Distinct pathways for export of silencing RNA in Caenorhabditis elegans systemic RNAi.

Dietary supplied double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) systemically in some animals, including the nematode Caenorhabditis elegans. Although this phenomenon has been utilized as a major tool for gene silencing in C. elegans, how cells spread the silencing RNA throughout the organism is largely unknown. Here, we identify two novel systemic RNAi-related factors, REXD-1 and TBC-3, and show that these two factors together with SID-5 act redundantly to promote systemic spreading of dsRNA. Animals that are defective in all REXD-1, TBC-3, and SID-5 functions show strong deficiency in export of dsRNA from intestinal cells, whereas cellular uptake and processing of dsRNA and general secretion events other than dsRNA secretion are still functional in the triple mutant animals. Our findings reveal pathways that specifically regulate the export of dsRNA in parallel, implying the importance of spreading RNA molecules for intercellular communication in organisms.

AMPK-FOXO-IP3R signaling pathway mediates neurological and developmental defects caused by mitochondrial DNA mutations.

Pathological mutations in human mitochondrial genomes (mtDNA) can cause a series of neurological, behavioral, and developmental defects, but the underlying molecular mechanisms are poorly understood. We show here that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway plays a key role in mediating similar defects caused by different mtDNA mutations in Caenorhabditis elegans, including loss or reduction of osmotic, chemical and olfactory sensing, locomotion, and associative learning and memory, as well as increased embryonic lethality. mtDNA mutations cause reduced ATP (adenosine triphosphate) levels, activation of C. elegans AMPK AAK-2, and nuclear translocation of the FOXO transcription factor DAF-16. Activated DAF-16 up-regulates the expression of inositol triphosphate receptor ITR-1, an endoplasmic reticulum calcium channel, leading to increased basal cytosolic Ca2+ levels, decreased neuronal responsiveness, compromised synapses, and increased embryonic death. Treatment of mtDNA mutants with vitamin MK-4 restores cellular ATP and cytosolic Ca2+ levels, improves synaptic development, and suppresses sensory and behavioral defects and embryonic death. Our study provides crucial mechanistic insights into neuronal and developmental defects caused by mtDNA mutations and will improve understanding and treatment of related mitochondrial diseases.

BCL7B, a SWI/SNF complex subunit, orchestrates cancer immunity and stemness.

Cancer is one of the main causes of human death. Here, we focus on the B-cell lymphoma 7 protein family member B (BCL7B) gene, an accessory subunit of the SWI/SNF chromatin-remodelling complex. To characterize the function of BCL7B, heterozygous BCL7B-deficient stomach cancer cell lines were generated with the CRISPR/Cas9 genome editing system. The comprehensive gene expression patterns were compared between parental cells and each ΔBCL7B cell line by RNA-seq. The results showed marked downregulation of immune-related genes and upregulation of stemness-related genes in the ΔBCL7B cell lines. Moreover, by ChIP-seq analysis with H3K27me3 antibody, the changes of epigenetic modification sequences were compared between parental cells and each ΔBCL7B cell line. After machine learning, we detected the centroid sequence changes, which exerted an impact on antigen presentation. The regulation of BCL7B expression in cancer cells gives rise to cancer stem cell-like characteristics and the acquisition of an immune evasion phenotype.

An endomembrane zinc transporter negatively regulates systemic RNAi in Caenorhabditis elegans.

Double-stranded RNA (dsRNA) regulates gene expression in a sequence-dependent manner. In Caenorhabditis elegans, dsRNA spreads through the body and leads to systemic RNA silencing. Although several genes involved in systemic RNAi have been genetically identified, molecules that mediate systemic RNAi remain largely unknown. Here, we identified ZIPT-9, a C. elegans homolog of ZIP9/SLC39A9, as a broad-spectrum negative regulator of systemic RNAi. We showed that RSD-3, SID-3, and SID-5 genetically act in parallel for efficient RNAi, and that zipt-9 mutants suppress the RNAi defects of all the mutants. Analysis of a complete set of deletion mutants for SLC30 and SLC39 family genes revealed that only zipt-9 mutants showed altered RNAi activity. Based on these results and our analysis using transgenic Zn2+ reporters, we propose that ZIPT-9-dependent Zn2+ homeostasis, rather than overall cytosolic Zn2+, modulates systemic RNAi activity. Our findings reveal a previously unknown function of zinc transporters in negative RNAi regulation.

Efficient collection of a large number of mutations by mutagenesis of DNA damage response defective animals.

With the development of massive parallel sequencing technology, it has become easier to establish new model organisms that are ideally suited to the specific biological phenomena of interest. Considering the history of research using classical model organisms, we believe that the efficient construction and sharing of gene mutation libraries will facilitate the progress of studies using these new model organisms. Using C. elegans, we applied the TMP/UV mutagenesis method to animals lacking function in the DNA damage response genes atm-1 and xpc-1. This method produces genetic mutations three times more efficiently than mutagenesis of wild-type animals. Furthermore, we confirmed that the use of next-generation sequencing and the elimination of false positives through machine learning could automate the process of mutation identification with an accuracy of over 95%. Eventually, we sequenced the whole genomes of 488 strains and isolated 981 novel mutations generated by the present method; these strains have been made available to anyone who wants to use them. Since the targeted DNA damage response genes are well conserved and the mutagens used in this study are also effective in a variety of species, we believe that our method is generally applicable to a wide range of animal species.

OFF-responses of interneurons optimize avoidance behaviors depending on stimulus strength via electrical synapses.

Optimization of the types and timing of avoidance behaviors depending on the intensity of a noxious stimulus is essential for survival; however, processing in the central nervous system and its developmental basis are largely unknown. Here, we report that Caenorhabditis elegans preferentially selects one of three different types of avoidance behaviors depending on the strength of the noxious stimulus. We screened 210 neuronal transcription factors using a combination of optogenetics and RNA interference methods and identified 19 candidates required for avoidance behaviors. One candidate, gene lin-32 (abnormal cell LINeage 32), which encodes an atonal homolog, is required for the neural fate determination of AIB interneurons and functions by regulating the expression of electrical and chemical synapse genes, namely, inx-1 (innexin 1) and AMPA-type ionotropic glutamate receptor glr-1. When examined by Ca imaging, AIB showed an OFF calcium increase to the noxious stimulus. The OFF calcium increase was provoked only by strong stimulation, suggesting a role for optimization of the avoidance behavior. However, lin-32 mutants showed an impaired AIB OFF calcium increase, concomitant with a reduced occurrence of the dynamic avoidance behavior called the "omega turn". The AIB neural responses may be transferred to downstream inter/motor neurons projecting to the neck muscles via electrical synapses comprising inx-1. Finally, we found a correlation between powerful contractions of the neck muscles and omega turns. Thus, the central regulation of the magnitude and timing of activation of the AIB interneurons optimizes the probability of omega turn depending on the stimulus context.

Somatic mutations and increased lymphangiogenesis observed in a rare case of intramucosal gastric carcinoma with lymph node metastasis.

BACKGROUND AND AIM: Intramucosal gastric adenocarcinoma of the well-moderately differentiated type only exhibits lymph node metastasis in extremely rare cases. We encountered such case and investigated both the lymphangiogenic properties and somatic mutations in the cancer to understand the prometastatic features of early-stage gastric cancer. METHODS: We quantitatively measured the density of lymphatic vessels and identified mutations in 412 cancer-associated genes through next-generation target resequencing of DNA extracted from tumor cells in a formalin-fixed and paraffin-embedded tissue. Functional consequence of the identified mutation was examined in vitro by means of gene transfection, immunoblot, and the quantitative real-time polymerase chain reaction assay. RESULTS: The intramucosal carcinoma was accompanied by abundant lymphatic vessels. The metastatic tumor harbored somatic mutations in NBN, p.P6S, and PAX8, p.R49H. The PAX8R49H showed significantly higher transactivation activity toward E2F1 than the wild-type PAX8 (P< 0.001). CONCLUSIONS: Our data suggest that increased lymphangiogenesis and somatic mutations of NBN and/or PAX8 could facilitate lymph node metastasis from an intramucosal gastric carcinoma. These findings may potentially inform evaluations of the risk of developing lymph node metastasis in patients with intramucosal gastric cancer.

An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans.

Balancer chromosomes are critical tools for genetic research. In C. elegans, reciprocal translocations that lead to aneuploidy have been widely used to maintain lethal and sterile mutations in stable stocks. Here, we generated a set of aneuploidy-free and structurally defined crossover suppressors that contain two overlapping inversions using the CRISPR/Cas9 system. The toolkit includes 13 crossover suppressors and covers approximately 63% of all C. elegans coding genes. Together with the classical intrachromosomal crossover suppressors, the system now covers 89% of the coding genes. We also labeled the created balancers with fluorescent and phenotypic markers. We show that the crossover suppressors are better for embryonic analysis compared with translocational balancers. Additionally, we demonstrate an efficient method to generate lethal alleles by targeting essential genes on a chromosome balanced with a crossover suppressor. The toolkit will allow more efficient experiments in which lethal and sterile mutants can be analyzed.

Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9.

Balancer chromosomes are convenient tools used to maintain lethal mutations in heterozygotes. We established a method for engineering new balancers in C. elegans by using the CRISPR/Cas9 system in a non-homologous end-joining mutant. Our studies will make it easier for researchers to maintain lethal mutations and should provide a path for the development of a system that generates rearrangements at specific sites of interest to model and analyse the mechanisms of action of genes.

Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization.

Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

Locus-specific integration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system.

We established a method to generate integration from extrachromosomal arrays with the CRISPR/Cas9 system. Multi-copy transgenes were integrated into the defined loci of chromosomes by this method, while a multi-copy transgene is integrated into random loci by previous methods, such as UV- and gamma-irradiation. The effects of a combination of sgRNAs, which define the cleavage sites in extrachromosomes and chromosomes, and the copy number of potential cleavable sequences were examined. The relative copy number of cleavable sequences in extrachromosomes affects the frequency of fertile F1 transgenic animals. The expression levels of the reporter gene were almost proportional to the copy numbers of the integrated sequences at the same integration site. The technique is applicable to the transgenic strains abundantly stored and shared among the C. elegans community, particularly when researchers use sgRNAs against common plasmid sequences such as ß-lactamase.

A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination.

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.

Genetic control of startle behavior in medaka fish.

Genetic polymorphisms are thought to generate intraspecific behavioral diversities, both within and among populations. The mechanisms underlying genetic control of behavioral properties, however, remain unclear in wild-type vertebrates, including humans. To explore this issue, we used diverse inbred strains of medaka fish (Oryzias latipes) established from the same and different local populations. Medaka exhibit a startle response to a visual stimulus (extinction of illumination) by rapidly bending their bodies (C-start) 20-ms after the stimulus presentation. We measured the rates of the response to repeated stimuli (1-s interval, 40 times) among four inbred strains, HNI-I, HNI-II, HO5, and Hd-rR-II1, and quantified two properties of the startle response: sensitivity (response rate to the first stimulus) and attenuation of the response probability with repeated stimulus presentation. Among the four strains, the greatest differences in these properties were detected between HNI-II and Hd-rR-II1. HNI-II exhibited high sensitivity (approximately 80%) and no attenuation, while Hd-rR-II1 exhibited low sensitivity (approximately 50%) and almost complete attenuation after only five stimulus presentations. Our findings suggested behavioral diversity of the startle response within a local population as well as among different populations. Linkage analysis with F2 progeny between HNI-II and Hd-rR-II1 detected quantitative trait loci (QTL) highly related to attenuation, but not to sensitivity, with a maximum logarithm of odds score of 11.82 on linkage group 16. The three genotypes (homozygous for HNI-II and Hd-rR-II1 alleles, and heterozygous) at the marker nearest the QTL correlated with attenuation. Our findings are the first to suggest that a single genomic region might be sufficient to generate individual differences in startle behavior between wild-type strains. Further identification of genetic polymorphisms that define the behavioral trait will contribute to our understanding of the neural mechanisms underlying behavioral diversity, allowing us to investigate the adaptive significance of intraspecific behavioral polymorphisms of the startle response.

A neural mechanism underlying mating preferences for familiar individuals in medaka fish.

Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

A new data-mining method to search for behavioral properties that induce alignment and their involvement in social learning in medaka fish (Oryzias latipes).

BACKGROUND: Coordinated movement in social animal groups via social learning facilitates foraging activity. Few studies have examined the behavioral cause-and-effect between group members that mediates this social learning. METHODOLOGY/PRINCIPAL FINDINGS: We first established a behavioral paradigm for visual food learning using medaka fish and demonstrated that a single fish can learn to associate a visual cue with a food reward. Grouped medaka fish (6 fish) learn to respond to the visual cue more rapidly than a single fish, indicating that medaka fish undergo social learning. We then established a data-mining method based on Kullback-Leibler divergence (KLD) to search for candidate behaviors that induce alignment and found that high-speed movement of a focal fish tended to induce alignment of the other members locally and transiently under free-swimming conditions without presentation of a visual cue. The high-speed movement of the informed and trained fish during visual cue presentation appeared to facilitate the alignment of naïve fish in response to some visual cues, thereby mediating social learning. Compared with naïve fish, the informed fish had a higher tendency to induce alignment of other naïve fish under free-swimming conditions without visual cue presentation, suggesting the involvement of individual recognition in social learning. CONCLUSIONS/SIGNIFICANCE: Behavioral cause-and-effect studies of the high-speed movement between fish group members will contribute to our understanding of the dynamics of social behaviors. The data-mining method used in the present study is a powerful method to search for candidates factors associated with inter-individual interactions using a dataset for time-series coordinate data of individuals.

Controlled Cre/loxP site-specific recombination in the developing brain in medaka fish, Oryzias latipes.

BACKGROUND: Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain. METHODOLOGY/PRINCIPAL FINDINGS: To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0-1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2-3 dpf embyos compared with 0-1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish. CONCLUSIONS/SIGNIFICANCE: We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.