Osteoclast precursor cells are present in the blood of preossification chick embryos

In an attempt to ascertain the time of appearance of circulating osteoclast precursor cells, we have transplanted quail bone rudiments into or onto the extraembryonic membranes of variously aged chick embryos. We observed that osteoclast precursor cells (1) are present in the embryonic circulation prior to the onset of osteogenesis, (2) differentiate precociously in response to a factor or factors present in developing bone rudiments, and (3) increase in number until about midway in embryonic life.     

Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus

Experiments involving sequential transplantations of the chick embryonic thymus at E9 to E12 into a first 3-day host quail embryo and then into a second chick host allowed demonstration of the cyclic periodicity of hemopoietic cell seeding of the embryonic thymus. After a first wave of colonization occurring between E6.5 and E8, the thymus becomes refractory to hemopoietic cell entry for about 4 days. It resumes its capacity to be seeded by a second wave of blood-borne stem cells at E12. After a second period of non receptivity starting at E14, a third wave of incoming cells reaches the thymus around E18. Therefore, with a slightly different periodicity, the same cyclic mechanism regulates the renewal of lymphocytes in chick and quail embryos. Quail hemopoietic cells were immunostained in the chimeric thymuses, with a species specific monoclonal antibody (anti-MB1) which recognizes a common surface antigenic determinant on all endothelial and blood cells of the quail (except erythrocytes). Two steps could thus be distinguished in the seeding process. When the thymus becomes receptive for hemopoietic cells, the latter first accumulate in the intrathymic blood vessels before penetrating massively in the thymic parenchyma. The quail chick-chimera system combined with the use of a species- and cell-type-specific antibody provides a unique tool for studying thymic colonization by lymphocyte precursors.     

Erythropoiesis in normal and mutant chick embryos

Hemoglobin D_Davis (Hb D_D), an autosomal codominant in chickens, the α^D-globin chain of Hb M of primitive cells and Hb D of definitive erythrocytes. Erythropoiesis and Hb synthesis was investigated in normal, heterozygous, and homozygous Hb D_D mutant embryos (stages 15–44) and adults. The time of appearance, morphology, relationships to developmental changes, and number of primitive and definitive cells were determined. Primitive hemoglobins between stages 17 and 44 showed four components, P1, P2, E, and M (or M_D), on high-resolution isoelectric focusing gels. Comparison of $\text{P1}\text{P2}$ ratios in the four phenotypes indicated that homozygous Hb D_D embryos had an increased proportion of Hb P2 relative to Hb P1 between stages 17 and 35. This difference coincided with an increase in the number of large primitive cells. In all phenotypes the proportions of primitive hemoglobins decreased after stage 25 and they were not detected after stage 40. Basophilic definitive erythroblasts were present in cell suspensions from all phenotypes between stages 24 and 25. Hb A, the major Hb and Hb D, the minor Hb, of definitive cells of embryos and adults were detected by isoelectric focusing of lysates by stage 29. Definitive cells from late embryos of all phenotypes had higher proportions of Hb D (or Hb D_D) than did red cells from corresponding adult birds. Heterozygous Hb D_D embroys and adults had both Hb D and Hb D_D. Hb D_D comprises about 30% of the total minor Hb rather than 50% expected for heterozygosity at a single locus. In this respect heterozygous Hb D_D chick embryos and adult birds are similar to certain heterozygous α-chain variants in humans. A minor Hb, H, found in lysates of later embryos disappears in lysates of normal chicks 65 days after hatching, but was present in the circulation of homozygous Hb D_D chicks until at least 195 days after hatching. Additionally, several minor Hb components which may be asymmetrical hybrids or derived precursors of Hb A and Hb D (or Hb D_D) were observed. This study provides the precise developmental stages when the switchover of erythroid cell populations and hemoglobins in the chick embryo occurs. This is the first investigation of an α-globin chain mutant which is synthesized during all stages of red cell development and may be a useful animal model for the study of hemoglobinopathies in vertebrates.     

Dihydroorotic acid dehydrogenase activity in actinomycin-D-treated and normal chick embryos

The effect of actinomycin D on chick embryos cultivated in vitro by New's culturing method was studied. Exposure of chick embryos to actinomycin D (0.05 micrograms/ml) at the primitive streak stage (stage 4; Hamburger and Hamilton) for 6 h showed interference in orotic acid formation. The assay of the enzyme dihydroorotic acid dehydrogenase was carried out in both treated and control embryos. No enzymic activity was observed in actinomycin-D-treated embryos in contrast to the considerable activity in the controls. These observations suggest an interference by actinomycin D in the biogenesis of the enzyme dihydroorotic acid dehydrogenase.     

Lipid composition of brain myelin from normal and hyperphenylalaninemic chick embryos

The influence of hyperphenylalaninemia on the lipid composition of brain myelin has been investigated in 19-day-old chick embryos. CNP-ase activity was used as myelin marker enzyme for myelin isolation. CNP-ase activity was significantly lower in hyperphenylalaninemic myelin when compared with control. No significant differences were observed after experimental treatment in the total lipid content of myelin as well as in the proportion of cholesterol:phospholipid:galactolipid. Nevertheless, a clear increase in the percentage of esterified cholesterol was found. No appreciable alterations were observed in the phospholipid composition of brain myelin from both control and hyperphenylalaninemic chick embryos. However, the ratio of unsaturated to saturated fatty acids in serine plasmalogen and sphingomyelin was considerably increased by this treatment. This ratio in choline and ethanolamine phosphatides from treated embryos did not differ from that of controls.     

The effects of methylxanthines on catecholamine-stimulated and normal chick embryos.

Dose of theophylline and caffeine which do not produce aortic arch anomalies in embryonic chicks have been shown to potentiate catecholamine-induced aortic arch malformations in that experimental animal. Theophylline (2.1 X 10(-5) mole per milliliter isotonic saline solution) potentiated the effective dose of norepinephrine more than 100 times. The greatest potentiation observed with epinephrine (2.5 X) was induced by 2.6 X 10(-5) mole caffeine. This study also demonstrated that both methylxanthines specifically induce aneurysms of the ascending aorta and complete absence (or nearly complete constriction) of the right ductus arteriosus. The incidences of these types of cardiovascular malformations proved to be dose dependent with theophylline a more potent teratogen than caffeine. The mobilization of calcium and/or cyclic nucleotide phosphodiesterase inhibition by the methylxanthines are suggested as significant actions in the potentiation of catecholamine-induced aortic arch anomalies.     

Vascular system in the developing wing bud of normal and talpid mutant chick embryos

The vascular limb mesoderm probably plays a prominent role in limb pattern formation in both normal and talpid chick embryos. The differential vascularization, or its consequences, is a factor controlling the initial stages of differentiation in the developing bud. The axial artery runs from the subclavian artery to the distal region of the normal limb bud, whereas in the talpid3 only secondary blood vessels develop. In the talpid, the gene permits the chondrogenic regions to grow and at the same time keeps the peripheral regions to a certain size. The mesenchyme tissue lies within the effective range of a metabolic gradient extending from either the ectodermal surface or the peripheral vessels to the limb axis.     

Subcellular localization of low-molecular RNA in the cells of chick embryos

The subcellular distribution of chick embryo low molecular weight RNAs has been studied by the thermal phenol fractionation procedure. The major part of 8SII RNA, earlier discovered in some oncornaviruses and normal cells, was extracted by phenol at 4%. Our results thus give evidence of the cytoplasmic localization of this RNA. Another part of 8SII RNA, which has been extracted at 65 degrees, is thought to consist of newly synthesized molecules located in the nuclei. A similarity in subcellular distribution of low molecular weight RNAs from chick embryo and rat liver has been obtained, i. e. such a distribution may be of universal nature.     

Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos.

Studies in Xenopus laevis and zebrafish suggest a key role for beta-catenin in the specification of the axis of bilateral symmetry. In these organisms, nuclear beta-catenin demarcates the dorsalizing centers. We have asked whether beta-catenin plays a comparable role in the chick embryo and how it is adapted to the particular developmental constraints of chick development. The first nuclear localization of beta-catenin is observed in late intrauterine stages of development in the periphery of the blastoderm, the developing area opaca and marginal zone. Obviously, this early, radially symmetric domain does not predict the future organizing center of the embryo. During further development, cells containing nuclear beta-catenin spread under the epiblast and form the secondary hypoblast. The onset of hypoblast formation thus demarcates the first bilateral symmetry in nuclear beta-catenin distribution. Lithium chloride exposure also causes ectopic nuclear localization of beta-catenin in cells of the epiblast in the area pellucida. Embryos treated before primitive streak formation become completely radialized, as shown by the expression of molecular markers, CMIX and GSC. Lithium treatments performed during early or medium streak stages cause excessive development of the anterior primitive streak, node and notochord, and lead to a degeneration of prospective ventral and posterior structures, as shown by the expression of the molecular markers GSC, CNOT1, BMP2 and Ch-Tbx6L. In summary, we found that in spite of remarkable spatiotemporal differences, beta-catenin acts in the chick in a manner similar to that in fish and amphibia.     

Mesenchymal precursor cells in the blood of normal individuals

Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).     

The formation of multivesicular structures in the adipose cells of chick embryos

Observation of the cytogenesis of adipose tissue of the chick embryo revealed a quantity of multiversicular structures (MVs) which were found in the intercellular space. Some of them were attached to the adipocytes and others were independently located in the intercellular space. The origin of those MVs appeared to be part of the degenerating mitochondria. Centrally located vesicles and vacuoles in degenerating mitochondria formed a group of short tubules and vacuoles which protruded through the cytoplasmic membrane or bulged out at the edge of the cytoplasmic process. The MVs then spread over the cytoplasmic membrane and finally were discharged from the cell surface as in the manner of apocrine secretion. An invisible barrier between the mass of vesicles and the rest of the cytoplasmic structures appeared to segregate the extruding MVs from the intercellular components such as ribosomes, microtubules, and microfilaments.     

Evidence for the presence of androgen receptors in human Leydig cells

Localization of androgen receptors (ARs) in the human testis Leydig cells was examined with an AR assay and Northern blot analysis. Leydig cells, highly purified on a Percoll gradient, were used for the experiments. AR concentration in the total cell extract containing both the cytosol and nuclear fractions in Leydig cells was measured using [3H]methyltrienolone. ARs in Leydig cells showed a high affinity for [3H]methyltrienolone and the Kd and Bmax of the receptors were 1.24 nM and 11.7 fmol/mg protein, respectively. Northern blot analysis, using a 32P-labeled full-length human AR complementary DNA (cDNA) detected a 9.5-kb hybridizing band in the total RNA extracted from Leydig cells. These data can be interpreted as evidence of the existence of ARs in human Leydig cells.     

Evidence for the association of prolyl hydroxylase from chick embryos with membranes through membrane-bound procollagen.

Proline hydroxylase of membrane pellets from tissues of chick embryo was solubilized by several extraction mediums. Three kinds of enzyme linkage with membranes were pointed out: a labile and non specific adsorption, a specific linkage on endogenous procollagen which is linked with the membranes, and a part of membranes themselves. Our results suggest that, in the liver, these three kinds of linkage exist. Whereas, in tibia bones only labile linkages appear by non specific adsorption or through the link with membrane procollagen.     

Studies on the differentiation capacity of neural epithelium cells in chick embryos

Cell and Tissue Research -