Cholinoreception of chick embryo myocardial explants]

The effects of acetylcholine, subecholine (selective nicotinomimetic) and methylfurmetide (selective muscarinomimetic) on the spontaneous contractions of atrial and ventricular explants from the heart of 408 day embryos as well as of mixed explants, have been studied by means of a special photoelectric device. In most of the experiments, acetylcholine reduced the rate of contractions, the sensitivity of preparations being increased with the age of embryos. Preliminary atropine treatment reversed or significantly decreased acetylcholine effect. Subecholine usually increased the contraction rhythm, whereas methylfurmetide decreased the spontaneous activity.     

Contraction-relaxation dynamics and mechanical restitution in the developing myocardium of the chick embryo

Parameters of the contraction-relaxation cycle and mechanical restitution (MR) were assessed in isolated ventricular preparations of 3- and 4-day chick embryos (EM) and posthatched (PH) chicks. Ryanodine reduced the relaxation rate in the EM but increased it in the PH chicks. It also suppressed a rest-induced potentiation and the MR in all the preparations. Low Na superfusion significantly suppressed the relaxation and decreased the rest potentiation in the myocardial preparations all ages. The findings substantiate existence of a sarcolemmal Ca pool which participates in regulation of twitch parameters and Ca outflux via the Na-Ca exchange.     

Tissue explants affecting extension and orientation of axons in cultured chick embryo ganglia

Various ganglia from 10-day-old chick embryos were cultured for 3 days in substrata of hydrated collagen lattices. Each ganglion was surrounded at a distance of about 1 mm by three tissue expiants which were identical in one series of cultures and taken from different organs in another. The extent of axon outgrowth towards the different explants was estimated by counting intersections between the axons and test lines arranged perpendicularly to the radial outgrowth direction. The various organs stimulated axon formation to distinctly different extents. Spinal cord, skeletal muscle, skin, liver, colon, kidney and heart had, in that order, increasingly stimulative influence on sympathetic chain ganglia. Colon, followed by heart and liver, had the strongest stimulative effect on Remak's colon ganglion. Spinal and trigeminal ganglia showed dense outgrowth of fibroblast-like cells and were not included in the calculations. However, they appeared to be stimulated to extend axons by exposure to heart explants. The results imply that the tissue explants release various amounts of stimulative factors that reach the ganglia by diffusion. When presented to different tissue explants, the same ganglion showed different extents of outgrowth towards the various tissues. Also, ganglia showed dense outgrowth of axons directed towards inserted capillary tubes containing nerve growth factor. The courses taken by the axons as revealed in silver impregnated whole mounted ganglia suggest that chemotaxis can account for the directed axon outgrowth.     

Muscle and cartilage differentiation in small and large explants from the chick embryo limb bud

Larger fragments of prospective chondrogenic or myogenic limb bud mesenchyme of the 4-day chick embryo differentiated primarily into cartilage in organ culture. Muscle sometimes was present in the peripheral areas adjacent to the larger central masses of cartilage. When the individual fragments of limb bud mesenchyme were cut into four smaller pieces and grown in organ culture, cartilage did not differentiate but muscle was present.Autoradiographic experiments with labeled thymidine and quantitative experiments with ³H-adenosine revealed a marked stimulation of DNA and RNA synthesis in the smaller explants, compared to the larger masses of limb bud mesenchyme.     

Induction of blood cells in Xenopus embryo explants

 A Xenopus-specific anti-leukocyte monoclonal antibody designated XL-2 was isolated and used to identify leukocytes in tailbud embryos and activin A-treated explants of blastula animal cap. XL-2 bound to a 135-kDa polypeptide in western blots of protein extracts from adult thymocytes, tailbud embryos, tadpoles, and explants. In cell suspensions, it immunostained the cell surface of all types of adult leukocytes including lymphocytes, monocyte/macrophages, thrombocytes, and granulocytes. At embryonic stage 24, immunocytochemistry revealed XL-2-positive leukocytes, the earliest time at which such cells have been recognized. Whole-mount staining of tailbud embryos and tadpoles showed a widely dispersed population of XL-2-reactive leukocytes, many of which had elongated shapes and ameboid pseudopodia. In activin A-treated animal caps, XL-2 recognized a subpopulation of cells within the lumen of the central fluid-filled cavity as well as cells in the interstitium of mesenchymal and mesothelial components of the explant. Together, activin A and human interleukin-11 induced 100% of explants to form lumenal blood cells. Compared to activin A alone, murine stem cell factor plus activin A significantly increased the numbers of XL-2-reactive leukocytes and erythrocytes. These results support the view that activin A induces leukocyte and erythrocyte progenitors during Xenopus embryogenesis. Received: 29 August 1997 / Accepted: 28 October 1997     

On auxotrophy for pyrimidines of respiration-deficient chick embryo cells

Chick embryo cells treated with chloramphenicol are inherently resistant to the growth-inhibitory effect of the drug when cultured in the presence of tryptose phosphate broth. The cells were found to be auxotrophic for pyrimidines and the presence in the broth of compounds of pyrimidine origin is demonstrated by chromatographic procedures and mass spectral analyses. They are in the form of ribonucleosides, ribonucleotides and pyrimidine-containing oligoribonucleotides. To understand the mechanism responsible for pyrimidine auxotrophy, the activity of enzymes involved in the pyrimidine biosynthetic pathway was determined. Measurement of the conversion of dihydroorotic acid to orotic acid in cell-free extracts revealed that chloramphenicol-treated chick embryo cells are deficient in dihydroorotate dehydrogenase activity. The data in vitro are supported by studies on the nutritional requirements of the respiration-deficient cells and by the incorporation in vivo of labelled dihydroorotic acid into the acid-insoluble fraction of the cells. Although the activity of the dehydrogenase in vitro is decreased by 95%, the enzyme is present in chloramphenicol-treated cells and its activity is unmasked by the artificial electron acceptor menadione. A study of the activity of other enzymes of the pyrimidine biosynthetic pathway demonstrated that their activity is comparable to that in control cells. The present results indicate that auxotrophy for pyrimidines results from the inhibition of the flow of electrons along the mitochondrial electron transport chain.     

Equilibrium-driven mechanism for preferential adhesion between chick embryo cells

The experiments presented here confirm the hypothesis according to which, in our experimental system of differential cell adhesion (where we studied the kinetics of the earliest period of adhesion of a suspension of chick embryo neuroblasts to layers of astroblasts or fibroblasts), the mechanism of adhesion appears to consist of two steps, the first of which is a short-term reversible phase corresponding to a binding equilibrium. In fact, adhesion of neuroblasts to each of the two cell layers occurs according to kinetic constants and attains levels which are characteristic for each of the two adhesion systems. In both systems, neuroblasts that have not adhered at equilibrium are able to adhere if inoculated over a fresh cell layer of the same type, as they do during the first inoculation; conversely, neuroblasts that have adhered to a cell layer can be made to de-adhere by substituting cell-free fresh medium to the inoculation medium containing non-adhering neuroblasts. This shows that, as predicted for a reversible equilibrium system, removal of adhering neuroblasts from the system at equilibrium provokes adhesion, and removal of non-adhered neuroblasts provokes de-adhesion. Furthermore the level of adhesion at equilibrium is, in all cases, the same. The reversibility of adhesion, which is almost quantitative during the onset of the equilibrium, gradually decreases with time, indicating the presence of a process of irreversible attachment between cells after the first reversible step. The developmental implications of the complete sequential mechanisms are briefly discussed.