Biology subjects

Werner, C.

Publications and source records attributed to Werner, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Soil viral response to drought disturbance and ecosystem recovery in an artificial rainforest

Anthropogenic climate change is driving increased temperature and altered precipitation patterns in tropical rainforests, yet the impact of severe drought on soil viral communities and their recovery capacity remains poorly understood. Soil viruses can exert strong controls on bacterial populations impacting microbially-mediated soil processes such as carbon cycling. At the same time, viruses are very sensitive to changes in the soil environment. We leverage meta-omics data from soils collected during a two-month drought and one-month recovery experiment carried in the Biosphere 2 Tropical Rainforest artificial biome. Our results indicate that soil viral community structure responds to small scale soil habitat heterogeneity more strongly than on overall changes in moisture. Additionally, the abundance of soil viruses was associated with a higher diversity of metabolites in the soil, suggesting viruses play a key role in soil biogeochemical processes. Altogether, our study suggests that soil viruses are an active part of tropical rainforest soil microbial communities and that is necessary to understand their response to drought.

microbiology↗

bioRxiv2026

13CO2 pulse labelling reveals species-specific alterations in carbon allocation and volatile organic compound emissions under heat stress

Temperate forests increasingly face extreme air temperature, but plant physiological responses, particularly alterations in carbon allocation or protection via volatile organic compound (VOC) emissions, remain poorly understood. We pulse-labelled well-watered saplings of Fagus sylvatica and Pseudotsuga menziesii in a controlled heat stress experiment with 13CO2 to quantify heat-induced shifts in CO2, VOC and C pool exchange, specifically analyzing compound-specific {delta}13C of terpenoids, water-soluble organic matter (WSOM) and dark respiration. Under heat stress, up to 50% of fresh assimilates were directed to maintenance respiration and 1-2% to VOC emissions, while net assimilation and water use efficiency decreased by 50-75% in both species. Heat directly affected metabolic processes and reduced turnover rates of fresh assimilates in F. sylvatica, but accelerated them in P. menziesii. Strong 13C labelling of some compounds, particularly acyclic ones, suggested increased de novo synthesis of specific terpenoids for heat stress protection. By tracing the fate of recently assimilated 13CO2 we demonstrate that heat stress reduces net carbon uptake and water use efficiency, disrupts turnover of C pools and increases carbon loss via respiration and de novo synthesis of specific VOCs, potentially diminishing net carbon uptake of forests under future heat extremes.

plant biology↗

bioRxiv2026

Early warning indicators for heat-induced mortality in temperate tree saplings

{middle dot} Globally, forest ecosystems face widespread mortality events. However, the independent impacts of distinct stressors, such as heat stress vs edaphic drought, remain poorly understood and physiological early warning indicators for tree mortality are urgently required. {middle dot} We exposed well-watered saplings of Fagus sylvatica, Pseudotsuga menziesii and Picea abies to summer heat waves and subsequent natural winter-desiccation. Physiological parameters (e.g. gas exchange, water uptake velocity via 2H labelling, and volatile organic compound emissions) were monitored throughout the growing season and survival was assessed regularly until subsequent spring to capture immediate and delayed mortality as a consequence of legacy effects. {middle dot} Heat exposure without soil water deficit, followed by winter desiccation, triggered species-specific mortality rates (51.8% F. sylvatica, 48.2% P. abies, 16.9% P. menziesii), with P. abies exhibiting significantly faster mortality response than the other species. Reduced water uptake, lower stomatal conductance, impaired photosynthetic efficiency, and altered VOC emissions distinguished non-surviving from surviving saplings months before visible damage in all three species. {middle dot} Heat stress drives mortality independent of edaphic drought, with sub-lethal physiological indicators detectable up to 10 months before visual signs. These early warning indicators could enable damage detection before lethal thresholds are crossed, offering new strategies for mitigating climate change-driven forest decline.

ecology↗

bioRxiv2026

The rhizosphere of Picea abies is a hotspot of terpenoid production

Forest soils are an important source of volatile organic compounds (VOCs), yet little is known about how different tree species influence soil VOC emissions and the role of rhizosphere processes in mediating VOC release form roots. We analysed soil VOC emissions from the soil surface and bulk soil as well as from roots with intact rhizosphere and washed roots of Picea abies and Fagus sylvatica. Tree saplings were grown on natural forest soil, and VOC emissions and gas exchange of soils and roots were measured under controlled conditions using online gas analysers integrated into an automated system. To assess the contribution of rhizosphere soil and microbial communities to root VOC emissions, roots were analysed (a) without washing, preserving the rhizosphere, (b) water-washed, and (c) ethanol-washed (70 vol%) to minimize microbial contributions. Species-specific VOC emission patterns were observed in both soils and roots. P. abies showed higher total emission rates and a more diverse, terpenoid-rich VOC profile dominated by -pinene, {beta}-pinene, {beta}-myrcene, and -phellandrene than F. sylvatica. Notably, these differences were evident not only at the soil surface but also in root and litter free bulk soil. Root washing further revealed that the rhizosphere is a hotspot of terpenoid production in P. abies, with significantly higher monoterpenoid emissions from unwashed roots than from water or ethanol-washed roots. This study demonstrates how tree species shape net soil VOC emissions, potentially leading to cascading effects on atmospheric VOC concentrations, and highlights the importance of the rhizosphere in regulating belowground VOC production.

plant biology↗

bioRxiv2026

Structural cell biology by mega-expansion microscopy

Structural characterization of macromolecular assemblies within intact cells remains one of the central challenges in cell biology. While cryo-electron tomography provides unparalleled structural information, its applicability is limited by sample thickness, imaging throughput, and accessibility. Fluorescence microscopy offers molecular specificity and compatibility with intact biological specimens but has so far lacked the spatial resolution required to visualize cellular ultrastructure. Here we introduce Mega-expansion microscopy (Mega-ExM), a fluorescence imaging approach that enables structural visualization of whole cells using conventional confocal microscopes. Mega-ExM combines iterative hydrogel expansion with whole-proteome NHS-dye labeling and post-expansion immunostaining to achieve tunable expansion factors of up to [~]1,500-fold while preserving ultrastructure. At expansion factors of 40-260x, Mega-ExM resolves centrioles, mitochondrial cristae, protein-dense domains within mitochondrial cristae consistent with respiratory-chain supercomplexes, the synaptonemal complex, and nuclear pore complexes (NPCs) with high fidelity. Particle averaging of [~]200x expanded NPCs yields reconstructions with a structural resolution of [~]35 [A], approaching what cryo-electron tomography has achieved for selected protein assemblies. By combining molecular specificity, large imaging volumes, and nanoscale structural resolution on conventional fluorescence microscopes, Mega-ExM establishes a broadly accessible platform for in situ structural biology.

biophysics↗

bioRxiv2026

Synergistic effect of heat and drought on leaf VOC emissions and root exudates in Norway spruce saplings

Compound droughts, i.e. the co-occurrences of heat and drought, represent a serious challenge for temperate forest trees leading to significant losses in forest biomass. We studied the physiological response of Norway spruce (Picea abies) saplings to heat and drought individually, and in combination. Continuous measurements of leaf gas exchange and VOC emission allowed us to identify fast-response reactions, while discrete VOC and root exudate samplings added qualitative information on compositional changes. Additionally, we used 13CO2 and 2H2O label pulses to investigate C-allocation and root water uptake in response to stress. Heat as well as drought reduced assimilation rates in the saplings, whereas transpiration, leaf VOC emission and root exudation rates increased in response to heat. Drought alone increased VOC emission but decreased exudation rates. Combined heat and drought triggered an amplified response in both processes despite negative net CO2 assimilation rates. Label incorporation showed compromised water uptake capacity of drought-stressed plants and illustrated de novo C-allocation to VOC emission and root exudates. The results point at the high susceptibility of Norway spruce saplings to drought and heat. Combined stress resulted in synergistic responses in VOC emissions and root exudates, showing the detrimental effect of compound droughts on Norway spruce. HighlightIn this study, we found synergistic effects of heat and drought on carbon losses from leaf VOC emission and root exudates despite negative assimilation rates in Norway spruce saplings.

plant biology↗

bioRxiv2026

Seasonal dynamics and sun/shade heterogeneity of leaf gas exchange and VOC emissions inside a tall temperate forest canopy

Leaf gas exchange is the key driver of forest carbon uptake and directly determines forest carbon sink activity. Additionally, plants release a variety of biogenic volatile organic compounds (VOCs) acting as stress signals of trees. However, continuous hourly resolved measurements of leaf gas exchange and VOC emissions in tall tree canopies are challenging and remain scarce. To this end, we developed a sophisticated in-situ leaf gas exchange measurement system with 24 cuvettes deployed on mature Fagus sylvatica (n=3) and Pseudotsuga menziesii (n=3) individuals in a mixed temperate forest. We additionally measured sap flux density (Js), radial growth and tree water deficit (TWD) to gain a holistic picture of seasonal leaf and stem water and carbon flux dynamics during the summer of 2024. During midsummer, we found a gradual reduction of stomatal conductance (gs) and VOC emissions of sun, but not shade branchlets of P. menziesii in response to moderate atmospheric and edaphic drying. Decreased gs led to a downregulation of transpiration (E), Js, and carbon isotope discrimination accompanied by an increase in TWD and intrinsic water used efficiency. Leaf gas exchange of shade branchlets remained unaffected due to microclimatic buffering effects. Contrarily, sun leaves of F. sylvatica, profited from sunny midsummer conditions and increased leaf gas exchange, whereas shade leaves benefitted from more diffuse light during early summer exhibiting similar carbon assimilation, transpiration and VOC emissions as sun leaves. For both species we found a clear time lag of four to five hours between maximum leaf and stem water fluxes and a delay of up to 20 hours for the recovery of TWD, highlighting the role of stem water reserves. Pronounced seasonal and diurnal differences of leaf gas exchange, stem water fluxes and VOC emissions showed, that continuous data are essential to better understand variability of ecosystem flux dynamics.

plant biology↗

bioRxiv2025

Fabrication of Short Polymeric μFibers as Building Blocks for Anisotropic High-Throughput Compatible 3D Tissue Models

Modeling the 3D microenvironment and cell growth of natively anisotropic human tissues in vitro constitutes a significant challenge in tissue engineering and biofabrication. Short polymeric fibers gain growing attention in this field due to their applicability as pipettable or injectable anisometric building blocks in 3D hydrogel-based cell culture systems or bioinks. However, the production of monodisperse short fibers with high production rates suitable for screening remains challenging. In this study, short, quasi-monodisperse, magneto-responsive, fluorescent poly({varepsilon}-caprolactone) {micro}fibers with variable dimensions in the micrometer range are produced in a scalable, semi-continuous two-step fabrication process combining controlled wet-dry spinning with subsequent cryosectioning. Influences of the spinning process parameters on fiber properties and process features, as well as boundary spinning conditions and upscaling potential, are explored using Design of Experiments approaches. Further, magnetic alignment of the {micro}fibers in a weak magnetic field and incorporation of nile red as fluorescent dye for facile analysis in 3D are demonstrated. Implementation of aligned {micro}fibers into a hydrogel-based 3D vasculogenesis model, produced in a high-throughput automated manner, is shown to stimulate oriented cell growth. This highlights the potential of our {micro}fibers as guiding elements inside tissue and disease models and their suitability for automated high-throughput applications.

bioengineering↗

bioRxiv2025

Continuous chlorophyll fluorescence measurements trace sudden cold spell effects on photosynthetic efficiency in a temperate mixed forest

Air temperature extremes and fluctuations are expected to become more frequent and have been shown to affect the physiological functioning of temperate forests across Europe. The exposure to sudden cold spells during summer combined with high light intensities can lead to photoinhibition of photosystem II (PSII) and thereby reduce photosynthetic efficiency. In this study, we aimed to analyse the dynamics of photoinhibition as well as related protection mechanisms of tall tree canopies during such cold spells. Therefore, we continuously assessed leaf level chlorophyll fluorescence (ChlF) and ecosystem carbon fluxes by eddy covariance in a temperate mixed forest in southern Germany during growing season 2024. While the deciduous, broadleaved F. sylvatica indicated chronic photoinhibition as a response to sudden cold spells, the evergreen, coniferous P. menziesii showed higher tolerance to low air temperatures. Both species increased thermal energy dissipation at PSII during cold spells indicating the activation of protective mechanisms. Likewise, both species exhibited mainly the sustained form of non-photochemical quenching (NPQs) as a reaction to chilling temperatures and high light intensities which maintained elevated during the recovery phases. However, the dynamics of upregulation of photoprotection and recovery processes differed between the two tree species. Furthermore, the integration of ecosystem carbon exchange and continuous leaf level ChlF measurements gave valuable insights into the photosynthetic dynamics of the mixed forest canopy. This study emphasises the importance of the assessment of dynamic responses to future climate impacts on forest ecosystem on different scales.

ecology↗

bioRxiv2025

Eavesdropping roots: Fagus sylvatica detects belowground stress signals from conspecific and heterospecific (Picea abies) neighbors, triggering increased shoot VOC emissions

O_LIVolatile organic compounds (VOCs) facilitate aboveground plant communication, but belowground signaling remains less understood. C_LIO_LIThis study explored root-root interactions between Picea abies and Fagus sylvatica saplings in monospecific (Fagus-Fagus) and heterospecific (Picea-Fagus) pairs (n=6), excluding shoot-level VOC communication. Sender plants were treated with jasmonic acid to simulate herbivory and labeled with 13CO2 and 15NH4NO3 to trace nutrient transfer in a split-root design. VOC emissions and gas exchange were measured over ten days using PTR-TOF-MS and 13CO2-spectroscopy and 13C and 15N were analyzed in roots and shoots via EA-IRMS. C_LIO_LIOur findings reveal, that (i) JA treatment induced strong de novo terpenoid emissions from P. abies and enhanced emissions of oxygenated VOCs and benzenoids from F. sylvatica, (ii) F. sylvatica receiver plants responded similarly to JA-treated neighbors, indicating belowground signaling, and (iii) responses of receiver plants were more pronounced in the heterospecific treatment. Furthermore, formic acid emissions from soils increased following JA treatment, suggesting altered soil microbial activity. Isotopic analysis revealed C exudation into the rhizosphere and N transfer to receiver plants. C_LIO_LIThese results suggest that belowground signaling enables early priming of herbivore-induced defenses in neighboring plants, and that the response intensity is modulated by species identity. C_LI

plant biology↗

bioRxiv2025

Cells dynamically adapt their nuclear volumes and proliferation rates during single to multicellular transitions

Tumour development and progression are associated with biophysical alterations that manifest across multiple spatial scales, from the subcellular to multicellular tissue scale. While cells dynamically regulate their biophysical properties like volumes and mechanics in dependence of cell state and function, it is unclear how these properties are controlled in the dense multicellular environment of a developing tumour. Here, we quantified cell and nuclear volumes of single cancer cells, while they grew into multicellular tumour spheroids within well-defined, tuneable biohybrid polymer hydrogels. We quantitatively showed that the formation of multicellular structures is associated with marked reductions of cellular and nuclear volumes, cell cycle delays as well as cell mechanical alterations, and that these changes are coupled. Single-to-multicellular transitions coincided with a drastic decrease in median nuclear volumes by up to 60%, as well as overall cell volume decrease. Nuclear volume decrease could not be explained by compression due to confining microenvironments. Instead, cell cycle adaptions were one significant contributor, with smaller-sized G1 cells accumulating in growing clusters, an effect that was reversed by CDK1 inhibition. Another contributor was nuclear volume decrease in cells within clusters that was associated with higher mass density and stiffness and could be abrogated upon cell release from clusters. In turn, multicellular-to-single cell transitions that happened in cells that invaded from a tumour spheroid into the surrounding matrix, were accompanied by nuclear volume increases and cell softening. Taken together, our study provides insights into how cells dynamically adapt their cellular/nuclear volumes, cell cycle progression and mechanics in dependence of the multicellular state.

biophysics↗

bioRxiv2025

Water stable isotopes reveal the ecohydrological importance of stemflow for mature and juvenile European beech

O_LIStemflow of forest trees can contribute a significant fraction of water to forests water fluxes; however, it is still unclear if and to what extent trees use stemflow for water supply and how much stemflow is lost by percolating below the root zone. C_LIO_LIWe applied deuterium-enriched stemflow equivalent to a throughfall depth of 23 mm to adult Fagus sylvatica trees to trace stemflow through the soil and trees. We continuously measured in-situ water stable isotope compositions in soil and xylem water and destructively sampled xylem water in the crowns of adult labelled (n=18), unlabelled F. sylvatica (n=15) and unlabeled Picea abies (n=9), complemented by destructive xylem water sampling of neighboring juvenile F. sylvatica (n=45). C_LIO_LIStemflow water supported 3.9 - 14.0% of daily sap flux of adult labelled F. sylvatica trees. In the soil, deuterium-enriched stemflow was detectable at a max. of 0 - 0.40 m to the labelled tree. However, unlabeled juvenile trees within a distance of [~]2 m showed label water uptake, indicating rooting into soil compartments affected by stemflow label. C_LIO_LIWe demonstrate the importance of stemflow as a water source for both adult and neighboring juvenile F. sylvatica, strongly profiting from stemflow infiltration. C_LI

plant biology↗

bioRxiv2025

Spatio-temporal plasticity of root exudation in three temperate tree species: effects of season, site and soil characteristics

Root exudation provides a constant carbon input to the rhizosphere and is therefore a very important factor in shaping this hotspot of biological activity. Nonetheless, root exudation data and its spatio-temporal plasticity is scarce. This study provides insights into compound-specific root exudation in three temperate tree species in two seasons (late spring and late summer) and two soil compartments (forest floor and the top mineral soil), including the effect of soil chemistry. At four sites with differing mean annual temperature and soil phosphorus level, root exudates were sampled using an in-situ cuvette-based system and analysed by gas chromatography-mass spectrometry. We found seasonally and spatially varying site- and species-specific exudation patterns. While the seasonal pattern was similar among species and sites, with higher exudation rates in late spring, soil compartment-specific exudation depended on species and site. Acer pseudoplatanus tended to exude more into the mineral soil at warmer sites, while Picea abies exuded more in the mineral soil at all sites. Exudation by Fagus sylvatica was independent from the soil compartment. Significant correlation between exchangeable soil cations and specific compounds exuded by F. sylvatica and P. abies were found. Exudation of specific compounds in F. sylvatica increased with the concentration of exchangeable Mg, Al and Fe, whereas exudation rates in P. abies decreased with most base cations concentration, while sugar exudation increased with the exchangeable non-base cations Al and Fe. These results demonstrate that root exudation is dynamically adjusted to the species-specific nutritional needs governed by site, season and soil characteristics.

plant biology↗

bioRxiv2025

AngleCam V2: Predicting leaf inclination angles across taxa from daytime and nighttime photos

O_LIUnderstanding how plants capture light and maintain their energy balance is crucial for predicting how ecosystems respond to environmental changes. By monitoring leaf inclination angle distributions (LIADs), we can gain insights into plant behaviour that directly influences ecosystem functioning. LIADs affect radiative transfer processes and reflectance signals, which are essential components of satellite-based vegetation monitoring. Despite their importance, scalable methods for continuously observing these dynamics across different plant species throughout day-night cycles are limited. C_LIO_LIWe present AngleCam V2, a deep learning model that estimates LIADs from both RGB and near-infrared (NIR) night-vision imagery. We compiled a dataset of over 4,500 images across 200 globally distributed species to facilitate generalization across taxa. Moreover, we developed a method to simulate pseudo-NIR imagery from RGB imagery to enable an efficient training of a deep learning model for tracking LIADs across day and night. The model is based on a vision transformer architecture with mixed-modality training using the RGB and the synthetic NIR images. C_LIO_LIAngleCam V2 achieved substantial improvements in generalization compared to AngleCam V1 (R2 = 0.62 vs 0.12 on the same holdout dataset). Phylogenetic analysis across 100 genera revealed no systematic taxonomic bias in prediction errors. Testing against leaf angle dynamics obtained from multitemporal terrestrial laser scanning demonstrated the reliable tracking of diurnal leaf movements (R2 = 0.61-0.75) and the successful detection of water limitation-induced changes over a 14-day monitoring period. C_LIO_LIThis method enables continuous monitoring of leaf angle dynamics using conventional cameras, enabling applications in ecosystem monitoring networks, plant stress detection, interpreting satellite vegetation signals, and citizen science platforms for global-scale understanding of plant structural responses. C_LI

ecology↗

bioRxiv2025

Resolving protein organization in cells with nanometer resolution

Advances in super-resolution microscopy have outpaced molecular labeling methods, impeding nanometer-scale imaging of endogenous multiprotein complexes in cells. To overcome these limitations, we developed an expansion microscopy (ExM) approach based on double-homogenized hydrogels that enables direct stochastic optical reconstruction microscopy (dSTORM) of 7-8-fold expanded immunolabeled samples. The resulting [~]4-fold increase in effective immunolabeling density resolved the 8 nm distance between neighboring -tubulin molecules in microtubules and the polyhedral lattice in clathrin-coated pits with nanometer precision in cells. Two-color Ex-dSTORM further revealed the molecular organization of RIM scaffolding protein and Munc13-1, an essential synaptic vesicle priming protein, in ring-like structures with diameters of 40-45 nm at the presynapse in hippocampal neurons. Our results demonstrate that Ex-dSTORM resolves the molecular organization of endogenous multiprotein complexes with nanometer spatial resolution in genetically unmodified cells. Thus, it provides a versatile method for the investigation of molecular protein distributions in their physiologically relevant context. One-Sentence SummaryThe optimized combination of dSTORM and double TREx ExM resolves previously unresolvable protein assemblies in cells

biophysics↗

bioRxiv2025

Scale-Specific Viscoelastic Characterization of Hydrogels: Integrated AFM and Finite Element Modeling

Viscoelastic hydrogels mimic the dynamic mechanical properties of native extracellular matrices, making them essential for biomedical applications. However, characterizing their scale-dependent mechanical properties remains challenging, despite their critical influence on cell-material interactions and biomaterial performance. Here, we present an integrated experimental-computational approach to quantify and model the viscoelastic behavior of interpenetrating polymer network hydrogels across micro- and macro-scales. Atomic force microscopy-based stress relaxation tests revealed that microgels exhibit rapid, localized relaxation, while macroscopic bulk gels displayed prolonged relaxation dominated by poroelastic effects. Finite element simulations accurately replicated experimental conditions, enabling the extraction of key parameters: instantaneous elastic modulus, relaxation modulus, and relaxation time constant. We further developed a novel analytical model to predict viscoelastic parameters from experimental data with minimal error (< 5%), significantly streamlining characterization. Our findings highlight the necessity of scale-specific mechanical analysis and provide a robust platform for designing biomaterials with tailored viscoelasticity for tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2025

Chiral monoterpene dynamics of shoots and roots of Norway spruce in response to drought

Although chiral monoterpenes emitted by plants above- and belowground shape the chemical landscape of many ecosystems, their biosynthesis and emissions, especially in response to drought, are poorly understood. We imposed a 6-week drought on two-year old, potted saplings of Norway spruce and analysed chiral monoterpene emissions and tissue concentrations from needles and roots. Isotopically labelled pyruvate was used to compare tissue-specific contributions of de novo synthesis to chiral monoterpene concentrations. While de novo synthesis of (-)--pinene and both enantiomers of limonene was apparent in needle emissions, no label was incorporated in roots. Drought reduced chiral monoterpene emissions to 30% of control levels, but increased needle and root tissue concentrations by 150 and 230%, respectively. Aboveground monoterpene concentrations were dominated by (-)-limonene, whereas belowground concentrations mainly consisted of the (-)-enantiomers of -pinene, {beta}-pinene, {beta}-phellandrene and camphene. Chiral composition in needles shifted in response to drought but remained stable in roots. We conclude that chiral monoterpene composition is tissue-specific and likely related to tissue-specific functioning. Instead of being passively emitted from storage pools, our results suggest active control mechanisms regulating chiral monoterpene emissions under drought conditions. Our findings imply important ramifications for understanding the regulation of emissions in relation to storage pools and plant-environmental interactions. Summary StatementEmissions of chiral monoterpenes and their composition is tissue-specific and regulated independent from storage pools in Picea abies. Chiral monoterpene ratios shift aboveground in response to drought, but are not affected belowground.

plant biology↗

bioRxiv2025

Mechanical control of tissue growth during limb regeneration

The axolotl is a highly regenerative species, capable of restoring full limbs, regardless of the amputation site. However, the regeneration rate is adjusted with the plane of amputation along the proximo-distal (PD) axis, leading to equivalent regeneration times regardless of the extent of tissue removal. We hypothesized that this phenomenon could be partly explained by differences in tissue mechanical properties. In this work, we describe tissue growth mathematically and evaluate cell cycle parameters of regenerating limbs amputated at different levels along the PD axis, demonstrating a linear correlation between the cell cycle length and the amputation site during early regeneration phases. We show as well, that blastema cells require their endogenous context to retain such proliferation differences. We measured mechanical properties in regenerating limbs with in vivo optical and standard indentation-based techniques and demonstrated that distal blastema cells are stiffer than proximal ones. Accordingly, we demonstrated that axolotl cells decrease their proliferation with increased extracellular matrix stiffness in vitro. Next, we evaluated the activity of the mechanotransducers YAP/TAZ in vivo by using a GTIIC-based reporter line combined with target gene expression data, which indicated that their activity peaks during the blastema stage, with higher activity after proximal amputations. Hence, our findings strongly suggest a mechanical dependence for the position-dependent regulation of cell proliferation during axolotl limb regeneration, where YAP/TAZ likely plays a role in the mechanotransduction mechanism.

biophysics↗