MicroRNA Expression in Zebrafish Embryonic Development Erno Wienholds, 1 Wigard P. Kloosterman, 1 Eric Miska, 2,3 Ezequiel Alvarez-Saavedra, 2 Eugene Berezikov, 1 Ewart de Bruijn, 1 H. Robert Horvitz, 2 Sakari Kauppinen, 4 Ronald H. A. Plasterk 1* 1 Hubrecht Laboratory, Centre for Biomedical Genetics, Utrecht, the Netherlands. 2 Howard Hughes Medical Institute, Department of Biology and McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, USA. 3 Wellcome Trust, Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom. 4 Wilhelm Johannsen Centre for Functional Genome Research, Institute of Medical Biochemistry and Genetics, University of Copenhagen, Denmark. *To whom correspondence should be addressed. E-mail: plasterk@niob.knaw.nl MicroRNAs (miRNAs) are small noncoding RNAs, approximately 21 nucleotides in length, that can regulate gene expression by base-pairing to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in situ hybridizations, using locked- nucleic-acid–modified oligonucleotide probes. Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, suggesting that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity. Current estimates of miRNA gene numbers in vertebrates are as high as 500 (1), of which many are conserved, and miRNAs may regulate up to 30% of genes (2). The miRNA first discovered, lin-4, is involved in developmental timing in the nematode C. elegans (3). In mammals, miRNAs have been implicated in hematopoietic lineage differentiation (4) and homeobox gene regulation (5). Zebrafish that are defective in miRNA processing arrest in development (6). Recently, miRNAs were shown to be dispensable for cell fate determination, axis formation and cell differentiation, but required for brain morphogenesis in zebrafish embryos (7). Together, these findings indicate that miRNAs can play essential roles in development. However, little is known about the individual roles of most miRNAs. To focus future miRNA studies, we determined the spatial and temporal expression patterns of 115 conserved vertebrate miRNAs (see online Material and Methods; table S1; table S2) in zebrafish embryos. First we determined the temporal expression of miRNAs during embryonic development by microarray analysis (Fig. 1A and fig. S1A). Up to segmentation (12 hours post- fertilization (hpf)) most miRNAs could not be detected. Most miRNAs became visible at 1 to 2 days post-fertilization and showed strong expression when organogenesis is virtually completed (96 hpf). In adults the majority of miRNAs remained expressed (Fig. 1A). In addition we determined the expression of miRNAs in dissected organs of adult fish. For some miRNAs a high degree of tissue specificity was observed (figs. S1B and S2, and table S3). In situ hybridization of miRNAs had thus far not been possible in animals. Recently LNA (locked-nucleic-acid)– modified DNA oligonucleotide probes have been shown to increase the sensitivity for the detection of miRNAs by northern blots (8). By northern blots analysis and in situ hybridization, using LNA probes, we detected predominantly mature miRNAs, which were reduced in dicer knockout zebrafish (fig. S3). We used these LNA probes for the whole- mount in situ detection of the conserved vertebrate miRNAs in zebrafish embryos and made a catalogue of miRNA expression patterns (fig. S4 and database S1). Most miRNAs (68%) were expressed in a highly tissue- specific manner. For example, miR-140 was specifically expressed in the cartilage of the jaw, head and fins, and its presence was entirely restricted to those regions (Fig. 1B and database S1). Fig. 1C shows representative examples of six miRNAs that were expressed in different organ systems: nervous system, digestive system, muscles, circulatory system, sensory organs and excretory system. Even within organs there is specificity, as exemplified in Fig. 1D, where miR-217 can be seen to be expressed in the exocrine pancreas, and miR-7 in the endocrine pancreas (Langerhans islets). More than half of the miRNAs (43) were expressed in (specific regions of) the central nervous system (fig. S4). Many miRNA genes are clustered in the genome and therefore probably expressed as one primary transcript, and indeed we observed that many such clustered genes showed identical or overlapping expression patterns (figs. S4 and S5). We compared the in situ data with microarray data for / www.sciencexpress.org /26 May 2005 / Page 1/ 10.1126/science.1114519