Sharon Krief

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.

The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf

Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, we provide evidence for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.

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 (S1P1) has been shown to mediate interaction between endothelial and mural cells during vascular maturation. In vitro studies have identified S1P1 as a pro-angiogenic factor. Here, we show that S1P1 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 S1P1-null mouse embryos before vessel maturation imply a previously unknown, mural cell-independent role for S1P1 as an anti-angiogenic factor. A similar phenotype observed when S1P1 expression was blocked specifically in ECs indicates that the effect of S1P1 on sprouting is EC-autonomous. Comparable vascular abnormalities in S1p1 knockdown zebrafish embryos suggest cross-species evolutionary conservation of this mechanism. Finally, genetic interaction between S1P1 and Vegfa suggests that these factors interplay to regulate vascular development, as Vegfa promotes sprouting whereas S1P1 inhibits it to prevent excessive sprouting and fusion of neovessels. More broadly, because S1P, the ligand of S1P1, 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.

Joint Development Involves a Continuous Influx of Gdf5-Positive Cells

Summary 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-CreERT2 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.

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. Highlighted article: Collagen I is deposited by osteoblasts onto endothelial cells within bone and serves as a template for mineralisation, with ossification thus spatially and temporally following vascular patterning.

The Proprioceptive System Regulates Morphologic Restoration of Fractured Bones

Summary 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.

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. Highlighted article: The vascularization of limb interdigital regions leads to an increase in tissue oxygenation that, in turn, triggers ROS production and cell death during mouse limb development.

On the Development of Sesamoid Bones

Sesamoid bones are a special group of small auxiliary bones that form in proximity to joints and contribute to their stability and function. Sesamoid bones display high degree of variability in size, location, penetrance and anatomical connection to the main skeleton across vertebrate species. Therefore, providing a comprehensive developmental model or classification system for sesamoid bones is challenging. Here, we examine the developmental mechanisms of three anatomically different sesamoid bones, namely patella, lateral fabella and digit sesamoids. Through a comprehensive comparative analysis at the cellular, molecular and mechanical levels, we demonstrate that all three types of sesamoid bones originated from Sox9+/Scx+ progenitors under the regulation of TGFβ and independent of mechanical stimuli from muscles. We show that BMP4 was necessary specifically for differentiation of patella but not of lateral fabella or digit sesamoids, whereas BMP2 regulated the growth of all examined sesamoids. Next, we show that whereas patella and digit sesamoids initially formed in juxtaposition to long bones, the lateral fabella formed independently at a distance. Finally, we provide evidence suggesting that while patella detached from the femur by formation of a synovial joint, digit sesamoids detached from the phalanx by a fibrocartilage joint. Collectively, these findings highlight both common and divergent molecular and mechanical features of sesamoid bone development, thereby advancing our understanding of their evolutionary plasticity.

Bone Morphology is Regulated Modularly by Global and Regional Genetic Programs

During skeletogenesis, a variety of protrusions of different shapes and sizes develop on the surfaces of long bones. These superstructures provide stable anchoring sites for ligaments and tendons during the assembly of the musculoskeletal system. Despite their importance, the mechanism by which superstructures are patterned and ultimately give rise to the unique morphology of each long bone is far from understood. In this work, we provide further evidence that long bones form modularly from Sox9+ cells, which contribute to their substructure, and from Sox9+/Scx+ progenitors that give rise to superstructures. Moreover, we identify components of the genetic program that controls the patterning of Sox9+/Scx+ progenitors 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. Additionally, by combining literature-based evidence and comparative transcriptomic analysis of different Sox9+/Scx+ progenitor populations, we identified genes potentially involved in patterning of bone superstructures. We present evidence indicating that Gli3 is 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 coordinately. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning that further supports the notion that long bone development is a modular process.

Development of migrating entheses involves replacement of progenitor populations

Attachment sites of tendons to bones, called entheses, are essential for proper musculoskeletal function. They are formed embryonically by Sox9+ progenitors and undergo a developmental process that continues into the postnatal period and involves Gli1 lineage cells. During bone elongation, some entheses maintain their relative positions by actively migrating along the bone shaft, while others, located at the bone’s extremities, remain stationary. Despite their importance, we lack information on the developmental transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and Gli1 lineage cells. Here, by performing a series of lineage tracing experiments, we identify the onset of Gli1 lineage contribution to different entheses during embryogenesis. We show that Gli1 expression is regulated by SHH signaling during embryonic development, whereas postnatally it is maintained by IHH signaling. Interestingly, we found that unlike in stationary entheses, where Sox9+ cells differentiate into the Gli1 lineage, in migrating entheses the Sox9 lineage is replaced by Gli1 lineage and do not contribute to the mature enthesis. Moreover, we show that these Gli1+ progenitors are pre-specified embryonically to form the different cellular domains of the mature enthesis. Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple, embryonic tissue, whereas another population is responsible for its maturation into a complex structure during its migration. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.