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Sesamoid bones are small auxiliary bones that form near joints and contribute to their stability and function. Thus far, providing a comprehensive developmental model or classification system for this highly diverse group of bones has been challenging. Here, we compare our previously reported mechanisms of patella development in the mouse with those of two anatomically different sesamoids, namely lateral fabella and digit sesamoids. We show that all three types of sesamoid bones originate from Sox9(+)/Scx(+) progenitors under the regulation of TGFβ and independently of mechanical stimuli from muscles. Whereas BMP2 regulates the growth of all examined sesamoids, the differentiation of lateral fabella or digit sesamoids is regulated redundantly by BMP4 and BMP2. Next, we show that whereas patella and digit sesamoids initially form in juxtaposition to long bones, lateral fabella forms independently and at a distance. Finally, our evidence suggests that, unlike the synovial joint that separates patella from femur, digit sesamoids detach from the phalanx by formation of a fibrocartilaginous joint. These findings highlight both common and divergent molecular and mechanical features of sesamoid bone development, which underscores their evolutionary plasticity.

Tendon-bone attachment sites, called entheses, are essential for musculoskeletal function. They are formed embryonically by Sox9+ progenitors and continue to develop postnatally utilizing Gli1 lineage cells. Despite their importance, we lack information on the transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and Gli1 lineage. Here, by performing a series of lineage tracing experiments, we identify the onset of Gli1 lineage contribution to different entheses. We show that Gli1 expression is regulated embryonically by SHH signaling, whereas postnatally it is maintained by IHH signaling. During bone elongation, some entheses migrate along the bone shaft, while others remain stationary. Interestingly, whereas in stationary entheses Sox9+ cells differentiate into the Gli1 lineage, in migrating entheses this lineage is replaced by Gli1 lineage. These Gli1+ progenitors are defined embryonically to occupy the different domains of the mature enthesis. Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple, embryonic tissue, whereas another population contributes to its maturation. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.

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.

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.

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.