Expression of adult globin mRNAs in hamster yolk-sac erythroid cells during ontogeny.

The embryonic-to-adult globin switch begins in intact yolk-sac erythroid cells (YSEC) of hamster embryos 2 days after initiation of erythropoiesis in the yolk-sac blood islands ("early" ontogeny), and is nearly completed 4 days later ("late" ontogeny). Rates of adult globin-chain synthesis, alpha and beta major, in intact YSEC are low in early ontogeny and peak in late ontogeny. The opposite phenomenon is observed when RNA procured from YSEC during the same ontogenic time interval is translated in cell-free wheat-germ extract. That is, peak rates of adult globin chain synthesis are observed in early ontogeny and decline thereafter. This information led us to search for, and find, a translational regulator, apparently protein, in YSEC cytoplasm that suppresses translation of adult globin mRNA in early ontogeny, thus explaining the discordant in vivo (intact cells) and in vitro (cell free) rates of synthesis of adult globins. To further characterize the globin switch at the level of RNA transcription, we quantified adult globin mRNAs (alpha and beta major) from YSEC on consecutive days of gestation by nuclear runoff, slot blot and Northern blot assays. Results of nuclear runoff assays showed maximal rates of transcription of alpha and beta major globin mRNAs in early ontogeny which gradually declined to near zero by late ontogeny. Results of slot blots and Northern blots showed that amounts of alpha and beta major globin mRNAs per YSEC were high in early ontogeny and gradually declined to near zero by late ontogeny. It is apparent that the embryonic-to-adult globin switch in intact YSEC correlates neither with the transcriptional activity of the adult globin genes nor with rates of adult globin-chain synthesis as assayed by cell-free translation of globin mRNA. These studies now provide information at the transcriptional level (1) complements the cited translational repressor work and (2) demonstrates that adult globin transcription and translation are pronouncedly discordant in hamster YSEC undergoing globin-chain ontogeny in the embryonic circulation.

Cloning of two adult hamster globin cDNAs (alpha and beta major).

A cDNA library was prepared from poly(A) mRNA extracted from adult anemic hamster spleen erythroid cells. cDNA clones containing inserts coding for adult alpha and beta major globin chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNA and (b) nucleotide sequence analysis of the inserts and comparison to the adult globin cDNAs of mouse, rabbit and human. Availability of sequences for embryonic (Li et al. (1992) Biochim. Biophys. Acta 1130, 218-220) and adult globin cDNAs (this report) will aid in investigations of the molecular mechanisms involved in the globin ontogeny of hamsters.

Cloning and sequence analysis of two embryonic beta-like globin cDNAs (y and z) of hamster.

A cDNA library was prepared from poly(A) mRNA extracted from 9-day hamster-yolk-sac erythroid cells. Two clones containing inserts coding for embryonic beta-like z or y globin-chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNAs and (b) nucleotide sequence analysis of the inserts and comparison to the embryonic beta-like globin genes of Balb/c mouse. Availability of sequences for embryonic and adult globin cDNAs will aid in investigations of the molecular mechanisms of the globin switch in hamster YSEC.

Translational control of globin chain ontogeny in hamster yolk sac erythroid cells.

Prior research has demonstrated that globin ontogeny of hamster proceeds nearly to completion during the several days that yolk sac erythroid cells (YSEC) circulate in the embryo; synthesis of embryonic globin chains gives way to synthesis of adult globin chains in these primitive cells. In the present study, we translated total cell RNA extracted from YSEC on days 9-13 of gestation in wheat germ cell-free extract, expecting to observe the same progressive rise that occurs in vivo in rates of translation of alpha- and beta-globin mRNA during ontogeny. The opposite occurred; translation rates of both globins decreased sharply. This disparity between synthesis of alpha- and beta-globins in vivo and in vitro suggested an element of control of translation attributable to the YSEC cytoplasm. We therefore assayed the effect of RNA-free clarified YSEC cytoplasm on cell-free translation of YSEC RNA. A repression of translation was detected of alpha- and beta-globin mRNA (not of embryonic globin mRNA), exercised strongly by cytoplasm from YSEC early in ontogeny (gestational day 9), and weakening as ontogeny progressed. The same effect was noted on alpha- and beta-globin mRNA of adult hamster and of rabbit. Heat treatment of cytoplasm abolished the greater part of the translation regulation, suggesting that the active agent is protein. Further characterization of this translational regulator included: (a) it binds to globin poly(A) mRNA but not to poly(A), (b) it was not detected in cell lysate of adult hamster brain, lung, or erythrocytes, and (c) it did not inhibit translation of adult hamster brain and liver RNA. We conclude that hamster globin ontogeny is substantially modulated by this translational regulation of alpha- and beta-globin expression.

Erythropoietin. Receptor characteristics during the ontogeny of hamster yolk sac erythroid cells.

Erythropoietin (Epo) binds specifically to receptors on the surface membrane of responsive erythroid cells. In search of ontogenic changes in Epo receptor behavior, we studied characteristics of specific binding to hamster yolk sac erythroid cells during hamster ontogeny. We detected receptors specific for Epo on these cells throughout the duration of their intravascular existence (hamster gestational days 8 through 13). These receptors are saturable at an Epo concentration of 1.2 nM in the incubation medium. Attainment of equilibrium of binding prior to hormone internalization, a requirement for receptor binding assays, was possible at 10 degrees C but not at 37 degrees C. Hence, all incubations of cells with Epo were carried out at 10 degrees C. Data on specific binding analyzed by the method of Scatchard demonstrated that yolk sac erythroid cells possess a single class of Epo receptors at each stage of gestation examined. Binding affinity and numbers of receptors per cell change as ontogeny progresses: Kd (the dissociation constant) increases, a phenomenon observed in other differentiating cell systems, whereas the number of receptors per cell peaks on gestational day 10. The variability in number of receptors per cell is consonant with up and down regulation controlled by Epo availability. We propose that the progressive increase in Kd might be best explained by ontogenic changes in cell membrane structure contiguous to the receptors themselves.

The globin gene expression program in the hamster embryo.

Molecular mechanisms involved in control of globin gene expression are a prominent target in current basic biologic research. A better understanding of these mechanisms might also impinge on a clinical goal: amelioration of the human hemoglobinopathies. Recent reports have established the coexistence of embryonic and adult globins in rodent yolk-sac erythroid cells, raising the possibility that globin ontogeny takes place in these cells. The present study was undertaken to define the extent of this putative ontogenic process. We measured daily rates of synthesis of individual globins in hamster yolk-sac erythroid cells from the earliest day in gestation that these cells are available (day 7) until the day they cease to replicate (day 13). Converted to a per-cell basis, the rates demonstrate an ontogenic progression in globin synthesis, from embryonic globins to adult globins, that encompasses nearly entirely the total globin ontogeny of this mammal. Synthesis of adult alpha globin is already detectable on day 7, whereas synthesis of the two adult beta globins does not appear until day 9. Synthesis of embryonic y globin stands in contrast to that of the other two embryonic globins (x and z), rising as they fall, a phenomenon reminiscent of the gamma globin of primates and certain ruminants. This physiologic primitive erythroid cell appears to be an unusually appropriate target for studies directed at globin ontogeny.

Ontogeny of hamster hemoglobins in yolk-sac erythroid cells in vivo and in culture.

During mammalian hemoglobin ontogeny, synthesis of the earliest globin chains (embryonic) is ultimately replaced by synthesis of globin chains (adult) characteristic of the fully formed organism. Elements of control of initiation, progression, and completion of globin-chain ontogeny are poorly understood. In search of a cell culture system in which ontogeny might be studied under closely controlled experimental conditions, we chose erythroid cells of the hamster embryo. First, the ontogeny of globin chains was defined in these yolk-sac-derived erythroid cells from day 10 through day 13 in gestation. Amounts of individual embryonic and adult globin chains were quantified, as were their rates of synthesis. Next, analogous studies were performed on yolk-sac erythroid cells from day 10 in gestation (prior to the appearance of fetal liver) grown in culture for 3 days, corresponding to days 10-13 in vivo. The ontogenic program in culture was virtually identical to that in vivo. Approximately 70% of active globin synthesis was embryonic at day 10 in gestation (day 0 of culture), declining to 30% by day 13 in gestation (day 3 of culture). Whereas only trace synthesis of the adult non-alpha chains (beta major and beta minor) were initially observed, their combined active synthesis achieved a level of approximately 30% 3 days later both in vivo and in culture. Cell hemoglobin content and cell morphology were similar in both systems. We conclude that an ontogenic program for globin-chain synthesis exists in these primitive erythroid cells, overriding possible influences of cell environment. Further, we suggest that these cells in culture provide a means of examining cell mechanisms associated with globin-gene ontogeny under controlled experimental conditions.

Studies on globin chain synthesis during hamster development.

Erythroid cells derived from the yolk-sac of the hamster embryos replicate in the embryonic circulation, actively synthesize hemoglobin, and are the dominant circulating erythroid cell through day 14 of gestation. Until day 11, at which time fetal liver rudiments appear, they are the only erythroid tissue present in the embryo. Because of their size (Fig. 1 and Table 1) they can be separated from erythrocytes produced by ontogenically later hemopoietic tissue (liver, spleen, bone marrow). The embryonic (three) and adult (three) globin chains of hamster hemoglobins separate cleanly by electrophoresis in polyacrylamide gel, (Fig. 2). This provides an advantage over mouse, whose x and z chains tend to co-migrate. During hamster embryonic development, yolk-sac erythroid cells first become available on day 7. Nine days later (birth), globin-chain ontogeny in the circulating blood is nearly complete (Fig. 3). When yolk-sac-derived erythroid cells are isolated at day 10 in gestation (prior to the appearance of fetal liver) and grown in culture for 3 days, changes in types of globin chains synthesized are identical to globin-chain ontogenic changes in vivo during gestational days 10 through 13 (Fig. 5). We conclude that the ontogenic "switch" of globin-gene expression in these erythroid cells is pre-programmed, and that the program is capable of full function despite a drastic change in cell environment from circulating blood to culture medium. The ability to study these primitive erythroid cells under the tightly controllable conditions of culture might facilitate acquisition of information on mechanisms of globin-gene ontogeny.

Simultaneous expression of globin genes for embryonic and adult hemoglobins during mammalian ontogeny.

The dominant hemoglobin of the adult hamster was detected in yolk-sac erythroid cells, and its identity was confirmed by peptide mapping and by analysis of relevant peptides. Both the presence and active synthesis of two embryonic hemoglobins presumed to exist only in yolk-sac erythroid cells were detected in neonatal liver and spleen. Thus the time span of expression of both embryonic and adult globin genes during mammalian ontogeny may be considerably broader than presently believed.

Intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and cell-free solutions.

We have measured the intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and in cell-free solutions, and have compared the results with similar data for liganded normal adult hemoglobin. The experiments involve the measurement of the spin-lattice relaxation time T1 of protons of solvent water molecules, as a function of an externally applied static magnetic field. From such data, one can derive a correlation time tauc, for each sample, which is a measure of the time taken for a hemoglobin molecule to randomize its orientation due to Brownian motion. Thus tauc is a measure of the freedom of rotational motion, on a molecular or microscopic level, of hemoglobin molecules. Intermolecular interactions will reduce this freedom of motion and lengthen tauc. We find that oxygenated sickle hemoglobin molecules have an additional intermolecular interaction not found for normal hemoglobin. This extra interaction is increased by the presence of either inorganic phosphate or diphosphoglycerate, and is greater for sickle hemoglobin within cells than in cell-free solutions. By comparing the present results with published data on the viscosity of oxygenated sickle and normal hemoglobin, we conclude that, at concentrations comparable to intracellular values, oxygenated sickle hemoglobin molecules form aggregates several tetramers in size. The possibility exists that these aggregates are the earliest stage of fiber formation itself, the physical basis of the sickling phenomena.