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First published online 13 June 2007
doi: 10.1242/dev.001933

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Regulation of tendon differentiation by scleraxis distinguishes force-transmitting tendons from muscle-anchoring tendons

¹ Shriners Hospital for Children, Research Division, Portland, OR 97239, USA.
² Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
³ Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
⁴ Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97239, USA.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Generation of an Scx null allele. (A) Schematic representation of alleles generated in the Scx locus. Black boxes represent coding regions of the Scx gene, and open boxes represent the 5' and 3' untranslated regions (UTRs). The recognition sequences for the Cre and FLP recombinases, the loxP (green boxes) and FRT (orange boxes) sites, respectively, are shown. The brown box represents the neomycin-resistance (Neo) cassette used for negative selection during targeting in embryonic stem cells (see Materials and methods for details). (B) Representation of the unique organization of the Scx locus. The two exons of the Scx gene (A) are located within the third intron of a second gene called block of proliferation 1 (Bop1), which is transcribed in the opposite orientation. (C) Scx in situ hybridization on cross sections through the digits of wild-type (WT) and Scx-/- embryos at E15.5.

View larger version (88K): [in this window] [in a new window]   Fig. 2. The long force-transmitting tendons and intermuscular tendons are severely disrupted in Scx-/- mice. Tendons in wild-type and mutant littermates were visualized directly in skinned tissues using the ScxGFP transgenic reporter (C-L). (A) The forelimbs of an Scx-/- newborn mouse at postnatal day (P)3 are locked in a dorsal flexure. (B) Forelimbs of P14 wild-type (WT) and Scx-/- mutant mice stained with Alcian blue and alizarin red. Black arrowheads point to the deltoid tuberosity, which is missing in the mutant. (C,D) The dorsal extensor tendons of the forelimb. Yellow arrowheads show the extensor digitorium communis (EDC). Long and short purple arrowheads show the extensor digiti quinti and extensor carpi ulnaris, respectively. (E,F) The ventral flexor tendons of the forelimb in wild type (E) and mutants (F). (G,H) Tail tendons at P14 visualized by direct illumination (G) or by fluorescence of the ScxGFP reporter (H). Yellow arrowheads show the tail tendons. Pink arrowheads show the annulus fibrosus. (I,J,M,N) Tendons of the trunk at E18.5. (I,J) Dorsal view of skinned trunks. (M,N) Sagittal trunk sections stained with an antibody to myosin heavy chain (MHC). Pink arrowheads show short-range tendons. Yellow arrowheads show long tendons. (K,L) The intermuscular tendons of the subscapularis muscle at E18.5 seen in a ventral view of the scapula. (O-T) Frontal sections through the trunk of E18.5 wild-type and Scx-/- embryos at the level of the diaphragm, carrying the ScxGFP reporter and counterstained with an MHC antibody. Yellow arrowheads show the middle tendon of the diaphragm. (O,P,S,T) Comparison of the signal of ScxGFP (O,P) and in situ hybridization with a collagen I probe (S,T) in the diaphragm tendon from a wild-type and a mutant embryo. (Q,R) Expression of tenascin C and collagen XII in the diaphragm tendon of a mutant embryo.

View larger version (127K): [in this window] [in a new window]   Fig. 3. Short-range anchoring tendons and ligaments are not affected in Scx-/- mice. (A-D) Frontal sections through the rib cage of wild-type (WT) and Scx-/- embryos at E18.5 stained with a myosin heavy chain (MHC; red) antibody to detect intercostal muscle attachments to the ribs. (A,B) ScxGFP signal (green) at the intercostal muscle attachments. (C,D) Section in situ hybridization (ISH) detecting collagen I expression in rib osteoblasts and in the tendinous attachment layer (yellow arrowheads) of wild-type and mutant embryos. (E,F) Sagittal sections through the knees of wild-type and Scx-/- embryos at E18.5. Collagen I was detected by section ISH followed by antibodies to MHC (red) and collagen II (green). Fluorescent antibody signals were merged with the ISH image in Photoshop. Yellow arrowheads show the cruciate ligaments of the knee; black arrowheads show the patellar ligament; purple arrowheads show the rectus femoris tendon and vastus medialis tendon. (G-I) Superficial sagittal sections through the tail of wild-type and Scx-/- embryos at E18.5 that are carrying the ScxGFP reporter. (G) The cartilage of tail bones visualized by DIC optics and overlaid with ScxGFP signal in the annulus fibrosis. (H,I) ScxGFP signal in the annulus fibrosis at high magnification. (J-L) Transmission electron microscope images of a medial sagittal section through the tail of an E18.5 Scx-/- embryo. (J) Low magnification reveals the nucleus pulposus (NP; the intervertebral disc), which is flanked laterally by cartilage cells and by dorsal and ventral tightly stacked layers of ligament cells of the annulus fibrosus (AF, red arrowheads). (K,L) The annulus fibrosus at a higher magnification. The collagen fibers (purple arrowheads) are aligned along the section plane (K) and perpendicular to the section plane (L). AF, annulus fibrosus; NP, nucleus pulposus; P, patella; Rf, rectus femoris muscle; Vm, vastus medialis muscle.

View larger version (118K): [in this window] [in a new window]   Fig. 4. The earliest tendon phenotype in Scx-/- embryos is detected at E13.5, concurrently with the overt formation of tendons. (A) Schematic representation of the major flexor tendons of the forelimb: FDP tendons (green), the FDS (red), and the lumbrical muscles and tendons (blue). Section levels are marked by black lines: (1) digit level, (2) metacarpal-phalangeal joint, (3) mid metacarpal, (4) proximal metacarpal. (B-M) Arrowhead assignments are: yellow, flexor digitorum profoundus (FDP); red, flexor digitorium superficialis (FDS); blue, lumbrical tendons; pink, extensor digitorium communis (EDC); white, flexor vinculum (B,E) or the palmar metacarpal ligament (J). (B-G) Sections through the autopod of wild-type (WT) and Scx-/- embryos at E18.5 highlighting the flexor and extensor tendons. The sections were taken at levels 1 (B,E), 2 (C,F) and 3 (D,G). (H,K) Scx in situ hybridization in limbs at E12.5 (H) and E14.0 (K), showing that, in wild-type mice, overt tendon formation occurs between E12.5 and E14.0. (I,L) ScxGFP detected by in situ hybridization in wild-type (I) and Scx-/- (L) forelimbs at E13.5. The EDC phenotype is first detected at E13.5 in mutant mice. (J,M) Sections through wild-type (J) and Scx-/- (M) forelimbs at E13.5 at a proximal metacarpal level (level 4 in A). The onset of the FDP phenotype occurs at E13.5. (N-Q) Cell proliferation in tendon cells of wild-type (N,O) and Scx-/- (P,Q) embryos at E14.5 in sections directly proximal to the wrist. BrdU was detected using a DAB stain and the images were overlaid with the ScxGFP signal from an immediate alternate section to highlight the tendons. (O,Q) Enlargements of the boxed areas in N and P, respectively. Arrowheads indicate tendons with BrdU-positive cells. (R-U) TUNEL staining in sections at digit level (level 1 in A) of wild-type (R,S) and Scx-/- (T,U) embryos at E14.5. (S,U) Enlargements of the boxed areas in R and T, respectively.

View larger version (91K): [in this window] [in a new window]   Fig. 5. The onset of the tail tendon phenotype in Scx-/- embryos occurs during the transition from progenitors to organized tendons at E13.5. (A-I) In situ hybridization to detect the Scx transcript (A,B) and ScxGFP transcript (F,G) in tails at E12.5 (A,F) and E13.5 (B,G) and ScxGFP in later stages (C-E,H,I). The rostral end of the tail is pointing right in A,B,F,G and pointing up in C,D,E. (C,E,H,I) Yellow arrowheads show the annulus fibrosus, pink arrowheads show tail tendons and white arrows show the insertion of individual tendons. (A-D,H) The development of tail tendons in wild-type (WT) embryos: (A,B) Scx in situ hybridization of whole tails. Black arrowheads show the splitting of the syndetome at E12.5; (C,D) detection of expression of the ScxGFP reporter in skinned tails at E18.5 (C) and at P14 (D); (H) cross section of a tail at E15.5. (E,F,G,I) The tail tendon phenotype in Scx-/- embryos. (F,G) In situ hybridization of tails to detect the transcript of the ScxGFP reporter. (E,I) The later tail phenotype, detected by ScxGFP fluorescence in a whole tail at E18.5 (E) and in a cross section at E15.5 (I). (J-M) Cell proliferation in tail tendons of wild-type (J,K) and Scx-/- (L,M) embryos at E13.5. BrdU was detected using a DAB stain and the images were overlaid with the ScxGFP signal from an immediate alternate section to highlight the tendons. (K,M) Enlargements of the boxed areas in J and L, respectively. Arrowheads show tendons with BrdU-positive cells.

View larger version (82K): [in this window] [in a new window]   Fig. 6. The ECM of the FDP tendon in the digits of Scx-/- embryos is also disrupted. (A-H) Yellow arrowhead, collagen fiber bundles; red arrowhead, cytoplasmic extensions of tenocytes; green arrowhead, microfibrils. (A,E) Hematoxylin and Eosin (H&E) staining of a section of wild-type (WT) and mutant digit at E18.5. (B,C,F,G) TEM images of wild-type and Scx-/- flexor digitorum profoundus (FDP) tendons at E18.5 amplified x7000 (B,F) and x38,000 (C,G). (B,H) TEM images of sections of wild-type and Scx-/- tail tendons at E18.5 magnified x4000.

View larger version (127K): [in this window] [in a new window]   Fig. 7. In Scx-/- mutant embryos, the endotenon cells fail to form a tendon sheath and are intermixed with the tenocytes in the FDP tendon. TEM (A,B) and antibody staining (C-G) of sections at the second digit phalange of wild-type (WT) and Scx-/- mouse embryos at E18.5. (A,B) TEM of the flexor digitorum profoundus (FDP) tendon amplified x550 to emphasize cellular aspects of the FDP. Green arrowheads, tenocytes; red arrowheads, the endotenon. (C,D) ScxGFP signal (green) and anti-EphA4 antibody (red) are overlaid on a DIC image of a cross section through the FDP tendon. (E-G) ScxGFP signal (green), and anti-tenascin C antibody (red) highlighting the endotenon in wild-type (E) and mutant (G,F) FDP tendon. Notice the collapse of the endotenon signal in the mutant tendon.

View larger version (74K): [in this window] [in a new window]   Fig. 8. Genes regulated by Scx. In situ hybridization on sections from the forelimb of an E16.5 Scx-/- mutant embryo (D-F) and a wild-type (WT) littermate (A-C) using probes for the collagen XIV (A,D), tenomodulin (B,E) and collagen I (C,F) genes.

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