Chick Retinal Pigment Epithelium Transdifferentiation Assay for Proneural Activities

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2012 Jun 13

PMID 22688708

Authors

Shu-Zhen Wang

Run-Tao Yan

Affiliations

Soon will be listed here.

Abstract

We describe a cell culture system for assaying proneural activities of genes hypothesized to play instrumental roles in neuronal fate specification during vertebrate retinal development. The retinal pigment epithelium (RPE) is collected from embryonic day 6 (E6) chick to establish a primary RPE cell culture. The culture is then infected with a replication competent retrovirus RCAS expressing the gene of interest. The presence of retinal neurons in the otherwise nonneural, RPE cell culture is examined between 4 and 10 days after the administration of the virus. Taking advantage of the plasticity and the relative simplicity of RPE cells, this method offers an informative assay for proneural activities prior to planning for large-scale in vivo experiments.

References

Xie W, Yan R, Ma W, Wang S . Enhanced retinal ganglion cell differentiation by ath5 and NSCL1 coexpression. Invest Ophthalmol Vis Sci. 2004; 45(9):2922-8. PMC: 1986831. DOI: 10.1167/iovs.04-0280. View

Opas M, Dziak E . bFGF-induced transdifferentiation of RPE to neuronal progenitors is regulated by the mechanical properties of the substratum. Dev Biol. 1994; 161(2):440-54. DOI: 10.1006/dbio.1994.1043. View

Liang L, Yan R, Li X, Chimento M, Wang S . Reprogramming progeny cells of embryonic RPE to produce photoreceptors: development of advanced photoreceptor traits under the induction of neuroD. Invest Ophthalmol Vis Sci. 2008; 49(9):4145-53. PMC: 2570253. DOI: 10.1167/iovs.07-1380. View

Ma W, Yan R, Xie W, Wang S . bHLH genes cath5 and cNSCL1 promote bFGF-stimulated RPE cells to transdifferentiate toward retinal ganglion cells. Dev Biol. 2004; 265(2):320-8. DOI: 10.1016/j.ydbio.2003.09.031. View

Guillemot F, Cepko C . Retinal fate and ganglion cell differentiation are potentiated by acidic FGF in an in vitro assay of early retinal development. Development. 1992; 114(3):743-54. DOI: 10.1242/dev.114.3.743. View

Zhao S, Thornquist S, Barnstable C . In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural retina. Brain Res. 1995; 677(2):300-10. DOI: 10.1016/0006-8993(95)00163-k. View

Araki M, Yamao M, Tsudzuki M . Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail. Dev Growth Differ. 1998; 40(2):167-76. DOI: 10.1046/j.1440-169x.1998.00006.x. View

Yan R, Wang S . neuroD induces photoreceptor cell overproduction in vivo and de novo generation in vitro. J Neurobiol. 1998; 36(4):485-96. PMC: 1764458. View

Reh T, Nagy T, Gretton H . Retinal pigmented epithelial cells induced to transdifferentiate to neurons by laminin. Nature. 1987; 330(6143):68-71. DOI: 10.1038/330068a0. View

10.

Grisanti S, Guidry C . Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype. Invest Ophthalmol Vis Sci. 1995; 36(2):391-405. View

11.

Ma W, Wang S . The final fates of neurogenin2-expressing cells include all major neuron types in the mouse retina. Mol Cell Neurosci. 2005; 31(3):463-9. PMC: 1876733. DOI: 10.1016/j.mcn.2005.10.018. View

12.

Pennesi M, Cho J, Yang Z, Wu S, Zhang J, Wu S . BETA2/NeuroD1 null mice: a new model for transcription factor-dependent photoreceptor degeneration. J Neurosci. 2003; 23(2):453-61. PMC: 6741880. View

13.

Yan R, Wang S . Expression of an array of photoreceptor genes in chick embryonic retinal pigment epithelium cell cultures under the induction of neuroD. Neurosci Lett. 2000; 280(2):83-6. DOI: 10.1016/s0304-3940(99)01003-4. View

14.

Yan R, Wang S . Differential induction of gene expression by basic fibroblast growth factor and neuroD in cultured retinal pigment epithelial cells. Vis Neurosci. 2000; 17(2):157-64. PMC: 1986837. DOI: 10.1017/s0952523800171172. View

15.

Eguchi G . Instability in cell commitment of vertebrate pigmented epithelial cells and their transdifferentiation into lens cells. Curr Top Dev Biol. 1986; 20:21-37. DOI: 10.1016/s0070-2153(08)60652-3. View

16.

COULOMBRE J, Coulombre A . Regeneration of neural retina from the pigmented epithelium in the chick embryo. Dev Biol. 1965; 12(1):79-92. DOI: 10.1016/0012-1606(65)90022-9. View

17.

Mao W, Yan R, Wang S . Reprogramming chick RPE progeny cells to differentiate towards retinal neurons by ash1. Mol Vis. 2008; 14:2309-20. PMC: 2603186. View

18.

Sakami S, Etter P, Reh T . Activin signaling limits the competence for retinal regeneration from the pigmented epithelium. Mech Dev. 2007; 125(1-2):106-16. PMC: 2254174. DOI: 10.1016/j.mod.2007.10.001. View

19.

Pittack C, Jones M, Reh T . Basic fibroblast growth factor induces retinal pigment epithelium to generate neural retina in vitro. Development. 1991; 113(2):577-88. DOI: 10.1242/dev.113.2.577. View

20.

Mao W, Yan R, Wang S . Proneural gene ash1 promotes amacrine cell production in the chick retina. Dev Neurobiol. 2008; 69(2-3):88-104. PMC: 2629813. DOI: 10.1002/dneu.20693. View