Changes in erythrocyte membrane antigens in developing fetal and postnatal mice.

A fetal antigen was detected by immunofluorescence on fetal erythrocytes of mice. The expression of this antigen decreases rapidly after birth and is no longer detectable 48 days later. Another antigen, called immature erythrocyte antigen, was detected on immature erythrocytes and appeared to be lost during cell maturation. The number of cells expressing this antigen reflects the mean age of the erythron at a given time. From the kinetics of variation of these antigens, it was concluded that: (1) The first cells lacking the embryonic antigen (adult cells?) were detected at birth; and (2) immature cells bearing the embryonic antigen were still produced after birth. The presence of this embryonic antigen before and after birth allows us to postulate the existence of a fetal erythropoiesis as observed in other species, although fetal hemoglobin has not been clearly demonstrated in the mouse.     

Antigenic characterization of chick erythrocytes and erythropoietic precursors: identification of several definitive populations during embryogenesis.

During ontogenesis of the chick, three normocyte populations can be resolved on the basis of their membrane antigens. Embryonic erythrocytes bear an embryonic antigen (E₁) which is not present on adult red cells. Conversely an adult antigen (A) is detected only on adult red cells. A third population bearing simultaneously both antigens develops transiently during the first month after hatching. These normocyte antigens were investigated at the level of erythrocytic stem cell identified by in vivo and in vitro assays. The antigens can be revealed only on erythrocytic precursors CFU-cE giving rise to erythrocytic clones in vitro in presence of avian erythropoietin. Three CFU-cE populations were defined therefrom. In marrow, all these populations are present in the embryo but only CFU-cE bearing A antigen subsist after hatching.     

Respiration and glycolysis in the liver of developing chick embryos and chicks]

Oxygen uptake in liver slices remains constant between the 12th and the 17th days of embryonic development, being equal to that in 30-60-day chicks. During the transition from allantoic respiration to the pulmonary one, oxygen consumption decreases, the decrease being observed up to the end of embryonic period. After hatching, oxygen consumption increases 4-5-fold to the 6-7th and decreases up to the initial level at the 10th day. Respiration of mitochondria isolated from the liver and concentration of cytochromes in mitochondria remain constant. The value P/O is the lowest, whereas catalase activity is the highest during hatching. The intensity of anaerobic glycolysis changes similarly to that of respiration.     

Keratan sulfate proteoglycan during embryonic development of the chicken cornea

Antibodies to corneal keratan sulfate proteoglycan (KSPG) were used to characterize the pattern of KSPG accumulation during differentiation of neural crest cells in the stroma of embryonic chick cornea. Immunohistochemistry with monoclonal antibody I22 to keratan sulfate found this KSPG antigen localized inside stromal cells at stage 29 (Day 6), ca. 12 hr after migration into the primary stroma. A 2- to 3-day lag then occurred before appearance of extracellular keratan sulfate, first seen on Day 9 (Stage 35) in the posterior stroma. Keratan sulfate antigen accumulated in a posterior to anterior direction during subsequent development. Uniform staining of the stroma for keratan sulfate did not occur until after Day 16. Among several tissues, only corneal stroma contained an extracellular matrix which stained for keratan sulfate, though intracellular staining of some cartilage cells was observed. Accumulation of KSPG antigens in developing cornea was measured in unfractionated guanidine extracts with a quantitative ELISA using three different antibodies against KSPG. Increases were first detected after Day 9 using monoclonal I22, and somewhat later with the other two antibodies. Assays with all three antibodies detected a sustained, exponential increase of KSPG throughout the 5 days prior to hatching. Keratan sulfate continued to accumulate after hatching, but an antibody with specificity to KSPG core protein, detected no relative increase in antigen after hatching. This suggests a modulation of KSPG primary structure late in development and after hatching. Overt differentiation of individual neural crest cells thus appears to begin ca. 12 hr after their arrival in the primary stroma; a lag of 2-3 days precedes active secretion of KSPG.     

Variation of DNA Polymerase Activities in Chick Neural Retina as a Function of Age

The activities of DNA polymerases α, β, and γ and of thymidine kinase were determined in the chick neural retina at different stages of embryonic development (starting at seven days) and after hatching (up to five years). Crude extracts of neural retinae were fractionated by centrifugation on sucrose gradients and the enzymatic activities measured using specific assays. The DNA polymerase a activity decreases greatly between 7 and 11 days of incubation. This decrease parallels the decline in mitotic activity. However, a constant residual activity remains after hatching, even in the oldest animals. DNA polymerase β activity increases slightly between 7 and 14 days of incubation; it then decreases slowly until seven days after hatching and remains constant thereafter. DNA polymerase γ activity is maximal between 7 and 14 days of incubation and then decreases until hatching. The activity of thymidine kinase increases slightly during the embryonic life until hatching and remains almost constant thereafter. The implication of these enzymes in DNA replication and repair processes is discussed.     

Variation of DNA polymerase activities in chick neural retina as a function of age

The activities of DNA polymerases alpha, beta, and gamma and of thymidine kinase were determined in the chick neural retina at different stages of embryonic development (starting at seven days) and after hatching (up to five years). Crude extracts of neural retinae were fractionated by centrifugation on sucrose gradients and the enzymatic activities measured using specific assays. The DNA polymerase alpha activity decreases greatly between 7 and 11 days of incubation. This decrease parallels the decline in mitotic activity. However, a constant residual activity remains after hatching, even in the oldest animals. DNA polymerase beta activity increases slightly between 7 and 14 days of incubation; it then decreases slowly until seven days after hatching and remains constant thereafter. DNA polymerase gamma activity is maximal between 7 and 14 days of incubation and then decreases until hatching. The activity of thymidine kinase increases slightly during the embryonic life until hatching and remains almost constant thereafter. The implication of these enzymes in DNA replication and repair processes is discussed.     

Changes of DNA Ligases in Chick Neural Retina as a Function of Age

In the course of chick neural retina development, several forms of DNA ligase have been found. During embryonic life the major DNA ligase activity that is found at seven days is form I (8.2 S) which gradually decreases and disappears by 14 days after incubation, whereas form II (6.2 S) increases to reach a maximum at the time of hatching. Form II then decreases reaching a constant level by Day 7 and from that time new slow sedimenting forms also appear (forms III and IV). Form III (2 S) is first detectable at seven days and increases up to 90 days, whereas form IV (3 S) is the only form detected in the 17- and 18-month-old and also in the 5-year-old birds. These four forms display different elution patterns on phosphocellulose column chromatography. They also differ in their thermal stability and sensitivity towards N-ethylmaleimide.     

Changes of DNA ligases in chick neural retina as a function of age

In the course of chick neural retina development, several forms of DNA ligase have been found. During embryonic life the major DNA ligase activity that is found at seven days is form I (8.2 S) which gradually decreases and disappears by 14 days after incubation, whereas form II (6.2 S) increases to reach a maximum at the time of hatching. Form II then decreases reaching a constant level by Day 7 and from that time new slow sedimenting forms also appear (forms III and IV). Form III(2 S) is first detectable at seven days and increases up to 90 days, whereas form IV (3 S) is the only form detected in the 17- and 18-month-old and also in the 5-year-old birds. These four forms display different elution patterns on phosphocellulose column chromatography. They also differ in their thermal stability and sensitivity towards N-ethylmaleimide.     

Early immunohistochemical detection of somatostatin-like and methionine-enkephablin-like neuropeptides in the brain of the migratory locust embryo

The two groups of neurosecretory cells producing neuropeptides related to somatostatin (SRIF) and methionine-enkephalin (met-enkephalin), previously high-lighted in the brain of adult migratory locusts, were detected by immunofluorescent techniques during the embryonic development of these insects. The earliest detection of these neurosecretory products occurred firstly in the terminal arborizations, then in the fibres, and finally in the perikarya. SRIF-like material is present in the corpora cardiaca already four days before hatching, i.e. at two-thirds of embryonic life, whereas immunoreactivity can be detected only after hatching in the perikarya located in the pars intercerebralis. The synthesis of met-enkephalin-like neuropeptide starts in the four cells of this system at least two days before hatching as shown by the immunofluorescence in the terminal arborizations along the tractus I to the corpora cardiaca. SRIF-like and met-enkephalin-like neurosecretory products are synthesized and carried to their release areas whilst the formation of brain structures and of the corpora cardiaca has not yet been completed.     

Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development

Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO₂ strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.     

Phosphorylation and degradation of ribosomes in starved Tetrahymena pyriformis.

We have characterized an embryonic antigen on the surface of chick erythrocytes using immunochemical electron microscopy. An indirect surface labeling technique (hemocyanin conjugated to goat antirabbit IgG and specific antisera prepared in rabbits) revealed that the antigenic sites, at hatching, nearly saturate the surface of erythrocytes with hemocyanin markers. The number of antigenic sites gradually decreases with age, and the antigen can no longer be detected at 7 months. Further, the antigen has been detected on the very earliest primitive erythrocytes which form in the extra-embryonic mesenchyme before circulation begins. The embryonic antigen appears to be firmly associated with the erythrocyte surface and cannot be removed by extensive washing either with phosphate-buffered saline or with EDTA. Labeling unfixed cells at 37 °C produces clustering of the surface markers, suggesting that the antigen is associated with a membrane component which is fairly free to move in the plane of the membrane. In addition, the erythrocytes from newly hatched chicks were found to agglutinate more readily with several different lectins, particularly Concanavalin A (ConA), than did the erythrocytes from adults. Three times more ConA is bound to chick erythrocytes than to adult erythrocytes, as estimated by electron microscopy. Although this difference in lectin binding suggests that the ConA-binding sites might be related to the embryonic antigen, the sugars known to block lectin-induced hemagglutination had no blocking effect on antiserum-induced agglutination or on antibody binding, as visualized by the electron microscope technique. Also, ConA binding was not inhibited by treatment of the chick erythrocytes with the specific antiserum.     

Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle

The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.     

Developmental changes in vitamin A level and lack of retinyl palmitate in chick lungs

1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation.     

The hemoglobins of the developing chicken embryos. Fractionation and globin composition of the individual component of total erythrocytes and of a single erythrocyte type.

The hemoglobins of the chicken embryo at several stages of development have been isolated in pure form by column chromatography and their relative amounts and globin compositions determined. The analyses on separated primitive and definitive erythrocytes show that the first contain four hemoglobins different from the adult ones. The two major ones at four days, decrease gradually and are no longer detectable from 15 days on. The two minor ones increase up to 6-7 days, then decrease but are still present at hatching. The definitive embryonic erythrocytes contain two hemoglobins identical to the adult ones but their ratios change gradually during development and approach that of the adult hemoglobins at hatching.     

Sex-related differences in the expression of chick enterocyte butyrylcholinesterase during embryonic and post-hatching development

Summary The butyrylcholinesterase activity of chick enterocytes was studied from day 15 in ovo up to day 90 after hatching. The activities detected in both sexes at the level of jejuno-ileum change in a parallel manner, but the activity is always higher in the female than in the male during embryonic development. After hatching, the differences are less apparent although the study of the enzyme distribution along the intestine showed sex-related variations, mainly at the level of the anterior and middle parts of jejuno-ileum in the young adult. Analysis of butyrylcholinesterase by sucrose gradient centrifugation allowed to identify two globular soluble species (G₁ and G₄ forms). The G₄/(G₁ + G₄) ratio decreases during the development but this variation in the female does not parallel that observed in the male. Besides, the molecular form distribution along the intestine, studied after hatching, differs according to the sex. Taken together our results lead to hypothesize that the ontogeny and the regulation of the chick enterocyte butyrylcholinesterase depend on hormones.Abbreviations AChE Acetylcholinesterase - BuChE Butyrylcholinesterase     

Ontogenetic changes of circulating erythroid cells and haemoglobin components in Esox lucius

Using light microscopy the morphology, the mitotic index and levels of erythroid cell types were detected from 48 h pike Esox lucius embryos before hatching to adult specimens. At the same developmental stages, the haemoglobins and globin chains expressed were electrophoretically characterized. The erythroid cells of the primitive generation were the most abundant from 48 h before hatching until 15–20 days after hatching, then their number decreased and only rare cells remained in the 3 month‐old juvenile specimens. These cells divided and differentiated in the blood and were substituted by the definitive erythrocyte series. As in other vertebrates, the immature cells of the two generations differed in morphological properties and in the synthetized haemoglobin. The circulating erythroid cells of the definitive population cell lineage were, at all differentiation stages, smaller than those of the primitive generation. The definitive erythrocytes appeared in blood smears of 7 days post‐hatching larvae, they increased rapidly and at 20 days they represented the predominant red blood cell population in the circulation of young pike. Electrophoretic analysis of haemolysates obtained from different developmental stages indicated the presence of distinct embryonic, larval and adult haemoglobins. The embryonic haemoglobins differed from those of the older larva and juvenile specimens and were detectable within the first week of post‐hatching development when only primitive erythrocytes were present in the blood.     

Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle

Three myosin heavy chain isoforms with unique peptide maps appear sequentially in the development of the chicken pectoralis major muscle. An embryonic isoform is expressed early and throughout development in the embryo. A second isoform appears just after hatching and predominates by 10 days ex ovo. A third isoform, indistinguishable from adult myosin heavy chain, predominates by 8 weeks after hatching. This sequence of myosin isoform change does not, however, appear during myogenesis in vitro. In cultures prepared from embryonic myoblasts only embryonic myosin heavy chain is expressed. This is true even in cultures maintained for 30 days. Myosin light chain expression also changes in vivo with a progressive increase in fast light chain 3 accumulation. In vitro, however, this shift to increasing fast light chain 3 accumulation does not occur. The results indicate that the myosin heavy chain and light chain pattern observed in vitro is identical to that of the embryonic muscle and that the conditions necessary for the shift in expression to a more mature myosin phenotype are not present in myogenic cultures. These cultures are therefore potentially of great value in probing further the neural and humoral determinants of muscle fiber maturation and growth.     

Ontogeny, regional distribution and properties of thyroid-hormone receptors in the developing chick brain

Studies on the thyroid-hormone receptors in the nuclei of developing chick brain revealed a single class of binding sites for tri-iodothyronine (T3) and thyroxine (T4) at all embryonic and adult ages. High-affinity [Ka = (1.85-3.3) X 10(9)M-1 and (0.3-0.6 X 10(9)M-1 for T3 and T4 respectively] receptors were detected in the brain as early as day 7 of embryonic development; their level increased progressively rapidly until day 13, and thereafter the value remained essentially constant during development. Occupancy of the receptor site with endogenous hormone was 75-90% at 7-11 days, 50-60% during the late phase of embryogenesis (13-17 days), and 80% after hatching. Comparison of the binding properties of the receptors with T3 and T4 indicates that, although the binding capacities per nucleus are almost identical, T4 has four to five times less binding affinity than T3. The half-lives of dissociation of solubilized T3- receptor complexes were 20-30h between 0 degrees and 7 degrees C, about 4h at 20 degrees C and less than 15 min at 37 degrees C. Studies of the regional distribution of receptors in the brain indicate that cerebrum has the highest concentration of T3 receptors (4000-7000 sites per nucleus); this concentration is 2-4-fold higher than that in the cerebellum, optic lobe or medulla oblongata. The overall results indicate that between 7 and 13 days of embryonic development the thyroid-hormone receptors in the embryonic chick brain, particularly in the cerebrum, assume a very high level and appear to be mostly saturated with endogenous hormone. This, and the temporal correspondence of the phenomenon with the period of neuronal growth and synaptogenesis, strongly indicate the influence of the hormone in the maturation of the developing brain.     

Embryonic-like myosin heavy chains in regenerating chicken muscle

An antibody to chicken ventricular myosin was found to cross-react by enzyme immunoassay with myosin heavy chains from embryonic chicken pectorials, but not with adult skeletal myosins. This antibody, which was previously shown to label cultured muscle cells from embryonic pectoralis (Cantini et al., J cell biol 85 (1981) 903), was used to investigate by indirect immunofluorescence the reactivity of chicken skeletal muscle cells differentiating in vivo during embryonic development and muscle regeneration. Muscle fibers in 11-day old chick embryonic pectoralis and anterior latissimus dorsi muscles showed a differential reactivity with this antibody. Labelled fibers progressively decreasgd in number during subsequent stages and disappeared completely around hatching. Only rare small muscle fibers, some of which had the shape and location typical of satellite elements, were labelled in adult chicken muscle. A cold injury was produced with dry ice in the fast pectoralis and the slow anterior latissimys dorsi muscles of young chickens. Two days after injury a number of labelled cells was first seen in the intermediate region between the outer necrotic area and the underlying uninjured muscle. These muscle cells rapidly increased in number and size, thin myotubes were seen after 3 days and by 4–5 days a superficial layer of brightly stained newly formed muscle fibers was observed at the site of the injury. Between one and two weeks after the lesion the intensity of staining of regenerated fibers progressively decreased as their size further increased. These findings indicate that an embryonic type of myosin heavy chain is transitorily expressed during muscle regeneration.     

Immunohistochemical analysis of proteoglycan biosynthesis during early development of the chicken cornea.

Antibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion.