Abstract
Purpose::
Previous work has shown that reaggregation of cells from Gerbil (Meriones unguiculatus) retina of postnatal day 1 (P1) leads to cellular spheres including constituents of the inner plexiform layer (IPL). This in vitro histogenesis was further promoted by secreted factors derived from the retinal pigmented epithelium (RPE). Since in a similar chicken system Wnt-2b was able to induce lamination and mimic retinal histology (1), we studied the involvement of the Wnt pathway in histogenesis of gerbil reaggregates by supplementation with Wnt conditioned media and GSK-3 inhibitors.
Methods::
3D-spheres derived from dissociated retinal cells of postnatal days 1-3 (P1-P3) gerbils were grown in reaggregation medium (DMEM plus 10% fetal calf serum, 0.1% penicillin/streptomycin, 0.01% gentamycin). Wnt-3a conditioned medium or, alternatively, GSK-3 inhibitors were added to activate the Wnt cascade. 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 and proliferation was studied by BrdU uptake.
Results::
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. Photoreceptors differentiated only in spheres derived from advanced stages (P2-P3), while they were absent in spheres from P1 retina. Wnt conditioned media induced a remarkable degree of laminar organisation. On the other side, treatment with LiCl increased sphere sizes. In this case, photoreceptors appeared already at earlier stages.
Conclusions::
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 it is still inferior to the chick system. Interestingly, IPL-like areas appeared earlier than photoreceptor expression. An involvement of Wnt signaling in the in vitro histogenesis of mammalian retinospheres was indicated. References: Nakagawa S, Takada S, Takada R, Takeichi M. Developmental Biology 260, 414-425, (2003)
Keywords: differentiation • retinal culture • retinal development