Calcium and neurulation in mammalian embryos. II. Effects of cytoskeletal inhibitors and calcium antagonists on the neural folds of rat embryos.

The role of calcium in neurulation in mammalian embryos has been studied by culturing rat embryos at 10.4 days of gestation, when the cephalic neural folds have elevated but not fused, in serum containing cytoskeletal inhibitors or calcium antagonists. The effects of these antagonists on the morphology of the cephalic neural folds have been examined by scanning electron microscopy. The different agents caused the cephalic neural folds to part to varying degrees. The neural folds were classified as intact (normal), open (folds parted up to 90 degrees with each other), flattened (folds parted from 90 degrees to 180 degrees) or collapsed (folds parted more than 180 degrees). The microtubule inhibitors colchicine and nocodazole at 10(-4) M respectively cause the cephalic neural folds of 10.4-day embryos to collapse after 60 min. At 5.2 X 10(-6)M the microfilament inhibitor cytochalasin B causes the folds to open after 60 min. Longer term culture of 9.5-day embryos for 24 h in diazepam, which is reported to inhibit myosin synthesis, causes general developmental retardation including a delay in the closure of the neural tube. Culture of 10.4-day rat embryos for 60 min in papaverine at 2.4 X 10(-4) M or gallopamil (D-600) at 5.0 X 10(-4) M, agents which reduce the entry of calcium into cells, causes opening of the elevated cephalic neural folds. In contrast TMB-8, which is purported to perturb some intracellular calcium-dependent functions, does not cause opening of the elevated cephalic neural folds, even at high concentrations. The results suggest that both microtubules and microfilaments are essential to the maintenance of the elevated cephalic neural folds in rat embryos. The results are also compatible with the idea that calcium ion flux across the membranes of the neuroepithelial cells might be important for the elevation of the neural folds, and thus for successful neurulation.

Ionophore-induced cell shape changes in Xenopus early embryos.

Local application of the Ca++ ionophore A23187 to the intact lateral ectoderm of Xenopus early neurulae causes changes in the shapes of the cells; ectoderm cells lose their relatively flat surfaces and become rounded. Some of the affected cells form microvilli. Ionophore was found to induce cell shape changes in ectoderm in the presence of cytochalasin-B, suggesting that microfilaments are not involved. Ionophore was also found to induce cell shape changes in neurula ectoderm when it was applied to embryos cultured in Ca++- and Mg++-free medium containing EDTA, suggesting that extracellular Ca++ is not utilized in the ionophore-induced cell shape changes. Similarly, the Ca++ antagonists D-600, which reduces the entry of Ca++ into cells, and TMB-8, which antagonises certain intracellular Ca++-dependent functions, did not inhibit the effects of A23187 on amphibian embryos.

Effects of calcium antagonists and calcium-buffered salines on wound healing in Xenopus early embryos.

The role of calcium in the process of wound closure in Xenopus early embryos was studied. Embryos were wounded in the presence of the calcium antagonists D-600 and TMB-8 or in calcium-buffered salines, and the effects on wound healing were observed by scanning electron microscopy. D-600 and TMB-8 inhibit wound closure and these antagonists appear to act synergistically since their combined effect is greater than their individual effects. Experiments with calcium-buffered salines suggest that wound closure can proceed in the presence of low extracellular calcium. In all conditions there is a correlation between the degree of wound closure and the shapes of the cells at the wound margin; closing wounds are accompanied by cells elongated radial to the wound, gaping (non-closing) wounds are accompanied by cells stretched tangential to the wound. Thus the results suggest that calcium influx may not be a requirement for the changes in cell shape which accompany, and probably effect, wound closure in Xenopus early embryos.

Calcium and neurulation in mammalian embryos.

The role of calcium in neurulation in rat embryos has been studied. Rat embryos at 10 X 4 days of gestation, when the cephalic neural folds have elevated but not fused, have been cultured in various media, and the effects of these media on the morphology of the cephalic neural folds have been observed by scanning and transmission electron microscopy. Embryos cultured in serum containing EDTA or EGTA, or in saline without divalent cations exhibit opening, then folding back ('collapse') of the cephalic neural folds. The neural cells lose their elongated shape and become rounded. Older embryos in which the cephalic neural folds have already fused do not show collapse of the neural tube. Culture of 10 X 4-day rat embryos with elevated but unfused cephalic neural folds in calcium- and magnesium-free saline to which either calcium or magnesium has been restored shows that calcium is the divalent cation which is essential for the maintenance of the elevated neural folds. In the presence of calcium, lanthanum, which competes for calcium sites, causes opening but not collapse of the elevated cephalic neural folds. Embryos treated with trypsin show dissociation of the lateral (non-neural) ectoderm but the neural folds remain elevated. If embryos in which the cephalic neural folds have been caused to collapse are further cultured in serum the folds re-elevate, although normal neural tube morphology is not completely regained. The possible implications of these observations to the understanding of the cellular mechanisms of normal neurulation, and of neural tube malformations are discussed.

Scanning electron microscopy of wound healing in rat embryos.

Wound healing in rat early embryos has been studied by scanning electron microscopy. Initially the wound gapes slightly and cells peripheral to the wound assume a cobble-stone appearance. Wound closure is quite rapid; some small wounds are almost closed within 10 min of incision. Wound closure is accompanied by the appearance of some elongated cells at the wound edge. These features are similar to, although less pronounced than, those which have been observed to accompany wound closure in amphibian and avian embryos. Healing of wounds made in the amnion is also accompanied by changes in the shapes of cells at the wound margins. Wound healing in embryos cultured in Hank's saline is similar to wound healing in embryos cultured in serum, suggesting that the macromolecular components of serum are not essential to wound healing. Cytochalasin B, which inhibits wound closure in amphibian embryos, does not inhibit wound healing in rat early embryos unless used at a concentration high enough to cause cell dissociation. Similarly chelation of the free calcium in the medium, which also prevents wound closure in amphibian embryos, does not inhibit wound closure unless the embryo is dissociating. Removal of free calcium does however cause collapse of the elevated neural folds. These observations suggest that the cellular mechanisms involved in wound healing are different in mammalian and amphibian embryos.