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M. Rieke, P.G. Layer; Histotypic Reaggregates From Postnatal Gerbil Retinae (Meriones Unguiculatus), and Their Dependance on LiCl Supplementation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5750.
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© ARVO (1962-2015); The Authors (2016-present)
Previous work has shown that reaggregation of cells from the E6 chick retina leads to cellular spheres including constituents of all retinal layers. This in vitro histogenesis was supported by secreted factors derived from the retinal pigmented epithelium (RPE) or Muller glia (1). To analyse the reorganisation ability of mammalian retinal cells, we here established a reaggregation system from gerbil retina (Meriones unguiculatus). Since in the chicken system Wnt–2b was able to mimic the histogenetic effects (2), we also studied the involvement of the Wnt pathway in histogenesis of gerbil reaggregates by LiCl supplementation.
3D–spheres derived from dissociated retinal cells of postnatal days 1–3 (P1–P3) gerbils were grown in reaggregation medium. LiCl was added to the medium to activate Wnt downstream signalling and also Wnt–3a conditioned medium was used. Reaggregates were collected after 10–15 days in rotation culture, before the spheres were fixed (PFA 4%) and cryosectioned. Differentiation and reorganisation were documented immunohistochemically with antibodies against specific cell–markers.
Distinctive histotypic reorganisation occurred in 3D–spheres of mammalian retinal cells, whereby most major retinal cell–types could be identified. Ganglion and amacrine cells surrounded IPL–like areas, which were strictly separated from areas where photoreceptors occurred. Photoreceptors differentiated only in spheres derived from advanced stages (P2–P3), while they were absent in spheres from P1 retina. Treatment with LiCl led to an increase of sphere sizes. Interestingly, now photoreceptors appeared already in spheres from earlier stages.
Dissociated retinal cells from Gerbil were able to form histotypic reaggregates; their degree of organisation was higher than in any mammalian system described before, but is still inferior to the chick system. Interestingly, IPL–like areas appeared earlier than photoreceptor expression. An involvement of Wnt signalling in the in vitro histogenesis of mammalian retinospheres was indicated by LiCl supplementation.
1) Layer PG, Robitzki A, Rothermel A, Willbold E. Trends Neurosci. 25(3), 131–134, 2002.
2) Nakagawa S, Takada S, Takada R, Takeichi M. Developmental Biology 260, 414–425, (2003)
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