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27 records · Page 2Linked to original sources
PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗
PubMed2026

Zebrafish as a versatile model in biomedical research, from disease modeling to regenerative medicine: a review.

Zebrafish are an effective animal model widely utilized in biomedical research. They are known for their rapid reproduction and substantial genetic similarity to humans. Their transparent embryos directly enable the visualization of developmental processes and disease progression. This makes zebrafish invaluable for studying a broad range of human diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions. Compared with other vertebrate models, zebrafish offer several advantages, including ease of genome editing, cost-effective maintenance, and suitability for high-throughput drug screening. Recent advancements have expanded the use of zebrafish in disease modeling and regenerative medicine, providing deeper insights into the genetic and cellular mechanisms underlying human pathologies. Zebrafish provide a robust platform for evaluating the safety, efficacy, and regenerative potential of both natural and synthetic biomaterials, including hydroxyapatite, bioactive glass nanoparticles, and bioceramics. This capability facilitates the creation of artificial tissues that closely resemble native structures. Additionally, integrating artificial intelligence technologies has improved automated data analysis and phenotyping in zebrafish studies, enhancing both accuracy and throughput. This review highlights current applications of zebrafish in disease modeling, drug discovery, regenerative medicine, and biomaterial assessment, emphasizing their evolving role as a versatile preclinical platform supported by advanced genetic and computational tools.

Animals↗
PubMed2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia differentiation and determination of cardiac left-right asymmetry via regulation of Arl13b splicing.

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephalophŏnus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

ARL13B↗
PubMed2026

Maternal transfer of nonylphenol drives oxidative, immune, and epigenetic dysregulation in zebrafish offspring.

Nonylphenol (NP), a widespread surfactant and endocrine-disrupting pollutant, poses significant ecological and public health risks globally; however, its transgenerational effects remain poorly understood. Using zebrafish (Danio rerio), we compared chronic maternal NP exposure (50 and 100 µg/L, 28 days) with acute embryonic exposure (0.22 µmol/L) during 0-3 days post-fertilization (dpf) to delineate mechanistic differences in toxicity. Maternal NP exposure produced severe developmental defects in offspring, including edema, axial curvature, impaired swim bladder inflation, reduced growth, cardiac dysfunction, and decreased viability. These phenotypes were accompanied by systemic molecular disruptions including oxidative stress, altered estrogen receptor (ER) expression, dysregulated mitogen-activated protein kinase (MAPK) signaling, and suppressed innate immune response characterized by attenuated neutrophil/macrophage density, reduced CD68 and complement protein C3 expression, diminished nitrite load, and downregulation of pro-inflammatory mediators at both transcript and protein levels. Maternal exposure further induced apoptosis and persistent epigenetic reprogramming (alterations in DNA methylation and histone-modifying enzymes), hallmarks of transgenerational toxicity. In contrast, direct embryonic NP exposure elicited morphological abnormalities without significant lethality, accompanied by induction of pro-inflammatory cytokines, nitric oxide (NO) synthesis, and MAPK activation, reflecting an augmented inflammatory response. These mechanistic contrasts reveal that maternal NP exposure is a potent driver of systemic, heritable molecular reprogramming, whereas embryonic exposure triggers acute inflammatory pathways. Together, our findings underscore the global relevance of NP as a transgenerational toxicant, advocating for its urgent inclusion in ecotoxicological risk assessments and regulatory frameworks.

Animals↗
PubMed2026

A quantitative coordinate system for developmental dynamics.

Quantitative comparison of morphogenesis across individuals remains a fundamental challenge, as developing embryos vary in shape, orientation and developmental tempo. Moreover, real-time three-dimensional imaging generates large, heterogeneous four-dimensional datasets that are difficult to directly align. As a result, developmental variability is typically described qualitatively rather than measured. Here we introduce STERN, a quantitative framework that learns continuous spatiotemporal representations of morphogenesis directly from in vivo 4D imaging data. By embedding embryos into a shared spatiotemporal space, STERN defines a quantitative developmental coordinate system that enables direct comparison of developmental trajectories across individuals without requiring explicit registration or staging. Applied to mouse embryogenesis, STERN reveals that embryos follow conserved developmental trajectories while progressing at distinct temporal rates, providing a quantitative measure of developmental heterochrony. Extending this framework to zebrafish neural crest light-sheet timelapse imaging, we further show that developmental order is preserved across distinct imaging views even with altered anatomical coverage, supporting the generality of the learned representation across vertebrate imaging contexts. Finally, in developing mouse hearts, where morphogenesis proceeds through subtle and continuously evolving structural changes, STERN resolves fine-scale developmental dynamics at minute-scale temporal resolution that are difficult to localize reproducibly using human experts or general-purpose multimodal AI. Together, these results establish a shared quantitative coordinate system for morphogenesis, in which developmental trajectories become directly comparable across individuals and developmental variability becomes a measurable property.

Journal Article↗
PubMed2026

The maternal-to-zygotic transition is a critical window for PFOA-induced disruption of developmental programming.

Early embryogenesis is governed by precisely timed gene regulatory programs that coordinate cell fate specification, tissue patterning, and morphogenesis. The maternal-to-zygotic transition (MZT) represents a pivotal developmental milestone during which regulatory control shifts from maternally deposited transcripts to activation of the zygotic genome. Disruption of this transition has the potential to alter developmental trajectories with lasting consequences. Per- and polyfluoroalkyl substances (PFAS), environmentally persistent contaminants, have been linked to developmental abnormalities, yet their impact on core embryonic gene regulatory networks especially with exposure during MZT is not well understood. Using zebrafish (Danio rerio), a tractable vertebrate model and New Approach Methodology (NAM), we investigated how PFAS exposure during the MZT alters early developmental programming. Embryos were exposed starting at different times before and within the MZT time window and collected at 24 h post-fertilization (hpf) for transcriptomic analysis. Targeted qRT-PCR revealed dysregulation of genes controlling transcriptional activation, lineage specification, proliferation, and differentiation. Whole-transcriptome RNA sequencing (RNA-seq) further identified widespread perturbations in gene networks governing transcriptional regulation, cell signaling, and embryonic morphogenesis. Temporal analysis revealed that exposure beginning at 3.5 hpf, followed by 8 hpf, corresponding to early zygotic genome activation and near completion of zygotic activation, respectively, resulted in the greatest differential gene expression changes at 24 hpf. Consistent with these early gene regulatory perturbations, larvae exposed starting at 8 hpf also exhibited altered behavior at 5 days post-fertilization. Together, these findings demonstrate that PFAS exposure during MZT disrupts the establishment of embryonic gene regulatory networks, linking environmental toxicant exposure to altered developmental patterning and organismal outcomes. This work underscores the vulnerability of early developmental transitions to environmental perturbation and positions MZT as a critical window of susceptibility during development.

NAMs (new approach methodologies)↗
PubMed2025

In vivo differentiation of embryonic cells devoid of key reprogramming factors.

Embryonic cell differentiation depends on reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this coincides with genome activation, which is regulated by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Here, we investigate the role of NPS in developmental reprogramming and differentiation by analyzing the fate of NPS mutant cells in a wild-type embryo using single-cell RNA-seq. We find that many cells fail to activate transcription or undergo cell death, while others acquire gene expression profiles that resemble germ cells, neural progenitors, and motoneuron states. These cells achieve intermediate transcriptional states, revealing the essential role of NPS in coordinating nuclear and cytoplasmic reprogramming and preventing the premature activation of lineage-specific differentiation programs. These results demonstrate that most developmental programs require developmental reprogramming by NPS, yet some cells can bypass transient totipotency to achieve intermediate developmental states resembling wild-type states in vivo.

Animals↗
PubMed2026

Discovery of NAT-6-321056 as a novel modulator of VEGFR2 signaling to suppress tumor angiogenesis.

Vascular endothelial growth factor receptor 2 (VEGFR2) is a master regulator of angiogenesis and cancer progression. However, current VEGFR2 modulators face significant challenges, including off-target toxicity and acquired resistance, underscoring the urgent need for novel therapeutic agents with improved efficacy and safety profiles. Here, we reported that virtual screening of 39,442 natural products from the ZINC natural products-derived library, coupled with molecular docking and molecular dynamics (MD) simulations to evaluate the binding stability of candidate compounds, identified NAT-6-321056 as a highly promising modulator of VEGFR2 signaling. Biological evaluations demonstrated that NAT-6-321056 exerted potent inhibition on the growth of a broad spectrum of cancer cells, including both solid tumors and hematological malignancies. In EA.hy 926 endothelial cells and SK-N-DZ neuroblast cells, the compound significantly suppressed proliferation, migration, and invasion. Microscale thermophoresis (MST) confirmed direct binding of NAT-6-321056 to VEGFR2 with favorable affinity. Kinase profiling against a panel of 33 kinases indicated that NAT-6-321056 exhibited a multi-kinase modulation profile. Mechanistic studies revealed that NAT-6-321056 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and was associated with reduced VEGFR2 phosphorylation and attenuation of the downstream ERK/JNK/AKT signaling pathways. Moreover, NAT-6-321056 exhibited robust in vivo anti-angiogenic effects in both the chick chorioallantoic membrane (CAM) assay and transgenic zebrafish vascular fluorescence imaging models. Computational absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction suggested acceptable drug-like properties. Collectively, these findings demonstrated that NAT-6-321056 is a promising modulator of VEGFR2 signaling with potent anti-angiogenic activity and represents a viable candidate for cancer therapy.

Vascular Endothelial Growth Factor Receptor-2↗
PubMed2004

Conserved function of medaka pink-eyed dilution in melanin synthesis and its divergent transcriptional regulation in gonads among vertebrates.

Medaka is emerging as a model organism for the study of vertebrate development and genetics, and its effectiveness in forward genetics should prove equal to that of zebrafish. Here, we identify by positional cloning a gene responsible for the medaka i-3 albino mutant. i-3 larvae have weakly tyrosinase-positive cells but lack strongly positive and dendritic cells, suggesting loss of fully differentiated melanophores. The region surrounding the i-3 locus is syntenic to human 19p13, but a BAC clone covering the i-3 locus contained orthologs located at 15q11-13, including OCA2 (P). Medaka P consists of 842 amino acids and shares approximately 65% identity with mammalian P proteins. The i-3 mutation is a four-base deletion in exon 13, which causes a frameshift and truncation of the protein. We detected medaka P transcripts in melanin-producing eyeballs and (putative) skin melanophores on embryos and an alternatively spliced form in the non-melanin-producing ovary or oocytes. The mouse p is similarly expressed in gonads, but not alternatively spliced. This is the first isolation of nonmammalian P, the functional mechanism of action of which has not yet been elucidated, even in mammals. Further investigation of the functions of P proteins and the regulation of their expression will provide new insight into body color determination and gene evolution.

Amino Acid Sequence↗