Download Determination of epithelial half-somites in skeletal

Development 116, 441-445 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
441
Determination of epithelial half-somites in skeletal morphogenesis
RONALD S. GOLDSTEIN* and CHAYA KALCHEIM †
Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School, P.O.B. 1172- Jerusalem 91010, Israel
*Present address: Department of Life Sciences, Bar-Ilan University, 52900 Ramat-Gan, Israel
†To whom correspondence should be addressed
Summary
The segmental body plan of vertebrates arises from the
metameric organization of the paraxial mesoderm into
somites. Each mesodermal somite is subdivided into at
least two distinct domains: rostral and caudal. The segmental pattern of dorsal root ganglia, sympathetic ganglia and nerves is imposed by differential properties of
either somitic domain. In the present work, we have
extended these studies by investigating the contribution
of rostral or caudal-half somites to vertebral development using grafts of multiple somite halves. In both rostral and caudal somitic implants, the grafted mesoderm
dissociates normally into sclerotome and dermomyotome, and the sclerotome further develops into vertebrae. However, the morphogenetic capabilities of each
somitic half differ. The pedicle of the vertebral arch is
almost continuous in caudal half-somite grafts and is
virtually absent in rostral half-somite implants. Simi-
larly, the intervertebral disk is present in rostral halfsomite chimeras, and much reduced or virtually absent
in caudal somite chimeras. Thus, only the caudal half
cells are committed to give rise to the vertebral pedicle,
and only the rostral half cells are committed to give rise
to the fibrocartilage of the intervertebral disk.
Each vertebra is therefore composed of a pedicle-containing area, apparently formed by the caudal halfsomite, followed by a pedicle-free zone, the intervertebral foramen, derived from the rostral somite. These
data directly support the hypothesis of resegmentation,
in which vertebrae arise by fusion of the caudal and rostral halves of two consecutive somites.
Introduction
halves from quail donors. Multiple half-somites retain their
rostral or caudal character since RS grafts resulted in continuous, non-segmented PNS structures, including larger
than normal DRG, and CS grafts in distorted segmentation
of PNS structures and smaller than normal DRG (Goldstein
et al., 1990; Kalcheim and Teillet, 1989).
Certain aspects of vertebral morphology related to axial
level, are also determined while the somite is still epithelial. For example, the dorsoventral axis of the sclerotome
is determined three hours after somite segmentation from
the segmental plate (Aoyama and Asamoto, 1988). In
addition, replacement of cervical somites with those from
thoracic levels, leads to the appearance of rib-like structures in the neck (Kieny et al., 1972). As to the possible
molecular mechanisms controlling somite determination,
members of the murine Hox gene family were shown to be
expressed in the somites and their derivatives, along defined
extents of the rostrocaudal axis (Kessel and Gruss, 1990;
Mahon et al., 1988). Moreover, ectopic expression of the
Hox 1.1 transgene caused specific variations of the cervical vertebra, suggesting that this homeobox gene plays a
regulatory role in vertebral development (Kessel et al.,
1990).
Several groups have investigated whether the early deter-
The somite is a segmentally repeating mesodermal structure common to vertebrates. It dissociates into the sclerotome, which gives rise to the axial skeleton, and the dermomyotome, which gives rise to the axial musculature and
dermis. Various aspects of sclerotomal differentiation are
determined while the somite is still an epithelial structure.
For example, the division of the epithelial somite into rostral and caudal domains has been shown to be responsible
for the segmental morphogenesis of the peripheral nervous
system (PNS) of the trunk, including the nerves (Keynes
and Stern, 1984), dorsal root ganglia (DRG; Rickmann et
al., 1985; Bronner-Fraser, 1986; Kalcheim and Teillet,
1989; Lallier and Bronner-Fraser, 1988; Teillet et al., 1987)
and sympathetic ganglia (SG; Goldstein and Kalcheim,
1991). The rostral somite is permissive to axonal outgrowth
and neural crest cell migration, while the caudal portion of
the somite inhibits axonal outgrowth by collapsing growth
cones (Davies et al., 1990) , and also prevents the entrance
of neural crest cells. This division into rostral and caudal
domains is already determined while the somite is epithelial, as demonstrated by replacing somites of chick hosts
with multiple rostral (RS ) or caudal (CS) epithelial somitic
Key words: avian embryos, peripheral nervous system, quailchick chimeras, vertebral development.
442
R. S. Goldstein and C. Kalcheim
mination of rostral and caudal half-somites is expressed in
vertebral morphogenesis. Studies in which the fate of halfsomites was followed by either the quail marker (Stern and
Keynes, 1987), or peanut lectin binding (Bagnall and
Sanders, 1989), led to conflicting conclusions. Replacement
of single chick half-somites by their quail counterparts
showed that each somitic half can give rise to all vertebral
components. In contrast, peanut lectin, which at early stages
preferentially stains the caudal half of the sclerotome (Stern
et al., 1986), at later stages stains specific vertebral components, such as the intervertebral (iv) disk.
As part of our continuing studies on the mutual interactions between the paraxial mesoderm and the nervous
system, we have reevaluated the issue of the contribution
of half somites to vertebral morphogenesis, using grafts of
multiple quail rostral or caudal somitic halves into chick
hosts. We find that both rostral and caudal somitic halves
can give rise to most parts of the axial skeleton, including
the rib. However, the ability to give rise to two components
of the spinal column is confined to distinct half-somites.
The pedicle is derived from only the caudal somitic-half,
and the iv disk from only the rostral. These results shed
light on fundamental processes involved in the segmentation of axial structures in the verterbral trunk.
Materials and methods
Embryos of chick (Gallus gallus) and Japanese quail (Coturnix
coturnix Japonica) were used for this study. Eggs from commercial sources were kept in a humidified incubator at 38 ± 1°C.
Embryonic microsurgery
Surgery in this study was essentially the same as has been
described (Kalcheim and Teillet, 1989). Briefly, rostral or caudal
halves of the three or four most recently formed somites were
excised from 20-24-somite stage quail embryos. The sclerotome
from somites at this level of the rostrocaudal axis generates cartilage that forms ribs (Kieny et al., 1972; Murillo-Ferrol, 1963;
Seno, 1961). A drop of 50% pancreatin (Gibco) in phosphatebuffered saline (PBS) pH 7.4, was applied locally to facilitate dissection and ensure complete removal of the somitic mesoderm.
Like somitic-halves from several donor quails were pooled in PBS
until implanting.
The three or four most recently formed somites on the right
side of chick recipients were removed, and the space produced
was filled with 2-5 quail rostral half-somites. In some animals,
unsegmented paraxial mesoderm equivalent in length to 2 somites
was removed, along with the last detached somite(s). Two types
of chimeras were produced. In the first group, recipient embryos
having 11-15 somites at the time of implantation were used, resulting in modification of the mesoderm at the cervical level of the
neuraxis. The second group of recipients had 20-22 somite pairs
at the time of the surgery, resulting in modification of the vertebra at the brachial level of the axis. Only embryos in which
replacement of host mesoderm was complete in the vertebral
column along the entire rostrocaudal extent of the graft were used
for analysis. Serial section analysis of 14 and 8 embryos receiving rostral and caudal half-somite grafts respectively, was performed. Three-dimensional computer reconstructions were made
from six chimeras, three with RS grafts, and three with CS grafts.
Histology
Embryos were fixed in Bouin’s fluid on E9-E10, and embedded
in paraplast. Serial 10 µm sections were mounted on gelatinized
slides, and the tissue stained by one of two procedures. Cervicallevel chimeras were stained with Feulgen and counterstained with
Fast green. Chimeras with brachial level half-somite grafts were
stained with the monoclonal antibody HNK-1, which at this stage
of development strongly labels both the central and peripheral nervous systems (Abo and Balch, 1981). The HNK-1 binding was
visualized both with a goat anti-mouse secondary antibody coupled to horseradish peroxidase (Sigma) followed by diaminobenzidine treatment. The tissue was counterstained with Meyer’s
hematoxylin, which permitted distinction of the donor quail cells
from the host cells almost as well as the more conventionally used
Feulgen staining. The hematoxylin staining, however, had the
advantage of being compatible with peroxidase cytochemistry,
unlike the Feulgen technique. After initial analysis, some preparations were restained in 1% aqueous toluidine blue to facilitate
distinction of hyaline and fibrous cartilage.
Data analysis
Three dimensional reconstructions of the neural and vertebral
structures were made from alternate serial sections using the
HVEM 3D program developed by the University of Colorado at
Boulder Laboratory for High Voltage Electron Microscopy, and
the images photographed from the computer monitor on Kodak
Ektar 25 film.
Results
Common properties of vertebrae in the chimeras
Dissociation of the epithelial somites into dermomyotome
and sclerotome occurs normally in multiple half-somite
chimeras, but vertebral morphogenesis is modified (like that
of PNS structures, Goldstein et al., 1990; Goldstein and
Kalcheim, 1991; Kalcheim and Teillet, 1989). Vertebrae in
both types of chimeras contained most of the appropriate
components such as body, neural arches and costal
processes, suggesting that both half-somite moieties can
contribute to these structures in vivo.
Both rostral and caudal chimeras prepared at cervical
levels, were obtained by implanting half somites from
brachial levels of the quail neuraxis. In both cases, long
and thin rib-like cartilaginous structures developed from the
implanted quail mesoderm (Fig. 1A, C) as observed by
others implanting whole brachial somites in the neck (Kieny
et al., 1972). These structures were caudal-pointing extensions of the small costal processes present in normal cervical vertebrae. This structure is homologous to the rib at
brachial levels, which, however projects caudal instead of
the anterior direction taken by normal ribs. Our observation of ectopic ribs in both types of grafts provides evidence that both rostral and caudal mesoderm can contribute
to their formation.
The caudal, but not the rostral half-somite, gives rise to the
pedicle
On the unoperated side of the chimeras, the DRG protrude
between adjacent pedicles of the vertebra (Fig. 1A, C), as
is the case in normal embryos. In contrast, the morpho-
Half somite grafts and vertebral development
genesis of the vertebrae in embryos containing grafts of
multiple half-somites is altered.
In the case of vertebrae formed from sclerotome derived
from rostral half-somites, the morphogenesis of the lateral
vertebral arch is incomplete due to the total absence of a
pedicle connecting the neural arch with the vertebral body
(n=13 out of 14 succesful replacements). Consequently, a
continuous opening remains in the vertebra throughout the
entire length of the graft (Figs 1A, 2A), creating a passage
for the axons to extend from the spinal cord to the periphery (data not shown). In addition, the non-segmented DRG
protrude through this lateral defect (Figs 1A, 2A).
In contrast, in chimeras with CS grafts, an almost continuous pedicle-like structure develops from the grafted
mesoderm throughout most of its length, enclosing the DRG
within the vertebra (n=8; Fig. 1B and see also Kalcheim
and Teillet, 1989). In each of the chimeras with CS grafts,
a discrete opening in the lateral aspect of the vertebra could
always be seen, through which peripheral nerves exited and
the DRG sometimes partially protruded (Fig. 1B, C).
The rostral half somite, but not the caudal, gives rise to the
intervertebral disk
The iv disk is composed of fibrocartilage containing more
densely packed cells than the hyaline cartilage of the vertebrae. In toluidine blue-stained material, the iv disk stains
blue, as opposed to the violet metachromatic stain of the
vertebra (Fig. 2).
In chimeras with multiple rostral half-somite grafts, the
disk formed on both the normal, unoperated side (not
shown) and on the operated side from cells of the grafted
quail mesoderm (n=6, Fig. 2). The disks on the grafted side
were at least as large (in terms of number of sections
present) as the normal disks, and completely separated adjacent vertebral bodies. In each graft, the position of the disk
sometimes matched that on the normal side, and sometimes
did not. This result shows that individual vertebrae can form
from a mesoderm composed exclusively of rostral halfsomites.
In chimeras with multiple caudal somite-grafts, the iv
disks were either much reduced, or entirely absent (n=5,
Fig. 3). For example, in one brachial chimera with a graft
replacing four chick somites, four disks were present on the
control side, occupying a total of 63, 10 µm thick sections,
whereas on the operated side, all cartilage in the area of the
vertebral body was of normal hyaline morphology (Fig.
3A). In most caudal grafts, short pieces of fibrocartilage
were present. These never completely separated the vertebral bodies, and were mostly confined to a small medial
zone between the notochord and the spinal cord. In several
grafts, small incursions of chick cells were seen in this
mediodorsal area of the vertebral body into the operated
side.
Discussion
We report here that experimental construction of a paraxial mesoderm composed exclusively of multiple rostral or
caudal epithelial somite-halves, leads to the formation of
443
Fig. 3. The caudal half of the somite does not give rise to the
intervertebral (iv) disk. Serial section analysis of two embryos
receiving caudal half-somite grafts in place of host somites 18-21.
Sections containing fibrocartilage (iv disk) are depicted by a thick
line, and those with hyaline cartilage (vertebral body) with a thin
line. In (A), no sections containing iv disk-like structures in the
operated side were observed, in contrast to 63 sections in 4 iv
disks on the control side. In (B), 3 short stretches of sections (a
total of 23) containing fibrocartilage were present on the operated
side of the chimera; however these cells were mostly confined to a
medial zone between the notochord and spinal cord and never
completely separated the vertebrae. On the control side of this
embryo there were 4 iv disks spanning a total of 75 sections.
vertebrae in which specific elements are missing. Multiple
rostral grafts do not contain pedicle, while multiple caudal
grafts lack iv disks. The differences in the morphogenesis
of the vertebrae composed of only caudal or rostral moieties is likely to reflect intrinsic differences in the commitment of epithelial somite-halves: epithelial CS halves contain cells with the potential to give rise to the pedicle of
the vertebrae, whereas the RS halves contain instead cells
with the potential to form the iv disk. Consistent with this
early somitic cell commitment to specific cartilaginous
structures, we find that both rostral and caudal chimeras,
prepared by implanting half somites from the rib-forming
level of the axis into the neck, contain rib-like structures
derived from the grafted mesoderm (see Fig. 1). This is in
agreement with previous observations by Kieny et al.
(1972) who performed implants of either unsegmented or
segmented somitic mesoderm from thoracic into cervical
regions and vice-versa.
The formation of the iv disk from the rostral half of the
somite is in apparent contradiction with another recent
study (Bagnall and Sanders, 1989), in which the iv disk was
heavily stained with peanut lectin, suggesting that it arises
from the caudal somitic half. Expression of carbohydrate
epitopes dynamically changes during ontogeny. Therefore,
the observed staining with peanut lectin in the iv disk may
be developmentally regulated rather than serving as a lineage marker to demonstrate the derivation of the iv disk
from the caudal half of the somites.
Although early determination of somite cells may be the
mechanism that accounts for the present observations, it is
still possible that the migration of sclerotomal cells and con-
444
R. S. Goldstein and C. Kalcheim
sequent skeletal morphogenesis also partly depend upon
specific interactions taking place between these progenitors
and neural elements in the rostral half of each somite. For
example, the lack of pedicle in the RS chimeras might be
due to competition for space between the expanding unsegmented DRG (Goldstein et al., 1990) and the sclerotomal
cells. However, in RS grafts there are a few sections in
which the pedicle is missing, although there is no DRG in
that section. Conversely, DRG develop within the closed,
almost continuous pedicle in CS chimeras, suggesting that
competition is not the mechanism responsible for the lack
of pedicle in RS chimeras.
The DRG normally lies within the intervertebral foramen, resting on the pedicle of the vertebral arch of the corresponding segment. Based on the present results, we
suggest that during normal development, the caudal somitic
domain forms the vertebral pedicle, whereas the rostral half
of a somite gives rise to the region corresponding to the
intervertebral foramen, due to its inability to develop a pedicle (Fig. 4). This is consistent with the finding that both
DRG and peripheral nerves exclusively form facing the rostral somitic domains in the early embryo, and lie in the
intervertebral foramina in the mature animal.
Since each normal vertebra contains a region with a pedicle located cranial to the corresponding pedicle-free, DRGcontaining zone, it may be inferred that each vertebra
derives from the caudal half of a given somite and the rostral half of the subsequent one, in agreement with the resegmentation hypothesis (Fig. 4). Although some studies have
questioned the validity of this hypothesis (Stern and
Keynes, 1987, and discussed in Bagnall and Sanders, 1989;
Keynes and Stern, 1988), others have supported it using
different techniques (Aoyama and Asamoto, 1988; Bagnall,
1992; Bagnall and Sanders, 1989; Bagnall et al., 1988;
Ewan and Everett, 1992). For example, in very recent
studies, injection of a fluorescent dye or recombinant retrovirus into single somites, resulted in the labeling of components of two contiguous vertebrae (Bagnall, 1992; Ewan
and Everett, 1992).
The presence of a well developed iv disk in RS chimeras
raises an important basic question about the source of segmentation cues in the development of axial structures of the
trunk. It is believed that axial segmentation is caused by
the alternation of RS and CS domains of the somites. The
iv disk that separates two contiguous vertebrae, is an
expression of this segmentation. The formation of the iv
disks between the vertebrae derived exclusively from RS
mesoderm in our chimeras, may be accounted for by several reasons. First, it is possible that the cue for segmentation of the grafted RS comes from the mesenchyme on the
unoperated side of the embryo. This seems unlikely because
in most chimeras there is a lack of bilateral correspondence
in the position of the iv disks and also, iv disks are present
on both sides of RS chimeras receiving bilateral grafts.
Interestingly, the rostrocaudal position of the iv disks in the
bilateral chimeras does not match (unpublished data). A
second possibility is that the other major axial structures,
the neural tube and/or notochord, provide specific segmentation cues to the paraxial mesoderm. This is also unlikely
because of the bilateral asymmetry in iv disk formation in
the RS chimeras. Finally, it is possible that the RS is sub-
Fig. 4. Early determination of half somites in vertebral
morphogenesis. Immediately upon individualization, the epithelial
somites are divided into at least two domains, rostral (R), and
caudal (C). According to the model of resegmentation, each
vertebra forms from the fusion of the caudal half of a given somite
and the rostral half of the subsequent one, separated by iv disks.
The vertebral body can be formed from both RS and CS halves
(Fig. 1), (the horizontal line we have drawn separating the RS and
CS contributions to the vertebral body, is an imaginary one, since
our transplants of multiple half somites do not adress the exact
position of this boundary, see Bagnall et al., 1988). In contrast, we
show that the pedicle (Ped) derives exclusively from the CS
(hatched) and the iv disk from the RS mesoderm (open). We
therefore propose the following model for vertebral formation:
each vertebra has a rostral portion containing a pedicle which, as
shown here, derives from the CS, and a caudal portion deriving
from the RS which lacks the pedicle and has, instead, an
intervertebral foramen (F) into which neural structures protrude.
According to this model, the iv disk is the caudalmost part of the
vertebra, deriving from the RS half. The horizontal stripes define
the spinous process of the vertebra whose somitic origin is
unknown. NT, neural tube; SC, spinal cord.
divided into smaller domains committed to give rise to distinct vertebral components, such as the iv disk. This last
notion extends the currently accepted view that vertebral
segmentation results from the alternation of early committed somitic halves.
We wish to express our gratitude to Professor A. Ornoy for
valuable instruction in the details of the histology of developing
cartilage and for helpful suggestions. We also thank Ms. Chana
Carmeli for excellent technical assistance, Michal Pollak for help
with the computer reconstructions and Gilat Brill for critical reading of the manuscript. This work was supported by grants from
the National Council for Research and Development and the Commission for European Communities, the Familial Dysautonomia
Foundation, and the Israel Academy of Sciences and Humanities
to Chaya Kalcheim, and a grant from the Israel Institute for Psychobiology- Charles Smith foundation to Ron Goldstein.
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dev1923 colour tip-in
Fig. 2. The rostral half of the somite
gives rise to the intervertebral (iv) disk.
A transverse section of an E9 chick
embryo receiving a graft of 6 quail
rostral half-somites in place of somites
18-21. (A) On the operated, left side of
the chimera, note the absence of the
pedicle and the presence of a DRG
(drg), in contrast to the closed vertebra
and lack of DRG on the control (right)
side. (B) The grafted mesoderm
developed into fibrocartilage of an iv
disk (blue) in place of the left half of
the vertebral body. The right, control
side is metachromatically stained
purple for hyaline cartilage. (C) High
magnification of the box in B showing
that the iv disk is composed of quail
(arrows), and the hyaline cartilage of
chick cells. ch, chick; drg, dorsal root
ganglion; N, notochord; SC, spinal
cord; P, pedicle; q, quail. Bars: A, 100
µm; B, 40 µm; C, 15 µm.