Biology subjects

Murphy, P.

Publications and source records attributed to Murphy, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Biological Signaling Pathways Associated with Late Tendon Development in Chicken Embryos and Their Dependence on Mechanical Stimulation

During embryonic and neonatal development, tendons exhibit rapid changes in mechanics and structure, including increases in collagen content, crosslinking, and fibril length. These functional and structural changes are partly dependent on mechanical stimulation. However, the specific mechanobiological mechanisms driving these changes remain poorly understood. The objective of this study was to identify the potential biological mechanisms and pathways driving tendon maturation into load-bearing materials using an embryonic chick model. Using bulk RNA sequencing, gene set enrichment analysis, and transcription factor analysis, we produced a transcriptomic profile of chick hindlimb tendons during late embryonic development (E16, E18, and E20) and tendons from rigid and flaccid paralysis-treated embryos. Additionally, we assessed the results from two differential expression analysis models to improve the replicability of our findings. Our results suggest that the largest transcriptomic response occurs between embryonic day 16 and 20, and that among paralysis conditions, rigid paralysis produces the greatest effect relative to controls. Across development, we found positive enrichment in processes related to mechanotransduction, extracellular matrix remodeling, cell adhesion, and contractility, and identified candidate pathways upregulated with development, including TGF-{beta}, PI3K-AKT-mTOR, Hippo, circadian rhythm, and focal adhesion signaling. In paralysis-treated embryos, our data suggest impairment in extracellular matrix remodeling and mechanotransduction events. Specifically, we found that both modes of paralysis are associated with a cellular stress response and downregulation of WNT signaling. Together, our findings identify candidate mechanotransduction pathways that are associated with late tendon maturation and altered by paralysis, which may inform tendon tissue engineering and regenerative approaches.

bioengineering↗

bioRxiv2026

CXCR4 coordinates adhesion, migration, and development of human NK cells

Natural killer (NK) cells undergo stepwise differentiation from multipotent progenitors within secondary lymphoid tissues. Despite the central importance of the tissue microenvironment in their development, little is known about cell-cell interactions that regulate human NK cell trafficking and maturation. Here, we identify the chemokine receptor CXCR4 and its lig- and CXCL12 as regulators of stromal-NK cell interactions required for NK cell maturation. We demonstrate that CXCR4 is expressed throughout human NK cell development in peripheral blood and tonsil, and CXCL12 is enriched in stromal niches containing developing NK cells. Pharmacologic blockade or genetic disruption of CXCR4 resulted in diminished adhesion to integrin ligands and high-resolution imaging demonstrated crosstalk between CXCR4 and integrins, providing a mechanistic basis for chemokine-dependent modulation of adhesion. Further, CXCR4 blockade resulted in altered contact-dependent motility on stromal cells and integrin ligands, with decreased stable stromal engagement and increased cell speed. Consistent with a requirement for these interactions, treatment with the CXCR4 antagonist plerixafor (AMD3100) impaired NK cell generation from CD34+ precursors. Analysis of NK cells from WHIM syndrome patients with CXCR4 gain-of-function mutations treated with plerixafor revealed similar defects in migration and adhesion, supporting the in-vivo relevance CXCR4-dependent regulation of NK cell adhesion and motility.

immunology↗

bioRxiv2025

Transcriptomic Profiles from Normal and Tumor Tissue Samples Reveal Distinct Venule Populations and Novel Tumor Endothelial Cell Markers in Breast Cancer

BackgroundThe breast tumor microenvironment (TME) is a complex milieu composed of many factors contributing to breast cancer (BC) heterogeneity and therapeutic resistance. Aberrant tumor vasculature in the TME limits nutrient and drug delivery, inhibits anti-tumor immunity, and contributes to a lack of cancer therapy efficacy. Utilizing publicly available scRNA-seq datasets, this study characterizes differences between normal breast and breast tumor endothelial cells (EC), provides insights into tumor endothelial cell subtypes, endothelial anergy, and identifies novel, tumor-specific vascular therapeutic targets. MethodsGene expression data from normal and breast tumor tissue samples were integrated, and the EC subset was extracted via canonical gene marker expression. The EC subset was clustered and evaluated for cell subtypes and differentially expressed genes (DEG). Normal EC (NEC) and tumor EC (TEC) markers were further assessed for correlation to bulk gene expression and patient survival outcomes in cBioPortal and Kaplan-Meier Plotter. Cell type gene expression specificity was evaluated in the 3CA single-cell RNA-seq datasets across multiple cancers. ResultsThis analysis revealed differences in NEC and TEC subtype populations. Breast NEC contained similar proportions of venule and capillary populations, while breast TEC demonstrated a majority of the venule subtype. Further, TEC venules were phenotypically distinct from the NEC venules. Consistent with endothelial anergy, suppression of the key adhesion protein SELE was noted, as well as several pro-inflammatory cytokines including IL6, CCL2, and CXCL8, likely downstream of aberrant NF-kB signaling. Differential gene expression analysis identified several TEC specific up-regulated genes compared to NEC, including CLEC14a, IGFBP4, EMCN, and ADM5. CLEC14a, EMCN, and ADM5 were further validated in the single-cell Curated Cancer Cell Atlas (3CA) to be highly specific to the endothelial cell clusters across multiple tumor types, while IGFBP4 was diversely expressed in endothelial, fibroblast, and some malignant cell types. ADM5, a novel tumor vascular marker, was enhanced in TEC venules and less so in arteriole or capillaries. High expression of ADM5 was associated with poor breast cancer patient survival in the basal PAM50 cancer subtype compared to normal and luminal subtypes. Further, across multiple cancer types, high ADM5 expression was associated with reduced patient survival in anti-PD1- and anti-CTLA4-treated patients but not in anti-PDL-treated patients. ConclusionsIntegration of single-cell RNA-seq data identified an anergic-like response in breast TEC and multiple, highly specific markers to TEC not found in normal breast tissue. CLEC14a and EMCN were validated as TEC markers, extending their annotation in breast TEC, and ADM5 identified as a novel TEC marker in breast and other cancers. Moreover, as ADM5 is associated with reduced patient overall survival, this data suggests that a better understanding of ADM5 and other TEC-specific response pathways may provide novel approaches to reactivate anergic TECs and lead to effective therapeutic interventions for cancer patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/661087v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@a81bf2org.highwire.dtl.DTLVardef@c2b983org.highwire.dtl.DTLVardef@216ab9org.highwire.dtl.DTLVardef@1e5bebb_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cancer biology↗

bioRxiv2025

Liver transplant rejection features memory cell priming, spatially differentiated networks and novel drug targets.

Acute T-cell-mediated transplant rejection, the earliest contributor to immunological graft failure responds variably to T-cell suppression. Early rejection can co-exist with donor-specific antibodies which also shorten graft survival, without affecting graft histology visibly. Here, during early liver transplant rejection, blood leukocytes manifest primed T-cells with heat shock protein (HSP) signaling and transcriptional programs for memory cell expansion. Corresponding biopsies demonstrate upregulated immune synapse and proteasomal genes in T-cell-infiltrated portal and central regions of the liver lobule. With donor-specific antibodies, the intervening intralobular region demonstrates a germinal-center-like allograft response with upregulated CD40, chemokine, T-follicular and complement signaling, which intensifies along the direction of sinusoidal blood flow from portal to central regions. Proteasomal and HSP90 inhibitors suppress donor-specific alloresponses of T- and B-cells in blood samples from patients with early rejection. The molecular injury spectrum of early rejection is complex, spatially differentiated and reveals novel early immunosuppressive strategies to extend graft survival.

immunology↗

bioRxiv2024

Architecture of the UBR4 complex, a giant E4 ligase central to eukaryotic protein quality control

Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Our cryo-EM analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3 MDa ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Structural data illustrate how UBR4 binds substrate and extends K48-specific ubiquitin chains. Importantly, efficient substrate targeting depends on both pre-ubiquitination and specific N-degrons, with KCMF1 acting as key substrate filter. Furthermore, we show that the architecture of the E4 megacomplex is conserved across eukaryotes but with species specific adaptations, allowing UBR4 to perform its precisely tuned quality-control function in diverse cellular environments.

biochemistry↗

bioRxiv2024

Genetic and Environmental Determinants of Streaming and Aggregation in Myxococcus xanthus

Under starvation conditions, a spot of a few million Myxococcus xanthus cells on agar will migrate inward to form aggregates that mature into dome-shaped fruiting bodies. This migration is thought to occur within structures called streams, which are considered crucial for initiating aggregation. The prevailing traffic jam model hypothesizes that intersections of streams cause cell crowding and jamming, thereby initiating the process of aggregate formation. However, this hypothesis has not been rigorously tested, in part due to the lack of a standardized, quantifiable definition of streams. To address this gap, we captured time-lapse movies and conducted fluorescent cell tracking experiments using wild-type and two motility-deficient mutant M. xanthus strains. By quantitatively defining streams and developing a novel stream detection mask, we show that streams are not essential for nascent aggregate formation, though they may accelerate the process. Moreover, our results indicate that streaming has a genetic component: disrupting only one of the two M. xanthus motility systems hinders stream formation. Together, these findings challenge the idea that stream intersections are required to drive aggregate formation and suggest that M. xanthus aggregation may be driven by mechanisms independent of streaming, highlighting the need for alternative models to fully explain aggregation dynamics.

microbiology↗

bioRxiv2024

Role of Non-Exponential Reversal times in Aggregation Models of Bacterial Populations

In this paper, we consider 1D agent-based and kinetic models of aggregation with reversals. In particular, we fit a Gamma distribution to represent the run times in myxobacteria and analyze numerically the importance of non-exponential reversal times. We demonstrate that non-exponential reversal times aid aggregation and result in tighter aggregates. We compare and contrast the behavior of agent-based and kinetic models, and also consider kinetic models with aggregation driven by chemotaxis. Thus, incorporating non-exponential reversal times into models of aggregation can be particularly important for reproducing experimental data, such as aggregate persistence and dispersal.

biophysics↗

bioRxiv2024

Convergence of orphan quality control pathways at a ubiquitin chain-elongating ligase

Unassembled and partially assembled subunits of multi-protein complexes have emerged as major quality control clients, particularly under conditions of imbalanced gene expression such as stress, aging, and aneuploidy. The factors and mechanisms that eliminate such orphan subunits to maintain protein homeostasis are incompletely defined. Here, we show that the UBR4-KCMF1 ubiquitin ligase complex is required for efficient degradation of multiple unrelated orphan subunits from the chaperonin, proteasome cap, proteasome core, and a protein targeting complex. Epistasis analysis in cells and reconstitution studies in vitro show that the UBR4-KCMF1 complex acts downstream of a priming ubiquitin ligase that first mono-ubiquitinates orphans. UBR4 recognizes both the orphan and its mono-ubiquitin and builds a K48-linked poly-ubiquitin degradation signal. The discovery of a convergence point for multiple quality control pathways may explain why aneuploid cells are especially sensitive to loss of UBR4 or KCMF1 and identifies a potential vulnerability across many cancers.

cell biology↗

bioRxiv2024

Embryo movement is required for limb tendon maturation

Following early cell specification and tenocyte differentiation at the sites of future tendons, very little is known about how tendon maturation into robust load-bearing tissue is regulated. Between embryonic day (E)16 and E18 in the chick, there is a rapid change in mechanical properties which is dependent on normal embryo movement. However, the tissue, cellular and molecular changes that contribute to this transition are not well defined. Here we profiled aspects of late tendon development (collagen fibre alignment, cell organisation and Yap pathway activity), describing changes that coincide with tissue maturation. We compared effects of rigid (constant static loading) and flaccid (no loading) immobilisation to gain insight into developmental steps influenced by mechanical cues. We show that YAP signalling is active and responsive to movement in late tendon. Collagen fibre alignment increased over time and under static loading. Cells organise into end-to-end stacked columns with increased distance between adjacent columns, where collagen fibres are deposited; this organisation was lost following both types of immobilisation. We conclude that specific aspects of tendon maturation requires controlled levels of dynamic muscle-generated stimulation. Such a developmental approach to understanding how tendons are constructed will inform future work to engineer improved tensile load-bearing tissues.

developmental biology↗

bioRxiv2024

PHARAOH: A collaborative crowdsourcing platform for PHenotyping And Regional Analysis Of Histology

Deep learning has proven to be capable of automating key aspects of histopathologic analysis, but its continual reliance on large expert-annotated training datasets hinders widespread adoption. Here, we present an online collaborative portal that streamlines tissue image annotation to promote the development and sharing of custom computer vision models for PHenotyping And Regional Analysis Of Histology (PHARAOH; https://www.pathologyreports.ai/). PHARAOH uses a weakly supervised active learning framework whereby patch-level image features are leveraged to organize large swaths of tissue into morphologically-uniform clusters for batched human annotation. By providing cluster-level labels on only a handful of cases, we show how custom PHARAOH models can be developed and used to guide the quantification of cellular features that correlate with molecular, pathologic and patient outcome data. Both custom model design and feature extraction pipelines are amenable to crowdsourcing making PHARAOH a fully scalable systems-level solution for the systematic expansion and cataloging of computational pathology applications.

bioinformatics↗

bioRxiv2023

Structural Determinants of Tendon Function During Development and Their Sensitivity to Mechanical Stimulation

The load-bearing capabilities of tendon are acquired during neonatal stages of development, characterized by an abrupt increase in multiscale mechanical properties. While prior work has identified numerous changes within the collagenous structure during these developmental periods, the primary structural elements that give rise to this abrupt mechanical functionality, and their mechanobiological sensitivity, remains unclear. To address this gap in knowledge, we leveraged a combination of ultrastructural imaging, biochemical/thermodynamic assays, multiscale mechanical testing, and shear lag modeling to probe the dynamic structure-function relationships and establish their sensitivity to mechanical stimulation during tenogenesis. Mechanical testing and modeling suggested that the rapid increase in multiscale mechanics can be explained by a increasing fibril length and intrafibrillar crosslinking. To test this, we inhibited collagen crosslinking during development and observed a drastic reduction in multiscale mechanical capabilities that was explained by a reduction in both fibril modulus and length. Using muscle paralysis to investigate mechanosensitivity, we observed a significantly impaired multiscale mechanical response despite small changes in fibril diameter and fibril area fraction. While there was no change in crosslinking density, there was a decrease in thermal stability with flaccid paralysis, and our shear-lag model suggested that flaccid paralysis produces a reduction in fibril length and intrafibrillar crosslinking. Together, these data suggest that both intrafibrillar crosslink formation and fibril elongation are critical to the formation of load-bearing capabilities in tenogenesis and are sensitive to musculoskeletal activity. These findings provide critical insights into the biological mechanisms that give rise to load-bearing soft tissue.

bioengineering↗

bioRxiv2023

Optogenetic Stimulation of Single Ganglion Cells in the Living Primate Fovea

Though the responses of the rich variety of retinal ganglion cells (RGCs) reflect the totality of visual processing in the retina and provide the sole conduit for those processed responses to the brain, we have much to learn about how the brain uses these signals to guide behavior. An impediment to developing a comprehensive understanding of the role of retinal circuits in behavior is the paucity of causal studies in the intact primate visual system. Here we demonstrate the ability to optogenetically activate individual RGCs with flashes of light focused on single RGC somas in vivo, with no evidence for activation of neighboring cells. The ability to selectively activate specific cells is the first step toward causal experiments that directly link retinal circuits to visual experience and behavior.

neuroscience↗

bioRxiv2023

Foveal RGCs develop abnormal calcium dynamics weeks after photoreceptor ablation

Objective or purposePhysiological changes in retinal ganglion cells (RGCs) have been reported in rodent models of photoreceptor (PR) loss but this has not been investigated in primates. By expressing both a calcium indicator (GCaMP6s) and an optogenetic actuator (ChrimsonR) in foveal RGCs of the macaque, we reactivated RGCs in vivo and assessed their response in the weeks and years following PR loss. DesignWe used an in vivo calcium imaging approach to record optogenetically evoked activity in deafferented RGCs in primate fovea. Cellular scale recordings were made longitudinally over a 10 week period following photoreceptor ablation and compared to responses from RGCs that had lost photoreceptor input more than two years prior. ParticipantsThree eyes received photoreceptor ablation, OD of a male Macaca mulatta (M1), OS of a female Macaca fascicularis (M2) and OD of a male Macaca fascicularis (M3). Two animals were used for in vivo recording, one for histological assessment. MethodsCones were ablated with an ultrafast laser delivered through an adaptive optics scanning light ophthalmoscope (AOSLO). A 0.5 s pulse of 25Hz 660nm light optogenetically stimulated RGCs, and the resulting GCaMP fluorescence signal was recorded using AOSLO. Measurements were repeated over 10 weeks immediately after PR ablation, at 2.3 years and in control RGCs. Main Outcome measuresThe calcium rise time, decay constant and sensitivity index of optogenetic mediated RGC were derived from GCaMP fluorescence recordings from 221 RGCs (Animal M1) and 218 RGCs (Animal M2) in vivo. ResultsFollowing photoreceptor ablation, the mean decay constant of the calcium response in RGCs decreased 1.5 fold (1.6{+/-}0.5 s to 0.6{+/-}0.3 s SD) over the 10 week observation period in subject 1 and 2.1 fold (2.5{+/-}0.5 s to 1.2{+/-}0.2 s SD) within 8 weeks in subject 2. Calcium rise time and sensitivity index were stable. Optogenetic reactivation remained possible 2.3 years after PR ablation. ConclusionsAltered calcium dynamics developed in primate foveal RGCs in the weeks after photoreceptor ablation. The mean decay constant of optogenetic mediated calcium responses decreased 1.5 - 2-fold. This is the first report of this phenomenon in primate retina and further work is required to understand the role these changes play in cell survival and activity.

physiology↗

bioRxiv2023

Cell behaviors underlying Myxococcus xanthus aggregate coarsening

The soil bacterium Myxococcus xanthus is a model organism with a set of diverse behaviors. These behaviors include the starvation-induced multicellular development program, in which cells move collectively to assemble multicellular aggregates. After initial aggregates have formed, some will disperse, with smaller aggregates having a higher chance of dispersal. Initial aggregation is driven by two changes in cell behavior: cells slow down inside of aggregates and bias their motion by reversing direction less frequently when moving towards aggregates. However, the cell behaviors that drive dispersal are unknown. Here we use fluorescent microscopy to quantify changes in cell behavior after initial aggregates have formed. We observe that after initial aggregate formation, cells adjust the bias in reversal timings by initiating reversals more rapidly when approaching unstable aggregates. Using agent-based modeling, we then show dispersal is predominantly generated by this change in bias, which is strong enough to overcome slowdown inside aggregates. Notably, the change in reversal bias is correlated with the nearest aggregates size, connecting cellular activity to previously observed correlations between aggregate size and fate. To determine if this connection is consistent across strains, we analyze a second M. xanthus strain with reduced levels of dispersal. We find that far fewer cells near smaller aggregates modified their bias. This implies that aggregate dispersal is under genetic control, providing a foundation for further investigations into the role it plays in the life cycle of M. xanthus. ImportanceUnderstanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacterium Myxococcus xanthus forms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregates size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions.

microbiology↗

bioRxiv2022

Genome-wide screening reveals metabolic regulation of translational fidelity

Translational quality control is critical for maintaining the accuracy of protein synthesis in all domains of life. Mutations in aminoacyl-tRNA synthetases and the ribosome are known to affect translational fidelity and alter fitness, viability, stress responses, neuron function, and life span. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified 30 nonessential genes that are critical for maintaining the fidelity of stop-codon readthrough. Most of these identified genes have not been shown to affect translational fidelity previously. Intriguingly, we show that several genes controlling metabolism, including cyaA and guaA, unexpectedly enhance stop-codon readthrough. CyaA and GuaA catalyze the synthesis of cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (GMP), respectively. Both CyaA and GuaA increase the expression of ribosomes and tRNAs, allowing aminoacyl-tRNAs to compete with release factors and suppress stop codons. In addition, the effect of guaA deletion on stop-codon readthrough is abolished by deleting prfC, which encodes release factor 3 (RF3). Our results suggest that nucleotide and carbon metabolism is tightly coupled with translational fidelity.

genetics↗

bioRxiv2022

Integrated analysis of Wnt signalling system component gene expression

Wnt signalling controls patterning and differentiation across many tissues and organs of the developing embryo via temporally and spatially restricted expression of multi-gene families encoding ligands, receptors, pathway modulators and intracellular components. Here we report an integrated analysis of key encoding genes in the 3D space of the mouse embryo across multiple stages of development. We applied a method for 3D/3D image transformation to map all gene expression patterns to a single reference embryo for each stage providing both visual analysis and volumetric mapping allowing computational methods to interrogate the combined expression patterns. We identify novel territories where multiple Wnt and Fzd genes are co-expressed and cross-compare all patterns, including all seven Wnt paralogous gene pairs. The comprehensive analysis allows regions in the embryo where no Wnt or Fzd gene expression is detected, and where single Wnt genes are uniquely expressed, to be revealed. This work provides insight into a level of organisation of the patterns not previously possible, as well as presenting a resource that can be utilised further by the research community for whole system analysis.

developmental biology↗

bioRxiv2022

WlzWarp: An Open-Source Tool for Complex Alignment of Spatial Data

BackgroundThe spatio-temporal organisation of many biological processes such as gene-expression and neuronal activity is critical to understanding the overall biological behaviour, phenotype and disease. This is especially true during embryonic development. During development spatial patterns of gene expression are key to segmentation, tissue differentiation and organ development. In situ techniques can reveal the activity of genes and the presence of proteins to a very high sub-cellular resolution but typically at high resolution only a few probes can be used on any sample therefore to compare many such patterns requires mapping of image-based data to a standard spatial framework. Once mapped those data can be collated, queried and analysed in purely spatial terms to reveal unknown combinatorial gene-activity that could not be discovered any other way. Mapping spatial data to image domains with systematic variation in shape and pose, such as embryos or elongated organs, presents special problems. Automated techniques available for more constrained systems can not deliver the mapping fidelity required to analyse these data therefore we have developed a manual editing tool, WlzWarp, for mapping 3D image data using the constrained distance transform (CDT) which uniquely can deliver the complex transforms required. ResultsWe have implemented a fully open-source tool (available on GitHub), WlzWarp to provide interactive complex spatial mapping of 3D image data. We have applied WlzWarp to map a set of gene-expression patterns in the developing mouse embryo that could not be mapped by any other technique. The transform procedure was tested by using multiple images of the same gene from independent samples and thereby testing the full end-to-end mapping process. WlzWarp is implemented in C++ using open-source packages Qt, Coin and SIMVoleon for cross-architecture compatibility. It has been developed under Linux but also tested on Mac OSX and MS Windows. ConclusionsWlzWarp is a freely available software tool for non-linear registration and alignment of complex 2D & 3D spatial patterns from one image to another or to an atlas model. It has been tested in the context of embryo data but can be used for any 3D or 2D image registration problem.

bioinformatics↗

bioRxiv2021

Mechanical Stimulation via Muscle Activity is Necessary for the Maturation of Tendon Multiscale Mechanics during Embryonic Development

During embryonic development, tendons transform into a hypocellular tissue with robust tensile load-bearing capabilities. Previous work suggests that this mechanical transformation is due to increases in collagen fibril length and is dependent on mechanical stimulation via muscle activity. However, the relationship between changes in the microscale tissue structure and changes in macroscale tendon mechanics is still unclear. Additionally, the specific effect of mechanical stimulation on the multiscale structure-function relationships of developing tendons is also unknown. Therefore, the objective of this study was to measure the changes in tendon mechanics and structure at multiple length scales during embryonic development with and without skeletal muscle paralysis. Tensile testing of tendons from chicken embryos was performed to determine the macroscale tensile modulus as well as the magnitude of the fibril strains and interfibrillar sliding with applied tissue strain. Embryos were also treated with either decamethonium bromide or pancuronium bromide to produce rigid or flaccid paralysis. Histology was performed to assess changes in tendon size, spacing between tendon subunits, and collagen fiber diameter. We found that the increase in the macroscale modulus observed with development is accompanied by an increase in the fibril:tissue strain ratio, which is consistent with an increase in collagen fibril length. Additionally, we found that flaccid paralysis reduced the macroscale tendon modulus and the fibril:tissue strain ratio, whereas less pronounced effects that were not statistically significant were observed with rigid paralysis. Finally, skeletal paralysis also reduced the size of collagen fibril bundles (i.e., fibers). Together, these data suggest that more of the applied tissue strain is transmitted to the collagen fibrils at later embryonic ages, which leads in an increase the tendon macroscale tensile mechanics. Furthermore, our data suggest that mechanical stimulation during development is necessary to induce structural and mechanical changes at multiple physical length scales. This information provides valuable insight into the multiscale structure-function relationships of developing tendons and the importance of mechanical stimulation in producing a robust tensile load-bearing soft tissue.

developmental biology↗