Caudalization by retinoic acid is correlated with inhibition of cell population growth and expansion of chick blastoderms cultured in vitro.

Full primitive streak stage chick embryos, cultured in vitro for 20 h in the presence of 10(-9) to 10(-7) moles of retinoic acid (retinol, all-trans), exhibit increasing extent of malformations. RA causes caudalization, suppression of telencephalon, formation of open and enlarged neural tube in the regions of midbrain, hindbrain and spinal cord, failure of fusion of heart tubes, and gives rise to winged or diffused, and even supernumerary somites. Extreme abnormalities include failure to form the head fold and foregut. Abnormal embryos were graded according to Rao and Chauhan (Teratology 4: 191-198, 1971), and we find that the larger the severity of malformation, the smaller the size of total cell population and blastoderm area. Concomitant to caudalization, retinoic acid suppresses the cell population growth.

Multiple neural inductions in area opaca by grafts of the Hensen's node do not retard host chick embryo development.

Four hundred and forty four full primitive streak stage chick embryos were cultured in vitro and 261 were transplanted with 1, 3 or 5 Hensen's nodes in the area opaca. Irrespective of the number of grafts, neural induction was observed in 90% cases. The development of control and grafted embryos and the size of blastoderm area were monitored at the time of grafting and after 20 hr. We find that the induced neural tissue and differentiated tissue of graft-origin neither fuse with the host embryonic axis, nor retard its development.

Cell population growth in chick blastoderms cultured in vitro.

Primitive streak stage chick blastoderms were cultured in vitro up to 30 hr by New's technique. Chick blastoderms reaching stages 4 to 12 in vitro cultures and in ovo were harvested and homogenized to release cell nuclei. Fluorescent ethidium bromide-stained nuclei in homogenates were counted in Neubauer's chamber and the size of total blastoderm cell population was determined. Linear regression analysis revealed that both in ovo and in vitro chick blastoderm cell population grows in a biphasic manner with comparable cell population doubling times and the morphogenesis is not affected in vitro during the culture period.

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.

Effects of cytochalasin H on chick embryo explants cultured in vitro.

Effects of the mould metabolite, cytochalasin H (CH) were studied in chick embryos explanted at stages 4, 5 and 8 and cultured in vitro. Morphological and histological observations revealed that the major effects of CH are inhibition of primary morphogenesis of heart and disaggregation of cells. Inhibition of neural tube closure, microcephaly and shortening of body axis was also noted. The effects were found to be dose- and stage-dependent. Histochemical studies demonstrated that CH brings about a reduction in the contents of glycoproteins and non-sulfated glycosaminoglycans important in cell adhesion. Effects of CH appeared due to its interference primarily with cell adhesion and secondarily with morphogenetic movements.

Inhibitory effect of cytochalasin H on cell reaggregation of the fresh water sponge, Ephydatia meyeni Carter.

Cytochalasin H (CH) has been shown to inhibit the reaggregation of dissociated cells of a fresh water sponge. The effect is dose-dependent and reversible. Even after 24 h of CH treatment, the cells show the formation of pseudopodia suggesting that the effect of CH may not be due to arrested cell motility. Exogenously added alpha-D-glucosamine, a precursor of complex carbohydrates, brings about reaggregation of CH-treated cells. Fluorescent Con A binding, both patching and capping, is shown by untreated as well as CH-treated cells. It is likely that CH, like cytochalasin B, may interfere with the production, release or binding of carbohydrate-containing macromolecules important in cell aggregation.

Ultrastructural changes in the embryonic cells of the frog Microhyla ornata after cytochalasin H treatment.

Biological effects of cytochalasin H (CH), a newly isolated mould metabolite, have been found to bring about disaggregation of embryonic cells and to inhibit cytokinesis. Disaggregation is known to be a phenomenon related to the cell surface. (The cells are held together by a mucopolysaccharide glycoprotein complex.) In the present work the fact that the mucopolysaccharide glycoprotein surface coat gets affected by CH treatment is confirmed by electron microscopy with the help of Lanthanum, a specific marker, which gets selectively absorbed to the cell surface material and renders it electron dense. The ultrastructural observations indicated the reduction of the cell surface material in treated embryos as compared to the controls. The reappearance of lanthanum-bound cell surface material in the recovered embryos was also observed. However, the exact mechanism of the action of CH on the cell surface remains to be clarified.

Effect of urethane on folate contents of chick embryos measured with a microbial assay.

In earlier studies it was found that the effects of urethane could be reversed by folic acid and thymidine but not by uridine. It was further shown that urethane-treated chick embryos were unable to convert serine to glycine, homocysteine to methionine and uridine to thymidine, all of which are folate-dependent reactions. Due to the sensitivity of folic acid auxotrophs towards small amounts of folates present in the biological material, the folic acid contents of urethane-trewated and untreated chick embryos are studied, using microbial assay. It is found that there is a statistically significant reduction in the total folate content of chick embryos treated with urethane as compared to those from the untreated controls. It is conluded that urethane has an antifolate action.

Effects of cytochalasin H on the embryonic endodermal cells of Microhyla ornata.

Cytochalasin H (CH) like CB causes disaggregation of embryonic endodermal cells and reduces their adhesivity to the glass surface. These cells reaggregate on removal of the drug from the medium. Reversibility depends on the duration of drug treatment. The mechanism of drug action is explained.