Horizontal cell differentiation in the retina of the Brazilian opossum, Monodelphis domestica.

Differentiation of many diverse neuronal phenotypes is an essential part of nervous system development. We have studied the differentiation of horizontal cells, one of the basic neuronal types in the vertebrate retina, in a small, easily maintained marsupial by immunocytochemistry using antineurofilament and antivimentin antibodies. At birth the retina consists of proliferating neural epithelial cells, with a few early ganglion cells. Horizontal cells were first detected in 12-day-old pups; somas were within the epithelial neuroblastic layer and processes extended radially. By 19 days there were tangentially oriented dendrites and a few longer processes, the beginning of the outer plexiform (first synaptic) layer. By the time of eye opening (about 34 days) the basic histological organization of the mature retina was established. In the mature retina and during development, horizontal cell neurites in the outer plexiform layer, as well as ganglion cell axons, reacted strongly with several antineurofilament antibodies and with antivimentin; horizontal cell somas were detected only with one antineurofilament antibody. Only one population was detected, which we identify as the short-axon subtype, by comparison with horizontal cells in other marsupials and in eutherian mammals. This is the first description of the putative absence in a marsupial of one of the two horizontal cell subtypes found in most amniotes, including mammals so far studied, except murid rodents, which have only the short-axon subtype. Absence of one subtype in Monodelphis supports the hypothesis that the short-axon cell is the basic conserved phenotype of this class and suggests that experimental analysis of differentiation of horizontal cells in Monodelphis and murid rodents, compared to marsupials and eutherian mammals which have the basic two subtypes, can help elucidate mechanisms for controlling differentiation of specific cellular phenotypes and the variations in neurons within and among species.

Horizontal cells in the rabbit retina: differentiation of subtypes at neonatal and postnatal stages.

We are investigating the differentiation of the major subtypes of horizontal cell in the rabbit retina in order to learn more about developmental controls responsible for the variety of neuronal phenotypes. Immunohistochemistry with anti-neurofilament and anti-calbindin-D antibodies, followed by epoxy resin embedding, has facilitated study of these neurons. In the mature rabbit retina, axonless (A-type) horizontal cells reacted strongly in procedures using either antibody; short axon (B-type) somas did not show a reaction with anti-neurofilament antibodies and stained moderately using anti-calbindin antibodies. In the immature neonatal retina the somas of all the horizontal cells seemed to be similar with regard to general morphology, but two populations could be distinguished on the basis of immunostaining. Some, identified as A-type horizontal cells (by comparison with mature retina), were stained using either antibody. Interspersed among these were similar cells with no detectable immunoreactivity, identified as B-type horizontal cells. By the end of the first postnatal week, faint reactivity to anti-calbindin-D was present in the somas of B-type horizontal cells; they stained moderately throughout the rest of the period studied. Thus differences in immunostaining indicate that the two horizontal cell subpopulations are established early in the rabbit, though some other distinguishing characteristics emerge only gradually as the retina matures. These results suggest that in mammals the determination of phenotypic subtype occurs early, possibly at the time that the cell is specified as a horizontal neuron, or shortly thereafter.

The temporal and spatial expression of basic fibroblast growth factor during ocular development in the chicken.

PURPOSE: The authors determined the temporal and spatial localization of basic fibroblast growth factor (bFGF) during ocular development in the chick embryo in an attempt to elucidate its role in this process. METHODS: These studies used monospecific polyclonal rabbit anti-human bFGF immunoglobulin G in conjunction with immunohistochemical techniques and western blot analysis. Embryonic eyes at 5-20 days of development were studied. RESULTS: The bFGF was not detected by western blot analysis until embryonic day 12. However, low levels of bFGF-immunoreactive material were present in pigmented and neural retina and the lenses of 5-day embryos. Lens epithelial and fiber cells remained weakly stained throughout development; neuroepithelial cells, ganglion cells, amacrine cells, and photoreceptors all contained significant levels of bFGF-immunoreactive material. Corneal epithelium consistently contained high levels of immunoreactive material. In the corneal endothelium, increasing levels of immunoreactive material were seen as development proceeded. CONCLUSION: It was hypothesized that bFGF may be involved in regulating the proliferation and differentiation of ocular cells in either an autocrine or paracrine fashion during development and that it may play a role in tissue maintenance in the adult eye. These conclusions are consistent with the temporal and spatial expression of bFGF described here.

Basic fibroblast growth factor in the chick embryo: immunolocalization to striated muscle cells and their precursors.

The identification of acidic and basic fibroblast growth factors (FGFs) in a number of embryonic tissue extracts has implicated these growth factors in the regulation of a variety of embryonic events including angiogenesis, eye development, and muscle differentiation. Lack of information concerning the cellular distribution of the growth factor within these tissues has made it extremely difficult to assign developmental roles to FGF. We have localized bFGF in the developing chick embryo using immunohistochemical techniques and our monospecific polyclonal rabbit anti-human bFGF IgG. The spatial pattern for bFGF localization was highly specific. The anti-human bFGF antibodies recognized striated muscle cells and their precursors in 2-6-d chick embryos. Myocardium, somite myotome, and limb bud muscle all stain positively for bFGF. In addition, the anti-human bFGF antibodies localized specifically to the cell, rather than to the extracellular matrix or nucleus of myotubes. The localization of bFGF demonstrated here provides further support for the hypothesis (Clegg et al., 1987; Seed et al., 1988) that this growth factor is involved in muscle development.

Ultrastructural cytochemistry of monoamines in cultured human fetal sympathetic neurons.

The presence and storage of adrenergic neurotransmitter (monoamines) in cultured human fetal sympathetic neurons was investigated by chromate-dichromate cytochemistry, formaldehyde-induced fluorescence and potassium permanganate fixation. Monoamines were specifically identified in the neurons by the presence of an electron dense precipitate following cytochemical treatment. Reaction product was found in cell somas and processes in all chromate-dichromate treated cultures. The size range and morphology of the precipitate indicated a vesicular storage site within large dense core vesicles. Neurons fluoresced after treatment with formaldehyde vapors, further confirming the presence of monoamines. When potassium permanganate was employed as the fixative, occasional positive dense core vesicles were found but their frequency was greatly reduced from that seen in the chromate-dichromate treated cultures. These findings show that cultured human fetal sympathetic neurons retain an adrenergic phenotype during long-term serum-free culture. In addition, the storage site for the adrenergic neurotransmitter in the developing neuron is within large dense core vesicles. The lack of dense core vesicles in potassium permanganate fixed material is believed to be due to the depletion of monoamines during fixation.

The fine structure of continuous human neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC.

Cell cultures of the continuous human neuroblastoma lines SK-N-SH, SK-N-BS(2), and SK-N-MC at exponential and stationary growth phase have been examined by electron microscopy. At the level of fine structure these cells did not show typical neuronal differentiation such as extensive granular endoplasmic reticulum or neurites with microtubules and neurofilaments. Instead they were characterized by abundant free ribosomes, moderate Golgi complexes, and usually scant granular endoplasmic reticulum, features similar to the fine structure of early normal embryonic autonomic neurons. However, in several respects appearance of differentiated features of the neuroblastoma cells did not follow the pattern observed for normal neurons, suggesting noncoordinate expression of neuronal phenotypic properties. First, an occasional neuroblastoma cell had as extensive granular endoplasmic reticulum as would be found at later stages in normal developing neurons. Second, the cellular processes of these neuroblastoma cells did not have the fine structure of developing or mature axons in vivo. Third, few dense core vesicles were found in SK-N-SH and SK-N-BE(2), though these organelles are numerous in early normal adrenergic neurons and the adrenergic character of these two lines is apparent from other studies that have demonstrated expression of neurotransmitter synthesizing enzymes (SK-N-MC is cholinergic). The fine structural characterization of these continuous human neuroblastoma cell lines will allow this parameter to be utilized with other approaches in future experimental studies.

Organ culture of human nervous system tumors.

Tumors of the human nervous system (neuroblastomas, an ependymoma, a medulloblastoma, and a Schwannoma) obtained during surgery have been cultured organotypically by the method of Wolff. The tumors retained characteristic morphology, organization and patterns of behavior in vitro, and one neuroblastoma gave rise to a growing long-term culture. Long-term organotypic culture, where maintenance of tissue organization and growth occur together, is recommended for the study of neoplasms of the nervous system.

Human nervous system tumors. Observations by high voltages electron microscopy.

Thick sections (0.5--2 mu) of biopsies from human nervous system tumors (Schwannoma, ependymoma, medulloblastoma), fixed in aldehydes followed by osmium, and stained with uranyl acetate and lead, have been studied at 2.5 MV, and compared to thin sections of the same material observed by ordinary low voltage electron microscopy. High voltage electron microscopy permits direct observation of cell fine structure in three dimensions, including the spatial relations of organelles. Details of contours of nuclear envelopes, shapes of mitochondria, fine aspects of the structure of cell surfaces and processes, such as the flat sheet-like and irregular cylindrical processes of Schwannoma cells, the small projections and ridges on their surfaces, and microvilli and cilia of ependymoma cells, and other features have been observed. These initial observations demonstrate the applicability of high voltage electron microscopy to further study of neural neoplasms.

Electron-microscopie observations of a human schwannoma in organotypic culture.

A malignant Schwannoma of the cauda equina has been studied in long-term organotypic culture. Electron-microscopic observation demonstrates that, along with tissue organization, characteristic cell differentiation was retained in this type of culture, but with some morphological changes. As in the biopsy, the tumor cells were elongated and had abundant cell processes in vitro. An increase in the quantity of cytoplasmic filaments and changes in the form of cell processes were observed. Thus organotypic culture of human Schwann cell tumors provides a method of investigating factors which affect their development and differentiation.

Microtubules and filaments in developing axons and optic stalk cells.

Fibrous structures have been studied in the developing optic nerve of chick embryos. The first ganglion cell axons (3-day embryos) were of moderate size, with both neurofilaments and microtubules. Subsequently (4- and 5-day embryos), very small axons were also present. In thesc embryos and in the 4-day hatched chick, the density of microtubules fell within the same range for all but the very small axons, which tended to have more microtubules per unit area. Filaments similar to those previously thought to represent neurofilaments in other parts of the embryonic nervous system were present in the early optic stalk cells, calling into question the reliability of identifying early nerve cells on the basis of 'neurofilaments'.