Glycoprotein complexes interacting with cellulose in the "cell print" zones of the Dictyostelium discoideum extracellular matrix.

Cellulose is one of the commonest structural biopolymers. How cellulose is organized in extracellular matrices is a mystery. Here we investigate a model system, the extracellular matrix (ECM) of Dictyostelium discoideum which is composed of proteins and cellulose. A group of glycoproteins, the sheathins, which colocalize with cellulose in the ECM of D. discoideum are characterized. Sheathins are dimeric or trimeric forms of molecular mass 53-68 kDa, where the monomers are 12-35 kDa. The sheathin subunits are similar but not identical proteins. The sheathin family comprises sheathin 68, (68-kDa trimer); sheathin 62, (62-kDa dimer); sheathin 55, (55-kDa dimer), and sheathin 53, (53-kDa dimer). The subunits which assemble into the four sheathins represent at least three gene products: ShC, ShD, and ShE which are linked by disulphide bonds. Protein sequence analysis shows two of the sheathin genes encode products ShC and ShD with very similar amino terminal sequences. This group of D. discoideum ECM glycoproteins has homology with two other much larger ECM proteins of D. discoideum, ST430 and ST310, which are located in a more dispersed fashion in the ECM. Sheathins are tightly but non-covalently associated with the ECM, and this association requires strong denaturing conditions for disruption, e.g., SDS or 8 M urea. Sheathins form a component of the "cell prints" which are believed to have a role in cell-ECM interactions and slug cell migration.

Length regulation in the Dictyostelium discoideum slug is a late event.

Time-lapse video light microscopy was used to study the emergence and maturation of the migratory slug from a D. discoideum aggregate. The anterior part, the tip of this simple multicellular organism, establishes migration prior to the definition of the rear, and hence the length of the slug. It was found that newly formed slugs of wild-type strain WS380B can reach lengths greater than 1 cm, yet mature slugs of this strain are rarely longer than 2-3 mumm. Often the tip extended out of the aggregation mound upon an arching pillar of cells. After the tip first touched the substratum, it commenced migration with a rapid succession of movement steps. Here we show that at the initiation of migration, a differential rate of cell movement along the developing slug axis results in a series of complicated changes, before the stable and mature shape of the slug is formed. Our results lead to new conclusions about D. discoideum slug formation and shape maintenance. Evidence is presented for regulation of slug length.

The distribution of myosin II in Dictyostelium discoideum slug cells.

While the role of myosin II in muscle contraction has been well characterized, less is known about the role of myosin II in non-muscle cells. Recent molecular genetic experiments on Dictyostelium discoideum show that myosin II is necessary for cytokinesis and multicellular development. Here we use immunofluorescence microscopy with monoclonal and polyclonal antimyosin antibodies to visualize myosin II in cells of the multicellular D. discoideum slug. A subpopulation of peripheral and anterior cells label brightly with antimyosin II antibodies, and many of these cells display a polarized intracellular distribution of myosin II. Other cells in the slug label less brightly and their cytoplasm displays a more homogeneous distribution of myosin II. These results provide insight into cell motility within a three-dimensional tissue and they are discussed in relation to the possible roles of myosin II in multicellular development.

Pathogenesis of Thalidomide Teratogenesis in the Marmoset (Callithrix jacchus): Evidence Suggesting a Possible Trophic Influence of Cholinergic Nerves in Limb Morphogenesis: (thalidomide/neurotransmitters/cholinergic/nerves/anticholinergics/limb development).

Thalidomide administered to the marmoset (Callithrix jacchus) will produce a pattern of deformities similar to those found in man. In the cervical and lumbar regions of the thalidomide malformed marmoset fetus, the spinal cord and dorsal root ganglia are smaller than those of the control. The reduction in size of the D.R.G.'s is due to a marked reduction in the number of neurons. Drugs which influence the function of cholinergic nerves in the chick and rabbit embryo interfere with development. Thalidomide produced peripheral neuropathy in adults by interference with the function of the cholinergic nerves. Thus it appears that thalidomide malformations are due in part, to an interference with numbers of, and probably the functions of, the cholinergic nerves in the embryo. It would appear that cholinergic nerves have a possible morphogenetic and trophic influence in embryogenesis.

Effects of scopolamine hydrobromide on the development of the chick and rabbit embryo.

When 100 or 200 microgram of scopolamine hydrobromide were injected into hen eggs after they had been incubated for 96 h the major malformations of the embryo found were gastroschisis, exencephaly, reduction deformities of the limbs, microphthalmia and buphthalmia when the eggs were opened on the 12th day of incubation. Eight foetuses of one of eight New Zealand white rabbit does given scopolamine hydrobromide at the rate of 473 microgram/kg in their drinking water from the 10th to the 14th day of gestation showed malformations consisting of microphthalmia, buphthalmia, exencephaly and hydrocephaly. A second doe which similarly received 520 microgram/kg of the hydrobromide had four malformed foetuses showing microphthalmia and had two foetuses which were resorbed. However, there were no malformations in foetuses of eight does given bolus doses of scopolamine hydrobromide ranging from 37 to 184 microgram/kg by intramuscular injection every 12 h from the 10th to the 14th day of gestation. Results indicate interference by the drug with the function of the cholinergic nerves on the development of the chick and rabbit embryo.

Evidence of a Neural Inductive Phase in Limb Development: (induction/limb development/neural tube/neural crest/thalidomide).

To demonstrate a causal relationship between neuronal damage and peripheral mesodermal anomalies, an operation was performed through oblique incisions in the dorsum of 247 chick embryos at stages 12-15 of development. In the region of the 24-30th presumptive somites, a three to four somite length of the right half of the neural tube was cut with fine glass sytlettes and removed with a 40µm glass pipette. Immediately after the operation and at intervals during continued incubation (37±, 75% R.H.), embryos were processed for histology. Fifty one embryos survived until day 14 when they were examined for malformations. Excision frequently resulted in a variety of reduction deformities: some embryos appeared normal; 31 displayed unilateral hemimelia or amelia. Examination of serially sectioned embryos fixed immediately and at varying intervals after the operation, demonstrated that this range of malformations was consistent with variation in the amount of neural tissue excised. Disruption of the mesoderm and regeneration of nervous tissue was minimal. It is concluded that the nervous system per se or some factor produced by the nervous system is essential for the development of the limb.

Maternal Thalidomide Treatment and Cell Division in Cultured Rabbit Embryos: Automated Image Analysis of Autoradiographs: (thalidomide/autoradiography/rabbit embryo culture.).

To determine whether thalidomide dysmorphogenesis was preceded by a changed pattern of cell division, embryos from thalidomide treated rabbits were cultured for four hours in a medium containing 3 H-thymidine. Automated image analysis of serial autoradiographs of the embryos revealed no focus of increased or decreased cell division in the neural tube or limb bud. Thalidomide pretreatment caused a general decrease in cell division at 10 days post coitum. A similar effect was observed only in those 10 3/4 day embryos whose status in culture was low. It was concluded that unlike methotrexate, the dysmorphogenic effect of thalidomide could not be attributed to a generally suppressed mitotic rate; rather, this teratogen preprogrammes dysmorphogenesis before the onset of the limb bud outgrowth (10 days) with the result that teratogenesis is manifest only during limb development after 11 days.

The kidney of Leptonychotes weddelli (Pinnipedia: Phocidae) with some observations on the kidneys of two other southern phocid seals.

The structure of the kidney of Leptonychotes weddelli was examined using corrosion casts, India ink injection, and histological methods. Some observations were made on the kidney of the crabeater seal (Lobodon carcinophagus) and the elephant seal (Mirounga leonina). The kidneys in all three species are reniculate, as in many other marine mammalian species. Features that have not been described previously in a phocid seal are a peripyramidal muscle, and venous drainage characterized by a large extrinsic system and a small intrinsic system. Examination of specialized fornices, relative medullary thickness, and the volumes of juxtamedullary relative to peripheral glomeruli (all of which relate to urine concentrating ability) revealed that each reniculus of Leptonychotes is similar to the unilobar kidney of a small mammal that produces only moderately concentrated urine. The high glomerular volume to cortical volume ratio may be related to high glomerular filtration rates after feeding observed in marine mammals. It is concluded that reniculation is more likely to be related to the large size of most marine mammals than to some factor related directly to the marine environment.

Advances in Rabbit Embryo Culture during Organogenesis.

Modified rodent embryo culture techniques were used to maintain 10 1/2 day rabbit embryos in vitro for up to 24 hr. In homologous, immediately centrifuged, heat inactivated serum containing 2 µCi/ml 3 H-thymidine, observed status was consistently high at 12 hr but fell thereafter. Liquid scintillation counting and autoradiography revealed rapid uptake of radioactivity during the first 12 hr. The splanchnopleure of the inverted yolk-sac is only partially vascularised in the rabbit and is thus inadequate as a medium of metabolic exchange in extended culture. Nevertheless, this technique will enable a study of the morphology and biochemistry of teratology in vitro in a sensitive species.