Biology subjects

Chihara, T.

Publications and source records attributed to Chihara, T..

7 recordsLinked to original sources

bioRxiv2025

Characterization of heat shock protein expression and its application to temporal gene expression in the nematode Pristionchus pacificus

Temporal gene expression systems are widely used to examine gene functions at specific developmental stages. One method applied in temporal gene expression systems in many organisms is the heat-inducible gene expression system, which utilizes a heat shock promoter with evolutionarily conserved heat shock elements. The nematode Pristionchus pacificus is a satellite model system comparable to Caenorhabditis elegans, with unique developmental traits but lacking genetic tools for temporal gene expression. To establish a temporal gene expression system in P. pacificus, we investigated genes that were highly induced by heat shock. RNA-seq analysis revealed thousands of differentially expressed genes after a 2-hour heat shock event. One of the highly induced genes, PPA12242, is an ortholog of C. elegans hsp-16.41, and reporter transgenic animals have indicated that the genomic fragment upstream of this gene can induce gene expression in response to heat shock. PPA12242 can be highly induced in specific larval stages, which seem to be vulnerable to heat stress in some phenotypes. Taken together, we identified a potential heat shock promoter in P. pacificus that is applicable to the temporal gene expression system in this species.

genetics↗

bioRxiv2025

Hippo-activated cells induce non-cell autonomous tumorigenesis in Drosophila

The Hippo pathway is known as a tumor-suppressor pathway, and most related studies have indicated that the inhibition of the Hippo pathway leads to tumorigenesis. However, recent studies have suggested that the activated Hippo pathway can potentially promote tumorigenesis in some contexts. Here, we show that the activated Hippo pathway induces non-cell-autonomous tumorigenesis characterized by tumor markers in the Drosophila wing epithelium. This suggests that the Hippo-activated cells behave similarly to "oncogenic niche cells." We found that the Hippo-activated cells distantly induced tumor-like cells exhibiting aberrant cell proliferation. Moreover, we identified the evolutionarily conserved amino acid transporters, Sat1/2, that redundantly work with the growth factors, Wingless and Spitz, for non-cell autonomous tumorigenesis. Our findings provide novel insights into the Hippo pathway and may be useful in developing cancer treatments targeting the Hippo pathway.

cell biology↗

bioRxiv2024

Glutamate, GABA, and dense-core vesicle secretion regulate predatory feeding in the nematode Pristionchus pacificus

Nematodes are one of the most diverse groups of organisms, found in various environments, and exhibit various feeding behaviours. Pristionchus pacificus displays two types of feeding behaviours: predatory and bacterial feeding. Previously, we showed that the neurotransmitter serotonin plays important roles in regulating predatory feeding in P. pacificus. However, the role of other neurotransmitters in predatory feeding remains unclear. Using the CRISPR/Cas9 system, we generated mutants of the dense-core vesicle (DCV)-based neuropeptide secretion system and genes related to seven neurotransmitters. Predatory assays revealed that the vesicular glutamate transporter Ppa-EAT-4, glutamic acid decarboxylase Ppa-UNC-25 which is involved in GABA synthesis, and the calcium-dependent activator protein for DCV secretion Ppa-UNC-31, play roles in predatory feeding behaviour. We assessed the pharyngeal movement necessary for predation as well as locomotion rate in these mutants. While the Ppa-eat-4 and Ppa-unc-31 mutants decreased predation movement, the Ppa-unc-25 mutant showed a reduction only in bacterial events compared to wild type animals. Additionally, Ppa-unc-25 and Ppa-unc-31 decreased in motor movement, potentially reducing predation efficiency. Together, these results suggest that glutamate, GABA and DCV secretion modulate feeding behaviours in P. pacificus. Our mutant collection of neurotransmitter-related genes will be useful for future analysis of neurobiology and behavioural evolution.

animal behavior and cognition↗

bioRxiv2024

cGMP-dependent pathway and a GPCR kinase are required for photoresponse in the nematode Pristionchus pacificus

Light sensing is a critical function in most organisms and is mediated by photoreceptor proteins and phototransduction. Although most nematodes lack eyes, some species exhibit phototaxis. In the nematode Caenorhabditis elegans, the unique photoreceptor protein Cel-LITE-1, its downstream G proteins, and cyclic GMP (cGMP)-dependent pathways are required for phototransduction. However, the mechanism of light-sensing in other nematodes remains unknown. To address this question, we used the nematode Pristionchus pacificus, which was established as a satellite model organism for comparison with C. elegans. Similar to C. elegans, illumination with short-wavelength light induces avoidance behavior in P. pacificus. Opsin, cryptochrome/photolyase, and lite-1 were not detected in the P. pacificus genome using orthology and domain prediction-based analyses. To identify the genes related to phototransduction in P. pacificus, we conducted forward genetic screening for light-avoidance behavior and isolated four light-unresponsive mutants. Whole-genome sequencing and genetic mapping revealed that the cGMP-dependent pathway and Ppa-grk-2, which encodes a G protein-coupled receptor kinase (GRK) are required for light avoidance. Although the cGMP-dependent pathway is conserved in C. elegans phototransduction, GRK is not necessary for light avoidance in C. elegans. This suggests similarities and differences in light-sensing mechanisms between the two species. Using a reverse genetics approach, we showed that GABA and glutamate were involved in light avoidance. Through reporter analysis and suppression of synapse transmission, we identified candidate photosensory neurons. These findings advance our understanding of the diversity of phototransduction in nematodes even in the absence of eyes. Author summaryNematodes are a highly diverse group of animals found in a wide variety of habitats and sensory systems. In particular, light-induced behavior has been found to differ among species. The photoreceptor and its downstream pathways in Caenorhabditis elegans have been identified, revealing unique and distinct characteristics compared to those in other animals. However, the mechanisms of photoreception in other nematodes remain largely unknown. This study focused on the analysis of the photoreception mechanisms in Pristionchus pacificus, a species for which many genetic and molecular tools are available. Similar to C. elegans, P. pacificus also exhibits light avoidance behavior towards short-wavelength light; however, known animal photoreceptor genes could not be identified in the P. pacificus genome using bioinformatic approaches. Using forward and reverse genetic approaches, we found that certain genes and neurons are required for light avoidance, some of which are conserved in C. elegans photoreception. These results suggest that the light-sensing mechanisms of C. elegans and P. pacificus are similar, yet there are differences between the two species. These findings highlight the various light-sensing mechanisms in nematodes.

genetics↗

bioRxiv2023

Executioner caspase is proximal to Fasciclin 3 which facilitates non-lethal activation in Drosophila olfactory receptor neurons

The nervous system undergoes functional modification independent on cell turn over. Caspase participates in reversible neuronal modulation via non-lethal activation. However, the mechanism that enables non-lethal activation remains unclear. Here, we analyzed proximal proteins of Drosophila executioner caspase in the adult brain using TurboID. We discovered that executioner caspase Drice is, as an inactive proform, proximal to cell membrane proteins, including a specific splicing isoform of cell adhesion molecule Fasciclin 3 (Fas3), Fas3G. To investigate whether sequestration of executioner caspase to plasma membrane of axons is the mechanism for non-lethal activation, we developed a Gal4-Manipulated Area-Specific CaspaseTracker/CasExpress system for sensitive monitoring of caspase activity near plasma membrane. We demonstrated that Fas3G-overexpression promotes caspase activation in olfactory receptor neurons without killing them, by inducing expression of initiator caspase Dronc, which also comes close to Fas3G. Physiologically, Fas3G overexpression-facilitated non-lethal caspase activation suppresses innate olfactory attraction behavior. Our findings suggest that subcellularly-restricted caspase activation, defined by caspase proximal proteins, is the mechanism for non-lethal activation, opening the methodological development of reversible modification of neuronal function via regulating caspase proximal proteins.

molecular biology↗

bioRxiv2022

The Vap33/Eph/Vav/Cdc42 complex confers temporal specification to the outgrowth of primary dendrites in Drosophila neurons

SUMMARYThe formation of primary dendrites (dendritogenesis) significantly affects the overall orientation and coverage of dendritic arborization, limiting the number and types of inputs a neuron can receive. Previously we reported how a Drosophila motoneuron spatially controls the positioning of dendritogenesis through the Dscam1/Dock/Pak1 pathway; however, how the neuron defines the timing of this process remains elusive. Here we show that the Eph receptor tyrosine kinase provides a temporal cue. We find that, at the onset of dendritogenesis, the Eph receptor recruits the Rho Family GEF Vav to the intracellular domain of Eph, which transiently activates the Cdc42 family of small GTPase. We also show that vap33 (vesicle-associated membrane protein-associated protein) mutants exhibit defects in Cdc42 activation and dendritic outgrowth, indicating Vap33 may play an upstream role in Eph signaling. Together, our result and previous studies argue that the formation of primary dendrites requires the proximity of active Cdc42 and membrane-anchored Pak1 driven by collaborative action between two distinct signaling complexes, Vap33/Eph/Vav and Dscam1/Dock. Signal integration from multiple input pathways would represent a general mechanism for the spatiotemporal precision of dendrite branch formation.

neuroscience↗

bioRxiv2018

Nanopore formation in the cuticle of the insect olfactory sensillum

Insects sense odorants through small (50-200-nm diameter) pores in the cuticle covering the olfactory sensilla. These nanopores serve as a filter, preventing the entry of larger airborne particles and limiting water loss. Here we show that the nanopores in Drosophila originate from a curved ultrathin film formed in the outermost layer of the cuticle, secreted from specialized plasma membrane protrusions. The gore-tex/Osiris23 gene, encoding an endosomal protein, is required for nanopore formation and odor receptivity, and is expressed specifically in developing olfactory shaft cells. The 24-member Osiris gene family is expressed in various cuticle-secreting cells, and is found only in insect genomes. Some Osiris mutants show defective cuticle-associated functions, suggesting that Osiris genes may provide a platform for investigating functional surface nano-fabrication in insects.\n\nOne Sentence SummaryAn insect-specific endosomal protein promotes the formation of nano-scale pore structures of the apical extracellular matrix.

developmental biology↗