To date, 192 ZF miRNAs have been identified using classical cloning and Sanger sequencing methodologies [19, 20]. In this study, 25 novel miRNAs were added to the ZF repertoire using massively parallel DNA pyrosequencing of miRNA cDNA libraries prepared from different time points of the ZF embryo development and from different tissues. This methodology retrieved 173 of the 192 known ZF miRNAs whose expression in different tissues and developmental stages were validated using Northern blot analysis and/or in situ hybridizations. This high degree of data overlapping between cloning/Sanger sequencing and DNA pyrosequencing, plus the existence of target genes for the novel miRNAs, validated our approach of miRNA libraries fractionation and provided strong support for the authenticity of the newly identified miRNAs. The pyrosequencing approach also produced important information about the relative abundance of the ZF miRNAs. During early development (24 hpf), the number of different miRNAs was low (13), but expression level was high. The number of miRNA reads was higher at 72 hpf and in young adult fish. Among differentiated organs, brain and eyes showed the highest number of miRNA reads. This confirmed previous data showing differences in temporal miRNA expression and raised the hypothesis that many miRNAs play a role in late development and are required for organ morphogenesis .
Zebrafish microRNAs expression profile
The pyrosequencing data allowed us to build a miRNA expression profile for developmental differentiation and for adult fish, based on the normalized number of reads. Most miRNAs were expressed in more than one tissue (Figure 4A), others were tissue specific or showed stronger expression in specific tissues (Figure 4B), while others were development specific. Dre-miR-135c and dre-miR-25 were highly enriched at 24 hpf, but their relative expression decreased during embryo development. The data confirmed previous studies showing that dre-miR-135 expression is higher in development than in adult fish . The miR-430 family was also present during development and was absent in adult ZF [19, 20]. The expression profile also highlighted results of Giraldez and colleagues  showing that miR-430 is essential for regulation of morphogenesis during development.
Some miRNAs were expressed ubiquitously. For example, dre-miR-124 was abundant during both development and in adult fish, and its expression increased slightly during late stages of development and in the central nervous system (both brain and eyes). This miRNA alone accounted for ~48% of the pyrosequencing reads, a result that may be explained, at least in part, by the high copy number of its gene (6 copies in various chromosomes). At 24 hpf, when a significant part of the brain development had already occurred, dre-miR-124 represented 42% of the miRNA pool and its relative abundance reached 80% at 5 dpf. In the adult tissues, it represented 80% of brain and 54% of eye miRNAs. This is in agreement with previous studies in ZF and other organisms showing that miR-124 is up-regulated during development of the nervous system and is the most abundant miRNA in the adult brain [19, 20]. Also, neuronal differentiation is enhanced followed transfection of mir-124 in mouse neuronal stem cells . Taken together, the data suggest that dre-miR-124 may play an important role in ZF development, neuronal differentiation and in regulation of brain functions [35, 58]. On the other hand, dre-miR-203a and dre-miR-203b appeared early in development and maintained high levels of expression in adult fish, in particular in gills and skin. Indeed, miR-203 is a skin-specific keratinocyte-derived miRNA involved in keratinocyte differentiation .
A subset of miRNAs was expressed in differentiated tissues only. For example, dre-miR-101a, dre-miR-130b, dre-miR-130c, dre-miR-221 and dre-miR-499 were highly enriched in the heart, in agreement with previous in situ and Northern blot studies . Dre-miR-1 and dre-miR-133a were expressed in muscle and heart, where they play an important regulatory role in other organisms [60, 61]. Indeed, deletion of miR-1 altered regulation of cardiogenesis, electrical conduction and cell cycle of cardiomyocites, and miR-133 plus miR-1 regulate cardiac hypertrophy, as their over expression inhibits it. Interestingly, dre-miR-133b and dre-miR-133c were mainly detected in muscle and were not present in the heart. Finally, dre-miR-103 was specific of gut and liver while dre-miR-122 was liver specific [40, 62]. This was not surprising because mir-122 plays important roles in regulation of metabolism and its silencing in high-fat fed mice resulted in a significant reduction of hepatic steatosis, decreased cholesterol synthesis and stimulated fatty-acid oxidation .
Expression and putative functions of the novel zebrafish miRNAs
Of the 25 novel miRNAs identified in this study, 9 belong to conserved miRNA families (existing in at least one more organism) according to the conservation criteria used in this study, and the others are non-conserved (ZF specific). Most of the novel miRNAs are encoded by a single gene, but 7 are multigenic. In the latter case, miRDeep, Ensembl and RNAfold analysis showed that different genes encoding a single miRNA produce identical miRNA hairpins. Most of the novel miRNAs produced lower number of reads than the majority of the conserved miRNAs. This was not surprising since there is good correlation between miRNA conservation and expression level . Therefore, the low abundance of the novel miRNAs identified by pyrosequencing combined with the retrieval of 90% of the known ZF miRNAs (identified by cloning/Sanger sequencing) suggests that the vast majority of miRNAs present in our samples were retrieved. However, one cannot exclude the hypothesis that new miRNAs present in our dataset escaped identification due to the high stringency of the methodology used. Also, it is possible that other low abundance and highly specific ZF miRNAs may still be discovered using other deep DNA pyrosequencing strategies, namely Solexa/Illumina or SOLiD™. Finally, cDNA libraries from tissues not screened in this study may still reveal new ZF miRNAs. Recent bioinformatics analysis of the ZF genome identified additional miRNAs , however we were unable to identify reads matching these putative miRNAs using miRDeep alone or our pipeline data analysis system. This may be due to their very low expression level. Again, other massively parallel DNA pyrosequencing approaches may overcome these limitations and uncover such putative miRNAs .
Our bioinformatics approach retrieved 41 candidate target genes of 15 novel miRNAs. Since we used stringent search criteria to minimize false positives one cannot exclude the possibility that some targets were missed. Despite this, comparative analysis of the targets of the conserved miR_15, miR_16 and miR_21 with those of known miRNAs produced significant overlapping, thus validating our target prediction approach. For example, miR_16, which belongs to the miR-107 family, has GFM2 and VOX genes as putative targets. The miRBase Targets Version 5 also retrieved these genes as candidate targets for dre-miR-107. Similar results were obtained for miR_21 where RNF11 gene was highlighted as a candidate target of this novel miRNA of the mir-222 family. This result is also supported by retrieval of miR-222 in a blast search for miRNAs that target RNF11.
Most of the predicted targets are involved in cellular and developmental processes, which is in agreement with their expression during development. Indeed, the NRP2A gene, a putative target of miR_8, is involved in the differentiation of the nervous system, neural crest cell migration  and in VEGF-mediated vessel development . This correlates with the expression pattern of this miRNA at 72 hpf, 96 hpf, 5 dpf and in the adult brain. Also, miR_9, expressed at 72 hpf and 96 hpf, is predicted to target the PRDM1 and zgc:85707 genes which play important roles in embryonic axis specification, embryonic pectoral fin morphogenesis, regulation of neuron specification, regulation of signal transduction and multicellular organism development . The SEC23B and MYST3 genes which are involved in cartilage development were retrieved as putative targets of miR_10, which was also expressed during development. MYST3 (or MOX) regulates HOX expression and segmental identity, including cartilage patterning . Finally, the gills specific miR_14 is predicted to target the MELK gene, which is also strongly expressed in the gills and is involved in erythrocyte development .
Zebrafish microRNA star sequences
Star sequences of miRNAs (miRNA*) are difficult to detect by conventional methods due to their rapid turnover. However, high throughput sequencing retrieved many of them and revealed their relative abundance in different organisms [19, 20, 57, 64]. Our DNA pyrosequencing approach identified 107 miRNA* sequences: 42 were identified previously by cloning and Sanger sequencing [19, 20], 60 were identified in this pyrosequencing study, but belong to known miRNAs, and other 5 miRNA* belong to the novel miRNAs identified in this study. Most star sequences retrieved fewer reads than the corresponding mature miRNAs which is consistent with the miRNA biogenesis model and strand selection by RISC. However, six miRNA* were more abundant than their corresponding mature miRNAs, namely dre-miR-129*, dre-miR-140*, dre-miR-142a*, dre-miR-202*, dre-miR-210* and dre-miR-214*. Similar results were observed before for dre-mir-129*, dre-mir-142a*, dre-mir-142b* and dre-mir-214* . Dre-miR-30e, dre-miR-199, dre-miR-219 and dre-miR-462 showed similar strand-bias of both mature and star strands. Since this was also observed in the chicken embryo for mir-30e and mir-219  and in Drosophila melanogaster where several miRNA* are present at physiologically relevant levels and associate with Argonaute proteins, it is likely that both strands are loaded into RISC and may guide target repression. Finally, observed alterations in the ratio of expression of mature/star molecules suggests that some star molecules are functional and their activity may vary according to cellular context [57, 64]. Obviously, the biological function of these star sequences can only be unravelled through experimental testing, but their high number of reads suggests their inclusion in future ZF miRNA chips and expression profiling studies.