Search

Analysis of genome-scale evolution has been difficult in large, endangered animals because opportunities to collect high-quality genetic samples are limited. There is a need for novel field-friendly, cost-effective genetic techniques. This study conducted an exome-wide analysis of a total of 42 chimpanzees (Pan troglodytes) across six African regions, providing insights into population discrimination techniques. Wild chimpanzee DNA was extracted noninvasively from collected fecal samples using the lysis-buffer storage method. To target genome-scale regions of host DNA, exome-capture sequencing was performed using cost-effective baits originally designed for humans (closely related to chimpanzees). Multivariate analysis effectively discriminated differences in local populations, aiding in the identification of samples' geographical origins. Exome-wide heterozygosity was negatively correlated significantly with genome-wide nonsynonymous-synonymous substitution ratios, suggesting that mutation loads exist at the local population level. Exome sequences revealed functional diversity and protein-coding gene divergence. Segregating pseudogenes were comprehensively annotated, with many being population-specific and others shared among populations. Focusing on multicopy chemosensory receptor genes, the segregating pseudogenes OR7D4 (an olfactory receptor) and TAS2R42 (a bitter taste receptor) were shared among western and eastern chimpanzees. Overall, our analytical framework offers ecological insights into chimpanzees and may be applicable to other organisms., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Symbiotic nitrogen fixation is a highly coordinated process involving legume plants and nitrogen-fixing bacteria known as rhizobia. In this study, we investigated a novel Fix - mutant of the model legume Lotus japonicus that develops root nodules with endosymbiotic rhizobia but fails in nitrogen fixation. Map-based cloning identified the causal gene encoding the filamentation temperature-sensitive H (FtsH) protein, designated as LjFtsH4. The LjFtsH4 gene was expressed in all plant organs without increased levels during nodulation. Subcellular localization revealed that LjFtsH4, fused with a fluorescent protein, localized in mitochondria. The Ljftsh4 mutant nodules showed signs of premature senescence, including symbiosome membrane collapse and bacteroid disintegration. Additionally, nodule cells of Ljftsh4 mutant displayed mitochondria with indistinct crista structures. Grafting and complementation tests confirmed that the Fix - phenotype was determined by the root genotype, and that protease activity of LjFtsH4 was essential for nodule nitrogen fixation. Furthermore, the ATP content in Ljftsh4 mutant roots and nodules was lower than in the wild-type, suggesting reduced mitochondrial function. These findings underscore the critical role of LjFtsH4 in effective symbiotic nitrogen fixation in root nodules., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The crustacean Daphnia magna is an emerging model for ecological and toxicological genomics. However, the lack of methods for spatial and temporal control of gene expression has impaired the elucidation of molecular mechanisms underlying responses to environments in vivo. Here we report local activation of the hsp70 promoter-driven gene cassette in D. magna by the infrared laser-evoked gene operator (IR-LEGO), a method for heating the target cells with infrared irradiation. We identified the heat-inducible promoter upstream of the D. magna hsp70-A gene. Using this promoter, we generated a transgenic Daphnia harboring the heat-shock responsive GFP reporter gene and confirmed that the GFP gene responds to heat treatment not only in juveniles and adults but also in embryos. We collected embryos from the reporter line and irradiated four different regions of interest in the embryos: a proximal region of the third thoracic segment, a part of the midline, a second maxilla, and a distal region of the endopodite of the second antenna, all of which increased GFP fluorescence with an infrared laser. Our results suggest that the IR-LEGO method is useful for spatial and temporal control of gene expression and would advance the functional genomics in D. magna., (© 2024. The Author(s).)

Notch signaling is an evolutionarily conserved mechanism required for numerous types of cell fate decisions in metazoans. It mediates short-range communication between cells with receptors and ligands, both of which are expressed on the cell surfaces. In response to the ligand-receptor interaction, the ligand and the extracellular domain of the Notch receptor (NECD) in the complex are internalized into ligand-expressing cells by endocytosis, a prerequisite process for the conformational change of the membrane proximal region of Notch to induce critical proteolytic cleavages for its activation. Here we report that overexpression of transmembrane 2 (TM2) domain containing 3 (TM2D3), a mammalian homologue of Drosophila melanogaster Almondex (Amx), activates Notch1. This activation requires the ligand-binding domain in Notch1 and the C-terminal region containing TM2 domain in TM2D3. TM2D3 physically associates with Notch1 at the region distinct from the ligand-binding domain and enhances expression of Notch1 on the cell surface. Furthermore, cell surface expression of Notch1 and Notch2 is reduced in Tm2d3-deficient cells. Finally, amx-deficient Drosophila early embryos exhibit impaired endocytosis of NECD and Delta ligand, for which surface presentation of Notch is required. These results indicate that TM2D3 is an element involved in Notch signaling through the surface presentation., (© 2023. The Author(s).)

The demand for recombinant proteins is rising dramatically, and effective production systems are currently being developed. The production of recombinant proteins in plants is a promising approach due to its low cost and low risk of contamination of the proteins with endotoxins or infectious agents from the culture serum. Plant seeds primarily accumulate seed storage proteins (SSPs), which are transcribed and translated from a few genes; therefore, the mechanism underlying SSP accumulation has been studied to help devise ways to increase recombinant protein production. We found that the 3'UTR of SSP genes are essential for SSP accumulation and can be used in the production of recombinant proteins in Arabidopsis. Fusion of the 3'UTR of SSP genes to the 3' ends of DNA sequences encoding recombinant proteins enables massive accumulation of recombinant proteins with enzymatic activity in Arabidopsis seeds. This method is also applicable to the production of human Interferon Lambda-3 (IFN-lambda 3), a candidate biopharmaceutical compound against hepatitis C infection. Considering the low cost and ease of protein production in Arabidopsis, as well as the rapid growth of this plant, our method is useful for large-scale preparation of recombinant proteins for both academic research and biopharmaceutical production., (© 2023. The Author(s).)

Gravitropism is the plant organ bending in response to gravity, while a straightening mechanism prevents bending beyond the gravitropic set-point angle. The promotion and prevention of bending occur simultaneously around the inflorescence stem tip. How these two opposing forces work together and what part of the stem they affect are unknown. To understand the mechanical forces involved, we rotated wild type and organ-straightening-deficient mutant (myosin xif xik) Arabidopsis plants to a horizontal position to initiate bending. The mutant stems started to bend before the wild-type stems, which led us to hypothesize that the force preventing bending was weaker in mutant. We modeled the wild-type and mutant stems as elastic rods, and evaluated two parameters: an organ-angle-dependent gravitropic-responsive parameter (β) and an organ-curvature-dependent proprioceptive-responsive parameter (γ). Our model showed that these two parameters were lower in mutant than in wild type, implying that, unexpectedly, both promotion and prevention of bending are weak in mutant. Subsequently, finite element method simulations revealed that the compressive stress in the middle of the stem was significantly lower in wild type than in mutant. The results of this study show that myosin-XIk-and-XIf-dependent organ straightening adjusts the stress distribution to achieve a mechanically favorable shape., (© 2023. The Author(s).)

The extracellular signal-regulated kinase (ERK) is a serine/threonine kinase that is known to regulate cellular events such as cell proliferation and differentiation. The ERK signaling pathway is activated by fibroblast growth factors, and is considered to be indispensable for the differentiation of primitive endoderm cells, not only in mouse preimplantation embryos, but also in embryonic stem cell (ESC) culture. To monitor ERK activity in living undifferentiated and differentiating ESCs, we established EKAREV-NLS-EB5 ESC lines that stably express EKAREV-NLS, a biosensor based on the principle of fluorescence resonance energy transfer. Using EKAREV-NLS-EB5, we found that ERK activity exhibited pulsatile dynamics. ESCs were classified into two groups: active cells showing high-frequency ERK pulses, and inactive cells demonstrating no detectable ERK pulses during live imaging. Pharmacological inhibition of major components in the ERK signaling pathway revealed that Raf plays an important role in determining the pattern of ERK pulses., (© 2023. The Author(s).)

The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism., (© 2023. The Author(s).)

The snow crab, Chionoecetes opilio, is a giant deep-sea brachyuran. While several decapod crustaceans generally continue to molt and grow throughout their lifetime, the snow crab has a fixed number of molts. Adolescent males continue to molt proportionately to their previous size until the terminal molt at which time an allometric increase in chela size occurs and an alteration of behavioral activities occurs, ensuring breeding success. In this study, we investigated the circulating concentrations of methyl farnesoate (an innate juvenile hormone in decapods) (MF) before or after the terminal molt in males. We then conducted eyestalk RNAseq to obtain molecular insight into the regulation of physiological changes after the terminal molt. Our analyses revealed an increase in MF titers after the terminal molt. This MF surge may be caused by suppression of the genes that encode MF-degrading enzymes and mandibular organ-inhibiting hormone that negatively regulates MF biosynthesis. Moreover, our data suggests that behavioral changes after the terminal molt may be driven by the activation of biogenic amine-related pathways. These results are important not only for elucidating the physiological functions of MFs in decapod crustaceans, which are still largely unknown, but also for understanding the reproductive biology of the snow crab., (© 2023. The Author(s).)

Termite castes express specialized phenotypes for their own tasks and are a good example of insect polyphenism. To understand the comprehensive gene expression profiles during caste differentiation, RNA-seq analysis based on the genome data was performed during the worker, presoldier, and nymphoid molts in Reticulitermes speratus. In this species, artificial induction methods for each molt have already been established, and the time scale has been clarified. Three different periods (before the gut purge (GP), during the GP, and after the molt) were discriminated in each molt, and two body parts (head and other body regions) were separately sampled. The results revealed that many differentially expressed genes (head: 2884, body: 2579) were identified in each molt. Based on the independent real-time quantitative PCR analysis, we confirmed the different expression patterns of seven out of eight genes in the presoldier molt. Based on the GO and KEGG enrichment analyses, the expressions of genes related to juvenile hormone titer changes (e.g., JH acid methyltransferase), nutrition status (e.g., Acyl-CoA Delta desaturase), and cell proliferation (e.g., insulin receptor), were shown to specifically fluctuate in each molt. These differences may have a crucial impact on caste differentiation. These data are important resources for future termite sociogenomics., (© 2022. The Author(s).)

The kidney plays a central role in body fluid homeostasis. Cells in the glomeruli and juxtaglomerular apparatus sense mechanical forces and modulate glomerular filtration and renin release. However, details of mechanosensory systems in these cells are unclear. Piezo2 is a recently identified mechanically activated ion channel found in various tissues, especially sensory neurons. Herein, we examined Piezo2 expression and regulation in mouse kidneys. RNAscope in situ hybridization revealed that Piezo2 expression was highly localized in mesangial cells and juxtaglomerular renin-producing cells. Immunofluorescence assays detected GFP signals in mesangial cells and juxtaglomerular renin-producing cells of Piezo2 GFP reporter mice. Piezo2 transcripts were observed in the Foxd1-positive stromal progenitor cells of the metanephric mesenchyme in the developing mouse kidney, which are precursors of mesangial cells and renin-producing cells. In a mouse model of dehydration, Piezo2 expression was downregulated in mesangial cells and upregulated in juxtaglomerular renin-producing cells, along with the overproduction of renin and enlargement of the area of renin-producing cells. Furthermore, the expression of the renin coding gene Ren1 was reduced by Piezo2 knockdown in cultured juxtaglomerular As4.1 cells under static and stretched conditions. These data suggest pivotal roles for Piezo2 in the regulation of glomerular filtration and body fluid balance., (© 2022. The Author(s).)

Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application., (© 2022. The Author(s).)

The sex determination gene doublesex (dsx) encodes a transcription factor with two domains, oligomerization domain 1 (OD1) and OD2, and is present throughout insects. Sex-specific Dsx splicing isoforms regulate the transcription of target genes and trigger sex differentiation in all Holometabola examined to date. However, in some hemimetabolous insects, dsx is not spliced sexually and its sequence is less conserved. Here, to elucidate evolutionary changes in dsx in domain organisation and regulation in termites, we searched genome and/or transcriptome databases for the dsx OD1 and OD2 in seven termite species and their sister group (Cryptocercus woodroaches). Molecular phylogenetic and synteny analyses identified OD1 sequences of termites and C. punctulatus that clustered with dsx of Holometabola and regarded them as dsx orthologues. The Cryptocercus dsx orthologue containing OD2 was spliced sexually, as previously shown in other insects. However, OD2 was not found in all termite dsx orthologues. These orthologues were encoded by a single exon in three termites for which genome information is available; they were not alternatively spliced but transcribed in a male-specific manner in two examined species. Evolution of dsx regulation from sex-specific splicing to male-specific transcription may have occurred at an early stage of social evolution in termites., (© 2021. The Author(s).)

Vertebrates usually exhibit gonochorism, whereby their sex is fixed throughout their lifetime. However, approximately 500 species (~ 2%) of extant teleost fishes change sex during their lifetime. Although phylogenetic and evolutionary ecological studies have recently revealed that the extant sequential hermaphroditism in teleost fish is derived from gonochorism, the evolution of this transsexual ability remains unclear. We revealed in a previous study that the tunica of the ovaries of several protogynous hermaphrodite groupers contain functional androgen-producing cells, which were previously unknown structures in the ovaries of gonochoristic fishes. Additionally, we demonstrated that these androgen-producing cells play critical roles in initiating female-to-male sex change in several grouper species. In the present study, we widened the investigation to include 7 genera and 18 species of groupers and revealed that representatives from most major clades of extant groupers commonly contain these androgen-producing cells, termed testicular-inducing steroidogenic (TIS) cells. Our findings suggest that groupers acquired TIS cells in the tunica of the gonads for successful sex change during their evolution. Thus, TIS cells trigger the evolution of sex change in groupers.

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Most male lepidopterans produce fertile eupyrene sperm and non-fertile apyrene sperm, both of which are transferred to the female in a spermatophore during mating. Apyrene sperm outnumbers eupyrene sperm and both sperm types migrate from the bursa copulatrix to the spermatheca after mating. While eupyrene sperm are maintained in the spermatheca until oviposition, the number of apyrene sperm decreases with time. It is unclear whether apyrene sperm disappear from all sperm storage organs in females because both sperm types are often observed in the spermathecal gland. To investigate this, the numbers of both sperm types were estimated in the spermatheca and spermathecal gland of female Byasa alcinous (a monandrous butterfly) 6, 12, 48, 96, and 192 h after mating terminated. Apyrene sperm arrived in the spermatheca earlier than eupyrene sperm; however, some eupyrene and apyrene sperm migrated to the spermathecal gland from the spermatheca at almost the same time. The number of apyrene sperm reached a peak 12 h after the termination of mating and then decreased with time in both the spermatheca and spermathecal gland. Our results suggest that the role of apyrene sperm might be completed early after arriving in the spermatheca of B. alcinous.

The head horn of the Asian rhinoceros beetle develops as an extensively folded primordium before unfurling into its final 3D shape at the pupal molt. The information of the final 3D structure of the beetle horn is prefigured in the folding pattern of the developing primordium. However, the developmental mechanism underlying epithelial folding of the primordium is unknown. In this study, we addressed this gap in our understanding of the developmental patterning of the 3D horn shape of beetles by focusing on the formation of furrows at the surface of the primordium that become the bifurcated 3D shape of the horn. By gene knockdown analysis via RNAi, we found that knockdown of the gene Notch disturbed overall horn primordial furrow depth without affecting the 2D furrow pattern. In contrast, knockdown of CyclinE altered 2D horn primordial furrow pattern without affecting furrow depth. Our results show how the depth and 2D pattern of primordial surface furrows are regulated at least partially independently during beetle horn development, and how both can alter the final 3D shape of the horn.

Behavioral responses to environmental factors at the planktonic larval stage can have a crucial influence on habitat selection and therefore adult distributions in many benthic organisms. Reef-building corals show strong patterns of zonation across depth or underwater topography, with different suites of species aggregating in different light environments. One potential mechanism driving this pattern is the response of free-swimming larvae to light. However, there is little experimental support for this hypothesis; in particular, there are few direct and quantitative observations of larval behavior in response to light. Here, we analyzed the swimming behavior of larvae of the common reef coral Acropora tenuis under various light conditions. Larvae exhibited a step-down photophobic response, i.e. a marked decrease in swimming speed, in response to a rapid attenuation (step-down) of light intensity. Observations of larvae under different wavelengths indicated that only the loss of blue light (wavelengths between 400 and 500 nm) produced a significant response. Mathematical simulations of this step-down photophobic response indicate that larvae will aggregate in the lighter areas of two-dimensional large rectangular fields. These results suggest that the step-down photophobic response of coral larvae may play an important role in determining where larval settle on the reef.

The rhizome is a plant organ that develops from a shoot apical meristem but penetrates into belowground environments. To characterize the gene expression profile of rhizomes, we compared the rhizome transcriptome with those of the leaves, shoots and roots of a rhizomatous Brassicaceae plant, Cardamine leucantha. Overall, rhizome transcriptomes were characterized by the absence of genes that show rhizome-specific expression and expression profiles intermediate between those of shoots and roots. Our results suggest that both endogenous developmental factors and external environmental factors are important for controlling the rhizome transcriptome. Genes that showed relatively high expression in the rhizome compared to shoots and roots included those related to belowground defense, control of reactive oxygen species and cell elongation under dark conditions. A comparison of transcriptomes further allowed us to identify the presence of an ER body, a defense-related belowground organelle, in epidermal cells of the C. leucantha rhizome, which is the first report of ER bodies in rhizome tissue.

Seeds of soybean (Glycine max L.) are a major source of plant-derived oils. In the past, improvements have been made in the quantity and quality of seed oil. Triacylglycerols (TAGs) are the principal components of soybean seed oil, and understanding the metabolic regulation of TAGs in soybean seeds is essential. Here, we identified four soybean genes encoding TAG lipases, designated as SUGAR DEPENDENT1-1 (GmSDP1-1), GmSDP1-2, GmSDP1-3 and GmSDP1-4; these are homologous to Arabidopsis thaliana SDP1 (AtSDP1). To characterize the function of these genes during grain filling, transgenic lines of soybean were generated via RNA interference to knockdown the expression of all four GmSDP1 genes. The seed oil content of the transgenic soybean lines was significantly increased compared with the wild type (WT). Additionally, fatty acid profiles of the WT and transgenic soybean lines were altered; the content of linoleic acid, a major fatty acid in soybean seeds, was significantly reduced, whereas that of oleic acid was increased in transgenic soybean seeds compared with the WT. Substrate specificity experiments showed that TAG lipase preferentially cleaved oleic acid than linoleic acid in the oil body membrane in WT soybean. This study demonstrates that the GmSDP1 proteins regulate both the TAG content and fatty acid composition of soybean seeds during grain filling. These results provide a novel strategy for improving both the quantity and quality of soybean seed oil.

The heat shock response is important for the viability of all living organisms. It involves the induction of heat shock proteins whose expression is mainly regulated by heat shock factor 1 (HSF1). Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. High water temperature (HT) inhibits the female-type proliferation of germ cells and induces the masculinisation of XX medaka in some cases during gonadal sex differentiation. Here, we investigated the roles of HSF1 on the proliferation of germ cells using HSF1 knockout medaka. Loss of HSF1 function under HT completely inhibited the female-type proliferation of germ cells, induced the expression of the anti-Mullerian hormone receptor type 2 (amhr2) and apoptosis-related genes, and suppressed that of the dead end (dnd) and heat shock protein-related genes. Moreover, the loss of HSF1 and AMHR2 function under HT recovered female-type proliferation in germ cells, while loss of HSF1 function under HT induced gonadal somatic cell apoptosis during early sex differentiation. These results strongly suggest that HSF1 under the HT protects the female-type proliferation of germ cells by inhibiting amhr2 expression in gonadal somatic cells. These findings provide new insights into the molecular mechanisms underlying environmental sex determination.

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.

The L strain of Japanese quail exhibits a plumage phenotype that is light yellowish in colour. In this study, we identified a nonsense mutation in the premelanosome protein (PMEL) gene showing complete concordance with the yellowish plumage within a pedigree as well as across strains by genetic linkage analysis of an F 2 intercross population using approximately 2,000 single nucleotide polymorphisms (SNPs) that were detected by double digest restriction site-associated DNA sequencing (ddRAD-seq). The yellowish plumage was inherited in an autosomal recessive manner, and the causative mutation was located within an 810-kb genomic region of the LGE22C19W28_E50C23 linkage group (LGE22). This region contained the PMEL gene that is required for the normal melanosome morphogenesis and eumelanin deposition. A nonsense mutation that leads to a marked truncation of the deduced protein was found in PMEL of the mutant. The gene expression level of PMEL decreased substantially in the mutant. Genotypes at the site of the nonsense mutation were fully concordant with plumage colour phenotypes in 196 F 2 offspring. The nonsense mutation was not found in several quail strains with non-yellowish plumage. Thus, the yellowish plumage may be caused by the reduced eumelanin content in feathers because of the loss of PMEL function.

Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H + -PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1 pro :IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H + -PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.

Crawling migration plays an essential role in a variety of biological phenomena, including development, wound healing, and immune system function. Keratocytes are wound-healing cells in fish skin. Expansion of the leading edge of keratocytes and retraction of the rear are respectively induced by actin polymerization and contraction of stress fibers in the same way as for other cell types. Interestingly, stress fibers in keratocytes align almost perpendicular to the migration-direction. It seems that in order to efficiently retract the rear, it is better that the stress fibers align parallel to it. From the unique alignment of stress fibers in keratocytes, we speculated that the stress fibers may play a role for migration other than the retraction. Here, we reveal that the stress fibers are stereoscopically arranged so as to surround the cytoplasm in the cell body; we directly show, in sequential three-dimensional recordings, their rolling motion during migration. Removal of the stress fibers decreased migration velocity and induced the collapse of the left-right balance of crawling migration. The rotation of these stress fibers plays the role of a "wheel" in crawling migration of keratocytes.

Coral reef ecosystems rely on stable symbiotic relationship between the dinoflagellate Symbiodinium spp. and host cnidarian animals. The collapse of such symbiosis could cause coral 'bleaching' and subsequent host death. Despite huge interest on Symbiodinium, lack of mutant strains and readily available genetic tools have hampered molecular research. A major issue was the tolerance to marker antibiotics. Here, we isolated Symbiodinium mutants requiring uracil for growth, and hence, useful in transformation screening. We cultured Symbiodinium spp. cells in the presence of 5-fluoroorotic acid (5FOA), which inhibits the growth of cells expressing URA3 encoding orotidine-5'-monophosphate decarboxylase, and isolated cells that require uracil for growth. Sequence analyses and genetic complementation tests using yeast demonstrated that one of the mutant cell lines had a point mutation in URA3, resulting in a splicing error at an unusual exon-intron junction, and consequently, loss of enzyme activity. This mutant could maintain a symbiotic relationship with the model sea anemone Exaiptasia pallida only in sea water containing uracil. Results show that the URA3 mutant will be a useful tool for screening Symbiodinium transformants, both ex and in hospite, as survival in the absence of uracil is possible only upon successful introduction of URA3.

The external organs of holometabolous insects are generated through two consecutive processes: the development of imaginal primordia and their subsequent transformation into the adult structures. During the latter process, many different phenomena at the cellular level (e.g. cell shape changes, cell migration, folding and unfolding of epithelial sheets) contribute to the drastic changes observed in size and shape. Because of this complexity, the logic behind the formation of the 3D structure of adult external organs remains largely unknown. In this report, we investigated the metamorphosis of the horn in the Japanese rhinoceros beetle Trypoxylus dichotomus. The horn primordia is essentially a 2D epithelial cell sheet with dense furrows. We experimentally unfolded these furrows using three different methods and found that the furrow pattern solely determines the 3D horn structure, indicating that horn formation in beetles occurs by two distinct processes: formation of the furrows and subsequently unfolding them. We postulate that this developmental simplicity offers an inherent advantage to understanding the principles that guide 3D morphogenesis in insects.

Leptin signaling in the hypothalamus plays a crucial role in the regulation of body weight. Leptin resistance, in which leptin signaling is disrupted, is a major obstacle to the improvement of obesity. We herein demonstrated that protein tyrosine phosphatase receptor type J (Ptprj) is expressed in hypothalamic neurons together with leptin receptors, and that PTPRJ negatively regulates leptin signaling by inhibiting the activation of JAK2, the primary tyrosine kinase in leptin signaling, through the dephosphorylation of Y813 and Y868 in JAK2 autophosphorylation sites. Leptin signaling is enhanced in Ptprj-deficient mice, and they exhibit lower weight gain than wild-type mice because of a reduced food intake. Diet-induced obesity and the leptin treatment up-regulated PTPRJ expression in the hypothalamus, while the overexpression of PTPRJ induced leptin resistance. Thus, the induction of PTPRJ is a factor contributing to the development of leptin resistance, and the inhibition of PTPRJ may be a potential strategy for improving obesity.

Most terrestrial carnivorous plants are specialized on insect prey digestion to obtain additional nutrients. Few species of the genus Nepenthes developed mutualistic relationships with mammals for nitrogen supplementation. Whether dietary changes require certain enzymatic composition to utilize new sources of nutrients has rarely been tested. Here, we investigated the role of urease for Nepenthes hemsleyana that gains nitrogen from the bat Kerivoula hardwickii while it roosts inside the pitchers. We hypothesized that N. hemsleyana is able to use urea from the bats' excrements. In fact, we demonstrate that 15 N-enriched urea provided to Nepenthes pitchers is metabolized and its nitrogen is distributed within the plant. As ureases are necessary to degrade urea, these hydrolytic enzymes should be involved. We proved the presence and enzymatic activity of a urease for Nepenthes plant tissues. The corresponding urease cDNA from N. hemsleyana was isolated and functionally expressed. A comprehensive phylogenetic analysis for eukaryotic ureases, including Nepenthes and five other carnivorous plants' taxa, identified them as canonical ureases and reflects the plant phylogeny. Hence, this study reveals ureases as an emblematic example for an efficient, low-cost but high adaptive plasticity in plants while developing a further specialized lifestyle from carnivory to coprophagy.

Females of social Hymenoptera only mate at the beginning of their adult lives and produce offspring until their death. In most ant species, queens live for over a decade, indicating that ant queens can store large numbers of spermatozoa throughout their long lives. To reveal the prolonged sperm storage mechanisms, we identified enriched genes in the sperm-storage organ (spermatheca) relative to those in body samples in Crematogaster osakensis queens using the RNA-sequencing method. The genes encoding antioxidant enzymes, proteases, and extracellular matrix-related genes, and novel genes that have no similar sequences in the public databases were identified. We also performed differential expression analyses between the virgin and mated spermathecae or between the spermathecae at 1-week and 1-year after mating, to identify genes altered by the mating status or by the sperm storage period, respectively. Gene Ontology enrichment analyses suggested that antioxidant function is enhanced in the spermatheca at 1-week after mating compared with the virgin spermatheca and the spermatheca at 1-year after mating. In situ hybridization analyses of 128 selected contigs revealed that 12 contigs were particular to the spermatheca. These genes have never been reported in the reproductive organs of insect females, suggesting specialized roles in ant spermatheca.

The unicellular alga Chlamydomonas reinhardtii contains many types of small RNAs (sRNAs) but the biological role(s) of bona fide microRNAs (miRNAs) remains unclear. To address their possible function(s) in responses to nutrient availability, we examined miRNA expression in cells cultured under different trophic conditions (mixotrophic in the presence of acetate or photoautotrophic in the presence or absence of nitrogen). We also reanalyzed miRNA expression data in Chlamydomonas subject to sulfur or phosphate deprivation. Several miRNAs were differentially expressed under the various trophic conditions. However, in transcriptome analyses, the majority of their predicted targets did not show expected changes in transcript abundance, suggesting that they are not subject to miRNA-mediated RNA degradation. Mutant strains, defective in sRNAs or in ARGONAUTE3 (a key component of sRNA-mediated gene silencing), did not display major phenotypic defects when grown under multiple nutritional regimes. Additionally, Chlamydomonas miRNAs were not conserved, even in algae of the closely related Volvocaceae family, and many showed features resembling those of recently evolved, species-specific miRNAs in the genus Arabidopsis. Our results suggest that, in C. reinhardtii, miRNAs might be subject to relatively fast evolution and have only a minor, largely modulatory role in gene regulation under diverse trophic states.

Under certain circumstances differentiated cells can be reprogrammed to form stem cells in land plants, but only a portion of the cells reprograms successfully. A long-distance inhibitory signal from reprogrammed cells to surrounding cells has been reported in some ferns. Here we show the existence of anisotropic inhibitory signal to regulate stem cell formation in the moss Physcomitrella patens. When single cells were isolated from a gametophore leaf, over 90% of them were reprogrammed to stem cells with characteristic nuclear expansion. By contrast, when two adjacent cells were isolated, the nuclei of both cells expanded, but successful reprogramming of both cells occurred only in approximately one fifth of the pairs. When three aligned cells were isolated, the reprogramming rate of both edge cells decreased with a living middle cell but did not with a dead middle cell. Furthermore, unequal conversion into stem cells was more prominent in cell pairs aligned parallel to the proximal-distal leaf axis than in those perpendicular to the axis. This study gives an insight into the role of the inhibitory signal in development and evolution as well as the efficient stem cell induction from differentiated cells.

Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used "dynamic" metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e.g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-to-lipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis-related genes (e.g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e.g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel.

The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.

RNG105 (also known as Caprin1) is a major RNA-binding protein in neuronal RNA granules, and is responsible for mRNA transport to dendrites and neuronal network formation. A recent study reported that a heterozygous mutation in the Rng105 gene was found in an autism spectrum disorder (ASD) patient, but it remains unclear whether there is a causal relation between RNG105 deficiency and ASD. Here, we subjected Rng105(+/-) mice to a comprehensive behavioral test battery, and revealed the influence of RNG105 deficiency on mouse behavior. Rng105(+/-) mice exhibited a reduced sociality in a home cage and a weak preference for social novelty. Consistently, the Rng105(+/-) mice also showed a weak preference for novel objects and novel place patterns. Furthermore, although the Rng105(+/-) mice exhibited normal memory acquisition, they tended to have relative difficulty in reversal learning in the spatial reference tasks. These findings suggest that the RNG105 heterozygous knockout leads to a reduction in sociality, response to novelty and flexibility in learning, which are implicated in ASD-like behavior.

Sex is pivotal for reproduction, healthcare and evolution. In the fish medaka, the Y-chromosomal dmy (also dmrt1bY) serves the sex determiner, which activates dmrt1 for male sex maintenance. However, how dmy makes the male decision via initiating testicular differentiation has remained unknown. Here we report that autosomal gsdf serves a male sex initiator. Gene addition and deletion revealed that gsdf was necessary and sufficient for maleness via initiating testicular differentiation. We show that gsdf transcription is activated directly by dmy. These results establish the autosomal gsdf as the first male sex initiator. We propose that dmy determines maleness through activating gsdf and dmrt1 without its own participation in developmental processes of sex initiation and maintenance. gsdf may easily become a sex determiner or other autosomal genes can be recruited as new sex determiners to initiate gsdf expression. Our findings offer new insights into molecular mechanisms underlying sex development and evolution of sex-controlling genes in vertebrates.

Despite identification of several sex-determining genes in non-mammalian vertebrates, their detailed molecular cascades of sex determination/differentiation are not known. Here, we used a novel RNAi to characterise the molecular mechanism of Dmy (the sex-determining gene of medaka)-mediated masculinity in XY fish. Dmy knockdown (Dmy-KD) suppressed male pathway (Gsdf, Sox9a2, etc.) and favoured female cascade (Rspo1, etc.) in embryonic XY gonads, resulting in a fertile male-to-female sex-reversal. Gsdf, Sox9a2, and Rspo1 directly interacted with Dmy, and co-injection of Gsdf and Sox9a2 re-established masculinity in XY-Dmy-KD transgenics, insinuating that Dmy initiates masculinity by stimulating and suppressing Gsdf/Sox9a2 and Rspo1 expression, respectively. Gonadal expression of Wt1a starts prior to Dmy and didn't change upon Dmy-KD. Furthermore, Wt1a stimulated the promoter activity of Dmy, suggesting Wt1a as a regulator of Dmy. These findings provide new insights into the role of vertebrate sex-determining genes associated with the molecular interplay between the male and female pathways.

Platelet derived growth factor (PDGF) plays a pivotal role in the remodeling of connective tissues. Emerging data indicate the distinctive role of PDGF receptor-α (PDGFRα) in this process. In the present study, the Pdgfra gene was systemically inactivated in adult mouse (α-KO mouse), and the role of PDGFRα was examined in the subcutaneously implanted sponge matrices. PDGFRα expressed in the fibroblasts of Pdgfra-preserving control mice (Flox mice), was significantly reduced in the sponges in α-KO mice. Neovascularized areas were largely suppressed in the α-KO mice than in the Flox mice, whereas the other parameters related to the blood vessels and endothelial cells were similar. The deposition of collagen and fibronectin and the expression of collagen 1a1 and 3a1 genes were significantly reduced in α-KO mice. There was a significantly decrease in the number and dividing fibroblasts in the α-KO mice, and those of macrophages were similar between the two genotypes. Hepatocyte growth factor (Hgf) gene expression was suppressed in Pdgfra-inactivated fibroblasts and connective tissue. The findings implicate the role of PDGFRα-dependent ECM and HGF production in fibroblasts that promotes the remodeling of connective tissue and suggest that PDGFRα may be a relevant target to regulate connective tissue remodeling.

A non-autonomous DNA transposon in rice, nDart1, is actively transposed in the presence of an autonomous element, aDart1, under natural conditions. The nDart1-promoted gene tagging line was developed using the endogenous nDart1/aDart1 system to generate various rice mutants effectively. While the dominant mutants were occasionally isolated from the tagging line, it was unclear what causes dominant mutations. A semidominant mutant, Bushy dwarf tiller1 (Bdt1), which has the valuable agronomic traits of multiple tillering and dwarfism, was obtained from the tagging line. Bdt1 mutant carried a newly inserted nDart1 at 38-bp upstream of transcription initiation site of a non-protein-coding gene, miR156d. This insertion caused an upstream shift of the miR156d transcription initiation site and, consequently, increased the functional transcripts producing mature microRNAs. These results indicate that the total amount of miR156d is controlled not only by transcript quantity but also by transcript quality. Furthermore, transgenic lines introduced an miR156d fragment that flanked the nDart1 sequence at the 5' region, suggesting that insertion of nDart1 in the gene promoter region enhances gene expression as a cis-element. This study demonstrates the ability of nDart1 to produce gain-of-function mutants as well as further insights into the function of transposable elements in genome evolution.

The "cock-a-doodle-doo" crowing of roosters, which symbolizes the break of dawn in many cultures, is controlled by the circadian clock. When one rooster announces the break of dawn, others in the vicinity immediately follow. Chickens are highly social animals, and they develop a linear and fixed hierarchy in small groups. We found that when chickens were housed in small groups, the top-ranking rooster determined the timing of predawn crowing. Specifically, the top-ranking rooster always started to crow first, followed by its subordinates, in descending order of social rank. When the top-ranking rooster was physically removed from a group, the second-ranking rooster initiated crowing. The presence of a dominant rooster significantly reduced the number of predawn crows in subordinates. However, the number of crows induced by external stimuli was independent of social rank, confirming that subordinates have the ability to crow. Although the timing of subordinates' predawn crowing was strongly dependent on that of the top-ranking rooster, free-running periods of body temperature rhythms differed among individuals, and crowing rhythm did not entrain to a crowing sound stimulus. These results indicate that in a group situation, the top-ranking rooster has priority to announce the break of dawn, and that subordinate roosters are patient enough to wait for the top-ranking rooster's first crow every morning and thus compromise their circadian clock for social reasons.

The sexual plasticity of the gonads is not retained after the completion of sex differentiation in vertebrates, except in some hermaphroditic species. Here, we report that the depletion of estradiol-17β (E2) by aromatase inhibitors (AI) for up to six months resulted in a functional female-to-male sex reversal in sexually-mature adults of two gonochoristic fish species, Nile tilapia and medaka. The sex-reversed fish showed a typical male pattern of E2 and androgen levels, secondary sexual characteristics, and male-like sex behavior, producing fertile sperm. Conversely, co-treatment of E2 inhibited AI-induced sex reversal. In situ hybridization of medaka gonads during AI-induced sex reversal indicated that cysts on the dorsal side of the adult ovaries are the origin of germ cells and Sertoli cells in the newly formed testicular tissue. Gonochoristic fish maintain their sexual plasticity until adulthood and E2 plays a critical role in maintaining the female phenotype.