CYTOCHROME-C OXIDASE IS ONE OF SEVERAL GENES ELEVATED IN MARGINAL RETINA OF THE CHICK EMBRYO L. E. PARAOANU,* B. WEIß, A. A. ROBITZKI AND P. G. LAYER Developmental Biology and Neurogenetics, Darmstadt University of Technology, Schnittspahnstrasse 3, D-64287 Darmstadt, Germany Abstract—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. Re- verse transcription–polymerase chain reaction differential display was used to identify genes that were differentially expressed in chick central and peripheral embryonic ret- ina. 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 prod- ucts. 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 periph- eral 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. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: cytochrome c-oxidase, ciliary margin, differential display, cell proliferation, retinal development, stem cells. The developing retinal neuroepithelium contains multipo- tential precursor cells that give rise to all of the neurons and the glial cell types present in the adult retina. The generation of such a highly organized laminar structure requires coordinated events of cell proliferation, migration and differentiation. In the chicken retina, the permanent withdrawal of individual cells from the mitotic cycle begins on embryonic day 3 (E3), and continues until E8 in the central retina and for a few more days at the periphery (Adler, 2000). Thus, retinal differentiation follows a spatio- temporal gradient from the center to the periphery (Layer and Kotz, 1983; Prada et al., 1999). The formation of a stratified structure begins at E6 and ends at E14 (Spence and Robson, 1989). The chicken retina shows also a higher differentiation level at early developmental stages compared with the whole organism. In any given retinal area, the first cells leaving the cycle are determined to become ganglion cells, and the last ones bipolar cells (Prada et al., 1991). However, all classes of neurons, at varying proportions, are being produced during most of the time. Fischer and Reh (2000) have found that new neurons are added to the retina of the chicken via proliferation and subsequent differentiation of neurons and glia at the retinal margin in a zone highly reminiscent of the ciliary marginal zone of lower vertebrates, e.g. fish and amphibians. The ciliary marginal zone is represented by a circumferential zone of cells that generates new retinal cells which are continually added at the anterior margin of the retina. The regenerative capacity of the ciliary margin in the chick embryo was clearly demonstrated by the reconstitution of organized laminar reaggregates from dispersed cells de- rived from the retinal periphery (Layer et al., 1990), which included pluripotent embryonic stem cells (Willbold and Layer, 1992). Most interestingly, stem cells still exist in the adult eye periphery of chicken (Fischer and Reh, 2000) and mice (Ahmad et al., 2000, 2004; Tropepe et al., 2000), stressing the significance of this tissue part for the topic of retinal regeneration. Patterning and differentiation within the retina is regulated by cell intrinsic as well as cell extrinsic mechanisms. This is reflected by differential expression of diverse genes, as has been demonstrated repeatedly, e.g. by microarray and in situ analyses for the chick retina (Hackam et al., 2003). In order to better understand the molecular basis of tissues that are still proliferative, e.g. as the ciliary retinal margin, or of tissues which are undergoing differentiation processes, like the more central retina, we have compared gene expression patterns of central and peripheral retinae by differential display– reverse transcription–polymerase chain reaction (DD-RT- PCR). Besides a multitude of other gene products, one of the genes up-regulated in the peripheral retina was the cytochrome-c oxidase I (COX-1; ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). In mature nervous tissues, which require much energy, this enzyme is an indicator of neuronal activity (Wong-Riley, 1989). Here, our genetic as *Corresponding author. Tel: +49-6151-166105; fax: +49-6151-166548. E-mail address: paraoanu@bio.tu-darmstadt.de (L. E. Paraoanu). Abbreviations: cDNA, complementary DNA; COX-1, cytochrome-c oxidase I; DD-RT-PCR, differential display–reverse transcription– polymerase chain reaction; DIG, digoxigenin; E, embryonic day; EST, expressed sequence tag; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; N-CAM, neuronal cell adhesion mol- ecule; ONL, outer nuclear layer; OPL, outer plexiform layer; PBS, phosphate-buffered saline; RPE, retinal pigment epithelium; SSC, standard saline citrate. Neuroscience 132 (2005) 665– 672 0306-4522/05$30.00+0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.11.055 665