Primordial germ cells (PGCs) are the population of cells that, in the human embryo, are initially specified at day 12 post-fertilization, and form the precursor cells for the future gametes. Although in vitro differentiation of PGCs from human stem cells has been achieved, these primordial germ cell-like cells (hPGCLCs) fail to completely mature without the use of ex vivo human or animal gonadal soma. Previous studies in mice revealed that several metabolic changes occur during the specification and maturation of these cells, which are essential for their developmental progress. However, little is known about the metabolic profile of human primordial germ cells. In the scarcity of human PGCs, particularly at the early specification stage, hPGCLCs serve as a research model to study PGC formation. To characterize the metabolic and proteomic profile of these cells, we differentiated hPGCLCs using induced-pluripotent stem cells and performed a mass spectrometry analysis to establish their metabolome and proteome. These cells revealed distinct metabolic profile, with changes particularly at the proteome level. This included a shift between canonical and non-canonical citric acid cycle in hPGCLCs, downregulation of late-stage glycolysis and reduction of nucleotide de novo synthesis. By providing an integrative map of these metabolic networks, we aim to provide insight on the metabolism of hPGCLC development that could help improve methods for fully in vitro differentiation and maturation of hPGCLCs.
ONSEN is a heat-activated Ty1/copia retrotransposon in Arabidopsis thaliana controlled by heat shock factors (HSFs) and epigenetic silencing. Heat shock element (HSE)-like sequences in ONSEN long terminal repeats (LTRs) contribute to heat responsiveness, but relationships among sequence architecture, basal DNA methylation and natural variation remain unclear. We combined transcription-factor motif prediction, transposable-element comparisons, methylome and RNA-seq data, and Arabidopsis genome assemblies. In silico disruption of five HSE cores eliminated HSF-family motif compatibility in the selected design and all 5,119 exact-GC alternatives. Across 16 curated Col-0 terminal windows, ONSEN contained 33-49 non-redundant HSF motif-coordinate placements per 800-bp window and was strongly enriched relative to 1,930 non-ONSEN transposable elements across score thresholds and continuous metrics. Direct comparison with 779 non-ONSEN LTR retrotransposons showed selectively elevated basal CHH methylation at ONSEN termini. Genome-wide RNA-seq analysis revealed broad heat-responsive gene and transposable-element changes, including strong ONSEN induction, whereas candidate-window analysis distinguished ONSEN from most HSF-rich non-ONSEN outliers. ONSEN-like variants across eight accessions generally retained HSF-compatible motifs while altering predicted DNA binding with one finger (DOF)-family motif composition. Together, these findings define ONSEN terminal regions as HSF-rich regulatory sequences that retain heat-responsive potential within a methylated chromatin context and identify candidates for functional analysis.
Epithelial tube morphogenesis requires coordinated interactions between epithelial cells and their extracellular environment, yet the extracellular mechanisms that regulate this process remain poorly understood. The Drosophila trachea provides an excellent model for investigating how extracellular remodeling contributes to epithelial tube morphogenesis. AdamTS-B, an extracellular ADAMTS protease, is expressed in the embryonic trachea from early to late stages of tracheal development. Loss of AdamTS-B caused mild defects in unicellular branch organization, whereas overexpression produced ectopic cyst-like structures. Additionally, AdamTS-B localized intracellularly and to basal protrusions of tracheal cells. Constitutively active EGFR phenocopied the AdamTS-B overexpression phenotype. Together, our findings identify AdamTS-B as a dosage-sensitive regulator of unicellular tracheal branch morphogenesis and support a model in which extracellular proteolysis contributes to epithelial tube architecture. The phenotypic similarity between AdamTS-B overexpression and constitutive EGFR activation further suggests a potential functional relationship between extracellular proteolysis and EGFR signaling during tracheal development.
Planarians are an important model system for studying whole-body regeneration. The visualization of gene expression patterns during this process is an essential experimental technique to understand the underlying mechanisms. Multi-colour fluorescent in situ hybridization (FISH) is currently the standard approach for gene expression analysis. However, the small number of available haptens limits probe multiplexing options, and the required sequential development of individual probes can be extremely time consuming. In situ hybridization chain reaction (HCR) is a newer technique that promises parallel hybridization of multiple genes via annealing of fluorescently labelled hairpins to target sequences. While HCR is well established in several model organisms, including mice and Drosophila, existing protocols perform poorly on whole-mount planarians. Here, we report a systematically optimized HCR protocol that is faster and less labor-intensive than traditional FISH. This method can be applied to dissociated planarian cells or combined with the established DEEP Clear method to label whole-mount animals. These improvements establish HCR as a useful addition to the planarian tool kit, particularly for multiplex gene expression analysis.
The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation is poorly understood. To address this, human induced pluripotent stem cells (hiPSC) were differentiated into cardiomyocytes (CM) that were wild type (WT) or contained a genomic deletion of Topoisomerase 2B (BKO). Both WT and BKO hiPSC could be induced to differentiate into sheets of beating cardiomyocytes. BKO hiPSC take slightly longer to differentiate into sheets of beating CM than WT iPSC. RNA was prepared from both undifferentiated and differentiated WT and BKO hiPSC. RNA-seq was used to examine gene expression changes when the WT and BKO hiPSC were differentiated into CM. Gene expression changes following differentiation of BKO cells were largely similar to those in WT cells. In addition, the differentiated WT CM were treated with dexrazoxane (ICRF-187), a TOP2 catalytic inhibitor that targets both TOP2A and TOP2B, or topobexin, a new TOP2B selective catalytic inhibitor. Topobexin inhibition partially phenocopied a TOP2B deletion and thereby providing an alternative to TOP2B gene knockout in many cell lines. In future, hiPSC derived CM with and without TOP2B and inhibition by topobexin ex vivo CM could be used to study anthracycline-induced cardiotoxicity and to screen for cardioprotectants.
Starvation induces robust food-seeking behavior in animals as a fundamental survival response. In Drosophila, biogenic amine octopamine (OA) plays a critical role in starvation-induced locomotor hyperactivity and awakeness, while the underlying signaling cascade remains not fully understood. Here, we demonstrated that the conserved transcription factor cAMP-Responsible Element Binding Protein (CREB), activated during starvation through the SIK2-CRTC pathway, contributes to starvation-induced sleep reduction. Mechanistically, we found that tyramine β-hydroxylase, the rate-limiting enzyme for OA synthesis, is upregulated by starvation in a CREB-dependent manner. Selective silencing of CREB in OA-producing neurons ameliorated OA-induced sleep suppression, and CREB loss-of-function mutations can partially rescue sleep fragmentation in a Drosophila model of Huntington's disease. Collectively, these findings reveal a novel regulatory role for CREB in sleep homeostasis through the transcriptional control of OA biosynthesis.
Marking the 20th anniversary of a well-established meeting series focused on the biological and clinical importance of SOX genes as regulators of cell fate, the 7th International Workshop on SOX Transcription Factors held from 8-11 September 2025 at the Prince Resort MICE Karuizawa in Nagano, Japan, brought together 83 participants from 17 countries. The meeting showcased the foundations of SOX gene research and recent technological and conceptual breakthroughs across diverse model systems and human research, illustrating the field's evolution and emerging directions for future research. Reflecting the breadth of SOX biology - from early embryonic development and sex determination to pathogenesis and sex testing policies in sports - this Meeting Review highlights the key scientific advances and messages presented at the meeting to encourage and inspire researchers in the field of SOX biology.
The pre-malignant precursor to oesophageal adenocarcinoma (OAC), Barrett's oesophagus (BO), is prevalent in 10-15% of patients with chronic gastroesophageal acid reflux. However, in most cases, the BO condition is stable to cancer promotion; i.e. less than 1% of patients develop OAC. Here, we investigate whether there are factors that can act to prevent BO patients from developing OAC. The receptor tyrosine kinase, cKIT, is expressed in Barrett's tissue, but sparingly so in either normal or OAC tissue. To investigate whether cKIT influences cellular function that might be consistent with sustaining BO, we knocked out the KIT gene. Gene Ontology analysis revealed that cells deleted for cKIT showed differentially expressed genes (DEGs) affecting functions associated with intercellular interactions and tight junction formation. Further analysis of DEGs showed that the absence of cKIT, as well as treatment with acid and bile salts, affected genes associated with oestrogen signalling. Under these conditions, the tight junction protein Occludin was found to translocate to the nucleus. Thus, the cKIT receptor, through regulating intracellular oestrogen signalling, affects cellular tight junction formation. Reducing cKIT function in BO tissue potentially leads to elevated permeability and exposure of basal and stromal cells to luminal noxious agents which could act as OAC promoters.
The vertebrate retina contains specialised regions for high-acuity vision, exemplified by the human fovea and its zebrafish analogue, the high-acuity zone (HAZ). Despite the widespread use of zebrafish to model retinal disease, a stage-resolved quantitative reference describing normal eye, photoreceptor layer (PRL) and lens growth has been lacking. Here, we apply contrast-enhanced micro-computed tomography (micro-CT) to construct the first three-dimensional micro-CT normative atlas of wild-type zebrafish eye development across five larval stages [3, 5, 7, 10 and 18 days post-fertilisation (dpf)], mapping circumferential PRL thickness, eye and lens morphology, and compartment growth rates. Regional PRL thickening within the temporo-ventral region of the expected HAZ emerged by 5 dpf and was sustained by a localised redistribution of growth, persisting and extending towards the optic nerve through 18 dpf. The PRL, lens and eye grew through four phases, alternating between disproportionate PRL expansion and coordinated growth, while the eye remodelled from a nasal-dominant to a temporo-ventral-dominant form. This regional specialisation was protracted relative to gross ocular growth and could proceed independently of it, paralleling the extended postnatal maturation of the human fovea. This atlas provides a quantitative baseline for distinguishing disease-induced changes from normal variation, supporting zebrafish models of foveal hypoplasia and related disorders.
Rapid climate change is increasing thermal extremes that may challenge developing endotherms. In altricial birds, early postnatal life is a sensitive period because thermoregulatory and physiological systems are still maturing. We experimentally manipulated nest temperature in a wild population of great tit to test whether mild, ecologically realistic heat (+2.3°C) and cold (-1.3°C) challenges affected development and physiology. Treatments were applied from days 2-8 post-hatching, and we measured growth, survival, hydration status (plasma osmolarity and uric acid), mitochondrial density, and immunoglobulin levels. Contrary to our predictions, thermal manipulation had no detectable effects on survival, morphology, or physiological traits at either day 8 or day 14. In contrast, all physiological markers varied significantly with age, reflecting normal osmoregulatory, metabolic, and immune development. These findings suggest that moderate, short-term thermal deviations during early development fall within the tolerance range of this population, or that parental behaviour buffered nestlings against temperature changes. However, stronger or prolonged extremes may exceed buffering capacity, and delayed effects cannot be excluded. Long-term studies are needed to assess latent fitness costs under increasingly variable climates.
With the rapid development of quantitative behavioral analysis, locomotion behaviors can be examined and described in greater detail. However, behavioral structures underlying the dysfunctions and locomotor defects observed with aging remain poorly understood. Here, we recorded and analyzed worms' locomotion movement for both young and aged individuals. The results revealed distinct locomotion patterns between the two groups. Compared to the young worms, the aged individuals exhibited more disorganized forward locomotion, characterized by reduced periodicity and lower cycle stability. The time duration and frequency of backward locomotion did not differ significantly between the two groups, whereas turn behavior following reversals was significantly reduced. Moreover, the aged worms showed increased individual heterogeneity in the usage of behavioral repertoire with age. In summary, this study systematically characterizes the behavior defects of aged animals by quantifying changes in locomotor states, event transitions, and behavior organization.
During vertebrate nervous system development, neurons are produced in excess and those receiving neurotrophin ligands are maintained, enabling neural circuit establishment. An apoptotic wave sweeps across the Drosophila pupal visual system, but whether neurotrophins participate in forming adult visual circuits remained unknown. Here, we show that Drosophila Neurotrophin-3 (spz-3) and DNT-2 (spz-5) are expressed in retinal cells and medulla neurons, and Toll receptors across the visual system. Using loss and gain of function conditions for DNT-3 (spz-3) and DNT-2 (spz-5) we show that they both can, and are required to, promote cell survival. Importantly, genetic interaction data show that DNT-2 can function together with Toll-2. DNT-2 neurons were identified as medulla Mi1 neurons that connect to lamina L1 neurons expressing Toll-2. Loss of function for DNT-2 or Toll-2 induced apoptosis, and Toll-2 knock-down prevented the pro-survival function of DNT-2. DNT-2 over-expression resulted in excess Toll-2 neurons, whereas most Toll-2 neurons were lost in DNT-2 mutants. Furthermore, DNT-2 and Toll-2 were required for appropriate L1 axonal columnar organisation and dendritic morphology. Altogether, evolutionarily conserved neurotrophin family ligands control neuronal number through Toll receptors during visual circuit development in Drosophila.
The Fucci mouse lines are valuable tools for tracing cell cycle stages during development and in postnatal tissues; however, the insertion loci for the transgenes remain unidentified. Here, we report the genomic insertion sites of both the Fucci-G1 and -S/G2/M transgenes, identified through whole-genome sequencing. Interestingly, the Fucci-G1 transgene is located proximal to the Shh lung gut enhancer (SLGE) on chromosome 5. Homozygous Fucci-G1 embryos are perinatal lethal but survive until embryonic day (E)18.5, exhibiting craniofacial and foregut-related defects. Expression of Shh and canonical targets are downregulated in the pharyngeal arches of E11.5 homozygous Fucci-G1 embryos. Furthermore, in situ hybridization analysis showed diminished Shh expression in the pharyngeal and foregut endoderm, consistent with disruptions to SLGE activity. Our results indicate that heterozygous Fucci-G1 mice are suitable for cell cycle studies; however, we caution potential genetic interference when investigating SHH-related pathways.
Sea urchins attach to intertidal substrates in the face of dynamic hydrodynamic forces using flexible, adhesive tube feet. Tube-foot-based adhesion varies between species and among populations with different habitats (i.e. temperature and hydrodynamic forces). While some sea urchin species have tube foot stem tissues (tested in anesthetized individuals) that better resist breaking under higher loading rates, it remains unclear whether this improves the whole-animal adhesive performance in unanesthetized animals. To test whether the loading rate and previous hydrodynamic exposure influence the adhesive performance of the intertidal purple sea urchin, Strongylocentrotus purpuratus, we measured the strain rate dependence of the tube foot disc, tube foot stem, and whole-animal adhesion, using three sets of sea urchins with different environmental histories. In unanesthetized S. purpuratus, strain rate influenced tube foot disc tenacity, but not the tube foot stem or whole-animal adhesion. Rather, sea urchins collected more recently had higher whole-animal adhesion, and reliance on the tube foot disc and the tube foot stem varied among animal sets. These results further support the hypothesis that hydrodynamic loading maintains adhesive performance in sea urchins and highlight the importance of investigating strain rate at levels of organization that impact organismal performance in situ.
Adult grey-headed albatrosses breeding on Marion Island experience highly variable near-ground wind vectors that can result in crash landings, some of which are fatal. This study quantifies the combinations of airspeed and wind direction that can lead to loss of lift or the generation of downforce sufficient to cause such crashes. Using a previously developed three-dimensional grey-headed albatross body geometry, we conducted numerical simulations of this rigid geometry across a wide range of flight conditions defined by airspeed, angle of attack, and sideslip angle. Lift and aerodynamic efficiency (lift-to-drag ratio) are then evaluated to identify conditions under which insufficient lift is produced. Simulations show that for airspeeds below 10 m·s⁻¹, the generated lift is lower than the average weight of an adult grey-headed albatross, with peak aerodynamic efficiency occurring at an angle of attack of approximately 5°. While the geometry generates lift effectively under either strong crosswinds or downdrafts alone, their combination can produce substantial downforce. Given that albatrosses preferentially exploit crosswinds at the meso-scale, transient gusts combining crosswind and downdraft components may force birds into the ground, particularly during low-altitude nest departure, increasing the likelihood of fatal crash landings.
The expression of metazoan replication-dependent histone genes is controlled by the nuclear protein at the ataxia-telangiectasia locus (NPAT) and U7 small nuclear ribonucleoprotein particle (snRNP). NPAT activates transcription of histone genes during S-phase, whereas U7 snRNP is a multi-subunit endonuclease that cleaves the resultant transcripts at the 3' end, yielding mature histone mRNAs. In cycling cells, NPAT and U7 snRNP with its four unique components, U7 snRNA, Lsm10, Lsm11 and FLASH, are highly enriched in histone locus bodies (HLBs), the nuclear condensates formed near histone gene loci. Here, we show that in muscle and neural cells that have ceased to replicate their chromatin and permanently exited the cell cycle, HLBs are dismantled and NPAT, FLASH and Lsm11 are detected in the cytoplasm. This observation suggests that in postmitotic cells, NPAT and U7 snRNP become repurposed for functions unrelated to generating histone mRNAs. We identified a highly conserved region in Lsm11 that engages in various protein-protein interactions and likely acts as a universal platform that controls the assembly, localization and function of Lsm11 complexes, including U7 snRNP, during cell growth and differentiation. Since the assembly of U7 snRNP requires survival motor neuron, the protein mutated in spinal muscular atrophy, our results may provide a new perspective on the pathophysiology of this neuromuscular disorder.
Male-specific peripubertal DNA demethylation in the liver has been reported in mice. Here, we investigated whether it also occurs in rats, the influence of maternal obesity and whether DNA demethylation changes contribute to observed sex-specific effects of maternal obesity in offspring. Female rats were fed a high-fat, high-sugar 'cafeteria' (Caf) diet before mating with standard chow-fed males. The offspring liver methylome and transcriptome were examined. Body weight was higher in Caf-fed dams prior to mating, during gestation and at parturition. Male and female offspring from Caf-fed dams had lower birth weights but higher adult weights and adiposity than offspring from chow-fed dams. A comparison of DNA methylation in 3-week-old weaner males versus female siblings from chow-fed dams did not reveal the male-specific DNA demethylation that was previously reported in mice. However, strong maternal diet effects in male weaner offspring methylation were observed. A comparison of female weaners from chow- versus Caf-fed dams showed a range of differences, with 39% of differentially methylated regions (DMRs) having higher methylation in Caf offspring and 61% of DMRs having higher methylation in chow offspring. In stark contrast, 99% of maternal-diet-induced DMRs in male weaner offspring had higher methylation in offspring from Caf-fed dams. This suggests that maternal obesity induces widespread hypermethylation in the male offspring liver at weaning. However, a comparison with RNA sequencing data revealed limited transcriptional changes at this developmental stage or in adult offspring. While these data highlight how environmentally sensitive DNA methylation is in the male rodent perinatal period, these methylation changes may not be a major contributor to sex differences in developmentally programmed liver disease.
Do memories acquired through learning by insects in the larval stage persist throughout metamorphosis into adulthood? There are contradictory reports to answer this question. After a larval stage, holometabolous insects undergo pupation and metamorphosis, during which the body is substantially transformed. This remodeling also affects the brain: neuronal connections degrade, axons and dendrites prune and regrow, new neurons are generated, and new synaptic connections are established. Such drastic remodeling appears incompatible with reports from several insect species, including Drosophila melanogaster, suggesting that larval memories are maintained throughout metamorphosis and can be observed in the adult imago. We use aversive associative olfactory conditioning of wild-type and pruning-defective D. melanogaster larvae to revisit this long-standing and unresolved question. In contrast to previous reports, we found no evidence that memories acquired through classical aversive conditioning in the larval stage survive the neuronal remodeling processes during metamorphosis. Even when pruning and regrowth of larval Kenyon cells of the mushroom body, a central brain structure essential for associative learning, are largely prevented by genetic intervention, no maintenance of larval memories could be detected in the adult fly. We conclude that at least larval olfactory memories acquired through aversive classical conditioning are erased over the course of metamorphosis.
Repeated measures designs are a common experimental setup in biology, from ecology to biomedical sciences. Traditional statistical techniques for analyzing such data, such as repeated measures ANOVA (RMANOVA), have limitations that prevent them from being used to answer many questions relevant to measurements taken over time. Response feature analysis (RFA) techniques can provide an ideal means for answering those questions by analyzing data based on a creatively chosen summary function of the individual subject's data. The process and rationale for the RFA is laid out in a step-by-step manner and is demonstrated on an example of behavioral data from a rodent spinal cord injury study. In the original analysis, a difference in average functional score was only found at one timepoint early in the study. Using RFA, we show that the female rats reached 90% of their peak recovery 15.3 days (95% CI 1.73, 28.86 days) earlier than the male rats. RFA enables answering the important question 'do the two sexes differ in the speed at which they recover functional ability?', and, by way of demonstration, illustrates its flexibility in answering questions that traditional repeated measures analyses cannot.
Predators have evolved sophisticated behavioral patterns that enable efficient prey capture. The scale-eating cichlid Perissodus microlepis exhibits pronounced morphological asymmetry in mouth-opening direction, corresponding to behavioral laterality in attack direction. Given that recognizing and tracking the flank of prey are crucial for successful predation, we hypothesized that the morphological asymmetry also influences their spatial positioning relative to prey during attack initiation. To test this idea, we simultaneously tracked predator and prey movements using DeepLabCut and analyzed the predatory sequence. Not only strike direction but also the lateralized approach initiation position was correlated with mouth asymmetry. Additionally, trajectory analyses revealed two distinct predation strategies based on the predator's relative position to the prey at the start of the approach: a rear approach, whereby predators move in from behind and strike at close range with strong body flexion, and a side approach, whereby predators initiate from lateral positions at shorter distances and rush forward. Although rear and side attacks differ in their kinematics and strike timing, both strategies achieve high predation success by applying strong forces to the prey's flank. Overall, this study advances our understanding of the predation strategies of scale-eating cichlids, providing insights into their evolutionary significance.
