69
Development 103, 69-85 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
The contribution made by a single somite to the vertebral column:
experimental evidence in support of resegmentation using the
chick-quail chimaera model
K. M. BAGNALL1, S. J. HIGGINS1 and E. J. SANDERS2
1
Departments of Anatomy and Cell Biology, and 2PhysioIogy University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Summary
The somitic involvement in the formation of the
vertebral column was examined using the chick-quail
chimaera model. Single cervical somites from quail
donor embryos were transplanted into similarly
staged chick host embryos. Following further incubation, serial sections of variously staged embryos
were stained with the Feulgen reaction to distinguish
the two cell populations.
Quail cells were generally located within a delimited
region in one half of each of the two adjacent vertebrae, as well as in the intervening disc. The horizon-
tal plane of division through each vertebra passed
approximately through the centre of the body and
divided the neural arch into rostral and caudal halves
through the rostral border of the caudal notch. These
results give support to the controversial theory of
resegmentation, in which it was suggested that there is
an apparent realignment of segmentation between the
somite stage and the subsequent vertebral stage of
development.
Introduction
The methodology usually employed in the study of
vertebral formation has consisted of serial sections of
staged embryos. The inability to identify the cells
derived from a single somite by this means has
resulted in the development of widely divergent
theories and has perpetuated the controversy regarding the details of vertebral formation. Although some
limited experimental evidence in support of the
theory of resegmentation has been obtained indirectly from studies of tetraparental mice (Moore &
Mintz, 1972), cell polarity during development (Trelstad, 1977) and somitic transfers between chick and
quail in investigations of-wing muscle determination
(Beresford, 1983), there is also some recent experimental evidence that appears to dispute the theory
(Noden, 1983; Dalgleish, 1985; Stern & Keynes,
1987). This study was undertaken to reexamine the
issue of the somitic involvement in vertebral formation. To this purpose, use of a permanent cell marker
system was employed to permit later identification of
descendent cells from heterospecific single-somite
transplants. We have replaced single somites in chick
embryos at 2 days of incubation with those of 2-day
While it is well accepted that somites contribute to
the formation of vertebrae and the intervening intervertebral discs, the precise relationship between them
has long been controversial. Remak (1855) was the
first to recognize an apparent realignment of material
between the somite stage and the subsequent vertebral stage of development and formulated the
theory of resegmentation that considers that a vertebra is formed by the combination of the caudal half
of one bilateral pair of somites with the rostral half of
the next caudal pair of somites. This theory has
received support from several other studies (Von
Ebner, 1888; Bardeen, 1905; Piiper, 1928; Dawes,
1930; Williams, 1942; Sensenig, 1943, 1949; Werner,
1971) although the precise contribution of the two
pairs of somites to specific parts of the vertebra and
disc is not clear (O'Rahilly & Meyer, 1979). The
theory of resegmentation has often been challenged,
however (most recently by Baur, 1969; Verbout,
1976,1985) and equally plausible alternatives theories
have been proposed.
Key words: somite, vertebrate, chick-quail,
segmentation, embryo, chimaera.
70
K. M. Bagnall, S. J. Higgins and E. J. Sanders
In preparation of the donor, the quail embryo was
removed from the surface of the yolk and placed in Pannett
and Compton's saline. The embryo was of a similar stage to
the previously prepared chick embryo (usually within one
to two somite pairs [see Table 1]). The equivalent somite to
the one removed from the chick (within two or three
somites) was isolated from the quail embryo and transferred to the operation site in the chick using a micropipette. It was then manoeuvered into the correct position,
using carbon particles as markers of somite orientation,
although this method was not always a completely reliable
one. The hole in the chick shell was then sealed using
adhesive tape and the eggs reincubated to allow further
development.
After further incubation ranging from 3-8 days, the
surviving embryos were fixed in Zenker's fluid, rinsed in
running tap water and stored in 70 % ethanol. The cervical
region of each embryo was then dissected from the rest of
the body and processed for embedding in paraffin. Serial
sections of 8^m thickness were made in various planes and
the sections stained with Feulgen method for demonstration of DNA to distinguish the quail from chick cells (Le
Douarin, 1973).
The slides were observed and photographed using a Leitz
orthoplan photomicroscope. The positions of the quail cells
quail embryos and observed the subsequent site of
the quail cells in the vertebrae of the chimaeras at 10
days of incubation. In this way, we have been able to
establish the somitic contribution to vertebral formation.
Materials and methods
Fertile chick (Callus domesticus) and quail (Coturnix coturnix japonica) eggs were incubated at 37 °C for 42 and 48 h,
respectively, to reach approximately stages 11 or 12 according to the system of Hamburger & Hamilton (1951) for
chick embryos. A window was made in each of the host
chick eggs and a small amount of black drawing ink diluted
1:1 with Pannett and Compton's saline containing antibiotics (penicillin and streptomycin, lOOOOi.u. ml"1,
SIGMA) was injected subendodermally to permit visualization of the embryos. The vitelline membrane covering the
embryo was then broken. A single somite from the future
cervical region, usually within two or three of the last
formed somite (see Table 1), was then excised using a
tungsten wire needle and a micropipette. Pannett and
Compton's saline was added whenever required to prevent
the embryo from drying out.
Table 1. A summary of the data relating to the host chick and quail embryos used during the transplantation
Donor
Host
Embryo no.
26/5
31/2
31/3
31/4
34/4
38/2
38/3
38/6
40/1
40/4
44/2
52/2
52/5
75/3
83/1
83/3
92/1
92/3
95/4
96/2
96/5
Som rem
Tot pr
Som tra
Tot pr
Stage
at fix
Plane of
section
8r
16r
15r
16r
9r
16r
16r
15r
llr
9r
16r
16r
15r
lOr
15r
13r
16r
llr
18r
13r
15r
14
19
16
20
11
17
18
17
13
9
18
17
18
11
17
15
17
12
19
13
16
u
14r
12r
13r
lOr
151
161
u
17
14
17
11
16
16
14
12
12
14
13
u
12
12
13
17
13
19
13
u
35
28
29
28
29
28
35
36
28
26
35
30
30
36
26
27
30
28
33
31
32
trans
sagit
coron
coron
coron
coron
trans
trans
coron
coron
coron
trans
coron
trans
trans
coron
coron
coron
sagit
coron
coron
Som rem = number of the somite removed
Tot pr
= total number of somite pairs present
Som tra
= number of the somite transplanted
Stage at fix = stage of the embryo at fixation (H & H)
r
= right
1
= left
u
= undetermined
= transverse
trans
= sagittal
sagit
= coronal
coron
u
lOr
9r
141
91
u
10r
81
13r
16r
llr
18r
13r
u
Single somite contribution to vertebral formation
within the vertebrae were noted and recorded for each of
the embryos. Specific attention was directed to the quail cell
location in each of the vertebral elements (centrum, costal
process, transverse process, lamina, pedicle, spinous process, articular processes and the intervertebral disc; Fig. 1).
71
Reconstructions using a series of contour drawings of the
regions that contained quail cells were made of nine
embryos that were considered to be representative of the
total data.
Results
From a total of 209 experiments, 21 embryos developed to stage 26-36 (Hamburger & Hamilton, 1951)
in which some chondrification of the vertebral elements could be distinguished. In these embryos,
quail cells were present in the cartilage of the developing vertebrae, in the paraxial musculature, in the
spinal cord meninges, in the dermis and in the general
connective tissue around the vertebral column. Integration of the quail cells was variable and in three
embryos (31/2, 38/6, 52/5) appeared to be minimal.
With few exceptions, the quail cells were found in
regions only on the operated side of the embryo and
in all embryos the quail cell regions were continuous
with the adjacent chick cell regions, with no interruptions between the two populations. In all cases, the
quail cells had formed portions of two consecutive
vertebrae (see Table 2), although considerable variation of quail cell location was observed.
A wide variety of abnormalities was observed in
the vertebral elements and in adjacent soft tissues
within the quail cell area. In fact, no single embryo
exhibited normal morphology of all of the structures
and tissues in the operated region. Some of the
abnormalities observed are indicated in Table 2 (Ab
or F). They generally included intervertebral fusion,
appearance of anomalous cartilaginous processes,
reduced proportions of elements and complete absence of some parts of the involved vertebrae.
List of abbreviations for figures
c, cervical;
ce, centrum;
ch, chick;
cp,
di,
g,
in,
costal process;
disc;
spinal ganglion;
caudal (inferior)
notch of pedicle;
ip, caudal (inferior)
articular process;
;, articular joint;
I,
lamina;
mu, muscle;
n,
notochord;
p,
pedicle;
q,
quail;
sc, spinal cord;
spc, spinal canal;
sp, rostral (superior)
articular process;
spp, spinal process;
t,
transverse foramen;
tcp, tip of costal process;
transverse process
tp,
Fig. 1. Illustrations of a cervical vertebra of a mature
chick. Rostral (A), lateral (B), ventral (C) and dorsal (D)
views of an ossified middle cervical vertebra are
presented. Note the elongated centrum and the caudally
projecting tip of the costal process noticeable in the
lateral view. The transverse foramen can be seen in the
rostral view.
Centrum and disc
Quail cell distribution patterns were found to be most
consistent in the centra and intervening intervertebral
disc. 20 of the 21 embryos contained quail cells which
comprised some or most of the adjacent quadrants of
two consecutive centra and most or all of the corresponding half of the intervening disc (Figs 2, 3). This
indicates that the central region of the medial sclerotome from a single pair of somites forms the intervertebral disc, and the cells rostral and caudal to the disc
region merge with cells from the adjacent somites to
form parts of the rostral and caudal centra. Each
centrum, then, is a composite of cells from two
consecutive somite pairs, each pair making approximately equal contributions and the disc forms bilaterally within one somite pair.
Pedicles
Ten of twenty embryos contained quail cells in the
pedicles of two adjacent vertebrae. However, of
72
K. M. Bagnall, S. J. Higgins and E. J. Sanders
Table 2. Distribution of quail cells in vertebral elements of chimaeras
Embryo no.
Cent
Vert w Qu
26/5
C2
C3
5
*
•
31/2
C9
CIO
6
*
31/3
C9
CIO
5
C9
CIO
6
C3
C4
5
38/2
CIO
Cll
5
*
*
38/3
Cll
C12
6
*
*
38/6
C9
CIO
5
•F
•F
C5
C6
5
C4
C5
4
44/2
CIO
Cll
5
52/2
CIO
Cll
5
C9
CIO
5
75/3
C5
C6
4
83/1
C9
CIO
5
83/3
C7
C8
5
92/1
CIO
Cll
5
92/3
C5
C6
5
C12
C13
5
C7
C8
5
C9
CIO
5
31/4
34/4
40/1
40/4
52/5
95/4
96/2
96/5
Vertebral components with quail cells
Disc
Ped
Lam
Spn
Cost
Tran
Artie
Ro Ca
u
u
•F
•
Ab
•F
•F
•F
*
*
Ab
•F
•F
•F
*
*
+F
•F
•F
•F
•F
•F
•F
*
•F
•F
•F
+F
+F
•F
•F
•F
u
u
•F
•F
•F
•F
*
•F
Ab
•F
•F
•F
*F
*
•F
»F
•
•F
•F
•F
*
*
*
*
*
*
*
*
*
•
+F
—
-
*
*
•F
•F
•F
+F
(—)
•F
,
•F
Ab
Ab
Ab
+
_
Ab
•F
•F
(-)
_
-
•F
Ab
•F
•F
•F
•F
*
*
_
*
*F
•F
•F
•F
•F
Vert w Qu = vertebra containing quail cells
#
= somite number minusi number of the caudal
vertebra
= centrum
Cent
= pedicle
Ped
Lam
= lamina
= Spinous process
Spn
= costal process
Cost
= transverse process
Tran
= articular process
Artie
= rostral
Ro
+F
+F
Ca
C
•
+
Ab
Ab
Ab
+
*
*
•F
•F
*
*
*
+F
•F
•F
•F
•F
*F
•F
= caudal
= cervical
= many quail cells forming significant portion of the element
= some quail cells present and confluent with other quail
areas
— = no quail cells or very few isolated quail cells present
( ) = element not yet completely formed
Ab = element absent
F = abnormal fusion of elements
u = undetermined
Single somite contribution to vertebral formation
73
these ten, only two exhibited relatively normal morphology of the two pedicles and continuous distribution of the quail cells. In two other embryos the
quail cell content was high but the pedicles were
fused. Five embryos displayed a large proportion of
quail cells in the caudal pedicle and only a few quail
cells in the rostral pedicle; however, two of these five
had fused pedicles. One other embryo contained only
a few quail cells in each pedicle. Of the remaining ten
embryos, six contained a large proportion of quail
cells in the caudal pedicle and none in the rostral
pedicle. Few contained quail cells in the rostral
pedicle when the caudal pedicle was missing. Therefore, in summary, eighteen of the twenty embryos
either contained few or no quail cells in the rostral
pedicle, or exhibited a major anomaly of the pedicles
(both fused or caudal pedicle absent). In particular,
whenever the caudal pedicle was present it contained
quail cells (16 of 16 embryos) (Table 2, Fig. 4).
These results suggest that cells from a single somite
contribute mostly to the caudal pedicle and to the
base of the rostral pedicle (dorsal extension of the
centrum). Somitic contribution to the rostral pedicle
normally would not extend past the rostral border of
the caudal notch of the rostral vertebra, consistent
with the pattern seen in other parts of the neural arch.
2A
sc
ce
n
di
V<.-:;» ; • - . ; . . . . . .
ee
B
Fig. 2. Low (A) and high (B) magnification of transverse
sections through the disc and adjacent centra in a
stage-35 embryo (38/3). As the intervertebral junction is
oblique, quail cells in the disc and adjacent centra in two
successive vertebrae can be noted. Some mixing of chick
and quail cells in muscle and connective tissue is apparent
(arrows indicate quail cells). Medially, quail cells extend
very slightly beyond the midsagittal plane of the spinal
cord, but ventral to the notochord the chick cell region
appears to have shifted correspondingly to the quail side.
Bar, 100 fim.
Laminae
Quail cells were present in portions of both of the
adjacent laminae in all twenty embryos (see Fig. 5).
Twelve embryos contained a large proportion of quail
cells in both rostral and caudal laminae but seven of
these demonstrated at least partial fusion of these
elements. In five embryos, the caudal lamina contained numerous quail cells and the rostral lamina
only a few. The reverse situation was present in only
one embryo. Two embryos contained only a small
amount of quail cells in each of the laminae. From the
serial reconstructions (Figs 7, 8) of the embryos, it
could be seen that the quail cell region extended only
a short distance into the more rostral lamina. In
particular, quail cells were found in only a few
sections (approximately 100 fxm or less) rostral to the
rostral border of the caudal notch. As this was the
original rostral boundary of the somite (position of
the spinal ganglion) most likely the normal contribution to the rostral lamina would be relatively small.
Conversely, the single somite contribution to the
caudal lamina should be relatively large. Cells from a
single somite, then, normally should form parts of
two adjacent laminae, but a far greater portion of the
more caudal lamina.
74
K. M. Bagnall, S. J. Higgins and E. J. Sanders
3A
Fig. 3. Low (A) and high (B) magnification of transverse sections through the disc and adjacent centra in a stage-36
embryo (75/3). As the intervertebral junction is oblique, quail cells can be seen forming the right half of adjacent centra
in two successive vertebrae and the intervening disc. Some quail cells in the perichondrium extend to the left of the
spinal cord. A few quail cells are possibly forming the meninges (arrows). Bar, 100f.im.
Single somite contribution to vertebral formation
75
4A
* *• .*
sc
B
Fig. 4. Low (A) and high (B) magnification of longitudinal sections through the pedicles of a stage-28 embryo (40/1).
Note that the caudal pedicle (right) is entirely quail, while the rostral pedicle (left) has only a few quail cells (arrows).
A small ganglion is present between the pedicles. Bar, 100/am.
Spinous processes
The distribution patterns in the spinous processes
were inconsistent, possibly related to the high incidence of abnormalities associated with these elements and to the lack of chondrification in ten
embryos. In nine embryos, quail cells were not
present in either of the two spinous processes or their
precursors. Seven other embryos, however, contained a large proportion of quail cells in both spines
or precursors but of these, four embryos displayed
some degree of fusion of the two spinous processes
and the other three had not yet formed complete
spinous processes on the involved vertebrae. In three
embryos, quail cells were present to a large extent in
the region of the caudal spine but only minimally, or
not at all, in the rostral spine. One embryo contained
just a few quail cells in each of the two spinous
processes. On the basis of these results no firm
distribution pattern of quail cells in the spinous
processes could be established although, perhaps, it
should be closely associated with that of the lamina,
as each spinous process seemed to be an extension
and fusion of the two bilateral laminae.
Costal processes
Fifteen of the twenty-one embryos contained quail
cells in each of the costal processes of the two
adjacent vertebrae. Of these fifteen, however, eleven
76
K. M. Bagnall, S. J. Higgins and E. J. Sanders
sc
.0
5A
, * » . * • • '
**
c/?-^
.•..•.:fl..-iA,"4-•'•"•••
,*/'
•
i*»
sr
;-;.
%
:
• * • • • • • . . •
•
"
:
-
.
>
Fig. 5. Low (A) and high (B) magnification of longitudinal sections through the laminae of a stage-35 embryo (44/2).
Quail cells occupy portions of two adjacent laminae. Chick cells are present in the extremities of the two quailcontaining laminae. Bar, 100 jim. Low (C) and high (D) magnification of longitudinal sections through the laminae of a
stage-32 embryo (96/2). The two involved laminae are fused (see C, /+/), the majority of cells being quail. Chick cells
are present at each extremity. Note the wider dispersal of quail cells (arrows) in the adjacent muscle which is otherwise
mainly chick. Bar, 100fjm.
Single somite contribution to vertebral formation
cases were found in which the rostral costal process
contained quail cells only in the caudally projecting
tip. In the remaining six embryos, four were missing
one of the two costal processes (the existing costal
process contained a large number of quail cells) and
two exhibited quail cells only in the caudal costal
process. These results suggest that cells from a single
somite make up almost the entire caudal costal
process and also make a small contribution to the
caudal tip of the rostral costal process.
Transverse processes
The situation here was very similar to that already
described for the pedicles. Adjacent transverse processes contained at least some quail cells in ten of
twenty cases; but in one of these embryos the
transverse processes were fused. In contrast, seven of
the remaining ten embryos demonstrated quail cells
in only the caudal transverse process, although the
rostral process was present, while each of the other
three embryos were missing the transverse process of
the caudal vertebra. Therefore, as in the pedicles, in
fifteen of twenty embryos examined, either few (five
cases) or no (seven cases) quail cells were found in the
rostral process, or the caudal transverse process was
absent (three cases) and whenever the caudal transverse process was present, it was found to contain
quail cells (17 of 17 embryos). Furthermore, it was
noted in many embryos that the quail cell distribution
in the transverse processes frequently matched that of
the pedicles, possibly indicative of developmental
synchrony in these two elements. It would appear,
therefore, that the contribution from a single somite
is mainly to the caudal transverse process with a
minimal contribution, if any, to the rostral transverse
process.
Articular processes
Fifteen of twenty embryos contained quail cells in
both of the adjacent articular processes (i.e. the
caudal articular process of the rostral vertebra and
the rostral articular process of the caudal vertebra)
(see Fig. 6). However, in twelve of these cases, the
involved elements demonstrated at least partial
fusion of the elements. Surprisingly, in six embryos,
quail cells were also noted in the caudal articular
process of the caudal vertebra, but were not present
in the rostral process of the rostral vertebra in any of
the embryos. Of two embryos without quail cells in
adjacent articular processes, one exhibited an unusual pattern of quail cell distribution in the centra
and the other contained no quail cells in any of the
articular processes. The continuity of the cell distribution across the facet joint was also apparent from
the composite reconstructions and it would appear
that adjacent articular processes, as well as all the
11
tissues that form the joint surfaces and joint capsule,
are formed of cells from a single somite.
The quail cell distribution was readily apparent
from the contour drawings made from the serial
sections of nine of the embryos. Examples of these
reconstructions from two of the embryos are shown in
Figs 7-10. Fig. 7 shows caudal views (A,C), a rostral
view (D), and two caudal oblique views (E,F) of the
caudal vertebra of two adjacent vertebrae that contained quail cells. It also shows a rostral view of the
region within the vertebra occupied by the quail cells
(B). The corrresponding views of the rostral vertebra
of the same embryo are shown in Fig. 8. Fig. 7 clearly
shows the quail cells contained only within a rostral
quadrant of the vertebral body and not extending
over the midline (C,D). The quail cells can be seen
occupying all of the pedicle adjacent to the centrum
(F) and are distributed further into the transverse
process (C,E). All of the accompanying costal process can be seen to contain quail cells apart from a
small area at its tip (E). As the quail cell distribution
enters the lamina, it gradually becomes more dorsal
in the rostral sections, although only a few quail cells
in this specimen can be seen in the spinous process
(E). The relative proportion of quail cells in each
section of the lamina also becomes less as the sections
become more rostral (E). There are no quail cells to
be found in the caudal articulating process (E,F)
whereas the rostral articulating process is formed
entirely of quail cells (E).
Fig. 8 shows the corresponding views of Fig. 7 of
the rostral vertebra of the two adjacent vertebrae that
contained quail cells. In this example, the quail cells
can be seen to occupy only a caudal quadrant of the
vertebral body (C,F) with few or no quail cells being
found in the pedicle and transverse process (B-E).
However, there are a few quail cells to be found in the
tip of the accompanying costal process (E). As the
quail cells continue around the neural arch, they
populate the caudal half of the lamina entirely,
including the spinous process (E,F). The caudal
articulating process is also composed entirely of quail
cells (E,F).
Fig. 9 is an expanded caudal oblique view of the
two vertebrae shown in Figs 7 and 8 when combined.
The distribution of the quail cells can clearly be seen
to be continuous between the two vertebrae when the
vertebral elements are aligned, particularly in relation to the lamina and articular processes. Also
conspicuous is the small contingent of quail cells
found in the tip of the costal process of the rostral
vertebra.
Fig. 10 is a contour drawing of coronal sections of
another embryo. The area of quail cell distribution is
K. M. Bagnall, S. J. Higgins and E. J. Sanders
shaded and clearly demonstrates that it is restricted
to one side of the vertebral column and is almost
equally divided between adjacent rostral and caudal
1 *•<•.* m
halves of two vertebral bodies. The distribution can
also be seen in the adjacent halves of the corresponding neural arches.
vp.-.-..
Fig. 6. Longitudinal sections through the laminae and articular processes of stage-35 (44/2) and -28 (40/1) embryos.
Above, low (A) and high (B) magnifications, adjacent articular processes and the joint space are quail. Chick cells are
present in the neural arch extensions. Below (C) fused articular processes show quail continuing rostrally (left). Note
chick-quail mixing in the muscle (arrows indicate quail cells). Bar, 100^m.
Single somite contribution to vertebral formation
Discussion
The results of this study clearly support the concept of
a resegmentation between the somitic stage of development and the resulting vertebrae as first proposed
79
by Remak (1855) and supported by a number of other
investigators, including Piiper (1928), Williams (1942)
and Sensenig (1949). A single somite has been shown
to contribute to the caudal half of one vertebral body,
the intervening disc and the rostral half of the next
Fig. 7. Serial reconstructions of C6 of embryo 75/3
(stage 36). Caudal views (A,C) and rostral view (D) of
the caudal vertebra. The quail region has been shaded
separately (B) and within the vertebra (C,D).
Bar, 100 jim. In E and F (both are rostral oblique views
E, left, F, right), successive sections have been separated
3 mm to demonstrate more clearly the location of the
quail cells. The quail region begins part way through the
centrum and continues dorsally and laterally to
encompass most of the pedicle and costal process, all of
the transverse and rostral articular process and part of
the lamina and spinous process.
80
K. M. Bagnall, S. J. Higgins and E. J. Sanders
vertebral body. This division applies not only to the
vertebral bodies but also to the neural arches with
single somites contributing to caudal and rostral
portions of adjacent neural arches, although the
division is not quite so clear as with the vertebral
bodies. In the neural arch, the major somitic contribution is to the caudal arch with contribution to the
rostral arch being limited to a transverse plane
Fig. 8. Serial reconstructions of C5 of
embryo 75/3 (stage 36). Caudal (A,C)
and rostral (D) views of the rostral
vertebra. Note that the quail region
(B) includes part of the tip of the
costal process, but the centrum and
neural arch quail regions are
discontinuous. Bar, 100^m. In E and
F (both are rostral views), successive
sections have been separated 5 mm to
demonstrate more clearly the location
of the quail cells. The quail region
recedes dorsally through progressively
more rostral sections. The location of
quail cells in the dorsal portion of the
tip of the costal process can be noted.
Single somite contribution to vertebral formation
81
C5
Fig. 10. Serial reconstruction of C8 to C,2 of embryo 96/5
(stage 32). The quail region has been shaded and outlined
(dotted line). The continuity of the quail region across
the two vertebrae including the disc is apparent.
Bar, 100 ;/m.
Fig. 9. Separated right caudal view of C5 and Q of
embryo 75/3. Successive sections of C5 and Q have been
separated 10 mm to illustrate the continuity of the quail
cell region. C6 is outlined in black to distinguish it from
the Cj elements. The region of partial fusion in the
lamina is indicated by a broken line (arrow).
situated approximately through the rostral border of
the caudal notch. Fig. 11 summarizes these conclusions and extrapolates them to the expected contribution made by a single somite to ossified vertebral
formation based on our results. It is interesting to
note that the single somitic contribution (the quail
cell distribution) has clearly defined limits which
appear to follow the geometry of the vertebral
column rather than the anatomy. This is particularly
evident where quail cells are found in the tip of the
costal process of the rostral vertebra. It suggests that
quail cells differentiate according to their spatial
position and warrants further study, particularly of
the associated intervening tissues that are somitically
derived.
The methodology employed in this study is not
without problems which may have affected the results
and their interpretation. For example, it has been
shown by Strudel (1966) and ourselves (Bagnall et al.
1986) that the embryo can recover completely from
the removal of single somites and produce a normal
vertebral column. The site and mechanism of this
regulation is unknown and, therefore, it could not be
determined whether the methodology employed here
produces a vertebra composed of quail somitic cells
82
K. M. Bagnall, S. J. Higgins and E. J. Sanders
Fig. 11. Proposed contribution of cells from a single somite to formation of two adjacent vertebrae. Midcervical
vertebrae from a 10-day chick embryo are shown on the left. On the right is a representation of mature ossified
midcervical chicken vertebrae. Note that the vertebral region formed by cells from one somite crosses two adjacent
centra. The limits of single somite contribution are formed by the caudal notches of the pedicles. Also note the
contribution to the caudal tip of the costal process.
and chick cells derived from regulation. Furthermore, it has also been reported (Kieny et al. 1972) and
shown (Bagnall et al. 1986) that the procedure for
removal of somites often results in small pieces of
somitic material being left behind inadvertently.
Further studies (Bagnall etal. 1987) have shown these
pieces to be capable of reorganization into a somitelike unit with a reduced volume. While every attempt
was made to remove all of the chick somite and
replace it solely by an intact quail somite, there is no
guarantee that this was achieved in every case. The
consequences of potential cell interactions between
Single somite contribution to vertebral formation
remnant chick somitic tissue and extraneous quail
somitic tissue are unknown.
Kieny et al. (1972) using somite transfer techniques
demonstrated that the somites are regionally determined prior to the time of their formation. The
degree of regional determination was restricted in
their study to differences between cervical and thoracic vertebrae, with vertebrae having cervical
characteristics being produced in the thoracic region
and vice versa. Whether or not this determination can
be applied to consecutive vertebrae is unknown but
does have implications for this study. The levels of the
extirpated and donor somites were generally within
two or three of each other. The effect of such a small
difference in vertebral level on future development is
not known, but we considered that it would be
minimal in the midcervical region where most of the
surgical procedures were performed. Transitional
differences between consecutive vertebrae at this
level are slight compared to those differences between more widely separated levels and between the
different regions of the vertebral column.
In a series of experiments, Stern & Keynes (1986,
1987) have shown that the somite is further differentiated into presumptive rostral and caudal sclerotome
halves early in its formation and have suggested that
this difference is determined at the time of segmentation (Stern & Keynes, 1986). Furthermore, in
experiments in which similar somite halves (rostral or
caudal) have been situated adjacent to each other,
Stern & Keynes (1987) have found that sclerotome
cells from like halves mix with each other while those
from unlike halves do not. This suggests that correct
orientation of the donor quail somite is important,
since incorrect orientation with subsequent fusion of
cells from similar sclerotome halves might produce
abnormal vertebrae perhaps similar to some that we
encountered. However, the stage of development at
which somitic cells become committed to forming
specific vertebral elements is not known, nor is the
stage at which they become committed to forming
specific vertebrae. In addition, Gallera (1966) showed
that the somite is not yet differentiated into sclerotome and dermatome at the time of its formation,
although it is possible that determination of rostral
and caudal halves of the somite on the one hand, and
dermatome and sclerotome, on the other, are separate events. While every attempt was made to maintain the correct orientation of the transferred quail
somite, it is uncertain and unlikely that this occurred
in every instance. The development of approximately
normal vertebrae, assuming incorrect orientation of
the somitic axes, raises questions concerning the cell
determination that has already occurred and warrants
further study.
83
Chevallier et al. (1977), when investigating muscle
function in the wings of chicks using chimaeras, found
a small but significant difference in results when
transferring donor chick to quail host as compared
with transferring donor quail to chick host. These
differences were attributed to differences in the
developmental rates between chick and quail. The
more mature quail cells might behave differently in a
less-mature chick environment. In addition, Sanders
(1986) found differences in cell surface properties
(adhesiveness) between homologous cells from these
two species and suggested that they could possibly
explain some of the occasional inconsistent results
using this methodology. The effects in the present
study of potential disparity in developmental rates
and cell adhesiveness are uncertain.
Kieny et al. (1972), in their experiments to investigate regional transfer of somites, suggested that, in
those cases where completely normal regional development had occurred, the transplanted somite had
been rejected and that the normal regulatory process
of chick embryos had been activated. The question of
selfregulation following injury and removal of somite
by the embryo has been discussed earlier, but the
question of rejection of the transplanted somite,
either in whole or in part, by the host must be
considered, even though it was not assessed in this
study. The one case (53/2) of complete absence of
donor quail cells in the developing chick embryo
might demonstrate complete rejection of the transplant by the host and subsequent regulation. Differing degrees of rejection, in whatever form, must be
considered to have affected the results.
A final problem relating to the validity of the
development of chimaeric vertebrae, centres on the
general trauma associated with surgical intervention
of this kind. It is conceivable that alteration of the
embryonic environment by the surgical procedure,
particularly that which is local to the site of extirpation, could affect subsequent vertebral development.
From the preceding discussion, it is clear that there
are many problems associated with the methodology
employed that could affect the validity of the results.
However, we believe that the general consistency of
our results indicate that they were valid and that the
methodological uncertainties could explain the relatively small differences found in quail cell distribution, the abnormalities in vertebral formation that
were found and the variation in the number of quail
cells present.
The phenomenon of resegmentation between the
obvious somitic segmentation in the early embryo and
84
K. M. Bagnall, S. J. Higgins and E. J. Sanders
the vertebral segmentation in the more mature embryo is intriguing, particularly as many authors consider this phenomenon to be common to all vertebrates. The resegmentation of a fundamental,
underlying metameric pattern has been studied in
other species. Lawrence and coworkers (Lawrence &
Martinez-Arias, 1985; Martinez-Arias & Lawrence,
1985), studying segmentation patterns in the fruit fly
(Drosophila melanogaster), have found that the most
obvious embryonic segmentation pattern is not the
true underlying metameric pattern. They have found
that the more obvious segmental embryonic pattern is
shifted by half a segment in relation to the metameric
pattern. If this concept is applied to somites and the
subsequent formation of vertebrae, then a remarkable similarity becomes apparent (Stern & Keynes,
1986). The most obvious embryonic segmental pattern (the somites) appears to be one half segment out
of alignment with the adult segmental pattern (the
vertebrae). Perhaps our attention should be shifted
one half segment from the somites to focus on the
intersomitic clefts coupled with a cluster of cells lying
rostral and another cluster lying caudal as being
representative of the metameric pattern in the mature
animal. In this way, a somite would contain cells from
two separate segments and the appearance of the
intersomitic cleft would be highlighted as it simultaneously defines the caudal half of one somite that
has just formed and the rostral half of the next one
(Stern & Keynes, 1986, 1987). Application of this
concept would mean that the vertebrae would not be
out of alignment with the underlying metameric
pattern of the embryo and the conceptual difficulty of
'resegmentation' will have been circumvented. The
implications of this concept are numerous and perhaps warrant further study particularly if the field of
genetic expression is based on segmentation patterns.
This work was supported by grants from the Natural
Sciences and Engineering Research Council of Canada
(K.M.B.), and the Medical Research Council of Canada
(E.J.S.).
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