• Animal cells and systems
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• v.2 no.4
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• pp.529-538
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• 1998

In order to analyze the intracellu1ar localization of specific RNA components of ribonucleoproteins (RNP) in Xenopus oocytes, a modified protocol of whole-mount in situ Hybridization is presented in this paper, Mitochondria specific 12S rRNA probe was used to detect the amplification and distribution of mitochondria in various stages of the oocyte life cycle, and the results were found to be consistent with previously known distribution of mitochondria. The results with other specific probes (U1 and U3 small nuclear RNAs, and 5S RNA) also indicate that this procedure is generally effective in localizing RNAs in RNP complexes even inside organelles. In addition, the RNA component of RNase MRP, the RNP with endoribo-nuclease activity, localize to the nucleus in various stages of the oocyte life cycle. Some of MRP RNA, however, were found to be localized to the special population of mitochondria near the nucleus, especially in the active stage of mitochondrial amplification. It suggests dual localization of RNase MRP in the nucleus and mitochondria, which is consistent with the proposed roles of RNase MRP in mitochondrial DNA replication and in rRNA processing in the nucleolus.

• Animal cells and systems
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• v.2 no.1
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• pp.113-116
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• 1998

A cDNA clone coding for ribosomal protein 46 (rp46) which is a component of 60S ribosomal large subunit has been identified from Drosophila melanogaster. A cDNA clone encoding S. cerevisiae rp46 was used as a probe to screen a Drosophila larvae cDNA library. The DNA sequence analysis revealed that the cDNA coding for Drosophils rp46 contains a complete reading frame of 153 nucleotides coding for 51 amino acids. The deduced amino acid sequence showed 71-75% homology with those of other eukaryotic organisms. Northern blot analysis showed that about 1-kb rp46 transcripts are abundant throughout fly development. Whole mount embryonic mRNA in situ hybridization also showed no preferential distribution of the transcripts to any specific region. The chromosomal in situ hybridization revealed that the identified gene is localized at position 60C on the right arm of the second polytene chromosome with a possibility of single copy.

• Animal cells and systems
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• v.1 no.2
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• pp.363-370
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• 1997

RNase MRP is a ribonucleoprotein complex with a site-specific endonuclease activity. Its original substrate for cleavage is the small mitochondrial RNA near the mitochondrial DNA replication origin, thus it was proposed to generate the primer for mtDNA replication. Recently, it has been shown to have another substrate in the nucleus, such as pre-S.8S ribosomal RNA in nucleolus. The gene for the RNA component of RNase MRP (MRP RNA) was found to be encoded by the nucleus genome, suggesting an interesting intracellular trafficking of MRP RNA to both mitochondria and nucleolus after transcription in nucleus. In this study, genomic DNA encoding MRP RNA was microinjected into the nucleus of Xenopus oocytes, to analyze promoter regions involved in the transcription. It showed that the proximal sequence element and TATA box are important for basal level transcription; octamer motif and Sp1 binding sites are for elevated level transcription. Most of Xenopus MRP RNA was exported out to the cytoplasm following transcription in the nucleus. Utilizing various hybrid constructs, export of MRP RNA was found to be regulated by the promoter and the 5' half of the coding region of the gene. Interestingly, the transcription in nucleus seems to be coupled to the export of MRP RNA to cytoplasm. Intracellular transport of injected MRP RNA can be easily visualized by whole-mount in situ hybridization following microinjection; it also shows possible intra-nuclear sites for transcription and export of MRP RNA.

• BMB Reports
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• v.44 no.3
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• pp.199-204
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• 2011

Ephrin signaling is involved in various morphogenetic events, such as axon guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain biochemical insight into the function of the EphrinB1 ICD. We identified the transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein. Whole-mount in situ hybridization of Xenopus embryos confirmed the co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during various stages of development. The EphrinB1/xTLE4 interaction was confirmed by co-immunoprecipitation experiments. Further characterization of the interaction revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP domain of xTLE4 are required for binding. Additionally, phosphorylation of EphrinB1 by a constitutively activated fibroblast growth factor receptor resulted in loss of the interaction, suggesting that the interaction is modulated by tyrosine phosphorylation of the EphrinB1 ICD.

• Molecules and Cells
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• v.37 no.4
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• pp.330-336
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• 2014

Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelial-mesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount in situ hybridization. Of the thymosin family members only zTB4 was expressed in the developing heart region. Cardiac valve development at 48 h post fertilization was defected in zebrafish TB4 (zTB4) morpholino-injected embryos (morphants). In zTB4 morphants, abnormal linear heart tube development was observed. The expressions of bone morphogenetic protein (BMP) 4, notch1b, and hyaluronic acid synthase (HAS) 2 genes were also markedly reduced in atrio-ventricular canal (AVC). Endocardial cells in the AVC region were stained with anti-Zn5 antibody reactive against Dm-grasp (an EMT marker) to observe EMT in developing cardiac valves in zTB4 morphants. EMT marker expression in valve endothelial cells was confirmed after transfection with TB4 siRNA in the presence of transforming growth factor ${\beta}$ ($TGF{\beta}$) by RT-PCR and immunofluorescent assay. Zn5-positive endocardial AVC cells were not observed in zTB4 morphants, and knockdown of TB4 suppressed TGF-${\beta}$-induced EMT in ovine valve endothelial cells. Taken together, our results demonstrate that TB4 plays a pivotal role in cardiac valve formation by increasing EMT.

• Development and Reproduction
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• v.17 no.4
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• pp.389-397
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• 2013

FGF9/16/20 signaling pathway specify the developmental fates of notochord, mesenchyme, and neural cells in ascidian embryos. Although a conserved Ras/MEK/Erk/Ets pathway is known to be involved in this signaling, the detailed mechanisms of regulation of FGF signaling pathway have remained largely elusive. In this study, we have isolated Hr-Erf, an ascidian orthologue of vertebrate Erf, to elucidate interactions of transcription factors involved in FGF signaling of the ascidian embryo. The Hr-Erf cDNA encompassed 3110 nucleotides including sequence encoded a predicted polypeptide of 760 amino acids. The polypeptide had the Ets DNA-binding domain in its N-terminal region. In adult animals, Hr-Erf mRNA was predominantly detected in muscle, and at lower levels in ganglion, gills, gonad, hepatopancreas, and stomach by quantitative real-time PCR (QPCR) method. During embryogenesis, Hr-Erf mRNA was detected from eggs to early developmental stage embryos, whereas the transcript levels were decreased after neurula stage. Similar to the QPCR results, maternal transcripts of Hr-Erf was detected in the fertilized eggs by whole-mount in situ hybridization. Maternal mRNA of Hr-Erf was gradually lost from the neurula stage. Zygotic expression of Hr-Erf started in most blastomeres at the 8-cell stage. At gastrula stage, Hr-Erf was specifically expressed in the precursor cells of brain and mesenchyme. When MEK inhibitor was treated, embryos resulted in loss of Hr-Erf expression in mesenchyme cells, and in excess of Hr-Erf in a-line neural cells. These results suggest that zygotic Hr-Erf products are involved in specification of mesenchyme and neural cells.

• Molecules and Cells
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• v.40 no.12
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• pp.945-953
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• 2017

We report the biological functions of a zebrafish homologue of RING-finger protein 152 (rnf152) during embryogenesis. rnf152 was initially identified as a brain-enriched E3 ligase involved in early embryogenesis of zebrafish. Expression of rnf152 was ubiquitous in the brain at 24 hpf but restricted to the eyes, midbrain-hindbrain boundary (MHB), and rhombomeres at 48 hpf. Knockdown of rnf152 in zebrafish embryos caused defects in the eyes, MHB, and rhombomeres (r1-7) at 24 hpf. These defects in rnf152-deficient embryos were analyzed by whole-mount in situ hybridization (WISH) using neuroD, deltaD, notch1a, and notch3 probes. NeuroD expression was abolished in the marginal zone, outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the eyes at 27 hpf. Furthermore, deltaD and notch1a expression was remarkably reduced in the ONL, INL, subpallium, tectum, cerebellum, and rhombomeres (r1-7) at 24 hpf, whereas notch3 expression was reduced in the tectum, cerebellum, and rhombomeres at 24 hpf. Finally, we confirmed that expression of Notch target genes, her4 and ascl1a, also decreased significantly in these areas at 24 hpf. Thus, we propose that Rnf152 is essential for development of the eyes, midbrain and hindbrain, and that Delta-Notch signaling is involved.

• Molecules and Cells
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• v.38 no.6
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• pp.580-586
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• 2015

While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide (MO)-based gene knockdown. We find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock-down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers, including cmlc2, vmhc, and amhc. Furthermore, double knock-down by coinjection of smyd3 and setd7 MOs caused the synergistic defects in heart development. As similar to knock-down effect, overexpression of these genes also caused the heart morphogenesis defect in zebrafish. These results indicate that histone modifying enzymes, SMYD3 and SETD7, appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development.

• Molecules and Cells
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• v.39 no.4
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• pp.352-357
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• 2016

Vertebrate neurogenesis requires inhibition of endogenous bone morphogenetic protein (BMP) signals in the ectoderm. Blocking of BMPs in animal cap explants causes the formation of anterior neural tissues as a default fate. To identify genes involved in the anterior neural specification, we analyzed gene expression profiles using a Xenopus Affymetrix Gene Chip after BMP-4 inhibition in animal cap explants. We found that the xCyp26c gene, encoding a retinoic acid (RA) degradation enzyme, was upregulated following inhibition of BMP signaling in early neuroectodermal cells. Whole-mount in situ hybridization analysis showed that xCyp26c expression started in the anterior region during the early neurula stage. Overexpression of xCyp26c weakly induced neural genes in animal cap explants. xCyp26c abolished the expression of all trans-/cis-RA-induced posterior genes, but not basic FGF-induced posterior genes. Depletion of xCyp26c by morpholino-oligonucleotides suppressed the normal formation of the axis and head, indicating that xCyp26c plays a critical role in the specification of anterior neural tissue in whole embryos. In animal cap explants, however, xCyp26c morpholinos did not alter anterior-to-posterior neural tissue formation. Together, these results suggest that xCyp26c plays a specific role in anterior-posterior (A-P) neural patterning of Xenopus embryos.