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First published online 13 June 2007
doi: 10.1242/dev.001933
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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.
* Author for correspondence (e-mail: Ronen{at}shcc.org)
Accepted 15 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Scleraxis, Tendon, Connective tissue, Mouse mutant
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Conversely, the origin tendons at the proximal end of the same arm or shank
muscles mainly serve to anchor the muscles at their skeletal origins. An
extreme example of anchoring tendons can be found in the monolayer of tendon
cells that surround the ribs and provide the matrix that connects the
intercostal muscles to the ribs.
Tendon studies have focused mostly on force-transmitting tendons and
considerable effort has been directed towards the analysis of the protein
composition and organization of these tendons and towards understanding how
this organization provides the physical properties necessary for tendon
function (Benjamin and Ralphs,
2000; Zhang et al.,
2005). By contrast, the ontogeny of the tendons has received far
less attention, and little is currently known about the molecules regulating
tendon specification, differentiation, patterning or matrix deposition.
Some insight into the requirements for tendon formation has been attained
via the analysis of mice harboring genetic mutations that result in discrete
tendon phenotypes (reviewed in Tozer and
Duprez, 2005). For example, mutations in the small leucine-rich
proteoglycan genes decorin (Dcn)
(Zhang et al., 2006),
fibromodulin (Fmod) (Svensson et
al., 1999) and biglycan (Bgn)
(Ameye and Young, 2002) result
in irregularities in the diameter of collagen fibers in the tendons. Tendon
disruptions have also been described in patients with conditions affecting
matrix molecules, such as the Ehler-Danlos syndrome, in which the collagen I
gene is mutated (Mao and Bristow,
2001). Although these studies highlight the obvious importance of
the structural elements that make up the tendon, to date, there is no
description of mutations affecting tendon differentiation and patterning.
Several years ago, we found that the bHLH transcription factor scleraxis
(Scx) is a distinct marker for tendon and ligament progenitors, and
differentiated cells (Cserjesi et al.,
1995; Schweitzer et al.,
2001). Scx is a highly specific marker of the tendon/ligament
lineage and appears to be induced at the earliest stage of specification of
this lineage (Brent et al.,
2003; Brent and Tabin,
2004; Schweitzer et al.,
2001). As the first robust marker for tendon progenitors,
Scx expression provided a unique opportunity to follow the origin and
regulation of tendon cell fate. We identified a tendon progenitor pool in the
limb subjacent to the ectoderm (Schweitzer
et al., 2001), and a somitic compartment of tendon progenitors,
the syndetome, was later identified at the dorsolateral edge of the early
sclerotome, arising in response to Fgf signaling from the adjacent myotome
(Brent et al., 2003;
Brent et al., 2005).
Analysis of Scx expression has thus proven to be an extremely
powerful tool in tendon studies, but Scx function in tendon formation
has not been elucidated. Although Scx misexpression is not sufficient
to alter the normal progression of tendon formation
(Schweitzer et al., 2001), as
a bHLH transcription factor expressed in all tendon cells from progenitor
stages through development, Scx is none-the-less a likely candidate to be a
transcription factor that is involved in regulating the tendon cell fate.
In a previous attempt to explore Scx function, embryos homozygous for a
targeted ScxKO allele died in the early stages of
embryogenesis, thus precluding the ability to address a possible role of Scx
in tendon differentiation (Brown et al.,
1999). Surprisingly, we found that the reported
ScxKO phenotype was largely due to an effect of the
neomycin-resistance (Neo) cassette on neighboring genes. We therefore
generated a new loss-of-function allele of Scx to re-evaluate the
phenotype of a simple Scx mutation.
Homozygous Scx-/- mice were viable but showed a dramatic disruption of tendon differentiation, manifesting in dorsal flexure of the forelimb paw, the limited use of all paws, reduced functionality of the back muscles and the complete loss of the ability to move the tail. Surprisingly, Scx loss did not affect all tendon categories equally. Whereas all force-transmitting and intermuscular tendons were affected in Scx-/- mutants (although the effects varied in severity), the muscle-anchoring tendons and the ligaments were not affected. The tendon defects were first detected at or close to E13.5 in all tendons, concurrent with the condensation and differentiation of tendon progenitors that result in emergence of discreet tendon morphologies. Scx however, has additional roles in tendon formation and, in tendons that persist in Scx-/- mice, we found that the tendon matrix was reduced and disorganized, and the cellular organization of the tendons was disrupted.
This is the first report of a genetic alteration producing a tendon differentiation phenotype. While emphasizing the diversity of tendon tissues, it lays the foundation for unraveling the genes required for tendon formation, and opens the door to the analysis of the transcriptional network regulating the tendon cell fate.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The 5' loxP sequence followed by a HindIII
site was introduced into a SacII restriction site in the 5' UTR
of Scx and the first exon of Scx was flanked by inserting
the FLP recombinase target (FRT)-Neo cassette appended with a 3' loxP
site (Meyers et al., 1998)
into an EcoRI restriction site in the single intron of the
Scx gene. Out of 180 embryonic stem cell (ES) clones assayed,
approximately 60 were targeted to the Scx locus and 27 retained both
loxP sites, as determined by Southern blot using the HindIII site in
the 5' loxP site. A single Scxfneo male was
generated by blastocyst injection and mated with FLPeR females
(Farley et al., 2000) to
produce the conditional Scxflox allele.
Scxflox males, in turn, were mated with prx1Cre
females, taking advantage of the stochastic ubiquitous Cre activity in females
from this line (Logan et al.,
2002) to generate the Scx-null allele (see
Fig. 1A). For genotyping
conditions, see Table 1.
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), anti-EphA4 antibody (1:50; R&D Systems) and the
anti-tenascin C antibody (1:100; gift from Harold Erickson, Duke University
Medical Center, Durham, NC). Images were captured using a Nikon Eclipse E800
compound microscope and a MicroPublisher cooled CCD camera from QImaging. Whole-mount and section in situ hybridizations were performed using protocols found on the Tabin laboratory web pages: http://genepath.med.harvard.edu/~cepko/protocol/ctlab/ish.ct.htm and http://genepath.med.harvard.edu/~cepko/protocol/insituprotocol.pdf.
Probes included Scx (Brown et al.,
1999) that was also used to detect the ScxGFP transcript
(Pryce et al., 2007) and
tenomodulin (Brandau et al.,
2001). The probe for collagen XIV was amplified from the fourth
exon of the gene using the following primers: Col14for,
5'-TCCTGATCAGCCTGAGTTCACACCG-3'; Col14rev,
5'-CAGGAGTGTGTTTCAGGGCTTGCCT-3'.
Cell proliferation was detected using the BrdU In-Situ Detection Kit (BD Pharmingen, #550803). The ScxGFP signal was captured from directly alternating sections and merged with the BrdU signal in Photoshop (Adobe). For apoptosis, a standard TUNEL protocol was followed performing the terminal transferase reaction with a biotin-16-dUTP, followed by strepavidin-Cy5 for detection.
Counting tenocytes in a cross section of the FDP tendon
ScxGFP-positive cells were counted in flexor digitorum profoundum
(FDP) tendons in sections at the level of the digit vinculum of both wild-type
and mutant embryos at E18.5 (Fig.
4B,E, white arrowhead). The sections were stained with an antibody
to beta-catenin to highlight the cell membranes and with DAPI to highlight the
nuclei and help identify the individual cells. Cells were counted in ten
sections from five different embryos and gave an average of 97 FDP
tenocytes/section in wild-type embryos and 56 FDP tenocytes/section in mutant
embryos.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), but
ScxKO embryos died before E8.5, precluding the evaluation
of the effects on tendon formation (Brown
et al., 1999). We therefore decided to generate a conditional
allele in the Scx locus (by flanking the first exon, which includes
most of the open reading frame, with loxP sites) and used the FRT-Neo
cassette (Meyers et al., 1998)
to enable selective removal of the Neo cassette
(Fig. 1A). Founder
Scxfneo mice were mated with mice expressing FLP
recombinase and Cre recombinase in the germline to generate a conditional
allele, Scxflox, and a null allele,
Scx- (Fig. 1A
and see Materials and methods). Homozygous Scxflox/flox
mice were viable, but, surprisingly, homozygous
Scxfneo/fneo embryos died at approximately E10.5 (data not
shown). The only difference between the two alleles is the Neo cassette
embedded in the single intron of the Scx gene, suggesting that the
Neo cassette in this locus is sufficient to cause embryonic lethality.
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;
Olson et al., 1996;
Sartorelli and Caretti, 2005).
The organization of the genomic locus of Scx provides a likely
explanation for the dramatic effect of the Neo cassette in this instance. The
entire Scx gene is located in the third intron of block of
proliferation 1 (Bop1; Fig.
1B), a housekeeping gene required for ribosome biogenesis
(Strezoska et al., 2000;
Strezoska et al., 2002). It is
therefore likely that the lethality observed in
Scxfneo/fneo embryos is caused by an effect on the
transcription levels and/or splicing of the Bop1 gene. Because the
original ScxKO allele
(Brown et al., 1999) also
introduced a Neo cassette in a similar genomic position, it is likely that the
phenotype observed in that study was also affected by the presence of the Neo
cassette. We therefore decided to re-evaluate the phenotype of a simple
deletion of the Scx gene using the Scx- allele
that we have generated.
Scx-/- mice are viable and display severe disruption of force-transmitting tendons
Contrary to the early lethality of ScxKO/KO embryos,
homozygous Scx-/- mice survived to term. Indeed,
homozygous Scx-/- embryos could not be distinguished from
their littermates (data not shown), and hence their identity was verified by
PCR (data not shown) and by in situ hybridization with an Scx probe
(Fig. 1C). Although
Scx-/- mutant mice were viable, they displayed phenotypes
that suggest a severe tendon disruption (see Movie 1 in the supplementary
material). In mutants examined immediately after birth, the autopod of the
forelimb was locked in a dorsal flexure, and the mice walked on their wrists
(Fig. 2A). Furthermore,
Scx-/- mutants could not grip the cage railing with the
paws of the fore or hind limbs and their tails were completely immobile. In
addition, Scx-/- mutants demonstrated a severe impairment
in the use of their back muscles and found it very difficult to turn over when
placed lying on their back (see Movie 1 in the supplementary material). In
principle, some of these impairments could be caused by skeletal
malformations, but we found no significant skeletal defects, with the
exception of a loss of the deltoid tuberosity - a lateral outgrowth of the
humerus whose formation is dependent on the activity of the attached muscles -
suggesting that the deltoid tuberosity may also be missing in
Scx-/- mice because of a tendon phenotype
(Fig. 2B) (E. Zelzer and R.S.,
unpublished results). To characterize possible tendon disruptions that cause
these phenotypes, we wanted to examine tendon morphology in the mutants.
Because we could not use Scx expression to detect the tendons in
Scx-/- mutant embryos, we took advantage of
ScxGFP, a transgenic tendon reporter mouse line, in which the
expression of GFP is regulated by Scx enhancers, resulting in strong
GFP expression in all tendon and ligament cells
(Pryce et al., 2007). Crossing
this transgene into the Scx-/- background enabled
visualization of tendons and tendon progenitors in the mutants. The tendon
phenotype was additionally corroborated by staining for the tendon structural
protein tenascin C (Chiquet and Fambrough,
1984) and by in situ hybridization for collagen I (data not
shown).
Using the ScxGFP reporter, we indeed found a severe disruption of tendon formation in Scx-/- mutants at E18.5. We first observed a significant loss of limb tendons; some tendons were completely missing in Scx-/- mutants, whereas others appeared small and rudimentary compared with those of wild-type littermates (Fig. 2C-F). For example, in wild-type embryos, the major extensor tendons of the forelimb, the extensor digitorium communis (EDC), originated in the EDC muscles close to the elbow and extended in close proximity up to the wrist, where they separated and continued along each digit, finally inserting at the tip of the digit (Fig. 2C, yellow arrowheads). In Scx-/- embryos, however, the EDC could be detected in the digits, but the tendons disappeared at the wrist level (Fig. 2D, yellow arrowheads). By contrast, the extensor digiti quinti and the extensor carpi ulnaris tendons were complete in the mutant, but were still significantly reduced compared with their wild-type counterparts (Fig. 2C,D, long and short pink arrowheads, respectively). In Scx-/- mutants, the flexor tendons were affected more severely than the extensors; not even rudimentary flexor tendons were apparent in the zeugopod and only some flexors were detected in the digits (Fig. 2E,F). Even more dramatically, the inability of the mutants to use their tails was the result of a complete loss of all tail tendons, as visualized in 2-week-old mice, when collagen fibers of the tendons can be detected by direct illumination and the cells in these tendons could be seen by ScxGFP fluorescence (Fig. 2G,H, yellow arrowheads). Finally, we found a similar scenario in the trunk (Fig. 2I,J,M,N): whereas short axial tendons were possibly somewhat disrupted or reduced (Fig. 2, pink arrowhead), the long tendons were completely absent (Fig. 2, yellow arrowheads), as was seen both in whole-mount GFP images (Fig. 2I,J) and in sections counterstained with an antibody directed against myosin heavy chain (MHC) to visualize the axial musculature (Fig. 2M,N).
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Ligaments, which connect bone to bone, are closely related to tendons and
also express Scx from early stages
(Schweitzer et al., 2001).
There are, however, distinct differences between tendons and ligaments in the
patterning, shape, composition and structure of their collagen fibers. We
therefore examined the integrity of ligaments in Scx-/-
mutants. In sagittal sections through the knee of Scx-/-
embryos, we found that the cruciate ligaments of the knee and the patellar
ligament that connects the patella to the tibia appeared normal in both
morphology, and in expression of ScxGFP (data not shown) and of
collagen I (Fig. 3E,F, yellow
and black arrowheads, respectively). Conversely, the rectus femoris tendon,
connecting the patella to the rectus femoris muscle, was almost entirely
missing in Scx-/- mutants, emphasizing the sharp contrast
between the phenotypic effects on tendons and ligaments
(Fig. 3E,F, purple
arrowheads).
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In addition to its expression in the tendon and ligament lineages, the
Scx gene is expressed in a number of other tissues, including in the
sertoli cells of the testes (Muir et al.,
2005), in the bronchi of the lungs and in the kidneys
(Pryce et al., 2007). Major
structural defects were not observed in these tissues in
Scx-/- mutants (data not shown), but potential roles for
Scx in these tissues might be revealed upon closer examination in future
studies.
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To determine which steps of tendon morphogenesis are affected in
Scx-/- mutants, we examined the limbs at earlier stages.
In wild-type mice, the distribution of tendon progenitors in limb buds is
highly dynamic up to E12.5 (Fig.
4H) (Schweitzer et al.,
2001). A sharp transition involving the condensation of the
progenitors occurs by E13.5 (Fig.
4I) and, by early E14.0 (Fig.
4K), distinct tendons can be detected. ScxGFP expression
in Scx-/- embryos was not different from that of wild type
up until E12.5, and distinct defects were first observed at E13.5. For
example, in wild type, the EDC tendons at the wrist level originated from a
patch of progenitors that transformed into a triangular tendon blastema at
E13.5 and into the distinct EDC tendons by E14.0
(Fig. 4H,I,K, pink arrowheads).
The triangular EDC tendon blastema did not form in the mutant at E13.5
(Fig. 4L, pink arrowhead),
representing the first manifestation of the EDC phenotype described above
(Fig. 2C,D). The dramatic
flexor phenotype at the metacarpal level was also initiated at this stage. In
cross sections of E13.5 forelimbs at the proximal metacarpal level in
wild-type mice (Fig. 4A, level
4), the ventral progenitors split into two distinct condensed layers
(Fig. 4J) that will give rise
to the FDP (Fig. 4J, yellow
arrowhead) and the palmar metacarpal ligament
(Fig. 4J, white arrowhead). In
the mutant, these condensations failed to form, and the tendon progenitors
remained as loosely organized mesenchyme
(Fig. 4M). Both phenotypes
indicate a specific requirement for Scx activity during the condensation of
the tendon progenitors to form the long tendons of the limb.
In addition to the dramatic tendon loss discussed above, other limb tendons that formed in the mutant appeared smaller than their wild-type counterparts (Fig. 2D), and this mutant phenotype could be detected as soon as these tendons were formed at E14.5. We therefore looked for changes in cell proliferation or cell death that may cause these size differences. TUNEL staining of limb sections at digit levels of E14.5 embryos identified the expected cell death in interdigital mesenchyme in mutant mice (Fig. 4R,T). Cell death was not detected in wild-type tendons (Fig. 4R,S), but distinct cell death was observed in the FDP of mutant embryos (Fig. 4T,U). Interestingly, events of cell death were not identified in other limb tendons, ruling out the possibility that cell death is responsible for the general rudimentary nature of tendons in Scx-/- embryos (data not shown). Next, we looked for changes in cell proliferation in limb tendons of Scx-/- embryos. Interestingly, in all proximal-distal levels that showed positive BrdU signals in tendons, we found proliferating cells in the same tendons in limbs from both wild-type and mutant embryos (Fig. 4N-Q, red arrowheads), demonstrating that Scx is not essential for cell proliferation in tendons. However, because of the rudimentary nature of mutant tendons, we could not faithfully compare rates of cell proliferation and therefore cannot exclude the possibility of slower rates of proliferation in mutant tendons, but the early detection of the tendon phenotype suggests that a mere reduction in proliferation would not be sufficient to account for the observed tendon phenotype in mutants. In conclusion, these results suggest that proliferation and cell death do not play a major role in determining the size of tendons in mutant embryos. Because the tendon phenotypes emerged concomitantly with overt tendon formation, we hypothesize that, similar to the dramatic tendon phenotypes described above, the reduced tendon size in mutant embryos might also be attributable to a tendon condensation defect in which a smaller number of progenitors are incorporated into the tendons in Scx-/- embryos.
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Axial tendon progenitors are induced in the syndetome, at the intersection
between two somites (Brent et al.,
2003). In somites of the wild-type tail, we observed that the
syndetome is subsequently separated into dorsal and ventral domains giving
rise to the progenitor populations of the two dorsal and two ventral tendons
(Fig. 5A, black arrowheads).
Subsequently, the syndetomal progenitor populations condensed and extended
rostrally (Fig. 5B). The dorsal
and ventral tendons elongated to the base of the tail, thus establishing the
individual tendons that connect each tail bone with the tail muscles
(Fig. 5B). In cross section at
E15.5, the tight individual tendon condensations could be clearly seen
(Fig. 5H, pink arrowhead). By
the end of embryogenesis, at E18.5, each vertebrae generated complete tendons
and these tendons were organized in four radial groups
(Fig. 5C,H) that coalesced
after birth to give rise to the massive tendons of the tail
(Fig. 5D).
In Scx-/- mutant embryos, the induction of the syndetome and the separation of the dorsal and ventral progenitor populations were not disrupted (Fig. 5F, black arrowheads). As in the limb, the tendon phenotype emerged at E13.5 with very limited condensation of tendon progenitors (Fig. 5G), and there was a continued presence of sparsely distributed tendon cells in later stages (Fig. 5I, pink arrowhead). Consequently, tendon elongation was very limited in mutant embryos and tendon cells from multiple segments seemed to get incorporated into one or a small number of tendons (Fig. 5G). Similar to limb tendons, we did not detect cell death in tail tendons from mutant embryos at these stages (data not shown), and cell proliferation could easily be detected in all the rudimentary tail tendons of mutant embryos (Fig. 5J-M). At the end of embryogenesis, the tails of Scx-/- embryos contained only a small number of tendon rudiments (Fig. 5E).
In conclusion, in tail as well as limb tendons, the stage that is affected most in tendons of Scx-/- embryos appears to be the condensation and incorporation of progenitors into mature tendons. The small number of tenocytes that persist in the tail retain a capacity to organize a tendon structure, suggesting that, as in the limb, this essential feature of tenocytes is not regulated by Scx. However, these tenocytes subsequently undergo cell death (Fig. 6H), leading to the complete loss of tail tendons (Fig. 2H).
Organization of the endotenon and of the tendon matrix is disrupted in Scx-/- mutants
We have so far shown an essential role for Scx in the early stages of
tendon differentiation. In wild-type embryos, once the tenocytes are properly
organized they produce the remarkably structured extracellular components of
the tendons, the tightly packed bundles of collagen fibers
(Benjamin and Ralphs, 2000). To
investigate the possible involvement of Scx in these later aspects of tendon
morphogenesis, we focused on the most robust tendon that does form in
Scx-/- mutants - the digit segment of the FDP tendon
(Fig. 4E,F). The FDP tendon in
the digit of Scx-/- embryos was significantly smaller
compared with a wild-type littermate (Fig.
4B,E and Fig.
6A,E). The size difference might result from lower cell numbers
caused by the cell death that we detected in these tendons
(Fig. 4R-U), and, indeed, we
found that mutant FDP tendons had approximately 60% of the number of cells
found in the wild-type FDP tendons (see Materials and methods). Moreover, the
cells in the mutant tendons appeared to be more compact in histological
staining (Fig. 6A,E),
indicating that matrix deposition is probably also affected. The reduced
matrix content was apparent in intermediate-magnification transmission
electron microscope (TEM) images, which showed the parallel collagen fibers in
cross section (Fig. 6B,F,
yellow arrowheads). Although bundles of collagen fibers could be detected in
the mutant, the amount of matrix was greatly reduced
(Fig. 6B,F), and unoccupied
spaces were common in the mutant, but not in the wild-type, tendon. Moreover,
in wild-type embryos, the tenocytes developed a complex network of cytoplasmic
extensions (Fig. 6B-D, red
arrowheads) that engulfed bundles of collagen fibers in order to regulate the
localized deposition of matrix components. These cytoplasmic extensions were
also severely decreased and appeared less complex in the mutant
(Fig. 6F,G). Another component
of the tendon matrix affected in the Scx-/- mutants were
the microfibrils (Fig. 6C,
green arrowhead). Normally found in discrete bundles in the wild-type tendon,
the microfibrils in mutant tendons were highly disorganized
(Fig. 6G, green arrowhead). The
decrease in collagen deposition and loss of cytoplasmic extensions was seen
even more dramatically in the highly rudimentary tail tendons of
Scx-/- mutants at E18.5
(Fig. 5E and
Fig. 6D,H). In tail sections,
the tenocytes could also be seen undergoing apoptosis in the mutant, as
evident by their highly enlarged nuclei
(Fig. 6H). Indeed, although
tendon rudiments could be seen in mutant embryos, they were completely absent
in 2-week-old mutant mice.
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; Kannus,
2000). Whereas endotenon cells were organized in a sheath of
stretched cells in the FDP of wild-type E18.5 embryos, in
Scx-/- embryos, the endotenon cells appeared disorganized
and failed to generate a continuous layer
(Fig. 7B). Staining for
endotenon markers further highlighted the disrupted organization of the
endotenon. In agreement with previous observations in chick embryos
(D'Souza and Patel, 1999), we
found that the ephrin receptor EphA4 was expressed in the endotenon of limb
tendons (Fig. 7C). In the
mutant, EphA4-expressing cells were intermixed with the tenocytes,
emphasizing a disruption of the regular organization of the endotenon
(Fig. 7D). Similarly, in the
digit flexors, the matrix protein tenascin C was exclusively organized in the
endotenon (Fig. 7E), but, in
Scx-/- embryos, tenascin C protein was fully intermixed
with the FDP tenocytes (Fig.
7F,G), again demonstrating that the structural integrity of the
endotenon is lost in Scx-/- mutants. It is important to note that Scx is exclusively expressed in tenocytes, suggesting that the effect on the sheath is indirect. However, because the lineage of the endotenon cells has not been determined, it is also possible that these cells may express Scx at earlier stages of their development.
Molecular characterization of the Scx-/- tendon phenotype
We have found that Scx has multiple essential roles in tendon formation,
affecting both tendon differentiation and organization. To identify genes
regulated by Scx that could be responsible for Scx function in tendon
formation, we therefore examined the expression of genes known to be expressed
specifically in all tendons [e.g. tenomodulin
(Brandau et al., 2001);
collagen I and collagen XIV (Walchli et
al., 1994; Young et al.,
2000); collagen XII (Dublet
and van der Rest, 1987); tenascin C
(Chiquet and Fambrough, 1984);
fibromodulin, lumican and decorin (Ezura
et al., 2000)] or genes described as being specifically expressed
in tendons within the context of the limb bud [e.g. Six2
(Oliver et al., 1995),
Eya1 (Xu et al.,
1997) and Fgf18 (Liu
et al., 2002)]. Of these, only two showed a complete loss of
expression in Scx-/- mutants: collagen XIV and tenomodulin
(Fig. 8A,B,D,E and data not
shown). Collagen XIV is a fascicle-associated collagen involved in the
regulation of the diameter of collagen fibers
(Gelse et al., 2003;
Young et al., 2002).
Tenomodulin is a single transmembrane protein
(Brandau et al., 2001), and a
mild proliferation defect has been detected in tendons of tenomodulin mutants
(Docheva et al., 2005).
Interestingly, the induction of tenomodulin gene expression by Scx
misexpression was recently demonstrated in chick
(Shukunami et al., 2006). We
also detected a slight but reproducible decrease in the levels of collagen I
gene expression in Scx-/- embryos
(Fig. 8C,D). Based on their
known properties, it is unlikely that the modulation of these three genes is
sufficient to explain the phenotypic effects that we observe upon the loss of
Scx activity.
The fact that tendon progenitors are present and many tendons still form in
the mutants suggests that, despite its early expression in tendon progenitors,
Scx is not necessary for tenocyte specification. We wanted to test whether
this could be explained by partial redundancy with other, related
transcription factors. Scx belongs to the small Twist subfamily of the bHLH
transcription factors (Atchley and Fitch,
1997). Within this subfamily, paraxis (also known as Tcf15 - Mouse
Genome Informatics) (Burgess et al.,
1995), is the closest relative to Scx and the twist genes
represent more-distant subfamily members. The small Hand subfamily of bHLH
transcription factors is also highly related to Scx
(Atchley and Fitch, 1997;
Firulli, 2003). We did not
detect a distinct tendon expression for any of these genes when examined by in
situ hybridization (data not shown). Furthermore, neither paraxis nor
Hand1 or Hand2 expression was upregulated in
Scx-/- mutants.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Tozer and Duprez, 2005). To
determine the role of Scx in tendon formation, we created a targeted null
allele of the Scx gene. Homozygous Scx-/- mice
were viable but showed a dramatic defect in tendon differentiation resulting
in a severe loss of intermuscular tendons and of the tendons responsible for
transmitting musculoskeletal force in the limbs, tail and trunk. Surprisingly,
there was no discernible defect in other categories of tendons whose function
is mostly to anchor muscles and dissipate the force generated by muscle
contraction. Tendon progenitors were induced in Scx-/- embryos and tendon phenotypes were first detected at E13.5, concomitant with the incorporation of tendon progenitors into histologically distinct tendons. The most striking phenotype in Scx-/- embryos was the failure of tendon progenitors to condense into morphologically distinct tendons. The variable effect on different tendon categories highlights the molecular and structural diversity within the broad definition of tissues as tendons. Moreover, we show that Scx plays a role in later aspects of tendon formation, including in the secretion of structural matrix components, in the production of extended cytoplasmic extensions that support matrix organization, and in the interplay between tenocytes and endotenon cells.
As a result of differential severity in the defects in tendon formation, the normal balance between extensor and flexor forces in the limbs was disrupted in mutant mice, resulting in non-physiological flexures and the complete loss of some functions of the muscular skeletal system. These phenotypic features are reminiscent of a congenital human malformation known as arthrogryposis, suggesting that mild Scx mutations may serve as a model for this condition.
Scx is required for the differentiation and formation of force-transmitting and intermuscular tendons
The definition of tendons as the connective tissues that connect muscle to
bone accounts for a highly diverse set of tissues. Most studies focus on the
robust tendons of the limbs and tail, which show a remarkable specialization
to support transmission of force over considerable distances. However, the
force-transmitting tendons are complemented by tendons whose physiological
role is mostly to anchor muscles to respective skeletal attachments. Anchoring
tendons include the origin tendons of limb muscles and many of the trunk
tendons, notably including the attachments of the intercostal muscles through
a monolayer of tendon cells that surround the ribs. Anchoring tendons
frequently exhibit broad contact areas with the muscles or skeletal elements,
reflecting the need to dissipate the force generated by the muscles. Finally,
a divergent group of tendons, the intermuscular tendons, connect muscle
segments such as the middle tendon of the diaphragm and probably play a role
in maintaining the integrity of the muscles in question.
When we first cloned Scx, we noticed that it was expressed in all
tendon tissues, reinforcing the notion that they all represent a single
well-defined cell and tissue identity
(Brent et al., 2003;
Schweitzer et al., 2001). It
is therefore fascinating that Scx function does not seem to be required for
that core identity of the tendon tissues, but rather is specific to a distinct
subset of tendon tissues, thus providing the first direct demonstration of
differences in the molecular processes that confer the cellular identities of
the different tendon categories.
Our analysis suggests that Scx function is required for the normal differentiation of force-transmitting tendons and of intermuscular tendons, and, noticeably, all the tendons in these categories are affected in Scx-/- mutants. Although there is no obvious similarity between these two tendon groups, it is important to note that, in both categories, in the mutants, most of the muscles remained attached to their targets, emphasizing that Scx function is crucial for the generation of the complex tendon structures, but not for the capacity of tendons to mediate tissue attachments. Moreover, despite the considerable variability observed in the phenotypes of force-transmitting tendons in mutants, in all cases, the phenotype was first detected in the transition from tendon progenitors to condensed and overtly distinct tendons, suggesting that the scleraxis function is likely to be related to the incorporation of tendon progenitors into discrete tendons. Consequently, the difference in phenotypic severity might represent the difference between the complete failure of progenitor condensation and partial condensation resulting in smaller rudimentary tendons.
Scx affects both cellular differentiation and extracellular matrix organization in tendons
The functional components of the mature tendon are highly organized bundles
of collagen fibers that transmit the forces between the muscles and the
skeleton. Little, however, is known about the molecular processes that enable
the tenocytes to coordinate the secretion and organization of these matrix
structures during tendon genesis (Benjamin
and Ralphs, 2000; Kannus,
2000). The most robust tendon in Scx-/-
mutants, the digit segment of the FDP, displayed a variety of defects in its
ability to produce the tendon matrix, manifesting in a dramatic decrease in
the number of collagen fibers and in their organization. Concomitantly, we
observed a decrease in the number of tenocytes present and a partial loss of
the network of cytoplasmic processes that enable the tenocytes to regulate the
local secretion and synthesis of collagen fibers. It will be interesting to
determine in future studies whether the effects on the organization of the
matrix and on the cellular morphology are both directly regulated by Scx or
whether these aspects of tendon structure are mutually dependent so that a
primary effect on one of them may secondarily disrupt the other.
Transcriptional regulation of tendon cell fate
The transcriptional regulation of the tendon cell fate has not been
addressed previously. This study provides for the first time two pivotal
observations regarding the tendon transcriptome. First, Scx is essential for
tendon differentiation and for the incorporation of tendon progenitors in
mature tendons. Future discovery of the identity and function of Scx targets
will therefore help unravel the molecular processes involved in tendon
differentiation. Second, the induction and maintenance of the tendon cell fate
are not dependent on Scx function, nor is the expression of Scx
itself, because expression of the ScxGFP reporter was maintained in
tendon cells of Scx-/- mutants. Transcriptional regulators
of the Scx gene may therefore represent key players in tendon
formation.
This study represents the first demonstration of a tendon differentiation phenotype. Moreover, the elucidation of Scx function and the tools that it allowed us to develop provide a powerful starting point for future studies of the transcriptional regulation of this important and often neglected tissue.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/14/2697/DC1
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ACKNOWLEDGMENTS |
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