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(Investigative Ophthalmology and Visual Science. 2006;47:2716-2725.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-1472
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Knock-Down of GFR{alpha}4 Expression by RNA Interference Affects the Development of Retinal Cell Types in Three-Dimensional Histiotypic Retinal Spheres

Andrée Rothermel,1,2 Katja Volpert,2,3 Mirjam Burghardt,3 Christina Lantzsch,1 Andrea A. Robitzki,1 and Paul G. Layer3

1From the Center for Biotechnology and Biomedicine, Molecularbiological Biochemical Processing Technology, University of Leipzig, Leipzig, Germany; and the 3Institute for Zoology, Developmental Biology and Neurogenetics, Darmstadt University of Technology, Darmstadt, Germany.

PURPOSE. To determine the role of glial cell line-derived neurotropic factor family receptor alpha 4 (GFR{alpha}4) during retinogenesis in a three-dimensional histiotypic in vitro model of the embryonic chicken retina.

METHODS. Retinal spheres were cultured from dissociated 6-day-old chicken retina under permanent rotation and transfected with GFR{alpha}4 siRNA at culture day 2. Alterations on proliferation, apoptosis, and differentiation were determined by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry after 24, 48, and 72 hours.

RESULTS. In contrast to control cultures, retinal spheres transfected with GFR{alpha}4 siRNA showed reduced GFR{alpha}4 mRNA expression of only 38% after 24 hours, 3% after 48 hours, and 5% after 72 hours. Based on the suppression of GFR{alpha}4, a decline in proliferating cells from 10% to 4.8% even after 24 hours and a reduction of sphere size by up to 25% at later culture stages were observed. Moreover, the number of Pax 6-positive amacrine, ganglion, and horizontal cells was significantly decreased from 36% to 16% in GFR{alpha}4 siRNA-transfected retinal spheres 72 hours after transfection. Additionally, GFR{alpha}4 gene silencing affected the development of different types of photoreceptors, as revealed by a significant decrease of blue opsin mRNA expression from 29% to 2%, whereas green opsin mRNA and the number rho4D2-positive photoreceptors were significantly increased.

CONCLUSIONS. These data showed for the first time that GFR{alpha}4 plays an essential role in regulating, at least in vitro, the development and differentiation of various cell types during retinogenesis.




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K. N. Volpert, A. Rothermel, and P. G. Layer
GDNF Stimulates Rod Photoreceptors and Dopaminergic Amacrine Cells in Chicken Retinal Reaggregates
Invest. Ophthalmol. Vis. Sci., November 1, 2007; 48(11): 5306 - 5314.
[Abstract] [Full Text] [PDF]


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