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The two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae), is an agricultural pest distributed almost worldwide. Because of the frequent use of synthetic pesticides for its control, it has developed resistance to almost all types of active ingredients, making its chemical control very difficult. RNA interference (RNAi) is a sequence-specific gene silencing process triggered by double-stranded RNA (dsRNA). Since RNAi is induced in TSSM by orally ingested dsRNA, the use of dsRNA with sequences specific to essential genes in TSSM has potential as an alternative to synthetic pesticides. Here, we evaluated the effects of RNAi targeting the TSSM gene encoding a β-adrenergic-like octopamine receptor (TuOctβ2R), a target of the synthetic pesticide amitraz, by oral administration of dsRNA for the gene (dsTuOctβ2R). Survival and fecundity rates and feeding activity in mites fed on dsTuOctβ2R were significantly lower than those in a control group fed on dsRNA targeting an intergenic region, even in an amitraz-resistant population. Mites fed on dsTuOctβ2R moved significantly faster than the controls, suggesting that downregulation of TuOctβ2R induces hyperactivity while suppressing feeding behavior. These results indicate that the TuOctβ2R gene is a possible candidate for RNAi-based biopesticides to control TSSM.

Self-renewal and differentiation are tightly controlled to maintain haematopoietic stem cell (HSC) homeostasis in the adult bone marrow1,2. During fetal development, expansion of HSCs (self-renewal) and production of differentiated haematopoietic cells (differentiation) are both required to sustain the haematopoietic system for body growth3,4. However, it remains unclear how these two seemingly opposing tasks are accomplished within the short embryonic period. Here we used in vivo genetic tracing in mice to analyse the formation of HSCs and progenitors from intra-arterial haematopoietic clusters, which contain HSC precursors and express the transcription factor hepatic leukaemia factor (HLF). Through kinetic study, we observed the simultaneous formation of HSCs and defined progenitors—previously regarded as descendants of HSCs5—from the HLF+precursor population, followed by prompt formation of the hierarchical haematopoietic population structure in the fetal liver in an HSC-independent manner. The transcription factor EVI1 is heterogeneously expressed within the precursor population, with EVI1hicells being predominantly localized to intra-embryonic arteries and preferentially giving rise to HSCs. By genetically manipulating EVI1 expression, we were able to alter HSC and progenitor output from precursors in vivo. Using fate tracking, we also demonstrated that fetal HSCs are slowly used to produce short-term HSCs at late gestation. These data suggest that fetal HSCs minimally contribute to the generation of progenitors and functional blood cells before birth. Stem cell-independent pathways during development thus offer a rational strategy for the rapid and simultaneous growth of tissues and stem cell pools.

We demonstrated the emission of multicolored light from a powder electroluminescent device using a thermochromic material through temperature control. At 20°C, the emission of a sapphire blue light was observed. Upon increasing the temperature, the color of the thermochromic materials changed, leading to sky‐blue emission. This study reports the preparation of a novel powder electroluminescent device, enabling control of the emission color through a thermal response. At room temperature, a weak sapphire‐blue emission was observed under the application of an alternative current voltage. With an increase in the temperature, the color appearance of the device changed, leading to strong sky‐blue emissions.

Although sequence variants in CD2-associated protein (CD2AP) have been identified in patients with focal segmental glomerulosclerosis (FSGS), definitive proof of causality in human disease is meager. By whole-exome sequencing, we identified a homozygous frame-shift mutation in CD2AP (p.S198fs) in three siblings born of consanguineous parents who developed childhood-onset FSGS and end stage renal disease. When the same frameshift mutation was introduced in mice by gene editing, the mice developed FSGS and kidney failure. These results provide conclusive evidence that homozygous mutation of CD2AP causes FSGS in humans.

•Indirect decompression of the lumbar canal in degenerative lumbar canal stenosis (LCS) with instability shows potential to decrease surgical invasion and eliminate the risk of dural tear and nerve injury.•Severe stenosis is considered a contraindication for indirect decompression because of the risk of insufficient decompression and restenosis in case of cage sinking.•Removing both facet joints and bilateral distraction allow for sufficient indirect decompression by ligamentotaxis; inserting multiple cages from both sides spreads the weight bearing area to prevent restenosis due to cage sinking.•Bilateral transforaminal lumbar interbody fusion (TLIF) with ligamentum flavum preservation allows for successful indirect decompression in cases who have contraindications to indirect decompression through anterior approaches or conventional TLIF.

Retinol-binding protein 4 (RBP4) is a specific carrier for retinol in the blood. In hepatocytes, newly synthesized RBP4 associates with retinol and transthyretin and is secreted into the blood. The ternary transthyretin–RBP4–retinol complex transports retinol in the circulation and delivers it to target tissues. Rbp4-deficient mice in a mixed genetic background (129xC57BL/6J) have decreased sensitivity to light in the b-wave amplitude on electroretinogram. Sensitivity progressively improves and approaches that of wild-type mice at 24 weeks of age. In the present study, we produced Rbp4-deficient mice in the C57BL/6 genetic background. These mice displayed more severe phenotypes. They had decreased a- and b-wave amplitudes on electroretinograms. In accordance with these abnormalities, we found structural changes in these mice, such as loss of the peripheral choroid and photoreceptor layer in the peripheral retinas. In the central retinas, the distance between the inner limiting membrane and the outer plexiform layer was much shorter with fewer ganglion cells and fewer synapses in the inner plexiform layer. Furthermore, ocular developmental defects of retinal depigmentation, optic disc abnormality, and persistent hyaloid artery were also observed. All these abnormalities had not recovered even at 40 weeks of age. Our Rbp4-deficient mice accumulated retinol in the liver but it was undetectable in the serum, indicating an inverse relation between serum and liver retinol levels. Our results suggest that RBP4 is critical for the mobilization of retinol from hepatic storage pools, and that such mobilization is necessary for ocular development and visual function.

The transcription factor c‐Myb was originally identified as a transforming oncoprotein encoded by two avian leukemia viruses. Subsequently, through the generation of mouse models that affect its expression, c‐Myb has been shown to be a key regulator of hematopoiesis, including having critical roles in hematopoietic stem cells (HSCs). The precise function of c‐Myb in HSCs although remains unclear. We have generated a novel c‐myballele in mice that allows direct observation of c‐Myb protein levels in single cells. Using this reporter line we demonstrate that subtypes of HSCs can be isolated based upon their respective c‐Myb protein expression levels. HSCs expressing low levels of c‐Myb protein (c‐MyblowHSC) appear to represent the most immature, dormant HSCs and they are a predominant component of HSCs that retain bromodeoxyuridine labeling. Hematopoietic stress, induced by 5‐fluorouracil ablation, revealed that in this circumstance c‐Myb‐expressing cells become critical for multilineage repopulation. The discrimination of HSC subpopulations based on c‐Myb protein levels is not reflected in the levels of c‐mybmRNA, there being no more than a 1.3‐fold difference comparing c‐Myblowand c‐MybhighHSCs. This illustrates how essential it is to include protein studies when aiming to understand the regulatory networks that control stem cell behavior. StemCells2015;33:479–490

Hepatocyte growth factor activator inhibitor type 1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-bound serine protease inhibitor expressed in epithelial tissues. Mutant mouse models revealed that HAI-1/SPINT1 is essential for placental labyrinth formation and is critically involved in regulating epidermal keratinization through interaction with its cognate cell surface protease, matriptase. HAI-1/SPINT1 is abundantly expressed in both human and mouse intestinal epithelium; therefore, we analyzed its role in intestinal function using mice with intestinal epithelial cell–specific deletion of Spint1generated by interbreeding mice carrying Spint1LoxPhomozygous alleles with transgenic mice carrying the Cre recombinase gene controlled by the intestine-specific Villinpromoter. Although the resulting mice had normal development and appearance, crypts in the proximal aspect of the colon, including the cecum, exhibited histologic abnormalities and increased apoptosis and epithelial cell turnover accompanied by increased intestinal permeability. Distended endoplasmic reticula were observed ultrastructurally in some crypt epithelial cells, indicative of endoplasmic reticular stress. To study the role of HAI-1/SPINT1 in mucosal injury, we induced colitis by adding dextran sodium sulfate to the drinking water. After dextran sodium sulfate treatment, intestine-specific HAI-1/SPINT1–deficient mice had more severe symptoms and a significantly lower survival rate relative to control mice. These results suggest that HAI-1/SPINT1 plays an important role in maintaining colonic epithelium integrity.

A carbon nanotube (CNT)-based force transducer designed to be embedded in the body of a live animal was fabricated and implanted into the stomach of a rat omit to measure contractile movement. The transducer comprised dispersed poly(ethylene glycol)-grafted multiwalled CNTs applied to a comb-like Au-electrode formed on a poly(dimethylsiloxane) sheet. The implanted rat was injected with acetylcholine to induce muscular contractions and changes in the resistance of the transducer were measured. Such changes arise owing to strain in the CNT network upon distortion. The measured resistance change was found to be proportional to the concentration of injected acetylcholine.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1+/+blastocysts with Hai-1/Spint1−/−embryonic stem cells successfully generated high-chimeric Hai-1/Spint1−/−embryos (B6Hai-1−/−High) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1−/−mice was caused by placental dysfunction. However, newborn B6Hai-1−/−Highmice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

A series of Li-doped lanthanoide silicates, LixLn1-x(SiO4)6O3-x (Ln=La, Nd), were prepared by the solid phase reaction and their ionic conductivities were evaluated. The Li-doping gave the apatite crystal structures (hexagonal, P63/m) analogous to Ln10(SiO4)6O3. The Li-doping was limited up to around x=2 for Ln=La and around x=3.5 for Ln=Nd. In the region of low Li-contents, oxide ion conduction characteristic for Ln10(SiO4)6O3 was still dominant. With an increase in the Li-content, the component of Li-ion conduction emerged due to the formation of lithium silicate glassy phase existing at the grain-boundaries. The O2 and CO2 sensor measurements using the sinters well supported the results of the conductivity analyses. Thus it was found that varying the Li-content could regulate an ionic conductivity and carrier. These properties of the Li-doped lanthanoid silicates might give a good compatibility into the two conduction layers junction, oxide ion conductor//alkali ion conductors fundamental to solid electrolyte gas sensors.

Mice heterozygous for the Otx2 mutation display a craniofacial malformation, known as otocephaly or agnathia-holoprosencephaly complex. The severity of the phenotype is dependent on the genetic background of a C57BL/6 (B6) strain; most of the offspring of Otx2 knock-out chimeras, which are equivalent to the F1 of CBA and B6 strains, backcrossed with B6 females display reduction or loss of mandible, whereas those backcrossed with CBA females do not show noticeable phenotype at birth. The availability of phenotypically disparate strains renders identification of Otx2 modifier loci possible. In this study, a backcross of chimera with B6 was generated and genome-wide scans were conducted with polymorphic markers for non-mendelian distribution of alleles in Otx2 heterozygous mutant mice displaying abnormalities in the lower jaw. We identified one significant locus, Otmf18, between D18Mit68 and D18Mit120 on chromosomes 18, linked to the mandibular phenotype (LOD score 3.33). A similar replication experiment using a second backcross (N3) mouse demonstrated the presence of another significant locus, Otmf2 between D2Mit164 and D2Mit282 on chromosome 2, linked to the mandibular phenotype (LOD score 3.93). These two modifiers account for the distribution of the craniofacial malformations by the genetic effect between B6 and CBA strains. Moreover, Otmf2 contain a candidate gene for several diseases in mice and humans. These genetic studies involving an otocephalic mouse model appear to provide new insights into mechanistic pathways of craniofacial development. Furthermore, these experiments offer a powerful approach with respect to identification and characterization of candidate genes that may contribute to human agnathia-holoprosencephaly complex diseases.

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

ABSTRACTA member of the mitogen-activated protein kinase superfamily, MAK, has been proposed to have an important role in spermatogenesis, since Makgene expression is highly restricted to testicular germ cells. To assess the biological function of MAK, we have established MAK-deficient (Mak−/−) mice. Mak−/−mice developed normally, and no gross abnormalities were observed. Spermatogenesis of the Mak−/−mice was also intact, and most of the mice were fertile. However, Mak−/−male-derived litter sizes and their sperm motility in vitro were mildly reduced. These data show that function of MAK is not essential for spermatogenesis and male fertility.

The development of the mammalian antero-posterior (A-P) axis is proposed to be established by distinct anterior and posterior signaling centers, anterior visceral endoderm and primitive streak, respectively. Knock-out studies in mice have shown that Otx2and Criptohave crucial roles in the generation and/or functions of these anterior and posterior centers, respectively. In both Otx2and Criptosingle mutants, the initial formation of the A-P axis takes place in a proximal–distal (P-D) orientation, but subsequent axis rotation fails to occur. To examine the developmental consequences of the lack of these two genes, we have analyzed the Otx2−/−;Cripto−/−double homozygous mutant phenotype. In the double mutants, the expression of the A-P axis markers Cer-l, Lim1, and Wnt3was not induced, while expression of Fgf8and Twas expanded throughout the epiblast, indicating that the double mutants could not form the A-P axis even in its initial P-D orientation. In addition, the double mutants displayed defects in differentiation of the visceral endoderm overlying the epiblast, as well as in the extraembryonic ectoderm. Furthermore, differentiation of neuroectoderm was accelerated as judged by the reduction of Oct4expression and emergence of Sox1and Gbx2expression in the double mutant epiblast. The resulting ectoderm only displayed characteristics of anterior hindbrain, implicating it as a ground state in the mammalian body plan. Our results indicate that complementary functions of Otx2and Criptoare essential for initial patterning of the A-P axis in the mouse embryo.

At around embryonic day 9, the primitive heart of a mouse embryo undergoes spectacular alterations within 24 hours. We created mice harboring an nls-lacZ gene in place of connexin45, which encodes the only known gap junction protein in the primitive heart before embryonic day 9, using the Cre-loxP system. Connexin45-deficient mice died of heart failure at around embryonic day 10. They initiated heart contractions, but conduction block appeared within 24 hours after the first contractions. Their cardiac walls displayed an endocardial cushion defect, while the cardiac jelly was present. These abnormalities were caused by impairment of the epithelial-mesenchymal transformation of the cardiac endothelium. Activation of the cardiac endothelium depended on the presence of the connexin45 gap junctions since signaling through Ca2+/calcineurin and NF-ATc1 (originally named NF-ATc) was disrupted in the mutant hearts. These results indicate a requirement for gap junction channels during early cardiogenesis and hence implicate connexin45 in congenital heart diseases. Movies available on-line: http://www.biologists.com/Development/movies/dev4369.html

Pathologic features of hemodialysisassociated spinal disorders were evaluated using preoperative radiographic images and histologic findings of the spinal lesions resected during surgery.

The associations between vertebral body collapse and the size or location of the metastatic lesions were analyzed statistically to estimate the critical point of collapse.

The polymerization of vinyl monomers initiated by carboxyl groups on the surface of carbon black has been investigated. Channel blacks were found capable of initiating the cationic polymerization of vinyl monomers such as vinyl ethers, indene, and acenaphthylene but unable to initiate that of styrene and vinylpyridines. The rate of polymerization, Rp, of isobutyl vinyl ether (IBVE) initiated by Carbolac 1 in polar solvent (CCl4/C6H5NO2=50 v/v%) is expressed by the following equation: Rp=const [IBVE]2[Carbolac 1]. The features are explained on the basis of a conventional cationic mechanism. The overall activation energy is estimated to be 27 kcal mol-1. This value is larger than that of the polymerization of IBVE in nonpolar solvent (CCl4). In addition, during polymerization, the polymer is grafted onto the surface of carbon black and the carbon black obtained from the reaction gives a stable colloidal dispersion in good solvents for the polymer. The grafting sites of the polymer were found to be carboxyl groups present on the surface.

“Gene trapping” in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses theneor gene which lacks a polyadenylation signal and has a splice donor signal. Expression of theneor gene as fusion transcripts with the 3′ end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3′ rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

In the presence of carbon black, the two-phase dehydrogenation of hydrazobiscyclohexanenitrile and hydrazobisisobutyronitrile in organic solvent by an aqueous solution of potassium ferricyanide gave the corresponding azo-compound in a good yield. It became apparent that the two-phase dehydrogenation of the hydrazines was catalyzed by phenolic hydroxyl groups on the carbon black surface.

The genes Emx1 and Emx2 are mouse cognates of a Drosophila head gap gene, empty spiracles, and their expression patterns have suggested their involvement in regional patterning of the forebrain. To define their functions we introduced mutations into these loci. The newborn Emx2 mutants displayed defects in archipallium structures that are believed to play essential roles in learning, memory and behavior: the dentate gyrus was missing, and the hippocampus and medial limbic cortex were greatly reduced in size. In contrast, defects were subtle in adult Emx1 mutant brain. In the early developing Emx2 mutant forebrain, the evagination of cerebral hemispheres was reduced and the roof between the hemispheres was expanded, suggesting the lateral shift of its boundary. Defects were not apparent, however, in the region where Emx1 expression overlaps that of Emx2, nor was any defect found in the early embryonic forebrain caused by mutation of the Emx1 gene, of which expression principally occurs within the Emx2-positive region. Emx2 most likely delineates the palliochoroidal boundary in the absence of Emx1 expression during early dorsal forebrain patterning. In the more lateral region of telencephalon, Emx2-deficiency may be compensated for by Emx1 and vice versa. Phenotypes of newborn brains also suggest that these genes function in neurogenesis corresponding to their later expressions.

Previous studies suggested that the Otx2 gene plays an essential role in the development of cranial skeletons and nerves of mesencephalic neural crest origin. To clarify this role, we have identified the cis-acting elements in mouse and pufferfish Otx2 genes responsible for the expression in the crest cells using a transgenic approach with the lacZ reporter gene. In mouse, 49 bp sequences in the proximal 5′ region upstream were essential and sufficient to direct the transgene expression in the cephalic mesenchyme. In pufferfish, the 1.1 kb distal region, located far downstream (from +14.4 to +15.5 kb), had almost identical activity. Between them, several DNA sequences were conserved, and mutational analyses indicated that motif A was critical for the transgene expression in the premandibular region while motif B was critical in both premandibular and mandibular regions. Motif B, CTAATTA, contains the core motif for binding of homeodomain proteins while motif A, TAAATCTG, does not match any known consensus binding sequences for transcriptional factors. The cephalic mesenchyme that expressed β-galactosidase under these cis-elements is most likely to correspond to mesencephalic crest cells. Thus the molecular machinery regulating Otx2 expression in these cells appears to be conserved between mouse and fish, implying a crucial role of the Otx2 gene in development of the neural-crest-derived structures of the gnathostome rostral head.

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The kinetochore is a multiprotein complex that connects centromeres to microtubules in meiosis and mitosis. It plays an essential role in chromosome segregation. During meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules from the same spindle pole (mono-orientation) and protect centromeric cohesion mediated by cohesin protein at the onset of anaphase. These 2 kinetochore functions, mono-orientation and centromeric cohesion protection, are widely conserved among eukaryotic organisms. The mechanism for conservation is unknown.

No relevant conflicts of interest to declare.