Biology subjects

Mochii, M.

Publications and source records attributed to Mochii, M..

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↗

bioRxiv2026

Visualization of peripheral nerves in developing and regenerating limbs using a novel peripherin reporter line of Xenopus laevis

Peripherin (PRPH) is a class III intermediate filament protein expressed in peripheral nerves and upregulated during axon outgrowth and regeneration. In this study, we developed a transgenic Xenopus laevis line for long-term in vivo visualization of the peripheral nervous system. Deletion and motif analyses identified cis-regulatory regions within the promoter and intron 1 that are important for neuronal expression of the X. laevis prph gene. Stable lines exhibited robust EGFP reporter activity in developing neural primordia in embryos and in the peripheral nerves of tadpoles. Transgenic tadpoles enabled in vivo imaging of peripheral nerves throughout limb development. During larval limb regeneration, we observed modest early nerve entry into the blastema, recapitulating that seen in early limb development. In contrast, post-metamorphic limb blastemas displayed extensive innervation from the early phase of regeneration. Moreover, increased reporter activity in the nerves of the regenerating adult forelimb suggests regeneration-associated regulation of peripheral innervation and its potential role in blastema formation. This transgenic line will serve as a versatile tool for analyzing such large-scale neural remodeling across development, metamorphosis, and regeneration.

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↗