Biology subjects

Kagawa, N.

Publications and source records attributed to Kagawa, N..

4 recordsLinked to original sources

bioRxiv2026

A Limbless Phenotype Uncovers an Essential Role for Shh in Amphibian Limb Initiation

Sonic Hedgehog (SHH) is a key signaling molecule controlling anterior-posterior patterning in vertebrate paired appendages, but its requirement for initial appendage outgrowth varies across lineages. Whether SHH is genetically required for early limb-bud outgrowth in amphibians has remained unresolved due to embryonic pleiotropy caused by systemic SHH disruption. Here, we exploited the duplicated homeologs (shh.L and shh.S) in the allotetraploid frog Xenopus laevis to bypass systemic pleiotropy and investigate the limb-specific function of shh. We found that the conserved limb enhancer MFCS1/ZRS is retained at the shh.L locus but degraded at shh.S, resulting in limb-bud expression strictly confined to shh.L. Individual CRISPR/Cas9 disruption of shh.L, but not shh.S, led to a complete failure of initial limb-bud outgrowth and the absence of appendicular skeletal elements, without causing severe systemic defects. Our findings provide direct genetic evidence that shh.L is essential for initial limb outgrowth in X. laevis, demonstrating that dependence on SHH for early appendage initiation is strongly conserved in teleosts and amphibians but was reduced during amniote evolution.

developmental biology↗

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↗

bioRxiv2025

Step-by-step protocol for making a knock-in Xenopus laevis to visualize endogenous gene expression

We established a novel knock-in technique, New and Easy Xenopus Targeted integration (NEXTi), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene (egfp) into target 5 untranslated region (UTR) of Xenopus laevis genome. It enables us to track eGFP expression under regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in vector-injected embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L, myod1.S, sox2.L and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFP signals were obtained by outcrossing the matured injected frogs with wild-type animals. Integrations of donor DNAs into target 5 UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection and genotyping of F1 animals for the NEXTi procedure. 1. HighlightO_LIIntended organism: Xenopus laevis C_LIO_LIPurpose of the protocol: Efficient knock-in for visualizing endogenous target gene expression using CRISPR-Cas9 system C_LIO_LIEssential equipment and materials: Microinjector, fluorescence microscopy, Cas9 protein, sgRNA, donor DNA C_LIO_LIFeatures: Expression of reporter genes depends on endogenous enhancer/promoter activities. C_LI

developmental biology↗

bioRxiv2019

Loss of multiple enzyme activities due to the human genetic variation P284T in NADPH cytochrome P450 oxidoreductase

Cytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Here we are reporting the effects of a POR genetic variant P284T which is located in the hinge region of POR that is necessary for the flexibility of domain movements. Human wild-type and P284T mutant of POR, as well as cytochrome P450 proteins, were expressed in bacteria, purified and then reconstituted in liposomes for enzyme kinetic assays. Quality of POR proteins was checked by cytochrome c, ferricyanide and tetrazolium dye reduction assay and measurements flavin content. We found that for the P284T variant of POR the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4 and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation.

pharmacology and toxicology↗