Complete dissipation of coherent clonal growth occurs before gastrulation in mouse epiblast.

Observations on chimaeric mice argue that there must be considerable dispersal and intermingling of the clonal descendants of epiblast founder cells at an early stage in the development of the tissue. However, it has not been established when or how this occurs. Here we have used a genetic marker that enables donor cells to be visualized in situ to examine the early postimplantation distribution of clones obtained by transplanting epiblast founder cells into host blastocysts. We have also determined the spatial relationship between sister cells in non-chimaeric postimplantation epiblast by ionophoretic injection of a fluorescent macromolecule. Both experimental approaches support the conclusion that breakdown of coherent clonal growth accompanies epithelialization of the epiblast and is essentially complete by the onset of gastrulation. Furthermore, the clonal analysis shows that descendants of different epiblast founder cells continue to intermingle extensively well into organogenesis. We suggest that this sustained intermingling of cells in the epithelial epiblast, which does not occur in the adjacent visceral endoderm, depends on cells losing contact with the basal lamina when they divide. These findings have implications both for patterning of the early amniote embryo and for the growth of tall columnar epithelia in general.

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.

Vitamin deficiencies and neural-tube defects: human and animal studies.

The evidence for an association between vitamin deficiency in early pregnancy and the prevalence of neural-tube defects in humans is reviewed, with particular emphasis on the methodological problems encountered in conducting such studies. It is apparent that there is strong evidence for an association between maternal vitamin status and the outcome of pregnancy, but this is not universally accepted, due to ethical and practical constraints on the design of the studies. There are also problems in obtaining precise information from experiments on animals in vivo. By culturing rat embryos in dialysed serum in which the micromolecular component is defined, it has been possible to demonstrate that significant numbers of neural-tube defects can be produced by the deficiency of a single vitamin (inositol), and that multiple vitamin deficiency produces a further significant increase in the frequency of such defects.

Culture media for postimplantation embryos.

The search for an effective culture medium for postimplantation mammalian embryos is considered from two viewpoints: identification of the medium that gives the best development in culture and of the problems associated with formulating a fully defined medium for such embryos. The types of media that have been applied to the culture of mammalian embryos are reviewed and their effectiveness assessed. At present, the media that support the best development of explanted embryos are composed partly or wholly of heat-activated rat serum, prepared by centrifugation of blood immediately after withdrawal from the donor. Experimental approaches to elaborating fully defined media are considered, and progress with identifying the nutrient requirements of postimplantation mammalian embryos in culture is reviewed. Particular attention is given to the use of extensively dialysed serum which, when supplemented with glucose, amino acids, and vitamins, supports good development of midgestation rodent embryos in vitro.

Changes with gestational age in the nutritional requirements of postimplantation rat embryos in culture.

Rat embryos explanted at 9.0, 9.5, and 10.5 days of gestation were cultured for periods of 61, 49, or 45 h, respectively, in extensively dialysed rat serum supplemented with various combinations of glucose, amino acids, and vitamins. Glucose was found to be a necessary and sufficient energy source for embryos of all three ages, and virtually no development took place in its absence. Only the youngest embryos required free amino acids for good development in dialysed serum, whereas at all three ages, vitamin supplementation was necessary. However, lack of vitamins had a much more marked deleterious effect on the younger embryos than on those explanted at 10.5 d. Experiments with media deficient in individual vitamins showed that for normal development, 9.0-d embryos required a number of vitamins--principally pantothenic acid, riboflavin, inositol, folic acid and niacinamide, whereas 10.5-d embryos needed only riboflavin. For embryos explanted at 9.5 d, the position was intermediate, with riboflavin and inositol the most significant vitamins. Inositol deficiency in embryos explanted at 9.5 d produced a characteristic neural tube defect--failure of closure at the level of the hindbrain. Thus it appears that both the range of micromolecular nutrients and the severity of developmental impairment in their absence decrease with advancing gestational age.

Growth and proliferation in mouse parietal yolk sac during whole embryo culture.

Use of the culture techniques for postimplantation rodent embryos, modified by explanting Day 9 or Day 10 embryos with the trophoblast cells removed but the remainder of the parietal yolk sac left intact, resulted in significant expansion of Reichert's membrane, accompanied by a marked increase in the numbers of the adherent parietal endoderm cells which synthesize the membrane. The surface area of the parietal endoderm/Reichert's membrane complex roughly doubled during culture, and the combined protein content of the cells and their basement membrane was also raised after incubation. Parietal endoderm cell numbers, calculated from area and cell density measurements on flattened membranes, increased by 54-190%, depending on the age of the embryo.

The 1986 McCollum award lecture. Fuel-mediated teratogenesis during early organogenesis: the effects of increased concentrations of glucose, ketones, or somatomedin inhibitor during rat embryo culture.

Whole rat embryos were explanted at head-fold, late pre-somite stage (day 9.5 of gestation) and cultured in rat sera varyingly supplemented with glucose (3, 6, 9, or 12 mg/mL), D,L sodium beta-hydroxybutyrate (2, 4, 8, or 16 mM), or both (6 mg/mL D-glucose plus 8 mM beta-hydroxybutyrate). During 48 h culture, increasing glucose alone or beta-hydroxybutyrate alone effected growth retardation and faulty neural and extraneural organogenesis in dose-dependent fashion. Synergistic dysmorphogenic effects occurred when minimally teratogenic concentrations of glucose and beta-hydroxybutyrate were combined. Sera from diabetic animals containing somatomedin inhibitor bioactivity were also able to produce growth retardation and major developmental lesions in presence of amounts of glucose and ketones which of themselves were not teratogenic. Thus, aberrant fuels and fuel-related products can impair growth and organogenesis in early post-implantation embryo. Such fuel-mediated teratogenesis may be multifactorial and include possibilities for synergistic and additive interactions.

Regional and temporal differences in the parietal endoderm of the midgestation mouse embryo.

The morphological appearance of parietal endoderm (PE) cells from ninth to fourteenth day mouse embryos was examined by light and scanning electron microscopy, and their behaviour in vitro was studied using time-lapse video recordings. At all ages, the PE cells proximal to the placenta had a predominantly blebby phenotype whilst, in all but the youngest embryos studied, the distal cells were smooth and rounded. In the older embryos, the cells in the middle and distal regions had numerous short filopodia and were sometimes very large and flattened. Time-lapse studies on tenth day preparations showed intense motile activity (though little net movement) of the PE cells in the proximal region, with relatively little activity in more distal regions. The cell activity was generally less in eleventh day preparations, though it was still significantly greater in the proximal zone than elsewhere. The significance of these observations for understanding the development of the parietal endoderm is discussed.

Abnormalities induced in cultured rat embryos by hyperglycaemia.

Rat embryos explanted at stages between primitive streak and head-fold were exposed in rotating bottle cultures to glucose levels raised by 3-15 mg/ml either throughout a 66-h culture period, or for shorter intervals near the start of culture (with the rest of the culture period at normal glucose levels). A high proportion of embryos explanted at 8 days 20 h or at 9 days and exposed to 12 mg/ml exogenous glucose for only the first or second 8-h periods of a 66-68-h culture were malformed, whereas embryos exposed for the third 8 h were relatively resistant, and embryos exposed later still were unaffected. There were indications that embryos at stages of development corresponding to the second 8-h periods in these cultures were particularly susceptible to hyperglycaemia. Embryos explanted at 9 days exhibited malformations when exposed to 6-9 mg/ml glucose throughout culture, compared with the 12-15 mg/ml shown previously to be necessary to induce anomalies in embryos explanted 12 h later.

Application of post-implantation embryo culture to problems in teratology.

Rat embryos explanted at 9 1/2 or 11 1/2 days of gestation and cultured in rotating bottles were subjected to a number of agents. Embryos explanted at 9 1/2 days (head-fold stage) and incubated for 48 h at 40.5 degrees C were retarded and abnormal and these effects were more severe in embryos cultured at 41 degrees C. Embryos exposed to 40 degrees over this period were superficially normal, but separate head and body protein determinations and careful measurement of the head dimensions showed them to be microcephalic. Addition of 12-15 mg/ml D-glucose to the culture medium of embryos grown in vitro over the same period also produces malformations. This teratogenic action of D-glucose is distinct from but exacerbated by its osmotic effect. Embryos explanted 12 h earlier (i.e. at 9 days of gestation) and cultured for 66 h were more sensitive to excess glucose--abnormalities were produced by 6-9 mg/ml exogenous D-glucose. Embryos explanted at 11 1/2 days of gestation and cultured for 18 h responded to both 10% CO and suboptimal oxygen levels with a reduction in growth rate and an increase in the ratio of lactate produced to glucose consumed, suggesting a shift to less efficient anaerobic energy metabolism.

Nutrient requirements of rat embryos undergoing organogenesis in vitro.

Rat embryos at the head-fold stage were cultured in rotating bottles for 2 days in rat serum extensively dialysed against glucose-free BSS, and to which various energy sources and nutrients were added. Optimum growth and differentiation, virtually indentical with that obtained in while serum and to that seen during the corresponding period in vivo, were obtained with the addition of glucose and certain vitamins: pantothenic acid and riboflavin had a general beneficial effect on development, i-inositol suppressed neural tube defects, and folic acid significantly improved growth of the embryos.

A rotating bottle culture method with continuous replacement of the gas phase.

A 'rotator' culture method is described which provides a continuous flow of oxygenating gas to cultures in rotating bottles. The system maintains constant O2 and CO2 levels in the culture medium throughout the incubation period. It also provides a more stable pH than systems with sealed culture bottles.

The effect of carbon monoxide on glucose metabolism and growth of rat embryos.

Rat embryos at days 11-12 of gestation cultured for 18 hours in vitro respond to reduced oxygen or to elevated levels of CO. Glucose consumption and lactate production increase while the growth rate decreases. The ratio of lactate produced to glucose consumed increases to values characteristic of earlier, more anaerobic, embryos. It is suggested that most of the energy consumed by the embryo goes to support growth and that the change in glucose metabolism results in a decrease in available energy which decreases the growth rate. The response to reduced oxygen levels is more marked than the response to elevated levels of carbon monoxide and we interpret the result to imply that oxygen transport in these embryos is primarily via solution rather than via combination with hemoglobin.

Abnormalities induced in cultured rat embryos by hyperthermia.

Rat embryos were explanted at nine and one-half days of gestation and cultured for 48 hours in rotating bottles containing rat serum and a gas phase, at temperatures of 38, 40, 40.5 and 41 degrees C. The embryo cultured at 40.5 degrees C were retarded and many of them were abnormal, and at 41 degrees C, all the embryos were malformed and retarded. The most frequent abnormalities occurring at both these temperatures were microcephaly and oedema of the pericardium. Development of the embryos cultured at 40 degrees C was similar to that of the controls at 38 degrees C, and superficially they appeared to be normal. However, measurement of the head dimensions, and separate determinations of head and body protein contents showed that the 40 degrees C embryos were microcephalic.

Teratogenic effects of excess glucose on head-fold rat embryos in culture.

Rat embryos explanted at nine and one-half days of gestation (early head-fold stages) were cultured for 48 hours in rotating bottles containing rat serum and a gas phase. Addition of 12-15 mg/ml of D-glucose to the culture medium resulted in severe abnormalities and retarded development in a high proportion of embryos, whether or not the osmolarity increase brought about by the addition of glucose was corrected. The raised osmolarity, however, increased the frequency and severity of malformations observed. L-glucose produced no abnormalities, though embryonic development was retarded.

Growth of opossum embryos in vitro during organogenesis.

Opossum embryos, explanted between primitive streak and late fetal stages, were grown in culture for periods of 20-30 H. Many of the explants had a good heartbeat and blood circulation in embryo and yolk sac after 12 h, and a few after 24 h. Growth of the embryos included formation of the neural tube and body flexures, increase in the number of somites, differentiation of the limbs and digits, and development of the amnion and allantois. Embryos explanted during the last day of gestation showed persistent and vigorous body movements in culture, particularly of the forelimbs, head and tongue.