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DEVELOPMENTAL DYNAMICS 215:179–189 (1999)
Role of Actin Stress Fibres in the Development
of the Intervertebral Disc: Cytoskeletal Control
of Extracellular Matrix Assembly
ANTHONY J. HAYES, MICHAEL BENJAMIN, AND JAMES R. RALPHS*
Connective Tissue Biology Laboratory, Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom
ABSTRACT
Orientation of collagen fibrils is
a key event in the development of many tissues.
In the intervertebral disc, the outer annulus
fibrosus comprises lamellae of parallel collagen
fibres, the direction of orientation of the long axis
of which alternates in angle between lamellae. In
development, this organisation is preceded by
the formation of sheets of oriented fibroblasts,
which then deposit the oriented lamellae. Here,
using fluorescent labelling, confocal and electron
microscopic techniques on developmental series,
we show that the orientation of cells in lamellae
is associated with the formation of adherens
junctions intercellularly, involving cadherins and
vinculin, and longitudinal stress fibres (label for
filamentous actin and tropomyosin) intracellularly. The stress fibres direct the initial elongation of cells and control the deposition of oriented
extracellular matrix via junctional complexes with
the matrix involving vinculin and ␣ 5 ␤ 1 integrins,
which in turn promote the formation of oriented
fibronectin at the cell surface; oriented collagen is
deposited between cells at the same stages. Shortly
after birth, the stress fibres disappear, probably
because cells now gain orientational cues from the
matrix, and are undergoing differentiation-related
changes to form fibrocartilage cells. Dev Dyn
1999;215:179–189. r 1999 Wiley-Liss, Inc.
Key words: intervertebral disc; development;
stress fibres; cell–cell interactions;
cell–matrix interactions
INTRODUCTION
Orientation of collagen fibres during their deposition
by fibroblasts is a key event in development of many
connective tissues. For example, fibrils must be more-orless parallel and run along the long axis of tensile
structures (e.g., tendon and ligament) or are interwoven in tissues experiencing load from various directions
(e.g., dermis). Some tissues consist of a series of collagenous lamellae, with parallel fibrils within lamellae but
with orientation being different in successive lamellae.
The most highly ordered and tightly controlled example
is probably in the cornea, but such organisation also
occurs in bone and in the intervertebral disc. Here, we
describe the sequence of events occurring in the deposir 1999 WILEY-LISS, INC.
tion of the lamellae of the annulus fibrosus of the
intervertebral disc.
The intervertebral disc is a composite structure,
consisting of the outer annulus fibrosus, of sclerotomal
origin, and inner nucleus pulposus, initially derived
from the notochord. Two distinct regions of the annulus
can be distinguished: the outer annulus, which is
fibrous in origin, and the inner annulus, which is highly
fibrocartilaginous and is initially derived from cartilage
(Peacock, 1951a,b; Walmsley, 1953; Humzah and
Soames, 1988; Rufai et al., 1995). The outer annulus is
constructed of lamellae of ordered collagen fibril bundles,
which are arranged concentrically around the nucleus,
and anchored firmly into the cartilage endplates of the
vertebral bodies (Humzah and Soames, 1988). Within
individual lamellae, collagen fibres run parallel to each
other and are inclined at a precise angle to the long axis
of the spine (Hickey and Hukins, 1980). The inclination
of this angle alternates in adjacent lamellae, conferring
cross-ply strength to the annulus fibrosus. Functionally, the annulus allows twisting and bending of the
spine, and the nucleus resists compressive loading;
some of that compressive loading is transferred to the
inner, fibrocartilaginous part of the annulus (Bogduk,
1997).
During development, the lamellar organisation of
oriented collagen fibres in the annulus is preceded by a
similar organisation of sheets of oriented fibroblasts
arranged concentrically around the early nucleus (Peacock,1951 a,b; Walmsley, 1953; Rufai et al., 1995). The
oriented sheets of fibroblasts clearly lay down the
organised lamellae, but how cells become oriented into
cell sheets, and how the cell sheets lay down an
oriented extracellular matrix (ECM) is unclear. Cells
lay down oriented collagen in association with a series
of cellular compartments (Birk and Zycband, 1994) and,
at least in tendon, appear to cooperate with one another
in depositing the oriented matrix (McNeilly et al.,
1996). However, as the developing annulus fibrosus
shows, there has to be a cellular orientation phase
Grant sponsor: Arthritis and Rheumatism Council; Grant number:
R0521.
*Correspondence to: James R. Ralphs, Connective Tissue Biology
Laboratory, Cardiff School of Biosciences, Cardiff University, Preclinical Buildings, Museum Avenue, P.O. Box 911, Cardiff CF1 3US, United
Kingdom.
Received 19 January 1999; Accepted 26 March 1999
180
HAYES ET AL.
before cells can deposit their collagen in the correct
lamellar orientation. Whilst considerable attention has
been paid to segmentation and patterning along the
axial skeleton (e.g., Wallin et al., 1994; Burke et al.,
1995; Christ and Ordahl, 1995; Smith and Tuan, 1995),
little is known about development downstream of patterning events, and how these highly ordered structures actually form: what causes cells to orient and
deposit oriented ECM?
Cell shape and movement is mediated by the cytoskeleton, under the control of various external influences
including cell–cell and cell–matrix interactions (e.g.,
Zanetti and Solursh, 1986; Hay, 1989; Ingber et al.,
1994; Mooney et al., 1995). In the present study, we
examine the role of the cytoskeletal elements actin and
vimentin and their associated cell–cell and cell–matrix
junctions in the formation of oriented cell sheets and
the subsequent deposition of collagen lamellae. We
show important roles for actin-associated cell–cell junctions in early formation of annulus fibrosus blastemas
and actin stress fibres and cell–matrix junctions in
early orientation of cells and the ECM.
RESULTS
Development of the Rat Intervertebral Disc:
Histological Overview
At embryonic day (E)15, the spine consisted of repeated perinotochordal condensations of mesenchyme,
the intervertebral disc anlagen, which were separated
by the early cartilage of the developing vertebral bodies
(Fig. 1). At this stage, the cells making up the disc
anlage were closely packed together, with no histologically detectable ECM (see also next paragraph). The
outermost cells were flattened and the innermost were
more rounded or polygonal. By E16, the notochord had
bulged at regular intervals along its length, clearly
defining the position of the future nuclei pulposi, and
the annulus had formed distinct inner and outer parts
(Figs. 2 and 3). The inner annulus was cartilaginous,
continuous with the vertebral bodies, and contained
round-to-oval cells that were separated by small
amounts of highly Alcian-blue-positive cartilage matrix. The outer annulus consisted of compact sheets of
elongate, parallel fibroblasts, again with no histologically discernible ECM (Figs. 3 and 4). These cells were
precisely oriented at a constant angle to the long axis of
the vertebral column. The inclination of this angle
alternated in neighbouring sheets, producing a complex
lattice-like arrangement of cells when seen in parasagittal sections (Fig. 4). With further development, ECM
accumulated between cell sheets, separating them from
one another. In the neonate, the lamellar arrangement
of cells and ECM was fully apparent, and by 2 weeks
the annulus consisted of rows of rounded cells separated by the prominent oriented collagenous lamellae
(Fig. 5).
Ultrastructurally, at E15 outer annulus cells were
somewhat flattened, with many cell–cell contacts (Fig.
6). Many of these contacts contained distinct cell junc-
tions, probably adherens junctions (Fig. 7; see below).
Spaces between cells contained an extremely sparse
ECM. By E16, cells had become very elongated and the
alternating orientation of cells in successive cell sheets
could easily be appreciated (Fig. 8). There was still very
little ECM, although small bundles of banded collagen
fibrils could be seen between the closely packed cells,
intimately associated with the cell surface (Fig. 9). The
cells themselves contained prominent longitudinal fibrillar rods, consistent in appearance with stress fibres
(Figs. 8–10) and positioned just beneath the plasmalemma (Figs. 9 and 10). Dense plaques were sometimes
observed immediately beneath the plasmalemma that
could have represented cell–matrix junctions (probably
focal adhesions; see below). In the neonate, cells were
much more rounded in outline, embedded in a densely
collagenous, highly ordered ECM and had no obvious
stress fibres in the cytoplasm (Fig. 11).
Cytoskeletal Organisation
Actin organisation was intimately associated with
the stage of development of the outer annulus. At E15,
actin label was diffuse throughout cells of the disc
anlagen (Fig.12). Starting at late E15, and fully by E16,
actin label was strong in the oriented fibroblasts of the
outer annulus (Fig. 13). This pattern of actin distribution persisted in the outer annulus until birth (Figs.
14–18). In sections tangential to the outer annulus,
parallel cables of actin ran along the long axes of the
elongated fibroblasts, with the change in cell orientation in neighbouring cell sheets giving the appearance
of a lattice-like arrangement of actin filaments (Figs.
14, 16, and 18). In dual labels, tropomyosin colocalised
with actin cables in the outer annulus in all embryonic
stages investigated, showing them to be actin/myosin
stress fibres (Fig. 16). In transverse sections of the disc,
the stress fibres were clearly seen to be restricted to the
closely packed fibroblasts of the outer annulus (Fig. 15).
In the neonate, the complex lattice of actin cables had
disappeared (Fig. 19), although, as at all stages, the
notochordal cells of the nucleus pulposus labelled
strongly. Actin label in the annulus at this stage often
appeared punctate, with few traces of the fibre organisation observed earlier, and there was no label for tropomyosin. Vimentin was absent from the annulus up to
E18/19, although it was strongly labelled in the notochord and the notochordally derived nucleus pulposus.
At E20, weak label was detected in the outer annulus,
along with strong label in the nucleus pulposus and in
chondrocytes of the vertebral body, notably hypertrophic chondrocytes (Fig. 20). In neonates, vimentin label
was more prominent, with longitudinally running filaments being present in the annular fibroblasts (Fig.
21).
Cell Junctions
Cadherins were detected at the E15 stage, in a region
of cells with bright label on their surfaces in the
position of the future annulus (Fig. 22) and in the
ACTIN AND MATRIX IN ANNULUS FIBROSUS DEVELOPMENT
Figs. 1–5.
Histology of the developing intervertebral disc.
Fig. 1. Resin section of E15 spine, midsagittal plane. Dense cellular
anlagen of the intervertebral discs (d) are positioned between the
cartilage of the developing vertebral bodies (vb), with the notochord (n),
which is of uniform diameter, running longitudinally through the centre of
all. The outer part of the disc anlagen appears dark and consists of
close-packed, flattened cells (see Fig. 6). The inner part consists of larger,
paler cells of more polygonal shape. (Toluidine blue.)
181
Fig. 3. Higher magnification view of E16 disc shows nucleus pulposus
(np) and differentiation of annulus into cartilaginous inner (ia) and fibrous
outer (oa) parts. Line indicates approximate position of section shown in
Fig. 4. (Haematoxylin, eosin and Alcian blue.)
Fig. 4. Section tangential to outer annulus of E16 disc, in the
approximate position indicated in Fig. 3. The section shows a lattice-like
arrangement of cells oriented into parallel sheets linking the vertebral
bodies (vb), with successive sheets lying at an angle to one another.
(Haematoxylin, eosin and Alcian blue.)
Fig. 2. Paraffin section of E16 spine, midsagittal plane. The notochord has
bulged at regular intervals within the developing discs to form the precursors of
the nuclei pulposi (np), and overt tissue differentiation has occurred in the
annulus (See Fig. 3). (Haematoxylin, eosin and Alcian blue.)
Fig. 5. Midsagittal section of outer annulus of 2-week disc. Layers of cells
have laid down oriented lamellae of collagen, with successive lamellae
showing alternating orientation of parallel collagen fibrils. The inner annulus (ia)
is at the lower edge of the figure. (Haematoxylin, eosin and Alcian blue.)
notochord. By E16, label was clearly restricted to the
region of the future outer annulus (Fig. 23); at this and
all subsequent stages, label could be detected in the
notochordally derived nucleus pulposus. By E17, label
could no longer be detected in the annulus (Fig. 24).
Vinculin could be detected in the annulus fibrosus at all
182
HAYES ET AL.
Figures 6–11.
ACTIN AND MATRIX IN ANNULUS FIBROSUS DEVELOPMENT
stages examined. At the late E15/early E16 stage when
cell orientation had begun, vinculin had distribution
similar to that of cadherins (Fig. 25). Later, dual labels
showed clusters of small, bright foci of vinculin label,
some forming streaks along the actin stress fibres (Fig
26), others as more rounded clusters that were on the
cell surface in the region of the cell around the nucleus.
Desmosomal antigen could not be detected at all in the
annulus at the stages examined, although it was prominent between notochordal cells of the nucleus pulposus
at all stages (Fig. 27). ␣5 and ␤1 integrin subunits were
present at all stages. At E15, they were associated with
cells but showed no obvious orientation. From E16
onwards, the integrins labelled as elongate streaks on
the surfaces of oriented annular fibroblasts, giving a
lattice-like appearance in glancing sections of outer
annulus (Figs. 28 and 29). Cultured annular fibroblasts
contained prominent stress fibres (Fig. 30) that were
associated with rods of fibronectin on the cell surface
(Fig. 31).
Extracellular Matrix
In vivo, fibronectin label was present pericellularly
throughout the disc tissue in all stages investigated.
Before E16, it had no obvious organisation; at E16 and
beyond, in the outer annulus it formed a bright, prominent lattice-like pattern, with long streaks of label
being associated with the cell surface and co-localising
with the submembranous actin bundles (Fig. 32). Type I
collagen label occurred only in the outer annulus. At
E16 it was weakly present, but labelling intensity
increased with age. It formed a lattice-like arrangement of fibres in the embryonic stages. By the neonate
stage, a well-developed lamellar arrangement of type I
collagen fibre bundles had developed (Fig. 33).
DISCUSSION
The developmental sequence for the annulus fibrosus
matches that previously described in human intervertebral discs (Peacock, 1951a,b; Walmsley, 1953) and
extends our previous observations on rat disc develop-
Figs. 6–11.
Ultrastructure of the developing annulus fibrosus.
Fig. 6. Low-power view of future outer annulus fibrosus cells at E15.
Cells are flattened and have many cell processes and cell–cell contacts.
There is a very sparse extracellular matrix between cells, with no visible
collagen fibrils.
Fig. 7. High-power view of cell–cell contacts at E15. Adherens
junctions (arrows) are prominent at points of contact between cells.
Fig. 8. Low-power micrograph of the E16 outer annulus. Cells are
elongated and spindle shaped, with the alternating orientation of cells in
successive sheets clearly visible by the differing cell outlines—highly
elongate in longitudinal section (white asterisks) and more rounded in
oblique section (black asterisks). Cells contain prominent fibrillar longitudinal stress fibres (arrowheads).
Fig. 9. High-power view of two cells from adjacent cell sheets at E16;
the cell on the left is cut in longitudinal section, on the right in transverse
section. The image is oriented at 90° to Fig. 8. The section passes close to
183
ment (Rufai et al., 1995). There is clearly the requirement for orientation of fibroblasts to deposit an oriented ECM structure. The results presented here give
indications of processes that may be important in initial
orientation of fibroblasts, and subsequently in the
deposition of the oriented collagenous lamellae. We
provide evidence that cell–cell and cell–matrix interactions involving the actin cytoskeleton are important in
cell organisation and subsequent matrix organisation.
Stress fibres are often regarded as artefacts of culture, and indeed for many cell types this is probably the
case; for example, chondrocytes do not contain them in
vivo (Durrant et al., in press), but contain them in
abundance if grown on tissue culture plastic surfaces
(e.g., Benya et al., 1988). However, stress fibres do occur
in vivo at some sites and under some circumstances,
e.g., in endothelial cells (White and Fujiwara, 1986;
Sugimoto et al., 1995) and repairing epithelia (Nodder
and Martin, 1997). There are also reports of contractile
actin filaments in some connective tissue cells in vivo:
prominent actin filaments have been associated with
matrix deposition in fibroblasts/myofibroblasts of granulation tissue (Gabbiani, 1994; Horiba and Fukuda,
1994; Desmouliere, 1995; Berry et al., 1998), including
that of healing and remodelling ligament scar (Faryniarz et al., 1996) and healing cornea (Petroll et al.,
1993), and stress fibres occur in tendon cells (Ralphs et
al., 1998). In the present study, the appearance of stress
fibres, as shown by the codistribution of filamentous
actin and tropomyosin, and actin-associated cell–cell
and cell–matrix junctions are intimately associated
with the onset of development and growth of the outer
part of the annulus fibrosus.
At the earliest stages, E15 and E16, the presence of
cadherins (important in cell–cell interactions and components of adherens junctions, e.g., Yonemura et al.,
1995) on annular fibroblasts suggests that cell–cell
attachment is of key importance in establishing cellular
orientation. The codistribution of vinculin (component
of adherens junctions and focal adhesions with the
ECM) with cadherins at these stages, along with ultra-
the midregion of both cells, and the nuclei (nu) occupy most of the
cytoplasm. A longitudinal stress fibre (sf) is clear on the left, between the
nucleus and the plasmalemma. A transverse section of a stress fibre
(white asterisk) is present on the right, again between the nucleus and
plasmalemma. Obliquely sectioned collagen fibrils are present in the
narrow space between the cells (black asterisk).
Fig. 10. Similar view of two more adjacent cells. The longitudinally
sectioned cell on the left contains a longitudinal stress fibre (sf). The cell
on the right has prominent focal densities on the cytoplasmic side of the
plasmalemma (arrows). Longitudinally running collagen fibrils are present
between the cells (asterisk). m, mitochondrion; er, rough endoplasmic
reticulum.
Fig. 11. Low-power micrograph of neonatal outer annulus. The annulus now contains prominent oriented lamellae of collagen fibrils with
alternating orientation—obliquely sectioned at the white asterisk, transversely at the black asterisk—with cells positioned between them. Cells
are more rounded with no stress fibres in the cytoplasm.
184
HAYES ET AL.
Figures 12–31.
ACTIN AND MATRIX IN ANNULUS FIBROSUS DEVELOPMENT
185
structural observations, suggests that cells are linked
together via adherens junctions. The integrins are also
present, indicating the presence of cell–matrix associations as well. Adherens junctions and focal adhesions
have been shown to mediate assembly of stress fibres
from the junctional structures (e.g., Chi-Rosso et al.,
1997; Mackay et al., 1997; Turnacioglu et al., 1998).
Therefore, it may be that the initial assembly of stress
fibres at the E15/16 stage is initiated by the formation
of cell–cell and/or cell–matrix junctions. The fact that
cadherin expression is lost after E16 suggests that the
cell–cell junctions are of key importance in establishing
the initial cellular pattern. Stress fibre formation probably drives the elongation of outer annulus cells within
the plane of their cell sheets after E15; thus, the
orientation of successive sheets of fibroblasts seen
clearly at E16 would be determined by the arrangement
of junctional contacts in the E15 disc. The continued
expression of vinculin, along with the organisation of
integrin subunits into streaks associated with stress
fibres, suggests that cell–matrix contacts are important
in subsequent organisation and deposition of the ECM.
Ultrastructural evidence supports this: cells at stages
where there are prominent stress fibres have collagen
fibrils at the cell surface, along with evidence of dark
plaques at the plasma membrane, suggestive of focal
contacts. To summarise, initial stages of annulus organisation require cell–cell interactions involving cadherins, resulting in the formation of adherens junctions
and stress fibres, but when cellular patterns are established and matrix synthesis begins, cells switch to
having their major interactions with the ECM.
The notochord is of obvious importance to disc development. It plays a key role in the onset of annulus
development; there is no overt differentiation until the
notochord starts to bulge in the regions of the future
nuclei pulposi. Recent work (Aszodi et al., 1998) indicates that the bulging of the notochord is driven by
Figs. 12–31. Confocal microscope images show cytoskeletal organisation and distribution of cell junctional components in the developing
annulus fibrosus and in vitro; all but Figs. 16 and 26 are counterstained
with propidium iodide to give cellular context.
chondrocytes of the vertebral bodies, especially hypertrophic chondrocytes (h). Weak label is present in the outer annulus; vimentin label was
not detected in the annulus before this stage.
Fig. 12. E15 disc anlagen labelled with phalloidin–fluorescein isothiocyanate. Label is prominent in cells of the disc (d), notochord (n) and
vertebral bodies (vb). It appears brighter in the disc anlagen because of
the high cell density.
Fig. 13. E16 disc labelled with phalloidin–fluorescein isothiocyanate.
Label is of particularly high intensity in the outer annulus, although still
present in other cell types, including the bulging notochord (n). The white
line indicates the approximate position of the section shown in Fig. 14.
Fig. 14. E16 disc labelled with phalloidin–fluorescein isothiocyanate.
High-power view of a tangential section, position indicated in Fig. 13. Cells
contain prominent longitudinal actin cables, consistent in position and
orientation with those seen ultrastructurally. At least two layers of cells are
present in this 20-µm cryosection, their alternating orientations giving rise
to the lattice pattern in the confocal reconstruction.
Fig. 15. Transverse section of E17 annulus, labelled with phalloidin–
fluorescein isothiocyanate. The section shows that actin cables are tightly
restricted to the outer annulus (oa): neither the cartilaginous inner annulus
(ia) nor the surrounding tissue (upper left of figure) contain them.
Fig. 16. Tangential sections of E18 outer annulus dual labelled with
phalloidin–fluorescein isothiocyanate (green; top) and indirect immunofluorescence for tropomyosin with Texas red second antibody (red; bottom).
Distributions in the outer annulus are identical, confirming identification of
actin cables as stress fibres.
Fig. 21. Tangential section of neonatal outer annulus, immunolabelled
for vimentin. Outer annular fibroblasts contain longitudinal vimentin
filaments.
Fig. 22. Sagittal (slightly oblique) section of E15 disc anlagen immunolabelled with pan-cadherin antibody. The notochord (n) passes centrally
through the vertebral body (vb) to the right and the disc anlagen (d) but
then leaves the section plane. Prominent label is present between cells in
the notochord and disc anlagen; there is none in the vertebral bodies.
Fig. 23. Sagittal section of E16 annulus immunolabelled with pancadherin antibody. Punctate label is prominent between cells of the
organising cell sheets in the outer annulus (oa).
Fig. 24. Sagittal section of E17 disc immunolabelled with pancadherin antibody. Label is restricted to the notochordal nucleus pulposus; none can be detected in the outer annulus (oa).
Fig. 25. Sagittal section of late E15/early E16 outer annulus immunolabelled for vinculin. Numerous small foci of label are present between
cells of the developing outer annulus (oa). ia, inner annulus.
Fig. 26. Tangential section of E19 outer annulus, dual labelled for
actin (phalloidin: green) and vinculin (immunolabel, Texas red: red).
Vinculin label occurs as clusters of foci, some of which line up along stress
fibres (presumably where these meet the cell surface), and others form
rounded clusters. These are associated with the cell surface in the vicinity
of the nucleus (not visible in this preparation).
Fig. 17. E19 disc labelled with phalloidin–fluorescein isothiocyanate.
Low-power view of developing disc. Actin label remains intense in the
outer annulus, and in the notochordal cells of the nucleus pulposus (np).
Fig. 27. Sagittal section of neonatal disc, immunolabelled for desmosomal antigen. No desmosomes are detected in the annulus; they are
present between the notochordal cells of the nucleus pulposus (n) and in
association with blood vessels at the periphery of the disc (arrow).
Fig. 18. Slightly oblique tangential section of E19 outer annulus,
labelled with phalloidin–fluorescein isothiocyanate. Successive layers of
cells with alternating orientation are clearly demonstrated by nuclear
angle and actin cable direction.
Figs. 28 and 29. Tangential sections of E18 outer annulus immunolabelled for ␣5 and ␤1 integrins, respectively. Both are distributed as
longitudinal streaks associated with the cells.
Fig. 19. Tangential section of neonatal outer annulus, labelled with
phalloidin–fluorescein isothiocyanate. Actin cables, prominent until the
day of birth, have dispersed, leaving a more punctate actin distribution.
Fig. 20. Midsagittal section of E20 disc immunolabelled for vimentin.
Label is prominent in the notochordal nucleus pulposus (np) and in
Figs. 30 and 31. Cultured outer annular fibroblasts labelled with
phalloidin for actin and immunolabelled for fibronectin, respectively. Cells
contain longitudinally oriented actin stress fibres, and longitudinally
oriented streaks of fibronectin at the cell surface. Fibronectin also can
appear as clusters of bright foci on the cell surface in the vicinity of the
nucleus, usually on the undersurface of the cell (Fig. 31, arrow).
186
HAYES ET AL.
Figs. 32 and 33. Extracellular matrix organisation in the developing
annulus fibrosus. Confocal reconstructions of immunolabelled 20-µm
cryosections.
studies (not shown) indicate that these run longitudinally in association
with outer annular fibroblasts.
Fig. 32. Tangential section of E21 foetal outer annulus immunolabelled for fibronectin. Label is in the form of thin, bright rods; dual label
Fig. 33. Tangential section of neonatal outer annulus immunolabelled
for type I collagen. Collagen fibres appear as a lattice in this reconstruction, which contains several alternately oriented lamellae.
pressure from the developing cartilage in the vertebral
bodies. The notochord narrows as it is squeezed circumferentially and stretched longitudinally by the developing vertebral bodies, its cells being pushed into the
notochordal bulges in the weaker, nonpressure-producing disc anlagen. This mechanism of notochordal movements explains the prominence of cytoskeletal elements and cell–cell junctions in notochordal cells: they
must remain attached firmly to one another so that all
end up in the early nucleus pulposus. If cartilage
development is prevented (by type II collagen knockout), the notochord does not bulge, and the intervertebral disc does not differentiate (Aszodi et al., 1998). It
would appear, therefore, that provided the axial pattern of repeated disc anlagen/vertebral body anlagen is
established, then disc differentiation will proceed, provided cartilage differentiation occurs in the vertebral
bodies. Within the disc anlagen, some changes are
evident before notochordal bulging, because there are
differences between the E15 inner and outer annulus;
this could potentially be mediated by signalling molecules from the notochord. The stimulus to promote disc
differentiation could simply be mechanical: the notochordal bulges will exert compressive loads on the inner
annulus precursors, enclosed as they are by outer
annulus precursors, which could lead to the chondrogenic differentiation of the inner annulus. Cells of the
outer annulus may experience different loads, because
they are attached to one another and are wrapped
around an enlarging structure; they would thus experience some tensile forces, which could trigger stress fibre
formation and deposition of fibrous matrix.
The organisation of ECM (fibronectin at the cell
surface and collagen fibrils of the developing lamellae)
mirrors that of the actin stress fibres in the cell sheets.
Indeed, we show that all of the elements are in place at
the appropriate developmental stages for there to be a
transmembrane link from the stress fibres to the
ECM—in sequence, stress fibre, vinculin, integrin, cell
surface fibronectin, and collagen. As further support for
this suggestion, the ultrastructural studies showed
intimate association between actin stress fibres with
the inside of the plasmalemma and collagen fibres with
the outside. The integrins examined together form the
fibronectin receptor, ␣5␤1 integrin (Cheresh and
Mecham, 1994). We suggest that this receptor is organised from the interior of the cell by stress fibres via
adhesion complexes, and thus organises fibronectin on
the cell surface. Similar events seem to occur in early
stages of bone formation (Winnard et al., 1995). Recent
in vitro studies also show close association of stress
fibres, integrins, and fibronectin, as seen with annulus
cells. Fibronectin accumulates on cell surfaces in vitro
via the ␣5␤1 integrin (Sechler et al., 1997; Bordoulous
et al., 1998) and then self-assembles into filaments
under the influence of other receptors, or ‘‘matrix
assembly sites’’ (Sechler et al., 1997; Hocking et al.,
1998). Fibronectin assembly at the cell surface leads to
stress fibre reorganisation and also modulates junction
formation and outside-in signalling processes (Sechler
and Schwarzbauer, 1997; Hocking et al., 1998). In the
developing disc, it seems likely that stress fibres organise the positions of the receptors and direction of
assembly of cell surface fibronectin. The organisation of
the receptor may be dependent on ligand binding; in
formation of typical focal contacts, ligand binding leads
to clustering of the receptor and interaction with the
cytoskeleton (e.g., Pfaff et al., 1998). It is unclear
whether unbound receptors can link with the cytoskeleton (Hughes and Pfaff, 1998). In the developing annulus, therefore, fibronectin may be ‘‘captured’’ by the
integrin and then organised along stress fibre–associ-
ACTIN AND MATRIX IN ANNULUS FIBROSUS DEVELOPMENT
ated focal contacts. This seems likely to be the sequence
of events because fibronectin is present throughout the
period studied, but only organises when stress fibres
are present. It is of crucial importance to tissue function
that cells and matrix orient correctly, and thus it seems
probable that a cell organisational event—stress fibre
formation based on cell–cell contact —precedes a matrix organisational event, i.e., fibronectin at the cell
surface. Once the pattern is established, integrinmediated signals will almost certainly feed back into
the cell, and can have profound effects on cell behaviour
(Bourdoulous et al., 1998; Sechler and Schwarzbauer,
1998). The individual integrin subunits examined can
also take part in forming receptors for other ECM
molecules (Cheresh and Mecham, 1994), and it seems
likely that other integrins will be present that will also
take part in matrix organisation.
The intermediate filament vimentin does not seem to
be involved in the cell and matrix orientation process,
because it appears too late. It certainly does not appear
to be involved in the cell–cell interactions because no
desmosomal contacts can be detected. At the later
stages, cells do label for vimentin, and here it could
thus be involved in cell–matrix interactions. Vimentin
could also be associated with annulus differentiation.
In tendons, fibrocartilage occurs at sites where the
tendon experiences compressive loads; at these sites,
fibrocartilage cells accumulate large quantities of vimentin (Ralphs et al., 1991, 1992). Intermediate filaments
are of clear importance in adaptation of tissues to
mechanical stress (Galou et al., 1997). It may be,
therefore, that the accumulation of vimentin in annulus cells relates to the onset of fibrocartilaginous differentiation, with increased compression being transferred from the nucleus pulposus at late foetal stages
and after birth.
The loss of stress fibres from annular fibroblasts after
birth suggests that they are no longer required for cell
orientation and matrix organisation. It may be that
with a well-ordered matrix already laid down, cells now
orient to the ECM, rather than requiring their own
internal organisers. Bone cells may behave similarly—at the earliest stages of membrane bone formation, the osteoblasts contain prominent actin fibres that
organise fibronectin at the cell surface (Winnard et al.,
1995). Fibronectin then loses its association with the
cell surface, presumably when the osteoblasts have
established their initial oriented ECM. There may be
further reasons for the disappearance of stress fibres
from annulus cells. If, as discussed above, the rapid
bulging of the notochord causes tensile stress in the
outer annulus and triggers stress fibre formation, then
the more static nature of the structure once it has
become established may lead to a reduction in tension
and thus disassembly of the stress fibres. Also, it should
be noted that cell shape changes occur subsequent to
birth, with cells becoming progressively more rounded
as they differentiate into fibrocartilage cells (Rufai et
al., 1995). The stress fibres would have to be dismantled
187
to allow cells to round up, but also possession of stress
fibres appears to preclude a chondrogenic phenotype.
Chondrocytes cultured on plastic flatten, form stress
fibres, and dedifferentiate to a fibroblastic phenotype; if
stress fibres are eliminated by treatment with cytochalasin, they redifferentiate to the chondrocyte phenotype
(e.g., Zanetti and Solursh, 1984; Benya et al., 1988).
The loss of stress fibres from the annulus fibroblasts
may thus be an important early stage in fibrocartilaginous differentiation.
EXPERIMENTAL PROCEDURES
Animals
Embryonic and young postnatal white Wistar rats
were used throughout. Pregnant females were killed by
cervical dislocation, and embryos were removed from
the uterus, decapitated, and processed for histology or
immunohistochemistry. For routine histology, the torsos were processed whole; for electron microscopy,
regions containing individual discs or their primordia
were dissected out; for immunohistochemistry, the whole
lumbar spine was dissected out before processing. At
least five animals were used for each procedure at each
of the following ages: Embryonic day (E) 15,16,17,18,
19, 20, 21; neonates; and 1 week after birth.
Histology
Specimens were fixed in 10% neutral buffered formol
saline for 5 days; those from animals older than E19
were in addition decalcified in 2% nitric acid, and then
all were dehydrated, cleared, and embedded in paraffin
wax. Serial 8-µm sections were cut in the sagittal plane
and stained with Alcian blue and haematoxylin and
eosin. Micrographs were taken using a Leica DM12
photomicroscope. For electron microscopy, specimens
were fixed in 2% glutaraldehyde/2% paraformaldehyde,
postfixed in osmium tetroxide, dehydrated, and embedded in araldite. Semithin sections were stained with
toluidine blue for light microscopy; ultrathin silver-gold
sections were stained with uranyl acetate and lead
citrate and examined with a Phillips EM 208 transmission electron microscope.
Immunohistochemistry
Specimens were frozen unfixed with dry ice, and
serial sagittal cryosections were cut at a thickness of 15
µm. Sections passing through intervertebral discs were
fluorescently labelled as follows.
Cytoskeletal components. Actin was directly labelled
using phalloidin/fluorescein isothiocyanate (FITC) (1µg/
ml; Sigma, Poole, UK), the actin stress fibre component
tropomyosin indirectly labelled using monoclonal antibody TM311 (1:400, Sigma; Das et al., 1993), and the
intermediate, filament vimentin using monoclonal antibody Vim13.2 (1:200, Sigma; Adams and Watt, 1988).
Junctional complexes. Cell junctions associated with
actin were labelled with antibodies to vinculin (associated with cell–cell adherens junctions and cell–matrix
focal contacts; hVin1, 1:200, Sigma; Goncharova et al.,
188
HAYES ET AL.
1992), and cadherins (pan-cadherin; monoclonal CH19, 1:100, Sigma; Takeichi, 1988), associated with
cell–cell adherens junctions. Desmosomes, associated
with cell–cell junctions involving intermediate filaments, were labelled using monoclonal antibody ZK31
(1:400, Sigma; Lang et al., 1986).
Matrix receptors. The components of the fibronectin
receptor ␣5␤1 integrin were labelled using rabbit polyclonal anti ␣5 integrin, (1:20, Chemicon, London, UK;
Rossino et al., 1990; Pena et al., 1994) and rabbit
polyclonal anti ␤1 integrin, 1:20, gift from Prof. R.O.
Hynes; Marcantonio and Hynes, 1988).
ECM components. Fibronectin and type I collagen
were labelled using rabbit polyclonal antihuman fibronectin (1:200; Sigma) and monoclonal type I collagen
(COL1, 1:2,000; Sigma), respectively.
Antibody labelling was performed using standard
procedures for indirect immunofluorescence, with labelling for collagen and fibronectin requiring enzyme
pretreatment of hyaluronidase (0.25 U/ml) and chondroitinase ABC (0.1 U/ml at 37°C for 30 min to unmask
epitopes. Controls were incubated with nonimmune
mouse immunoglobulins (10 µg/ml), rabbit serum
(1:100), or the primary antibody incubation step was
omitted. Primary antibody binding was detected using
FITC-conjugated goat anti-mouse IgG (1:100; Sigma).
Dual labels for actin and vinculin, and actin and
tropomyosin, were performed by first immunolabelling
sections with the primary antibodies and a Texas-red
conjugated second antibody (10 µg/ml; Vector Laboratories, Peterborough, UK) followed by direct labelling for
actin as above. Where appropriate, nuclei were counterstained with propidium iodide (0.5 µg/ml; Molecular
Probes, Leiden, The Netherlands). All sections were
mounted in Vectashield fade-retarding mountant (Vector Laboratories) and examined using a Molecular
Dynamics Sarastro 2000 confocal laser scanning microscope, set up either for single- or dual-channel fluorescence recordings. Specimens were scanned with a
25-mW argon ion laser with appropriate excitation and
emission filters for fluorescein (excitation 494 nm/
emission 518 nm), fluorescein/propidium iodide or fluorescein/Texas red (excitation 488 nm, primary beamsplitter 510 nm, secondary beamsplitter 595 nm,
detector filters 600 nm and 530 nm). Optical section
series were taken through the whole cryosection thickness using ⫻40 and ⫻60 objectives, and three-dimensional projections were prepared from the section series
using Molecular Dynamics ImageSpacey software running on a Silicon Graphics XS24 4000 workstation.
Cell Culture
Intervertebral discs were dissected from E19 embryos by cutting the endplates off the vertebral bodies.
The endplate was then punctured and the nucleus
pulposus squeezed out whole with gentle pressure. The
remaining annulus fibrosus and endplates were incubated for 30 min in 1% collagenase (Sigma) to remove
the cartilage of the inner annulus and the endplate.
This treatment left just the fibrous annulus from which
cells were disaggregated by further collagenase treatment. Cells were washed and plated into 30-mm tissue
culture dishes in high glucose Dulbecco’s Modified
Eagle’s Medium (DMEM) containing 10% foetal bovine
serum (all tissue culture reagents from Gibco, Paisley,
UK). After reaching confluence they were passaged and
then 3 hr after replating were fixed in 90% methanol at
4°C and labelled for actin and fibronectin as above.
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