Separation of primitive and definitive erythroid cells of the chick embryo

The primitive and definitive erythroid cells of the chick embryo are separated preparatively by means of velocity sedimentation at unit gravity in BSA gradients. Analyses of the hemoglobins contained by the fractionated cells show a segregation of different hemoglobins between the primitive and definitive cells. Studies of the incorporation of [³H]leucine show that the fractionated cells are normal with respect to their protein synthetic activities and that their relative rates of incorporation are markedly different.     

Some observations on the primitive and definitive erythrocytes of the developing chick

Summary The cytological changes in the primitive and definitive erythrocytes of the incubating chick have been followed. Observations have been made on the nucleoli, vital granules, mitochondria,Golgi apparatus, reticulum ofSinigaglia and the reticulation patterns of the basophilic substance. The cells of the primitive and definitive lines are ordinarily readily distinguished from one another. Data are included on the rate of disappearance of the primitive cells from the circulation. They may persist as long as two weeks after hatching. Giant primitive erythrocytes are common during the first week of incubation. The cells have one, two three or four nuclei. The nuclearplasma relationship is maintained somewhere near a constant. These atypical cells are due to aberrations in mitosis. Data on the percentage of mitosis in both types of erythrocytes are also included. The initial activity of the spleen and bone-marrow is reflected in the blood stream. There is a distinct rise in the proportion of young definitive erythrocytes. An attempt is made to correlate the findings ofHall (1934) on the changing affinity of the hemoglobin for oxygen with the changing blood picture. The primitive line does not persist long enough to account for the phenomenon. It is suggested, however, that the hemoglobin of the erythrocytes produced by the yolk sac may differ from that of the cells produced by the spleen and bone-marrow. With Plates I–III.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.     

Changes in intermediate haemoglobins during autoxidation of haemoglobin.

Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.     

Organic phosphates increase the solubility of avian haemoglobin D and embryonic chicken haemoglobin.

The isolated minor haemoglobin fractions (haemoglobin D) of ostrich, chicken and duck haemoglobin, which constitute about 30% of total intracellular haemoglobin, form crystalline aggregates upon deoxygenation at physiological temperature, ionic strength and pH and at haemoglobin concentrations even well below those present in the red cell. The aggregation is reversed by oxygenation, and can be inhibited by addition of organic phosphates or the corresponding major haemoglobin fraction in a stoichiometric ratio of 1:1. Embryonic haemoglobin from chicken has similar characteristics with respect to its solubility. The results indicate close functional homology of alpha D and embryonic pi-chains as well as a novel role for organic phosphates in the regulation of haemoglobin function.     

The cytoskeleton of isolated murine primitive erythrocytes

Summary Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among the cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitve erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.     

The development of vestibulo-ocular circuitry in the chicken embryo.

This article reviews studies of the organization and development of the vestibulo-ocular reflex arc in the chicken embryo. It summarizes some of the principal features that characterize the development of this circuit, including the gradual clustering of motoneurons in the oculomotor nucleus into functionally identifiable motoneuron pools, the patterning of vestibular projection neurons into coherent clusters with specific axonal trajectories and terminations onto the oculomotor motoneuron pools, the reverse order of synapse formation during development (motoneuron to muscle, then vestibular projection neuron to motoneuron), and the selectivity of initial synaptic termination at both the ultimate and penultimate relays within the reflex arc. Reference to studies in other vertebrate species is made to provide a comparative context, and potential mechanisms are discussed that may contribute to the underlying synaptic specificity in this circuit.     

The chicken stathmin gene and its expression in the embryo.

Stathmin, which functions as an intracellular relay in signal transduction pathways, has been suggested as a potential indicator of pluripotent cells in the early mouse embryo. In this study, chicken stathmin cDNA and genomic DNA were analyzed. In mammals stathmin consists of five exons and four introns; exons 3, 4, and 5 in the mammalian stathmin gene are equivalent to one relatively large exon in the chicken stathmin gene. Introns equivalent to introns 3 and 4 in the mammalian stathmin gene are not present in the counterpart gene in chickens and, although intron 2 was shown to be present in both mammals and birds, it is smaller in the chicken stathmin gene. Despite differences in the genomic organization of the gene and its smaller size in chickens compared with that in humans and mice, similarities in the coding sequences and in the expression of the chicken and mouse stathmin genes at certain stages of embryo development, as determined by whole-mount in situ hybridization experiments, suggest that their products are functional homologues. The argument is thus substantiated for further investigations into the use of regulatory regions of the stathmin gene in a system for the establishment of long-term cultures of germline competent chicken embryonic stem (ES) cells by the selective ablation of differentiated cells in culture using drug selection.     

Sex determination in the chicken embryo.

The chicken embryo represents a suitable model for studying vertebrate sex determination and gonadal sex differentiation. While the basic mechanism of sex determination in birds is still unknown, gonadal morphogenesis is very similar to that in mammals, and most of the genes implicated in mammalian sex determination have avian homologues. However, in the chicken embryo, these genes show some interesting differences in structure or expression patterns to their mammalian counterparts, broadening our understanding of their functions. The novel candidate testis-determining gene in mammals, DMRT1, is also present in the chicken, and is expressed specifically in the embryonic gonads. In chicken embryos, DMRT1 is more highly expressed in the gonads and Müllerian ducts of male embryos than in those of females. Meanwhile, expression of the orphan nuclear receptor, Steroidogenic Factor 1 (SF1) is up-regulated during ovarian differentiation in the chicken embryo. This contrasts with the expression pattern of SF1 in mouse embryos, in which expression is down-regulated during female differentiation. Another orphan receptor initially implicated in mammalian sex determination, DAX1, is poorly conserved in the chicken. A chicken DAX1 homologue isolated from a urogenital ridge library lacked the unusual DNA-binding motif seen in mammals. Chicken DAX1 is autosomal, and is expressed in the embryonic gonads, showing somewhat higher expression in female compared to male gonads, as in mammals. However, expression is not down-regulated at the onset of testicular differentiation in chicken embryos, as occurs in mice. These comparative data shed light on vertebrate sex determination in general.     

The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2‐expressing populations using Tie2‐Cre;Rosa26R‐EYFP mice. In Tie2‐Cre;Rosa26R‐EYFP mice, analysis of bone marrow cells showed Cre‐mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ～ 84% of each lineage was EYFP⁺, and nearly all cells that come from Tie2‐expressing lineages are CD45⁺, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2⁺ origin. Our findings of high levels of Tie2‐Cre recombination in the hematopoietic lineage have implications for the use of the Tie2‐Cre mouse as a lineage‐restricted driver strain. genesis 48:563–567, 2010. © 2010 Wiley‐Liss, Inc.     

Comparative study of the erythrocytes and haemoglobins in nototheniid fishes from Antarctica

The blood of seven Antarctic nototheniid species and representatives from three other families contained low haemoglobin concentrations compared with non-polar marine teleosts. Haematocrit values were slightly lower than values from other teleosts, while haemoglobin and erythrocyte counts were substantially reduced by comparison. Interspecific variation in haemoglobin concentration seemed to be a function of activity level. Mean corpuscular haemoglobin concentration of all Antarctic species was strikingly low by comparison with species from lower latitudes and was not correlated with the habits of the species. Haemoglobin componentry was compared using celluslose-acetate electrophoresis and, unlike many temperate species, only one major haemoprotein was isolated from each benthic species, but four components were evident in the pelagic species Trematomus borchgrevinki . The possible functional significance of these findings was discussed in relation to the ecology of each species.     

Taurine uptake in chicken leukocytes and erythrocytes.

1. The intracellular taurine concentration and rate of taurine uptake of chicken erythrocytes and two leukocyte populations were determined from one to six weeks of age. 2. Plasma taurine concentrations increased significantly from the time of hatching to week 2 and remained constant thereafter. 3. Intracellular taurine concentrations in both leukocyte populations increased significantly with age without any significant change in the erythrocytes. 4. Taurine uptake rate for erythrocytes was significantly higher at weeks 1-3 while both leukocyte populations showed no significant change during the six week period studied.