Development of Haemoglobin by De-embryonated Chick Blastoderms Cultured in vitro and the Effect of Abnormal RNA upon its Synthesis

Abstract

ABSTRACT A simple in vitro histogenesis is described in which the protein haemoglobin is synthesized in the developing blood-islands of a de-embryonated blastoderm. The spread of the blastoderm which follows de-embryonation when it is cultured on the vitelline membrane according to the New technique is eliminated by transfer to an agar gel medium. Cell-division and differentiation in the blood-islands was not inhibited to the same extent as it was in the other tissues present. The erythroblasts multiply, differentiate, and synthesize haemoglobin. The RNA base analogue and antimetabolite 8-azaguanine effectively blocks the synthesis of haemoglobin up to but not including developmental stage 8 on the Hamburger-Hamilton scale. Stages 9 and 10 synthesize haemoglobin in the presence of this analogue. The inhibition by 8-azaguanine is in part removed when the normal base guanine is included with it in the medium. The inhibition is not removed to any extent by incubation with guanine either before or after contact with azaguanine. It is concluded that there is a period in developmental stage 8 of only 1 or 2 hours that represents the border line between RNA analogue inhibition and non-inhibition. It is considered that during this period the RNA associated mechanisms or templates for haemoglobin production begin to operate. The amino-acid analogue fluorophenylalanine but not thienylalanine inhibited haemoglobin synthesis. Incubation following de-embryonation in RNAse had little effect on the synthesis. Chloramphenicol proved to be ineffective as an inhibitor of protein synthesis in this system. The data presented are discussed in relation to the sequence in which haemoglobin becomes detectable in the nucleolus, the nucleus, and the cytoplasm of the developing erythroblast. Haemoglobin development is observed directly in the living system and histochemically by the peroxidatic oxidation of o-dianisidine.