The stony coral, Acropora tenuis, possesses progenitor/undifferentiated cells similar to other anthozoans; however, their developmental potential and fate remain obscure. The present study initially showed that nerve progenitors and undifferentiated-like cells were both tagged with histone H3 trimethylation at Lys 4 and Lys 27, suggesting that Acropora progenitor/undifferentiated cells might be multipotent. A rabbit anti-reverse transcriptase (anti-AtRTase) antibody recognized undifferentiated-like cells in the middle region of the ectodermal layer in embryos 60-85 h post-fertilization. At the larval stages, AtRTase signals were concentrated around the oral pole where the larval ectoderm invaginated to form the pharyngeal stomodeum. Undifferentiated-like cells, but not the apical-most glandular epithelium in the ectoderm, appeared to enter the pharynx. Consistent with this hypothesis, AtRTase signals were continuous from undifferentiated-like cells to the stomodeum. Nerve progenitors (neuroblasts) expressed nerve-specific tubulin beta-III (TUBB3). They differentiated into nerve cells and expressed a neuroblast differentiation-associated protein around the aboral pole of larvae. On the other hand, non-aboral neuroblasts expressed the autophagy-related protein 10 in addition to TUBB3 and developed into vacuolated hyaline cells (VHCs) that penetrated through the ectoderm apicobasally. The present study suggests strongly that ectodermal undifferentiated cells in A. tenuis have the potential to form the stomodeum. In contrast, nerve progenitors possess the dual potential to differentiate into nerve cells and VHCs, which appears to depend on aboral and non-aboral positions.
Genetically encoded ATP biosensors enable monitoring of cellular energy status, but their application in multicellular organisms remains limited. QUEEN is a ratiometric ATP biosensor consisting of a bacterial ATP-binding protein fused to a circularly permuted fluorescent protein and has been primarily validated in cultured cells. Here, we generated transgenic Drosophila melanogaster lines expressing QUEEN-7μ, enabling tissue-specific expression through the GAL4/UAS system. We characterized its performance in motor neurons and muscles. QUEEN-7μ responses were validated using pharmacological and genetic perturbations of mitochondrial function. Mitochondrial inhibition decreased the QUEEN-7μ ratio, consistent with reduced ATP levels, whereas acute treatment with mitochonic acid-5 (MA-5), a small molecule that enhances mitochondrial ATP synthesis, increased the QUEEN-7μ ratio, with a larger effect at higher concentration. Consistently, genetic manipulations, including the mitochondrial Complex I knockdown and Mitofilin/MIC60 overexpression, produced corresponding decreases and increases in the QUEEN-7μ ratio. These results establish QUEEN-7μ as a reliable ratiometric ATP reporter for quantitative in vivo analysis in Drosophila. This system provides a versatile platform for investigating energy metabolism and mitochondrial function in Drosophila.
The lateral line is a mechanosensory organ found in cyclostomes, teleosts, and amphibians. Hair cells within its neuromasts have kinocilia and stereocilia that sense water flow, and the lateral line nerve transduces this sensory input. Hair cells in the lateral line are analogous to those in the mammalian inner ear. Lateral line development and regeneration have been studied extensively in zebrafish. However, lateral line formation in other species remains poorly understood. The Iberian ribbed newt (Pleurodeles waltl) is a versatile model for regeneration with a short period of sexual maturation and aseasonal/prolific ovulation. Here, we investigated the features, regeneration processes, and additional neuromast formation in the posterior lateral line of Iberian ribbed newts using Brainbow reporters. The lateral line in the Iberian ribbed newts shares common features with other species. Laser ablation of hair cells or transection of the posterior lateral line nerve induced additional neuromast formation, to which epidermal cells contributed at least in part. We refer to it as "replicative neuromast regeneration" to allow distinction from several modes of neuromast formation in normal development. Based on these results, we propose that in response to injury, the posterior lateral line nerve induces epidermal cells to contribute to newly formed neuromasts in a replicative manner. Our results provide insights into lateral line morphogenesis.
Fat2 is an atypical cadherin with a large extracellular domain containing multiple cadherin repeats. Although members of the Fat family have been implicated in organ development, the role of Fat2 in neural development remains poorly understood. Here, we investigated the function of Fat2 in zebrafish, focusing on its involvement in the development of cerebellar granule cells. Granule cells in the rostromedial cerebellum project their axons, known as parallel fibers, to Purkinje cell dendrites. In contrast, granule cells in the caudolateral cerebellum project their axons to Purkinje cells and further extend them caudally to innervate crest cells, Purkinje-like neurons located in the dorsal hindbrain region called the medio-octaval lateralis. We found that in fat2 mutants, axons of caudolateral cerebellar granule cells projecting to crest cells exhibit abnormal branching and frequently overshoot their targets. These results indicate that Fat2 is required for proper axon formation in at least a subset of cerebellar granule cells.
The Notch signaling pathway is essential for the self-renewal, differentiation, and maintenance of neural stem cells (NSCs). While genetic studies in Drosophila have implicated Strawberry Notch in Notch signaling, the role of its vertebrate homologue, Sbno1, in mammalian NSCs remains largely unexplored. Here we show that Sbno1 interacts with Rbpj, the central transcriptional mediator of Notch, as well as with the deubiquitinating enzyme Usp8. Promoter analyses revealed that Sbno1 and Usp8 synergistically enhance expression of Hes5, a well-known Notch target in NSCs. Using in utero electroporation to overexpress or knock out Usp8 in embryonic cortical NSCs, we demonstrate that Usp8 is essential for NSC maintenance whereas loss of Sbno1 leads to NSC depletion and premature neuronal differentiation. Mechanistically, our results suggest that Sbno1 facilitates Usp8-mediated stabilization of the Notch intracellular domain and resolves R-loops at the Hes5 promoter, thereby promoting target gene expression. Together, these findings identify Sbno1 and Usp8 as critical, previously unrecognized regulators of Notch signaling in NSCs and provide the first in vivo evidence of their physiological roles in mammalian cortical development.
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.
Three-dimensional (3D) genome organization is dynamically restructured during early vertebrate development, yet how chromatin domains are established remains poorly understood. In particular, the contribution of individual cohesin regulators to this process during embryogenesis is unclear. PDS5 proteins are key modulators of cohesin dynamics, but their depletion has been reported to cause context-dependent and sometimes contrasting architectural effects in cultured cells. Here, we investigated the roles of the cohesin regulators Pds5a and Pds5b during early development using the medaka embryo. Developmental transcriptome analysis revealed distinct but overlapping expression dynamics of pds5a and pds5b around the transition from zygotic genome activation to gastrulation. Morpholino-mediated depletion of either paralog resulted in only mild morphological phenotypes, whereas simultaneous depletion caused more severe developmental defects. In situ Hi-C analysis showed that single depletion of pds5a or pds5b induced only modest changes in 3D genome organization. In contrast, double depletion led to pronounced architectural alterations, including increased long-range chromatin contacts and de novo formation of extended chromatin loops. Transcriptome analysis revealed largely shared, with some condition-specific, gene expression changes in both single- and double-knockdown embryos, indicating that transcriptional effects can occur even in the absence of major architectural disruption. Together, our findings demonstrate that Pds5a and Pds5b act cooperatively to constrain cohesin-mediated long-range interactions during embryogenesis and highlight the importance of analyzing cohesin regulator function within a developmental context to understand how 3D genome organization is established in vivo.
Germ cells in zebrafish are specified by a preformation mechanism, unlike in mice. Max (Myc associated factor X) and its associated PRC1.6 (polycomb repressive complex 1.6) act as negative regulators of germ cell- and meiosis-related genes in mice; however, their function in zebrafish remains unknown. Here, we generated max mutants in zebrafish using genome-editing techniques to investigate the role of Max in germ cell development and meiosis. No obvious morphological abnormalities were observed in the mutants up to 4 days post fertilization (dpf), likely due to maternally supplied max mRNAs. However, the mutants failed to form an inflated swim bladder and gradually died after 10 dpf. The number of primordial germ cells (PGCs) did not differ between the max mutant and wild-type larvae up to 12 dpf. In contrast, analysis of protein expression levels of germ cell-related and meiosis-related genes in PGCs at 9 dpf revealed that the levels of Sycp2, Sycp3, and Dmc1 proteins were significantly increased in max mutants, whereas the expression of Ddx4 and Piwil1 remained unchanged. Together, these results suggest that zebrafish Max selectively represses the expression of a subset of meiotic genes in PGCs.
The Joint Meeting of the 77th Japan Society for Cell Biology (JSCB) and the 58th Japan Society of Developmental Biologists (JSDB) was held from July 16 to 18, 2025, in Nagoya, Japan. With the goal of fostering discussion across disciplinary boundaries and envisioning future directions in cell biology, developmental biology, and related areas of life science, the conference featured various presentations over the course of 3 days: plenary lectures by Keiko Torii (University of Texas/HHMI) and Tian Xu (Westlake University), an educational lecture by Masayuki Miura (National Institute for Basic Biology), 404 poster presentations, and 220 oral presentations. Special panel discussions were organized by the conference chairs, titled "Our Mistakes," followed by "This is My Path in Research," which sparked discussions on life science research through the lens of the Nobel Prize, highlighting dreams, hopes, and passion in scientific careers.
Tile patterns are fundamental organizational principles of multicellular epithelial tissues. The Drosophila compound eye provides a striking example, in which ommatidia are arranged in a highly regular hexagonal lattice, while tetragonal patterns emerge in specific small-eye mutants. Although increased dorsoventral tension has been implicated in this hexagonal-to-tetragonal transition, conventional vertex models fail to reproduce the observed pattern transformation, indicating the presence of additional uncharacterized force-generating mechanisms. Here, we demonstrate that anisotropic cellular forces driven by radial actin fibers are a key determinant of ommatidial tiling geometry. By extending the vertex model to incorporate both dorsoventral stretching and anisotropic forces that generate rotational torque at cell boundaries, we successfully recapitulate the hexagonal-to-tetragonal transition observed in mutant eyes. Experimental disruption of radial actin fibers suppressed tetragonal pattern formation and induced irregular tiling, providing in vivo support for the model predictions. Importantly, in silico analyses further revealed that anisotropic forces play a dual role: while they drive tetragonalization under symmetry-breaking conditions in mutant eyes, they stabilize regular hexagonal tiling in the wild-type context. These findings identify anisotropic cellular forces as an essential component of epithelial pattern formation and establish an extended vertex model framework for understanding force-driven morphogenetic transitions during development.
RNA sequencing (RNA-seq) has become an essential tool for analyzing gene expression and exploring cell type-specific transcriptomes. However, sample preparation and quality control remain challenging, as current approaches typically rely on dissecting tissues containing mixed cell populations or using flow cytometry to isolate fluorescently labeled cells. Here we present a simple and reliable method for RNA-seq of chromatophores (pigment cells) by manually isolating cells based on their natural pigmentation. We analyzed four chromatophore types-melanophores, xanthophores, iridophores, and leucophores-in medaka (Oryzias latipes). Remarkably, as few as 100 cells per type yielded reasonably high-quality transcriptomes sufficient to identify differentially expressed genes (DEGs). Furthermore, this method was successfully applied to a non-model medaka species, O. woworae, which shares the same four chromatophore types. Our approach enables efficient, low-cost, and cross-species transcriptome analysis of chromatophores without requiring transgenic markers or flow cytometry.
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.
During anuran metamorphosis, rapid and extensive morphological transformations occur throughout the body, and these changes are triggered by the thyroid hormone. The thyroid hormone receptor (TR) is a nuclear receptor that is present in all vertebrates. TR binds specific DNA sequences to repress target genes in the absence of ligand and to activate them when ligand-bound. This dual regulatory function has been proposed to underlie the rapid pace of anuran metamorphosis. One TR subtype, TRα, suppresses hindlimb (HL) development in pre-metamorphic Xenopus tropicalis. However, the genes repressed by TRα remain unidentified, and it is unclear whether TRα-mediated developmental suppression occurs in specific organs. This study aimed to identify HL genes regulated by TRα during developmental suppression and to determine whether this suppression is widespread in the pre-metamorphic tadpole. We analyzed temporal changes in morphology and gene expression in the HL buds and intestines of TRα-knockout (KO) pre-metamorphic X. tropicalis tadpoles. HL buds appeared earlier in KO tadpoles than in the wild type. Whole-mount in situ hybridization showed that the interval from fertilization to initial expression of shh was shorter in KO HLs. However, the expression pattern in HL buds with comparable morphology was essentially identical between genotypes. On the other hand, there was no significant acceleration in the growth of the intestine or body (from snout to vent) of the KO tadpoles. Our findings suggest that unliganded TRα delays the onset of HL development prior to metamorphosis.
Zinc finger protein 281 (Znf281) plays important roles in human malignancies, stem cell pluripotency, and placental and embryonic development. However, the function of Znf281 during early neural development remains unclear. Here, we investigated the role of Znf281 in the formation of neural tissue in Xenopus embryos. znf281 transcripts are expressed in the animal hemisphere of the embryo at the blastula and gastrula stages and gradually localize in neural tissue after gastrulation. Overexpression of Znf281 induces neural tissue with anterior-posterior patterning and inhibits epidermal differentiation in ectodermal explants and embryos. Mechanistically, Znf281 reduces the levels of phosphorylated Smad1/5/8 proteins, the downstream effectors of bone morphogenetic protein (BMP) signaling, to promote neural development. Moreover, knockdown of Znf281 in embryos results in the reduced expression of neural markers, indicating that Znf281 is required for early neural development. These results suggest that Znf281 plays an important role in the establishment of the central nervous system by modulating BMP signaling during vertebrate embryogenesis.
In medaka ( Oryzias latipes ), the first morphological sex difference is germ cell number before hatching, which is determined by the dmy gene on the Y chromosome. This study aimed to clarify whether zygotically synthesized estrogen influences the kinetics of germ cell number during early gonadal sex differentiation. We established disruptive mutants of the estrogen-synthesizing enzyme aromatase by knocking out cyp19a1a (Δ cyp19a1a ) and cyp19a1b (Δ cyp19a1b ) as well as double disruptive mutants (Δcyp19a1s DKO) from each individual knockout using CRISPR/Cas9. Δcyp19a1s DKO XY and XX adult fish at 90 days post-hatching (dph) exhibited basal levels of estradiol-17β. At hatching (0 dph: stage 39), WT XX fry had significantly more germ cells than WT XY fry, and gonial cells were the most advanced germ cell stage across both sexes. Germ cell number and gonadal histology in Δcyp19a1s DKO mutants resembled those of WT fry. At 10 dph, germ cell number and gonadal histology were also similar between WT and Δcyp19a1s XY fry. In Δcyp19a1s DKO XX fry, diplotene oocytes and the total number of germ cells were significantly lower compared with WT. Exposure to 17α-ethynylestradiol rescued the reduction in diplotene oocytes in Δcyp19a1s DKO mutants to levels comparable to the control, resulting in the rescue of total germ cell number. Overall, our findings suggest that zygotically synthesized estrogen does not affect sex differences in germ cell number as the initial morphological sex difference but partly facilitates the differentiation from pachytene to diplotene oocytes.
Plant growth is intricately linked to the development of a robust and extensive root system, a process that is finely tuned by the plant's ability to sense and respond to environmental nutrient cues. Among these, nitrate and photosynthetically derived sucrose stand out as key regulators of root architecture, guiding plants in their foraging efforts to maximize resource acquisition. However, the mechanisms by which plants integrate these signals to modulate root growth, particularly lateral root development, remain only partially understood. This study employs differential growth analysis to determine the degree of interplay between nitrate and sucrose sensing pathways mediating root growth, specifically refining the role for CEP (C-terminally Encoded Peptide) Receptor 1 (CEPR1). Pathways modulating root growth in response to perception of nitrate and sucrose do not operate independently and rely on CEPR1 to dynamically inhibit lateral root growth based on nitrate availability in a sucrose dependent manner. These findings highlight the interplay between distinct nutrient sensing pathways in adjusting plant root architecture and accentuate the sophisticated adaptive strategies plants employ in nutrient foraging.
Programmed cell death during embryonic development plays a vital role in shaping limb morphology in amniotes. BMP (bone morphogenetic protein) signaling has been shown to be essential for inducing interdigital cell death, but its relationship with reactive oxygen species (ROS) production, another driver of this process, remains unclear. Here, we show that BMP signaling modulates ROS production, which is required for subsequent cell death in the interdigital regions of chicken hindlimbs. Through transcriptome analyses, we identify the candidate genes encoding molecular machinery potentially involved in ROS production in response to changes in BMP signaling. Our findings suggest that BMP signaling may influence the redox balance by upregulating the genes encoding ROS-generating enzymes such as Nox2 and Nox4 (components of NADPH oxidase), and downregulating the ROS-scavenging enzyme Sod1. Pharmacological inhibition of NADPH oxidase reduces ROS levels and cell death, indicating that ROS production in the chicken interdigital cell regions is at least partially NADPH oxidase-dependent. Together, these results support a model in which BMP signaling is required for the regulation of programmed cell death, at least in part by modulating redox homeostasis.
The RNA-binding protein NANOS2 plays a crucial role in male gonocyte development and the maintenance of spermatogonial stem cells. In the absence of the Nanos2 gene (Nanos2-KO), germ cells fail to enter G0 arrest and initiate the male differentiation program (including DNA methylation and the piRNA pathway), ultimately undergoing apoptosis before birth. Nanos2 transcription begins at embryonic day 12.5 (E12.5) and terminates at E15.5. However, as the NANOS2 protein continues to be stably expressed beyond E15.5, it is important to elucidate the function of NANOS2 during this post-E15.5 period in germ cell fate determination. To address the functional significance of sustained NANOS2 protein expression, we employed an auxin-inducible degron (AID2) system to achieve rapid degradation of NANOS2 after E15.5. Within 24 h of 5-Ph-IAA administration, NANOS2 protein was efficiently depleted. As a result, germ cells resumed the cell cycle, exhibited aberrant gene expression patterns similar to Nanos2-KO gonocytes, and underwent apoptosis if NANOS2 depletion occurred at E15.5 or E16.5. Although some surviving cells expressed undifferentiated spermatogonial markers PLZF and GFRA1 after birth, and entered meiotic division, most germ cells disappeared before completing meiosis. These findings reveal that sustained NANOS2 protein expression during the embryonic stage is essential for establishing functional spermatogonial stem cells, highlighting a previously unrecognized regulatory mechanism in male germ cell development.
Temporal gene expression systems are widely used to examine gene functions at specific developmental stages. The heat-inducible gene expression system, which uses a heat shock promoter with evolutionarily conserved heat shock elements, is used in temporal gene expression systems in many organisms. The nematode, Pristionchus pacificus, is a satellite model system comparable to Caenorhabditis elegans, with unique developmental traits but lacking genetic tools for assessing temporal gene expression. To establish a temporal gene expression system in P. pacificus, we investigated the genes that were highly induced by heat shock. RNA-sequencing analysis revealed many differentially expressed genes after a 2-h heat shock event. One of the highly induced genes, PPA12242, is an ortholog of C. elegans hsp-16.41, and transgenic animals harboring a reporter system have shown that the genomic fragment upstream of this gene can induce gene expression in response to heat shock. Using the PPA12242 promoter, gene expression can be induced at all larval stages, and some phenotypes appear to be vulnerable to heat stress. Taken together, we identified a potential heat shock promoter in P. pacificus that is applicable to the temporal gene expression system of this species.
