Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.     

Immunohistochemical detection of subunit A(M) of lactate dehydrogenase in tissues of the developing chick

Summary The distribution of A(M) subunits of lactate dehydrogenase (mainly LDH₅) in developing muscle, heart, liver, lung, kidney and cartilage tissue of chicken embryos was examined by the indirect fluorescent antibody technique. Antibodies against porcine LDH₅, purified by affinity chromatography, were used for this purpose. In special areas of newly formed myofibrils in somitic myoblasts fluorescence was already detected after 4 days of incubation, and located at the same place in muscle tissue of all advanced developmental stages examined. During the myotube stage of muscle development staining was also located in the peripheral thickened cytoplasma of the myotubes. The myocardium did not exhibit any fluorescent staining in the developmental stages examined. Endocardium, epicardium and pericardium, however, were fluorescent in young developmental stages. The liver showed fluorescence in 5- to 8-day embryos mainly in the endothelial cells of the blood sinusoids. In 9- to 12-day embryos the bile ducts became fluorescent. In lungs after 9- to 12-day development the epithelium and the surrounding tissues of bronchi exhibited strong immunofluorescence. The mesonephros exhibited faint granular fluorescence in tubule-forming cells and their membranes after 4–9 days of incubation. Advanced developmental stages only exhibited fluorescent blood cells. This latter staining is at least partly due to non-specific reactions of blood cell membranes with FITC-conjugated anti-rabbit IgG. Cartilage is characterized by non-specific fluorescence, but in embryos older than 8 days strong granular fluorescence of chondrocytes and staining of the perichondrium distinguished sections treated with anti-LDH₅ antibodies from control sections reacted only with FITC-conjugated anti-rabbit IgG. In addition, strong fluorescent staining was detectable in certain areas of the 5-day neural tube and faint staining in the mucosa of the intestine from embryos older than 10 days.     

ESR measurement of dissolved oxygen in 4-day chick embryo blood

The magnetic interactions between dissolved oxygen molecules and nitroxide radical spin probes lead to broadening of electron spin resonance (ESR) lines. Based on this property we described an ESR methodology to measure PO2 values in 4-day chick embryo and adult human blood. The total blood volume required for the measurement was only 9 microliter. Using this method PO2 for adult human mixed venous blood was found to be 36.7 mmHg. This is within the established clinical range of 25-40 mmHg for mixed venous blood PO2. The range of mean PO2 values for 4-day chick embryo blood was 27.3- 35.0 mmHg. This is the first time that PO2 values have been reported for individual chick embryos at such an early stage.     

Identification of components of phospholipids metabolism in automated phosphate ester chromatography.

Transport accross the cell membrane of 3-O-methylglucose, a non-phosphorylatable glucose analogue, was measured in primary cultures of fibroblasts from 8-, 12- and 16-day chick embryos. Transport of this hexose was found to be 3.5 times and 2 times faster in fibroblasts from 16-day embryos than in fibroblasts from 8- and 12-day embryos, respectively. Compared with 8- and 12-day embryos, the rate of efflux in fibroblasts from 16-day embryos was found to be increased. 3-O-methylglucose transport in these cells did not result in an accumulation of the hexose against a concentration gradient. It was concluded that in fibroblasts from older embryos a facilitated diffusion system for hexose transport was stimulated. Embryo differentiation could be associated with a change in the plasma membrane by increasing either the number or the mobility of the glucose carriers, since the Vmax of the transport system for 3-O-methylglucose increased in fibroblasts from older embryos, while the affinity or Km of the system remained unchanged.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

Ultrastructure of the neural basal lamina in loop-tail mice

Ultrastructural features of the neural basal lamina were studied by means of the tannic acid and ruthenium red techniques in normal and abnormal dysraphic loop-tail mice at 9-11 days of gestation. With ruthenium red, the configuration of the neural basal lamina is similar in both normal and abnormal embryos at 9-11 days. However, differences were detected in the abnormal 9-day embryos processed with tannic acid, as compared with normal littermates. These include irregularities in the lamina rara externa, as well as differences in the staining pattern of the neuroepithelial cell plasma membrane. By 11 days of gestation, the lamina rara externa of the normal embryos shows features similar to those observed in the 9-day abnormal embryos.     

Development of the cerebrospinal fluid in chick embryos. Physicochemical properties.

The development of the physicochemical properties of the cerebrospinal fluid (CSF) was studied in chick embryos from the 9th day of incubation up to hatching. Some of these properties were compared with the corresponding blood or blood plasma properties. During the second half of incubation the CSF pressure rose from 13.2 plus or minus 0.18 mm H2O in 9-day-old embryos to 80.7 plus or minus 0.48 mm H2O just prior to hatching. The critical stages of this development were the 13th to 15th and the 19th to 21st day of incubation. In 13- and 15-day-old embryos, CSF pressure fell sharply after the intracerebral injection of ouabain, but in 19-day embryos it was unaffected. Except for the 15th and 19th incubation day, the CSF pH was always lower than the plasma pH. From the 11th day of incubation up to hatching, the CSF pH fell from 7.36 plus or minus 0.002 to 7.2 plus or minus 0.005. On the 11th and 13th day, specific CSF resistance was higher than plasma resistance, whereas from the 17th incubation day it was significantly lower than the plasma value. During the second half of incubation, specific CSF resistance fell from 1.059 times 10(6) to 0.824 times 10(6) omega mm.m(-1). A difference between the D.C. potential of the venous blood and the CSF appeared for the first time in 15-day-old embryos, the CSF being negative in relation to the blood. By the end of the incubation period this potential difference rose to 10.82 times 0.07 mv.     

Gene activation of alcohol dehydrogenase in Japanese quail and chicken-quail hybrid embryos

No preferential activation of the maternally derived alcohol dehydrogenase (ADH) allele was found in any of the chicken male x Japanese quail female hybrids examined. ADH activity in the liver was, in fact, found to exist in two different cathodal zonal regions on starch gel electropherograms; the zone II bands appeared at day 5 of incubation in the quail embryo (day 6 in the hybrid embryo) and the zone I bands appeared in 9-day quail embryos (10-day hybrid embryos). By day 13 of incubation, only the faster-migrating zone I bands could be detected in both quail and hybrid embryos.     

Changes in hybridizable RNA in winter wheat embryos during germination and vernalization

DNA-RNA hybridization-competition experiments were performedto analyze RNA heterogeneity in wheat embryos during germinationand vernalization. A significant difference was found betweenRNA populations of 24-hr and 3-day germinated embryos, whileminor differences were detected between 3- and 5-day germinatedembryo RNAs and between 20- and 40-day cold-treated embryo RNAs.RNA populations in 30-day cold-treated embryos were significantlydifferent from those in 50-day ones. The RNA species presentin 50-day cold-treated embryos were not similar to those in3- and 5-day germinated embryos. A great portion of the RNAspecies in 3-day germinated embryos was found in 30-day cold-treatedembryos. These results suggested that some new RNA species aresynthesized in wheat embryos during 30 to 50 days of cold treatment. ¹ Present address: Department of Biochemistry, Faculty of PharmaceuticalSciences, Higashi Nippon Gakuen University, Ishikari-Tobetsu,Hokkaido 061–02, Japan. (Received February 21, 1977; )     

Origin of the iris sphincter muscle in chick embryo

The origin of the iridial sphincter muscle in chick embryo was investigated by means of immunohistochemistry. Desmin immunoreactive cells are shown in the mesenchymal stroma overlying the anterior epithelial layer of the iris in 4 1/2-day chick embryos. In 9-11-day chick embryos also some cells of the posterior epithelium near the pupillary margin, and of the iridial lamella show a slighter desmin-immunoreactivity. This finding agrees with a double origin of the iridial sphincter muscle: an early mesenchymal one and a later epithelial other.     

The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.     

The action of cisplatin on the developing meso- and metanephros of chick embryos

Injection of various doses of cisplatin to 2-14-day chick embryos showed that within 2-8 days of incubation cisplatin produces total toxic effect, the number of dead embryos being dependent on a dose of the drug. Within 9-16 days of incubation, i.e. a period when both the mature mesonephros and the developing metanephros are in action, no significant changes were observed in the content of urea and uric acid, the weight of the meso- and metanephros, their water content, and ion content of the blood. Electron microscopic studies revealed no structural changes in the renal tubules. The data obtained suggest that cisplatin does not produce any nephrotoxic effect in chick embryos irrespectively of their age.     

A comparative study of the DNA structural damages and synthesis in embryos of the silkworm Bombyx mori irradiated with gamma rays at different developmental stages]

Studies have been made on formation and reparation of apurine-apyrimidine (AP) regions, monothread DNA ruptures, as well as on inhibition and recovery of DNA synthesis in gamma-irradiated 3- and 7-day embryos of the silkworm which sharply differ in their radiosensitivity. It was shown that in 3-day embryos, the number of AP regions and DNA ruptures immediately after irradiation is significantly higher than in 7-day embryos. DNA synthesis is more radiosensitive in 3-day embryos as compared to that in 7-day ones. Kinetics of postradiation recovery of regions and DNA ruptures in 3- and 7-day embryos is heterogeneous and significantly different. However, radiation inhibition and postradiation recovery of DNA synthesis in irradiated 3- and 7-day embryos are associated mainly with postradiation survival of these embryos.     

Histological changes induced in developing limb buds of C57BL mouse embryos submitted in utero to the combined influence of acetazolamide and cadmium sulphate

Microscopic defects in limb buds of C57BL mouse embryos after the combined teratogenic action of acetazolamide plus cadmium sulphate administered on day 9 of gestation were studied in serial sections. Postaxial deficiencies observed in 12-15-day embryos and affecting preferentially the right forelimbs were classified in nine morphological types according to increasing amounts of missing parts. Type X defect consists of a nearly complete amelia in which all four limbs are represented only by the girdle and proximal end of the stylopod. Type XI abnormality appears as an intermediate reduction affecting the area of digit IV. In addition to modifications of the forelimb bud shape detected from the 10-day stage onwards, observations made 24 and 48 hr after treatment confirmed that the postaxial defects result from an absolute lack of postaxial mesoderm occurring without cell necrosis as a consequence of a postaxial shortening of the apical ectodermal ridge (aer). In 10-day embryos, the latter appears shortened and hypertrophied; it is later fragmented into alternate thick and thin portions in 11-day affected limb buds. These ectodermal changes might account for the genesis of all types of defects observed. Untreated 9-day embryos with 12-25 pairs of somites display a number of asymmetries between their right and left forelimb territories: Until the 19-somite stage, the vascular supply to that area is provided exclusively by the umbilical vein, which is larger on the right side; the initial amount of somatopleural limb mesoderm is greater in the right rudiment and the genesis of its aer is slightly protracted as compared to the left one. These asymmetries might contribute to the right side predominance of the forelimb defects induced by acetazolamide and cadmium.     

Microtubules and filaments in developing axons and optic stalk cells

Fibrous structures have been studied in the developing optic nerve of chick embryos. The first ganglion cell axons (3-day embryos) were of moderate size, with both neurofilaments and microtubules. Subsequently (4- and 5-day embryos), very small axons were also present. In thesc embryos and in the 4-day hatched chick, the density of microtubules fell within the same range for all but the very small axons, which tended to have more microtubules per unit area. Filaments similar to those previously thought to represent neurofilaments in other parts of the embryonic nervous system were present in the early optic stalk cells, calling into question the reliability of identifying early nerve cells on the basis of 'neurofilaments'.     

Nerve growth factor in skeletal tissues of the embryonic chick

Summary This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6–9 of incubation at 12 h intervals, and on days 10–18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24–36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.     

The cholinergic reaction of the amnion in the chick embryo

Studies have been made of the spontaneous contractions of the amnion and acetylcholine sensitivity of amniotic membrane in 8--14-day chick embryos. In 12--14-day embryos, the spontaneous rhythmic contractions were rather rare as compared to those in 8--9-day ones, their frequency being also lower. On the basis of kinetic analysis, it was concluded that both the dissociation constant (K) and the value of Pmax do not exhibit significant changes for tonotropic reaction from the 8th to the 14th day and for chronotropic reaction--from the 8th to the 10th day of incubation. After the 10th day of incubation, dose-effect chronotropic reaction not expressed. The spontaneous activity of the amnion and acetylcholine sensitivity of the amniotic membrane depend on the temperature being maximal at 38 degrees C. Possible regulatory mechanisms of contractile activity in chick amnion are discussed.     

Mechanisms of changes in blood flow distribution between organs in hens during ontogenesis

Anatomical and histological investigations of large arteries feeding head, extremities and chorioallantoic membrane were carried out in 11-, 14-, and 18-day old chicken embryos and in 1- and 40-day old Shaver chickens. The numerical relationships between an arterial diameter and a body mass; a vessel length, its external diameter and a wall thickness, were determined. Our data demonstrate that walls of large arteries in the anterior and the posterior parts of the chicken embryo body are different in quantity, composition, and maturity of their tissue components, determining the lumen size and hemodynamic conditions in the blood bed. The vessel growth velocity have been estimated. Ontogenic widening of gastrocnemius and pectoralis muscle capillary beds and of their supplying trunks, was compared. Calculations using the data on organ portions of cardiac output in chicken embryos show the maintenance of similar liner blood flow velocities in different large arteries suring ontogenesis.