Characterization and expression of embryonic globin in the rainbow trout, Oncorhynchus mykiss: Intra-embryonic initiation of erythropoiesis

When fractionated by reverse-phase high performance liquid chromatography ( HPLC), the embryonic hemoglobin of the rainbow trout, Oncorhynchus mykiss, consisted of eight globins different from adult globins in terms of retenti on time. Amino acid sequences of the N-terminal regions of some globins wer e determined. In addition, four cDNA clones for embryonic globins from 10-d ay embryos were isolated (at 15 degrees C), sequenced and the amino acid se quences predicted. In comparison with the sequences of previously character ized globins, they corresponded to two alpha-type and two beta-type globins and therefore were named em.alpha-1, em.alpha-2, em.beta-1 and em.beta-2. The N-terminal 36 amino acids of one (E2) of the embryonic globins isolated by HPLC were identical to those of the sequence deduced from a cDNA, em.be ta-2. The phylogenetic relationship between the embryonic globins and other globins previously reported was discussed. The present study is the first demonstration of amino acid sequences of embryonic globins in a teleost. To understand the initiation of erythropoiesis in the early development of th e rainbow trout, histochemistry using o-dianisidine/hydrogen peroxide, immu nohistochemistry using an antibody against embryonic hemoglobin, and northe rn blotting and whole embryo in situ hybridization using antisense RNA prob e for em.beta-2 were performed. Embryonic globin mRNA, globin and hemoglobi n appeared first in the anterior part of the intermediate cell mass (ICM) l ocated in the median line beneath the notochord of embryos 6-7 days after f ertilization at 15 degrees C (Vernier's stages 16-20). Shortly after that, the expression signal extended to the posterior part of the ICM and spread out laterally to blood islands on the posterior yolk sac. Thus, the initiat ion of erythropoiesis in the early embryo of rainbow trout is intraembryoni c.