Hidenobu Tsujimura

Reduction of Background Mutations by Low-Dose X Irradiation of Drosophila Spermatocytes at a Low Dose Rate

Abstract Koana, T., Okada, M. O., Ogura, K., Tsujimura, H. and Sakai, K. Reduction of Background Mutations by Low-Dose X Irradiation of Drosophila Spermatocytes at a Low Dose Rate. Radiat. Res. 167, 217–221 (2007). A sex-linked recessive lethal mutation assay was performed in Drosophila melanogaster using immature spermatocytes and spermatogonia irradiated with X rays at a high or low dose rate. The mutation frequency in the sperm irradiated with a low dose at a low dose rate was significantly lower than that in the sham-irradiated group, whereas irradiation with a high dose resulted in a significant increase in the mutation frequency. It was obvious that the dose–response relationship was not linear, but rather was U-shaped. When mutant germ cells defective in DNA excision repair were used instead of wild-type cells, low-dose irradiation at a low dose rate did not reduce the mutation frequency. These observations suggest that error-free DNA repair functions were activated by low dose of low-dose-rate radiation and that this repaired spontaneous DNA damage rather than the X-ray-induced damage, thus producing a practical threshold.

Precise control of fasciclin II expression is required for adult mushroom body development in Drosophila.

Fasciclin II (FASII) is a cell adhesion molecule that participates in axonal pathfinding, fasciculation and divergence in the Drosophila nervous system. Here, we examined spatio‐temporal control of fasII expression during the development of adult mushroom body (MB) and found that suppression of fasII in α′/β′ neurons is essential for the formation of adult α′/β′ and α/β lobes. Of γ, α′/β′ and α/β neurons, which are derived sequentially from the same four MB neuroblasts, only γ and α/β neurons expressed fasII. When fasII was misexpressed in developing MB neurons, defects resulted, including loss or misdirection of adult α′/β′ lobes and concurrent misdirection of α/β lobes. Although no gross anatomical defects were apparent in the larval MB lobes, α′/β′ lobes collapsed at the pupal stage when the larval lobe of γ neurons degenerated. In addition, α/β lobes, which developed at this time, were misdirected in close relationship with the collapse of α′/β′ lobes. These defects did not occur when fasII was overexpressed in only γ and α/β neurons, indicating that ectopic expression of fasII in α′/β′ neurons is required for the defects. Our findings also suggest that the α′/β′ lobe play a role in guiding the pathfinding by α/β axons.

Metamorphosis of wing motor system in the silk moth, Bombyx mori L. (Lepidoptera: Bombycidae): anatomy of the sensory and motor neurons that innervate larval mesothoracic dorsal musculature, stretch receptors, and epidermis

Abstract Anatomy of dorsal mesothoracic structures, such as muscles, sensory organs, and innervation, was studied in the silkworm, Bombyx mori L. (Lepidoptera : Bombycidae), and compared with the adult wing motor system. Musculature and nerve innervation were investigated by dissection and electron micrograph; and central projection of sensory fibers and morphology of somata and dendrites of motor neurons by cobalt back-filling, followed by silver intensification. There are 23 muscle bundles (DLM) and 2 stretch receptors (SR). The DLMs, SRs, and epidermis are innervated by a branch of the dorsal nerve trunk emerging from the mesothoracic ganglion (MSG). The branch bifurcates into a dorsal sensory branch of about 300 sensory fibers and a dorsal motor branch of 14 fibers. The sensory fibers project mainly to a longitudinal portion near the mid line in the ventral neuropil of MSG and the metathoracic ganglion. Several fibers extend into the prothoracic ganglion (PG) and a few into the subesophageal and 1st abdominal ganglia. At least 13 (probably 14) motor neurons send axons to DLMs: 9 (probably 10) in PG, and 4 in MSG. Their dendrites are located mostly on the dorsoipsilateral side of the neuropil, but several branches cross the mid line and give rise to many fine branches on the contralateral side. Comparison between the larval (present study) and adult motor system shows a significant similarity in the musculature, peripheral nerve pattern, and motor neurons with some peculiarities.

Spatio-temporal pattern of programmed cell death in the developing Drosophila optic lobe.

A large number of cells die via programmed cell death during the normal development of the Drosophila optic lobe. In this study, we report the precise spatial and temporal pattern of cell death in this organ. Cell death in the developing optic lobe occurs in two distinct phases. The first phase extends from the start of metamorphosis to the mid‐pupal stage. During this phase, a large number of cells die in the optic lobe as a whole, with a peak of cell death at an early pupal stage in the lamina and medulla cortices and the region of the T2/T3/C neurons, and a smaller number of dead cells observed in the lobula plate cortex. The second phase extends from the mid‐pupal stage to eclosion. Throughout this period, a small number of dying cells can be observed, with a small peak at a late pupal stage. Most of the dying cells are neurons. During the first phase, dying cells are distributed in specific patterns in cortices. The lamina cortex contains two distinct clusters of dying cells; the medulla cortex, four clusters; the lobula plate cortex, one cluster; and the region of the T2/T3/C neurons, one cluster. Many of the clusters maintain their distinct positions in the optic lobe but others extend the region they cover during development. The presence of distinct clusters of dying cells at different phases suggests that distinct mechanisms control cell death during different stages of optic lobe development in Drosophila.

Drosophila CTLA-2-like Protein (D/CTLA-2) Inhibits Cysteine Proteinase 1 (CP1), a Cathepsin L-like Enzyme

Abstract In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine pro-teinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.

Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila

The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. Here, we analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. We examined expression patterns of two EcR isoforms, EcR-A and EcR-B1, in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, we genetically analyzed EcR function in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. βFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression.

Reduction of Spontaneous Somatic Mutation Frequency by a Low-Dose X Irradiation of Drosophila Larvae and Possible Involvement of DNA Single-Strand Damage Repair

The third instar larvae of Drosophila were irradiated with X rays, and the somatic mutation frequency in their wings was measured after their eclosion. In the flies with normal DNA repair and apoptosis functions, 0.2 Gy irradiation at 0.05 Gy/min reduced the frequency of the so-called small spot (mutant cell clone with reduced reproductive activity) compared with that in the sham-irradiated flies. When apoptosis was suppressed using the baculovirus p35 gene, the small spot frequency increased four times in the sham-irradiated control group, but the reduction by the 0.2-Gy irradiation was still evident. In a non-homologous end joining-deficient mutant, the small spot frequency was also reduced by 0.2 Gy radiation. In a mutant deficient in single-strand break repair, no reduction in the small spot frequency by 0.2 Gy radiation was observed, and the small spot frequency increased with the radiation dose. Large spot (mutant cell clone with normal reproductive activity) frequency was not affected by suppression of apoptosis and increased monotonically with radiation dose in wild-type larvae and in mutants for single- or double-strand break repair. It is hypothesized that some of the small spots resulted from single-strand damage and, in wild-type larvae, 0.2 Gy radiation activated the normal single-strand break repair gene, which reduced the background somatic mutation frequency.

A U-Shaped Dose–Response Relationship between X Radiation and Sex-Linked Recessive Lethal Mutation in Male Germ Cells of Drosophila

Abstract We reported previously that low-dose X irradiation of DNA repair-proficient immature sperm of wild-type Drosophila melanogaster at a low dose rate (50 mGy/min) resulted in a mutation frequency that was lower than that in the sham-irradiated group. Therefore, a U-shaped dose–response relationship was suggested. Here we show that the dose–response curve is actually U-shaped by carrying out a large-scale sex-linked recessive lethal assay using Drosophila. No reduction of the mutation frequency was observed in a strain mutant for the nucleotide excision repair gene mei-9a (Drosophila homologue of human XPF). Introduction of a chromosome fragment containing mei-9+ into the mei-9a mutant strain restored the reduction of the mutation frequency in the low-dose-irradiated group. These results showed that DNA repair was responsible for the U-shaped dose–response relationship in Drosophila.

Metamorphosis of Wing Motor System in the Silk Moth, Bombyx mori: Origin of Wing Motor Neurons

The origin of the peripheral nerve and motor neurons that innervate the adult mesothoracic dorsal longitudinal muscles (DLMs) was examined in the silk moth, Bombyx mori. The anatomical features of the peripheral nerve and motor neurons were investigated by dissection, electron microscopy, and cobalt back‐fill staining at different pupal stages. These studies showed that the peripheral nerve (IIN1c) that innervates the adult DLMs originates from a branch (db branch) of the larval mesothoracic dorsal nerve that innervates the larval DLMs. During metamorphosis the larval nerve shortens or lengthens locally without change in its basic branching pattern, and the db branch moves towards the mesothoracic ganglion to become the IIN1c. All the adult DLM motor neurons are from larval ones. Nine of the 14 larval DLM motor neurons survive during metamorphosis to become adult DLM motor neurons, and 5 disappear in early pupal stages.

A light-regulated bio-micro-actuator powered by transgenic Drosophila melanogaster muscle tissue

This paper reports the first demonstration of a light-regulated bio-micro-actuator powered by a transgenic Drosophila melanogaster DV tissue (insect heart muscle). We expressed a mutant form of blue light-sensitive cation channel, channelrhodopsin-2, into the cell membrane of the DV tissue. The DV tissue was assembled into a micro-structure made of poly-dimethylsiloxane (PDMS). The light-sensitive DV tissue contracted synchronously with blue light irradiation, and the micro-actuator was driven in this way. The moving frequency of the micro-actuator increased as irradiation intensity and duration increased. These results demonstrate that the light-driven muscle-powered actuator will have various applications.