The expression and function of microRNAs in vertebrate embryonic development

Abstract

MicroRNAs regulate gene expression at the posttranscriptional level by binding to the 3'UTR of mRNAs. These small RNA molecules (~22 bases in length) are processed from long primary transcripts (pri-miRNA). In animals, microRNAs bind with imperfect complementarity to their target mRNA. This leads to relocalization of the mRNA to cytoplasmic bodies, where the mRNA is degraded and translationally repressed. Although the mechanism of inhibition is still unclear, the work in this thesis introduces one important aspect of miRNA regulation, which is the recognition of the target mRNA. Using zebrafish embryos as an in vivo system, the activity of the let-7 miRNA was determined. Analysis of all possible point mutant derivatives of let-7 showed that the first 8 nucleotides are most important for its activity. This part of the microRNA is known as the microRNA seed. As a consensus, many mRNA that are targeted by microRNAs are regulated by perfect seed pairing. MicroRNA target prediction algorithms use the microRNA seed as the basis for computational target identification. Such predictions have shown that every microRNA may regulate hundreds of mRNAs. The regulation of these mRNAs is essential for embryonic development, since animals without microRNAs cannot live. To understand the role of individual microRNAs in vertebrate embryonic development, this thesis describes a method to visualize microRNAs in the embryo, based on Locked Nucleic Acid (LNA) probes. MicroRNA expression analysis was thus far limited to low resolution Northern blotting and micro-arrays. Determining the expression of 115 conserved microRNAs with LNA probes revealed striking tissue-specific expression patterns, suggesting that microRNAs play a role in tissue development or maintenance of tissue identity. The complete microRNA repertoire of an animal is estimated to run into the thousands. To extend the set of zebrafish microRNAs, this thesis describes a cloning approach combined with deep-sequencing to identify new microRNAs. This showed that the conserved and highly expressed microRNAs in zebrafish are now known. Also 66 novel microRNAs were discovered, but these are generally poorly conserved and expressed at low levels. Vertebrate embryonic development is most easily studied in zebrafish, but genetically disrupting miRNA genes to see which miRNA does what is technically challenging. In this thesis, a method is described to transiently interfere with miRNA function during the first few days of zebrafish embryonic development by introducing specific antisense morpholino oligonucleotides (morpholinos have been used previously to interfere with the synthesis of the much larger mRNAs). Morpholinos targeting the miRNA precursor can block processing of the pri-miRNA or directly inhibit the activity of the mature miRNA. Morpholino-mediated microRNA knockdown did not reveal gross developmental defects for many microRNAs. However, knockdown of zebrafish miR-375 showed that this microRNA is essential for formation of the insulin-secreting pancreatic islet. Loss of miR-375 results in dispersed islet cells by 36 hours postfertilization, representing one of the first vertebrate miRNA loss-of-function phenotypes. Morpholinos will be widely applied for studying microRNAs in embryonic development.