embryos are shown (rsc1-4), each exhibiting representative levels of partial rescue of hematopoietic progenitors (arrowheads). The hematopoietic rescue in scl injected clo embryos was more pronounced (arrowhead with asterisk). (D) Overexpression of gata1 led to normal level of globin transcripts in the anterior ICM of wild-type embryos, but no globin expression was detected in the bls mutants.
