MicroRNA Expression in Zebrafish Embryonic Development

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Science  08 Jul 2005:
Vol. 309, Issue 5732, pp. 310-311
DOI: 10.1126/science.1114519


MicroRNAs (miRNAs) are small noncoding RNAs, about 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, which suggests 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 Caenorhabditis 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 are 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 1 to 2 days after 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).

Fig. 1.

miRNA expression in zebrafish embryonic development. (A) Microarray expression levels of 90 (of the 115) miRNAs during embryonic development. Colors indicate relative and mean-centered expression for each miRNA: blue, low; black, mean; yellow, high. (B) Ventral view of miR-140 whole-mount in situ expression in cartilage of pharyngeal arches, head skeleton, and fins at 72 hpf. (C) Lateral views of miRNA whole-mount in situ expression in different organ systems at 72 hpf: miR-124a, nervous systems; miR-122, liver; miR-206, muscles; miR-126, blood vessels and heart; miR-200a, lateral line system and sensory organs; miR-30c, pronephros. (D) Histological analysis of miRNA in situ expression in the pancreas 5 days after fertilization. Abbreviations: e, exocrine pancreas; i, pancreatic islet; gb, gall bladder; g, gut.

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 catalog 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). Representative examples are shown (Fig. 1C) 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, are 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 zebrafish and mammals (fig. S2 and table S3). Up to 77% of the in situ expression patterns were confirmed by at least one of the microarray data sets. In addition, miRNA in situ data showed patterns that cannot easily be detected by microarrays. For example, some miRNAs were expressed in hair cells of sensory epithelia (fig. S6).

In conclusion, we here describe the first comprehensive set of miRNA expression patterns in animal development. We found these patterns to be remarkably specific and diverse, which suggests highly specific and diverse roles for miRNAs. Most miRNAs are expressed in a tissue-specific manner during segmentation and later stages but were not detected during early development. Although we cannot exclude a role for undetectable early miRNAs, this observation indicates that most miRNAs may not be essential for tissue fate establishment but rather play crucial roles in differentiation or the maintenance of tissue identity.

Supporting Online Material

Materials and Methods

Figs. S1 to S6

Tables S1 to S3

Database S1

References and Notes

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