Requirement for scleraxis in the recruitment of mesenchymal progenitors during embryonic tendon elongation

Abstract

The transcription factor scleraxis (Scx) is required for tendon development; however, the function of Scx is not fully understood. Although Scx is expressed by all tendon progenitors and cells, only long tendons are disrupted in the Scx^-/- mutant; short tendons appear normal and the ability of muscle to attach to skeleton is not affected. We recently demonstrated that long tendons are formed in two stages: first, by muscle anchoring to skeleton via a short tendon anlage; and second, by rapid elongation of the tendon in parallel with skeletal growth. Through lineage tracing, we extend these observations to all long tendons and show that tendon elongation is fueled by recruitment of new mesenchymal progenitors. Conditional loss of Scx in mesenchymal progenitors did not affect the first stage of anchoring; however, new cells were not recruited during elongation and long tendon formation was impaired. Interestingly, for tenocyte recruitment, Scx expression was required only in the recruited cells and not in the recruiting tendon. The phenotype of Scx mutants can thus be understood as a failure of tendon cell recruitment during tendon elongation.

Keywords: Mouse; Musculoskeletal; Scleraxis; Tendon development.

Fig. 1.

Long tail tendons develop via anchoring and elongation. (A) Whole-mount confocal and (B) transverse section images of long tail tendons from ScxGFP embryos at E15.5. (C) Whole-mount confocal images of short-range tail tendon anlage from ScxGFP embryos at E13.5. (C′,C″) Whole-mount staining with MHC for muscle and ScxGFP and MHC overlays. (D) Schematic of a short tail tendon integrating muscle with skeleton at E12.5/E13.5 and elongated long tail tendons at E15.5. Blue arrow in A indicates an example vertebral body (V in B). Yellow and white arrows highlight proximal and distal tendons, respectively. Scale bars: 200 µm for whole-mount tails; 100 µm for E15.5 transverse section in B.

Fig. 2.

Tail tendon elongation is enabled by recruitment of new cells. (A) Schematic of Sox9^lin cell composition of short-range tail tendons labeled by Sox9^Cre at E12.5 (yellow color indicates both ScxGFP+ and RosaT+). (B-C″) Whole-mount confocal (B-B″) and transverse section (C-C″) images of Sox9^Cre; RosaT; ScxGFP tail tendon anlage at E12.5. (B′,C′) RosaT signal; (B″,C″) ScxGFP and RosaT overlays. (D) Schematic of Sox9^lin composition of long tail tendons from Sox^CreERT2 embryos at E16.5 (tamoxifen at E12.5) highlight RosaT labeling of skeletal-tendon insertions. (E) Transverse sections through the distal tail tip of Sox9^CreERT2; RosaT; ScxGFP embryos at E16.5; tamoxifen was given at E12.5. Sections collected through level planes shown in D (L1-L4); 1-5 labels follow specific dorsal tendons through the sections. (F) Schematic of Sox9^lin composition of long tail tendons from Sox9^CreERT2 embryos at E16.5 (tamoxifen at E12.5) highlight the absence of RosaT in the tendon body. (G-G″) Transverse sections through tail base of Sox9^CreERT2; RosaT; ScxGFP embryos at E16.5; tamoxifen was given at E12.5. Section collected through level plane L5 shown in F. (G′,G″) RosaT, and ScxGFP/RosaT overlays. Scale bars: 200 µm for E12.5 whole-mount tail (B-B″); 25 µm for all transverse sections.

Fig. 3.

Scx is required for mesenchymal cell recruitment during tail tendon elongation. (A) Schematic of Sox9^lin cell composition of dorsal tail tendons labeled by Sox9^Cre; RosaT at E18.5. (B-B″) Transverse section images of Sox9^Cre; RosaT; ScxGFP long dorsal tail tendons at E18.5. (B′,B″) RosaT and ScxGFP/RosaT overlays. (C) Schematic of Sox9^lin cell composition of dorsal tail tendons of mutant Scx^Sox9Cre; RosaT; ScxGFP embryos at E18.5. (D-D″) Transverse section images of Scx^Sox9Cre; RosaT; ScxGFP dorsal tail tendons at E18.5. (D′,D″) RosaT and ScxGFP/RosaT overlays. Red cells indicate Sox9^lin tendon cells; green cells indicate non-Sox9^lin tendon cells. Transverse sections show tendon midsubstance regions. Orange arrows highlight RosaT+/ScxGFP− cells. Scale bars: 25 µm.

Fig. 4.

Limb tendon elongation is also fueled by recruitment of new progenitors. (A) Whole-mount confocal image of Sox9^Cre; RosaT; ScxGFP forelimb at E12.5. (B) Whole-mount confocal image of Sox9^Cre; RosaT; ScxGFP forelimb at E15.5. (C) Transverse section images of wild-type E16.5 Sox9^Cre labeled tendons at the wrist (L1) and midsubstance (L2) levels shown in B. (D) Transverse section images of wild-type Sox9^CreERT2; RosaT; ScxGFP zeugopod tendons at E16.5 (tamoxifen at E12.5) highlight RosaT labeling of skeletal-tendon insertions. Comparison of Sox9^Cre and Sox9^CreERT2 images show that Sox9^lin cells in the tendon midsubstance are not derived from the short-range tendon but are derived from newly recruited cells. Scale bars: 200 µm (whole-mount images); 100 µm (transverse sections).

Fig. 5.

Scx is required for mesenchymal cell recruitment during limb tendon elongation. (A) Schematic image of extensor tendons. (B,C) Transverse section images of wild-type Sox9^Cre-labeled tendons at the section levels L1 and L2 shown in A. (D,E) Transverse section images of mutant Scx^Sox9Cre tendons at the section levels L1 and L2 shown in A. Scale bar: 20 µm.

Fig. 6.

Transuterine microinjection of limb mesenchymal cells into stage-matched E12.5 limb buds confirms that positive cell recruitment depends on Scx function. (A) Schematic of RosaT-labeled wild-type donor cell isolation and microinjection into wild-type host limbs. (B) Transverse section through a representative tendon showing positive recruitment of donor cells (yellow arrows, RosaT+/ScxGFP+) into host tendon. (B′) RosaT only. White arrows indicate RosaT+/ScxGFP-negative expression. (C) Transverse section through a representative tendon as an example of non-recruitment, indicated by RosaT+/ScxGFP-negative expression (white arrow). (C′) RosaT only. (D) Schematic of RosaT-labeled wild-type donor cell isolation and microinjection into mutant Scx^−/− host limbs. (E-E″) Whole-mount Scx^−/− limb shows extensive recruitment of wild-type RosaT donor cells into tendon (yellow arrows). (F-G′) Transverse sections through mutant (F,F′) and wild-type (G,G′) tendons show massive and minimal recruitment of wild-type RosaT+/ScxGFP+ donor cells, respectively (yellow arrows). Data are representative of 26 wild-type embryos and four Scx^−/− mutant embryos. Scale bars: 25 µm.

Fig. 7.

Model of cell recruitment and Scx dependency during long tendon elongation. Schematic of Scx-independent anchoring and Scx-dependent elongation phases of long tendon development. Red cells indicate Sox9^lin/ScxGFP tendon cells that make up the early anchoring tendon. Subsequently, tendon cells (green) are recruited to enable long tendon growth.