Exposure to diazinon alters in vitro retinogenesis: retinospheroid morphology, development of chicken retinal cell types, and gene expression.

Developing embryos are more vulnerable than adults to acute cholinergic intoxication by anticholinesterases, including organophosphorus pesticides. These agents affect the process of neural development itself, leading to permanent deficits in the architecture of the nervous system. Recent evidence on direct roles of acetylcholinesterase (AChE) on neuronal differentiation provides additional grounds for investigating the developmental toxicity of anticholinesterases. Therefore, the effect of the organophosphate diazinon on the development of chick retinal differentiation was studied by an in vitro reaggregate approach. Reaggregated spheres from dissociated retinal cells of the E6 chick embryo were produced in rotation culture. During the whole culture period of 10 days, experimental cultures were supplemented with different concentrations of the pesticide, from 20 to 120 microM diazinon. The pesticide-treated spheres were reduced in size, and their outer surface was irregular. More importantly, inner structural distortions could be easily traced because the structure of control spheroids can be well characterized by a histotypical arrangement of laminar parts homologous to the normal retina. Acetylcholinesterase activity in diazinon-treated spheres was reduced when compared with controls. As a dramatic effect of exposure to the pesticide, inner plexiform layer (IPL)-like areas in spheroids were not distinguishable anymore. Similarly, photoreceptor rosettes and Müller radial glia were strongly decreased, whereas apoptosis was stimulated. The expression of transcripts for choline-acetyltransferase and muscarinic receptors was affected, revealing an effect of diazinon on the cholinergic system. This further proves the significance of cholinesterases and the cholinergic system for proper nervous system development and shows that further studies of debilitating diazinon actions on development are necessary.

On the multifunctionality of cholinesterases.

Here we present novel information on non-classical functions of cholinesterases and on a cross-talk linking the two enzymes AChE and BChE. The first part of the article is focussed on the regulation of ChEs and the effects acquired when one of the proteins is knocked down (siRNA for BChE, AChE knock-out mouse). In the second part evidence is presented showing that AChE may exert adhesive properties through its binding to laminin, thus being involved in cell-matrix or cell-cell communication.

Cytochrome-c oxidase is one of several genes elevated in marginal retina of the chick embryo.

The retinal ciliary margin is particularly relevant for the correct generation and regeneration of vertebrate retinae, since pluripotent stem cells are located there throughout development, and--at least in some species--even until adult stages. Our aim was to identify factors (genes) which are involved in processes of proliferation and differentiation in the developing chicken retina. Reverse transcription-polymerase chain reaction differential display was used to identify genes that were differentially expressed in chick central and peripheral embryonic retina. Candidate genes analyzed through sequencing and database searches were confirmed by Northern blot analysis and histochemistry. A series of differentially expressed genes were detected, including a neuronal cell adhesion molecule, an esterase, and homeobox gene products. One of the sequenced products was identified as subunit I of cytochrome-c oxidase (COX-1), an enzyme which is central to energy metabolism and particularly relevant for developing nervous systems. Northern blot analysis confirmed its up-regulation in the chick peripheral retina, being maximal at embryonic day 7. In the retinal pigmented epithelium its expression is lower than in the retinal periphery but higher than in central retina. COX histochemistry revealed distinct laminar patterns in central retina, but also an elevated level of activity in the peripheral retina throughout development. These data not only show that the developing ciliary margin of the chick retina has high energy requirements, but also indicate that COX-1 could play essential roles in developing cells and in stem cells of the eye periphery.