A fundamental question in biomineralization is the nature of the first-formed mineral phase. In vertebrate bone formation, this issue has been the subject of a long-standing controversy. We address this key issue using the continuously growing fin bony rays of the Tuebingen long-fin zebrafish as a model for bone mineralization. Employing high-resolution scanning and transmission electron microscopy imaging, electron diffraction, and elemental analysis, we demonstrate the presence of an abundant amorphous calcium phosphate phase in the newly formed fin bones. The extracted amorphous mineral particles crystallize with time, and mineral crystallinity increases during bone maturation. Based on these findings, we propose that this amorphous calcium phosphate phase may be a precursor phase that later transforms into the mature crystalline mineral.biomineralization ͉ fish fin

SUMMARY 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.

SUMMARYThe 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, Scxpositive, 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 eminenceforming 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.

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.