Knock-down of GFRalpha4 expression by RNA interference affects the development of retinal cell types in three-dimensional histiotypic retinal spheres.
OBJECTIVE
To determine the role of glial cell line-derived neurotropic factor family receptor alpha 4 (GFRalpha4) 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 GFRalpha4 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 GFRalpha4 siRNA showed reduced GFRalpha4 mRNA expression of only 38% after 24 hours, 3% after 48 hours, and 5% after 72 hours. Based on the suppression of GFRalpha4, 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 GFRalpha4 siRNA-transfected retinal spheres 72 hours after transfection. Additionally, GFRalpha4 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 GFRalpha4 plays an essential role in regulating, at least in vitro, the development and differentiation of various cell types during retinogenesis.
Rothermel
Andrée
A
Center for Biotechnology and Biomedicine, Molecularbiological Biochemical Processing Technology, University of Leipzig, Leipzig, Germany. andree.rothermel@bbz.uni-leipzig.de
Volpert
Katja
K
Burghardt
Mirjam
M
Lantzsch
Christina
C
Robitzki
Andrea A
AA
Layer
Paul G
PG
D025042:Amacrine Cells / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D030161:Avian Proteins / Q000235:genetics*; D002452:Cell Count; D002454:Cell Differentiation; D049109:Cell Proliferation; D002642:Chick Embryo; D047108:Embryonic Development / Q000502:physiology*; D005136:Eye Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing / Q000502:physiology*; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000235:genetics*; D018398:Homeodomain Proteins / Q000378:metabolism; D017403:In Situ Hybridization; D008562:Membrane Glycoproteins / Q000235:genetics*; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism; D015347:RNA Probes; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D012160:Retina / Q000166:cytology / Q000196:embryology*; D051245:Retinal Bipolar Cells / Q000166:cytology / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism; D051248:Retinal Horizontal Cells / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017299:Rod Opsins / Q000235:genetics; D014162:Transfection
D030161:Avian Proteins; D005136:Eye Proteins; C113146:GFR receptor alpha 4, Gallus gallus; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D018398:Homeodomain Proteins; D008562:Membrane Glycoproteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D015347:RNA Probes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D017299:Rod Opsins