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

Abstract

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.

Fig. 1.

Different stages in Drosophila muscle-tendon interactions during embryogenesis. (A) In the first stage, tendon progenitors are defined in the ectoderm by the induction of StripeB (SrB) expression by the products of segment polarity genes, such as Hedgehog (Hh) and Wingless (Wg). StripeB expression is maintained at a low level as a result of post-transcriptional repression by the RNA-binding protein How(L). SrB regulates its own expression positively, as well as the expression of its inhibitor How(L). Tendon progenitors secrete Slit and provide initial cues for directing muscle bipolar migration. The muscle responds to Slit through Robo receptors. In addition, Kon-tiki (Kon) contributes to the migration of the muscles. (B) In a second step, the muscle signals to the tendon through epidermal growth factor receptor (Egfr) activation by means of the neuregulin-like secreted ligand Vein to initiate tendon differentiation and elevate SrB expression. SrB further induces the expression of Slit and Lrt; Lrt is required to arrest muscle migration. Tendon precursors that do not bind muscles lose SrB expression and become ectoderm cells. (C) In a third step, the myotendinous junction (black) is formed through muscle-specific αPS2βPS integrin association with the tendon-secreted ECM component Thrombospondin (Tsp) and its regulator Slow. Laminin (Lam) binds the αPS1βPS tendon-specific integrin. At this stage, How(S) is elevated in the cytoplasm of the tendon cell, promoting the expression of StripeA (SrA), which is essential to induce tendon terminal differentiation by induction of Short stop (Shot), Delilah (Dei), and β1 Tubulin (β1Tub).

Fig. 2.

Tissue interactions required for tendon progenitor induction in vertebrate embryos. Induction of tendon progenitors, identified as Scx-expressing cells, depends on a unique set of tissue interactions in different parts of the embryo. Each panel shows tendon progenitor distribution by whole-mount in situ hybridization (ISH) with an Scx probe. The line across each upper image shows the orientation of the section schematized beneath, which highlights the relevant tissue interactions (with tendon progenitors shown in green; muscle progenitors in red; cartilage in yellow). (A) Whole-mount Scx ISH on E10.5 mouse embryo and a schematic of a frontal trunk section, showing somite pairs (squares) and the neural tube (gray). Skeletal tissue derives from the sclerotome (Sc) of somites, whereas the musculature arises from the myotome (m). The tendon progenitors are found in the syndetome (S, green), a stripe of sclerotome cells at the junction between two adjacent myotomes. Scx expression in syndetome cells is induced by FGFs secreted from the adjacent myotomes (arrows). (B) Whole-mount Scx ISH on E10.5 mouse limb bud and a schematized sagittal section through the limb bud. In the early limb bud, Scx is expressed in mesodermal cells directly under the dorsal and ventral ectoderm. Scx expression at this stage depends on ectoderm (curved arrows) and not on a signal from the myoblasts or from prechondrogenic cells. (C) Whole-mount Scx ISH on E12.5 mouse limb and a schematized sagittal section through the autopod. In the differentiating autopod, Scx is expressed in sub-ectodermal mesoderm along the differentiating skeletal elements. Scx expression along the differentiating digits can be induced by a signal from the skeletal condensations (straight arrow), and the sub-ectodermal position of the tendon progenitors suggests a role for the ectoderm (curved arrows) in tendon induction as well. A, anterior; D, distal; P, posterior; Pr, proximal; nt, neural tube.

Fig. 3.

The main stages and regulators of tendon induction and differentiation in vertebrate embryos. The induction and differentiation of tendon progenitors occur in three distinct stages (A-C, in which Scx-expressing tendon progenitors are represented in green, and mesenchymal cells in white). (A) Induction. The initial induction of Scx-expressing tendon progenitors is associated with FGF signaling, but the myotome in somites is the only identified source to date. In somites and digits, the progenitors are induced at or near their functional position between the myogenic and skeletogenic cells, but in the early limb bud and branchial arches the site of progenitor induction is not related to their final destination. (B) Organization. In an E12.5 mouse embryo, tendon progenitors throughout the embryonic body organize as loose cellular aggregations between the differentiating muscle and skeletal tissues. This transition depends on TGFβ signaling, which mediates the recruitment of additional tendon progenitors by the muscle and cartilage tissues to position and integrate the tendon progenitors with their interacting musculoskeletal tissues (white arrows). In addition, TGFβ ligands expressed by the tendon progenitors are likely to contribute to the maintenance of the tenoblastic identity of the tendon progenitors (black arrow). (C) Aggregation and differentiation. By E13.5, the tendon progenitors condense and organize into structurally distinct tendons that connect to the muscle and cartilage. In some, but not all, tendons tenocyte differentiation depends on Scx function. In most tissues, tendon differentiation depends on the presence of muscles (arrow), but the extensor and flexor tendons that extend into the autopod differentiate as structurally distinct tendons even in the absence of muscles.

Fig. 4.

Bone ridge formation proceeds via a biphasic process. (A,B) Schematics of the mouse forelimb skeleton, illustrating the involvement of tendons and muscles in bone ridge formation. The attachment site area (circled) is magnified beneath. According to the biphasic model, tendons regulate the initiation of tuberosity (see Glossary, Box 2) (A) and muscles control its subsequent growth (B). (A) The molecular mechanism that underlies tendon regulation of bone ridge initiation involves the bHLH transcription factor Scx, which regulates Bmp4 expression in tendon cells. Next, upon binding of Bmp4 to its receptor Alk3 (Bmpr1a) in chondrocytes, a signaling cascade is activated, eventually leading to the initiation of a bone ridge. (B) The mechanism whereby muscle contraction promotes bone ridge growth remains to be uncovered.