Abstract
Abstract: :
Purpose: We previously reported that various photoreceptor–specific genes show dissimilar temporal and/or spatial patterns of expression, suggesting that they may be regulated through different mechanisms. We have now tested this hypothesis by investigating the effects of growth factors and second messenger molecules on the expression of visual pigments and other photoreceptor–specific genes. Methods: At embryonic day (ED) 5–6, when photoreceptors are being generated, retinas were isolated from chick embryos, dissociated, and grown in low–density cultures either in control medium or in the presence of CNTF, activin, or staurosporin. After 4 days in vitro the cultures were analyzed by in situ hybridization (ISH), RT–PCR and real time PCR for the expression of visual pigments and other photoreceptor–specific genes involved in phototransduction, retinoid metabolism and outer segment formation. Results: The calcium–binding protein visinin was detected by ISH in nearly all cultured photoreceptors under each of the conditions tested. In control cultures, the red cone pigment (iodopsin) was practically the only VP detected by ISH, being present in over 50% of the photoreceptors; the green pigment was seldom seen by ISH, but was detectable by PCR. Staurosporin, a protein kinase inhibitor, caused induction of rhodopsin expression, accompanied by a suppression of iodopsin expression. Iodopsin expression was also suppressed by activin, but without concomitant changes in rhodopsin. Green mRNA levels increased 10 fold in CNTF treated cultures, becoming detectable by ISH in over 50% of the photoreceptors; it appeared co–expressed with iodopsin in some cells. Most of the remaining photoreceptor–specific genes investigated under the various experimental conditions failed to show changes equivalent to, or correlated with, those observed for visual pigment genes. Conclusions: The data show that CNTF, activin, and modulators of protein kinase activity have powerful but dissimilar effects on different photoreceptor subtypes, and support the notion that various photoreceptor–specific genes are independently regulated through different mechanisms.
Keywords: gene/expression • color pigments and opsins • signal transduction