Biology subjects

Galloway, J. L.

Publications and source records attributed to Galloway, J. L..

5 recordsLinked to original sources

bioRxiv2025

Decoding menopause-induced tissue fibrosis using pan-tissue network inference

Menopause drives fibrotic remodeling and consequent tissue dysfunction across multiple organs, yet the tissue-conserved versus tissue-specific mechanisms underlying this phenomenon remain poorly defined. Here, we employed a network inference framework to uncover how menopause triggers coordinated shifts in intercellular signaling cascades that promote fibrosis. We leveraged publicly archived single-cell RNA-sequencing data from the liver, lung, pancreas, and skeletal muscle of ovariectomized and control mice. Given the central role of immune cells in orchestrating inflammation and fibrosis, we focused our analysis on immune cell populations. Using canonical markers of immune cells, we annotated major cell types and reconstructed cell-cell interaction networks to map transcriptional responses to hormonal shifts. Network analysis revealed pervasive reshaping of intercellular signaling common to all tissues evaluated in the setting of menopause. Within this immune-centered framework, we found that estrogen-responsive macrophages consistently function as major signaling hubs across all tissues evaluated, exhibiting extensive interactions with myofibroblasts--key drivers of extracellular matrix production and fibrotic remodeling. Notably, these shared signaling patterns were not detectable using conventional differential gene expression analysis, which revealed minimal overlap in gene-level responses in macrophages across tissues. In addition to conserved patterns, we identified tissue-specific interaction networks that reflect unique immune adaptations to hormonal loss. As an example, natural killer cells acted as a signaling hub in muscle-specific patterns, suggesting their direct contribution to menopausal skeletal muscle adaptation. Tissue-specific patterning was also evident in the liver, lung, and pancreas, where other immune cell types, such as CD8+ T cells and endothelial cells, functioned as prominent signaling hubs, indicating diverse remodeling of the immune microenvironment. The network approach introduced here represents a systems-level framework for mapping multicellular network rewiring following hormonal depletion and highlights conserved immune-stromal modules as potential therapeutic targets to prevent menopause-associated dysfunction.

systems biology↗

bioRxiv2024

Dynamic transcriptional and epigenetic changes define postnatal tendon growth

Tendons are dynamic structures that efficiently transmit forces and enable movement. From birth, tendons undergo dramatic changes from a principally cellular tissue to a hypocellular one characterized by a dense and highly ordered extracellular matrix. During this time, tendon cells change morphology from rounded to stellate in appearance and their proliferative rates decline. There is also significant expansion and maturation of the extracellular matrix (ECM) as tendons grow in length and diameter and alter their biomechanical properties to sustain increased physical activities. Surprisingly, for such an important stage of tendon maturation, we understand very little about the transcriptional and epigenetic regulators that direct these processes. Here, we present a roadmap of genes that are differentially regulated during the early neonatal and postnatal time period. We find differentially expressed genes fall into specific transcriptional modules, representing expression increases, decreases, or gene sets undergoing dynamic changes over postnatal time. By pairing our transcriptomic data with epigenetic data, we were able to perform an integrative analysis of the datasets and further define modules with highly correlated changes in gene expression and chromatin accessibility. From this analysis, several new pathways emerge. Among them, we focus on Yap1, a transcriptional co-activator of the Hippo signaling pathway. We observe accessible regions near to differentially expressed genes, containing motifs for TEAD, the transcription factor that binds Yap to regulate transcription. Conditional loss of Yap1 at postnatal stages results in altered expression of Col1a1 and disrupted matrix organization and density, suggesting that Yap is important for refining tendon ECM maturation. Together, our analyses identify a regulator of matrix maturation and provides a rich dataset with which to interrogate transcriptional networks and pathways during this poorly understood time in tendon growth.

developmental biology↗

bioRxiv2023

Endogenous Tenocyte Activation Underlies the Regenerative Capacity of Adult Zebrafish Tendon

Tendons are essential, frequently injured connective tissues that transmit forces from muscle to bone. Their unique highly ordered, matrix-rich structure is critical for proper function. While adult mammalian tendons heal after acute injuries, endogenous tendon cells, or tenocytes, fail to respond appropriately, resulting in the formation of disorganized fibrovascular scar tissue with impaired function and increased propensity for re-injury. Here, we show that unlike mammals, adult zebrafish tenocytes activate upon injury and fully regenerate the tendon. Using a full tear injury model in the adult zebrafish craniofacial tendon, we defined the hallmark stages and cellular basis of tendon regeneration through multiphoton imaging, lineage tracing, and transmission electron microscopy approaches. Remarkably, we observe that the zebrafish tendon can regenerate and restore normal collagen matrix ultrastructure by 6 months post-injury (mpi). We show that tendon regeneration progresses in three main phases: inflammation within 24 hours post-injury (hpi), cellular proliferation and formation of a cellular bridge between the severed tendon ends at 3-5 days post-injury (dpi), and re-differentiation and matrix remodeling beginning from 5 dpi to 6 mpi. Importantly, we demonstrate that pre-existing tenocytes are the main cellular source of regeneration. Collectively, our work debuts the zebrafish tendon as one of the only reported adult tendon regenerative models and positions it as an invaluable comparative system to identify regenerative mechanisms that may inspire new tendon injury treatments in the clinic.

developmental biology↗

bioRxiv2022

A quiescent resident progenitor pool is the central organizer of tendon healing

A tendons ordered extracellular matrix (ECM) is integral for transmitting force and highly prone to injury. How tendon cells, or tenocytes, embedded within this dense ECM mobilize and contribute to healing is unknown. Here, we identify a specialized Axin2+ population in mouse and human tendons that remains latent in homeostasis yet initiates the healing response and serves as a major source of tendon progenitors. Axin2+ tenocytes readily expand in vitro and express stem cell markers. In vivo, Axin2+-descendants are major functional contributors to repair: Axin2+ tenocytes proliferate, acquire injury responsive states, and re-adopt a tenocyte fate post-injury. Specific loss of Wnt secretion in Axin2+ cells alters their progenitor identity, disrupts their activation upon injury, and severely compromises any healing response. Our work highlights an unusual paradigm, wherein quiescent Axin2+ tenocytes self-regulate their identity and mobilization upon injury and provide the key initiating signal to organize a tendon-wide healing response.

cell biology↗

bioRxiv2018

A robust method for RNA extraction and purification from a single adult mouse tendon

BackgroundMechanistic understanding of tendon molecular and cellular biology is crucial towards furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.\n\nMethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between 3-5 months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.\n\nResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons.\n\nDiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RNA-seq and RT-qPCR. We show this can reduce biological variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues as well as precious human samples.

molecular biology↗