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Many proteins are assumed to mediate post-transcriptional regulation of mRNAs. However, the lack of information about their target mRNAs and functional domains hampers the detailed analysis of their molecular function. Here we describe a method to analyze the post-transcriptional effects of proteins of interest by artificially tethering the protein to a reporter mRNA in zebrafish embryos.

Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish., (Copyright © 2017 Elsevier Inc. All rights reserved.)

MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5' cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.

Many studies have demonstrated the functions of individual genes associated with embryogenesis and have determined the genome sequences of several organisms. Despite the availability of enormous amount of genetic information, dynamic changes that occur during embryogenesis have not yet been completely understood. In order to understand the dynamic processes involved in embryogenesis, we employed the metabolomic approach. The results of our study indicated that there is a close correlation between metabolomes and developmental stages. Our method enables the identification of embryonic stages using metabolomes as "fingerprints." In this manner, we could successfully predict embryonic development on the basis of metabolomic fingerprints. This is the first report describing a model for predicting vertebrate development by using metabolomics.

In oocytes, many maternally supplied products are stored, and these products play important roles in cell cycle regulation and early development. Mos protein, which is coded on the c-mos gene, promotes oocyte maturation and is involved in MAP-kinase signaling pathway. In Xenopus, maternally supplied c-mos mRNA undergoes poly(A) addition, and translational activation via CPE (cytoplasmic polyadenylation element) and CPEB (CPE binding protein). The elongated poly(A) is shortened and the c-mos mRNA is degraded during early embryogenesis via EDEN (embryo deadenylation element) and EDEN-BP (EDEN-binding protein). We cloned the full-length zebrafish c-mos gene, which is conserved at the protein coding region in vertebrates. c-mos mRNA has two putative CPE sequences in its 3'UTR, which binds to zebrafish CPEB homologous protein, Zor-1. We could not observe EDEN sequence, and could not detect interaction between c-mos mRNA and zebrafish EDEN-BP homologous protein, Brul, even though immuno precipitation and RT-PCR experiments suggested that c-mos mRNA interacts with Zor-1 in vivo. Interestingly, we found c-mos mRNA is located in the animal cortex of zebrafish oocyte, where Zor-1 protein exists. Taken together, these results suggest that the animal cortex is the central core of oocyte maturation in zebrafish.

In zebrafish, primordial germ cells (PGCs) are determined by a specialized maternal cytoplasm, the germ plasm, which forms at the distal ends of the cleavage furrows in 4-cell embryos. The germ plasm includes maternal mRNAs from the germline-specific genes such as vasa and nanos1, and vegetally localized dazl RNA is also incorporated into the germ plasm. However, little is known about the distributions and assembly mechanisms of germ plasm components, especially during oogenesis. Here we report that the germ plasm RNAs vasa, nanos1, and dazl co-localize with the mitochondrial cloud (MC) and are transported to the vegetal cortex during early oogenesis. We found that a mitochondrial cloud localization element (MCLE) previously identified in the 3' untranslated region (3'UTR) of Xenopus Xcat2 gene can direct RNA localization to the vegetal cortex via the MC in zebrafish oocytes. In addition, the RNA-binding protein Hermes is a component of the MC in zebrafish oocytes, as is the case in Xenopus. Moreover, we provide evidence that the dazl 3'UTR possesses at least three types of cis-acting elements that direct multiple steps in the localization process: MC localization, anchorage at the vegetal cortex, and localization at the cleavage furrows. Taken together, the data show that the MC functions as a conserved feature that participates in transport of the germ plasm RNAs in Xenopus and zebrafish oocytes. Furthermore, we propose that the germ plasm components are assembled in a stepwise and spatiotemporally-regulated manner during oogenesis and early embryogenesis in zebrafish.

The ribosomal proteins (RPs) form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases.

Early in development, primordial germ cells (PGCs) are set aside from somatic cells and acquire a unique gene-expression program . The mechanisms underlying germline-specific gene expression are largely unknown. Nanos expression is required during germline development and is posttranscriptionally restricted to PGCs . Here we report that the microRNA miR-430 targets the 3' untranslated region (UTR) of nanos1 during zebrafish embryogenesis. A miR-430 target site within the nanos1 3' UTR reduces poly(A) tail length, mRNA stability, and translation. Repression is disrupted in maternal-zygotic dicer mutants (MZdicer), which lack mature miRNAs , and is restored by injection of processed miR-430. Although miR-430 represses other genes equally in germline and soma, specific regions in the nanos1 3' UTR compensate for microRNA-mediated repression in PGCs and allow germline-specific expression. We show that the 3' UTR of an additional PGC-specific gene, TDRD7, is also targeted by miR-430. These results indicate that miR-430 targets the 3' UTRs of germline genes and suggest that differential susceptibility to microRNAs contributes to tissue-specific gene expression.

Maternally supplied germ plasm is essential for germ lineage establishment in many species, but the molecular details are still largely unknown, especially in vertebrates, and identification of novel factors that localize to germ plasm is desirable. We previously reported that one of the components of zebrafish germ plasm is mRNA of the bruno-like (brul) gene, a homologue of bruno, which, in Drosophila, is known to participate in germ lineage establishment. Here, we show that not only mRNA but also protein of brul is localized to the zebrafish germ plasm at the ends of the cleavage furrows. In 4- and 8-cell stage embryos, Brul protein is localized to the periphery of the blastomeres, as well as to the ends of the cleavage furrows, forming numerous minute particles. These particles appear at the cortex of the fertilized egg within 10 min after fertilization. Surprisingly, these distinctive localizations, as well as the minute particles, completely disappeared by the 16-cell stage, although relatively weak expression was detected ubiquitously throughout embryogenesis. This is the first report of a protein that localizes to the germ plasm in zebrafish.

Maternally supplied factors in fertilized eggs play essential roles in the establishment of primordial germ cells. In zebrafish, cytoplasm at the distal ends of the first and second cleavage furrows has been assumed to contain germ lineage determinants, since maternal transcripts of germ lineage-specific genes are localized to ends of the cleavage furrows. To investigate whether these parts of cytoplasm are required for germ cell formation, we removed all four regions of the cytoplasm by glass capillary at the 4-cell stage. Histological analysis revealed that the ablation of cytoplasm at the ends of the cleavage planes resulted in a severe reduction in the number of germ cells. In addition, the expression of germ lineage markers was eliminated by cytoplasmic ablation. These results demonstrated that cytoplasm at the distal ends of cleavage furrows is essential for germ cell formation. We also found novel localization patterns for zDazl and brul mRNAs along the cleavage planes. Our findings provide the first direct evidence that localized cytoplasmic factors are indispensable for germ cell establishment in zebrafish.

Background: In many species, DAZ homologous genes encode RNA-binding proteins containing two conserved motifs, namely the RNA-recognition motif (RRM) and the DAZ motif. Genetic analysis and gene disruption studies have demonstrated that DAZ family proteins play important roles in gametogenesis. However, little is known about the biochemical functions of DAZ family proteins., Results: Using in vitro selection and UV-crosslinking experiments, we identified the sequence 'GUUC' as the target RNA sequence of zebrafish DAZ-like protein (zDAZL). In transfection experiments, zDAZL protein activated translation in a manner dependent on the binding sequence in the 3'UTR of the Drosophila twine gene or zDazl gene. Moreover, it is highly likely that the zDAZL protein associates with polysomes through the DAZ motif in vivo, and that the association with polysomes is indispensable for translational activation., Conclusions: This is the first report that the DAZ family protein directly promotes the translation of the target mRNAs in vertebrates. This study provides important insights into the molecular mechanisms underlying the post-transcriptional regulation of DAZ family proteins in gametogenesis.