Effects of the major formaldehyde catalyzer ADH5 on phenotypes of fanconi anemia zebrafish model.

BACKGROUND: Fanconi anemia (FA) is a devastating hereditary disorder for which we desperately need a novel therapeutic strategy. It is caused by mutations in one of at least 22 genes in the FA pathway and is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. The FA pathway is required for the efficient repair of damaged DNA, including interstrand cross-links (ICL). Recent studies indicate formaldehyde as an ultimate endogenous cause of DNA damage in FA pathophysiology. Formaldehyde can form DNA adducts as well as ICLs by inducing covalent linkages between opposite strands of double-stranded DNA. METHODS AND RESULTS: In this study, we generated a disease model of FA in zebrafish by disrupting the ube2t or fancd2 gene, which resulted in a striking phenotype of female-to-male sex reversal. Since formaldehyde is detoxified from the body by alcohol dehydrogenase 5 (ADH5), we generated fancd2-/-/adh5-/- zebrafish. We observed a body size reduction and a lower number of mature spermatozoa than wild-type or single knockout zebrafish. To evaluate if increased activity in ADH5 can affect the FA phenotype, we overexpressed human ADH5 in fancd2-/- zebrafish. The progress of spermatogenesis seemed to be partially recovered due to ADH5 overexpression. CONCLUSIONS: Our results suggest potential utility of an ADH5 enzyme activator as a therapeutic measure for the clearance of formaldehyde and treatment of FA.

Toward more accurate prediction of caspase cleavage sites: a comprehensive review of current methods, tools and features.

As one of the few irreversible protein posttranslational modifications, proteolytic cleavage is involved in nearly all aspects of cellular activities, ranging from gene regulation to cell life-cycle regulation. Among the various protease-specific types of proteolytic cleavage, cleavages by casapses/granzyme B are considered as essential in the initiation and execution of programmed cell death and inflammation processes. Although a number of substrates for both types of proteolytic cleavage have been experimentally identified, the complete repertoire of caspases and granzyme B substrates remains to be fully characterized. To tackle this issue and complement experimental efforts for substrate identification, systematic bioinformatics studies of known cleavage sites provide important insights into caspase/granzyme B substrate specificity, and facilitate the discovery of novel substrates. In this article, we review and benchmark 12 state-of-the-art sequence-based bioinformatics approaches and tools for caspases/granzyme B cleavage prediction. We evaluate and compare these methods in terms of their input/output, algorithms used, prediction performance, validation methods and software availability and utility. In addition, we construct independent data sets consisting of caspases/granzyme B substrates from different species and accordingly assess the predictive power of these different predictors for the identification of cleavage sites. We find that the prediction results are highly variable among different predictors. Furthermore, we experimentally validate the predictions of a case study by performing caspase cleavage assay. We anticipate that this comprehensive review and survey analysis will provide an insightful resource for biologists and bioinformaticians who are interested in using and/or developing tools for caspase/granzyme B cleavage prediction.

Chordin and dickkopf-1b are essential for the formation of head structures through activation of the FGF signaling pathway in zebrafish.

The ability of the Spemann organizer to induce dorsal axis formation is dependent on downstream factors of the maternal Wnt/ß-catenin signaling pathway. The fibroblast growth factor (FGF) signaling pathway has been identified as one of the downstream components of the maternal Wnt/ß-catenin signaling pathway. The ability of the FGF signaling pathway to induce the formation of a dorsal axis with a complete head structure requires chordin (chd) expression; however, the molecular mechanisms involved in this developmental process, due to activation of FGF signaling, remain unclear. In this study, we showed that activation of the FGF signaling pathway induced the formation of complete head structures through the expression of chd and dickkopf-1b (dkk1b). Using the organizer-deficient maternal mutant, ichabod, we identified dkk1b as a novel downstream factor in the FGF signaling pathway. We also demonstrate that dkk1b expression is necessary, after activation of the FGF signaling pathway, to induce neuroectoderm patterning along the anteroposterior (AP) axis and for formation of complete head structures. Co-injection of chd and dkk1b mRNA resulted in the formation of a dorsal axis with a complete head structure in ichabod embryos, confirming the role of these factors in this developmental process. Unexpectedly, we found that chd induced dkk1b expression in ichabod embryos at the shield stage. However, chd failed to maintain dkk1b expression levels in cells of the shield and, subsequently, in the cells of the prechordal plate after mid-gastrula stage. In contrast, activation of the FGF signaling pathway maintained the dkk1b expression from the beginning of gastrulation to early somitogenesis. In conclusion, activation of the FGF signaling pathway induces the formation of a dorsal axis with a complete head structure through the expression of chd and subsequent maintenance of dkk1b expression levels.

An accessibility-incorporated method for accurate prediction of RNA-RNA interactions from sequence data.

MOTIVATION: RNA-RNA interactions via base pairing play a vital role in the post-transcriptional regulation of gene expression. Efficient identification of targets for such regulatory RNAs needs not only discriminative power for positive and negative RNA-RNA interacting sequence data but also accurate prediction of interaction sites from positive data. Recently, a few studies have incorporated interaction site accessibility into their prediction methods, indicating the enhancement of predictive performance on limited positive data. RESULTS: Here we show the efficacy of our accessibility-based prediction model RactIPAce on newly compiled datasets. The first experiment in interaction site prediction shows that RactIPAce achieves the best predictive performance on the newly compiled dataset of experimentally verified interactions in the literature as compared with the state-of-the-art methods. In addition, the second experiment in discrimination between positive and negative interacting pairs reveals that the combination of accessibility-based methods including our approach can be effective to discern real interacting RNAs. Taking these into account, our prediction model can be effective to predict interaction sites after screening for real interacting RNAs, which will boost the functional analysis of regulatory RNAs. AVAILABILITY AND IMPLEMENTATION: The program RactIPAce along with data used in this work is available at https://github.com/satoken/ractip/releases/tag/v1.0.1 CONTACT: : ykato@rna.med.osaka-u.ac.jp or shingo@i.kyoto-u.ac.jpSupplementary information: Supplementary data are available at Bioinformatics online.

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages.

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta-actin promoter. The aNLS-EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time-lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.

Induction and patterning of trunk and tail neural ectoderm by the homeobox gene eve1 in zebrafish embryos.

In vertebrates, Evx homeodomain transcription factor-encoding genes are expressed in the posterior region during embryonic development, and overexpression experiments have revealed roles in tail development in fish and frogs. We analyzed the molecular mechanisms of posterior neural development and axis formation regulated by eve1. We show that eve1 is involved in establishing trunk and tail neural ectoderm by two independent mechanisms: First, eve1 posteriorizes neural ectoderm via induction of aldh1a2, which encodes an enzyme that synthesizes retinoic acid; second, eve1 is involved in neural induction in the posterior ectoderm by attenuating BMP expression. Further, eve1 can restore trunk neural tube formation in the organizer-deficient ichabod(-/-) mutant. We conclude that eve1 is crucial for the organization of the antero-posterior and dorso-ventral axis in the gastrula ectoderm and also has trunk- and tail-promoting activity.

Correct anteroposterior patterning of the zebrafish neurectoderm in the absence of the early dorsal organizer.

BACKGROUND: The embryonic organizer (i.e., Spemann organizer) has a pivotal role in the establishment of the dorsoventral (DV) axis through the coordination of BMP signaling. However, as impaired organizer function also results in anterior and posterior truncations, it is of interest to determine if proper anteroposterior (AP) pattern can be obtained even in the absence of early organizer signaling. RESULTS: Using the ventralized, maternal effect ichabod (ich) mutant, and by inhibiting BMP signaling in ich embryos, we provide conclusive evidence that AP patterning is independent of the organizer in zebrafish, and is governed by TGFß, FGF, and Wnt signals emanating from the germ-ring. The expression patterns of neurectodermal markers in embryos with impaired BMP signaling show that the directionality of such signals is oriented along the animal-vegetal axis, which is essentially concordant with the AP axis. In addition, we find that in embryos inhibited in both Wnt and BMP signaling, the AP pattern of such markers is unchanged from that of the normal untreated embryo. These embryos develop radially organized trunk and head tissues, with an outer neurectodermal layer containing diffusely positioned neuronal precursors. Such organization is reflective of the presumed eumetazoan ancestor and might provide clues for the evolution of centralization in the nervous system. CONCLUSIONS: Using a zebrafish mutant deficient in the induction of the embryonic organizer, we demonstrate that the AP patterning of the neuroectoderm during gastrulation is independent of DV patterning. Our results provide further support for Nieuwkoop's "two step model" of embryonic induction. We also show that the zebrafish embryo can form a radial diffuse neural sheath in the absence of both BMP signaling and the early organizer.

The zebrafish dorsal axis is apparent at the four-cell stage.

A central question in the development of multicellular organisms pertains to the timing and mechanisms of specification of the embryonic axes. In many organisms, specification of the dorsoventral axis requires signalling by proteins of the Transforming growth factor-beta and Wnt families. Here we show that maternal transcripts of the zebrafish Nodal-related morphogen, Squint (Sqt), can localize to two blastomeres at the four-cell stage and predict the dorsal axis. Removal of cells containing sqt transcripts from four-to-eight-cell embryos or injection of antisense morpholino oligonucleotides targeting sqt into oocytes can cause a loss of dorsal structures. Localization of sqt transcripts is independent of maternal Wnt pathway function and requires a highly conserved sequence in the 3' untranslated region. Thus, the dorsoventral axis is apparent by early cleavage stages and may require the maternally encoded morphogen Sqt and its associated factors. Because the 3' untranslated region of the human nodal gene can also localize exogenous sequences to dorsal cells, this mechanism may be evolutionarily conserved.

Single-embryo metabolomics and systematic prediction of developmental stage in zebrafish.

Metabolites, the end products of gene expression in living organisms, are tightly correlated with an organism's development and growth. Thus, metabolic profiling is a potentially important tool for understanding the events that have occurred in cells, tissues, and individual organisms. Here, we present a method for predicting the developmental stage of zebrafish embryos using novel metabolomic non-target fingerprints of "single-embryos". With this method, we observed the rate of development at different temperatures. Our results suggest that this method allows us to analyse the condition, or distinguish the genotype, of single-embryos before expression of their ultimate phenotype.

Different effects of methylphenidate and atomoxetine on the behavior and brain transcriptome of zebrafish.

Attention deficit-hyperactivity disorder (ADHD) is a prevalent neuropsychiatric disorder found in children. It is characterized by inattention, hyperactivity, and impulsivity. Methylphenidate (MPH) and atomoxetine (ATX) are commonly prescribed for the treatment of ADHD. In the present study, we examined the behavioral and brain transcriptome changes in MPH-treated and ATX-treated zebrafish. In behavioral analysis, zebrafish showed opposite response to each treatment. MPH-treated fish showed higher anxiety-like behavior while ATX-treated fish showed lower anxiety-like behavior. Further, we performed RNA sequencing analysis of zebrafish brain to elucidate the underlying biological pathways associated with MPH and ATX treatment. Interestingly, we found that shared differentially expressed genes in MPH-treated and ATX-treated fish were instrumental in cholesterol biosynthesis pathway and were regulated in opposite manner. Our findings highlight the contrast between MTH and ATX, and may suggest the alterations in clinical practice for these medications and drug development for ADHD.

A novel application of metabolomics in vertebrate development.

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.

Wetland environmental bioreactor system contributes to the decomposition of cellulose.

Recently, numerous species of aquatic invertebrates inhabiting wetlands have been shown to possess endogenous cellulase, following the discovery that termites have cellulase genes encoded in their own genome rather than relying on symbiotic bacteria for decomposing cellulose. Wetlands have been empirically shown to play an important role in the decomposition of land-originating hard-to-degrade polysaccharides such as cellulose. However, the mechanism that connects the cellulase producer and the wetlands remains unknown, which makes it very difficult to evaluate the ecological function of wetlands. Here we found that a macrobenthic bivalve, Corbicula japonica, secretes its cellulase to the wetland sediment. Secreted cellulases are immobilized in the components of the sediment. Moreover, adding cellulose or glucose to C. japonica could trigger its cellulase secretion level. These findings suggest a novel wetland cellulose decomposition mechanism. The decomposition ability of wetlands was previously ascribed only to microbes and/or invertebrates that contain cellulases. Our findings suggest that benthic animals supply wetlands with their enzymes as decomposition agents, while wetland sediments serve as immobilization scaffolds for the enzymes. This system, which was named by us an "environmental bioreactor system," could provide a key function in wetlands.

Chordin expression, mediated by Nodal and FGF signaling, is restricted by redundant function of two beta-catenins in the zebrafish embryo.

Using embryos transgenic for the TOP-GFP reporter, we show that the two zebrafish beta-catenins have different roles in the organizer and germ-ring regions of the embryo. beta-Catenin-activated transcription in the prospective organizer region specifically requires beta-catenin-2, whereas the ventrolateral domain of activated transcription is abolished only when both beta-catenins are inhibited. chordin expression during zebrafish gastrulation has been previously shown in both axial and paraxial domains, but is excluded from ventrolateral domains. We show that this gene is expressed in paraxial territories adjacent to the domain of ventrolateral beta-catenin-activated transcription, with only slight overlap, consistent with the now well-known inhibitory effects of Wnt8 on dorsal gene expression. Eliminating both Wnt8/beta-catenin signaling and organizer activity by inhibition of expression of the two beta-catenins results in massive ectopic circumferential expression of chordin and later, by formation of a distinctive embryonic phenotype ('ciuffo') that expresses trunk and anterior neural markers with correct relative anteroposterior patterning. We show that chordin expression is required for this neural gene expression. The Nodal gene squint has been shown to be necessary for optimal expression of chordin and is sufficient in some contexts for its expression. However, chordin is not normally expressed in the ventrolateral germ-ring despite robust expression of squint in this domain. We show the ectopic circumferential expression of chordin and other dorsal genes to be completely dependent on Nodal and FGF signaling, and to be independent of a functional organizer. We propose that whereas the axial domain of chordin expression is formed by cells that are derived from the organizer, the paraxial domain is the result of axial-derived anti-Wnt signals, which relieve the repression that otherwise is set by the Wnt8/beta-catenin/vox,vent pathway on latent germ-ring Nodal/FGF-activated expression.

In vivo targeted single-nucleotide editing in zebrafish.

To date, several genome editing technologies have been developed and are widely utilized in many fields of biology. Most of these technologies, if not all, use nucleases to create DNA double-strand breaks (DSBs), raising the potential risk of cell death and/or oncogenic transformation. The risks hinder their therapeutic applications in humans. Here, we show that in vivo targeted single-nucleotide editing in zebrafish, a vertebrate model organism, can be successfully accomplished with the Target-AID system, which involves deamination of a targeted cytidine to create a nucleotide substitution from cytosine to thymine after replication. Application of the system to two zebrafish genes, chordin (chd) and one-eyed pinhead (oep), successfully introduced premature stop codons (TAG or TAA) in the targeted genomic loci. The modifications were heritable and faithfully produced phenocopies of well-known homozygous mutants of each gene. These results demonstrate for the first time that the Target-AID system can create heritable nucleotide substitutions in vivo in a programmable manner, in vertebrates, namely zebrafish.

Highly sensitive avoidance plays a key role in sensory adaptation to deep-sea hydrothermal vent environments.

The environments around deep-sea hydrothermal vents are very harsh conditions for organisms due to the possibility of exposure to highly toxic compounds and extremely hot venting there. Despite such extreme environments, some indigenous species have thrived there. Alvinellid worms (Annelida) are among the organisms best adapted to high-temperature and oxidatively stressful venting regions. Although intensive studies of the adaptation of these worms to the environments of hydrothermal vents have been made, little is known about the worms' sensory adaptation to the severe chemical conditions there. To examine the sensitivity of the vent-endemic worm Paralvinella hessleri to low pH and oxidative stress, we determined the concentration of acetic acid and hydrogen peroxide that induced avoidance behavior of this worm, and compared these concentrations to those obtained for related species inhabiting intertidal zones, Thelepus sp. The concentrations of the chemicals that induced avoidance behavior of P. hessleri were 10-100 times lower than those for Thelepus sp. To identify the receptors for these chemicals, chemical avoidance tests were performed with the addition of ruthenium red, a blocker of transient receptor potential (TRP) channels. This treatment suppressed the chemical avoidance behavior of P. hessleri, which suggests that TRP channels are involved in the chemical avoidance behavior of this species. Our results revealed for the first time hypersensitive detection systems for acid and for oxidative stress in the vent-endemic worm P. hessleri, possibly mediated by TRP channels, suggesting that such sensory systems may have facilitated the adaptation of this organism to harsh vent environments.

Visualization of primordial germ cells in vivo using GFP-nos1 3'UTR mRNA.

In some teleost fish, primordial germ cells (PGCs) inherit specific maternal cytoplasmic factors such as vasa and nanos 1 (nos1) mRNA. It has been shown that the 3'untranslated regions (UTRs) of vasa and nos1 have critical roles for stabilization of these RNAs in zebrafish PGCs. In this study, to determine whether this role of the nos 1 3'UTR is conserved between teleost species, we injected artificially synthesized mRNA, combining green fluorescent protein (GFP) and the zebrafish nos 1 3'UTR (GFP-nos 1 3'UTR mRNA), into the fertilized eggs of various fish species. The 3'UTR of the Oryzias latipes vasa homologue (olvas ) mRNA was assayed in the same manner. We demonstrate that the PGCs of seven teleost species could be visualized using GFP-nos 1 3'UTR mRNA. GFP-olvas 3'UTR mRNA did not identify PGCs in herring or loach embryos, but did enable visualization of the PGCs in medaka embryos. Our results indicate that the 3'UTR of the zebrafish nos1 mRNA can promote maintenance of RNAs in the PGCs of different fish species. Finally, we describe and compare the migration routes of PGCs in seven teleost species.

Deficiency of Serotonin in Raphe Neurons and Altered Behavioral Responses in Tryptophan Hydroxylase 2-Knockout Medaka (Oryzias latipes).

Serotonin (5-hydroxytryptamine [5-HT]) is a bioactive monoamine that acts as a neurotransmitter in the central and peripheral nervous system of animals. Teleost fish species have serotonergic neurons in the raphe nuclei of the brainstem; however, the role of 5-HT in the raphe neurons in teleost fish remains largely unknown. Here, we established a medaka (Oryzias latipes) strain with targeted disruption of tryptophan hydroxylase 2 (tph2) gene that is involved in the 5-HT synthesis in the raphe nuclei. Immunohistochemistry and mass spectrometry analysis revealed that the homozygous mutants (tph2Δ13/Δ13) lacked the ability to synthesize 5-HT in the raphe neurons. To investigate the effects of 5-HT deficiency in adult behaviors, the mutant fish were subjected to five behavioral paradigms (diving, open-field, light-dark transition, mirror-biting, and two-fish social interaction). The homozygous mutation caused a longer duration of freezing response in all examined paradigms and reduced the number of entries to the top area in the diving test. In addition, the mutants exhibited a decreased number of mirror-biting in the males and an increased contact time in direct social interaction between the females. These results indicate that this tph2-knockout medaka serves as a good model to analyze the effects of 5-HT deficiency in the raphe neurons.

The formation of primordial germ cells from germline cells in spherical embryos derived from the blastodisc of 2-cell embryos in goldfish, Carassius auratus.

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nos1 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.

Chronic fluoxetine treatment induces anxiolytic responses and altered social behaviors in medaka, Oryzias latipes.

Medaka (Oryzias latipes) is a small freshwater teleost that is an emerging model system for neurobehavioral research and toxicological testing. The selective serotonin reuptake inhibitor class of antidepressants such as fluoxetine is one of the widely prescribed drugs, but little is known about the effects of these drugs on medaka behaviors. To assess the behavioral effects of fluoxetine, we chronically administrated fluoxetine to medaka adult fish and analyzed the anxiety-related and social behaviors using five behavioral paradigms (diving, open-field, light-dark transition, mirror-biting, and social interaction) with an automated behavioral testing system. Fish chronically treated with fluoxetine exhibited anxiolytic responses such as an overall increased time spent in the top area in the diving test and an increased time spent in center area in the open-field test. Analysis of socially evoked behavior showed that chronic fluoxetine administration decreased the number of mirror biting times in the mirror-biting test and increased latency to first contact in the social interaction test. Additionally, chronic fluoxetine administration reduced the horizontal locomotor activity in the open-field test but not the vertical activity in the diving test. These investigations are mostly consistent with previous reports in the other teleost species and rodent models. These results indicate that behavioral assessment in medaka adult fish will become useful for screening of effects of pharmaceutical and toxicological compounds in animal behaviors.

Determining Associations between Human Diseases and non-coding RNAs with Critical Roles in Network Control.

Deciphering the association between life molecules and human diseases is currently an important task in systems biology. Research over the past decade has unveiled that the human genome is almost entirely transcribed, producing a vast number of non-protein-coding RNAs (ncRNAs) with potential regulatory functions. More recent findings suggest that many diseases may not be exclusively linked to mutations in protein-coding genes. The combination of these arguments poses the question of whether ncRNAs that play a critical role in network control are also enriched with disease-associated ncRNAs. To address this question, we mapped the available annotated information of more than 350 human disorders to the largest collection of human ncRNA-protein interactions, which define a bipartite network of almost 93,000 interactions. Using a novel algorithmic-based controllability framework applied to the constructed bipartite network, we found that ncRNAs engaged in critical network control are also statistically linked to human disorders (P-value of P = 9.8 × 10(-109)). Taken together, these findings suggest that the addition of those genes that encode optimized subsets of ncRNAs engaged in critical control within the pool of candidate genes could aid disease gene prioritization studies.