Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK
Author for correspondence (e-mail:
dt2{at}sanger.ac.uk)
Accepted 29 October 2003
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SUMMARY |
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Key words: Segmentation, Somites, Notochord, Vertebrae, Bone, Zebrafish
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Introduction |
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Despite these insights at the molecular level, the relationship between
somite patterning and vertebral segmentation remains to be firmly established.
Somites give rise to both axial muscles (from the myotome) and vertebrae (from
the sclerotome) but, as Remak first observed, vertebrae are displaced by half
a segment relative to muscle segments
(Remak, 1850). To account for
this frameshift, he proposed that a single vertebra is formed from a
combination of the anterior (cranial) half of one sclerotome with the
posterior (caudal) half of the next-anterior sclerotome
(Remak, 1850
;
Verbout, 1976
). Thus a single
somite gives rise to a single muscle element yet contributes to two adjacent
vertebrae. This `resegmentation' model has been widely accepted for amniotes,
both from early descriptive studies (e.g.
Goodrich, 1930
;
Gadow, 1933
) and more recent
cell lineage studies (e.g. Bagnall et al.,
1988
; Goldstein and Kalcheim,
1992
; Huang et al.,
1996
; Aoyama and Asamoto,
2000
; Huang et al.,
2000
). Resegmentation has remained somewhat controversial
nonetheless (Verbout, 1976
;
Keynes and Stern, 1988
), and
it has also been unclear whether the model is applicable to all vertebrates.
In zebrafish, sclerotome cells constitute only a small proportion of the
somite (Stickney et al., 2000
)
and can give rise to muscle
(Morin-Kensicki and Eisen,
1997
). Moreover, a recent lineage analysis in the fish has shown
that cells from one half-sclerotome can contribute to two consecutive
vertebrae, rather than only to one vertebra as predicted by resegmentation
(Morin-Kensicki et al., 2002
).
This has been termed `leaky' resegmentation, and is consistent with a similar
finding made in a previous study using the chick embryo
(Stern and Keynes, 1987
).
If the classical resegmentation model does not apply in zebrafish, the question remains as to the source of segmental patterning of centra in this species. In particular, although the notochord is known to be critical for sclerotome development, its possible participation in the segmental patterning of vertebrae has been neglected, and it has generally been regarded as a passive player in this process. With this in mind, we have therefore undertaken a study of zebrafish vertebral patterning, and our results suggest that the notochord plays a central role in segmental patterning.
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Materials and methods |
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Skeletal staining
Embryos and larvae were fixed in 4% paraformaldehyde (PFA), then labelled
with alcian green and alizarin red as described
(Kelly and Bryden, 1983). In
vivo labelling was achieved by rearing fish in either 0.05% quercetin or
alizarin red in embryo medium. Both quercetin and alizarin red were seen to
label bone matrix; quercetin was found to be most suitable because of its low
toxicity, utility at physiological pH and narrow fluorescence spectrum
(Simmons and Kunin, 1979
).
Antibody and alkaline phosphatase labelling
Larvae at 20 days post-fertilization (dpf), previously reared in alizarin
red, were fixed in 4% PFA and stained in wholemount as described
(Westerfield, 1995) with minor
modifications. The zns5 monoclonal antibody (gift of Chuck Kimmel, University
of Oregon, OR, USA) was used at 1:200 dilution; Alexafluor 488 (Molecular
Probes, Eugene, OR) was used as a secondary antibody. Larvae were embedded in
Sakura Tissue-Tek OCT Compound (Bayer, Newbury, UK) and frozen sections were
cut at 10 µm thickness and counterstained using Vectamount containing DAPI
(Vector Laboratories, Peterborough, UK). Sections were viewed using a Leica
TCS-NT confocal microscope. Alkaline phosphatase staining was performed using
an ELF97 cytological labelling kit (E6602) as described by the manufacturer
(Molecular Probes) and visualised by fluorescence microscopy on a Zeiss
Axioplan 2 microscope. With a DAPI filter set, the positive signal appears
green. Centra at all axial levels were examined, and representative images
were taken.
Transmission electron microscopy
Larvae at 20 dpf were fixed in 4% glutaraldehyde in cacodylate buffer
containing 0.006% hydrogen peroxide for 3 hours, then washed in cacodylate
buffer before post-fixing in osmium tetroxide. Samples were bulk stained with
uranyl acetate, dehydrated in ethanol and embedded in Spurr's resin. Thin
sections (5 nm) were prepared with a Leica Ultracut UCT, stained with uranyl
acetate and lead citrate and viewed in a Philips CM100 electron microscope at
80 KV.
Notochord dissection
Embryos at 2-6 dpf were anaesthetised in 0.2 mg/ml 3-amino benzoic acid
ethylester (MS222) and then decapitated. Notochords were dissected using
tungsten needles. Some were fixed immediately after dissection in 4% PFA and
labelled with DAPI and/or 0.05% quercetin. Others were cultured in L-15
containing 10% FCS, 0.1% gentamycin at 32°C for 10-20 days before being
labelled with quercetin and viewed using a Zeiss Axioplan 2 microscope.
Notochord cell ablation
Embryos and larvae were anaesthetised in 0.2 mg/ml MS222, mounted on a
depression slide and viewed on a Zeiss Axioplan 2 microscope with Micropoint
laser system (Photonic Instruments, St. Charles, IL, USA). Individual cells
were ablated with pulses of 440 nm laser light. Targeted notochord cells were
located either at the myoseptal border, or at the centre of the prospective
centra; individual notochord cells are large and vacuolated, with a clear
boundary, and are readily distinguished. Single cells were targeted with
sub-micron resolution using the cross-hairs of the eyepiece (see
Fig. 4C,D); targeted cells
could be seen to burst, and surrounding undamaged notochord cells expanded to
fill the available space. In most cases only one cell was ablated; cells
outside the focal plane of the beam were not damaged because the beam energy
was only sufficient to damage cells lying in the focal plane itself. Sometimes
a second cell partially overlapped the targeted cell, and this was also
ablated by a second laser pulse refocused on this second cell. Fish were
allowed to recover and later labelled with 0.05% quercetin as described
above.
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Results |
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Osteoblasts are not present in centrum bone matrix
A second mechanism of bone formation, intramembranous ossification, is
recognized, where bone forms directly from mesenchymal cells that
differentiate into osteoblasts, with no cartilage intermediate
(Gilbert, 2000). We therefore
looked for the presence of osteoblasts within the developing zebrafish centra.
As a control we confirmed that zns5
(Johnson and Weston, 1995
) and
alkaline phosphatase (Simmons and Kunin,
1979
) label osteoblasts in developing skull bones, where
intramembranous ossification takes place
(Fig. 2A,C). However, no
osteoblasts were seen in the developing centra using these markers
(Fig. 2B,D), suggesting that
bone formation in developing centra is atypical. The thick section in
Fig. 2D shows several nuclei in
the notochord vicinity, and these probably arise from paraxial mesoderm and/or
neural tube.
|
The notochord is a source of bone matrix in vitro
To investigate whether the notochord is a source of bone matrix, we
dissected notochords free of surrounding tissues prior to centrum formation,
and cultured them in vitro for 10-20 days, by which time centrum bone would
have formed in vivo. Matrix deposition was assessed by labelling with
quercetin (Simmons and Kunin,
1979). In cultured notochords strong labelling for bone matrix was
seen between individual cells (Fig.
3A,B). Fully developed centra were not seen, presumably reflecting
diffusion of matrix in vitro when the notochord is no longer confined by the
presence of surrounding tissues. To exclude the presence of residual adherent
osteoblasts on the outside surface of the notochord sheath, a nuclear label
was also used, and no nuclei were seen external to the notochord
(Fig. 3C-E).
|
These results confirm that the notochord directs centrum bone synthesis in vivo. Because ablation of notochord cells in defined, periodic locations was required to prevent centrum development, our results also raise the possibility that the notochord has an inherent periodicity that may impart segmental patterning to the vertebral column.
An instructive role for the notochord in segmental patterning
According to the resegmentation hypothesis, disrupting somite segmentation
should perturb vertebral segmentation, because the reorganization of
half-sclerotomes would be abnormal. However, if the notochord also plays an
instructive role in segmental patterning, its presence may be sufficient to
maintain segmentation following somite disruption. In fused somites
(fss) embryos, somites lose their anterior-posterior polarity and
their overt boundaries are irregular (van
Eeden et al., 1996). In these embryos, the neural and haemal
arches are highly disorganized, but centra develop nonetheless with normal
shape, size and periodicity (Fig.
5A-D) (see also van Eeden et
al., 1996
). This observation is consistent with a primary role for
the notochord in centrum segmentation.
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Discussion |
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A previous morphological investigation of vertebral development in another
teleost, medaka, has shown that bone matrix in this species is secreted by a
layer of osteocytes that lie over the outer surface of the centrum, with no
deposition on the inner surface (Ekanayake
and Hall, 1987; Ekanayake and
Hall, 1988
). Matrix-producing cells avoid becoming trapped in
their own secretion because of its polarized nature, where cells only generate
matrix in the direction of previously deposited bone. We have found that
zebrafish centra also lack osteoblasts embedded in the bone matrix, but the
secretion process resembles an inverted version of that in medaka; matrix is
deposited from inside the bony ring, by notochord cells, rather than from
outside.
Such a mechanism raises the intriguing problem of how zebrafish centra
increase in size during later development. One possibility is that the
sclerotome-derived osteoblasts invade the centra at a later developmental
stage. In keeping with this, the presence of some sclerotome cells has
recently been noted in the developing zebrafish vertebra, particularly in the
neural arches, showing that sclerotome cells can contribute vertebral bone
(Morin-Kensicki et al., 2002).
The relative contribution of sclerotome cells to centra is less clear,
however, and requires further investigation.
A duality in vertebral patterning
Our results also suggest that the notochord may make an important
contribution to the segmental patterning of the vertebral column. In
particular, we find that segmentation is lost after ablation in vivo of
notochord cells lying at precise, segmentally reiterated positions along the
developing trunk, adjacent to the somite boundaries. This suggests that
notochord cells adjacent to boundaries initiate centrum formation; subsequent
expansion of the centrum may result from diffusion of this matrix along the
long axis, and/or an additional contribution of matrix from neighbouring
notochord cells at later stages.
In fss mutant embryos, where sclerotome patterning is abnormal,
the subsequent patterning of the neural and haemal arches is also
disorganized, yet the centra develop normally. In combination with the
ablation studies, these findings suggest the existence of a duality in
vertebral patterning, in which the notochord and somites impart complementary
segmental information to the vertebral column. The notochord is necessary to
generate the metameric arrangement of centra, whereas the somites direct
patterning of the arch elements attached to them. It is interesting to note
here that, like the centra, the arches also develop in the absence of a
cartilage intermediate, implying that sclerotome-derived chondrocytes are also
absent in this vertebral region. Although it is possible that residual
manifestations of trunk segmentation may remain in fss mutant embryos
(van Eeden et al., 1998), our
use of these embryos to test the resegmentation hypothesis is appropriate. The
key feature of this model is the subdivision of the sclerotome into anterior
and posterior halves, with a subsequent recombination of adjacent halves; the
finding that the segmental organization of the sclerotome is destroyed in
fss mutants, yet centra are normal, presents a major difficulty for
any simple version of this model.
A further difficulty in applying classical resegmentation in the zebrafish
comes from a recent lineage study testing the hypothesis directly. Clonal
analysis of the zebrafish sclerotome has shown that cells from one
half-sclerotome can contribute to two adjacent vertebrae. There is, therefore,
no tight correlation between sclerotome fate and vertebral origins in the
zebrafish (Morin-Kensicki et al.,
2002), and a similar finding has been reported previously using
quail-to-chick half-somite grafts (Stern
and Keynes, 1987
). Morin-Kensicki et al. have proposed a `leaky'
resegmentation model, based on a continuing primacy for the sclerotome in
vertebral patterning. This modified model does predict the extensive vertebral
arch defects seen in fss mutants, but has greater difficulty in
explaining the normal appearance of centra in these mutants. An alternative
view, supported by the findings of this study, is that the notochord plays a
central and instructive role in centrum segmentation.
Notochord segmentation
Our results suggest that the notochord is functionally segmented, raising
the question of whether it is also segmented at the molecular level. To date,
no periodic patterns of gene expression have been described in the early
notochord, but these may exist nonetheless. Regionally restricted patterns of
Hox gene expression have been described in the zebrafish notochord
(Prince et al., 1998),
suggesting that this morphologically uniform structure is at least subdivided
regionally along the anterior-posterior axis. Further evidence for regional
subdivision is provided by the observation that the anterior notochord in
Xenopus is a more powerful inducer of En-2 in neural
ectoderm than posterior notochord
(Hemmati-Brivanlou et al.,
1990
); the regional inducing capacity of the early chordamesoderm
is also well known (Spemann,
1938
).
If the notochord has an instructive role in segmental patterning, the question arises as to how this is acquired. Segmentation of both notochord and somite mesoderm may originate simultaneously during gastrulation. Alternatively, notochord segmentation may be secondary to somite segmentation, being transmitted by unknown mechanisms between these adjacent tissues.
Overall, our results support the view that the notochord is an archetypal
segmental structure in the vertebrate trunk
(Stern, 1990;
Fleming et al., 2001
).
Consistent with this view, extirpation of the notochord in amphibian and avian
embryos results in the formation of a rod of vertebral cartilage lacking overt
segmental features (Kitchin,
1949
; Strudel,
1955
). This raises the intriguing possibility that the notochord
has a conserved role in the instruction of vertebral patterning in all
vertebrate classes.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge CB10 1SA, UK
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