The morphology of physiological cell death and of necroses caused by treatment with various embryotoxic substances (cyclophosphamide, actinomycin D, vitamin A, vincristine, 6-aminonicotinamide, 6-mercaptopurine) and Mg deficiency were studied electron microscopically in rat and mouse embryos and fetuses. Three types of necroses were distinguished in control tissues.(1) Condensation and fragmentation of single cells undergoing phagocytosis, with lysosomal disintegration of the fragments in neighboring cells. (2) Primary formation of lysosomes in dying cells, with activation and subsequent destruction and phagocytosis of the fragments by neighboring cells. This type of cell death in most instances was found during destruction of organs and large cell units.(3) Disintegration of cells into fragments, which were optically no longer detectable, without involvement of the lysosomal system, e.g., in embryonic and epiphyseal cartilage before ossification. In cases of embryolethal toxic agents, e.g., Mg deficiency, cell necroses of large areas prevailed, which were characterized by typical pyknoses (rough, dense chromatin) and lack of lysosomes. In the case of teratogenic effects, however, substances that disturb replication, transcription, and translation (cyclophosphamide, actinomycin D, 6-mercaptopurine) caused an increase in the number of necroses of type 1. It can, therefore, be assumed that under these conditions similar processes take place in physiological and experimentally induced necroses. After administration of vitamin A, labilization of lysosomes and necroses of type 2 were observed.

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Chondrocytes grown in monolayer culture at low density, with serum added, either dedifferentiate after several days whereby their cell shape or they are overgrown by fibroblast-like cells. The aim of this study was to optimize the cultivation of chondrocytes in monolayer culture and to slow down their transformation or their overgrowth by fibroblast-like cells. For this purpose freshly isolated chondrocytes of cartilage anlagen from 17-day-old mouse embryos were grown on plastic or collagen type II-coated substrates. With this model: (a) chondrocytes grown on plastic substrates had almost completely changed to fibroblast-like cells after 5 days in culture. (b) When grown on collagen type II, the chondrocytes maintained their round phenotype for more than 2 weeks in culture. (c) Immunomorphological investigations showed that chondrocytes produce collagen type II and fibronectin and express specific surface receptors (integrins of the beta 1-group) on the membrane from day 1 until the end of the culture period when grown on collagen type II. (d) Treatment with beta 1-integrin antibodies clearly reduces chondrocyte adhesion on collagen type II by about 70%. Hence, these data indicate that the most probable influence of collagen type II on cellular behaviour depends on the integrins participating in a chondrocyte-collagen type II interaction, and this model represents a pure chondrocyte culture which allows cell growth for an extended period.

Prenatal exposure to the antiepileptic drug valproic acid (VPA) has been associated with the formation of spina bifida aperta, meningocele, and meningomyelocele in the human. Until now, a direct relationship between VPA application and spina bifida has not been experimentally demonstrated. VPA was known only to induce exencephaly in mice, a defect of the anterior neural tube. Maximal sensitivity toward production of this defect was on day 8 of gestation (plug day = day 0). The closure of the posterior neuropore occurs later in the development of mice than the closure of the anterior neuropore. To investigate whether there is a direct relationship between VPA application during pregnancy and induction of spina bifida in mice, we administered various doses of the drug on day 9 of gestation, at three time intervals (at 0, 6, and 12 hr). This administration of VPA produced spina bifida aperta and spina bifida occulta in mice. High doses of VPA (3 x 450 and 3 x 500 mg/kg) induced a low rate of spina bifida aperta in the lumbosacral region. High incidences of spina bifida occulta, a less serious form of spina bifida, were induced with lower doses. This malformation was demonstrated in double-stained fetal skeletons by measurements of the distance between the cartilaginous ends of each vertebral arch. The occurrence of this defect and its localization was dose-dependent. The lumbar region was affected by all doses investigated (3 x 300, 3 x 350, 3 x 400, 3 x 450, and 3 x 500 mg/kg). The sacral/coccygeal region was affected additionally, but with higher doses (3 x 400, 3 x 450, and 3 x 500 mg/kg). A comparison of the results obtained with day 16 and 17 control fetuses showed that the pattern of gaps present in the lumbar and sacral region of the spinal cord in treated groups was drug-specific and not related to a developmental delay. Our results indicate that multiple administrations of VPA on day 9 of gestation in mice result in a low incidence of spina bifida aperta and a high incidence of spina bifida occulta, and provides a relevant model for the study of human spina bifida defects.

The aim of this contribution is to summarize our knowledge of the morphology of the basement membrane (BM). The first step in this direction is the attempt to define this term. The BM is composed of the Lamina lucida, densa, and fibroreticularis. Subsequently, the historical development of this term is discussed. Our main interest is, of course, focused on the description of the BM-structure up to the macromolecular level and the special forms of this structure. This is supplemented by discussing its chemical composition and establishing a relationship between morphology and biochemistry. The obtained findings yielded some indications as to the molecular composition of the BM which may serve for the construction of "models." The composition of the Lamina lucida (L.l.) and the Lamina or Pars fibroreticularis (L.f.) must be discussed separately, since, if present, they show a different and strongly varying structure (L.f.). An important aspect is the function of this extracellular layer which comprises mechanical tasks up to inductive effects. Finally, the concepts of the formation of the BM, especially of the Lamina densa (L.d.), are summarized. It obviously consists of a sequence of individual steps which starts with expression and secretion of the L.d.-components and is followed by an induction of integrin expression.