Abstract
Abstract: :
Purpose: All cells must make ribosomes, in which rRNA transcription is the rate–limiting step; however, some cells may require more ribosomes than others. Cell–type specific regulation of rRNA synthesis has been largely ignored in the past, because of the inability to measure rRNA transcription rates in situ. Here we describe a map of rRNA transcription rates in ocular tissues detected by a novel in situ hybridization technique. We believe this is the first map of its kind. Methods: Traditionally, the rate of transcription has been measured by metabolic labeling (e.g. the run–on assay), which can be carried out only in isolated or cultured cells. It has been difficult, if not impossible, to assess the rate of gene transcription of a gene in a specific cell type in situ. We described recently an in situ hybridization technique, which detects the quantity of 47S precursor rRNA (pre–rRNA), the nascent transcript of rRNA genes. We showed that the quantity of 47S pre–rRNA correlated positively with the rate of rRNA transcription in vivo. We found that 47S pre–rRNA was a far more sensitive indicator for perturbation in rRNA synthesis than the mature 18S or 28S rRNA. Results: Using this technique, we constructed a map of RNA transcription activity in individual cells in the entire mouse eye during its embryonic and neonatal development, adulthood and aging. We found that rRNA transcription rate varies widely in the different ocular tissues. The highest levels of rRNA transcription were detected in adult corneal epithelium, lens transitional zone, and the ganglion cells and the outer nuclear layers of retina. Within these tissues, rRNA transcription was also regulated developmentally, especially during the neonatal development prior to eye opening. We also detected a decline in rRNA synthesis in the lens transitional zone in aged eyes. Analyses of cellular proliferation in parallel revealed that rRNA transcription was correlated with cell proliferation in cornea and lens epithelia. However, no such correlation was observed in retinal ganglion cells and lens cells in the transitional zone. Conclusions: These findings provide a base–line for future studies of cell–type specific regulation of rRNA synthesis in ocular tissues. It is not surprising that actively proliferating cells have a higher ribosomal need; however, it is puzzling that some of the quiescent retinal cells synthesize rRNA at a high rate throughout their life. Because these cells do not divide, their ribosomes, which usually have a long–half life, would not be diluted. Our observation therefore suggests the existence of an unappreciated biochemical need of these cells.
Keywords: gene/expression • in situ hybridization • transcription