Biology subjects

Chihara, A.

Publications and source records attributed to Chihara, A..

3 recordsLinked to original sources

bioRxiv2025

A safer fluorescent in situ hybridization protocol for cryosections

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows for multiplexed, simultaneous detection of distinct gene expression patterns spatiotemporally, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of various model organisms, including mouse (Mus musculus), amphibians (Xenopus laevis and Pleurodeles waltl), and medaka (Oryzias latipes). Our results demonstrate successful multiplexed detection of morphogenetic and cell-type marker genes in these model animals using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment, thus preserving tissue integrity. Furthermore, we show that this protocol is fully compatible with EGFP immunostaining, allowing for the simultaneous detection of mRNAs and reporter proteins in transgenic animals. This protocol retains the benefits of highly sensitive, multiplexed, and multimodal detection afforded by integrating in situ HCR and SABER-FISH with immunohistochemistry, while providing a safer option for researchers, thereby offering a valuable tool for basic biology.

molecular biology↗

bioRxiv2023

Fgf10 mutant newts can regenerate normal limbs despite severe developmental hindlimb defects

In the amniote limb, FGF10 is essential for limb bud initiation and outgrowth. However, whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established an Fgf10 null mutant line in the newt Pleurodeles waltl which have amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs exhibit severe digit reduction, fail to ossify the zeugopod, and downregulate FGF target genes like Sall1, Runx1 and Hoxa11/d11. Despite these developmental defects, Fgf10 mutants were able to regenerate near-normal hindlimbs. Together, our results suggest an important role for Fgf10 in hindlimb digit formation and zeugopod ossification during development, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

developmental biology↗

bioRxiv2021

A novel capsid protein network allows the characteristic inner membrane structure of Marseilleviridae giant viruses

Marseilleviridae is a family of the new order of giant viruses, which exhibit a characteristic inner membrane. Here, we investigated the entire structure of tokyovirus, a species of Marseillevirus at 7.7 [A] resolution using 1 MV high-voltage cryo-EM and single particle analysis. The minor capsid lattice formed by five proteins, shows a novel structure compared to other icosahedral giant viruses. Under the minor capsid proteins, scaffold proteins connect two five-fold vertices and interact with the inner membrane. Previously reported giant viruses utilise "tape measure" proteins, proposed to control its capsid size, which could not be identified in tokyovirus, but scaffold proteins appear to perform a similar role. A density on top of the major capsid protein was identified, which suggested to be a 14kDa glycoprotein. Our observations suggest that the icosahedral particle of Marseilleviridae is constructed with a novel capsid protein network, which allows the characteristic inner membrane structure.

microbiology↗