Biology subjects

Millay, D.

Publications and source records attributed to Millay, D..

10 recordsLinked to original sources

bioRxiv2026

Single nucleus RNA sequencing of juvenile dermatomyositis skeletal muscle identifies altered angiogenic signaling

Juvenile dermatomyositis (JDM) is a chronic multisystem vasculopathy and inflammatory myopathy characterized by proximal muscle weakness, distinct rash, and risk of complications such as calcinosis cutis, skin ulceration, and mortality. Molecular insight from diagnostic muscle biopsy histology is limited, and the mechanistic pathoetiology of JDM remains poorly defined. We used single nuclei transcriptomics to assess muscle samples from patients with newly diagnosed treatment-naive JDM. As a control, we assessed muscle samples from patients with congenital (nemaline) myopathy (CM), a non-inflammatory disorder. A total of 25,794 high quality nuclei were analyzed and clustered into various muscle-resident or infiltrating cellular populations. JDM tissue was characterized by an enriched interferon (IFN) response signature across endothelial, stromal, and immune cell compartments. Endothelial and perivascular populations showed increased inflammatory and angiogenic programs. Intercellular communication inference analysis identified dysregulated vascular endothelial growth factor (VEGF)-related signaling involving endothelial, stromal, and myonuclear populations as a possible mechanism for myonuclear-driven modulation of the muscle microvasculature. Spatial RNA in situ hybridization supported increased expression of selected IFN responsive and angiogenesis signaling genes in JDM tissue. Collectively, these data provide a cell type-resolved view of treatment-naive JDM muscle and highlight vascular and IFN pathways for follow-up in larger cohorts.

genomics↗

bioRxiv2025

mRNA transcription in skeletal muscle drives growth and determines nuclear accretion

Multinucleation of skeletal muscle cells (myofibers) is a determinant of size and fundamental for function. While it is established that myofibers need to accrue adequate numbers of nuclei for optimal growth, the molecular circuitry linking myonuclei to growth and why myofibers need additional nuclei remains unknown. We found that growth is still possible after restriction of nuclear content in myofibers and this was associated with increased levels of RNA Polymerase II (Polr2a) leading to elevated mRNA content. Through development of a genetic mouse model where endogenous Polr2a is upregulated in myofibers, we established that increased transcriptional output is sufficient to drive functional growth of myofibers. Notably, we discovered that Polr2a overexpression curtails the need for additional nuclei for myofiber growth. These data reveal a previously neglected driver of functional muscle growth and highlight that increasing Polr2a-mediated transcription from the vast numbers of nuclei within myofibers could be leveraged to combat muscle wasting conditions.

cell biology↗

bioRxiv2025

Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

Transforming growth factor {beta} (TGF{beta}) is a secreted growth factor that is sequestered to the extracellular matrix (ECM) as a latent complex. In adult disease TGF{beta} release in the heart transforms fibroblasts into a differentiated state that synthesizes more ECM. However, it is not known how TGF{beta} functions in the early developing heart to impact resident fibroblasts. Here, we observe that deletion of the Tgfb1, Tgfb2, and Tgfb3 genes (TGF{beta} ligands) from cardiomyocytes in the early developing heart results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and altered ECM content. Early postnatal hearts from Tgfb1/2/3 cardiomyocyte-deleted mice are dysmorphic and cardiac fibroblasts have incorrect activity and produce inappropriate ECM with reduced stiffness. Gene expression profiling of hearts from myocyte-specific Tgfb1/2/3 deleted mice reveal defects in both cardiomyocyte and fibroblast maturation with ectopic expression of multiple skeletal muscle-specific genes beginning at embryonic day 17.5 and progressing with age. However, cardiomyocyte-specific deletion of TGF{beta} receptors I/II encoding genes (Tgfbr1/2) or Smad2/3 encoding genes (Smad2/3) do not recapitulate this phenotype suggesting that TGF{beta} directly programs early heart fibroblast development that in turn specifies cardiomyocyte maturation. Importantly, Col1a2-/-;Col6a2-/- mice with defective cardiac ECM stiffness, mice lacking cardiomyocyte Itgb1 with reduced ECM load sensing, and Tcf21-/- embryos at E17.5 lacking cardiac fibroblasts each fail to generate the same pathologic ECM program with ectopic cardiomyocyte differentiation observed with Tgfb1/2/3 myocyte-specific deletion. These and additional results indicate that TGF{beta} generated by cardiomyocytes in the embryonic heart mediates fibroblast differentiation that co-evolves the ECM environment that in turn programs cardiomyocyte maturation to establish their identity.

developmental biology↗

bioRxiv2024

The multimodal transcriptional response of denervated skeletal muscle involves alterations in cholesterol homeostasis impacting muscle size

The development and maintenance of the neuromuscular junction (NMJ) requires reciprocal signals between the nerve terminals and the multinucleated skeletal muscle fiber (myofiber). This interaction leads to highly specialized transcription in the sub-synaptic or NMJ myonuclei within mature myofibers leading to clustering of acetylcholine receptors (AChRs). Here, we utilized single-nucleus RNA sequencing (snRNA-seq) to delineate the transcriptional response of myonuclei to denervation. Through snRNA-seq on skeletal muscle from two independent mouse models of denervation, sciatic nerve transection and amyotrophic lateral sclerosis, we identify a multimodal transcriptional response of NMJ-enriched genes and an alteration in cholesterol homeostasis in both slow and fast myofibers. Gramd1, a family of genes involved in non-vesicular cholesterol transport, are enriched at the NMJ in innervated muscle and upregulated in both models of denervation by the NMJ and extrasynaptic myonuclei. In vivo gain and loss of function studies indicate that NMJ-enriched Gramd1 genes regulate myofiber sizes independent of an obvious impact on AChR clustering. We uncovered a dynamic transcriptional response of myonuclei to denervation and highlight a critical role for cholesterol transport to maintain myofiber sizes.

cell biology↗

bioRxiv2023

Lineage tracing of newly accrued nuclei in skeletal myofibers uncovers distinct transcripts and interplay between nuclear populations

Multinucleated skeletal muscle cells have an obligatory need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in syncytial cells that already harbor hundreds of nuclei. To begin to answer this long-standing question, we utilized nuclear RNA-sequencing approaches and developed a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the myogenic environment to which they fuse. Moreover, accrual of new nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-inducing stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclear populations influence each other to maintain optimal functional growth.

cell biology↗

bioRxiv2023

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (Dys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

cell biology↗

bioRxiv2021

The epigenetic landscape in purified myonuclei from fast and slow muscles

Muscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the functional properties of the two muscles each with a distinct regulatory program involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles are also regulated by different sets of transcription factors; e.g. in soleus binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SOX1 binding sites were found to be overrepresented. In addition, novel factors for muscle regulation such as MAF, ZFX and ZBTB14 were identified.

physiology↗

bioRxiv2020

Fibroblast fusion to the muscle fiber regulates myotendinous junction formation

Vertebrate muscles and tendons are derived from distinct embryonic origins yet they must interact in order to facilitate muscle contraction and body movements. How robust muscle tendon junctions (MTJs) form to be able to withstand contraction forces is still not understood. Using techniques at a single cell resolution we reexamined the classical view of distinct identities for the tissues composing the musculoskeletal system. We identified fibroblasts that have switched on a myogenic program and demonstrate these dual identity cells fuse into the developing muscle fibers along the MTJs facilitating the introduction of fibroblast-specific transcripts into the elongating myofibers. We suggest this mechanism resulting in a hybrid muscle fiber, primarily along the fiber tips, enables a smooth transition from muscle fiber characteristics towards tendon features essential for forming robust MTJs. We propose that dual characteristics of junctional cells could be a common mechanism for generating stable interactions between tissues throughout the musculoskeletal system.

developmental biology↗

bioRxiv2020

Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers

While the majority of cells contain a single nucleus, cell types such as trophoblasts, osteoclasts, and skeletal myofibers require multinucleation. One advantage of multinucleation can be the assignment of distinct functions to different nuclei, but comprehensive interrogation of transcriptional heterogeneity within multinucleated tissues has been challenging due to the presence of a shared cytoplasm. Here, we utilized single-nucleus RNA-sequencing (snRNA-seq) to determine the extent of transcriptional diversity within multinucleated skeletal myofibers. Nuclei from mouse skeletal muscle were profiled across the lifespan, which revealed the emergence of distinct myonuclear populations in postnatal development and their reactivation in aging muscle. Our datasets also provided a platform for discovery of novel genes associated with rare specialized regions of the muscle cell, including markers of the myotendinous junction and functionally validated factors expressed at the neuromuscular junction. These findings reveal that myonuclei within syncytial muscle fibers possess distinct transcriptional profiles that regulate muscle biology.

cell biology↗

bioRxiv2017

Myomerger Induces Fusion Of Non-Fusogenic Cells And Is Required For Myoblast Fusion

Despite the importance of cell fusion for mammalian development and physiology, the factors critical for this process remain to be fully defined1. This lack of knowledge has severely limited our ability to reconstitute cell fusion, which is necessary to decipher the biochemical mechanisms driving plasma membrane merger. Myomaker (Tmem8c) is a muscle-specific protein required for myoblast fusion2,3. Expression of myomaker in fibroblasts drives their fusion with myoblasts, but not with other myomaker-fibroblasts, highlighting the requirement of additional myoblast-derived factors for fusion. Here, we demonstrate that Gm7325, named myomerger, induces the fusion of myomaker-expressing fibroblasts. Cell mixing experiments reveal that while myomaker renders cells fusion-competent, myomerger induces fusogenicity. Thus, myomaker and myomerger confer fusogenic activity to normally non-fusogenic cells. Myomerger is skeletal muscle-specific and only expressed during developmental and regenerative myogenesis. Disruption of myomerger in myoblast cell lines through Cas9-mutagenesis generated non-fusogenic myocytes. Genetic deletion of myomerger in mice results in a paucity of muscle fibers demonstrating a requirement for myomerger in normal muscle formation. Myomerger deficient myocytes exhibit an ability to differentiate and harbor organized sarcomeres, however remain mono-nucleated. These data identify myomerger as a fundamental myoblast fusion protein and establishes a system that begins to reconstitute mammalian cell fusion.

developmental biology↗