Ganglioside patterns and cholera toxin-peroxidase labeling of aggregating cells from the chick optic tectum

The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. G_D and G_D1b were the predominant gangliosides early in development, while G_D1a and several other multisialogan gliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin-peroxidase binding site G_M1 showed conjugate binding correlates with increasing levels of G_M1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of G_M1 in the synaptic cleft region.     

Ganglioside, protein, hexose, and sialic acid changes in the trisected optic tectum of the chick embryo.

Spatiotemporal changes in membrane constituents of cells from the optic tectum of the chick embryo were analyzed during the period of maximum differentiation and synaptogenesis. Each tectum from 6-, 8-, 10-, and 12-day embryos was cut into three subregions along the topological gradient of differentiation. Electrophoretic analysis of proteins revealed an already complex population by Day 6 which remained relatively unchanged through later stages, with little if any topological variations. In contrast, chromatographic analysis of gangliosides showed an increasingly complex pattern as differentiation proceeded, with a growing preponderance of multisialogangliosides. Total membrane protein increased symmetrically with tissue mass in each subregion. However, hexose concentration and sialic acid/hexose ratios showed strikingly asymmetrical topological distributions as early as Day 8, and tended to fluctuate reversibly within brief (1 day or less) time periods. These results suggest that during the period of maximal differentiation and retino-tectal synaptogenesis in the optic tectum of the chick, the membrane protein population remains relatively stable and topologically invariant, whereas the polysaccharide chains of membrane macromolecules fluctuate according to topological position and developmental state in a complex, relatively rapid, and apparently oscillatory fashion.     

GFAP-immunoreactive perivascular glia in the chick optic tectum.

Immunocytochemical staining of the glial fibrillary acidic protein (GFAP) was utilized to characterize the processes of the astrocytes enveloping the vessel wall in the central nervous system. The study was carried out in the mesencephalic lobes of 18 and 20 incubation-day chick embryos and of 20 day chickens. A perivascular GFAP positivity was mainly detectable in the vessel portions running within the tectum white layers, while it was scarce, or absent, in the grey ones. The perivascular GFAP negativity in the tectum cellular layers was not considered result of the absence of astrocytic endfeet since our previous electronmicroscopical studies evidenced an almost complete perivascular astrocytic ring throughout the tectum layers at hatching time. Present data rather suggest that the expression of the GFAP-made intermediate filaments in developing astrocytes might be controlled by the surrounding microenvironment.     

Vasculogenesis in the chick embryo optic tectum

The initial formation and further development of the intraneural blood vessel network in the tectum opticum of the chick from the 4th to the 14th incubation day have been analyzed and some quantitative data morphometrically recorded. Vessels have been filled by intracardial injection of India ink in vivo. As inferred from our previous investigations on the vasculogenesis of several districts of the central and peripheral nervous system in the chick embryo, also in the developing optic tectum growth and distribution pattern of the vessels seem to unfold step by step under the local influence of earlier occurring morpho-histogenetic processes of the corresponding neural substratum.     

The contribution of axoplasmic flow in optic nerve and protein synthesis within the optic tectum to synaptic membrane proteins of the chick optic tectum

In the 5-day-old chick, radioactive leucine was incorporated into proteins of synaptosomal and subsynaptosomal fractions both by fast axoplasmic flow and synthesis within the optic tectum. The distribution of radioactivity in subsynaptosomal fractions suggested that both pathways contribute to the protein constituents of each fraction. The relative contributions to each fraction were similar except for the supernatant proteins, for which fast axoplasmic flow contributed less than the synthesis within the optic tectum. The qualitative contribution of fast flow and synthesis within the optic tectum to the synaptic membrane fraction was distinctive. Fast axoplasmic flow preferentially labelled the high molecular weight proteins, whereas synthesis within the optic tectum labelled a larger percentage of smaller molecular weight proteins.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Cholinergic development in chick optic tectum and retina reaggregated cell cultures

The developmental profiles of acetylcholinesterase and choline acetyltransferase in chick optic tectum and retina cell aggregates, over a 30-day period, have been determined and compared with the corresponding developmental curves obtained in vivo. Both acetylcholinesterase and choline acetyltransferase activities in retina cell aggregates and the acetylcholinesterase activity in optic tectum cell aggregates usually lie between 40 and 90% of the values measured in vivo for the same cell (tissue) type and developmental age. However, the choline acetyltransferase activity in tectum aggregates increases only during the first 7 days of culture, and then decreases to reach a low value of 8% of that measured in vivo, by day 24. This fact, which is associated with widespread degeneration and cell death, could be attributed to the condition of natural deafferentiation occurring in a tectum cell aggregate. A parallel has been drawn between this behavior of a tectum cell aggregate and the effect of early embryonic eye removal on the development of the contralateral optic tectum in vivo. Thus, the tectum may have a biphasic pattern of development, with an autonomous period of growth of about 2 wk, followed by an afference-dependent phase, while the retina behaves, from a cholinergic point of view, as a relatively self-sufficient structure.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - ACh acetylcholine - BW284 C51 dibromide 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide     

Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion

The effect of intercellular interactions on the determination and differentiation of early embryonic brain cells was tested by immunomagnetic cell separation techniques. Using the A2B5 monoclonal antibody, which in chick brain reacts with a neuron-specific surface ganglioside, we produced initially pure populations of optic tectum cells devoid of the antigen. A coincident depletion of neurofilament(+) cells (95%) and nonneuronal growth characteristics of the separated A2B5(-) cells indicated that the vast majority of neurons had been removed initially. Surprisingly, A2B5(+) cells rapidly appeared in separated A2B5(-) cell cultures. After 1 day, the percentage of A2B5(+) cells in separated cell cultures equalled those in unseparated cultures (approximately 50%). By a week in culture, A2B5(+) cells developed neuronal morphology and contained neurofilaments. A2B5(-) to (+) conversion was a regulated phenomenon in that removal of different proportions of the (+) cells resulted in different numbers of (-) to (+) conversions. New DNA synthesis was not required for the acquisition of cell surface A2B5 antigen or for differentiation of cells into definitive A2B5(+) neurons. Our results demonstrate that postmitotic embryonic brain contains cells which are capable of replacing depleted neurons in vitro.     

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 M) was abolished by 8-phenyltheophylline (15 uM). Glutamate and the glutamatergic agonists kainate or trans-d,l-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 M, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 M 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (rs)-alpha-methyl-4-carboxyphenylglycine [(rs)M-CPG], a metabotropic, antagonist, while 1 mMl-2-amino-3-phosphonopropionate (l-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 M and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.Abbreviations ADO adenosine - DNQX 6,7-dihydroxy-2,3-dinitro-quinoxaline - KA kainate - l-AP3 l-2-amino-3-phosphonopropionate - mGluRs metabotropic glutamate receptors - P-THEO 8-phenyltheophylline - (rs)M-CPG (rs)-alpha-methyl-4-carboxyphenyl-glycine - trans-ACPD trans-d,l-1-aminocyclopentane-1,3-dicarboxyho acid     

Migratory paths and phenotypic choices of clonally related cells in the avian optic tectum.

We used retrovirus-mediated gene transfer to study the migration of clonally related cells in the developing chicken optic tectum. Clonal cohorts initially form radial arrays in the ventricular zone (approximately E5), but eventually divide into three separate migratory streams. In the first migration, a minor population of cells migrates tangentially along axon fascicles in medio-laterally directed files (approximately E6-E7); these eventually differentiate into multipolar efferent cells. After E7, the majority of cells in each clone migrate radially along fascicles of radial glia to form the tectal plate, wherein they differentiate into neurons and astrocytes. Around E9, a set of small cells leaves the radial arrays in superficial layers to form a second tangential migration; at least some of these differentiate into astrocytes. Thus, as the tectum develops, cells derived from a single multipotential precursor migrate along three separate pathways, follow separate guidance cues, and adopt distinct phenotypes.     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.