Cholinesterases in avian neurogenesis.
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Acetylcholinesterase↗
Biomedical subjects
E Willbold
Publications and source records attributed to E Willbold.
Butyrylcholinesterase regulates laminar retinogenesis of the chick embryo in vitro.
During chicken neurogenesis, the sequential expression of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) between final cell proliferation and differentiation is functionally not understood. Recently, cholinesterases have been shown to regulate neurite growth in vitro. Here, we investigated the effects of inhibition of BChE on laminar histogenesis in retinospheroids that arise from dissociated embryonic chicken retinal cells in rotation culture. In the presence of the BChE inhibitor iso-OMPA (tetraisopropyl pyrophosphoramide), the number of spheroids/dish is increased, and their diameter is decreased by about 20%, corresponding to about 50% volume size. As a corollary, the course of histotypical differentiation is dramatically accelerated. Thus as a consequence of BChE inhibition both, organization of nuclear cell layers and of plexiform-like (neuropile) areas, as detected by an antibody to the fiber fasciculation protein F11, is temporally advanced by at least two days. Moreover, AChE is almost fully diminished in these areas. The results further demonstrate novel roles of cholinesterases during laminar histogenesis of coherent neural networks in vitro.
Acetylcholinesterase↗
Histogenesis of the avian retina in reaggregation culture: from dissociated cells to laminar neuronal networks.
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Animals↗
Formation of neuroblastic layers in chicken retinospheroids: the fibre layer of Chievitz secludes AChE-positive cells from mitotic cells.
The significance of the classical subdivision of the retinal primitive neuroepithelium into an outer and an inner neuroblastic layer by the transient fibre layer of Chievitz (LOC) is little understood. We examine here the formation of neuroblastic layers by regenerating fully laminated retinospheroids from dissociated cells of the embryonic chick eye margin in rotary culture. By tracing cellular processes with the fibre-specific F11-antibody in retinospheroids, we occasionally find, in addition to an outer and an inner plexiform layer, a cell-free F11-positive LOC homologue, subdividing the inner nuclear layer. Moreover, we demonstrate that the LOC precisely separates postmitotic AChE-positive cells of the inner retina from an AChE-negative outer part holding all BrdU-labelled mitotic cells. These in vitro data suggest that the inner neuroblastic layer is exclusively composed of AChE-positive cells, thus representing a primary differentiation zone of the retina.
Acetylcholinesterase↗
Chicken retinospheroids as developmental and pharmacological in vitro models: acetylcholinesterase is regulated by its own and by butyrylcholinesterase activity.
The phylo- and ontogenetically related enzymes butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are expressed consecutively at the onset of avian neuronal differentiation. In order to investigate their possible co-regulation, we have studied the effect of highly selective inhibitors on each of the cholinesterases with respect to their expression in rotary cultures of the retina (retinospheroids) and stationary cultures of the embryonic chick tectum. Adding the irreversible BChE inhibitor iso-OMPA to reaggregating retinal cells has only slight morphological effects and fully inhibits BChE expression. Unexpectedly, iso-OMPA also suppresses the expression of AChE to 35%-60% of its control activity. Histochemically, this inhibition is most pronounced in fibrous regions. The release of AChE into the media of both types of cultures is inhibited by iso-OMPA by more than 85%. Control experiments show that AChE suppression by the BChE inhibitor is only partially explainable by direct cross-inhibition of iso-OMPA on AChE. In contrast, the treatment of retinospheroids with the reversible AChE inhibitor BW284C51 first accelerates the expression of AChE and then leads to a rapid decay of the spheroids. After injection of BW284C51 into living embryos, we find that AChE is expressed prematurely in cells that normally express BChE. We conclude that the cellular expression of AChE is regulated by the amount of both active BChE and active AChE within neuronal tissues. Thus, direct interaction with classical cholinergic systems is indicated for the seemingly redundant BChE.
Acetylcholinesterase↗
Müller glia endfeet, a basal lamina and the polarity of retinal layers form properly in vitro only in the presence of marginal pigmented epithelium.
Dissociated embryonic chicken retinal cells regenerate in rotary culture into cellular spheres that consist of subareas expressing all three nuclear layers in an inside-out sequence (rosetted vitroretinae). However, when pigmented cells from the eye margin (peripheral retinal pigment epithelium) are added to the system, the sequence of layers is identical with that of an in-situ retina (laminar vitroretinae). In order to elucidate further the lamina-stabilizing effect exerted by the retinal pigment epithelium, we have compared both systems, laying particular emphasis on the ultrastructure of the basal lamina and of Müller glia processes. Ultrastructurally, in both systems, an outer limiting membrane, inner segments of photoreceptors and the segregation of cell bodies into three cell layers develop properly. Synapses are detectable in a premature state, although only in the inner plexiform layer of laminar vitroretinae. Although present in both systems, radial processes of juvenile Müller glia cells are properly fixed at their endfeet only in laminar vitroretinae, since a basal lamina is only expressed here. Large amounts of laminin are detected immunohistochemically within the retinal pigment epithelium and along a basal stalk that reaches inside the laminar vitroretinae. We conclude that the peripheral retinal pigment epithelium is essential for the expression of a basal lamina in vitro. Moreover, the basal lamina may be responsible both for stabilizing the correct polarity of retinal layers and for the final differentiation of the Müller cells.
Animals↗
Regeneration of a chimeric retina from single cells in vitro: cell-lineage-dependent formation of radial cell columns by segregated chick and quail cells.
We report here that similar to E6-chicken retinal cells, dissociated cells from 5.5-day-old (E5.5) quail retinae reaggregate in rotary culture, multiply about tenfold and reestablish histotypical areas. These cellular aggregates include all nuclear layers either with inversed or correct laminar polarity, depending on the local origin of the cells (called "rosetted" and "laminar" in-vitro-retinae (IVR), respectively; Layer and Willbold 1989). In combined cultures, chick and quail cells are evenly mixed only during the first two days of culture. Along with the assembly of single cells into rosettes and then into discrete laminae, sectors of chick and quail cells begin to segregate. They are delineated by borders running radially through all three nuclear layers. Thus, interspecies migration of cells at this advanced stage of differentiation is strongly inhibited. Concomitant with this segregation, coherent radial columns spanning all three layers but containing cells from either species only, can be traced histologically. We conclude that a weak segregation of chick and quail retinal cells takes place already at the single cell level, but that the permanent segregation of entire tissue parts must be due to clonal cellular proliferation within the IVR in conjunction with some developmental-structure mechanism retaining clonal progenies within a columnar order.
Animals↗
Embryonic chicken retinal cells can regenerate all cell layers in vitro, but ciliary pigmented cells induce their correct polarity.
The capacities of retinal and pigmented cells to regenerate histotypic in-vitro-retinae (IVR) in rotary culture were investigated by dividing the eye cups of 6-day-old chicken embryos into a central and a peripheral part; they were cut along the ora serrata, and the retinal and the pigmented constituents of both parts were isolated. The 4 dissociated cell populations were cultured separately and in all double combinations. Two different types of IVR's were generated; one developed from central or peripheral retinal cells, the other required the addition of pigmented cells from the ciliary margin of the eye. The shape of these IVR's was examined using scanning electron microscopy, and they were also characterized histologically. The acetylcholinesterase pattern marked the inner half of the retina; F11-antibody and a peanut agglutinin marker revealed both plexiform layers and a radial fiber system. In both types, organized histotypical areas consisted of complete sets of retinal layers. In the type containing pigmented cells from the eye periphery, the sequence of layers was identical with that of an in-situ-retina ("laminar IVR"). In IVR's derived from retinal cells only, the sequence of layers was reversed ("rosetted IVR").
Animals↗