Autopod tendon development depends on cartilage. (A-D) Whole-mount in situ hybridization for Scx expression in WT and Sox9^Prx1Cre limbs at E12.5 and E13.5. Analogous zeugopod tendons are outlined in A,B. Brackets delineate wrist tendon progenitors at E12.5 and zeugopod tendons derived from these progenitors at E13.5. (E,F) Transverse sections of ScxGFP WT and Sox9^Prx1Cre zeugopod stained for MHC at E13.5. (G,H) Whole-mount in situ hybridization for Scx expression of conditional Bmp2^f/+;Bmp4^f/f;Prx1^Cre limbs and Gli3^Xt/Xt limbs at E13.5. Arrows (G,H) highlight missing or extra tendons.

Distinct regulatory patterns of tenocyte proliferation in autopod and zeugopod tendon segments. BrdU was detected using DAB staining and the images were overlaid with ScxGFP signal from an adjacent section to highlight cell proliferation in tendons. (A) Autopod and (B) zeugopod tendons at E12.5. (C) Autopod and (D) zeugopod tendons at E14.5. Enlarged views of the boxed areas in A-D are shown in A′-D′. Arrows highlight representative proliferative behavior in tendons.

The autopod and zeugopod segments of the FDP tendons are derived from separate progenitor populations. (A) Whole-mount in situ hybridization for Six2 at E13.5. (B,C) Six2 and (D,E) Scx in situ hybridization of transverse sections (levels indicated in A). (F-G′) Transverse sections through limb of Six2^CreERT2;RosaT;ScxGFP embryos at E16.5; tamoxifen was given at E12.5. The Six2 cell lineage highlighted by the RosaT signal (F,G) was overlaid with ScxGFP signal to identify the affected tendons. (H) Schematic showing lineage distribution in FDP tendons. Blue and purple arrowheads and outlines indicate FDP and EDC tendons, respectively. FDS muscles and tendons are also derived from Six2 lineage cells (non-outlined red tissues in G,G′). Scale bars: 50 μm.

Zeugopod tendons develop via muscle anchoring, followed by elongation in parallel with skeletal growth. (A-D) Whole-mount in situ hybridization of WT limbs for Scx from E10.5 to E13.5. (E) Sagittal and (F) transverse sections of E12.5 limbs stained for muscle (MHC) and cartilage (collagen type II). Arrow (E) indicates integration of tendon to muscle. (G,H) Scx expression in whole-mount Sp^d limbs, as compared with WT (C,D), at E12.5 and E13.5. Brackets delineate wrist tendon progenitors at E12.5 and zeugopod tendons derived from these progenitors at E13.5. (I) Whole-mount MHC-stained Col2GFP limb at E12.5 and whole-mount ScxGFP limb at E12.5 (left); whole-mount ScxGFP limbs with muscle labeled by MHC staining at E12.5 and E13.5 (center); or Pax7Cre at E14.5 (right). Yellow arrows highlight the wrist. Individual tendons in I can be identified using the schematic and numerical assignments in Fig. 1.

FDS tendons are formed via distinct cell populations. (A) Schematic of FDS tendon and muscle development. Adapted from Huang et al. (2013). (B-C″) Lineage tracing with Sox9^Cre and Rosa26-TdTomato at E16.5. TdTomato (B′,C′) and ScxGFP and TdTomato (B″,C″) overlays are shown. Orange and blue arrowheads highlight FDS and FDP tendons, respectively.

FDS tendon development is also modular and depends on muscle and cartilage. Transverse sections stained for MHC from (A,B) WT, (C,D) Sp^d and (E,F) Ihh^−/− embryos at E16.5. Enlarged views of the boxed areas in B,D,F are shown in B′,D′,F′. Digit FDS tendons in (G) paralyzed mdg mutants and (H) Scx^−/− mutants. (I) Schematic indicates digit and metacarpal levels and depicts the modular development of FDS tendons. Orange and blue arrowheads highlight FDS and FDP tendons, respectively. Scale bars: 50 μm.

Model of zeugopod tendon formation as a distinctly regulated developmental process. Muscle-independent tendon induction of wrist progenitors occurs at E12.5, thus integrating the musculoskeletal tissues (muscle-tendon-cartilage). Whereas autopod tendon development requires signals from cartilage, zeugopod tendons undergo a muscle-dependent elongation phase in parallel with skeletal growth, such that the extent of skeletal growth dictates the extent of tendon elongation. The requirement for muscle for elongation lies in early attachment to the wrist tendon anlagen, and subsequent individuation and robustness of tendon depend on muscle forces. Blue highlights autopod tendon progenitors and tendons; purple highlights the wrist anlagen and the wrist-derived zeugopod tendons. Although the EDC tendons are shown here, this model of tendon formation applies to all autopod tendons with the exception of the FDS tendons, which are highlighted separately in Fig. 9.