Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments
Ronen Schweitzer1,
Jay H. Chyung2,
Lewis C. Murtaugh2,*,
Ava E. Brent1,
Vicki Rosen4,
Eric N. Olson3,
Andrew Lassar2 and
Clifford J. Tabin1,
1 Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
3 Department of Molecular Biology, The University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75235, USA
4 Genetics Institute, 87 Cambridge Park Drive, Cambridge MA 02140, USA
* Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
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Fig. 1. Comparison of chick and mouse Scleraxis proteins. Alignment of the putative amino acid sequences of the chick and mouse Scleraxis proteins. The two proteins are identical in the bHLH domain and flanking amino acids. Long stretches of identity are also found both in the N-terminal region and in a 31 amino acid stretch at the C terminus. The overall amino acid identity between the two proteins is 75%.
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Fig. 2. Scleraxis is expressed in all axial and limb tendons. Expression of scleraxis in chick embryos at day 9 (stage 35; A,E,F) and day 10 (stage 36; B,C,D)of development , visualized by whole-mount in situ hybridization. In all panels, anterior is upwards. (A) Scleraxis expression at stage 35 marks the complex network of limb tendons. (B,C) Scleraxis expression in tendons of the primary axis. In the trunk (B), the attachments of the scapular (red arrowhead) and pelvic (yellow arrowhead) muscles, which include the broad fasciae of these muscles express scleraxis. Similarly, long axial muscles and related fasciae are also stained. (C) In the neck (ventral view), scleraxis marks the forming tendons at the anterior and posterior edges of each vertebrae. (D-F) Details of scleraxis expression in limb tendons. (D) In a stage 36 wing, scleraxis expression marks all tendons, including an aponeurosis (red arrowhead) and the muscle-associated wing fascia (yellow arrowhead). (E) All details of the foot tendons are also marked by scleraxis. (F) A single dissected flexor tendon from a stage 35 foot highlights scleraxis expression throughout the tendon including the myotendinous junction.
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Fig. 3. Scleraxis is expressed in putative progenitors of the axial tendons. (A) Scleraxis is expressed in the intersomitic mesenchyme at stage 26. The yellow arrowhead points to the extension of scleraxis expression into the rib primordium. (B) Scleraxis expression becomes more elaborate after the initial patterning of the axial muscles and cartilage at stage 29. (C-E) The domain of scleraxis expression in the somite was analyzed by comparison with other probes in hybridization to alternating coronal longitudinal sections through the back of a stage 25 embryo. Black arrowheads mark somite edges and red arrowheads point to the morphologically distinct myotome. (C) Pax1 is expressed in the sclerotome but excluded from the early cartilage condensations. Although expression extends to the intersomitic mesenchyme expression levels are higher in the center of the somite. (D) Scleraxis is expressed in cells adjacent and medial to the junction between the myotomes of consecutive somites. (E) MyoD is expressed specifically in the myotome. (F-H) Two color in situ hybridization for scleraxis and MyoD. Scleraxis (black) and MyoD (red) mark two adjacent but non overlapping cell population seen in a lateral view (F) and a dorsal view (G). (H) Scleraxis expression at the junction of adjacent myotomes is demarcated in a longitudinal coronal section (25 µm) of the stained embryos. (I) Schematic representation of a transverse section through a late somite. The sclerotome, the origin of axial cartilage, is at the ventromedial region of the somite. The myotome, composed of differentiated myofibrils, which will give rise to the axial muscles, lies directly underneath the dermatome. (J,K) In situ hybridization to transverse sections through the trunk at a thoracic level of a day 12 chick embryo. (J) MyoD marks all the axial and intercostal muscles. (K) In an adjacent section, scleraxis is expressed specifically only in a single row of cells connecting an intercostal muscle to a rib.
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Fig. 4. Scleraxis is expressed in putative limb tendon progenitors. Scleraxis expression in developing wings and legs was analyzed by whole-mount and section in situ hybridization. All limbs are shown in dorsal view. (A,B) Scleraxis is first detected in stage 21 leg buds in a superficial proximomedial domain and expression in this domain is enhanced by stage 23. (C) In situ hybridization to sections of a leg bud at stage 23 illustrates that the expression is superficial in both the dorsal and ventral mesenchyme. (D) This expression domain overlaps partially with the domain of migrating myoblasts, detected in an adjacent section by expression of Pax3. By stage 25, the scleraxis-expressing cells coalesce to form more discrete, limb-specific patterns in the leg (E) and wing (H). By stage 27, the dynamic expression of scleraxis continues to change in both leg (F) and wing (I). The first fibrous tendon elements can be seen in the proximal leg bud (F), concurrent with the onset of scleraxis expression in the forming autopod. The expression is elaborated by stage 29 to include in both leg (G) and wing (J) much longer tendon fibers and the phalangeal tendon blastemas. (K-M) To determine the spatial relationship between the scleraxis-expressing cells and the related tissues alternating transverse limb bud sections were hybridized with probes to scleraxis, a muscle marker, MyoD, and an early cartilage marker, autotaxin. In stage 27 leg buds, the domain of scleraxis-expressing cells (M) is still largely overlapping with that of the now differentiating MyoD-expressing cells (L). However, neither overlaps with the differentiating cartilage elements in the deeper limb mesenchyme (K).
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Fig. 5. Scleraxis is expressed in mouse limb tendons and their progenitors. Scleraxis expression detected by whole-mount in situ hybridization is shown in a developmental series of mouse forelimbs. The expression is shown in a dorsal view (A,B,D-G) and lateral view (C). As in chick, scleraxis expression is first detected in the proximomedial (A) and superficial (C) limb mesenchyme between E10 and E11. (B) Scleraxis expression is not altered in E10 splotch mutant embryos. (D,E) By E12 the expression is much more complex with distinct fibrous elements, and by E12.5 further elaboration of the proximal pattern and the early autopod expression can be detected. (F,G) At E13.5 the digit related expression extends over the whole length of the growing digits and by E14.5 the mature and complex tendon pattern including the phalangeal insertions can be detected.
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Fig. 6. The ectoderm and mesenchymal BMPs define the early domain of scleraxis expression. (A,B) Early ectoderm removal results in a complete loss of scleraxis expression. The medial ectoderm was removed from stage 21 right wing buds, and the embryos were harvested 14-20 hours later and processed for whole-mount in situ hybridization using a scleraxis probe. While scleraxis expression is clearly detected in the control left wing bud, expression is not seen in the operated wing bud (A). The area where the ectoderm was removed is easy to detect in a lateral view of the operated limb bud (arrows in B). Note the normal expression of scleraxis in the ventral limb bud and lack of expression in the dorsal mesenchyme. (C,D) To determine whether the ectoderm is required for maintenance of scleraxis expression, ectoderm was removed from wing buds at stage 24, after the onset of scleraxis expression, and the embryos again harvested after 14-20 hours. Low scleraxis expression is detected in the operated wing bud compared with the robust expression in the control left wing bud (C). In a lateral view of the operated wing bud (D), the region of ectoderm removal can again be easily detected (arrows in D) and residual scleraxis expression in the exposed mesenchyme can be seen. (E) Tbx5 expression is maintained in the exposed mesenchyme of a stage 22 wing bud 14 hours after ectoderm removal. (F-I) The early, proximomedial scleraxis expression domain in the wing bud is mutually exclusive to that of BMPs. Comparison of the early expression of Bmp2, Bmp4, Bmp7 and scleraxis; in wing buds from stage 22 embryos.
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Fig. 7. BMP signaling restricts scleraxis expression. BMP protein (A-D) and the BMP antagonist Noggin (E-J) were applied to wing buds and the effects on scleraxis expression were monitored by whole-mount in situ hybridization. (A-D) Scleraxis expression is repressed by BMP signaling. (A,B) Presumptive limb and adjacent somites of stage 10 embryos were infected with a BMP4 retrovirus. Repression of scleraxis is apparent in the somites (arrowheads in A) and in the limbs – compare in B the expression in the infected right wing bud (red arrowhead) and the control left wing bud. (C,D) Beads soaked in BMP4 protein (75 µg/ml) were implanted into the right wing bud of a stage 23 embryo and the embryos harvested after 3 hours (C) or into stage 25 embryos, which were harvested after 6 hours (D). A dramatic down regulation of scleraxis expression is detected in both cases. (E-G) Upregulation of scleraxis expression by antagonizing BMP signaling. (E) Embryo were infected at stage 10 with a Noggin-expressing retrovirus and harvested after 2 days at stage 22. Scleraxis expression, seen here in ventral view, was induced throughout most of the limb bud mesenchyme. (F) Beads soaked in recombinant Noggin protein were implanted into stage 23 wing buds, and the embryos were harvested after 6 hours. Scleraxis is induced, but only in the ventral mesenchyme adjacent to the bead (see inset in F). (G) Weak but distinct induction of scleraxis can be seen 3 hours after a Noggin-soaked bead was implanted into a stage 21 wing bud. (H-I) Noggin cannot replace the ectodermal inducing signal. (H) The right wing buds of a stage 10 embryo was infected with a Noggin retrovirus at stage 10. The ectoderm was subsequently removed at stage 21 and the embryos harvested after 24 hours. Scleraxis expression is not detected in the exposed mesenchyme (arrowhead), but broad induction of scleraxis can be seen in the regions where ectoderm healing had occurred. (I,J) The ectoderm was removed from the right wing bud of a stage 21 embryo and a bead soaked in Noggin protein was implanted into the exposed mesenchyme. The embryos were harvested after 10 hours. (I) Scleraxis expression was not induced in the dorsal exposed mesenchyme. The dark background around the bead is a reflection of scleraxis induction by the Noggin protein in the ventral mesenchyme, which is clearly seen in a ventral view of the same embryo (arrowhead in J).
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Fig. 8. Endogenous noggin contributes to the induction of autopod tendons. Induction of scleraxis expression in the autopod (B) is concurrent with noggin expression in the condensing digit cartilage (A) and high BMP expression in the interdigital mesenchyme (C). (D) Mild infection of limb buds at stage 19 with the Noggin retrovirus, resulted in limited spread of the virus by stage 29. In such limbs, which appear grossly normal, ectopic induction of scleraxis in the autopod was seen (see arrowhead in D). (E) Beads soaked in Noggin protein were implanted in the autopod at stage 28 and the embryos were harvested 24 hours later. Limited scleraxis induction (arrowhead in E) was detected in roughly half of the embryos (n=10). (F-J) Comparison of scleraxis expression in wild-type and mutant mouse embryos homozygous for a targeted deletion in the noggin gene. (G-J) Expression is shown in E13.5 hind limbs. While dorsal autopod expression of scleraxis is already broad and extends along the forming digits of wild-type limbs (G), the expression is very low in the dorsal side of a mutant autopod (H). In the ventral side of mutant forelimbs scleraxis is expressed and its domain is broader than the expression in the ventral autopod of a wild-type hindlimb (compare I with J). (F) By E14.5, dorsal tendons do form in the noggin mutant, though they are thinner and less developed compared with wild type (compare F with Fig. 5G).
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Fig. 9. Regulation of the tendon cell fate. The tendon progenitors, represented by the early limb expression of scleraxis, are found in the superficial proximomedial limb bud mesenchyme. Scleraxis expression is induced by a ubiquitous signal emanating from the ectoderm. However, the induction of progenitors is restricted to the proximomedial domain by the presence of localized BMP signaling, which represses scleraxis expression in other regions. Antagonizing the BMP signal by misexpression of Noggin results in a much broader induction of tendon progenitors. Nevertheless, the presence of excess progenitors does not lead to the production of excess tendons, suggesting that other signals regulate mature tendon formation. The same set of signals also appear to be required for distal tendon formation, where endogenous Noggin expressed by the condensing cartilage appears to contribute to the induction of the tendon cell fate.
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© The Company of Biologists Ltd 2001
