| MicroRNAs (miRNAs) constitute an abundant class of small RNAs that negatively regulate specific target mRNAs at the posttranscriptional level. Several miRNAs are expressed in a tissue-specific or developmentally restricted manner and have been implicated in tissue specification, neuronal differentiation, sensory cell fate specification, and stem cell maintenance. To evaluate whether miRNAs might also regulate specific aspects of olfactory neurogenesis, miRNAs were isolated from several olfactory tissues including the main olfactory epithelium (MOE), vomeronasal organ (VNO), and olfactory bulb (OB). This led to the identification of 18 novel miRNAs; 11 of which are restricted to olfactory tissues by Northern analysis. Using microarray hybridization, we examined the profile of miRNA expression during olfactory development and detected 4 additional miRNAs with olfactory-specific expression. To explore the spatial patterns of miRNA expression, we have developed a mammalian miRNA in situ hybridization protocol. The majority of olfactory-specific miRNAs were detected in olfactory neurons and two appeared restricted to olfactory precursors. The miRNA expression analysis also unveiled a temporal and spatial association of olfactory fate specification and miRNA expression. In order to evaluate the function of the olfactory-specific miRNAs, we took a computational approach to predict potential target genes. We found that over 30% of the predicted targets are involved in transcriptional regulation and development. Three predicted targets with well-characterized roles in neurogenesis including Notch 1, Zinc finger homeobox1a, and Zinc finger homeobox1b, were validated in vitro. To assess the global role of small RNAs in olfaction, we used mice carrying a conditional loss of function Dicer allele. Dicer is an enzyme required for the production of mature miRNAs and other small RNAs. When Dicer was eliminated from mature olfactory neurons, no morphological, developmental, or behavioral phenotype could be detected. We are currently in the process of evaluating the effects of Dicer deletion in olfactory precursors. In conclusion, this work has uncovered the existence of several miRNAs which, based on spatiotemporal expression patterns and target predictions, may be involved in olfactory development and neurogenesis. |
