Jan Langman

Ultrastructural observations on closure of the neural tube in the mouse

SummaryThe fusion of the neural walls in the cephalic part of mouse embryos varying in age from 9 to 20 somites was examined with the electron microscope. In the rhombencephalic region the rim of the neural wall was formed from outside inward by ectodermal surface cells, a row of flattened cells without surface projections and neuroepithelial cells. At the junction of the surface ectoderm and the flat cells were seen large projections containing a cytoplasmic matrix without organelles and previously referred to as “ruffles”. The initial contact between the walls was made by the large cytoplasmic arms and numerous finger-like projections interdigitating with similar projections from the opposite wall. The projections originated from the surface ectoderm and possibly neural crest cells. During further fusion the surface ectoderm cells formed dense membrane specializations, thus establishing a firm contact.The initial contact in the mesencephalon was formed by extensions from the surface ectoderm and was followed by the formation of specialized membrane junctions, as seen between the surface ectoderm in the rhombencephalon. The neuroepithelial cells facing the gap between the neural walls with their apical ends made contact with the cells from the opposing wall by numerous finger-like projections but membrane specializations failed to develop.The closing mechanism in the prosencephalon and anterior neuropore regions differed from the previous areas in that the initial contact was established by the neuroepithelial cells. Only after this contact had been formed did the surface ectoderm cells close the gap. In contrast with the other areas many phagocytosed particles were seen in the prosencephalon and in the region of the anterior neuropore. Many particles from degenerated cells were found inside healthy surrounding cells. Some of these particles contained nuclear material and cytoplasmic organelles.

The influence of excess vitamin A on neural tube closure in the mouse embryo

SummaryThe effect of excess vitamin A on the closure of the neural tube in mouse embryos was examined with light microscopy, transmission and scanning electronmicroscopy. The embryos were treated with the vitamin just before closure of the brain vesicles and examined during the following 24 h, a period during which under normal conditions the brain completely closes.At 18–24 h after treatment the external features of the treated specimens began to differ from those of the controls. In the treated embryos the neural walls folded laterally and became widely separated, whereas those of the controls folded dorsomedially and fused in the midline. Histologically, the first difference between treated and control embryos was noted at two hours after treatment, when large intercellular spaces appeared between the neuroepithelial cells of the treated embryos. These spaces were mainly present between the apical ends of the wedge-shaped neuroepithelial cells. This accumulation of intercellular spaces interfered with the normal morphogenetic movement of the neural walls, which remained convex instead of becoming concave. This convex bending resulted in non-closure of the neural tube.In addition to the appearance of large intercellular spaces some neuroepithelial cells as well as some mesenchymal, endothelial, and surface ectoderm cells showed swelling and degeneration as a result of the vitamin A treatment. This cell degeneration probably contributes to failure of the neural tube to close due to loss of cohesion at the luminal surface and the lack of mesenchymal support needed for the elevation of the neural walls. However, the increase of intercellular spaces at the apical side of the neuroepithelium is in all probability the major cause for the failure of the neural tube to close.

The uptake of 5-bromodeoxyuridine by the chicken embryo and its effects upon growth

SummaryWhen embryonic cells in vitro are exposed to bromodeoxyuridine (BUdR) the duration of exposure can be made to last for several cell generation times. Such exposure is known to prevent embryonic cells undergoing terminal differentiation while leaving cell division and basic cell function unaffected. When BUdR is injected into pregnant mammals, it remains available for incorporation in the DNA for only a fraction of one S phase and causes foetal anomalies that are apparently the result of cell death and a transient slowing of the cell generation time but not of failure of cell lines to differentiate.The objectives of our experiments were to ascertain the availability time of BUdR in the chicken embryo in ovo, to assess its teratogenicity and to examine its effects on the growth of the embryo.When 3H-BUdR (0.02 mg) was injected into the albumen space on day 3 of incubation, subsequent scintillation spectrometry and autoradiography showed that the drug was incorporated into the DNA of the embryo for more than 8 h or more than one cell generation time at this stage of development. On the other hand, a trace amount of tritiated thymidine (3H-TdR) was available for only one hour, the difference being probably due to an expansion of the nucleotide precursor pool in the case of BUdR.The injection of 0.02 mg BUdR on day 3 caused growth retardation as manifested by differences in weight and in DNA content between BUdR and saline treated embryos. The difference in DNA content was evident 24 h after treatment and was probably due in part of the cell necrosis in the developing CNS that began 10 h after injection. Differences in weight did not become apparent until 4 days after treatment and were thus thought to be due to factors other than cell necrosis.On day 11 of incubation, the mortality of BUdR treated embryos became significantly greater than that of controls and many survivors after this time had ventral body wall defects. When treatment was delayed until days 4 or 6, the subsequent development of BUdR and saline treated embryos was indistinguishable. The sensitivity of day 3 was thought to be due to the fact that embryo DNA content quadrupled between days 3 and 4 whereas it only doubled per 24 h period thereafter.

Floxuridine and Its Influence on Postnatal Cerebellar Development

Extract: Human infants with cytomegalovirus and herpes simplex are presently treated with floxuridine (5-FUDR), a DNA synthesis blocking agent. Since in the cerebellum of the newborn infant neuron formation continues for some time after birth, the question arises whether 5-FUDR might cause permanent damage to the cerebellum. In our experiment 21 2-day-old mice were treated by three injections of 5-FUDR (50 mg/kg body weight per injection), and particular attention was given to damage and subsequent repair of the external granular layer, the site of postnatal neuron formation. In the anterior lobes of the vermis most proliferating external granular cells died and repair was minimal. Purkinje cells were dispersed and few if any basket cells were formed. In the posterior lobes, including the uvula, considerable repair of the external granular layer was noted. The final cerebellar architecture, however, was never normal, and heterotopic granule cell nests were found in the molecular layer. In the intermediary lobes repair occurred, but Purkinje cells were found throughout the granular layer. Since the human cerebellum at birth is characterized by a thick external granular layer, it is not impossible that 5-FUDR may also cause disturbances in the human cerebellar architecture and function.Speculation: In the cerebellum of the newborn mouse and newborn infant important neurons are formed after birth. Therapy with drugs interfering with DNA, RNA, or protein synthesis should therefore be avoided. Although repair may occur in the gastrointestinal tract, experiments with mice suggest that repair of damage in the cerebellum is minimal. Whether ectopic cell nests in the molecular layer will start cell proliferation during further life is presently not known.

Fusion of nasal swellings in the mouse embryo. DNA synthesis and histological features.

SummaryFusion between the epithelial linings of the medial and lateral nasal swellings transforms the nasal groove into a primitive nasal cavity and forms an epithelial seam, the nasal fin, in the line of contact. Epithelial contact occurs between a restricted group of opposing epithelial cells; adjacent eithelial cells do not fuse but form the linings of the nasal and oral cavities. After its formation, the epithelial nasal fin regresses and is replaced by mesenchymal cells, except for a small posterior portion which remains as the bucconasal membrane.DNA synthesis at 3 different periods (20, 10 and 5 h) before contact on day 11 3/4 was examined in the fusing epithelia and adjacent non-fusing epithelia. DNA synthetic activity decreased in both regions at successive stages of development. Howerer, the decrease in the presumptive fusing epithelia at 10 and 5 h before contact was noteworthy in that it was significantly greater than in the non-fusing epithelia. In the fusing epithelia this decrease of DNA synthetic activity occurred not only in prospective degenerating cells, but was a general phenomenon involving viable cells also.To analyze the regression of the nasal fin, it was studied in serial sections. The majority of the cells were viable and only few degenerating cells were seen, suggesting that not all cells of the nasal fin undergo necrosis. Since the epithelial cells of the nasal fin always appeared to be separated from the surrounding mesenchymal cells, the transformation of surviving cells into mesenchymal cells appears unlikely. It is postulated that surviving epithelial cells are incorporated into the adjacent epithelia of the primitive oral and nasal cavities.

The immunologic specificity of human lens proteins.

Human lens extract was examined by electrophoretic and immunochemical methods. Paper electrophoresis failed to give any separation of the protein fractions, but electrophoresis in polyacrylamide gel resulted in the appearance of five distinct bands. The agar diffusion as well as the immunoelectrophoretic methods showed the presence of seven precipitin bands, indicating that the human lens contains a minimum of seven antigenic components. When the species specificity of the human lens was examined, it was found that its antigenic composition greatly resembled that of the monkey lens, except for minor differences of quantitative nature. From all other vertebrates, however, it differed by the presence of one species specific component, which is characterized by a high electrophoretic mobility. When the tissue specificity of the human lens was examined, cornea, retina, iris, aqueous humor and vitreous body were found to have a number of antigenic components in common with the lens. Despite the fact that these intraocular lens antigens are capable of reacting with lens antibodies in vitro, no evidence is presently available suggesting a similar reaction in vivo.