Use of suppression subtractive hybridization to identify genes regulated by ciliary neurotrophic factor in postnatal retinal explants.
OBJECTIVE
The retinal progenitors are multipotential, and the decision taken by a progenitor to differentiate along a particular path depends on both cell-intrinsic and cell-extrinsic factors. Ciliary neurotrophic factor (CNTF), a member of the interleukin-6 (IL-6) family, added to rat postnatal retinal progenitors inhibits rod photoreceptor cell differentiation, promotes Müller glia genesis and enhances the expression of bipolar neuron markers. We hypothesized that those transcripts regulated during CNTF-influenced retinal differentiation may be involved in the choice of progenitor cell fate. Our aim was to isolate these genes, characterize their expression in the retina, and to subsequently focus on candidates that may promote photoreceptor cell differentiation.


METHODS
Retinas were cultured in vitro as explants at postnatal day 0 (P0) in the absence or presence of CNTF for six days. Transcripts regulated by CNTF after six days in vitro (DIV) were selected by subtraction suppressive hybridization (SSH) and cloned as two libraries. The UC6 and DC6 libraries contained those genes upregulated and downregulated, respectively, in the presence of CNTF at 6DIV.


RESULTS
In the first library, UC6, eight clones representing seven different genes were isolated as up-regulated by CNTF. In the DC6 library, 21 clones, representing 17 different genes appeared as down-regulated by CNTF. Genes were classified in six categories, such as protein modification, signal transduction, and regulation of transcription according to the Gene Ontology Annotation.


CONCLUSIONS
Among the 24 selected genes, our study revealed 11 genes (two upregulated and nine downregulated) potentially involved in CNTF biological effects.
Roger
Jérôme
J
1INSERM U592, Paris, France. roger@st-antoine.inserm.fr <roger@st-antoine.inserm.fr>
Goureau
Olivier
O
Sahel
José-Alain
JA
Guillonneau
Xavier
X
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics; D020934:Ciliary Neurotrophic Factor / Q000502:physiology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D015723:Gene Library; D009693:Nucleic Acid Hybridization / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014018:Tissue Distribution
D020934:Ciliary Neurotrophic Factor; D005136:Eye Proteins