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.
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.
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.
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.
Primordial germ cells (PGCs), the precursors of the germline, have unique cellular characteristics to undergo long-distance migration to the embryonic gonads and have the potential to differentiate into somatic cells. Among the animal models studying PGC development, the chicken PGCs are an ideal model, since it is a rare model in which long-term PGC cultivation is applicable. Although the cultural applicability of chicken PGC makes it attractive for revealing the PGC character and its developmental processes, some differences from in vivo PGCs are known, such as the remarkable upregulation of cell proliferation and a lesser ability to reach the gonads. Understanding these differences at the molecular level is crucial. To this end, we first performed SMART-seq-based single-cell RNA sequencing to compare transcriptomes between in vivo PGCs and cultured PGCs. Our results revealed that PGC cultivation causes a shift from a MYC-dependent to a MYCN-dependent gene regulatory network (GRN) in PGCs, suggesting that this reprogramming contributes to the acquisition of proliferation ability and stem cell characteristics in cultured PGCs. Additionally, our results suggest that the MYCN-dependent GRN increases the risk of somatic differentiation, particularly in neural fate, in cultured PGCs. In addition, our transcriptome analysis identified a new cell population that shows molecular characteristics of germline-biased undifferentiated cells. Thus, our study provides fundamental molecular information to understand both the effects of PGC cultivation and the developmental process of chicken PGCs.
The corpus callosum (CC) is the large axon bundle connecting the telencephalic hemispheres. The CC is formed exclusively in placental mammals, and the lack of comparable structures in other amniotes obscures the evolutionary origin of the CC. We here demonstrate that interhemispheric remodeling, a prior developmental step for CC formation, is highly conserved in nonmammalian amniotes, such as reptiles and birds. In these animal groups, the spatio-temporal dynamics of interhemispheric remodeling are tightly connected with distinct commissural formations. We observed a high degree of similarity between the mammalian CC and reptilian rostral pallial commissure (RPC) and significant modifications in the avian pallial projection. Furthermore, we determined that Satb2 plays crucial roles in interhemispheric remodeling, which is associated with proper formation of both the CC and RPC in mice and geckoes, via the use of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-mediated gene targeting. Our findings suggest that developmental mechanisms for midline remodeling were already present in the common ancestor of amniotes, which contributed to the evolution of eutherian-specific CC formation.
The germline of Drosophila melanogaster originates from primordial germ cells (PGCs). The sex of PGCs is determined during mid-to-late embryogenesis, and PGCs subsequently begin to differentiate into eggs or sperm. Several genes involved in the sex determination of PGCs have been identified, but the mechanisms underlying sex differentiation remain unclear. To clarify these mechanisms, identifying the sex-specific differences in PGCs and elucidating how these differences arise is necessary. We previously found that translational activity in PGCs was higher in males than in females at the late embryonic stage. However, it remains unclear when male-biased translational activity is initiated and the mechanisms that induce this bias are unknown. Here, we show that male-biased translational activity in PGCs is observed from the stage when the JAK/STAT pathway, a known masculinizing pathway in PGCs, is activated. Furthermore, this male-biased translational activity depended on the sex of the surrounding somatic cells. Moreover, the JAK/STAT pathway did not affect male-biased translational activity in embryonic PGCs, although it did affect larval germline cells. These findings suggest that embryonic PGCs respond to previously unidentified signals, rather than to the JAK/STAT pathway, from somatic cells, which in turn induces male-biased translational activity.
The GLI3 gene, a pivotal component of the hedgehog (HH) signaling pathway, plays a fundamental role in the development and patterning of various body structures, including the brain and limbs. Mutations in the GLI3 gene, particularly in the C-terminal domain, are implicated in congenital anomalies such as Greig cephalopolysyndactyly syndrome and Pallister-Hall syndrome. Recent studies have also suggested an archaic human-type mutation in the C-terminal end, which altered downstream gene regulations and anatomical structures in mice. However, the biological effects of the disruption in the Gli3 C-terminal end have not been studied well. Here we report novel Gli3 mutant mice with nonsense mutations in the C-terminal end using CRISPR/Cas12a-mediated genome editing. Analysis of the genotype-phenotype correlations has revealed that the C-terminal end of Gli3 is critical for functional protein synthesis; therefore, the disruption of this region causes severe abnormalities in brain and digit formation. These results provide insight into the mechanisms by which GLI3 mutants can cause adverse consequences during human development or result in diverse phenotypes during evolution.
Throughout our lifespan, sustaining orderly morphological structures for ensuring proper functioning of organs is imperative. Among these structures, the extracellular matrix (ECM) plays a pivotal role in sustaining organ and tissue homeostasis. Nevertheless, elucidating the role of abnormal ECM-induced biomechanical microenvironmental changes associated with transition from chronic inflammation to cancer warrants further investigation. Additionally, the temporal and spatial dynamics of the extracellular environment and immune cell populations within inflammatory regions of the body remain inadequately understood. In this review, we critically present recent analytical techniques and biomechanical approaches to elucidate how the disordered distribution of cell populations, extracellular environment heterogeneity, and changes in tissue stiffness could be interrelated in the progression from ulcerative colitis to cancer.
The avian pallium is comprised of several nuclear regions (four main areas) and differs significantly from the mammalian telencephalon, which is a six-layered structure. Although it is known that cells with identical features at the gene expression level can exist in different regions, it remains unclear whether cells with identical features arise from the same region. In this study, we examined the cell lineages produced from each region by employing the recently developed integration-coupled gene expression On (iOn) switch and performing whole-brain imaging by light-sheet fluorescence microscopy (LSFM). We found that cells born in each area migrate across regions and, therefore, cells with identical characteristics are not always generated from the same progenitor regions. Furthermore, an examination of cell migration patterns in the dorsal ventricular ridge revealed that, as in mammals, cells preferably migrate along radial fibers in the early stages of migration, but radial fibers are not required for later migration. These results support the developmental convergence models recently proposed to describe cell lineage.
Although the differentiation of cardiac progenitors during embryogenesis has been characterized in detail in the ascidian Ciona robusta, heart development after metamorphosis remains unclear. The sub-terminal regions at both ends of the Ciona heart harbor pacemaker cells, as well as undifferentiated cells located within the growth zone. We performed an RNA-Seq analysis to identify transcription factors predominantly expressed in the sub-terminal regions of the Ciona heart. Among the 17 transcription factors predominantly expressed in one or both of the sub-terminal regions, Hox3 showed the strongest expression in both. Since the role of Hox3 during Ciona heart development remained unknown, we investigated the spatial expression and function of Hox3. In situ hybridization revealed the expression of Hox3 in undifferentiated cells within the growth zone at both ends of the heart tube. The TALEN-mediated disruption of Hox3 in cardiac progenitors resulted in irregularly swollen or shortened heart tubes. These results suggest that Hox3 plays a crucial role in heart tube formation by regulating the activity of growth zone cells. Similar Hox3 expression in the terminal regions of ascidian and vertebrate hearts suggests the partial conservation of cardiac patterning and pacemaker localization.
