Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments (original) (raw)
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Involvement of long- and short-range signalling during early tendon development
Anatomy and Embryology, 1999
Tendons connect muscle to skeletal elements. Although tendons have been shown to originate from the lateral plate mesoderm, very little is known at the molecular level about how they are formed. We have found that two genes, Follistatin and Eph-A4, are expressed in regions associated with tendon formation in developing chick limbs. Follistatin is expressed near the tip of the digits and subsequently around the tendon, whereas Eph A4 transcripts were localized in a slightly more proximal region and later in the body of the tendon. Previous work has demonstrated that application of TGFβ1 or TGFβ2 to inter-digital regions or the removal of ectoderm in the foot plate induces ectopic cartilage formation, while removal of ectoderm or application of FGF to tips of developing digits leads to truncation. Here we show that TGFβ1 or removal of ectoderm is also able to induce the expression of both Eph-A4 and Follistatin and that manipulations that cause truncations affect these genes. Thus cartilage and tendon development appear to be coordinated. Ectopic application of recombinant human Follistatin, an antgaonist of certain TGFβ super-family proteins including Activin and Bmp-4, results in the loss of tendon, implicating signalling by TGFβ super-family in the development of tendon during chick embryogenesis. Signalling by TGFβ family members, antagonised by Noggin is known to regulate skeletal development. Thus we suggest that parallel pathways govern both skeletal and tendon patterning.
Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix
Development (Cambridge, England), 2015
Tendons and ligaments are extracellular matrix (ECM)-rich structures that interconnect muscles and bones. Recent work has shown how tendon fibroblasts (tenocytes) interact with muscles via the ECM to establish connectivity and strengthen attachments under tension. Similarly, ECM-dependent interactions between tenocytes and cartilage/bone ensure that tendon-bone attachments form with the appropriate strength for the force required. Recent studies have also established a close lineal relationship between tenocytes and skeletal progenitors, highlighting the fact that defects in signals modulated by the ECM can alter the balance between these fates, as occurs in calcifying tendinopathies associated with aging. The dynamic fine-tuning of tendon ECM composition and assembly thus gives rise to the remarkable characteristics of this unique tissue type. Here, we provide an overview of the functions of the ECM in tendon formation and maturation that attempts to integrate findings from develop...
Cellular and molecular maturation in fetal and adult ovine calcaneal tendons
Journal of Anatomy, 2014
Processes of development during fetal life profoundly transform tendons from a plastic tissue into a highly differentiated structure, characterised by a very low ability to regenerate after injury in adulthood. Sheep tendon is frequently used as a translational model to investigate cell-based regenerative approaches. However, in contrast to other species, analytical and comparative baseline studies on the normal developmental maturation of sheep tendons from fetal through to adult life are not currently available. Thus, a detailed morphological and biochemical study was designed to characterise tissue maturation during mid-(2 months of pregnancy: 14 cm of length) and late fetal (4 months: 40 cm of length) life, through to adulthood. The results confirm that ovine tendon morphology undergoes profound transformations during this period. Endotenon was more developed in fetal tendons than in adult tissues, and its cell phenotype changed through tendon maturation. Indeed, groups of large rounded cells laying on smaller and more compacted ones expressing osteocalcin, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were identified exclusively in fetal mid-stage tissues, and not in late fetal or adult tendons. VEGF, NGF as well as blood vessels and nerve fibers showed decreased expression during tendon development. Moreover, the endotenon of midand late fetuses contained identifiable cells that expressed several pluripotent stem cell markers [Telomerase Reverse Transcriptase (TERT), SRY Determining Region Y Box-2 (SOX2), Nanog Homeobox (NANOG) and Octamer Binding Transcription Factor-4A (OCT-4A)]. These cells were not identifiable in adult specimens. Ovine tendon development was also accompanied by morphological modifications to cell nuclei, and a progressive decrease in cellularity, proliferation index and expression of connexins 43 and 32. Tendon maturation was similarly characterised by modulation of several other gene expression profiles, including Collagen type I, Collagen type III, Scleraxis B, Tenomodulin, Trombospondin 4 and Osteocalcin. These gene profiles underwent a dramatic reduction in adult tissues. Transforming growth factor-b1 expression (involved in collagen synthesis) underwent a similar decrease. In conclusion, these morphological studies carried out on sheep tendons at different stages of development and aging offer normal structural and molecular baseline data to allow accurate evaluation of data from subsequent interventional studies investigating tendon healing and regeneration in ovine experimental models.
ACS Chemical Biology, 2014
Repairing damaged cartilage and tendons is a major challenge of regenerative medicine. There has been great progress in the past decade toward obtaining stem cells for regenerative purposes from a variety of sources. However, the development of procedures to direct and maintain the differentiation of progenitors into cartilage or tendon is still a hurdle to overcome in regenerative medicine of the musculoskeletal system. This is because connective tissues often lack stable phenotypes and retain plasticity to return to the initial stages of differentiation or to transdifferentiate into another connective tissue cell lineage. This makes it necessary to unravel the molecular basis that is responsible for the differentiation of connective tissue cell lineages. In this review, we summarize the investigations performed in the past two decades to unravel the signals that regulate the differentiation of skeletal cell progenitors into cartilage and tendons during embryonic limb development. The data obtained in those studies demonstrate that Tgfβ, BMP, FGF, and Wnt establish a complex signaling network that directs the differentiation of skeletal cell progenitors. Remarkably, in the embryonic digit model, the divergent differentiation of progenitors depends on the temporal coordination of those signals, rather than being specified by an individual signaling pathway. Due to its potential medical relevance, we highlight the importance of the coordinate influence of the Tgfβ and BMP pathways in the differentiation of cell progenitors into tendon or cartilage.
Histology and Ultrastructure of the Developing Superficial Digital Flexor Tendon in Rabbits
Anatomia Histologia Embryologia, 2008
This study was designed to investigate the developmental changes in the superficial digital flexor tendon (SDFT) of the white New Zealand rabbit, from 5 to 7 days pre-natal to 112 days post-natal (PN), at histological and ultrastructural levels. The tendons changed from being highly cellular with 13079 ± 2538 cell/mm2 and little directional organization to a longitudinally oriented, predominantly connective tissue structure with relatively few, mature tenocytes (2384 ± 365 cell/mm2). Fibrillogenesis was seen in isolated vacuoles of fibroblast cytoplasm during fetal life as well as after birth, but less frequently with increasing age. At the ultrastructural level, there was a progressive increase in the mean diameter of collagen fibrils with age throughout the population, until PN day 28. A bimodal distribution of collagen fibril size was first observed on PN day 56 while at day 112, the fibrils were fully differentiated and showed a multimodal size distribution. The development of elastic fibres preceded that of collagen fibres and accompanied progressively more marked sinusoidal crimping in collagen fibres on PN days 7 and 14. The tendons of older animals became less tightly crimped. The cells of the epi- and endotenon were less mature and relatively undifferentiated compared with the cells in the body of the tendon of the same age, which might explain their ability to initiate healing of tendon injuries in older animals. In conclusion, extensive changes were observed in tendons during growth and maturation, with diameters of collagen fibrils, tissue orientation and cellularity being the parameters affected, probably due largely to increased physical activity.
Developmental Dynamics, 2008
Tendon is one of the least understood tissues of the musculoskeletal system in terms of development and morphogenesis. Collagen fibrillogenesis has been the most studied aspect of tendon development, focusing largely on the role of matrix molecules such as collagen type III and decorin. While involvement of matrix molecules in collagen fibrillogenesis during chick tendon development is well understood, the role of growth factors has yet to be elucidated. This work examines the expression patterns of TGF-ß1, -ß2, and -ß3, and their receptors with respect to expression patterns of collagen type III, decorin, and fibronectin. We focus on the intermediate stages of tendon development in the chick embryo, a period during which the tendon micro-and macro-architecture are being established. Our findings demonstrate for the first time that TGF-ß1, -ß2, and -ß3 have distinct spatiotemporal developmental protein localization patterns in the developing tendon and strongly suggest that these isoforms have independent roles in tendon development.
Lineage Tracing of Resident Tendon Progenitor Cells during Growth and Natural Healing
PLoS ONE, 2014
Unlike during embryogenesis, the identity of tissue resident progenitor cells that contribute to postnatal tendon growth and natural healing is poorly characterized. Therefore, we utilized 1) an inducible Cre driven by alpha smooth muscle actin (SMACreERT2), that identifies mesenchymal progenitors, 2) a constitutively active Cre driven by growth and differentiation factor 5 (GDF5Cre), a critical regulator of joint condensation, in combination with 3) an Ai9 Cre reporter to permanently label SMA9 and GDF5-9 populations and their progeny. In growing mice, SMA9+ cells were found in peritendinous structures and scleraxis-positive (ScxGFP+) cells within the tendon midsubstance and myotendinous junction. The progenitors within the tendon midsubstance were transiently labeled as they displayed a 4-fold expansion from day 2 to day 21 but reduced to baseline levels by day 70. SMA9+ cells were not found within tendon entheses or ligaments in the knee, suggesting a different origin. In contrast to the SMA9 population, GDF5-9+ cells extended from the bone through the enthesis and into a portion of the tendon midsubstance. GDF5-9+ cells were also found throughout the length of the ligaments, indicating a significant variation in the progenitors that contribute to tendons and ligaments. Following tendon injury, SMA9+ paratenon cells were the main contributors to the healing response. SMA9+ cells extended over the defect space at 1 week and differentiated into ScxGFP+ cells at 2 weeks, which coincided with increased collagen signal in the paratenon bridge. Thus, SMA9-labeled cells represent a unique progenitor source that contributes to the tendon midsubstance, paratenon, and myotendinous junction during growth and natural healing, while GDF5 progenitors contribute to tendon enthesis and ligament development. Understanding the mechanisms that regulate the expansion and differentiation of these progenitors may prove crucial to improving future repair strategies.
Archives of Biochemistry and Biophysics, 1998
for the postfibril growth stage of collagen fibrillogenesis. The large number of isolated genes differentially Collagen fibril growth is a very rapid and abrupt expressed during the rapid phase of fibril growth reprocess, resulting in a 4-to 5-fold increase in fibril veals a fine and possibly tissue-specific control of filength between 16 and 18 days of chicken metatarsal brillogenesis. ᭧ 1998 Academic Press tendon development. This fibril growth is due to a post-Key Words: subtractive hybridization; chicken tendepositional fusion/association of preformed intermedon development; collagen fibril growth. diates, termed fibril segments. We propose that the regulated assembly of collagen fibrils from the segment intermediates is mediated by interactions of structural macromolecules. The cells could modulate this process by responding to cytokines and altering cell-Collagen stabilizes the structure of most organs and matrix signaling, transcription, and translation. To is the major component of tissues such as tendon, coridentify the genes involved in this process a subnea, dermis, and bone. In tendon, type I collagen is the tractive hybridization procedure was utilized. Genes most abundant collagen and is organized into fibrils of cell proliferation were excluded as major contributhat are organized into fibers (1, 2). The fibers are furtors to differential gene expression in avian tendon on ther organized into tissue-specific aggregates (i.e., the days 14 and 19 of development after analysis of BrdUr large bundles typical of tendon) (3, 4). Collagen fibril incorporation. The BrdUr incorporation studies reformation is a multistep process that includes molecuvealed little, if any, tendon fibroblast proliferation at lar assembly, fibril growth, and matrix assembly steps both stages. This suggested that observed alterations (5, 6). These steps overlap and occur throughout develin gene expression would be related to the pre-and opment.
Transcriptomic analysis of mouse limb tendon cells during development
Development, 2014
The molecular signals driving tendon development are not fully identified. We have undertaken a transcriptome analysis of mouse limb tendon cells that were isolated at different stages of development based on scleraxis (Scx) expression. Microarray comparisons allowed us to establish a list of genes regulated in tendon cells during mouse limb development. Bioinformatics analysis of the tendon transcriptome showed that the two most strongly modified signalling pathways were TGF-β and MAPK. TGF-β/SMAD2/3 gain-and loss-offunction experiments in mouse limb explants and mesenchymal stem cells showed that TGF-β signalling was sufficient and required via SMAD2/3 to drive mouse mesodermal stem cells towards the tendon lineage ex vivo and in vitro. TGF-β was also sufficient for tendon gene expression in late limb explants during tendon differentiation. FGF does not have a tenogenic effect and the inhibition of the ERK MAPK signalling pathway was sufficient to activate Scx in mouse limb mesodermal progenitors and mesenchymal stem cells.