Abstract. M\Iouse tumor C1300 has been established in tissue culture. The cells have a round cell morphology in both the subcutaneous tumor and in suspension culture. However, when given a surface on which to attach, they send out processes up to 3 mm in length and assume the morphology of mature neurons. The attached cells are stained by the Bodian silver procedure for neurons, whereas the cells grown in suspension are not. Electron microscopy reveals that the attached cells contain neurofilaments, neurotubules, and densecore vesicles indicative of nerve fibers. Both free-floating and attached cells have tyrosine hydroxylase activity characteristic of sympathetic nervous tissue. Apparently cell attachment can induce morphological differentiation from an anaplastic round cell to a cell which has many properties of a mature neuron.The analysis of differentiated function is hampered by the fact that the end cell usually does not divide. In the case of extremely heterogeneous cell populations such as those found in the endocrine, nervous, immune, and hepatic tissues, further advances require a way of establishing clones of end cells which express their differentiated phenotype in tissue culture.1' 2 Such cells are most easily derived from neoplasms and have been used successfully in the study of endocrine,3 hepatic,4 and endoreticularl cell function. This communication extends these studies to the nervous system by describing a cloned, tissue-culture adapted, mouse cell line which can be induced to differentiate to a cell which has some properties of a mature neuron.Materials and Methods.-Tumor lines: Mouse tumor C1300 was obtained from Jackson Laboratories. This cell line has been previously described as a round cell tumor, possibly a neuroblastoma.5' 6 The spontaneous neoplasm originated in the body cavity in mouse strain A/J in 1940 and has been maintained by subcutaneous transfer in A/J mice. The solid tumor was adapted to tissue culture conditions by dispersing the cells in modified Eagle's medium' containing 20% fetal calf serum. The cell cultures were maintained at 370C in an 85% air, 15% CO2 incubator. When grown in plastic Petri dishes, the established cell line had a doubling time of 17 hr. Cells were cloned twice by spreading dilute cell suspensions on solid agar (0.5% agar, 4.5 mgm/ml trypticase soy broth in Eagle's modified medium plus 20% fetal calf serum) and picking visible colonies with a platinum loop after a 2-week incubations Clones had doubling times comparable to the initial explant, and were homogeneous with respect to cell morphology, staining characteristics, and karyotype.9Staining and electron microscopy: For light microscopy, cells were grown in Petri dishes with glass cover slips. After 2 to 4 weeks, the cover slips were washed several times in Eagle's medium and the attached cells stained according to the Giemsa,10 and Bodian10 11 procedures. Cells growing in suspension were centrifuged, washed twice in modified Eagle's medium, spread on a slide, and stained as described above.

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Aggregation factor, the macromolecular complex which mediates species-specific aggregation of dissociated sponge cells, was isolated from several species, partially characterized, and visualized by electron microscopy. All factors were large fibrous complexes with a backbone and side chains or arms. In some factors, the backbone is linear. In others it is circular and the complex appears as a sunburst with arms extending like rays from the circle. The size and location of the polysaccharide chains have been studied using purified preparations of Microciona prolifera. "Sunbursts" treated with ethylenediaminetraacetate (EDTA) for 4 weeks at 0 degrees C dissociate into 3 protein- and polysaccharide-containing components. Sodium dodecyl sulfate does not cause the sunburst to dissociate nor does it inhibit dissociation in the presence of EDTA suggesting that dissociation is not due to hydrolytic enzymes. The dissociation products were fractionated on a 977-A pore size micropore glass column. Fifteen percent of the material is excluded and appears in the electron microscope as the central circle of the sunburst. Digestion of the circles with 10(-3) M dithiothreitol (DTT) and 0.5 mg/ml proteinase K for 72 h at 37 degrees C produces 2 polysaccharide chains of 65,000 and 6,000 daltons as fractionated and sized on a 233-A pore size micropore glass column using Pharmacia dextrans as standards. The included fractions of the EDTA-treated material are subunits of the arms which contain 70% of the polysaccharide. A single polysaccharide of 6,000 daltons as measured on 233-A size glass beads and Sephadex G-75 is released from these subunits by proteinase digestion. Pharmacia dextrans are used as standard on both columns. We calculate that there would be four 65,000-dalton chains and one hundred 6,000-dalton chains per circle and fifty 6,000-dalton chains per arm. The third component of the EDTA-treated preparation is partially included on the column. It appears as linear fibrils in the electron microscope and contains polydisperse polysaccharides of several-hundred-thousand daltons. It may be an impurity since there is apparently less than 1 of the large polysaccharide chains per sunburst.

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The mature annelid cuticle contains orthogonally oriented collagen in a matrix capped superficially by a dense epicuticle with external corpuscles. The underlying epidermis is a simple columnar epithelium with two major cell types, mucous-secreting cells which secrete through channels in the cuticle to the exterior of the worm, and "supportive" cells which presumably produce and increase the cuticle by secreting into it. The structures of supportive cells, previously interpreted as specialized for establishing interfibrillar collagen order, are revealed by glutaraldehyde fixation as common cellular components without the qualities deemed useful to align collagen. Cell processes which penetrate and sometimes pass completely through the cuticle are not stable, not in geometric order, and lack cilia-like structure. Cilia, unlike the ubiquitous cellular processes, are highly restricted to regions of the epidermis with specialized functions. Cellular control, or other control, of collagen fibrillogenesis remains unestablished.

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