Department of Biomedical Engineering and Orthopaedic Research Center, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA
* Author for correspondence (e-mail: lefebvrv{at}bme.ri.ccf.org)
Accepted 9 December 2002
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SUMMARY |
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In Sox5//Sox6/ embryos, the notochord formed a typical rod-like structure. It fulfilled its inductive functions, as indicated by expression of sonic hedgehog and sclerotome specification. However, the notochord failed to become surrounded with an extracellular matrix sheath. This phenotype was associated with a downregulation of extracellular matrix genes, including the genes for collagen 2, aggrecan and perlecan in both notochord cells and surrounding chondrocytic cells of presumptive inner annuli and vertebral bodies. The mutant notochord then underwent an aberrant, fatal dismantling after sclerotome cell migration. Its cells became removed first from intervertebral spaces and then from vertebral bodies, and it progressively underwent apoptosis. Meanwhile, the development of inner annuli and vertebral bodies was dramatically impaired. Consequently, the vertebral column of Sox5//Sox6/ fetuses consisted of a very deficient cartilage and was devoid of nuclei pulposi. In Sox5//Sox6+/ and more severely in Sox5+//Sox6/ embryos, the notochord sheath was thinner, but cells survived. By birth, nuclei pulposi were rudimentary, and its cells poorly swelled and still expressing sonic hedgehog.
Hence, Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and formation of nuclei pulposi. Through these roles and essential roles in cartilage formation, they are central transcriptional regulators of vertebral column development.
Key words: Sox5, Sox6, Notochord, Nucleus pulposus, Intervertebral disc, Cartilage, Vertebral column, Mouse
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INTRODUCTION |
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The notochord is a midline structure of mesodermal origin that forms during
gastrulation in chordate embryos (Hogan et
al., 1994). By mouse embryonic day 9.5-11.5 (E9.5-E11.5), it
consists of a solid rod underlying the neural tube. The cells are small,
condensed and enveloped altogether within a sheath of extracellular matrix.
This sheath contains many collagens, proteoglycans and glycoproteins,
typically found in cartilage, basement membranes, or mesenchymal and fibrous
tissues (Gotz et al., 1995
;
Hayes et al., 2001
).
The notochord has crucial roles in inducing ectoderm, endoderm, and
mesoderm derivatives in the early embryo
(Cunliffe and Ingham, 1999;
Cleaver and Krieg, 2001
). It
may also have an important mechanical role, and thereby constitute a primitive
axial skeleton (Adams et al.,
1990
). In vertebrate embryos, the notochord has major roles later
on during vertebral column formation
(Christ et al., 2000
;
Pourquié et al., 1993
).
Intervertebral discs are joint-like structures that connect vertebrae. They
feature a highly hydrated nucleus pulposus core, surrounded with a
cartilaginous inner annulus and a fibrous outer annulus. At E10.5-E11.5,
signals from the notochord induce sclerotome cell migration, condensation, and
differentiation around the notochord and neural tube. The soformed
perinotochordal tube has a metameric pattern with the alternation of more
condensed and less condensed zones. Between E12.5 and E15.5, the more
condensed zones develop into outer and inner annuli and the less condensed
zones into vertebrae. The notochord thus has indirect roles in the formation
of vertebrae and annuli. Simultaneously, it undergoes profound changes.
Notochord cells located in vertebral bodies are removed and probably relocated
into intervertebral regions (Aszódi
et al., 1998
; Rufai et al., 1999). In these latter regions, they
proliferate and undergo hypertrophy to form the nuclei pulposi. The notochord
thus also directly participates in vertebral column formation.
Several molecules are known to control the early notochord
(Cunliffe and Ingham, 1999).
For example, the T-box transcription factor brachyury (T) determines notochord
cell differentiation and survival
(Herrmann and Kispert, 1994
),
and the secreted factors sonic hedgehog (Shh) and noggin mediate inductive
actions (Chiang et al., 1996
;
McMahon et al., 1998
;
Teillet et al., 1998
). In
vitro studies have suggested that the notochord sheath maintains the
structural scaffold and internal pressure of the notochord
(Carlson and Kenney, 1980
;
Adams et al., 1990
), but its
exact roles in vivo have not been determined. The cellular origin and
molecular control of this sheath remain unknown. Moreover, the mechanisms that
underlie the transformation of the notochord into nuclei pulposi are also
largely unknown. Several mouse mutants have an abnormal development of the
notochord and vertebral column. These include mice that lack collagen 2
(Li et al., 1995
;
Aszódi et al., 1998
),
the paired box transcription factors Pax1 and Pax9
(Wallin et al., 1994
;
Peters et al., 1999
), and the
homeobox transcription factor Bapx1
(Tribioli and Lufkin, 1999
;
Lettice et al., 1999
;
Akazawa et al., 2000
). In these
mice, vertebral body cartilages fail to develop properly. The notochord forms
normally, but fails to be removed from vertebral bodies and to develop into
nuclei pulposi. Bapx1, Pax1 and Pax9 are expressed in
sclerotome cells, but not in notochord cells. It was therefore proposed that
the transformation of the notochord into nuclei pulposi requires mechanical
pressure from the vertebral body cartilage matrix. As of today, no regulatory
factor expressed in notochord cells and controlling cell fate and
differentiation beyond early embryonic stages has been identified.
Sox5 and Sox6 encode two highly identical transcription
factors, respectively, L-Sox5 and Sox6
(Lefebvre, 2002). These
factors feature an Sry-related HMG box domain, which mediates DNA binding, and
a coiled-coil domain, which mediates homo- and heterodimerization
(Lefebvre et al., 1998
).
Sox5 also encode a short protein (Sox5) that lacks the N-terminal
half of L-Sox5, including the dimerization domain. The transcripts for L-Sox5
and Sox6 are co-expressed in all cartilages, and expressed in a few other
tissues. The transcript for Sox5 is expressed exclusively in testis. In vitro
experiments have suggested that L-Sox5 and Sox6 cooperate with Sox9, a distant
relative and master chondrogenic factor (Bi
et al., 1999
; Akiyama et al.,
2002
), in the direct activation of the collagen 2 gene
(Col2a1) (Lefebvre et al.,
1998
). The roles of SOX5 and SOX6 in humans are
unknown.
We recently inactivated Sox5 and Sox6 in the mouse and
revealed that the two genes have essential, redundant roles in chondrogenesis
(Smits et al., 2001). Whereas
Sox5/ mice and
Sox6/ mice are born with minor cartilage
defects,
Sox5//Sox6/
fetuses develop a generalized and dramatic chondrodysplasia.
Sox5//Sox6/
prechondrocytes develop normal precartilage condensations, but their
differentiation into chondroblasts is delayed and impaired. These cells
express cartilage extracellular matrix genes at low or undetectable levels and
poorly proliferate. They are unable to persist as a pool of proliferating
chondroblasts in epiphyses and to establish cartilage growth plates in
metaphyses. Instead, they precociously undergo an aberrant maturation into
hypertrophic chondrocytes. By E16.5, when the fetuses die, cartilages are
rudimentary and matrix deficient, and are starting to be invaded by
bone-forming cells.
We show here that Sox5 and Sox6 are expressed in notochord cells and surrounding sclerotome-derived cells, and that Sox5//Sox6/ embryos have severe defects in notochord development. Sox5 and Sox6 are dispensable for early inductive functions of the notochord, but indispensable for notochord sheath formation, cell survival and development into nuclei pulposi. Hence, Sox5 and Sox6 are required to direct the fate and differentiation of notochord cells and chondrocytes, two cell types with major roles in vertebral column formation.
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MATERIALS AND METHODS |
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RNA in situ hybridization
RNA in situ hybridization was performed using 35S-labeled
antisense probes (Smits et al.,
2001). Pictures were taken using a red filter for RNA signals and
under blue fluorescence for nuclei stained with the Hoechst 33258 dye.
T and Car3 cDNA probes were generated by RT-PCR of
500
bp of the 3' untranslated sequences. The Fmod cDNA probe
corresponded to 100 bp of the open reading frame and 200 bp of the 3'
untranslated sequence. The Col3a1
(Metsäranta et al.,
1991
), Lamc1 (Laurie
et al., 1989
), Pax1
(Deutsch et al., 1988
),
Shh (Echelard et al.,
1993
) and other cDNA probes
(Smits et al., 2001
) were as
described.
Cell proliferation assay
Pregnant mice were injected with 5-bromo-2'-deoxyuridine (BrdU)
(Zymed Laboratories) (10 µl/g of mouse) and sacrificed 2 hours later.
Embryos were fixed in 4% paraformaldehyde, dehydrated through a graded ethanol
series and embedded in paraffin wax. BrdU-labeled DNA was detected in 7 µm
sections using a BrdU immunostaining kit (Zymed laboratories). Sections were
counterstained with Hematoxylin.
TUNEL assay
The Alkaline Phosphatase In Situ Cell Death Detection kit (Roche) was used
according to the manufacturer's instructions, with the following
modifications. Before incubation with the terminal deoxynucleotidyl
transferase (TdT), sections were blocked for 20 minutes with 0.3 mg/ml bovine
serum albumin in phosphate buffered saline. The TdT enzyme was used at a 1:20
dilution and the converter alkaline phosphatase at a 1:2 dilution.
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RESULTS |
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Sox5 and Sox6 are co-expressed in the notochord,
nucleus pulposus and cartilage
The expression pattern of Sox5 and Sox6 during vertebral
column development was analyzed by RNA in situ hybridization of mouse embryo
sections (Fig. 2). At E11.5, at
the onset of vertebral column formation, Sox5 and Sox6 were
both expressed in notochord cells and in sclerotome cells surrounding the
notochord and neural tube. From E11.5 to E15.5, and until birth (data not
shown), Sox5 and Sox6 remained expressed in notochord cells
throughout differentiation into nucleus pulposus cells. They also remained
expressed in sclerotome-derived prechondrocytes and chondroblasts in vertebral
bodies and inner annuli, but not in outer annuli. At all stages, the signal
for Sox5 RNA was slightly weaker in the notochord and nucleus
pulposus than in precartilage and cartilage, whereas the signal for
Sox6 RNA was similar in all these tissues.
|
Sox5 and Sox6 are required for notochord sheath
formation
At E11.5, the notochord of both control and
Sox5//Sox6/
embryos was a rod of condensed cells, with the mutant notochord generally
containing fewer cells (Fig.
3A). Control embryos had started to deposit a notochord sheath,
which stained with Alcian Blue, whereas sclerotome-derived cells had not yet
accumulated any cartilage matrix. The notochord sheath was totally missing in
mutant embryos. By E13.5, the control notochord sheath had become much thicker
(Fig. 3B). It was evenly thick
in vertebral and intervertebral areas, despite a strong difference in
abundance of cartilage matrix between these two areas. In mutant littermates,
vertebral bodies had started to deposit some cartilage matrix, but the
notochord sheath was still undetectable. Sox5 and Sox6 thus
control notochord sheath formation.
|
We tested the RNA levels for notochord sheath components in E11.5 to E14.5
embryos (E13.5, Fig. 3C). As
previously described (Smits et al.,
2001),
Sox5//Sox6/
chondroblasts expressed Col2a1 at a partially reduced level, and the
genes for aggrecan (Agc1) and perlecan (Hspg2) at virtually
undetectable levels. Expression of these genes was similarly reduced in
Sox5//Sox6/
notochord cells. Control embryos expressed the gene for laminin
1
(Lamc1) in notochord cells and intervertebral mesenchyme, but not in
chondroblasts. Mutant notochord cells expressed Lamc1 at a similar or
slightly reduced level. We also tested the expression level of genes for other
basement membrane proteins, including collagen 4 (Col4a1) and
nidogen, and for other mesenchymal and fibrous tissue proteins, including
fibronectin, collagen 1 (Col1a1) and collagen 3 (Col3a1),
which were detected by immunolocalization in the notochord sheath of mouse or
rat embryos (Gotz et al.,
1995
; Hayes et al.,
2001
). However, these genes were not expressed in control
notochord cells, and their expression in other cells was not affected by the
Sox5//Sox6/
mutation (data not shown).
We also tested whether the
Sox5//Sox6/
mutation affected expression of non-matrix genes in the notochord. We chose
genes highly expressed in notochord cells, but not expressed in chondrocytes:
Shh (Echelard et al.,
1993), T (Wilkinson
et al., 1990
) and Car3 (carbonic anhydrase 3)
(Lyons et al., 1991
). Their
RNA levels were normal in the mutant notochord
(Fig. 3D).
Hence, Sox5 and Sox6 are not needed for the initial formation of the notochord, but are required for notochord sheath formation. They are required in notochord cells and chondrocytes to express genes for common components of the cartilage matrix and notochord sheath.
Sox5//Sox6/
notochord cells are removed from intervertebral spaces and vertebral
bodies
From E13.5 to E15.5, the control notochord developed into nuclei pulposi,
and sclerotome cells into vertebral body and inner annulus chondroblasts.
Because these transformations started earlier in the thoracolumbar region than
in the cervical and caudal regions, all steps could be visualized
simultaneously in E14.5 embryos (Fig.
4A). In the distal tail, the notochord was still a continuous rod,
intervertebral regions highly condensed, and vertebral bodies starting to
accumulate some cartilage matrix. In the proximal tail, the notochord was
starting to bulge in intervertebral regions. In the thoracic region and in the
E15.5 lumbar region, notochord cells were completely removed from the
vertebral bodies, but an acellular notochord sheath was still visible. Nuclei
pulposi, vertebral body and inner annulus cartilage were fully developing.
During the same time period, the notochord of Sox5//Sox6/ embryos underwent an aberrant dismantlement. First, notochord cells disappeared from presumptive intervertebral areas, but not from vertebral bodies (Fig. 4A, top two panels). The vertebral bodies were underdeveloped, but the intervertebral mesenchyme was normally condensed. Next, notochord cells formed trains that seemed to be moving out of the vertebral column between vertebral and intervertebral spaces (Fig. 4A, bottom two panels). The vertebral bodies featured some cartilage matrix, but the intervertebral mesenchyme was still condensed. The hybridization of Sox5//Sox6/ embryo sections with a Car3 RNA probe convincingly illustrated the disruption of the notochord and diversion of its cells (Fig. 4B). As shown earlier (Fig. 1B), Sox5//Sox6/ notochord cells were removed from vertebral bodies by E15.5, thus with a delay of approximately 2 days compared with control cells, and never developed nuclei pulposi.
We also analyzed transverse sections to ascertain that the notochord interruptions seen in sagittal sections were not due to waviness of the notochord (Fig. 4C,D). At E12.5, the notochord was still visible and expressing Shh in the cervical and caudal intervertebral spaces in both control and Sox5//Sox6/ embryos. In the thoracolumbar intervertebral spaces, notochord cells were identifiable in control embryos, but not in Sox5//Sox6/ embryos. Sox5//Sox6/ notochord cells were therefore vanishing from intervertebral regions.
Hence, Sox5 and Sox6 are needed to maintain the notochord in intervertebral regions and to remove the notochord from vertebral bodies in a timely manner.
Sox5 and Sox6 are needed for notochord cell
survival
We tested whether the reduced numbers of
Sox5//Sox6/
notochord cells in E11.5 embryos, and the removal of notochord cells from
intervertebral spaces in E12.5-E14.5 embryos resulted from abnormal cell
proliferation or from cell death. As we have shown previously that
Sox5 and Sox6 are required to promote chondroblast
proliferation, we first tested notochord cell proliferation
(Fig. 5A). In both control and
mutant embryos, notochord cells were proliferating actively at E11.5, and less
actively at E13.5. At both stages, mutant notochord cells were proliferating
at a similar or higher rate than control cells. Thus, the loss of mutant
notochord cells was not due to a decreased rate in cell proliferation.
|
Cell death was assessed by the TUNEL assay (Fig. 5B). At E11.5, few apoptotic cells were detected in control notochords, but many dying cells were seen in mutant notochords. Both control and mutant intervertebral mesenchymes featured dying cells, whereas only mutant vertebral mesenchymes featured some. At E13.5, control notochord cells were not dying, whereas Sox5//Sox6/ notochord cells were undergoing massive apoptosis in prevertebral and intervertebral regions. Cells were not dying in control cartilage, but a few cells were still dying in Sox5//Sox6/ intervertebral mesenchyme. Hence, apoptosis explains the progressive loss of all Sox5//Sox6/ notochord cells, but not the selective loss in intervertebral spaces.
Notochord disintegration occurred in embryos lacking T
(Herrmann and Kispert, 1994)
and Itn5a (integrin-
5)
(Goh et al., 1997
). However,
we found that Itn5a was expressed in intervertebral cells, but not in
notochord and prevertebral cells. Moreover, its expression was not affected by
the
Sox5//Sox6/
mutation (data not shown). As shown above, T was expressed at normal
levels in
Sox5//Sox6/
notochord cells (Fig. 3D). Therefore, the cause of apoptosis of
Sox5//Sox6/
notochord cells was not an altered expression of T or
Itn5a.
Sox5 and Sox6 promote inner annulus chondroblast
differentiation
The exclusion of
Sox5//Sox6/
notochord cells from intervertebral spaces could result either from a
notochord defect or from an intervertebral defect. In favor of the latter,
histological analysis suggested that the
Sox5//Sox6/
intervertebral mesenchyme was impaired in its development
(Fig. 4A). To test this
hypothesis further, we analyzed expression of cell-specific markers.
Pax1 and Pax9 are indispensable for cell fate
specification and differentiation of sclerotome cells. As expected
(Deutsch et al., 1988),
Pax1 was highly expressed in presumptive intervertebral regions in
E11.5 wild-type embryos (Fig.
6A). It was correctly expressed in
Sox5//Sox6/
sclerotome cells, indicating that these cells were correctly specified. From
E11.5 to E13.5, control cells underwent chondrogenesis, and Pax1
expression became confined to narrow zones of intervertebral mesenchyme, to
perichondrium and to outer annuli. By E15.5, it remained expressed only in
outer annuli, whereas, in mutant embryos, it was still highly expressed in
lower lumbar intervertebral areas.
|
By E15.5, the genes for the mesenchymal markers collagen 3 (Fig. 6B), collagen 1 and fibronectin (data not shown) were no longer expressed in control and mutant vertebral body chondroblasts and in control inner annulus chondroblasts. In the mutant intervertebral regions, they were being inactivated in the thoracic and higher lumbar regions (data not shown), but were still expressed in the lower lumbar region (Fig. 6B). The differentiation process of mutant inner annulus cells was thus severely delayed, but not blocked.
The RNAs for general cartilage markers, such as aggrecan (Fig. 6B) and link protein (data not shown), were highly expressed in the cartilages of control embryos, but almost undetectable in mutant intervertebral and vertebral cells. The RNA for fibromodulin, which is expressed at high levels in inner annulus chondroblasts, was undetectable in mutant intervertebral cells (Fig. 6B). This virtual absence of expression of general and inner annulus-specific chondroblast markers revealed that the cells were not only delayed, but also impaired in their differentiation process. These defects may therefore have contributed to remove notochord cells from intervertebral regions.
Sox5 and Sox6 are both needed for notochord
development
To assess the relative contribution of Sox5 and Sox6 to
notochord development, we analyzed compound mutants. At E12.5, the notochord
sheath was normal in
Sox5//Sox6+/+ embryos,
and slightly thinner in
Sox5+//Sox6+/ and
Sox5+/+/Sox6/ embryos
(Fig. 7A). It was significantly
thinner in
Sox5//Sox6+/
embryos, and very thin in
Sox5+//Sox6/
embryos. Thus, Sox5 and Sox6 were both needed for notochord
sheath formation, and Sox6 was slightly more important than
Sox5.
|
Notochord cells started to bulge in intervertebral spaces from E13.5 in Sox5+//Sox6/ embryos, as well as in control littermates, but mutant cells started to be removed from vertebral bodies between E14.5 and E16.5 with a short delay relative to control cells (Fig. 7B). This delay coincided with a delay in cartilage development. By E16.5, control nuclei pulposi were fully developing, and its cells were swollen, whereas nuclei pulposi were underdeveloped in Sox5+//Sox6/ embryos, and its cells were hardly swollen (Fig. 7B,C).
At birth, the nuclei pulposi of control mice appeared as swollen, translucent structures in skeletal preparations (Fig. 7D). Those of Sox5+//Sox6+/, Sox5//Sox6+/+ and Sox5//Sox6+/ mice were less expanded in width, but swollen and translucent. The nuclei pulposi of Sox5+/+/Sox6/ and Sox5+//Sox6/ mice were small, often fragmented and opaque. The cells in Sox5+//Sox6/ nuclei pulposi were still incompletely swollen (Fig. 7E) and still expressing Shh (Fig. 7F), whereas wild-type notochord cells had deactivated Shh expression by E15.5 upon transformation into nucleus pulposus cells (Fig. 7G).
The few Sox6/ mice that survived postnatally featured a kinked tail in addition to an overall growth delay (Fig. 7H). Histological analysis revealed that the tail kinks were always associated with an eccentric, small nucleus pulposus (Fig. 7I). Sox5/ mice died at birth, but Sox5+//Sox6+/ mice survived postnatally and all had a straight tail (data not shown).
Thus, Sox5 and Sox6 have mostly redundant functions in notochord development, but Sox6 is slightly more important than Sox5.
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DISCUSSION |
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Sox5 and Sox6 are required for notochord sheath
formation
Many types of matrix molecules are present in the notochord sheath, but
little is known about their function and gene regulation. The short tail
(Ts) mouse lacks the notochord sheath
(Center et al., 1988), but its
genetic alteration is unknown.
The first genetic alterations causing abrogation of notochord sheath
formation were identified in the laminin ß1 and laminin 1 genes in
the zebrafish (Parsons et al.,
2002
). Lamc1-null mouse embryos die too early (E5.5) to
study the role of laminin
1 in notochord sheath formation in this
species (Smyth et al., 1999
),
but crucial functions of laminin 1 have likely been conserved through
evolution. We found that notochord cells express Lamc1 at the time of
sheath formation, but Lamc1 expression is not or hardly affected by
the
Sox5//Sox6/
mutation, ruling out that Sox5 and Sox6 mediate notochord
sheath formation by controlling Lamc1 expression.
Although the notochord sheath may contain mesenchymal, fibrous tissue and basement membranes components, the genes for such components were not expressed in wild-type notochord cells at the time of sheath formation, and those that were expressed in surrounding tissues were not affected by the Sox5//Sox6/ mutation. Hence, Sox5 and Sox6 must mediate notochord sheath formation by controlling other genes.
The notochord sheath also contains cartilage matrix components. The
expression of genes for these components is partially or severely
downregulated in
Sox5//Sox6/
chondroblasts (Smits et al.,
2001) and we found here that their expression is similarly
affected in
Sox5//Sox6/
notochord cells. Col2a1 expression is partially downregulated.
However, as Col2a1/ mouse embryos have a
thick notochord sheath (Aszódi et
al., 1998
), the notochord phenotype of
Sox5//Sox6/
embryos must be caused by the altered expression of more genes. The wild-type
notochord sheath intensely stains with Alcian Blue, indicating that it
contains sulfated glucosaminoglycans. Accordingly, expression of the genes for
the sulfated proteoglycans aggrecan and perlecan is high in notochord and
surrounding chondrocytic cells. It is virtually abrogated in
Sox5//Sox6/
embryos. Mouse embryos that lack either gene develop a chondrodysplasia
similar to that of
Sox5//Sox6/
embryos, but it has not been described whether they have a similar notochord
phenotype (Rittenhouse et al.,
1978
; Watanabe and Yamada,
1999
; Arikawa-Hirasawa et al.,
1999
; Costell et al.,
1999
). Nevertheless, it is reasonable to propose that these
proteoglycans have crucial roles both in cartilage and the notochord sheath,
and that Sox5 and Sox6 mediate notochord sheath formation by
controlling these genes and possibly other genes in notochord and surrounding
cells.
The cellular origin of the notochord sheath is unknown. Transplantation
experiments in zebrafish embryos have shown that a normal notochord sheath
forms when laminin 1-deficient notochord precursor cells are transplanted into
a wild-type embryo, and vice versa (i.e. when wild-type notochord precursor
cells are transplanted into a mutant embryo)
(Parsons et al., 2002). Hence,
both notochord cells and surrounding cells may contribute notochord sheath
components. Other observations, however, indicate that notochord cells must
have a preponderant role in notochord sheath formation. First, we have shown
that the notochord sheath starts to form in wild-type embryos before any
cartilage matrix in surrounding tissues, and later, accumulates evenly in
cartilaginous vertebral bodies and mesenchymal intervertebral areas. Second,
embryos that lack Bapx1 (Akazawa
et al., 2000
), Pax1 and Pax9
(Peters et al., 1999
) fail to
form any cartilage matrix around the notochord, but appear to form a normal
notochord sheath. Finally, we have shown that Sox6 is expressed,
relative to Sox5, at a slightly higher level in notochord cells than
in surrounding cells, and that the inactivation of Sox6 is more
detrimental for notochord sheath formation than the inactivation of
Sox5. Based on these data, we believe that Sox5 and
Sox6 mediate notochord sheath formation by controlling extracellular
matrix genes mainly in notochord cells.
Sox5 and Sox6 are required for notochord cell
survival
Notochord cell death occurs in
Sox5//Sox6/
embryos later than in T/
(Herrmann and Kispert, 1994),
Itn5a/
(Goh et al., 1997
) and
Danforth's short tail embryos (Asakura and
Tapscott, 1998
). However, T is expressed at a normal
level in
Sox5//Sox6/
notochord cells. Itn5a is not expressed in wild-type notochord cells,
and not affected by the
Sox5//Sox6/
mutation in surrounding cells. Therefore, T and Itn5a are
not involved in the death of
Sox5//Sox6/
notochord cells. The gene altered in the Danforth's short tail mouse is
unknown.
Increased apoptosis of notochord cells occurs in mouse embryos with a
deletion of Jun in the notochord and sclerotome
(Behrens et al., 2003). It
occurs at the same stage of development as in
Sox5//Sox6/
embryos. However, Jun-deficient mice form a normal notochord sheath,
and most notochord cells survive to form nuclei pulposi. Jun must
therefore act independently of Sox5 and Sox6 in the
notochord.
The phenotypic comparison of Sox5//Sox6/ embryos and laminin 1-deficient zebrafish embryos strongly suggests that Sox5 and Sox6 mediate notochord cell survival essentially through their roles in notochord sheath formation. The primary defect of both mutants is the lack of notochord sheath, and it is directly followed by notochord cell apoptosis upon sclerotome cell condensation. The sheath may act on notochord cells in one or several ways. It may provide vital cell-matrix interactions, store essential growth factors or establish a barrier against death signals or mechanical pressure from surrounding tissues.
Sox5 and Sox6 are required for proper relocation of
notochord cells
Sox5//Sox6/
notochord cells are aberrantly removed from intervertebral areas, while their
rates of apoptosis and proliferation are not different between vertebral and
intervertebral regions, and mutant sclerotome cells are normally condensed and
specified. We therefore propose that
Sox5//Sox6/
notochord cells may be removed from intervertebral regions because of their
specific lack of notochord sheath.
Sox5//Sox6/
embryos remove notochord cells from vertebral bodies later than wild-type
embryos. Previous studies have suggested that the wild-type notochord is
removed upon swelling pressure from the vertebral body cartilage matrix. The
notochord persists in the cartilage-deficient
Col2a1/ vertebral bodies
(Aszódi et al., 1998),
and in the undifferentiated Bapx1/ vertebral
bodies (Lettice et al., 1999
;
Tribioli and Lufkin, 1999
;
Akazawa et al., 2000
). The
delay in removing
Sox5//Sox6/
notochord cells from vertebral bodies must result from the delay in cartilage
development. It may also be facilitated by the lack of notochord sheath and
apoptosis of notochord cells.
Wild-type notochord cells are believed to take refuge in intervertebral areas upon removal from vertebral bodies, but Sox5//Sox6/ notochord cells remain excluded from these areas and relocate between vertebral bodies and intervertebral areas. The repositioning of the cells, although ectopic, supports the relocation model proposed for wild-type notochord cells, and indicates that Sox5 and Sox6 are needed to maintain and relocate notochord cells in intervertebral areas.
Sox5 and Sox6 promote nucleus pulposus cell
differentiation
Little is known about the genetic program of nucleus pulposus cells. They
continue to express Sox5, Sox6 and T, but not Shh.
They undergo hypertrophy, but do not activate the hypertrophic chondrocyte
marker Col10a1 (data not shown). Their genetic program thus only
partially overlaps with that of chondrocytes.
Sox5//Sox6/
notochord cells die before developing nuclei pulposi, but the nuclei pulposi
of Sox5+//Sox6/
fetuses are severely impaired in their development. They remain rudimentary
and their cells swell poorly and maintain Shh expression.
Sox5 and Sox6 are the first genes for transcription factors
shown to be expressed in these cells and needed for their differentiation.
Hypertrophic chondrocyte differentiation is also impaired in
Sox5//Sox6/
embryos (Smits et al., 2001).
Chondrocytes activate Cbfa1 and VEGF, but weakly express
Col10a1, maintain Sox9 expression and poorly enlarge. These
defects must be secondary consequences of the
Sox5//Sox6/
mutation because Sox5 and Sox6 are no longer expressed in
hypertrophic chondrocytes. Similarly, some of the defects of
Sox5+//Sox6/
nuclei pulposi may be secondary to defects in the earlier notochord, or in
surrounding inner annuli.
Sox5 and Sox6 promote inner annulus cell
differentiation
The vertebral column of
Sox5//Sox6/
fetuses is mostly unsegmented by E15.5, even though the earlier vertebral
column is properly patterned, with sclerotome cells condensing correctly and
expressing Pax1. As previously described, the development of
vertebral body cartilage is delayed and impaired
(Smits et al., 2001) such
that, by E15.5, it features no hypertrophic chondrocytes yet, and no growth
plates. We have shown here that the differentiation process of inner annulus
cartilage is similarly delayed and impaired, such that both cartilages were
indistinguishable by E15.5. Moreover, mutant inner annulus cells do not
activate Fmod, an abundant marker of both inner annulus and
periarticular chondroblasts. We previously showed that periarticular
chondroblasts in
Sox5//Sox6/
limb cartilages also fail to activate Fmod
(Smits et al., 2001
).
Therefore, the impaired differentiation of
Sox5//Sox6/
inner annulus cells is a cell-autonomous defect, not a consequence of
notochord defects.
Sox5 and Sox6 have redundant roles in notochord
development
Sox5/ and
Sox6/ embryos have limited notochord defects
compared with
Sox5//Sox6/
embryos, indicating that Sox5 and Sox6 have mostly redundant
functions in notochord development, as in chondrogenesis
(Smits et al., 2001).
Nevertheless, the analysis of compound mutants has revealed that Sox6
is slightly more important than Sox5 for both notochord sheath
formation and nucleus pulposus development. This functional difference between
the two genes probably reflects the fact that Sox6 is expressed,
relatively to Sox5, at a slightly higher level in notochord cells
than in surrounding chondrocytes. This conclusion is consistent with the
observation that L-Sox5 and Sox6 have indistinguishable DNA binding and
transactivation activities in vitro
(Lefebvre et al., 1998
).
Sox5 and Sox6 have critical roles in vertebral
column formation
This study and our previous study
(Smits et al., 2001) have
identified critical roles for Sox5 and Sox6 in the
transcriptional control of notochord cells and chondrocytes
(Fig. 8).
|
The differentiation of early notochord cells does not require Sox5 and Sox6, but requires T. Shh and noggin, secreted by notochord cells, contribute to induce sclerotome cell differentiation into prechondrocytes. Bapx1, Pax1, Pax9 and Sox9 act downstream of Shh at this step in the vertebral column.
Both pathways crucially rely on Sox5 and Sox6 at the next step of cell differentiation. Sox5 and Sox6 promote expression of extracellular matrix genes, and are thereby required for notochord sheath and cartilage matrix formation. Sox5 and Sox6 are required for proper proliferation of chondroblasts, but not notochord cells. By contrast, they are needed for the survival of notochord cells, but not chondroblasts. Jun also is needed for notochord cell survival, and Sox9 for chondroblast differentiation.
Later, mechanical pressure from cartilage likely induces notochord cell
relocation from vertebral bodies into intervertebral areas. Sox5 and
Sox6 promote notochord cell differentiation into nucleus pulposus
cells. By contrast, Sox5 and Sox6 prevent chondroblast
differentiation into hypertrophic chondrocytes. Runx2 may promote
hypertrophic chondrocyte maturation in vertebrae
(Inada et al., 1999;
Kim et al., 1999
) and
Sox9 may prevent chondrocyte hypertrophy
(Bi et al., 2001
).
In conclusion, Sox5 and Sox6 have essential functions in the development of cartilage, the notochord and nuclei pulposi. They are thereby critical transcriptional regulators of vertebral column scaffold formation.
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ACKNOWLEDGMENTS |
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