Cholinesterases and cell proliferation in “nonstratified” and “stratified” cell aggregates from chicken retina and tectum

Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - iso-OMPA specific inhibitor of BChE - BW 284C51 specific inhibitor of AChE - IPL inner plexiform layer - OPL outer plexiform layer     

Regeneration of a chimeric retina from single cells in vitro: cell-lineage-dependent formation of radial cell columns by segregated chick and quail cells

Summary 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-structural mechanism retaining clonal progenies within a columnar order.Abbreviations ECM extracellular matrix - E5.5 days of embryonic age - GCL ganglion cell layer - GC's ganglion cells - i.c. in culture - INL inner nuclear layer - rosetted in-vitro-retina retinal cell organoid aggregated from single cells of the central retina - IPL inner plexiform layer - MRE marginal retinal epithelium - ONL outer nuclear layer - OPL outer plexiform layer - OS ora serrate - PR photoreceptor cell - laminated in-vitro-retina fully laminated retinal cellorganoid resembling an E15-retina aggregated from cells of the eye periphery including RPE - RPE retinal pigment epithelium     

The receptor protein tyrosine phosphatase mu, PTPmu, regulates histogenesis of the chick retina

The formation of laminae within the retina requires the coordinate regulation of cell differentiation and migration. The cell adhesion molecule and member of the immunoglobulin superfamily, receptor protein tyrosine phosphatase Mu, PTPmu, is expressed in precursor and early, differentiated cells of the prelaminated retina, and later becomes restricted to the inner plexiform, ganglion cell, and optic fiber layers. Since the timing of PTPmu expression correlates with the peak period of retinal lamination, we examined whether this RPTP could be regulating cell adhesion and migration within the retina, and thus influencing retinal development. Chick retinal organ cultures were infected with herpes simplex viruses encoding either an antisense sequence to PTPmu, wild-type PTPmu, or a catalytically inactive mutant form of PTPmu, and homophilic adhesion was blocked by using a function-blocking antibody. All conditions that perturbed PTPmu dramatically disrupted retinal histogenesis. Our findings demonstrate that catalytic activity and adhesion mediated by PTPmu regulate lamination of the retina, emphasizing the importance of adhesion and signaling via receptor protein tyrosine phosphatases in the developing nervous system. To our knowledge, this is the first demonstration that an Ig superfamily RPTP regulates the lamination of any neural tissue.     

Embryonic chicken retinal cells can regenerate all cell layers in vitro,but ciliary pigmented cells induce their correct polarity

Summary 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).     

Lamination of the cerebral cortex is disturbed in Gli3 mutant mice

The layered organization of the cerebral cortex develops in an inside-out pattern, a process which is controlled by the secreted protein reelin. Here we report on cortical lamination in the Gli3 hypomorphic mouse mutant Xt^J/Pdn which lacks the cortical hem, a major source of reelin⁺ Cajal Retzius cells in the cerebral cortex. Unlike other previously described mouse mutants with hem defects, cortical lamination is disturbed in Xt^J/Pdn animals. Surprisingly, these layering defects occur in the presence of reelin⁺ cells which are probably derived from an expanded Dbx1⁺ progenitor pool in the mutant. However, while these reelin⁺ neurons and also Calretinin⁺ cells are initially evenly distributed over the cortical surface they form clusters later during development suggesting a novel role for Gli3 in maintaining the proper arrangement of these cells in the marginal zone. Moreover, the radial glial network is disturbed in the regions of these clusters. In addition, the differentiation of subplate cells is affected which serve as a framework for developing a properly laminated cortex.     

The zebrafish Pard3 ortholog is required for separation of the eye fields and retinal lamination

The vertebrate retina develops from a sheet of neuroepithelial cells. Because adherens and tight junctions are critical for epithelial and neuronal differentiation in a variety of eukaryotic systems, we examined the role of Par-3, a PDZ scaffold protein that is critical in cellular membrane junction formation. We cloned the zebrafish Par-3 ortholog (pard3), which encodes two Pard3 proteins (150 and 180 kDa) that differ in their carboxyl-terminus. Immunohistochemistry revealed that Pard3 localized to the apical region of the retinal and brain neuroepithelium, partially overlapping the adherens junction-associated actin bundles. After retinal lamination, the Pard3 protein was restricted to the outer limiting membrane and the outer and inner plexiform layers in the retina. Reducing Pard3 expression with antisense morpholinos caused loss of the retinal pigmented epithelia, disruption of retinal lamination, and cell death in the ventral diencephalon, which resulted in cyclopia. Overexpressing Pard3 by injection of wild-type pard3 mRNA resulted in cyclopia and eyeless embryos. Thus, Pard3 plays a critical role in the origination and separation of zebrafish eye fields and retinal lamination.     

An examination of dental development in Graecopithecus freybergi (=Ouranopithecus macedoniensis)

This study examined enamel thickness and dental development in Graecopithecus freybergi (=Ouranopithecus macedoniensis), a late Miocene hominoid from Greece. Comparative emphasis was placed on Proconsul, Afropithecus, Dryopithecus, Lufengpithecus, and Gigantopithecus, fossil apes that vary in enamel thickness and patterns of development. In addition, comparisons were made with Paranthropus to investigate reported similarities in enamel thickness. Several sections of a right lower third molar were generated, from which enamel thickness and aspects of the enamel and dentine microstructure were determined. Data from parallel sections shed light on the effects of section obliquity, which may influence determination of both enamel thickness and crown formation time. Graecopithecus has relatively thick enamel, greater than any fossil ape but less than Paranthropus, with which it does show similarity in prism path and Hunter-Schreger band morphology. Aspects of enamel microstructure, including the periodicity and daily secretion rate, are similar to most extant and fossil apes, especially Afropithecus. Total crown formation time was estimated to be 3.5 years, which is greater than published values for modern Homo, similar to Pan, and less than Gigantopithecus. Data on dentine secretion and extension rates suggest that coronal dentine formation was relatively slow, but comparative data are very limited. Graecopithecus shares a crown formation pattern with several thick-enamelled hominoids, in which cuspal enamel makes up a very large portion of crown area, is formed by a large cell cohort, and is formed in less than half of the total time of formation. In Paranthropus, this pattern appears to be even more extreme, which may result in thicker enamel formed in an even shorter time. Developmental similarities between Paranthropus and Graecopithecus are interpreted to be parallelisms due to similarities in the mechanical demands of their diets.