The proteasome subunit psmb1 is essential for craniofacial cartilage maturation and morphogenesis.

Craniofacial dysmorphisms are among the most common birth defects. Proteasome mutations frequently result in craniofacial dysmorphisms, including lower jaw malformations; however, the underlying mechanisms are unknown. Here, we used a zebrafish proteasome subunit ß 1 (psmb1) mutant to define the cellular mechanisms underlying proteasome mutation-induced craniofacial dysmorphisms. psmb1 mutants exhibited a flattened ceratohyal and smaller Meckel's and palatoquadrate cartilages. Ceratohyal flattening was a result of failed chondrocyte convergent extension, accompanied by reduced numbers of chondrocytes in the lower jaw due to defects in chondrocyte differentiation. Morphogenesis of craniofacial muscles and tendons was similarly perturbed. psmb1 mutants lacked the hyohyal muscles, and craniofacial tendons were shortened and disorganized. We additionally identified a critical period for proteasome function in craniofacial development, specifically during chondrocyte and muscle differentiation. psmb1 overexpression in sox10+ cells of mutant embryos rescued both cartilage and tendon phenotypes but induced only a partial rescue of the muscle phenotype, indicating that psmb1 was required in both tissue-autonomous and nonautonomous fashions during craniofacial development. Overall, our work demonstrates that psmb1 is required for craniofacial cartilage, tendon, and muscle differentiation and morphogenesis.

Formation of tendinous intersections in the human fetal rectus abdominis.

Previous studies have poorly described the initial development process of the tendinous intersections of the rectus abdominis muscle (RAM). The present study aimed to observe the formation of tendinous intersections in the RAM during the early fetal period using diffusion tensor imaging (DTI). Fifteen human fetal specimens (crown-rump length [CRL]: 39.5-93.7 mm) were selected. Three-dimensional measurements revealed that Zone-4 (i.e., the zone between the pubic symphysis and the caudal base of the umbilical ring in the RAM) had a smaller width and was thicker than Zone-1 and Zone-2 (i.e., the zones between the costal arch and the cranial base of the umbilical ring) and Zone-3 (i.e., the zone at the umbilical ring). Characteristics of tendinous intersections in the RAM during the early fetal period were assessed according to number, size, type, laterality, and sex. The mean number of tendinous intersections on both sides was 3.1 (range: 2.0-4.0), and 21% of specimens had only two tendinous intersections, which was higher than that reported in previous adult studies. The present data suggest that the formation of tendinous intersections was still in progress in specimens with two tendinous intersections in the RAM and that the third tendinous intersection was formed in Zone-2. Ordinal logistic regression via generalized estimating equations revealed that the odds for a higher type of tendinous intersections in Zone-1 and Zone-2 were significantly higher than those in Zone-4 (adjusted odds ratio: 14.85, 8.84). The odds for the presence of incomplete types (tendinous intersections that could not completely transverse the RAM) in Zone-3 were significantly higher than those in Zone-1 (adjusted odds ratio: 7.4). The odds for missing tendinous intersections in Zone-4 were significantly higher than those in Zone-1 (adjusted odds ratio: 20.5). These zonal differences in the formation of tendinous intersections were consistent with those observed in previous adult studies. In this study, DTI detected tendinous intersections in a sample with a CRL of 45.8 mm (approximately 11 weeks of gestation), which is earlier than that in previous histological findings, indicating that the RAM does not have mature tendinous intersections until the 17th week of gestation. In conclusion, DTI could detect the premature differentiation of tendinous intersection formation. Our data may aid in elucidating the developmental processes of tendinous intersections in the RAM.

Growing stylohyoideus muscle insertion to the hyoid bone with special reference to its topographical relation to the intermediate tendon of digastricus muscle: A histological study using human fetuses.

BACKGROUND: In adults, the intermediate tendon of digastricus muscle usually runs along the medial or lateral side of the stylohyoideus muscle insertion. To provide a better understanding of the variations, we examined the topographical anatomy of the muscle and tendon in fetuses. METHODS: We examined histological sections from six early-term, 26 mid-term and six near-term fetuses (approximately 8-9, 12-18 weeks and 25-33 weeks). RESULTS: At early-term, an initial sheath of intermediate tendon of digastricus muscle received the stylohyoideus muscle at the superior aspect. The muscle and tendon was distant from the hyoid. At mid-term, near the insertion to the hyoid greater horn, the stylohyoideus muscle consistently surrounded more than 2/3 of the intermediate tendon circumference. In contrast, we found no near-term specimen in which the stylohyoideus muscle surrounded the intermediate tendon. The multilayered tendon sheath was fully developed until near-term and connected to the body of hyoid by an intermuscular septum between the thyrohyoideus muscle and one or two of suprahyoid muscles. Therefore, the hyoid insertion of the styloglossus muscle was a transient morphology at mid-term. CONCLUSION: The stylohyoideus muscle insertion was appeared to move from the tendon sheath to the hyoid greater horn and, until near-term, return to the tendon sheath. A fascia connecting the tendon sheath to the body of hyoid was strengthened by the suprahyoid and infrahyoid muscles. The latter muscles seemed to regulate fixation/relaxation of the intermediate tendon to the hyoid. The stylohyoideus muscle slips sandwiching the intermediate tendon might be a rare morphology.

Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments.

BACKGROUND: The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. RESULTS: We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. CONCLUSION: Taken together, we discovered that Fgf9 may play an influential role in muscle-bone cross-talk during embryonic and postnatal development.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation.

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.

Development and maintenance of tendons and ligaments.

Tendons and ligaments are fibrous connective tissues vital to the transmission of force and stabilization of the musculoskeletal system. Arising in precise regions of the embryo, tendons and ligaments share many properties and little is known about the molecular differences that differentiate them. Recent studies have revealed heterogeneity and plasticity within tendon and ligament cells, raising questions regarding the developmental mechanisms regulating tendon and ligament identity. Here, we discuss recent findings that contribute to our understanding of the mechanisms that establish and maintain tendon progenitors and their differentiated progeny in the head, trunk and limb. We also review the extent to which these findings are specific to certain anatomical regions and model organisms, and indicate which findings similarly apply to ligaments. Finally, we address current research regarding the cellular lineages that contribute to tendon and ligament repair, and to what extent their regulation is conserved within tendon and ligament development.

The twisted structure of the fetal calcaneal tendon is already visible in the second trimester.

INTRODUCTION: The progress in morphological science results from the greater possibilities of intra-pubic diagnosis and treatment of congenital disabilities, including the motor system. However, the structure and macroscopic development of the calcaneal tendon have not been investigated in detail. Studies on the adult calcaneal tendon showed that the calcaneal tendon is composed of twisted subtendons. This study aimed to investigate the internal structure of the fetal calcaneal tendon in the second trimester. MATERIALS AND METHODS: Thirty-six fetuses fixed in 10% formaldehyde were dissected using the layer-by-layer method and a surgical microscope. RESULTS: The twisted structure of the calcaneal tendon was revealed in all specimens. The posterior layer of the calcaneal tendon is formed by the subtendon from the medial head of the gastrocnemius muscle. In contrast, the anterior layer is formed by the subtendon from the lateral head of the gastrocnemius muscle. The subtendon from the soleus muscle constitutes the anteromedial outline of the calcaneal tendon. The lateral outline of the calcaneal tendon is formed by the subtendon originating from the medial head of the gastrocnemius muscle. In contrast, the medial outline is formed by the subtendon from the soleus muscle. In most of the examined limbs, the plantaris tendon attached to the tuber calcanei was not directly connected to the calcaneal tendon. CONCLUSIONS: The twisted structure of the subtendons of the fetal calcaneal tendon is already visible in the second trimester and is similar to that seen in adults.

Loss of Smad4 in the scleraxis cell lineage results in postnatal joint contracture.

Growth of the musculoskeletal system requires precise coordination between bone, muscle, and tendon during development. Insufficient elongation of the muscle-tendon unit relative to bone growth results in joint contracture, a condition characterized by reduction or complete loss of joint range of motion. Here we establish a novel murine model of joint contracture by targeting Smad4 for deletion in the tendon cell lineage using Scleraxis-Cre (ScxCre). Smad4ScxCre mutants develop a joint contracture shortly after birth. The contracture is stochastic in direction and increases in severity with age. Smad4ScxCre mutant tendons exhibited a stable reduction in cellularity and a progressive reduction in extracellular matrix volume. Collagen fibril diameters were reduced in the Smad4ScxCre mutants, suggesting a role for Smad4 signaling in the regulation of matrix accumulation. Although ScxCre also has sporadic activity in both cartilage and muscle, we demonstrate an essential role for Smad4 loss in tendons for the development of joint contractures. Disrupting the canonical TGFß-pathway in Smad2;3ScxCre mutants did not result in joint contractures. Conversely, disrupting the BMP pathway by targeting BMP receptors (Alk3ScxCre/Alk6null) recapitulated many features of the Smad4ScxCre contracture phenotype, suggesting that joint contracture in Smad4ScxCre mutants is caused by disruption of BMP signaling. Overall, these results establish a model of murine postnatal joint contracture and a role for BMP signaling in tendon elongation and extracellular matrix accumulation.

Examination of the Annular Tendon (Annulus of Zinn) as a Common Origin of the Extraocular Rectus Muscles: 2. Embryological Basis of Extraocular Muscles Anomalies.

Purpose: Many reports have described anomalous connections of the superior rectus (SR) with other extraocular rectus muscles, in which additional heads of the other three rectus muscles likely provided the connections. We examined how these connections are established during fetal development. Methods: We analyzed paraffin-embedded horizontal sections from 25 late-stage fetuses. Horizontal sections are best suited for understanding the mediolateral relationships of muscle origins. Results: We confirmed a common tendinous origin of the lateral rectus (LR), inferior rectus (IR) and medial rectus (MR) muscles that was separated from the SR origin. Notably, eight fetuses (32%) had tendinous or muscular connections between the SR and other rectus muscles that had one of four morphologies: (a) a thin tendon from the SR to the common tendon of the three rectus muscles (2 fetuses), (b) a thin tendon to the LR (one fetus), (c) a thin tendon to the inferior rectus muscle origin (two fetuses), and (d) SR muscle fibers arising from an additional head of the LR (three fetuses). Conclusions: The SR seemed to issue a thin tendon that passed along the inferior or lateral side of the oculomotor nerve. Conversely, the LR and inferior rectus muscle were likely to carry a supernumerary bundle that reached the SR. The accessory head of the medial rectus muscle showed a stable morphology in that it seemed to also provide an anomalous double head. However, the presence of an accessory head in the LR was rare. In contrast with our previously published diagram of the orbital apex, the accessory head of the medial rectus muscle passed along the lateral side of the superior oblique.

Regeneration of Damaged Tendon-Bone Junctions (Entheses)-TAK1 as a Potential Node Factor.

Musculoskeletal dysfunctions are highly prevalent due to increasing life expectancy. Consequently, novel solutions to optimize treatment of patients are required. The current major research focus is to develop innovative concepts for single tissues. However, interest is also emerging to generate applications for tissue transitions where highly divergent properties need to work together, as in bone-cartilage or bone-tendon transitions. Finding medical solutions for dysfunctions of such tissue transitions presents an added challenge, both in research and in clinics. This review aims to provide an overview of the anatomical structure of healthy adult entheses and their development during embryogenesis. Subsequently, important scientific progress in restoration of damaged entheses is presented. With respect to enthesis dysfunction, the review further focuses on inflammation. Although molecular, cellular and tissue mechanisms during inflammation are well understood, tissue regeneration in context of inflammation still presents an unmet clinical need and goes along with unresolved biological questions. Furthermore, this review gives particular attention to the potential role of a signaling mediator protein, transforming growth factor beta-activated kinase-1 (TAK1), which is at the node of regenerative and inflammatory signaling and is one example for a less regarded aspect and potential important link between tissue regeneration and inflammation.

Pacinian corpuscles in the human fetal foot: A study using 3D reconstruction and immunohistochemistry.

PURPOSE: Our group had recently described human hand Pacinian corpuscles (PCs): the hand PCs are not simply arranged along the digital palmar nerves but often exhibited specific morphologies known uncommonly. However, there is still no or few information about human foot PCs. MATERIALS AND METHODS: We observed transverse sections of all five toes including the interdigital area obtained from 12 feet of eight fetuses at 28-33 weeks (crown-rump length 230-290mm). Serial sections were prepared for 3D reconstructions and measurement. RESULTS: Foot PCs were characterized by (1) a dense distribution in the interdigital area in contrast to a few PCs in the distal tip of the all five toes; (2) abundant dorsal PCs including those in the nail bed and: (3) a long chain of PCs in the flexor tendon sheath of all five toes. Therefore, a distal dominance was not evident in the foot in contrast to the hand and, a tendon sheath contained much greater numbers of PCs than the hand. A tree-like or bouquet-like arrangement of PCs along a short perforating artery to the palmar digital skin was seen in the foot as we had described in the hand. The tree of foot PCs was sometimes seen laying transversely along the digital skin surface, not toward the skin. CONCLUSION: It is still unknown that, in utero, how the PCs distribution became different between the hand and foot: it might be determined genetically in a region-specific manner.

Examination of the Topographical Anatomy and Fetal Development of the Tendinous Annulus of Zinn for a Common Origin of the Extraocular Recti.

Purpose: The aim was to clarify the topographical anatomy of the common tendinous ring for the four rectus muscles in both adults and fetuses. Methods: We histologically examined the annular ligament for a common origin of the extraocular rectus muscles using 10 specimens from elderly individuals and 31 embryonic and fetal specimens. Results: At 6 to 8 weeks, each rectus carried an independent long tendon, individually originating from the sphenoid. Notably, we found additional origins from the optic or oculomotor nerve sheath. At 12 to 15 weeks, the lateral, inferior, and medial recti muscles were united to provide a C-shaped musculofibrous mass that was separated from the superior rectus originating from the edge of the optic canal opening. Morphologic features at 31 to 38 weeks were almost the same as those at 12 to 15 weeks, but the long and thick common tendon of the three recti reached the sphenoid body in the parasellar area. In adults, a ring-like arrangement of the rectus muscles ended at a site 8.1 to 12.0 mm anterior to the optic canal opening and independent of the superior rectus origin, the lateral, inferior, and medial recti formed a C-shaped muscle mass. The united origins of the three recti changed to a fibrous band extending along the superomedial wall of the orbital fissure. Conclusions: Consequently, none of the specimens we examined exhibited an annular tendon representing a common origin of the four recti, suggesting that the common tendinous ring includes only medial, lateral, and inferior rectus muscles with the superior rectus taking its origin independently.

Bone morphology is regulated modularly by global and regional genetic programs.

Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.

Embryonic and postnatal tendon cells respond differently to interleukin-1ß.

Adult tendons heal as scar tissue, whereas embryonic tendons heal scarlessly via unknown mechanisms. Scarred tendon healing results from inflammation-driven imbalances in anabolic and catabolic functions. To test scarless versus scarring age tendon cell responses to inflammatory conditions, we treated embryonic and postnatal tendon cells with interleukin (IL)-1ß and characterized expression of collagens, matrix metalloproteinases (MMPs), inflammatory mediators, and phosphorylation of signaling molecules. At baseline, postnatal cells expressed significantly higher levels of inflammatory mediators. When treated with IL-1ß, both postnatal and embryonic cells upregulated inflammatory mediators and MMPs. Notably, postnatal cells secreted inflammatory factors up to 12.5 times the concentration in embryonic cultures. IL-1ß activated NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) pathways in both cell types, but phosphorylated p38 MAPK levels were two times higher in postnatal than embryonic cells. Our results suggest that scarred healing tendon cells respond to proinflammatory cytokines by promoting an imbalance in anabolic and catabolic functions, and that the heightened response involves p38 MAPK signaling activity. In contrast, embryonic cell responses are smaller in magnitude. These intriguing findings support a potential role for tendon cells in determining scarless versus scarred healing outcomes by regulating the balance between anabolic and catabolic functions during tendon healing.

Importance of the circadian clock in tendon development.

Tendons are remarkable tissues that transmit force from muscle to bone during joint movement. They are remarkable because they withstand tensile forces that are orders of magnitude greater than can be withstood by isolated cells. The ability of the cells to survive is directly attributable to the stress shielding properties of the collagen-rich extracellular matrix of the tissue. A further remarkable feature is that the vast majority (>98%) of the collagen is never turned over; it is synthesized during embryonic through early adult development and persists for the lifetime of the person. How the collagen is synthesized, and importantly, how it is protected from fatigue failure for decades of countless loading cycles, remains a mystery. A recent discovery is that tendons are peripheral circadian clock tissues in which the expression of ~5% of the transcriptome is rhythmic during 24h. Evidence is emerging that a fraction of the total amount of collagen is synthesized and removed on a daily basis without being incorporated into the lifelong permanent collagen. This review provides some of the background, and summarizes the findings, of these latest discoveries. Detailed descriptions of tendon development, collagen synthesis and collagen fibrillogenesis can be found in excellent reviews (cited here) and will not be a major part of this review.

Development, repair, and regeneration of the limb musculoskeletal system.

The limb musculoskeletal system provides a primary means for locomotion, manipulation of objects and protection for most vertebrate organisms. Intricate integration of the bone, tendon and muscle tissues are required for function. These three tissues arise largely independent of one another, but the connections formed during later development are maintained throughout life and are re-established following injury. Each of these tissues also have mesenchymal stem/progenitor cells that function in maintenance and repair. Here in, we will review the major events in the development of limb skeleton, tendon, and muscle tissues, their response to injury, and discuss current knowledge regarding resident progenitor/stem cells within each tissue that participate in development, repair, and regeneration in vivo.

Differences in foetal topographical anatomy between insertion sites of the iliopsoas and gluteus medius muscles into the proximal femur: a consideration of femoral torsion.

BACKGROUND: Prenatal twisting of the femoral neck seems to result in an angle of anteversion or torsion, but the underlying process has not been elucidated. MATERIALS AND METHODS: This study analysed sagittal, frontal and horizontal sections of 34 embryo and foetal specimens of gestational age (GA) 6-16 weeks (crown-rump length 21-130 mm). At GA 6-7 weeks, the iliopsoas (IP) and gluteus medius (GME) muscles were inserted into the anterior and posterior aspects of the femur, respectively, allowing both insertions to be viewed in a single sagittal section. RESULTS: At GA 8 weeks, the greater trochanter and the femoral neck angle became evident, and the GME tendon was inserted into the upper tip of the trochanter. At GA 9 weeks, the location of IP insertion was to the medial side of the GME insertion. After 9 weeks, the IP insertion consisted of a wavy, tendino- us part of the psoas muscle and another part of the iliacus muscle, with many fibres of the latter muscle attached to the joint capsule. After GA 12 weeks, the IP was inserted into the anteromedial side of the greater trochanter, while the aponeurotic insertion of the GME wrapped around the trochanter. At GA 15-16 weeks, a deep flexion at the hip joint caused an alteration in the relative heights of the lesser and greater trochanter, with the former migrating from the inferior to the slightly superior side. CONCLUSIONS: These findings indicate that twisting of the femoral neck started at GA 8-9 weeks.

Stereotypic generation of axial tenocytes from bipartite sclerotome domains in zebrafish.

Development of a functional musculoskeletal system requires coordinated generation of muscles, bones, and tendons. However, how axial tendon cells (tenocytes) are generated during embryo development is still poorly understood. Here, we show that axial tenocytes arise from the sclerotome in zebrafish. In contrast to mouse and chick, the zebrafish sclerotome consists of two separate domains: a ventral domain and a previously undescribed dorsal domain. While dispensable for sclerotome induction, Hedgehog (Hh) signaling is required for the migration and maintenance of sclerotome derived cells. Axial tenocytes are located along the myotendinous junction (MTJ), extending long cellular processes into the intersomitic space. Using time-lapse imaging, we show that both sclerotome domains contribute to tenocytes in a dynamic and stereotypic manner. Tenocytes along a given MTJ always arise from the sclerotome of the adjacent anterior somite. Inhibition of Hh signaling results in loss of tenocytes and enhanced sensitivity to muscle detachment. Together, our work shows that axial tenocytes in zebrafish originate from the sclerotome and are essential for maintaining muscle integrity.