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Elazar Zelzer - Top 30 Publications

The Proprioceptive System Regulates Morphologic Restoration of Fractured Bones.

Successful fracture repair requires restoration of bone morphology and mechanical integrity. Recent evidence shows that fractured bones of neonatal mice undergo spontaneous realignment, dubbed "natural reduction." Here, we show that natural reduction is regulated by the proprioceptive system and improves with age. Comparison among mice of different ages revealed, surprisingly, that 3-month-old mice exhibited more rapid and effective natural reduction than newborns. Fractured bones of null mutants for transcription factor Runx3, lacking functional proprioceptors, failed to realign properly. Blocking Runx3 expression in the peripheral nervous system, but not in limb mesenchyme, recapitulated the null phenotype, as did inactivation of muscles flanking the fracture site. Egr3 knockout mice, which lack muscle spindles but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types, as well as muscle contraction, are required for this regulatory mechanism. These findings uncover a physiological role for proprioception in non-autonomous regulation of skeletal integrity.

The Proprioceptive System Masterminds Spinal Alignment: Insight into the Mechanism of Scoliosis.

Maintaining posture requires tight regulation of the position and orientation of numerous spinal components. Yet, surprisingly little is known about this regulatory mechanism, whose failure may result in spinal deformity as in adolescent idiopathic scoliosis. Here, we use genetic mouse models to demonstrate the involvement of proprioception in regulating spine alignment. Null mutants for Runx3 transcription factor, which lack TrkC neurons connecting between proprioceptive mechanoreceptors and spinal cord, developed peripubertal scoliosis not preceded by vertebral dysplasia or muscle asymmetry. Deletion of Runx3 in the peripheral nervous system or specifically in peripheral sensory neurons, or of enhancer elements driving Runx3 expression in proprioceptive neurons, induced a similar phenotype. Egr3 knockout mice, lacking muscle spindles, but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types may be required for this regulatory mechanism. These findings uncover a central role for the proprioceptive system in maintaining spinal alignment.

Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology.

Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix.

Development of a subset of forelimb muscles and their attachment sites requires the ulnar-mammary syndrome gene Tbx3.

In the vertebrate limb over 40 muscles are arranged in a precise pattern of attachment via muscle connective tissue and tendon to bone and provide an extensive range of motion. How the development of somite-derived muscle is coordinated with the development of lateral plate-derived muscle connective tissue, tendon and bone to assemble a functional limb musculoskeletal system is a long-standing question. Mutations in the T-box transcription factor, TBX3, have previously been identified as the genetic cause of ulnar-mammary syndrome (UMS), characterized by distinctive defects in posterior forelimb bones. Using conditional mutagenesis in mice, we now show that TBX3 has a broader role in limb musculoskeletal development. TBX3 is not only required for development of posterior forelimb bones (ulna and digits 4 and 5), but also for a subset of posterior muscles (lateral triceps and brachialis) and their bone eminence attachment sites. TBX3 specification of origin and insertion sites appears to be tightly linked with whether these particular muscles develop and may represent a newly discovered mechanism for specification of anatomical muscles. Re-examination of an individual with UMS reveals similar previously unrecognized muscle and bone eminence defects and indicates a conserved role for TBX3 in regulating musculoskeletal development.

Joint Development Involves a Continuous Influx of Gdf5-Positive Cells.

Synovial joints comprise several tissue types, including articular cartilage, the capsule, and ligaments. All of these compartments are commonly assumed to originate from an early set of Gdf5-expressing progenitors populating the interzone domain. Here, we provide evidence that joints develop through a continuous influx of cells into the interzone, where they contribute differentially to forming joint tissues. Using a knockin Gdf5-CreER(T2) mouse, we show that early labeling of Gdf5-positive interzone cells failed to mark the entire organ. Conversely, multiple Cre activation steps indicated a contribution of these cells to various joint compartments later in development. Spatiotemporal differences between Gdf5 and tdTomato reporter expression support the notion of a continuous recruitment process. Finally, differential contribution of Gdf5-positive cells to various tissues suggests that the spatiotemporal dynamics of Gdf5 expression may instruct lineage divergence. This work supports the influx model of joint development, which may apply to other organogenic processes.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development.

During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself.

Isometric Scaling in Developing Long Bones Is Achieved by an Optimal Epiphyseal Growth Balance.

One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.

On the development of the patella.

The current view of skeletal patterning fails to explain the formation of sesamoid bones. These small bones, which facilitate musculoskeletal function, are exceptionally embedded within tendons. Although their structural design has long puzzled researchers, only a limited model for sesamoid bone development has emerged. To date, sesamoids are thought to develop inside tendons in response to mechanical signals from the attaching muscles. However, this widely accepted model has lacked substantiation. Here, we show that, contrary to the current view, in the mouse embryo the patella initially develops as a bony process at the anteriodistal surface of the femur. Later, the patella is separated from the femur by a joint formation process that is regulated by mechanical load. Concurrently, the patella becomes superficially embedded within the quadriceps tendon. At the cellular level, we show that, similar to bone eminences, the patella is formed secondarily by a distinct pool of Sox9- and Scx-positive progenitor cells. Finally, we show that TGFβ signaling is necessary for the specification of patella progenitors, whereas the BMP4 pathway is required for their differentiation. These findings establish an alternative model for patella development and provide the mechanical and molecular mechanisms that underlie this process. More broadly, our finding that activation of a joint formation program can be used to switch between the formation of bony processes and of new auxiliary bones provides a new perspective on plasticity during skeletal patterning and evolution.

A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation.

Decades of work have identified the signaling pathways that regulate the differentiation of chondrocytes during bone formation, from their initial induction from mesenchymal progenitor cells to their terminal maturation into hypertrophic chondrocytes. Here, we review how multiple signaling molecules, mechanical signals and morphological cell features are integrated to activate a set of key transcription factors that determine and regulate the genetic program that induces chondrogenesis and chondrocyte differentiation. Moreover, we describe recent findings regarding the roles of several signaling pathways in modulating the proliferation and maturation of chondrocytes in the growth plate, which is the 'engine' of bone elongation.

Vascular patterning regulates interdigital cell death by a ROS-mediated mechanism.

Blood vessels serve as key regulators of organogenesis by providing oxygen, nutrients and molecular signals. During limb development, programmed cell death (PCD) contributes to separation of the digits. Interestingly, prior to the onset of PCD, the autopod vasculature undergoes extensive patterning that results in high interdigital vascularity. Here, we show that in mice, the limb vasculature positively regulates interdigital PCD. In vivo, reduction in interdigital vessel number inhibited PCD, resulting in syndactyly, whereas an increment in vessel number and distribution resulted in elevation and expansion of PCD. Production of reactive oxygen species (ROS), toxic compounds that have been implicated in PCD, also depended on interdigital vascular patterning. Finally, ex vivo incubation of limbs in gradually decreasing oxygen levels led to a correlated reduction in both ROS production and interdigital PCD. The results support a role for oxygen in these processes and provide a mechanistic explanation for the counterintuitive positive role of the vasculature in PCD. In conclusion, we suggest a new role for vascular patterning during limb development in regulating interdigital PCD by ROS production. More broadly, we propose a double safety mechanism that restricts PCD to interdigital areas, as the genetic program of PCD provides the first layer and vascular patterning serves as the second.

A mechanical Jack-like Mechanism drives spontaneous fracture healing in neonatal mice.

Treatment of fractured bones involves correction of displacement or angulation, known as reduction. However, angulated long-bone fractures in infants often heal and regain proper morphology spontaneously, without reduction. To study the mechanism underlying spontaneous regeneration of fractured bones, we left humeral fractures induced in newborn mice unstabilized, and rapid realignment of initially angulated bones was seen. This realignment was surprisingly not mediated by bone remodeling, but instead involved substantial movement of the two fragments prior to callus ossification. Analysis of gene expression profiles, cell proliferation, and bone growth revealed the formation of a functional, bidirectional growth plate at the concave side of the fracture. This growth plate acts like a mechanical jack, generating opposing forces that straighten the two fragments. Finally, we show that muscle force is important in this process, as blocking muscle contraction disrupts growth plate formation, leading to premature callus ossification and failed reduction.

Endothelial cells regulate neural crest and second heart field morphogenesis.

Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio-craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio-craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio-craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

Tendon-to-bone attachment: from development to maturity.

The attachment between tendon and bone occurs across a complex transitional tissue that minimizes stress concentrations and allows for load transfer between muscles and skeleton. This unique tissue cannot be reconstructed following injury, leading to high incidence of recurrent failure and stressing the need for new clinical approaches. This review describes the current understanding of the development and function of the attachment site between tendon and bone. The embryonic attachment unit, namely, the tip of the tendon and the bone eminence into which it is inserted, was recently shown to develop modularly from a unique population of Sox9- and Scx-positive cells, which are distinct from tendon fibroblasts and chondrocytes. The fate and differentiation of these cells is regulated by transforming growth factor beta and bone morphogenetic protein signaling, respectively. Muscle loads are then necessary for the tissue to mature and mineralize. Mineralization of the attachment unit, which occurs postnatally at most sites, is largely controlled by an Indian hedgehog/parathyroid hormone-related protein feedback loop. A number of fundamental questions regarding the development of this remarkable attachment system require further study. These relate to the signaling mechanism that facilitates the formation of an interface with a gradient of cellular and extracellular phenotypes, as well as to the interactions between tendon and bone at the point of attachment.

Nonradioactive in situ hybridization on skeletal tissue sections.

Spatial and temporal visualization of RNA transcripts in tissue is a key tool in studying both developmental and pathological processes. In situ hybridization is a highly sensitive method for RNA transcript detection. It is based on sequence complementation between a labeled RNA probe and the RNA transcript of interest. The labeled probe is then detected by immunohistochemical methods using an antibody conjugated to an enzyme that catalyzes the generation of chromogenic or fluorescent signals, which indicate the location of the transcript.

Repositioning forelimb superficialis muscles: tendon attachment and muscle activity enable active relocation of functional myofibers.

The muscles that govern hand motion are composed of extrinsic muscles that reside within the forearm and intrinsic muscles that reside within the hand. We find that the extrinsic muscles of the flexor digitorum superficialis (FDS) first differentiate as intrinsic muscles within the hand and then relocate as myofibers to their final position in the arm. This remarkable translocation of differentiated myofibers across a joint is dependent on muscle contraction and muscle-tendon attachment. Interestingly, the intrinsic flexor digitorum brevis (FDB) muscles of the foot are identical to the FDS in tendon pattern and delayed developmental timing but undergo limited muscle translocation, providing strong support for evolutionary homology between the FDS and FDB muscles. We propose that the intrinsic FDB pattern represents the original tetrapod limb and that translocation of the muscles to form the FDS is a mammalian evolutionary addition.

One load to rule them all: mechanical control of the musculoskeletal system in development and aging.

The musculoskeletal system functions because of the precise and coordinated assembly of its components, namely bones and joints, muscles, tendons and ligaments. This coordination requires cross-talk between the tissues, which is mediated by various molecular and mechanical cues. In this review, we summarize the progress that has been made in understanding the involvement of mechanical loads exerted by the musculature in the development of skeletal and tendinous tissues, in their integration into one functional unit and in the maintenance of this system. In addition, we discuss the possible role of muscle load in aging and propose new directions for future studies of the musculoskeletal system.

Tendon-bone attachment unit is formed modularly by a distinct pool of Scx- and Sox9-positive progenitors.

The assembly of the musculoskeletal system requires the formation of an attachment unit between a bone and a tendon. Tendons are often inserted into bone eminences, superstructures that improve the mechanical resilience of the attachment of muscles to the skeleton and facilitate movement. Despite their functional importance, little is known about the development of bone eminences and attachment units. Here, we show that bone eminence cells are descendants of a unique set of progenitors and that superstructures are added onto the developing long bone in a modular fashion. First, we show that bone eminences emerge only after the primary cartilage rudiments have formed. Cell lineage analyses revealed that eminence cells are not descendants of chondrocytes. Moreover, eminence progenitors were specified separately and after chondroprogenitors of the primary cartilage. Fields of Sox9-positive, Scx-positive, Col2a1-negative cells identified at presumable eminence sites confirm the identity and specificity of these progenitors. The loss of eminences in limbs in which Sox9 expression was blocked in Scx-positive cells supports the hypothesis that a distinct pool of Sox9- and Scx-positive progenitors forms these superstructures. We demonstrate that TGFβ signaling is necessary for the specification of bone eminence progenitors, whereas the SCX/BMP4 pathway is required for the differentiation of these progenitors to eminence-forming cells. Our findings suggest a modular model for bone development, involving a distinct pool of Sox9- and Scx-positive progenitor cells that form bone eminences under regulation of TGFβ and BMP4 signaling. This model offers a new perspective on bone morphogenesis and on attachment unit development during musculoskeletal assembly.

Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.

HIF1α is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development.

Collagen production is fundamental for the ontogeny and the phylogeny of all multicellular organisms. It depends on hydroxylation of proline residues, a reaction that uses molecular oxygen as a substrate. This dependency is expected to limit collagen production to oxygenated cells. However, during embryogenesis, cells in different tissues that develop under low oxygen levels must produce this essential protein. In this study, using the growth plate of developing bones as a model system, we identify the transcription factor hypoxia-inducible factor 1 α (HIF1α) as a central component in a mechanism that underlies collagen hydroxylation and secretion by hypoxic cells. We show that Hif1a loss of function in growth plate chondrocytes arrests the secretion of extracellular matrix proteins, including collagen type II. Reduced collagen hydroxylation and endoplasmic reticulum stress induction in Hif1a-depleted cells suggests that HIF1α regulates collagen secretion by mediating its hydroxylation and consequently its folding. We demonstrate in vivo the ability of Hif1α to drive the transcription of collagen prolyl 4-hydroxylase, which catalyzes collagen hydroxylation. We also show that, concurrently, HIF1α maintains cellular levels of oxygen, most likely by controlling the expression of pyruvate dehydrogenase kinase 1, an inhibitor of the tricarboxylic acid cycle. Through this two-armed mechanism, HIF1α acts as a central regulator of collagen production that allows chondrocytes to maintain their function as professional secretory cells in the hypoxic growth plate. As hypoxic conditions occur also during pathological conditions such as cancer, our findings may promote the understanding not only of embryogenesis, but also of pathological processes.

S1P1 inhibits sprouting angiogenesis during vascular development.

Coordination between the vascular system and forming organs is essential for proper embryonic development. The vasculature expands by sprouting angiogenesis, during which tip cells form filopodia that incorporate into capillary loops. Although several molecules, such as vascular endothelial growth factor A (Vegfa), are known to induce sprouting, the mechanism that terminates this process to ensure neovessel stability is still unknown. Sphingosine-1-phosphate receptor 1 (S1P(1)) has been shown to mediate interaction between endothelial and mural cells during vascular maturation. In vitro studies have identified S1P(1) as a pro-angiogenic factor. Here, we show that S1P(1) acts as an endothelial cell (EC)-autonomous negative regulator of sprouting angiogenesis during vascular development. Severe aberrations in vessel size and excessive sprouting found in limbs of S1P(1)-null mouse embryos before vessel maturation imply a previously unknown, mural cell-independent role for S1P(1) as an anti-angiogenic factor. A similar phenotype observed when S1P(1) expression was blocked specifically in ECs indicates that the effect of S1P(1) on sprouting is EC-autonomous. Comparable vascular abnormalities in S1p(1) knockdown zebrafish embryos suggest cross-species evolutionary conservation of this mechanism. Finally, genetic interaction between S1P(1) and Vegfa suggests that these factors interplay to regulate vascular development, as Vegfa promotes sprouting whereas S1P(1) inhibits it to prevent excessive sprouting and fusion of neovessels. More broadly, because S1P, the ligand of S1P(1), is blood-borne, our findings suggest a new mode of regulation of angiogenesis, whereby blood flow closes a negative feedback loop that inhibits sprouting angiogenesis once the vascular bed is established and functional.

Muscle contraction controls skeletal morphogenesis through regulation of chondrocyte convergent extension.

Convergent extension driven by mediolateral intercalation of chondrocytes is a key process that contributes to skeletal growth and morphogenesis. While progress has been made in deciphering the molecular mechanism that underlies this process, the involvement of mechanical load exerted by muscle contraction in its regulation has not been studied. Using the zebrafish as a model system, we found abnormal pharyngeal cartilage morphology in both chemically and genetically paralyzed embryos, demonstrating the importance of muscle contraction for zebrafish skeletal development. The shortening of skeletal elements was accompanied by prominent changes in cell morphology and organization. While in control the cells were elongated, chondrocytes in paralyzed zebrafish were smaller and exhibited a more rounded shape, confirmed by a reduction in their length-to-width ratio. The typical columnar organization of cells was affected too, as chondrocytes in various skeletal elements exhibited abnormal stacking patterns, indicating aberrant intercalation. Finally, we demonstrate impaired chondrocyte intercalation in growth plates of muscle-less Sp(d) mouse embryos, implying the evolutionary conservation of muscle force regulation of this essential morphogenetic process.Our findings provide a new perspective on the regulatory interaction between muscle contraction and skeletal morphogenesis by uncovering the role of muscle-induced mechanical loads in regulating chondrocyte intercalation in two different vertebrate models.

The heart endocardium is derived from vascular endothelial progenitors.

The embryonic heart is composed of two cell layers: the myocardium, which contributes to cardiac muscle tissue, and the endocardium, which covers the inner lumen of the heart. Whereas significant progress has been made toward elucidating the embryonic origins of the myocardium, the origins of the endocardium remain unclear. Here, we have identified an endocardium-forming field medial to the cardiac crescent, in a continuum with the endothelial plexus. In vivo live imaging of quail embryos revealed that endothelial progenitors, like second/anterior heart field progenitors, migrate to, and enter, the heart from the arterial pole. Furthermore, embryonic endothelial cells implanted into the cardiac crescent contribute to the endocardium, but not to the myocardium. In mouse, lineage analysis focusing on endocardial cells revealed an unexpected heterogeneity in the origins of the endocardium. To gain deeper insight into this heterogeneity, we conditionally ablated Flk1 in distinct cardiovascular progenitor populations; FLK1 is required in vivo for formation of the endocardium in the Mesp1 and Tie2 lineages, but not in the Isl1 lineage. Ablation of Flk1 coupled with lineage analysis in the Isl1 lineage revealed that endothelium-derived Isl1(-) endocardial cells were significantly increased, whereas Isl1(+) endocardial cells were reduced, suggesting that the endocardium is capable of undergoing regulative compensatory growth. Collectively, our findings demonstrate that the second heart field contains distinct myocardial and endocardial progenitor populations. We suggest that the endocardium derives, at least in part, from vascular endothelial cells.

Muscle force regulates bone shaping for optimal load-bearing capacity during embryogenesis.

The vertebrate skeleton consists of over 200 individual bones, each with its own unique shape, size and function. We study the role of intrauterine muscle-induced mechanical loads in determining the three-dimensional morphology of developing bones. Analysis of the force-generating capacity of intrauterine muscles in mice revealed that developing bones are subjected to significant and progressively increasing mechanical challenges. To evaluate the effect of intrauterine loads on bone morphogenesis and the contribution of the emerging shape to the ability of bones to withstand these loads, we monitored structural and mineral changes during development. Using daily micro-CT scans of appendicular long bones we identify a developmental program, which we term preferential bone growth, that determines the specific circumferential shape of each bone by employing asymmetric mineral deposition and transient cortical thickening. Finite element analysis demonstrates that the resulting bone structure has optimal load-bearing capacity. To test the hypothesis that muscle forces regulate preferential bone growth in utero, we examine this process in a mouse strain (mdg) that lacks muscle contractions. In the absence of mechanical loads, the stereotypical circumferential outline of each bone is lost, leading to the development of mechanically inferior bones. This study identifies muscle force regulation of preferential bone growth as the module that shapes the circumferential outline of bones and, consequently, optimizes their load-bearing capacity during development. Our findings invoke a common mechanism that permits the formation of different circumferential outlines in different bones.

Tendon homeostasis: the right pull.

Mechanotransduction, the conversion of a biophysical force into a cellular response, allows cells and tissues to respond to their mechanical milieu. How muscle force is translated through TGF-β signaling to regulate tendon homeostasis offers an interesting in vivo example of mechanotransduction.

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study.

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.

Connecting muscles to tendons: tendons and musculoskeletal development in flies and vertebrates.

The formation of the musculoskeletal system represents an intricate process of tissue assembly involving heterotypic inductive interactions between tendons, muscles and cartilage. An essential component of all musculoskeletal systems is the anchoring of the force-generating muscles to the solid support of the organism: the skeleton in vertebrates and the exoskeleton in invertebrates. Here, we discuss recent findings that illuminate musculoskeletal assembly in the vertebrate embryo, findings that emphasize the reciprocal interactions between the forming tendons, muscle and cartilage tissues. We also compare these events with those of the corresponding system in the Drosophila embryo, highlighting distinct and common pathways that promote efficient locomotion while preserving the form of the organism.

Bone vascularization and trabecular bone formation are mediated by PKB alpha/Akt1 in a gene-dosage-dependent manner: in vivo and ex vivo MRI.

PKBalpha/Akt1, a protein kinase, is a major mediator of angiogenic signaling. The purpose of this study was to determine the role of PKB alpha/Akt1 in bone vascularization and development. For that aim, macromolecular dynamic contrast enhanced MRI was applied to examine in vivo vascular changes in long bones of 40-day-old growing PKB alpha/Akt1-deficient, heterozygous, and wild-type mice. Ex vivo microMRI and microCT were applied to monitor the impact of PKB alpha/Akt1 gene dosage on trabecular bone formation during endochondral bone growth. PKB alpha/Akt1-deficient mice and, remarkably, also heterozygous mice showed significantly reduced blood volume fraction in the humerus compared to wild-type mice. Moreover, PKB alpha/Akt1-deficient mice showed a more severe vascular deficiency with reduced permeability. microCT and microMRI of trabeculae revealed impaired bone formation in both PKB alpha/Akt1-deficient and heterozygous mice, whereas cortical bone parameters were only reduced in PKB alpha/Akt1-deficient mice. Reduction of metaphyseal blood vessel invasion, concomitant with aberrant trabeculae and shorter long bones, demonstrates a gene-dose-dependent role for PKB alpha/Akt1 in regulation of overall size and endochondral bone growth. MRI proved to provide high sensitivity for in vivo detection of subtle gene dose effects leading to impaired bone vascularity and for uncovering changes in trabecular bone.

Bone ridge patterning during musculoskeletal assembly is mediated through SCX regulation of Bmp4 at the tendon-skeleton junction.

During the assembly of the musculoskeletal system, bone ridges provide a stable anchoring point and stress dissipation for the attachment of muscles via tendons to the skeleton. In this study, we investigate the development of the deltoid tuberosity as a model for bone ridge formation. We show that the deltoid tuberosity develops through endochondral ossification in a two-phase process: initiation is regulated by a signal from the tendons, whereas the subsequent growth phase is muscle dependent. We then show that the transcription factor scleraxis (SCX) regulates Bmp4 in tendon cells at their insertion site. The inhibition of deltoid tuberosity formation and several other bone ridges in embryos in which Bmp4 expression was blocked specifically in Scx-expressing cells implicates BMP4 as a key mediator of tendon effects on bone ridge formation. This study establishes a mechanistic basis for tendon-skeleton regulatory interactions during musculoskeletal assembly and bone secondary patterning.

Muscle contraction is necessary to maintain joint progenitor cell fate.

During embryogenesis, organ development is dependent upon maintaining appropriate progenitor cell commitment. Synovial joints develop from a pool of progenitor cells that differentiate into various cell types constituting the mature joint. The involvement of the musculature in joint formation has long been recognized. However, the mechanism by which the musculature regulates joint formation has remained elusive. In this study, we demonstrate, utilizing various murine models devoid of limb musculature or its contraction, that the contracting musculature is fundamental in maintaining joint progenitors committed to their fate, a requirement for correct joint cavitation and morphogenesis. Furthermore, contraction-dependent activation of beta-catenin, a key modulator of joint formation, provides a molecular mechanism for this regulation. In conclusion, our findings provide the missing link between progenitor cell fate determination and embryonic movement, two processes shown to be essential for correct organogenesis.