/. Embryol. exp. Morph. Vol. 49, pp. 73-88, 1979
Printed in Great Britain © Company of Biologists Limited 1979
73
Role of the somitic mesoderm in the development
of the thorax in bird embryos
II. Origin of thoracic and appendicular musculature
By ALAIN CHEVALLIER 1
Equipe de recherche associee au CNRS no. 621 ' Morphogenese experimentaie\
Laboratoire de Zoologie et Biologie animate, Universite Scientifique
et Medicale de Grenoble
SUMMARY
The regional embryonic origin of trunk and limb musculature was determined through
heterospeciflc homotopic or heterotopic transplantations of quail somitic or somiticsomatopleural mesoderm into chick hosts, and through localized X-irradiation of the somitic
mesoderm. Experiments were performed on 2-day embryos.
Results show that the myoblastic component of all truncal and appendicular muscles is of
somitic origin. Intra- and perimuscular connective tissue as well as tendons are of somatopleural origin. X-ray destruction of the somitic mesoderm at and beyond the wing level
resulted in the complete or almost complete absence of musculature in the wing and corresponding truncal region.
The mapping of the cephalocaudal origin of the various muscles was found to be as
follows:
Intrinsic and extrinsic muscles of the wing and
scapular girdle
Giand pectoral muscle
Intercostal muscles
Abdominal muscles
Intrinsic and extrinsic muscles of the leg and pelvic
girdle
Dorsal and intervertebral muscles
Somites
Somites
Somites
Somites
12-20
12-22
19-26
27-29
Somites 26-32
Metameric level, specific origin
In the heterotopic transplantations, the grafted somitic mesoderm gave rise to site-specific
morphogenesis, irrespective of the cephalocaudal level of its origin. This result demonstrates
that, at 2 days of incubation, the myogenic somitic cells are not regionalized.
INTRODUCTION
The origin of limb musculature in tetrapod vertebrates has long been controversial. Recent experimental investigations by Bourgeois & Houben (1975)
onPIeurode/es, by Milaire (1976) and Houben (1976) on the mole and the mouse,
by Agnish (1976) on the mouse, by Gumpel-Pinot (1974), Christ, Jacob &
1
Author's address: Laboratoire de Zoologie et Biologie animale, Universite scientifique et
medicale de Grenoble, B.P. 53, 38041 Grenoble Cedex, France.
74
A. CHEVALLIER
Jacob (1974a, b, 1977) and Chevallier, Kieny & Mauger (1976, 1977, 1978) on
the chick have brought evidence of the penetration of mesodermal somitic
cells into the somatopleural mesoderm of the limbs.
Chevallier (1978) recently showed that dispersed somitic cells start to penetrate into the limb somatopleure at the time when somites begin their
differentiation into dermo-myotome and sclerotome; the immigration stops
when dermatome and myotome become histologically distinct. Chevallier et al.
(1977) showed that somitic cells give rise to limb myoblasts and that these cells,
by the time they leave the somite, are not regionally determined along the
cephalocaudal axis of the embryo. These authors (1978) completed their analysis
by demonstrating that the limb musculature was deficient or absent when the
somitic mesoderm had been locally destroyed by X-irradiation.
In the present work, I wish to extend this analysis to the musculature of the
trunk and the girdles, since for these structures the controversy persists regarding the origin of several muscles. Indeed some authors (Straus & Rawles, 1953)
contend that the ventral half of the body wall is of somatopleural origin, while
others (Seno, 1961; Sweeney & Watterson, 1969; Pinot, 1969) suggest that the
somitic mesoderm gives rise to all lateroventral muscles of the body wall
(intercostal muscles and abdominal muscles) with the exception however of
the grand pectoral muscle. In opposition to the latter authors, Christ et al.
(1976, 1978) demonstrated that the grand pectoral muscle (1976) and the
musculature of the abdominal wall (1978) are also of somitic origin.
Xenoplastic transplantations of quail somitic mesoderm or of quail somitic
and somatopleural mesoderm into a chick host (combinations in which quail
nuclei may easily be distinguished from chick nuclei (Le Douarin & Barq, 1969))
and localized destruction through X-irradiation of definite portions of the
somitic mesoderm have allowed: (1) the localization along the cephalocaudal
axis of the embryo of the level of origin of the various muscular complexes to
which the somitic mesoderm gives rise; (2) the further verification that the
somitic mesoderm is able to give rise to muscle fibres and that the somatopleural mesoderm forms the tendinous tissue and the connective tissue surrounding the muscle bundles or penetrating between them; (3) the demonstration
that the somitic mesoderm is not regionalized along the cephalocaudal axis of the
embryo, either for the limb or trunk musculature and, for this reason, it is
capable of regulation at the stage at which the operations are performed;
(4) the demonstration that the destruction of a portion of the somitic mesoderm
through localized X-irradiation produces similar defects in the trunk and girdle
musculature as in that of the zeugopod.
MATERIAL AND METHODS
Experiments were performed on 2- to 2-5-day chick embryos (either from
a White Leghorn breed or from a cross between Rhode Island Red and
Origin of limb musculature
75
Wyandotte) (10-26 pairs of somites, stages 10-15 of Hamburger & Hamilton
(195.1)) and on quail embryos {Coturnix coturnix japonicd) of equivalent stages
(10-26 pairs of somites).
Two sets of experiments were performed. Set 1 consisted in taking a piece of
somitic mesoderm or of somitic and adjacent somatopleural mesoderm from
a 2- to 2-5-day quail embryo and implanting it either ortho- or heterotopically
at various levels of the cephalocaudal axis of a chick host embryo of the same
age, in place of a previously eliminated piece of somitic or somitic and somatopleural mesoderm. Set 2 consisted in destroying, through a localized X-irradiation, the somitic mesoderm along a length of 12 somites and/or presumptive
somites at a level comprising the presumptive area of the wing rudiment.
(A) Surgical techniques
1. Hetero specific grafts
(a) Preparation of the grafts
Quail blastoderms were excised according to the usual method which has
been described before (Chevallier, 1975, 1977; Chevallier et al. 1977; Kieny,
1972; Mauger, 1972). The length of the graft (somitic mesoderm or somitic
and somatopleural mesoderm) corresponds to that of a train of three to seven
somites or presumptive somites. The grafts were obtained from donor embryos
with 10-26 pairs of somites, between the levels of somites (or presumptive
somites) 5 and 36 inclusive.
Two different types of grafts have been prepared in this way. For an
illustration of the operations, refer to fig. 1 p. 278 of Chevallier (1977). A type-1
graft is composed exclusively of somitic mesoderm; a type-2 graft comprises
not only the somitic mesoderm, but also the adjacent somatopleural mesoderm
in its integrity or almost complete integrity (over a width equal to once or twice
that of a somite).
(b) Preparation of the host embryo
The hosts were all chick embryos. Unilateral excisions were performed at
various levels of the cephalocaudal axis between the level of the 10th somite and
that of the 36th presumptive somite. The excisions eliminated either the somitic
mesoderm alone to receive type-1 graft or the somitic mesoderm and part of the
adjacent somatopleural mesoderm to receive type-2 graft.
(c) Transplantation of the graft
A carbon marking was placed on the dorsal anterior end of the graft in order
to permit its orientation with respect to that of the host. All grafts were
implanted so that their three axes of polarity coincided with those of the host.
The various categories of grafts were as follows (the numbers in parentheses
indicate the somitic or presumptive somitic levels).
76
A. CHEVALLIER
Orthotopic grafts
Series 1: type-1 grafts implanted at the posterior cervical level (12-17).
Series 2: type-1 grafts implanted at the anterior cervico-thoracic level (15-20).
Series 3: type-1 grafts implanted at the middle cervico-thoracic level (17-22).
Series 4: type-1 grafts implanted at the thoracic level (19-26).
Series 5: type-1 grafts implanted at the lumbar level (26-32).
Series 6: type-2 grafts implanted at the middle cervical level (10-15).
Series 7: type-2 grafts implanted at the posterior cervical level (12-17).
Series 8: type-2 grafts implanted at the anterior cervico-thoracic level (15-20).
Series 9: type-2 grafts implanted at the middle cervico-thoracic level (17-22).
Series 10: type-2 grafts implanted at the lumbar level (26-32).
Series 11: type-2 grafts implanted at the lumbo-sacral level (30-36).
Heterotopic grafts
In these series the graft was always of type 1.
Series 12: graft of anterior cervical level (5-10) at the anterior cervico-thoracic
level (15-20).
Series 13: graft of thoracic level (20-26) at the anterior cervico-thoracic level
(15-20).
Series 14: graft of lumbar level (26-32) at the anterior cervico-thoracic level
(15-20).
Series 15: graft of anterior thoracic level (19-21) at the lumbar level (26-30).
Series 16: graft of posterior thoracic level (22-26) at the lumbar level (26-30).
2. Localized X-irradiation
Localized X-irradiation was used to destroy definite portions of the somitic
mesoderm in situ. For an illustration of the operating procedure, refer to fig. 1
p. 265 of Chevallier et al (1978).
The irradiation was performed under 20 kV and 30 mA, during 10 min,
through a rectangular slit (1-6 x 0-1 mm) perforated through a tantalum screen
of 0-1 mm thickness which protected the rest of the embryo and part of the area
pellucida from the X-rays. The embryo was placed at a distance of 37 cm from
the anticathod. The irradiation was localized on a portion of the still unsegmented or already partially segmented somitic mesoderm. The irradiated zone
always comprised somites 15-20 and extended at least over six somites or six
presumptive somites behind the presumptive area of the wing. The irradiated
embryos had between 11 and 21 pairs of somites at the time of irradiation.
(B) Observation of results
Embryos which had received a heterospecific graft were fixed between 4 and
11 days after the operation, between 6 and 13 days of incubation, in Helly's
Origin of limb musculature
11
fluid, and were sectioned for histology at 7-5 /mi thickness. The sections were
stained with the nuclear reaction of Feulgen and Rossenbeck for the identification of chick and quail cells. The irradiated embryos were fixed at 12 or 13 days
of incubation, sectioned for histology at 7-5 /tm and stained with haemalumeosin.
RESULTS
I. General results after transplantation experiments
(a) Anatomical constitution of the musculature
The grafts were composed either of somitic mesoderm only (type 1) or of
somitic and somatopleural mesoderm (type 2). Despite this difference in the
constitution of the grafts, the development of the muscular masses considered
as a whole did not appear to be disturbed, as the musculature at the operation
level was always present and normally developed. Their myoblastic component
was made up of quail cells and therefore of graft origin (Figs. 1, 3, 5). However,
it was observed that some muscles were anatomically of mixed constitution,
made up of myoblastic quail cells at the level of the graft, and of myoblastic
chick cells in front or in the rear of that region. This was the case particularly
for the dorsal muscles (Fig. 5) which extend over a long cephalocaudal distance,
such as the grand pectoral, the anterior or the posterior region of which might
be constituted of myoblastic chick cells while the rest of the muscle was made
up of myoblastic quail cells.
(b) Histological constitution
The implantation of a type-2 graft comprising somitic and somatopleural
mesoderm led to the formation of a musculature made up wholly (muscle, fibres,
tendons, connective tissue) of quail cells (Fig. 4). Contrariwise in the case of
type-1 grafts comprising somitic mesoderm only, the musculature was of bispecific constitution, the muscle fibres (myotubes) being constituted of quail
cells, while the connective and tendinous parts were made up of chick cells
(Fig. 2). These results confirmed those which had been obtained previously for
the wing musculature (Chevallier et ah 1977) and thus allowed generalization
of the notion of the multisomitic constitution of many muscles at the anatomical
level, and of the dual constitution of the trunk and limb muscles at the histological
level.
II. Orthotopic transplantations
Results are summarized in Table 1 (series 1-5) and Table 2 (series 6-11).
After implantation of a graft at the level of somites 10-15 (middle cervical
level, series 6), the axial (dorsal and intervertebral) muscles of that level were
made up of quail cells. However, the intrinsic and extrinsic musculature of the
limb, the musculature of the joint between girdle and limb were formed either
exclusively by chick cells (eight out of twelve cases for the grand pectoral, nine
6
E MB AQ
78
A. CHEVALLIER
Chick tissue except muscle and cartilage
Quail connective tissue
Chick cartilage
[Quail cartilage
I
Chick muscle
plljlii Chimcric muscle
iSllll Quail muscle
Origin of limb musculature
79
out of twelve cases for the other muscles of the scapular girdle and of the wing,
or by quail and chick cells (respectively four and three cases)).
When the graft was implanted at the level of somites 12-17 (series 1 and 7), in
all cases the muscles of the girdle and of the wing were entirely constituted of
quail cells. This was the case also for the axial (dorsal and intervertebral)
muscles of that level.
When the graft was implanted at the level of somites 15-20 (series 2 and 8),
the results were comparable to those of series 1 and 7. In all cases, the
musculature was entirely constituted of quail cells, except in one case (series 8)
where the graft had probably been rejected in part.
When the transplantation was performed at the level of somites 17-22
(series 3 and 9), the intervertebral and dorsal muscles were formed of quail
cells, but the intrinsic and extrinsic wing musculature was again heterogeneous
in its specific constitution, in a way similar to that which was observed in
series 6 at an anterior level. A certain number of embryos had their musculature
entirely constituted of quail cells (11 out of 17 cases for the grand pectoral,
9 out of 17 cases for the other muscles of the girdle, and 8 out of 17 cases for
the intrinsic wing musculature). In the remaining embryos the two species of
myoblasts were present.
When the graft was implanted at the level of somites 19-26 (series 4), the
muscles made of quail cells were the dorsal and intervertebral muscles of that
FIGURES
1-5
Results of heterospecific, homotopic transplantations of somitic or
somitic-somatopleural mesoderm.
Figs. 1-2. Donor (quail), 18 pairs of somites; type-1 graft, somitic mesoderm of
prospective wing level (somites 15-20); host (chick), 16 pairs of somites; host fixed
at 9 days of incubation. Transverse section. Anatomy on the operated (left) side is
normal, as compared to the non-operated (right) side (Fig. 1). The grand pectoral
muscle (GP) is chimaeric, composed of quail (Q) muscle fibres and chick (C)
intramuscular connective tissue (Fig. 2).
Figs. 3-4. Donor (quail), 16 pairs of somites; type-2 graft, somitic and somatopleural mesoderm of prospective wing level (somites 15-20); host (chick), 14 pairs
of somites; host fixed at 9 days of incubation. Transverse section. Anatomy on the
operated (right) side is normal as compared to the non-operated (left) side (Fig. 3).
The grand pectoral muscle (GP) is entirely (muscle fibres and connective tissue) constituted of quail (Q) cells (Fig. 4).
Fig. 5. Donor (quail) 22 pairs of somites; type-1 graft, somitic mesoderm of level
19-26; host (chick), 17 pairs of somites; host fixed at 10 days of incubation. Frontal
section shows m. latissimus dorsi (LD) of anatomically mixed constitution, the
anterior part of which is located within the operated region (left side) and of chimaeric
composition (quail muscle fibres and chick intramuscular and tendinous connective
tissue), and the posterior part of which, located outside the operated region, is made
up entirely by chick cells. I, ilium; S, spinal cord, Sc, scapula; V, vertebra. For the
nomenclature of main skeletal and muscular elements in transverse sections, see
Figs. 6-8.
6-2
12-17
15-20
17-22
19-26
26-32
1
1
1
1
1
Orthotopic
Orthotopic
Orthotopic
Orthotopic
Orthotopic
5 C Q
7 C Q
7 CQ
15 CQ
4
C
Of
C C Q Q C C
C C
Q
C
C
C
CQ
QC
QC
C
C
Q
Q
C
C
C
Q
C
C
C
C
Of
wing
Of
C
C
C
C
Q
pelvic Of Inter- Abdogirdle leg costal minal
Q
Q
Q
C C C
Q
Q
Q
C C C
5Q 2CQ 5Q 2CQ 2Q 5CQ C
C
C
6CQ 9C
C
C
C
C
Q
C
C
C
Q
Q
C
-v Grand scapular
15-20 19-26 26-32 pectoral girdle
Intervertebral of levels
Ant. Post. 10-15
Dorsal
Constitution of musculature
6
7
8
9
10
11
10-15
12-17
15-20
17-22
26-32
30-36
2
2
2
2
2
2
Orthotopic
Orthotopic
Orthotopic
Orthotopic
Orthotopic
Orthotopic
12
5
12
10
7
5
Level
of
Host
origin
(in soLevel of
Series mites) Type implantation Nb
Graft
C
C
C
C
Q
CQ
QC
CQ
CQ
CQ
C
C
Q
CQ
C
C
C
C
C
QC
Q
CQ
C
C
C
C
C
QC
C
C
C
C
C
C
Q
CQ
Intervertebral of levels
4QC8C
Q
11Q1C
6Q4CQ
C
C
Of
wing
C
C
C
C
C
Q
Q
1CQ4C
C
C
C
C
C
C
C
C
Q
C
Inter- Abdocostal minal
C
C
C
Of
pelvic Of
girdle leg
QC
3QC9C
Q
Q
C
11Q1QC
Q
C
4 Q 6 C Q 6Q 4 C Q
C
C
C
C
CQ
Of
Grand scapular
Ant. Post. 10-15 15-20 19-26 26-32 pectoral girdle
Dorsal
Constitution of musculature
Species-specific anatomical constitution of muscles is indicated by C (chick), Q (quail), CQ (anterior part of muscle of chick oiigin,
posteiior part of quail origin), QC (reverse composition). Numbers in front of these letters indicate the number of cases of corresponding constitution. When no numbers aie given, results were the same for all cases of the series.
Table 2. Results of orthotopic transplantations of quail somitic-somatopleural {type-2 grafts) mesoderm into chick hosts
1
2
3
4
5
Level
of
Host
origin
(in soLevel of
Series mites) Type implantation Nb
Graft
Species-specific anatomical constitution of muscles is indicated by C (chick), Q (quail), CQ (anterior part of muscle of chick origin,
posteiior part of quail origin), QC (reverse composition). Numbers in front of these letters indicate the number of cases of corresponding constitution. When no numbers are given, results were the same for all cases of the series.
Table 1. Results of orthotopic transplantations of quail somitic (type-1 grafts) mesoderm into chick hosts
w
O
E
w
oo
O
Origin of limb musculature
81
level, and in 6 out of 15 cases the grand pectoral showed a few muscle fibres
constituted by quail cells, while all other muscles of the girdle and the wing
were exclusively constituted by chick cells. The grand pectoral thus appeared to
originate from a region a little more extended than that of the other muscles of
the scapula and girdle.
Finally the implantation of the graft at the level of somites 26-32 (series 5
and 10) resulted in the development of muscles formed by quail cells in the
axial musculature of that level, the intrinsic and the extrinsic musculature of the
hind limb and of the pelvic girdle, and in the lateroventral musculature of the
abdomen, namely, m. rectus abdominis, m. transversus abdominis, and m.
obliquus externus and internus abdominis.
After implantation at the level of somites 30-36 (series 11), the abdominal
muscles were predominantly constituted by chick cells; only few quail muscle
fibres were visible in the muscular masses of the limbs and the pelvic girdle.
The axial musculature of that level was formed by a mixed population of chick
and quail cells.
III. Localized X-irradiation
This experimental method was used to verify and consolidate the results
obtained by xenoplastic transplantations. Indeed to ascertain that the musculature of a given cephalocaudal level (in the present study, the wing level)
originates from a certain cephalocaudal level of the somitic mesoderm, it is
necessary to stop the somitic mesodermal cells from penetrating into the
somatopleural mesoderm. This can be achieved by killing the somitic mesoderm through X-irradiation in a zone extending sufficiently in front and at
the rear of the wing areas so that the possibilities of an antero-posterior regulation are limited to a minimum. Among the 68 irradiated embryos of which
27 have already been studied histologically for the constitution of the intrinsic
musculature of the forearm (Chevallier et al. 1978), I have chosen eight embryos
which were either totally or partially deprived of muscles at the level of the
forearm. In the two cases where the musculature of the forearm was reduced
but not absent, the irradiated embryos showed similarly a partial development
of the extrinsic musculature of the upper arm and of the shoulder as well as of
the axial (dorsal and intervertebral) musculature. In the five cases where the
intrinsic muscles of the forearm were completely missing, a complete (Figs. 6, 7)
or an almost complete absence (Fig. 8) of musculature in the scapular zone
was observed.
IV. Heterotopic transplantations
The heterotopic transplantations were performed in order to analyse the
regionalization of the somitic mesoderm during the formation of the trunk
musculature. (Table 3, series 12-16).
We have shown previously (Chevallier et al. 1977, 1978) that the intrinsic
musculature of the wing and particularly that of the forearm is not regionalized,
82
A. CHEVALLIER
Origin of limb musculature
83
since somitic mesoderm from the neck level (somites 5-10), the flank level
(somites 20-26) and the leg level (somites 26-32) is able to give rise to a normal
and well developed intrinsic zeugopodial musculature. The 38 cases analysed
in the present report (series 12-16) confirm the results obtained previously. The
muscles originating from the grafted cells are muscles which strictly correspond
in each case and at each level to the level of implantation and not to the level
of origin of the donor embryo. Thus somitic mesoderm obtained from the level
of somites 5-10, 20-26 and 26-32, implanted at the level of the wing (somites
15-20), contribute to the formation of the muscle fibres of the dorsal muscle,
grand pectoral muscle and of the muscles of the pectoral girdle and of the wing
(series 12-14). Similarly, somitic mesoderm obtained from the flank region
(somites 19-26), implanted at the level of somites 26-30 (series 15 and 16) take
part in the formation of the musculature of the pelvic girdle, of the leg and of the
abdominal wall. Thus it is clearly the site of implantation which controls the
destiny of the somitic presumptive muscle cells.
DISCUSSION AND CONCLUSION
The implantation of a type-1 graft, composed of somitic mesoderm only, or
of a type-2 graft, composed of somitic and somatopleural mesoderm, leads in
all cases to a completely normal development of the musculature in the operated
region. This musculature is made of cells which originate from the graft.
At the histological level, there is an important difference between the constitution of the muscles after type-1 or type-2 grafts. After type-1 graft, the
musculature is found to be of bispecific composition: the muscle fibres (the
myoblasts and the myotubes) are of quail (graft) origin, while the tendons, the
intra- and peri-muscular connective tissue cells are of chick (host) origin.
Contrariwise after type-2 graft, the musculature (myoblasts, myotubes and
associated connective tissue) is entirely formed by grafted quail cells. This shows
quite clearly that the somitic mesoderm is able to give rise only to themyoblasts
and to the future muscle fibres proper, while the somatopleural mesoderm gives
F I G U R E S 6-8
Effects of localized X-irradiation of a longitudinal portion of the right somitic
mesoderm of chick embryos at the stage of 20 pairs of somites.
Fig. 6. Irradiation between levels of somite 12 and prospective somite 24. Embryo
fixed at 9 days of incubation. Transverse section at wing level. The musculature on the
operated side is completely missing. Note innervation (Ne) of amyogenic wing.
Figs. 7 and 8. Irradiation between levels of somite 15 and prospective somite 28.
Embryos fixed at 13 days of incubation. Transverse sections at wing level. In the
specimen of Fig. 7, the musculature on the operated side is completely lacking,
except at the intervertebral level (arrow). In the example of Fig. 8, the only muscles
that have formed on the operated side are inserted on the sternum (arrows). C,
coracoid; GP. grand pectoral muscle; H, humerus; N, notocord; Ne brachial nerve;
S, spinal cord, St. sternum.
12
13
14
15
16
5-10
20-26
26-32
19-21
22-26
15-20
15-20
15-20
26-30
26-30
8
19
3
4
4
Level
Host
of
origin
Level of
(in soimplantation
Series mites) Type (in somites) Nb
Graft
CQ
CQ
CQ
C
C
C
C
C
QC
QC
C
C
C
C
C
Q
Q
Q
C
C
C
C
C
C
C
Q
Q
c
C
C
Intervertebral of levels
Q
Q
Q
C
C
Q
Q
Q
C
C
Of
Grand scapular
Ant. Post. 10-15 15-20 19-26 26-32 pectoral girdle
Dorsal
Constitution of musculature
Q
Q
Q
C
C
wing
Of
C
C
C
QC
Q
C
C
C
QC
Q
c
c
c
C
C
Q
Q
c
c
c
Of
pelvic Of Inter- Abdogirdle leg costal minal
Species-specific anatomical constitution of muscles is indicated by C (chick), Q (quail), CQ (anterior part of muscle of chick origin,
posterior part of quail origin), QC (reveise composition). Numbers in front of these letters indicate the number of cases of corresponding constitution. When no numbers are given, results were the same for all cases of the series.
Table 3. Results of heterotopic transplantations of quail somitic (type-1 grafts) mesoderm into chick hosts
|-i
2
w
oo
Origin of limb musculature
85
rise to the connective matrix from which the tendons and the muscular
envelopes originate. This is in accord with the results concerning the mixed
somitic-somatopleural origin of the limb musculature (Chevallier et al. 1911 \
Christ et al. 1977) and the truncal musculature of avian embryos (Christ et al.
1914a, b, 1978).
The heterotopic transplantations show that, at the time of transplantation
(stage 13-15 of Hamburger & Hamilton) the future myoblasts of the somitic
mesoderm are not regionalized: they are not yet determined to give rise to level
specific muscles. On the contrary, the muscles that develop from the transplanted
somitic mesoderm are always in conformity with the site of implantation in
the host. This result is in conformity with that which was obtained for the
intrinsic musculature of the zeugopod of the wing (Chevallier et al. 1977). Both
the orthotopic and heterotopic transplantation experiments lead to the conclusion that the whole trunk musculature has a precise and well localized
origin along the cephalocaudal axis of the embryo. The intrinsic and extrinsic
wing musculature and that of the scapular girdle originate from the somites
12-20, while the grand pectoral originates from somites 12-22, that is from
a region which extends somewhat behind the presumptive region of the wing
bud. The musculature of the leg and that of the pelvic girdle originate from
somites 26-32; the abdominal muscles derive from somites 27 and 29; the
intercostal muscles derive from somites 19-26; the segmental intervertebral
muscles derive from the somites located at their corresponding level, while the
dorsal muscles which extend over a long distance longitudinally are formed
piece by piece by all somites in the studied regions (somites 10-36).
The data on the origin of the musculature are confirmed by the results of the
localized X-irradiation. Contrary to the excision, the irradiation prevents the
processes of regulation (Mauger, 1970), in so far as the dying cells left in place
preclude the invasion of anterior or posterior cells with regulatory capacities
into the operated region. Thus X-irradiation of a portion of the somitic mesoderm leads to a more or less complete deficiency of the trunk, girdle, and limb
musculature. The partial formation of muscles in some of the irradiated
embryos can be explained in three ways: either a certain regulation, probably
from the region anterior to the irradiated zone, had occurred, or all the somitic
cells of the irradiated zone were not destroyed by X-rays, some of them being
then able to continue their development and proliferate to form a deficient
musculature, or somatopleural cells might in these conditions give rise to
myoblasts.
These results are in conformity with those of Hamilton (1965) which showed
that somites 15-20 participate in the formation of the scapular girdle musculature
and with the opinions of Fischel (1895) and Starck (1975) for whom the girdle
musculature originates from the somitic mesoderm. Furthermore, they contribute to the understanding of how the various muscles and muscle complexes
differentiate along the cephalocaudal axis of the embryo.
86
A. CHEVALLIER
The dorsal muscles which extend along the whole length of the vertebral
column are formed level by level by each of the corresponding somites. Indeed
in the operated embryos these muscles are constituted of quail cells in the zone
corresponding to the implantation of the graft, while in front or in the rear of
that region they are formed by chick cells.
In other words, there is no progressive lengthening towards the rear of the
muscles starting from an anterior rudiment, but rather formation and differentiation, level by level, of each part of the muscle, from each somite.
The muscles of metameric origin, like the intervertebral and intercostal
muscles, are constituted either by quail or by chick cells according to whether
the somite from which they originate belongs to the operated zone or not.
These muscles undergo a transverse lengthening, concomitant with the development of the ribs in lateral and ventral direction.
The abdominal muscles (m. rectus abdominis, m. transversus abdominis, and
m. obliquus externus and internus abdominis) contrary to the preceding
muscles, not only have a transverse dorso-ventral extension but they also
extend antero-posteriorly. Indeed, according to our results, they originate from
somites 27-29 and they become lengthened to give rise to the muscular ventral
body wall, and to the inferior and posterior third of the thorax musculature.
The latter conclusion is in agreement with the results of Seno (1961) and Pinot
(1969).
Two regions of the somitic mesoderm have a particular morphogenetic fate.
These are the regions of somites 26-32 and 12-20 which give rise to the
musculature of the girdles and the limbs. This musculature attains a very large
development. I have not attempted to link the origin of one particular limb or
girdle muscle with one particular somite, but simply to locate globally this
musculature with respect to the presumptive area of the limbs. The musculature
of the pelvic girdle and of the leg originates from somites 26-32. For the
scapular girdle and the wing, experiments showed that their musculature was
constituted of quail cells when the grafts were implanted at the level of somites
12-17 and 15-20 (series 1, 7 and 2, 8. Contrariwise grafting at the level of somites
10-15 (series 6) and 17-22 (series 3 and 9) resulted in a heterogeneous constitution of the musculature. This shows that the graft was not implanted
exactly at the level of the presumptive zone specifically corresponding to the
girdle and the wing, but that it had been grafted in a zone either somewhat
anterior to the wing in series 6 or somewhat posterior to it in series 3 and 9.
In the first case quail cells were found predominantly in the preaxial region of
the wing, while, in the second case, they were found mostly in the postaxial
region (see Tables 1-2 series 6, 3 and 9, CP and PC).
Regarding the grand pectoral, which in birds attains a considerable cephalocaudal development, it is worth mentioning that it originates not only from
somites 12-20 like the other extrinsic wing muscles, but also from two or three
somites posterior to that zone as shown by the results of series 8. Moreover,
Origin of limb musculature
87
histological sections frequently show a grand pectoral muscle of quail type in
a chick environment and vice versa. This phenomenon is due to the fact that
the grand pectoral in its morphogenesis covers a region much more extended
than that of its origin; indeed it extends posteriorly and ventrally very far along
the sternum beyond the level of somite 26. For this reason, until recently, it was
thought that the grand pectoral was of somatopleural origin. Thus, when Pinot
(1969), for example, destroyed through X-irradiation the somitic mesoderm of
somites 19-26, she observed that the grand pectoral was normal. This may be
explained by the fact that the X-irradiation did not hit the more anterior region
from which the grand pectoral effectively originates. In the same manner, when
Strauss & Rawles (1953) applied carbon particles on somites 15-20, the grand
pectoral was not marked.
Finally, the present experiments show that somitic cells, at the stage when
somites become segmented, already possess the ability to differentiate into
myoblasts and to give rise to myotubes and muscle fibres. However, this
property is not regionalized and does not imply the capacity to undergo regionspecific morphogenesis. It appears plausible that the somite-originated myoblasts and myotubes become anatomically organized into muscles and muscle
complexes under the influence of the somatopleural tendinous and perimuscular connective tissue.
It is noteworthy that the somitic mesoderm which has essentially a homogeneous and later repetitive structure all along the cephalocaudal axis of the 2-day
embryo, displays morphogenetic potentialities which differ so much from level
to level. Indeed, at all levels, it gives rise to vertebral and axial muscles, while
in three specialized regions its cells possess the capacity to form ribs and intercostal muscles (in the flank region), the scapula and the musculature of pectoral
girdle and wing (in the forelimb region), or the musculature of pelvic girdle and
leg (in the hind limb region).
Ce memoire represente une partie de la these qui sera soutenue par A. Chevallier devant
I'Universite scientifique et medicale de Grenoble pour l'obtention du grade de docteur d'Etat
es Sciences.
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