Biology subjects

Umesono, Y.

Publications and source records attributed to Umesono, Y..

4 recordsLinked to original sources

bioRxiv2026

The CoP Assay: A Robust Method for Quantitative Assessment of Food Intake in the Planarian Dugesia japonica

Planarians exhibit a variety of behaviors in response to external stimuli and have emerged as a promising animal model for studying the neural regulation of behavior. Although their feeding behavior has been well documented, robust experimental methods for quantifying their food intake remain limited. In this study, we developed a novel methodology for the quantitative assessment of feeding termed the "Complete-or- Partial (CoP) assay." This assay evaluates feeding responses as a binary outcome (complete consumption versus partial ingestion) in a volume-dependent manner at the individual level. Using this method, we examined the effects of food quality on feeding responses by comparing two distinct diets: a "basal diet" containing the minimum concentration of liver homogenate necessary to induce ingestion and support growth, and a "high-density diet" with a ten-fold higher concentration. The CoP assay enabled the robust estimation of food intake volume and successfully detected a significant overconsumption of the high-density diet compared to the basal diet, demonstrating that planarians regulate their food intake in a quality-dependent manner. Furthermore, individuals exhibiting partial ingestion did not immediately feed again even when presented with the same diet freshly prepared, indicating that they had reached satiety. Thus, we propose that the CoP assay offers a robust and scalable platform for evaluating both food intake volume and satiety status, providing a powerful tool to facilitate future molecular analyses of feeding regulation in planarians.

animal behavior and cognition↗

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

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↗

bioRxiv2021

Proliferation maintains the undifferentiated status of stem cells: the role of the planarian cell cycle regulator Cdh1

The coincidence of cell cycle arrest and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since it possesses a quite simple cell cycle regulation in which cdh1 is the only factor that arrests the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the ablation of cdh1 caused pronounced propagation of the stem cells, the progenitor and differentiated cells were decreased. Further analysis indicated that the stem cells without cdh1 expression did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle arrest. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation. Summary statementBy using planarians, which have quite simple cell cycle regulation, we revealed that stem cells with cell cycle progression could not undergo differentiation even though an induction signal was activated.

developmental biology↗