Biology subjects

Shukunami, C.

Publications and source records attributed to Shukunami, C..

6 recordsLinked to original sources

bioRxiv2026

A Col22a1-EGFP reporter reveals dynamic three-dimensional organization of the myotendinous junction

The myotendinous junction (MTJ) is a specialized extracellular matrix-rich interface that transmits contractile force generated by skeletal muscle to tendon, but its three-dimensional organization and dynamics remain poorly characterized. Here, we generate a Col22a1-EGFP knock-in mouse that enables direct visualization and quantitative analysis of MTJs from embryonic development to adulthood. Tissue-clearing and three-dimensional imaging reveal extensive variation in MTJ morphology and spatial distribution among skeletal muscles and between myofiber types. During embryonic MTJ formation, COL22A1-EGFP undergoes a striking transition from intracellular puncta within developing myofibers to extracellular accumulation at myofiber tips, associated with polarized Golgi organization. We further identify complex tendon protrusions that emerge during postnatal growth and connect tendon bodies with subsets of MTJs. These interfaces contain morphologically distinct Scx-lineage cells that express Col22a1, identifying a specialized tendon-associated population at the MTJ. Finally, muscle unloading and overload differentially alter COL22A1-EGFP signal intensity and area, respectively, demonstrating that the reporter provides a quantitative readout of MTJ responses to altered mechanical conditions. Thus, Col22a1-EGFP reveals previously unrecognized spatial, developmental, and adaptive features of the muscle-tendon interface and provides an experimental platform for investigating MTJ biology in vivo.

developmental biology↗

bioRxiv2025

Hox - Meis-relayed topographical genetic switch underlies cardiopharyngeal neural crest diversification, revealed by multimodal analysis

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks (GRNs) underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and GRN architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2-Sox9-Scx GRN defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

developmental biology↗

bioRxiv2025

Functional impact of pathogenic Runt domain mutations in Runx2 in vivo: Insights into the skeletal and dental anomalies of cleidocranial dysplasia

Runt-related transcription factor 2 (RUNX2) is essential for skeletogenesis, and mutations in its gene cause cleidocranial dysplasia (CCD), an autosomal dominant skeletal disorder. The evolutionarily conserved 128-amino acid Runt homology domain (RHD) of human RUNX2 is essential for DNA binding and heterodimerization, and serves as a mutation hotspot associated with severe CCD phenotypes. To elucidate the functional impact of pathogenic RHD mutations in vivo, we generated two novel mouse lines: one carrying a missense mutation, c.695G>A (p.R232Q) (Runx2m/+), corresponding to the human RUNX2 c.674G>A (p.R225Q), and the other harboring a frameshift mutation, c.697_698delGA (p.E233TfsTer9) (Runx2112/+), causing a premature stop codon. Homozygous Runx2m/mand Runx2112/112 mice lacked membranous ossification, whereas heterozygous Runx2m/+ and Runx2112/+ mice displayed typical CCD-like skeletal features, including an open anterior fontanelle and clavicle hypoplasia. Unexpectedly, heterozygotes carrying pathogenic mutations in RHD developed an accessory root-like protrusion at the furcation of three-rooted maxillary first molars, representing a previously unrecognized dental phenotype during root development. Dual luciferase assays revealed impaired transactivation of the p.R232Q mutant Runx2 on the osteocalcin enhancer/promoter. Wild-type Runx2 was robustly expressed in osteoblasts and hypertrophic chondrocytes during bone formation, but the mutant Runx2 exhibited reduced expression in hypertrophic chondrocytes and partially impaired nuclear localization, resulting in arrested osteoblast and chondrocyte maturation. Our mutant mouse model provides a valuable in vivo platform to study CCD pathogenesis, mechanisms of tooth root furcation, and therapeutic interventions targeting dysfunctional RHD.

genetics↗

bioRxiv2023

Sclerostin modulates the degree of mineralization and the stiffness profile of the fibrocartilaginous enthesis for mechanical tissue integrity

Fibrocartilaginous entheses consist of four graded tissue layers including tendon, the unmineralized and mineralized fibrocartilage, and subchondral bone with varying degrees of stiffness. Here we examined the functional role of sclerostin that is expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of the unmineralized fibrocartilage and parallel replacement of epiphyseal hyaline cartilage by bone, the unmineralized fibrocartilage re-expanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin in the bottom of the mineralized fibrocartilage adjacent to subchondral bone. In Scx deficient mice with less mechanical loading due to defects of the Achilles tendon, the number of sclerostin+ fibrochondrocytes was significantly reduced in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, coding for sclerostin, caused increased mineral density in the mineralized zones of the fibrocartilaginous enthesis. Atomic force microscopy analysis revealed the higher stiffness of fibrocartilage. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of the fibrocartilaginous enthesis.

developmental biology↗

bioRxiv2022

Cardiac neural crest lineage diversity and underlying gene regulatory networks revealed by multimodal analysis

Neural crest cells (NCCs), a multipotent stem cell population, contribute to cardiac development as a source of the outflow septum, vascular smooth muscle and semilunar valves. However, genetic programs underlying lineage diversification of cardiac NCCs remain largely unknown. Using single-cell (sc) and spatial transcriptomics, we demonstrate multiple NCC subpopulations with distinct gene expression signatures; smooth muscle(-like), non-muscle mesenchymal, and Schwann cell progenitor/melanoblast-like cells. Integrative scRNA-seq and scATAC-seq analyses predict lineage trajectories starting from immature NCCs, which bifurcate into smooth muscle(-like) and non-muscle mesenchymal lineages in association with hierarchical transcription factor networks. Combinatory analyses with Cre-mediated genetic lineage tracing characterize intermediate NCCs at the bifurcation as Sox9+/Scx+ tendon and cartilage progenitor-like cells with genetic programs, some of which are common to skeletal tissues whereas others are unique to cardiac NCCs. These findings provide a basis for understanding the roles of NCCs in cardiac development and pathogenesis particularly associated with calcification.

developmental biology↗

bioRxiv2021

Scx-positive tendon cells are required for correct muscle patterning

The elaborate movement of the vertebrate body is supported by the precise connection of muscle, tendon and bone. Each of the >600 distinct skeletal muscles in the human body has unique attachment sites; however, the mechanism through which muscles are reproducibly attached to designated partner tendons during embryonic development is incompletely understood. We herein show that Screlaxis-positive tendon cells have an essential role in correct muscle attachment in mouse embryos. Specific ablation of Screlaxis-positive cells resulted in dislocation of muscle attachment sites and abnormal muscle bundle morphology. Step-by-step observation of myogenic cell lineage revealed that post-fusion myofibers, but not migrating myoblasts, require tendon cells for their morphology. Furthermore, muscles could change their attachment site, even after the formation of the insertion. Our study demonstrated an essential role of tendon cells in the reproducibility and plasticity of skeletal muscle patterning, in turn revealing a novel tissue-tissue interaction in musculoskeletal morphogenesis. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

developmental biology↗