Kazuhiro E. Fujimori

Infrared laser-mediated gene induction in targeted single cells in vivo.

We developed infrared laser–evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock–mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

Evidence that Sema3A and Sema3F regulate the migration of GABAergic neurons in the developing neocortex

The ganglionic eminence (GE) supplies neurons containing γ‐aminobutyric acid (GABA) to the pallium of the telencephalon. We investigated the molecular guidance mechanisms of GE cell migration in the neocortex and found neuropilin‐1 (Npn‐1) or neuropilin‐2 (Npn‐2) on the GE cells. Ectopic Sema3A or ‐3F expression by COS1 cell clusters placed on embryo neocortical slices reduced the cell migration but did not block it completely. However, the cell migration was almost completely blocked by COS1 cell clusters expressing both Sema3A and ‐3F. The direction of cell migration could be reversed by placing Sema3A‐ and ‐3F‐coexpressing COS1 cell clusters at the distal cut end of the neocortical slices. Further slice experiments revealed that migration of half of the GE cells in the neocortex was regulated by Sema3A and that migration of the other half of the GE cells in the neocortex was regulated by Sema3F. When the cells responding to Sema3A were diverted by ectopic Sema3A expression in vivo, Dlx2‐positive cells were found predominantly in the lower intermediate zone (IZ). When the cells responding to Sema3F were diverted by ectopic Sema3F expression in vivo, Dlx2‐positive cells were found predominantly in the upper IZ. It was speculated that the semaphorin–neuropilin interactions distribute the GABAergic GE cells evenly in the neocortex as well as guide the GE cells from the GE to the neocortex. The Sema3A expression site under the subplate extended dorsally as the embryo developed. The Sema3A expression seemed to block the Npn‐1‐positive GE cells in the neocortex from entering the cortical plate (CP) and guide them to the dorsal cortex and the hippocampus. Sema3F expression in the CP continued through the embryonic stages. The expression seemed to block Npn‐2‐positive GE cells in the neocortex from entering the CP and make them migrate into the lower IZ. Finally, the semaphorin–neuropilin interactions sorted GABAergic inteneurons into the CP and white matter neurons into the IZ. J. Comp. Neurol. 455:238–248, 2003.

Trafficking of a Ligand-Receptor Complex on the Growth Cones as an Essential Step for the Uptake of Nerve Growth Factor at the Distal End of the Axon: A Single-Molecule Analysis

The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nm Cy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied <5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones with a diffusion constant of 0.3 μm2 s-1. The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, ∼4 μm/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

Expression of Hex mRNA in early murine postimplantation embryo development

The onset of Hex expression and its role in early murine development was analyzed using in situ hybridization. Hex mRNA was first detected in the chorion of the ectoplacental cavity and weakly at the visceral endoderm of the future yolk sac at embryonic age (E) 7.5. Expression in embryonic tissues was detected exclusively in the hepatic anlage and thyroid primordium at E 9.5. At E 12.5 and E 15.5, Hex expression persisted in the fetal liver and thyroid, and was also detected in the fetal lung. These results suggest that Hex has its role in differentiation and/or organogenesis of several embryonic tissues.

Neuronal Roles of the Integrin-associated Protein (IAP/CD47) in Developing Cortical Neurons

Little is known about the role of the integrin-associated protein (IAP, or CD47) in neuronal development and its function in the central nervous system. We investigated neuronal responses in IAP-overexpressing cortical neurons using a virus-gene transfer system. We found that dendritic outgrowth was significantly enhanced in IAP (form 4)-transfected neurons. Furthermore, synaptic proteins including synaptotagmin, syntaxin, synapsin I, and SNAP25 (25-kDa synaptosomal associated protein) were up-regulated. In accordance with this finding, the release of the excitatory transmitter glutamate and the frequencies of Ca2+ oscillations (glutamate-mediated synaptic transmission) were increased. Interestingly, the overexpression of IAP activated mitogen-activated protein kinase (MAPK), and this activation was required for the IAP-dependent biological effects. After down-regulation of the endogenous IAP by small interfering RNA, MAPK activity, synaptic protein levels, and glutamate release decreased. These observations suggest that the IAP plays important roles in dendritic outgrowth and synaptic transmission in developing cortical neurons through the activation of MAPK.

Expression of L1 and TAG‐1 in the corticospinal, callosal, and hippocampal commissural neurons in the developing rat telencephalon as revealed by retrograde and in situ hybridization double labeling

In the telencephalon, the corticospinal (CS), callosal, and hippocampal commissural neurons are the major types of neurons that have axons crossing the midline of the brain. To understand the mechanisms involved in crossing the midline structure and to examine whether the expression patterns of L1 and TAG‐1 in the commissural neurons are similar to those in the spinal cord, we investigated L1 and TAG‐1 expression in these neurons in rats by using a double‐labeling technique involving retrograde labeling and in situ hybridization. Expression of L1 messenger RNA was detected in the retrogradely labeled CS projection neurons by 1,1`‐dioctadecyl‐3,3,3`,3`‐tetramethylindocarbocyanine perchlorate (DiI) injection into the pons at embryonic day (E) 19, but expression of TAG‐1 messenger RNA was not detected in these neurons. Also, after their axons crossed the pyramidal decussation, continued expression of L1 but no expression of TAG‐1 in the CS projection neurons was shown by an additional double‐labeling experiment involving DiI injection into the spinal cord at postnatal day (P) 1. An immunohistochemical study showed that L1 was continuously present in each level of the CS tract at E21 and P3, but TAG‐1 immunoreactivity was not found in any level at any stage. Finally, we examined the expression of L1 and TAG‐1 messenger RNAs in the callosal and hippocampal commissure neurons after their axons had crossed the midline by using the double‐labeling technique. In both cases, hybridization signals of the L1 and TAG‐1 messenger RNAs were observed in the retrogradely labeled neurons at P3. These results suggest that the roles of L1 and TAG‐1 in the formation of the commissures in the forebrain are different from their roles in the spinal cord. J. Comp. Neurol. 417:275–288, 2000.

Neurocrescin: a novel neurite-outgrowth factor secreted by muscle after denervation.

REGENERATION of injured axons at neuromuscular junctions has been assumed to be regulated by extracellular factors that promote neurite outgrowth. We report here the cloning of a novel neurite outgrowth factor, designated neurocrescin, from chick denervated skeletal muscle. A recombinant neurocrescin promoted neurite outgrowth from cultured neurons of spinal cord and telencephalon of chick embryo. It was expressed predominantly in neural tissue and muscle, and was secreted extracellularly after intramolecular cleavage. This truncated form was detected in denervated muscle but not in innervated muscle. Thus, neurocrescin appears to be a novel neurite outgrowth factor that is secreted in an activity-dependent fashion. A highly homologous counterpart was also cloned from mouse brain.

Comparative Genome Analysis Between Aspergillus oryzae Strains Reveals Close Relationship Between Sites of Mutation Localization and Regions of Highly Divergent Genes among Aspergillus Species

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.

Identification of a functional medaka heat shock promoter and characterization of its ability to induce exogenous gene expression in medaka in vitro and in vivo

Heat shock protein promoters (hsp promoters) are powerful tools for investigating gene functions, as the expression of targeted genes can be controlled simply by heating. However, there have been no reports of the utilization of an endogeneous medaka (Oryzias latipes) hsp promoter to induce exogenous gene expression in medaka. We identified and cloned a functional medaka hsp promoter (olphsp70.1) and verified its ability to act as an inducible promoter both in vitro and in vivo. The hsp promoter efficiently induced exogenous gene expression in cultured cells, developing embryos, and also in adult fishes. When used to control the expression of Venus, a variant of yellow fluorescent protein, in transgenic medaka, the hsp promoter was functional in all tissues except for the gonads of adults. These results indicate that the medaka hsp promoter can be a powerful tool for inducing exogenous gene expression and investigating gene functions both in vitro and in vivo in medaka.

Pharmacological characterization of isoproterenol-treated medaka fish

The transparent bodies of see-through medaka fish has led to their use as models for in vivo cell and tissue imaging. However, these fish may also prove useful as models for pathological processes. Accumulating reports show that pathology in fish is similar to that in mammals, including humans. In this study, pathological characterization, comparison of data from rodents and in vivo pharmacological analyses of isoproterenol (Iso)-treated fish were performed. Larval fish showed cardiac hypertrophy-like changes after exposure to > 10 microg/ml Iso for more than one day. Hearts of Iso-treated fish had a larger size and a thicker wall, especially in the ventricle, compared with normal hearts. Several messenger RNA levels dose-dependently increased in Iso-treated fish in a manner similar to that in rodents. Symptoms that accompany mammalian cardiac hypertrophy, such as congestion, were also observed. Propranolol (Pro), a non-selective beta-adrenoceptor blocker (AR), significantly protected the increase in heart rate and volume and other simultaneous symptoms induced by Iso exposure. Athletic capability, measured by optomotor response, showed a dose-dependent decrease after Iso treatment, but was protected by Pro. In contrast, metoprolol (Met) and butoxamine (Btx), selective beta1- and beta2-AR blockers, had little effect in this analysis. These results show that Iso-treated fish develop pathologies analogous to those found in mammalian systems, suggesting that medaka fish can be used for investigation of cardiac hypertrophy; however, drug affinity differences should be considered between species.