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.     

A Switching Controller System for a Wheeled Mobile Robot

A wheeled mobile mechanism with a passive and/or active linkage mechanism for rough terrain environment is developed and evaluated. The wheeled mobile mechanism which has high mobility in rough terrain needs sophisticated system to adapt various environments.We focus on the development of a switching controller system for wheeled mobile robots in rough terrain. This system consists of two sub-systems: an environment recognition system using link angles and an adaptive control system. In the environment recognition system, we introduce a Self-Organizing Map (SOM) for clustering link angles. In the adaptive controllers, we introduce neural networks to calculate the inverse model of the wheeled mobile robot.The environment recognition system can recognize the environment in which the robot travels, and the adjustable controllers are tuned by experimental results for each environment. The dual sub-system switching controller system is experimentally evaluated. The system recognizes its environment and adapts by switching the adjustable controllers. This system demonstrates superior performance to a well-tuned single PID controller.     

Crystal structure of chondroitin polymerase from Escherichia coli K4

Elongation of glycosaminoglycan chains, such as heparan and chondroitin, is catalyzed by bi-functional glycosyltransferases, for which both 3-dimensional structures and reaction mechanisms remain unknown. The bacterial chondroitin polymerase K4CP catalyzes elongation of the chondroitin chain by alternatively transferring the GlcUA and GalNAc moiety from UDP-GlcUA and UDP-GalNAc to the non-reducing ends of the chondroitin chain. Here, we have determined the crystal structure of K4CP in the presence of UDP and UDP-GalNAc as well as with UDP and UDP-GlcUA. The structures consisted of two GT-A fold domains in which the two active sites were 60 Å apart. UDP-GalNAc and UDP-GlcUA were found at the active sites of the N-terminal and C-terminal domains, respectively. The present K4CPstructures have provided the structural basis for further investigating the molecular mechanism of biosynthesis of chondroitin chain.     

Critical Role of an Antiviral Stress Granule Containing RIG-I and PKR in Viral Detection and Innate Immunity

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.     

Directionality of stripes formed by anisotropic reaction-diffusion models

Turing mechanism explains the formation of striped patterns in a uniform field in which two substances interact locally and diffuse randomly. In a twin paper, to explain the directionality of stripes on fish skin in closely related species, we studied the effect of anisotropic diffusion of the two substances on the direction of stripes, in the cases in which both substances have high diffusivity in the same direction. In this paper, we study the direction of stripes in more general situations in which the diffusive direction may differ between the two substances. We derive a formula for the direction of stripes, based on a heuristic argument of unstable modes of deviation from the uniform steady state. We confirm the accuracy of the formula by computer simulations. When the diffusive direction is different between two substances, the directions of stripes in the spatial pattern change smoothly with the magnitude of anisotropy of two substances. When the diffusive direction of the two substances is the same, the stripes are formed either parallel or perpendicular to the common diffusive direction, depending on the relative magnitude of the anisotropy. The transition between these two phases occurs sharply.     

Monoterpenoids and their glycosides from the leaf of thyme

From the polar portion of the methanol extract of thyme (leaf of Thymus vulgaris; Labiatae), which has been used as an important stomachic, carminative, a component of prepared cough tea, and a spice, seven monoterpenoid glycosides were isolated together with two known monoterpenoids and three known monoterpenoid glucosides. Structures of the seven monoterpenoid glycosides were determined by spectral analysis.     

Functional dissection of a mercuric ion transporter, MerC, from Acidithiobacillus ferrooxidans

Topological analysis with a phoA gene fusion suggested that Acidithiobacillus ferrooxidans MerC, a mercury transporter, has two periplasmic loops and four transmembrane domains. Cys-23 and Cys-26 of the protein were involved in Hg(2+)-recognition/uptake, but Cys-132 and Cys-137 were not. Escherichia coli cells producing the MerC were hypersensitive to CdCl(2). In this case, mutation of His72 rendered the host cells less CdCl(2) sensitive, whereas none of the Cys residues affected it. E. coli cells expressing the gene encoding a mercuric ion transporter (merC)-deletion mutant, in which the coding-sequence of the carboxy-terminal cytoplasmic region was removed, retained Hg(2+) hypersensitivity and showed about 55% HgCl(2) uptake ability compared to that of the one expressing the intact merC, indicating that the region is not essential for Hg(2+) uptake. Coexpression of A. ferrooxidans the gene encoding mercuric reductase (merA) and the merC deletion mutation conferred HgCl(2) tolerance to E. coli host cells. Under this condition, the merC deletion gene product was exclusively present as a monomer.     

Correlation between Level of Phenylpropanoids in Cotyledons and Flowering in Pharbitis Seedlings under High-Fluence Illumination

Strain Violet of Pharbitis nil flowers under continuous lightwhen exposed to fluence rate greater than 30 W m^–2 (15,000lux) for 12 days or longer, but strain Kidachi does not flower.A high fluence rate promoted the accumulation of chlorogenicacid (CGA), pinoresinol-ß-glucoside (PRG) and p-coumaroylquinicacid (COQ) in strain Violet, while in strain Kidachi the levelof these compounds did not increase even under the highest fluencerate tested. The level of CGA, PRG and COQ in the cotyledonsof strain Violet increased in pararell with induction of floweringunder high-fluence illumination. Aminooxyacetic acid (AOA) inhibitedflowering simulutaneously suppressing the accumulation of CGAand PRG. (Received November 30, 1993; Accepted May 7, 1994)