Calcium, microfilaments and morphogenesis.

Morphogenesis, the generation of tissue form, is important not only in the embryogenesis of a new individual, but also because a change in morphogenesis may be involved in the establishment of differences between individuals during evolution. Morphogenetic movements are effected in part by coordinated changes in the shapes of individual cells and over the past decade the cellular organelles responsible for cell shape have been identified as microfilaments and microtubules. In non-embryonic systems the contraction of microfilaments is controlled by the level of intracellular free calcium, and so calcium is implicated as an intermediate control mechanism in morphogenisis. Through techniques which perturb the calcium balance of cells, or which measure calcium ion concentration directly, evidence is accumulating that calcium is involved in morphogenetic movements such as gastrulation and neurulation, and related phenomena such as wound healing. Thus fundamental questions about the control of morphogenesis in embryogenesis and evolution may now be couched in more precise terms of the control of intracellular calcium ion balance.

The effects of mannose on rat embryos grown in vitro.

Rat embryos have been cultured in vitro from 9.5 days of gestation for different times in serum containing mannose, and the embryos have been observed by scanning electron microscopy. Embryos cultured in 3 mg/ml (1.7 X 10(-2) M) or 6 mg/ml (3.3 X 10(-2) M) mannose for 48 h showed inhibition of the expansion of the yolk sac and were smaller than the control embryos. Mannose-treated embryos also showed delayed development according to morphological criteria, and a range of abnormalities including abnormalities of the neural tube. Embryos cultured in 6 mg/ml mannose for 24 h also showed significant inhibition of yolk-sac expansion and were smaller and less advanced than the control embryos. Abnormalities were seen, including a delay in the closure of the neural folds. Abnormalities were also observed in embryos cultured in mannose for 10 h; embryos at the neural groove stage showed irregularities in the neural groove. Mannose did not inhibit the re-elevation of neural folds which had been caused to collapse by exposure to medium containing low calcium. These results are compatible with the idea that mannose retards development and thereby perturbs the morphogenesis of the neural tube.

Adolescents' ideas about the health of the fetus.

OBJECTIVE: To determine the preponderance of ideas in adolescents about the relationship between maternal habits and the health of the fetus. DESIGN: Quantitative survey using a precoded questionnaire, the content of which was derived from the transcripts of interviews and the responses to open-form questionnaires. SETTING: North West Region Health Authority area, UK. PARTICIPANTS: 674 adolescents in British National Curriculum Year 10 (age 14/15) from 6 Community Comprehensive Schools. FINDINGS: Most of the adolescents were aware of the dangers to the fetus of alcohol and smoking, including passive smoking. However, they were less aware of the potential hazards during pregnancy of eggs (Salmonella), soft cheeses (Listeria), liver (Vitamin A excess) or handling cats (Toxoplasmosis). Most of the respondents thought that the optimum time to initiate actions for a healthy pregnancy was when pregnancy had been confirmed, suggesting that the benefits of preconceptual care are not well known. KEY CONCLUSIONS: Adolescents lack knowledge about some of the specific hazards to the fetus of maternal diet and behaviour during pregnancy and are unaware of the importance of the early stages of pregnancy in this context. IMPLICATIONS: There is a need for the provision of education about the importance of a healthy maternal lifestyle before conception and during early pregnancy for adolescents. Midwives may have a proactive role in such education, but should be aware that young people may have specific areas of ignorance and misconceptions which will need addressing.

Outcomes of pregnancy in insulin dependent diabetic women: results of a five year population cohort study.

OBJECTIVE: To monitor pregnancies in women with pre-existent insulin dependent diabetes for pregnancy loss, congenital malformations, and fetal growth in a geographically defined area of north west England. DESIGN: Population cohort study. SETTING: 10 maternity units in Cheshire, Lancashire, and Merseyside which had no regional guidelines for the management of pregnancy in diabetic women. SUBJECTS: 462 pregnancies in 355 women with insulin dependent diabetes from the 10 centres over five years (1990-4 inclusive). MAIN OUTCOME MEASURES: Numbers and rates of miscarriages, stillbirths, and neonatal and postneonatal deaths; prevalence of congenital malformations; birth weight in relation to gestational age. RESULTS: Among 462 pregnancies, 351 (76%) resulted in a liveborn infant, 78 (17%) aborted spontaneously, nine (2%) resulted in stillbirth, and 24 (5%) were terminated. Of the terminations, nine were for congenital malformation. The stillbirth rate was 25.0/1000 total births (95% confidence interval 8.9 to 41.1) compared with a population rate of 5.0/1000, and infant mortality was 19.9/1000 live births (5.3 to 34.6) compared with 6.8/1000. The prevalence of congenital malformations was 94.0/1000 live births (63.5 to 124.5) compared with 9.7/1000 in the general population. When corrected for gestational age, mean birth weight in the sample was 1.3 standard deviations greater than that of infants of non-diabetic mothers. Infants with congenital malformations weighed less than those without. CONCLUSION: In an unselected population the infants of women with pre-existent insulin dependent diabetes mellitus have a 10-fold greater risk of a congenital malformation and a fivefold greater risk of being stillborn than infants in the general population. Further improvements in the management of pregnancy in diabetic women are needed if target of the St Vincent declaration of 1989 is to be met.

Calcium and wound healing in Xenopus early embryos.

The role of calcium in the healing of wounds made in the ectoderm of Xenopus neurulae has been studied. Embryos have been wounded in the presence of calcium inhibitors, and the effects on wound healing observed by scanning electron microscopy. In addition, unwounded embryos have been exposed to a local application of ionophore A23187 to simulate the possible calcium fluxes following wounding. Lanthanum, which competes for calcium channels, inhibits wound healing. EDTA, which binds divalent cations, also inhibits wound healing, but its effect can be reversed by the addition of excess calcium. Local application of ionophore A23187, which promotes transport of calcium across biological membranes, results in a local change in cell shapes. These observations lend support to the hypothesis that wound healing in amphibian early embryos, which is effected by changes in cell shapes similar to those seen in certain examples of normal morphogenesis, is initiated by a local influx of calcium into cells.

Effect of the calmodulin inhibitor R24571 (calmidazolium) on rat embryos cultured in vitro.

The possible effects of inhibition of the calcium-binding protein, calmodulin, on mammalian morphogenesis have been investigated by culturing rat embryos in vitro from 9 1/2 to 11 1/2 days of development in the presence of R24571 (calmidazolium), a specific inhibitor of calmodulin. Embryos cultured in 10(-2) mM R24571 for 48 h show inhibited development and exhibit a range of morphogenetic abnormalities including assymetry and neural tube defects. Embryos exposed to R24571 for the first 24 h of a 48 h culture are more severely affected than embryos exposed to R24571 for the last 24 h.

Effects of colchicine, cytochalasin-B and papaverine on wound healing in Xenopus early embryos.

The effects of colchicine, cytochalasin-B and papaverine on wound healing in Xenopus early embryos have been studied. Colchicine does not prevent wound healing, whereas cytochalasin-B does. Papaverine, under conditions which prevent the completion of neurulation, does not prevent wound healing. A model is given which might explain these observations.

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 puromycin and cycloheximide on properties of cells from Xenopus laevis early embryos.

The effects have been studied of puromycin and cycloheximide on the reaggregation of ectoderm cells dissociated from Xenopus laevis blastulae. Puromycin or cycloheximide can inhibit reaggregation, suggesting that cell reassociation is dependent upon protein synthesis. If the cells are allowed a 3 h 'recovery' period in culture medium following dissociation, before being exposed to either puromycin or cycloheximide, higher concentrations of the inhibitors are required to prevent cell aggregation, suggesting that significant synthesis of the proteins required for reaggregation occurs in the 3 h immediately following dissociation. Lower concentrations of puromycin permit cell reaggregation but reduce the normal formation of cilia. The effects have also been observed of puromycin on the scanning electron microscopical appearance of Xenopus blastula ectoderm cells cultured singly in vitro. Puromycin reduces the normal formation of pseudopodia, suggesting that puromycin might inhibit reaggregation partly by inhibiting cell movement. Puromycin also produces some elongated cells, possibly by inhibition of cytokinesis.

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.

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.

Computer modelling of neural tube defects.

Neurulation, the curling of the neuroepithelium to form the neural tube, is an essential component of the development of animal embryos. Defects of neural tube formation, which occur with an overall frequency of one in 500 human births, are the cause of severe and distressing congenital abnormalities. However, despite the fact that there is increasing information from animal experiments about the mechanisms which effect neural tube formation, much less is known about the fundamental causes of neural tube defects (NTD). The use of computer models provides one way of gaining clues about the ways in which neurulation may be compromised. Here we employ one computer model to examine the robustness of different cellular mechanisms which are thought to contribute to neurulation. The model, modified from that of Odell et al (Odell, G.M., Oster, G., Alberch, P. and Burnside, B., (1981)) mimics neurulation by laterally propagating a wave of apical contraction along an active zone within a ring of cells. We link the results to experimental evidence gained from studies of embryos in which neurulation has been perturbed. The results indicate that alteration of one of the properties of non-neural tissue can delay or inhibit neurulation, supporting the idea, gained from observation of embryos bearing genes which predispose to NTD, that the tissue underlying the neuroepithelium may contribute to the elevation of the neural folds. The results also show that reduction of the contractile properties of a small proportion of the neuroepithelial cell population may have a profound effect on overall tissue profiling. The results suggest that the elevation of the neural folds, and hence successful neurulation, may be vulnerable to relatively minor deficiencies in cell properties.

Scanning electron microscopy of cells isolated from amphibian early embryos.

Cells have been dissociated from Xenopus and Ambystoma late blastulae, allowed to adhere to glass coverslips, and studied by scanning electron microscopy. Xenopus ectoderm cells initially show filopodia; later larger single pseudopodia are formed. Ambystoma ectoderm cells show fewer filopodia than Xenopus ectoderm, but later form pseudopodia. Ectoderm cells of both Xenopus and Ambystoma show links between adjacent cells. Xenopus endoderm cells do not show filopodia initially, but later show large pseudopodia.

Perturbation of in vitro development of rodent embryos by calcium antagonist Quin-2.

Tissue shaping during embryogenesis is driven, at least in part, by changes in the shapes of the cells of the tissue undergoing morphogenesis. These cell shape changes are effected by microfilaments, and appear, as do microfilament-mediated events in non-embryonic cells, to be calcium-dependent. To explore the possible role of calcium in morphogenesis in mammalian embryos, the effects of Quin-2, a calcium-binding agent which can be loaded into cells as the acetomethyl tetraester (Quin-2-AM), on rat embryos cultured in vitro were determined. Quin-2-AM at 5 x 10(-5) M concentration delays the development of, and subsequently causes the abnormal development of, rat embryos cultured in vitro. In addition, Quin-2-AM inhibits the normal re-elevation of the cephalic neural folds which have been caused to collapse by exposure to medium containing a low concentration of calcium. These results suggest that morphogenetic movements, such as neurulation, depend upon an intracellular relocation of calcium, which can be perturbed by the calcium-binding capacity of Quin-2-AM.

The effect of concanavalin-A on the reaggregation of cells dissociated from Xenopus laevis early embryos.

The effects of concanavalin-A on the reaggregation and sorting of cells from Xenopus laevis early embryos have been studied. The results suggest that at high concentrations, concanavalin-A can prevent reaggregation.

The effects of bromodeoxyuridine and bromouracil on regeneration inHydra.

The effects on tentacle regeneration inHydra of two DNA-antimetabolites, bromouracil and bromodeoxyuridine, have been studied. Bromouracil inhibits tentacle regeneration inH. vulgaris andH. viridis; bromodeoxynridine inhibits tentacle regeneration inH. vulgaris but notH. viridis over the range of concentrations studied. Thus these two species appear to differ in their susceptibilities to these drugs, and the drugs may have different efficacies in this system.

Scanning electron microscopy of cells from hydroxyurea-arrested blastulae of Xenopus laevis.

Cells from Xenopus embryos blocked at the blastula stage by treatment with hydroxyurea have been isolated and cultured in vitro. The morphology of these cells has been compared with that of cells from normal embryos using scanning electron microscopy. Cells from such hydroxyurea-blocked embryos do not show the features, or changes in features, in culture shown by cells from normal embryos.