![]() ![]() Radioactively labeled probes were detected in wax tissue sections as silver grains in a photographic emulsion that coated the section. When intracellular mRNA distribution was being established as a mechanism for creating embryonic asymmetry, ISH in fixed samples was the only available method for examining the distribution of transcripts in fixed samples. In this review, we discuss key advances in mRNA labeling and detection approaches, imaging instrumentation, post acquisition analysis and the impact this has made on the field. More recently, technical advances have allowed the visualization and quantitation of mRNA movement in living cells, enabling more effective analysis of the molecular mechanisms involved. Initially, only in situ hybridization (ISH) on fixed samples was available to study intracellular mRNA localization. Since the 1980s, when the link between mRNA localization and protein targeting was established, there has been considerable interest in intracellular imaging of the distribution of mRNAs. Specific RNA binding proteins within RNPs play essential roles in mRNA localization, translational regulation and degradation. Such transcripts associate in the nucleus with RNA binding proteins to form ribonucleoprotein complexes (RNPs), whose composition is then thought to be extensively remodeled during export from the nucleus and over the subsequent life cycle of the mRNAs in the cytoplasm. mRNA starts its life as nascent transcripts that are first processed and then exported from the nucleus into the cytoplasm. Intracellular localization of mRNA is now thought to be a very common mechanism to target protein function, occurring in most eukaryotic model organisms and for a very wide range of transcripts in the genome. Although the study of the spatial positioning of transcripts initially focused on differences in expression levels between tissues, approximately 30 years ago it was realized that transcripts can also localize asymmetrically within cells. The question of when and where genes are expressed has been of major interest in biology for at least 50 years. These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization. These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components. Most significantly, methods for following the movement of mRNA in living cells are now in widespread use. ![]() In addition to increased ease and specificity of detection in fixed tissue, in situ hybridization methods now enable examination of mRNA distribution at the ultrastructural level with electron microscopy. Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms. To explore the mechanisms involved, considerable effort has been spent in establishing new and improved methods for visualizing mRNA. Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology.
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