Changes in sperm motility can serve as an early indicator of reproductive effects caused by environmental chemicals or genetic perturbations. However, sperm motility is highly sensitive to external factors such as osmolarity, ionic composition, and the timing of measurement after activation, making it challenging to obtain consistent and reproducible measurements. Here, we present a standardized protocol for assessing sperm motility in Japanese medaka (Oryzias latipes) using a sperm motility analysis system (SMAS), an application for computer-aided sperm motility analysis (CASA). This protocol details the procedures for sperm collection, activation, and quantitative motility assessment, with particular focus on changes in the percentage of motile sperm post activation and the effects of sperm cryopreservation. We demonstrate time-dependent declines in sperm motility and velocity, and highlight the importance of early post-activation measurements to accurately capture peak motility. Notably, cryopreservation significantly accelerated the decline in sperm motility rate without affecting the initial proportion of motile sperm. To enable reliable comparisons among experimental groups, we recommend standardizing the initiation time after sperm activation by using CASA, and show that measurements should be initiated within 1 min after activation to obtain consistent and reliable data. This standardized SMAS-based protocol provides a robust and reproducible framework for sperm motility analysis in medaka and will be valuable not only for studies in reproductive biology, toxicology, and environmental risk assessment but also for applied research, such as breeding of aquacultural fishes.

In rodents, estrogens aromatized from androgens in the brain are essential for the development of male-typical behaviors. In many other vertebrates, including humans and teleost fish, however, androgens facilitate these behaviors directly via the androgen receptor without aromatization into estrogens. Here, we report that mutagenesis-derived male medaka fish lacking Cyp19a1b (a subtype of aromatase predominantly expressed in the brain) exhibit severely impaired male-typical mating and aggression, despite elevated brain androgen levels. These phenotypes can be rescued by estrogen administration, indicating that brain-derived estrogens are pivotal for male-typical behaviors even in teleosts. Our results further suggest that these estrogens facilitate male-typical behaviors by potentiating androgen action in the brain via the direct stimulation of androgen receptor transcription. Taken together, these findings reveal a previously unappreciated mode of action of brain-derived estrogens in facilitating male-typical behaviors.

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses liver conditions not caused by alcohol, including metabolic dysfunction-associated steatohepatitis (MASH), which involves inflammation and fibrosis, with rising global prevalence. While research to explore MASLD/MASH prevention or cure has largely relied on rodent models, ethical concerns regarding animal welfare have prompted the exploration of alternatives. This study examines the application of a recently developed medaka fish MASH/MASLD model to assess the impact of food components on liver pathology. Medaka were divided into groups and fed for 12 weeks with either a normal diet (ND), a high-fat diet (HFD), or HFD supplemented with γ-oryzanol (Ory). Liver samples were analyzed morphologically, histologically, and biochemically using GC/MS and real-time PCR, with gut microbiota composition also assessed. We show that γ-oryzanol supplementation resulted in smaller, fewer lipid droplets in the liver compared with the HFD group, while GC/MS analysis showed a decreasing trend in total and individual fatty acid content. Additionally, gut microbiota diversity improved in the Ory group. These findings align with previous rodent studies, suggesting that γ-oryzanol may suppress hepatic fat accumulation and inflammation. The study demonstrates the potential of γ-oryzanol as a functional food ingredient for preventing MASLD. Furthermore, this study supports the use of medaka as a cost-effective and ethical alternative to rodent models in food science research.

Cell migration frequently requires cells to traverse tissue environments with distinct physical and biochemical properties. How migrating cells preserve a common migratory program while adapting to these heterogeneous environments remains poorly understood. Here, we show that chick primordial germ cells (PGCs) preserve a common bleb-based migratory mode throughout embryogenesis despite migrating through mechanically distinct tissues. PGCs formed membrane blebs during both intravascular crawling and migration through the dorsal mesentery. However, nuclear envelope (NE) unfolding and activation of the NE-cPLA2 pathway occurred specifically during migration through the mechanically confined dorsal mesentery, where this pathway was required for bleb formation and efficient migration. In contrast, bleb formation during vascular crawling occurred independently of the NE-cPLA2 pathway, demonstrating that distinct molecular mechanisms can generate the same migratory behavior in different tissue environments. Together, these findings suggest that diverse environmental cues converge on a conserved bleb-forming machinery. We propose a hierarchical model in which migrating cells preserve a common migratory program by flexibly switching the upstream molecular mechanisms that initiate bleb formation according to the tissue environment.

Manipulating gene expression in a tissue-specific and temporally controlled manner is essential for understanding the function of the focal genes. Still, in many cases, the limited availability of specific promoters to drive ectopic manipulation remains a restricting factor in developing organs, even in Drosophila. Developing external genitalia is one such organ with a complex anatomical structure shaped by a joint regulatory network of many transcription factors. To overcome the restriction, we employed the infrared laser-evoked gene operator system (IR-LEGO), in which infrared laser (1,480 nm) irradiation induces gene expression under the control of a heat shock promoter. Pupal genital structures were irradiated at approximately 24 or 48 h after puparium formation. We tested a range of laser power and depth to the target structure by a reporter assay using green fluorescent protein, which was induced under the control of the heat shock protein 70 promoter (hs-GAL4). In previous studies, the IR-LEGO has been used as a tool to induce ectopic transgene expression. In this study, we attempted to knock down genes such as yellow (y) and odd-paired (opa) ectopically by RNAi using the GAL4/UAS system. The results demonstrated that this technique has a high potential in manipulating transcript abundance levels in small groups of cells in specific genital structures to unravel novel functions of genes involved in the morphogenesis of species-specific and rapidly evolving anatomical structures.

BACKGROUND:Mesothelioma is an aggressive malignancy with limited therapeutic options. Genetic alterations involving the Hippo pathway are commonly observed. O-GlcNAcylation is frequently elevated in cancer and drives tumour progression. However, its relationship with Hippo pathway dysfunction in mesothelioma remains unclear. METHODS:O-GlcNAcylation levels were examined in mesothelioma samples and cell lines, and O-GlcNAcylated proteins were detected by mass spectrometry. The functional impact of O-GlcNAcylation was determined by quantifying nuclear transport dynamics using light-induced live-cell imaging. Genetic and pharmacological inhibition of O-GlcNAcylation was evaluated in vitro. Treatment with the nuclear export inhibitor KPT-330 (Selinexor) was assessed in vitro and in a mouse xenograft model. RESULTS:O-GlcNAcylation was markedly increased in mesothelioma cells with Hippo pathway inactivation. This modification primarily targeted nuclear pore complex proteins, including NUP214 and NUP62, and significantly accelerated nuclear export rates. Suppression of O-GlcNAcylation diminished nuclear export and inhibited cell proliferation. Importantly, pharmacological blockade of nuclear export using KPT-330 suppressed cell growth in vitro and produced significant antitumour effects in vivo. CONCLUSIONS:These findings demonstrate O-GlcNAcylation-driven enhancement of nuclear export as a therapeutically actionable vulnerability in mesothelioma with inactivation of the Hippo pathway.

Amphibians exhibit two remarkable biological phenomena: regeneration and metamorphosis. The ability to regenerate damaged body parts, such as the limbs, and to remodel organs-such as tail resorption during metamorphosis-is both fascinating and enigmatic. However, until recently, it has been difficult to manipulate gene expression in amphibians after embryogenesis, hindering molecular studies of these processes. Over the past two decades, the development of a simple and reproducible gene expression method-the heat-shock-inducible system-has helped overcome this limitation. This system involves generating transgenic animals carrying gene(s) of interest under the control of a heat shock promoter, typically the hsp70 promoter, followed by heat shock treatment to induce expression. Recent advancements have enabled not only the application of heat shock to the whole body but also spatially restricted gene induction in specific cell populations. In particular, laser irradiation allows for highly precise gene activation and lineage tracing, even at the single-cell level. One current limitation of this system is unintended "leaky" gene expression in the absence of heat shock. The recent availability of an alternative inducible system, Tet-on, in amphibians holds promise for overcoming this drawback and achieving tighter control of gene expression. In this review, we discuss potential refinements to the heat-shock-inducible system-including improvements in laser irradiation techniques and optimization of heat shock promoters (e.g., hsp70 promoter)-to address current limitations, and explore how this system may become an even more powerful tool for studying regeneration and metamorphosis in amphibians.

Latitudinal variation plays an important role in driving local adaptation and intraspecific polymorphism across a wide range of organisms. Among teleost fishes, populations from higher latitude tend to have more vertebrae than those from lower latitude, which is known as “Jordan’s rule”. Vertebral number is determined during embryogenesis by the number of somites formed, but the genetic mechanisms underlying this latitudinal variation remain largely unclear. The medaka (Oryzias latipes) species complex, including O. latipes, O. sakaizumii, O. sinensis and an undescribed species from East Korea, is a freshwater teleost species group distributed across a latitudinal range from 25° to 40°N. This wide geographical distribution makes medaka an ideal model system for studying latitudinal variation in morphological trait. In this study, we found a positive correlation between abdominal vertebral number and the latitude of original sampling sites of 90 wild-derived stocks of the O. latipes species complex. To further examine the environmental and genetic contributions to this trait, we reared 10 selected stocks originating from different latitudes at four incubation temperatures (22, 24, 26, 28 °C) until somite stage. Across all temperatures within each phylogenetic subgroup, stocks from higher latitudes consistently developed a greater number of abdominal vertebrae. Finally, we performed a genome-wide association study (GWAS) to identify genomic loci associated with vertebral number variation in these wild-derived stocks. This analysis revealed a single nucleotide polymorphism (SNP) on chromosome 10 that was significantly associated with total vertebral count. Taken together, our findings demonstrate that wild-derived stocks of the O. latipes species complex retain latitudinal variation in vertebral number. This system provides a powerful framework for investigating the evolutionary and genetic bases of geographic trait variation in teleost fishes.

Wnt chaperon Wntless (Wls) mediates the intracellular transport of Wnts and plays important roles in early vertebrate brain development. Spatially restricted induction of Wls denotes the earliest differentiation of non-telencephalic cells in human brain organoids. In zebrafish developing diencephalon, loss-of-Wls reduces the formation of habenula (HA) neurons but how Wls influences HA neurogenesis is unclear and whether Wls regulates gliocyte development is unknown. Here we report that the formations of cholinergic, substance P-ergic or glutamatergic neurons in HA are reduced differentially but the generation of gliocyte-derived choroid plexus (ChP) epithelia is increased in wls null mutants. At earlier stage, three-dimensional gene expression analyses revealed that while neurog1 expressions in HA progenitor zones are reduced, the expressions of Notch downstream effector her6 are increased and expanded into HA progenitor zones in wls mutants. Over-expressing Her6 in neurog1 -positive cells reduced neurog1 expressions in HA progenitor zones. These results indicate that Wls restricts the expressions of Notch effector her6 to promote the specification of neurog1 proneurons and demotes the generation of ChP epithelia in zebrafish embryonic dorsal diencephalon. ### Competing Interest Statement The authors have declared no competing interest. JSPS KAKENHI, 20K22572, 20H05886, 23H01817 NIBB Collaborative Research Program, 21-404, 24NIBB505 National Science and Technology Council, Taiwan, 111-2311-B-002-027, 112-2311-B-002-010, 113-2311-B-002-014

Xenopus laevis tadpoles and newts regenerate a limb almost completely after amputation, with recapitulation of the patten formation of the limb. Metamorphosed Xenopus froglets form a cone-shaped regenerating blastema, similar to tadpoles and newts, but ultimately regenerate only a hypomorphic cartilaginous spike. Previous study suggested that excessive chondrogenesis, distinct from Xenopus tadpoles, may occur in the regenerating limb of a froglet and may prevent pattern formation during regeneration. However, it remains unclear whether excessive chondrogenesis actually occurs in froglet blastemas. If it does, when does it initiate and how does it progress in the blastemas? To answer these questions, we examined the extent of chondrogenesis in regenerating blastemas which have the common morphological shapes observed in newts ( Pleurodeles waltl ), Xenopus laevis tadpoles, and froglets. To evaluate excessive chondrogenesis, we developed a simplified procedure using immunofluorescence for cartilage markers (Sox9 or Col2a1) and quantitative image analysis. Our analysis revealed that signs of excessive chondrogenesis were detected not in newts and tadpoles but in froglets blastemas. During limb regeneration in froglets, the first sign of excessive chondrogenesis was detected in the cone-shaped blastema at the medium bud (MB) stage, and excessive chondrogenesis progressed to a more severe state as the blastema grew. These results indicate that excessive chondrogenesis initiates specifically in froglet blastemas at the MB stage at the latest and progresses in a definite spatio-temporal manner. Further elucidation of the mechanisms underlying froglet-specific excessive chondrogenesis in the blastema may lead to the recovery of patterned limb regeneration in froglets with adequate inhibition of chondrogenesis. ### Competing Interest Statement The authors have declared no competing interest.

Type I collagen is a major component of the dermis and is formed by dermal fibroblasts. The development of dermal collagen structures has not been fully elucidated despite the major presence and importance of the dermis. This lack of understanding is due in part to the opacity of mammalian skin and it has been an obstacle to cosmetic and medical developments. We reveal the process of dermal collagen formation using the highly transparent skin of the axolotl and fluorescent collagen probes. We clarify that epidermal cells, not dermal fibroblasts, contribute to dermal collagen formation. Mesenchymal cells (fibroblasts) play a role in modifying the collagen fibers already built by keratinocytes. We confirm that collagen production by keratinocytes is a widely conserved mechanism in other model organisms. Our findings warrant a change in the current consensus about dermal collagen formation and could lead to innovations in cosmetology and skin medication.

The pancreas exhibits diverse structures and roles across vertebrates. The pancreas has evolved to include both endocrine and exocrine cells, a change that occurred during the transition from fish to amphibian. This event emphasizes the evolutionary significance of amphibians. However, research has focused predominantly on anuran amphibians, with urodeles, such as newts, remaining underexplored. In this study, we investigated the development of the pancreas using Pleurodeles waltl as a model species of urodele. The newt pancreas consists of a single organ with exocrine tissue characterized by acinar structures and endocrine tissue forming islets. Notably, the newt possesses unique pancreas-like tissues on their intestines. We found that disruption of the newt Pancreatic and Duodenal Homeobox (Pdx) 1 gene resulted in an underdeveloped pancreas. Conversely, disruption of the Pdx2 paralog in newt had no significant impact on pancreatic development. The newt pancreas shows a morphology similar to that of the mammalian pancreas, which includes both exocrine and endocrine tissues. These results highlight the intermediate evolutionary position of the newt in the context of the evolution of pancreatic development. Our findings indicate that characterization of the newt pancreas will be crucial for understanding the evolutionary progression of pancreatic function in vertebrates.

Temperature within the range of 15°C-37°C plays a pivotal role in modulating cellular processes and is essential for understanding the complex mechanisms underlying axonal transport and function in myelinated regions of Schwann cells (SCs). This review presents a comprehensive overview of the current knowledge on the impact of temperature on various aspects of axonal function, including saltatory conduction, ion channel activity, molecular motor dynamics, and Schwann cell function. We also delve into the potential implications of these findings in the context of neurological disorders and their treatment. The temperature-dependent nature of saltatory conduction and action potential propagation in myelinated axons is of particular interest, as it directly affects the efficiency of nerve signal transmission. Additionally, the activity of ion channels in the nodes of Ranvier is subject to modulation by temperature, further emphasizing the importance of understanding temperature's influence on neuronal function. This review concludes with a discussion of various unresolved questions in the field, and ideas are suggested for future research. Studying the precise molecular mechanisms underlying the temperature-dependent regulation of ion channels, molecular motors, and cytoskeletal components may lead to the development of novel strategies for the diagnosis and treatment of neurological disorders, which are commonly observed in demyelinating diseases and hereditary neuropathies. A deeper understanding of the role of temperature in neuronal function has the potential to significantly advance our knowledge of thermoregulation and neurologic function, ultimately leading to breakthroughs in the diagnosis and treatment of various neurological disorders.

A spatiotemporal understanding of gene function requires the precise control of gene expression in each cell. Here, we use an infrared laser-evoked gene operator (IR-LEGO) system to induce gene expression at the single-cell level in the moss Physcomitrium patens by heating a living cell with an IR laser and thereby activating the heat shock response. We identify the laser irradiation conditions that provide higher inducibility with lower invasiveness by changing the laser power and irradiation duration. Furthermore, we quantitatively characterize the induction profile of the heat shock response using a heat-induced fluorescence reporter system after the IR laser irradiation of single cells under different conditions. Our data indicate that IR laser irradiation with long duration leads to higher inducibility according to increase in the laser power but not vice versa, and that the higher laser power even without conferring apparent damage to the cells decelerates and/or delayed gene induction. We define the temporal shift in expression as a function of onset and duration according to laser power and irradiation duration. This study contributes to the versatile application of IR-LEGO in plants and improves our understanding of heat shock-induced gene expression.

The crustacean Daphnia magna is an emerging model for ecological and toxicological genomics. However, the lack of methods for spatial and temporal control of gene expression has impaired the elucidation of molecular mechanisms underlying responses to environments in vivo. Here we report local activation of the hsp70 promoter-driven gene cassette in D. magna by the infrared laser-evoked gene operator (IR-LEGO), a method for heating the target cells with infrared irradiation. We identified the heat-inducible promoter upstream of the D. magna hsp70-A gene. Using this promoter, we generated a transgenic Daphnia harboring the heat-shock responsive GFP reporter gene and confirmed that the GFP gene responds to heat treatment not only in juveniles and adults but also in embryos. We collected embryos from the reporter line and irradiated four different regions of interest in the embryos: a proximal region of the third thoracic segment, a part of the midline, a second maxilla, and a distal region of the endopodite of the second antenna, all of which increased GFP fluorescence with an infrared laser. Our results suggest that the IR-LEGO method is useful for spatial and temporal control of gene expression and would advance the functional genomics in D. magna.

5'Hox genes regulate pattern formation along the axes of the limb. Previously, we showed that Hoxa13/Hoxd13 double-mutant newts lacked all digits of the forelimbs during development and regeneration, showing that newt Hox13 is necessary for digit formation in development and regeneration. In addition, we found another unique phenotype. Some of the Hox13 crispant newts showed hindlimb defects, in which whole or almost whole hindlimbs were lost, suggesting a novel function of Hox13 in limb development. Using germline mutants, we showed that mutation in Hox13 led to hindlimb defects. The limb buds of Hox13 crispants formed, however, did not show outgrowth. Expression of Fgf10 and Tbx4, which are involved in limb outgrowth, decreased in the hindlimb buds of Hox13 crispants. In addition, hindlimb defects were observed in both Fgf10 and Tbx4 crispant newts. Finally, Fgf10 and Tbx4 interacted with Hox13 genetically. Our results revealed a novel function of Hox13 in regulating the outgrowth of the newt hindlimb bud through interaction with Fgf10 and Tbx4.

Background The behavioral photosensitivity of animals could be quantified via the optomotor response (OMR), for example, and the luminous efficiency function (the range of visible light) should largely rely on the repertoire and expression of light-absorbing proteins in the retina, i.e., the opsins. In fact, the OMR under red light was suppressed in medaka lacking the red (long-wavelength sensitive [LWS]) opsin. Results We investigated the ultraviolet (UV)- or blue-light sensitivity of medaka lacking the violet (short-wavelength sensitive 1 [SWS1]) and blue (SWS2) opsins. The sws1/sws2 double or sws1/sws2/lws triple mutants were as viable as the wild type. The remaining green (rhodopsin 2 [RH2]) or red opsins were not upregulated. Interestingly, the OMR of the double or triple mutants was equivalent or even increased under UV or blue light (λ = 350, 365, or 450 nm), which demonstrated that the rotating stripes (i.e., changes in luminance) could fully be recognized under UV light using RH2 alone. The OMR test using dichromatic stripes projected onto an RGB display consistently showed that the presence or absence of SWS1 and SWS2 did not affect the equiluminant conditions. Conclusions RH2 and LWS, but not SWS1 and SWS2, should predominantly contribute to the postreceptoral processes leading to the OMR or, possibly, to luminance detection in general, as the medium-wavelength-sensitive and LWS cones, but not the SWS cones, are responsible for luminance detection in humans.

Complex structures in living cells and tissues induce wavefront errors when light waves pass through them, and images observed with optical microscopes are undesirably blurred. This problem is especially serious for living plant cells because images are strikingly degraded even within a single cell. Adaptive optics (AO) is expected to be a solution to this problem by correcting such wavefront errors, thus enabling high-resolution imaging. In particular, scene-based AO involves wavefront sensing based on the image correlation between subapertures in a Shack-Hartmann wavefront sensor and thus does not require an intense point light source. However, the complex 3D structures of living cells often cause low correlation between subimages, leading to loss of accuracy in wavefront sensing. This paper proposes a novel method for scene-based sensing using only image correlations between adjacent subapertures. The method can minimize changes between subimages to be correlated and thus prevent inaccuracy in phase estimation. Using an artificial test target mimicking the optical properties of a layer of living plant cells, an imaging performance with a Strehl ratio of approximately 0.5 was confirmed. Upon observation of chloroplast autofluorescence inside living leaf cells of the moss Physcomitrium patens, recovered resolution images were successfully obtained even with complex biological structures. Under bright-field illumination, the proposed method outperformed the conventional method, demonstrating the future potential of this method for label- and damage-free AO microscopy. Several points for improvement in terms of the effect of AO correction are discussed.

Axolotls have been considered to be able to regenerate their skin completely. Our recent study updated this theory with the finding that the lattice structure of dermal collagen fibers was not fully regenerated after skin injury. We also discovered that nerves induce the regeneration of collagen fibers. The mechanism of collagen fiber regeneration remains unknown, however. In this study, we focused on the structure of collagen fibers with collagen braiding cells, and cell origin in axolotl skin regeneration. In the wounded dermis, cells involved in skin repair/ regeneration were derived from both the surrounding dermis and the subcutaneous tissue. Regardless of cell origin, cells acquired the proper cell morphology to braid collagen fiber with nerve presence. We also found that FGF signaling could substitute for the nerve roles in the conversion of subcutaneous fibroblasts to lattice-shaped dermal fibroblasts. Our findings contribute to the elucidation of the fundamental mechanisms of true skin regeneration and provide useful insights for pioneering new skin treatments.