J. Embryo/, exp. Morph. Vol. 43, pp. 263-278, 1978
Printed in Great Britain © Company of Biologists Limited 1978
Limb-somite relationship :
effect of removal of somitic mesoderm
on the wing musculature
By A L A I N C H E V A L L I E R , M A D E L E I N E K I E N Y AND
ANNICK MAUGER1
From the Laboratoire de Zoologie et de Biologie animale,
Université scientifique et médicale de Grenoble
Equipe de recherche associée au CNRS, n° 621,
' Morphogenèse expérimentale '
SUMMARY
The aim of this study is to test the ability of the intrinsic wing musculature to develop in the
absence of somitic mesoderm.
The experiments were performed on 2- to 2-5-day chick embryos either by replacing the
somitic mesoderm adjacent to the wing field with a piece of 9-day chick embryonic midgut or
by destroying, through local X-irradiation, not only the somitic mesoderm of the wing level,
but also at least three somites (or presumptive somites) anterior and/or three presumptive
somites posterior to the wing level.
The replacement of somitic tissue scarcely affected the organogenesis of the forearm
musculature, at least when both bones were present. In the other experiments, radio-destruction severely impaired the development of the forearm muscles, which were seldom all present
and in most cases were entirely missing. The absence of a given muscle involves the simultaneous absence of the corresponding tendons.
The possible origins of the muscles that formed despite the removal of the somitic mesoderm
are discussed.
INTRODUCTION
Up to a recent date, the outgrowing avian limb-bud has been thought to
consist of a homogeneous somatopleural population of multipotential mesodermal cells. But more recently, investigations performed by Christ, Jacob &
Jacob (1974, 1977), and by our group (Chevallier, Kieny & Mauger, 1976;
Chevallier, Kieny, Mauger & Sengel, 1977; Chevallier, Kieny & Mauger, 1977;
Chevallier, 1977), have shown that limb-bud mesodermal tissues originate from
two different sources. Using the quail/chick marker system advocated by Le
Douarin & Barq (1969) both groups came to the same conclusions when chick
somitic mesoderm from the wing level was orthotopically replaced by quail
1
Authors' address: Laboratoire de Zoologie et Biologie animale, Université scientifique et
médicale de Grenoble, 38041 Grenoble-Cedex, France.
263
264
A. CHEVALLIER, M. KIENY AND A. MAUGER
somitic mesoderm, i.e. cells originating from the quail somitic mesoderm were
found in the adjacently developing chick wing-bud and their location was
restricted to the muscle bulk. Hence, the anatomical muscle appeared as a
composite structure: tendons and connective tissues (perimysium and endomysium) were of somatopleural origin, whereas the muscle cells were of somitic
origin.
Furthermore, we showed that the same conclusions held for the development
of the leg-bud and that there was no regionalization in the participation of
somitic myogenic cells during the formation of the limb (wing or leg) musculature.
Nevertheless, the findings of the exclusively somitic origin of the limb muscular
cells could not be confirmed in the reverse grafting experiments of chick somitic
mesoderm into quail hosts. In these cases, the myogenic tissue comprised both
chick and quail cells in various proportions. This result raises the question of
the origin of the host-type limb muscle cells. Are they of somitic origin, due to a
compensatory migration of somitic mesoderm from levels anterior or posterior
to the implantation site? Or are they of somatopleural origin, implying that, in
certain particular conditions at least, the somatopleural mesoderm would be
able to express myogenic properties? It is known, indeed, that mesenchymal
cells of the limb-bud can switch from a chondrogenic to a myogenic fate, i.e.
they express myogenic or chondrogenic properties according to their spatial
location (Searls & Janners, 1969) or according to metabolic gradients (oxygen
tension) (Caplan & Koutroupas, 1973).
In order to test whether a somitic contribution is actually required for the
development of a normal limb musculature, we undertook new series of experiments to disrupt the relationships between the somitic mesoderm and the
adjacent somatopleural mesoderm. This disruption can be produced by two
means, either through the translocation of the wing somatopleure into an abnormal environment or through the elimination of the somitic mesoderm
adjacent to the wing somatopleure. In this paper we report the experiments
dealing with the second of the above-mentioned methods.
Previous findings have shown that somitic mesoderm regulates to a very great
extent its dermatomal [spinal feather tract (Mauger, 1972A)] as well as sclerotomal [vertebrae and ribs (Kieny, Mauger & Sengel, 1972)] and myotomal
[trunk musculature (Chevallier, unpublished data)] derivatives, when a portion
of its length is excised. Therefore, in order to study the effects of the absence of
the portion of the somitic mesoderm adjacent to the wing level, it was necessary
to impede these regulative processes. For that purpose we used two procedures
which we had already tested. The first one involved replacing the somitic tissues
by a piece of non-somitic tissue (Mauger, 1972e; Kieny et al. 1972); the second
one involved the local destruction of a segment of somitic tissue left in place
(Mauger, 1970).
Limb-somite
relationship in chick
265
Fig. 1. Experimental schemes of somitic mesoderm removal at age of 16 pairs of
somites, (a) Replacement of the left somitic mesoderm adjacent to the wing field
(hatched area) by a strip of midgut, (b), (c), Destruction by local X-irradiation of a
1-6 mm long portion of right somitic mesoderm behind the 14th somite (1st category) (b) or behind the 11th somite (2nd category) (c). Stippled area: irradiated
somitic mesoderm; hatched area: presumptive wing territory.
MATERIALS AND METHODS
The experiments were carried out on 2- to 2-5-day chick embryos (Wyandotte
x Rhode Island Red). The exact stage reached at the time of operation was
specified by the number of pairs of somites.
The experiments were designed to prevent the somatopleural mesoderm of
the wing field (level 15-20) from being invaded by myogenic cells from the
adjacent somitic mesoderm. Two kinds of experiments were performed.
In the first series, the unsegmented or partially segmented somitic mesoderm
of level 15-20 from the left side was excised and replaced by an equal length of
9-day chick embryonic gut according to the experimental procedure outlined
in Fig. 1 (a). It was easy to completely remove the piece of somitic mesoderm
when it was unsegmented. However, when the somitic mesoderm was excised
in a partially segmented state, it was impossible to exclude the possibility that a
few ventral and medial somitic cells were left in place within the already segmented portion. The midgut was cut longitudinally into quarters before strips
were grafted with their intestinal epithelium facing the host's endoderm.
In the second series, somitic mesoderm was destroyed on the right side by
local X-irradiation, which was performed through a rectangular (1-6 mm
long x 0-1 mm wide) slot cut in an 0-1 mm thick tantalum shield that protects
the rest of the embryo and part of the area pellucida. The irradiation, made
under 20 kV and 30 m A during 10 min, the embryo being placed at a distance
266
A. CHEVALLIER, M. KIENY AND A. MAUGER
of 37 cm from the anticathode, was localized on a long portion of un segmented
or partially segmented somitic mesoderm. The irradiated part always included
somite level 15-20 and extended at least three somites or presumptive somites
in front of and/or in the rear of the wing somite level (Fig. 1 b, c). For histological control of the X-irradiated somitic mesoderm, embryos were fixed 24,
48 and 96 h after irradiation, paraffin-embedded, sectioned at 5 jam and stained
with haemalum-eosin.
The operated embryos of both series were sacrificed between days 9 and 13 of
incubation, the majority at days 12 or 13. The operated areas were photographed
(if necessary after part of the feather filaments of the spinal pteryla and those of
the experimental and contralateral wings had been plucked). These embryos
were classified according to both the effects on the morphology of the spinal
tract, and the effects on the morphology of the wing on the operated side. For
each category of results, experimental (and often also contralateral) wings of a
certain number of embryos, taken at random, were paraffin-embedded, sectioned at 7-5 fim and stained with haemalum-eosin.
RESULTS
Since the purpose of this study was to analyse the organogenesis of the wing
musculature after elimination of the somitic mesoderm of the wing level, it was
necessary to perform the experiments before the normally invading somitic
cells are supposed to start their emigration, that is, as shown by one of us
(Chevallier, 1977) before the somitic mesoderm of the wing level is wholly
metamerized (stage 21 pairs of somites). Two kinds of experiments were undertaken: (1) the excised portion of the somitic mesoderm of level 15-20 was
replaced by a piece of non-somitic (and non-somatopleural) tissue; (2) the
somitic mesoderm was left in place, but was destroyed by local X-irradiation.
The study of the muscular organogenesis has been limited to the intrinsic
musculature of the wing, in particular to the muscles contained in the forearm.
(1) Replacement of somitic mesoderm by non-somitic tissue
The grafts used were strips of 9-day embryonic midgut, cut to the appropriate
size so they would fit as tightly as possible within the gap created by the excision
of the unsegmented or partially segmented somitic mesoderm. The grafts were
placed in embryos which ranged in age from stages 13 to 17 pairs of somites.
Nineteen embryos constitute this series; 3 were sacrificed at 9 days and 16
were sacrificed at 12 days, the stage at which the muscular organogenesis is
qualitatively completed. All of them presented plumage deficiencies at the
operated level, which means that the graft had not been rejected. Indeed, each
embryo presented an unilateral featherless area extending over a length of three
to six lateral feather rows within the narrow postcervical and thoracic portion
of the spinal pteryla (Fig. 2). Except in one case, all embryos developed a wing
Limb-somite relationship in chick
Forearm muscle organogenesis at 12 days of incubation after replacement of the
wing-level somitic mesoderm (level 15-20) by a piece of 9-day midgut at stage 13-17
pairs of somites. All histological sections are given at the same magnification.
Figs. 2 and 3. Host: 16 pairs of somites. Note on the left side the featherless notch
(Fig. 2). The left experimental wing is normally developed. Its forearm musculature
is complete (Fig. 3) (1st line of Table 1).
Fig. 4. Host: 15 pairs of somites. This histological section passes through the
proximal portion of the forearm of a reduced wing in which only the distal half of
the radius was formed. The musculature is nearly complete (4th line of Table 1).
Fig. 5. Host: 13 pairs of somites. Déficient wing, the preaxial portion of which
contains neither the radius nor its surrounding muscles, but only a loose unorganized mesenchyme.
Figs. 6 and 7. Host: 17 pairs of somites. Deficient wing in which only the postaxial
portion of the forearm was formed (Fig. 6). Compare with a section through the
contralateral control forearm (Fig. 7). R, radius; U, ulna. For the muscle abbreviations, see Table 1.
267
16
13
15
15
14
15
14
13
17
14
15
i
,
i
(U, ulna; R, radius)
inc lurcdrm
equipment of
ni
r
FDP
U, R
+++
U, R
++
U, R
+ ++
U, R (distal half)
+++
U, R
+ ++
U, R
+++
U, R (filiform)
U —
+++
U —
+++
u —
+++
u —
+++
FDP flexor digitorum profundus
ANC anconeus
EMU extensor metacarpi ulnaris
EDC extensor digitorum communis
FCU flexor carpi ulnaris
UMV ulnimetacarpalis ventralis
(According
Normal
Reduced
Reduced
Reduced
Reduced
Reduced
Deficient
Deficient
Deficient
Deficient
Deficient
External
Stage of morphology
operation
of the
in pairs
experimental
of somites
wing
+++
+++
+++
+++
++
++
++
+++
+++
+++
PS
PP
EIL
i
EML
+++ +++ +++ +++ +++ +++ +++ +++
+ + + + + + ++
++
++
++
++
++
+++ +++ +++ +++ +++ +++ +++ +++
+++ +++ +++ — +++ +++ ++
++
++ + + + ++
++
++
++
++
++
++ + + + ++
++
++
++
++
++
++
++
++
++
++ + + + ++
—
+++ +++ +++
—
+++ +++ +++
—
+++ +++ +++
EMR extensor metacarpi radialis
PS
pronator superficialis (longus)
PP
pronator profundus (brevis)
EIL
extensor indicis longus
EML extensor medius longus
to Sullivan, 1962)
+++
+++
+++
+++
++
++
++
+++
+++
+++
FCU UMV EMR
A
A
ANC EMU EDC
The radius
The ulna
A
Muscles surrounding
Table 1. Muscle organogenesis in 12-day wings after replacement of somitic mesoderm of the wing level by a piece of
midgut ( + + + , normal or subnormal; + +, half-reduced; —, absent)
W
0
0
5d
w
r
1—
w
<
H*
O
bo
oo
16,80
15,00
15
15,84
16,10
15, 16
Range
14-20
11-18
15
13-20
12-21
12-21
10
5
2
13
40
12
Normal
Deficient, with
unilateral apterium
Mean
stage
Number
Aspect of
spinal tract
Stages at irradiation
A
Surviving embryos
Total
Normal
Reduced
Deficient
Normal
Reduced
Deficient
External
morphology
of the
experimental
wing
Total
-^
27
7
14
6
3
2
1
Number of
wings
examined
2
2
0
0
3
2
1
Normal or
subnormal
7
0
5
2
0
0
0
Deficient
18
5
9
4
0
0
0
Absent
Number of wings in which the
musculature \vas
Histological analysis of the forearmi musculature
Table 2. Effect of local irradiation of a portion of somitic mesoderm including level 15-20 on the
intrinsic forearm musculature of the adjacent wing
to
1
5"
nb-somite relat
270
A. CHEVALLIER, M. KIENY AND A. MAUGER
on the operated side. Their humeral tracts were distorted, usually rotated
anticlockwise by ca. 45° and spreading into the bare indentation of the spinal
tract (Fig. 2). Their extrinsic musculature that serves to attach the humerus to
the scapular girdle was missing or very deficient (Chevallier, unpublished data).
These 18 experimental wings were classified as deficient, reduced or normal
according to the following criteria. Cylindrical wings (nine cases) in which the
prepatagium was absent or quasi-absent were designated as deficient (see Fig.
20); the autopod was malformed in three cases (embryos fixed at 9 days) either
by lacking digit II (one case) or by the development of an extra digit II (two
cases). In the other nine wings, the characteristic wing shape was retained ; their
normality or reduction was attested by comparing the number of their parallel
rows of secondary remiges and coverts, running transversely on the dorsal face
of the forearm, to the number of those of the contralateral wings. A normal
12-day wing bearing 13 or 14 rows, when the number of rows differed by more
than one unit (two or three) between the experimental and the contralateral
wings, the experimental wing was designated as reduced. Thus six wings were
reduced and three were normal.
Eleven (one normal, five reduced, five deficient) of the 15 12-day wings were
examined histologically (Table 1). The forearms of the normal and reduced
wings not only contained muscles but their muscular equipment was nearly
normal (Figs. 3, 4). In four out of the five deficient wings (Figs. 5, 6) which
contained only the ulnar bone, the forearm possessed six fully developed
muscles, which characteristically surround the ulna in normal development (on
the dorsal side: anconeus, extensor metacarpi ulnaris and extensor digitorum
communis ; on the ventral side : flexor digitorum profundus, flexor carpi ulnaris
and ulnimetacarpalis ventralis) (Figs. 6, 7). Those which normally accompany
the radius were missing (Figs 5, 6). In the remaining deficient wing equipped
FIGURES
8—14
Forearm musculature of normal-looking experimental wings in 13-day embryos
whose right somitic mesoderm had been X-irradiated at stages of 13-18 pairs of
somites. All histological sections are at the same magnification.
Figs. 8 and 9. Case of irradiation behind the 11th somite at stage 18 pairs of somites.
Note the unilateral apterium that extends, at right, over eight feather rows and
in which the distorted humeral tract tends to spread (Fig. 8). The forearm musculature is completely developed (Fig. 9) (compare with Figs. 7 and 14).
Figs. 10 and 11. Case of irradiation behind the 11th somite at stage 13 pairs of
somites. The right humeral tract spreads within the featherless spinal tract indentation (Fig. 10). Note the absence of muscles and tendons (Fig. 11).
Figs. 12-14. Case of irradiation behind the 14th somite at stage 15 paiis of somites.
The humeral tract of the right wing spreads into the right spinal apterium that
extends over ten feather rows (Fig. 12). Neither muscles nor tendons formed in the
forearm of the experimental wing (Fig. 13). For comparison, see Fig. 14, a section
through the contralateral wing. R, radius; U, ulna. For the muscle abbreviations,
see Table 1.
Limb-somite relationship in chick
271
18-2
272
A. CHEVALLIER, M. KIENY AND A. MAUGER
with an ulna and a filiform radius, only few slender muscles differentiated,
surrounding both the ulna and radius.
It is clear that the replacement of the portion of somitic mesoderm strictly
adjacent to the wing level does not lead to muscleless wings.
(2) Destruction of somitic mesoderm by local X-irradiation
In order to test whether the results just described could be attributed to a
compensatory migration of the somitic mesoderm anterior and/or posterior to
the wing level, - the somitic limb-myogenic properties being not regionalized, - it was necessary to eliminate more than the wing-level somitic
mesoderm. Because a long-length microsurgical tissue elimination and subsequent replacement leads, in our hands, to a too high mortality or to a too
severely disturbed development, we proceeded to an in situ destruction of a
portion of the somitic mesoderm by means of a local X-irradiation.
The irradiation was performed at ages ranging from stage 11 to 21 pairs of
somites. Its histological effects on the somitic mesoderm were in complete concordance with those previously described by one of us (Mauger, 1970) for
smaller areas under the same irradiation conditions, i.e. within 24 h, the
X-irradiation produces a disorganization of the somitic mesoderm; the myotome-dermatome-sclerotome organogenesis does not occur, and the whole
tissue becomes necrotic. Two days after the irradiation, the site of the radiolesions become gradually repopulated by healthy mesenchymal cells, which
however do not become reorganized into a somitic structure.
A total of 82 irradiated chick embryos were sacrificed at 13 days of incubation.
The irradiated portion always exceeded the wing level portion. In 30 cases, the
slotted tantalum shield was placed in such a way that a 1-6 mm long segment of
FIGURES
15-22
Forearm musculature of reduced (Figs. 15-19) and deficient (Figs. 20-22) wings
in 13-day embryos whose right somitic mesoderm had been X-irradiated at stages
of 17-20 pairs of somites. All histological sections are at the same magnification.
Figs. 15-17. Case of irradiation behind the 14th somite at stage of 17 pairs of
somites. In Fig. 15, at right, the spinal apterium extends over seven feather rows.
Two sections of the right forearm are shown in Figs. 16 and 17. Note the sporadic
muscles, mainly located between the two forearm bones (1st line, Table 3).
Figs. 18 and 19. Case of irradiation behind the 11th somite at stage of 17 pairs of
somites. The spinal tract bears a featherless notch that extends over six feather
rows (Fig. 18), and the intrinsic forearm muscles are missing (Fig. 19).
Figs. 20 and 21. Case of irradiation behind the 14th somite at stage of 20 pairs of
somites. The featherless notch extends over nine feather rows (Fig. 20) and the
forearm of the cylindrical deficient wing is non-muscled (Fig. 21).
Fig. 22. Histological illustration of a case of irradiation behind the 11th somite at
stage 18 pairs of somites. The muscleless forearm contains only the ulna and loose
unorganized mesenchyme. R, radius; U, ulna. For the muscle abbreviations see
Table 1.
Limb-somite relationship in chick
273
15
15
12
12
12
12
12
Reduced
Reduced
Reduced
Reduced
Reduced
Deficient
Deficient
External
*
morphology
anterior
level of
of the
wing
irradiation
U, R
U, R
U, R
U, R
U, R(filiform)
U, R (filiform)
U, R (filiform)
Skeletal
equipment
of the forearm
(U, ulna;
R, radius)
++
++
+++
+
ANC
+
FDP
EDC
+++ +++
EMU
The ulna
+
++
FCU
++
UMV
++
+
+
++
-f +
+
+++
EMR
++
++
++
PS or
PP$
The radius
+
++
+
++
++
EIL or
EML*
* This case is illustrated in Figs. 15-17.
f For the nomenclature of the abbreviations used, see Table 1.
t For the groups ' P S or P P ' and ' E I L or E M L ' , which are groups of two neighbouring muscles, it was impossible to ascertain the exact
quality.
17*
14
15
15
12
15
14
A
X-irradiation
Stage in
pairs of
somites
r
Muscles surroundingf
Table 3. Detailed description of the deficient forearm musculature in the seven reduced or deficient wings {see Table 2)
(+ + +, normal or subnormal; + +, half-reduced; +, more than half reduced; —, absent)
o
>
d
ö
>
m
%
50
m
<
>
O
X
Limb-somite
relationship in chick
275
somitic mesoderm behind the 14th somite or presumptive somite was irradiated
(1st category: level 15-20 plus three to seven presumptive somites behind). In
the remaining 52 cases, a length of 1-6 mm somitic mesoderm behind the 11th
somite was irradiated (2nd category: level 15-20 plus three somites or presumptive somites ahead of and one to four somites or presumptive somites behind the
wing level).
Because there were no major differences between these two categories, the
embryos were pooled and classified according to the same criteria as those
applied in the preceding series (Table 2). Thus, the spinal feather tracts of 17
embryos were undisturbed (13 cases of the 1st category and four cases of the
2nd category) and the wing on the operated side was generally normal or
slightly reduced. Histological investigation (three cases of 1st category) showed
the presence of a complete intrinsic and extrinsic wing musculature.
Whereas in 65 embryos (17 cases of the 1st category and 48 cases of the 2nd
category), the spinal tract presented an unilateral apterium at the irradiated postcervico-thoracical level, that extended over a length of 6-12 feather rows (Figs
8, 10, 12,15, 18, 20). The clockwise distorted right humeral tract protruded into
this spinal featherless notch, so that the dorsal and caudal borderlines of the
humeral tract were often in continuity with the mid-dorsal feather row of the
spinal tract. Forty of these 65 wings were harmoniously reduced (Figs 15, 18);
the other wings were in the same proportions either normal (13 cases) (Figs 8,
10, 12) or deficient (12 cases) (Fig. 20).
Of these 65 wings 27 (8 of the 1st category; 19 of the 2nd category) were
examined at histology. Only two of them (one of each category) that were
classified as normal-looking wings had a complete skeleton and intrinsic
forearm (and arm) musculature (Figs. 8, 9). In the 25 remaining wings the
skeleton was normal in the normal-looking and reduced wings. But in the
deficient ones, besides a subnormal ulna, the radius was filiform (five cases) or
even totally missing (one case) (Fig. 22). Regardless of the external morphology
of the wing, the forearm did not contain any muscle at all (18 cases) (Figs. 11,
13, 19, 21, 22) or contained only some (not more than seven) of the 11 main
muscles that normally (Fig. 14) constitute its musculature (seven cases) (Figs.
16, 17). The analysis of the poorly developed musculature (Table 3) suggests
that the few forearm muscles that developed were not randomly located. Some
of them were always present, while others were generally missing. Indeed, the
extensor metacarpi radialis (EMR) and the flexor carpi ulnaris (FCU) appeared
to be predominantly formed.
This experimental series shows clearly that local X-irradiation of wing-level
somite portions and beyond leads, in the majority of the cases, to muscleless
wings on the irradiated side. The remaining cases are in general poorly muscled;
and a complete forearm-muscle organogenesis is seldom obtained.
276
A. CHEVALLIER, M. KIENY AND A. MAUGER
DISCUSSION AND CONCLUSIONS
The results of the two experimental series are not identical. The replacement
of the wing level somitic mesoderm by a strip of midgut did not prevent the
adjacent wing from being muscled. Except one of the eleven cases that were
examined histologically, the musculature developed in accordance with the
skeleton formed. When both bones, ulna and radius, were present, the intrinsic
forearm musculature developed normally. When the forearm contained only the
ulna, the muscular equipment was limited to the six muscles that normally
surround this bone. The forearm, then nearly cylindrical, was generally
reduced to its proximal half. But the former may also have formed a preaxial
half; in such a case, the preaxial mesoderm comprised exclusively a loose
totally unorganized mesenchyme.
In contrast, the in situ destruction of a portion of the somitic mesoderm that
exceeded the wing level anteriorly and/or posteriorly led to different results.
Whether or not the embryo presented external signs of the radiolesions, the wing
musculature was hypomorphic or normal. These signs appear in the spinal
feather tract as a featherless notch at the wing level in which the distorted
humeral tract tends to spread.
Previous findings by one of us (Mauger, 1970) have shown that within the
X-irradiated somitic mesoderm, the dermatome fails to differentiate, and the
corresponding portion of the spinal tract forms glabrous skin. Moreover, these
spinal plumage deficiencies are always associated with more internal vertebral
(and ribs) deficiencies : the anterior levels of these plumage and axial alterations
are concordant with one another, whereas the posterior level of the axial
deficiencies, which corresponds to the posterior edge of the irradiated surface,
always extends more caudally than that of the plumage deficiencies (Mauger
and Kieny, unpublished data). Therefore, the length of plumage deficiency can
certainly be considered as a guarantee of the destruction of the corresponding
length of the somitic mesoderm.
Thus, embryos (20 %) with an apparent undisturbed plumage and with
normally muscled experimental wings could not be considered for the present
analysis. Only those showing the above-mentioned plumage abnormalities were
retained. Their experimental wings were normally muscled in 7 %, poorly
muscled in 26 % and had no muscles in 67 % of the 27 histologically examined
cases.
The muscular reduction was qualitative as well as quantitative. No more
than 7 of the 11 forearm muscles were recognizable and their volume seldom
reached half the normal volume of the corresponding muscles. Yet, even
reduced to about 25 % of its volume, the muscle bulk was accompanied by its
tendons; while, when totally absent, the corresponding tendons were missing,
too. The myogenic area was then occupied by a loose unorganized mesenchyme.
In the cases of total muscle absence, the forearm comprised cartilage, loose
Limb-somite
relationship in chick
277
mesenchyme, dermis and epidermis. These observations held also for the
reduced and absent muscles of the previously described replacement series.
Tendons being of somatopleural origin (Chevallier et al. 1976, 1977; Christ
et al. 1974, 1977), the simultaneous absence of tendons and muscle observed
at 12-13 days of incubation raises the question whether, during preceding
development, the tendon blastemae did not form or whether they did form and
disappeared because of the absence of myogenic cells. This point will be the
object of new investigations.
In general, it seems that the shape of the wing does not depend on the differentiation of all of its constituting tissues. It is obvious that the skeleton
represents the shape's framework, whereas the presence or absence of the
musculature has no or few repercussions on the wing's form.
As regards the cellular origin of the muscles in the experimental wings,
different possibilities can be postulated in the replacement series where the
wings are normally or subnormally muscled.
(1) The muscle cells may originate from the few somitic cells that may have
been left behind after the extirpation of the somitic mesoderm. As the histologically examined cases were operated at stages of 13-17 pairs of somites
(Table 1) and led to the same results whether the wing level somite mesoderm
was excised in an unsegmented or partially segmented state, this possibility
cannot reasonably account for the development of a complete wing musculature.
(2) The muscle cells can originate from the midgut external muscle layers.
This point can be rejected. Experiments in which, under the same conditions,
chick somitic mesoderm of the wing level had been replaced by a piece of quail
midgut, showed that only some scattered quail cells had penetrated the 4- to
6-day chick wing. They were not selectively located in the muscle areas, but
found in the vicinity of the nerves; they were supposed to be Schwann cells
(Chevallier, 1977).
(3) The muscle cells can originate from the somitic mesoderm situated ahead
of/or behind the implantation site. Because the somitic replacement concerns only
the narrow portion adjacent to the wing territory, such compensatory migration
of somitic cells may take place, particularly since the myogenic potentialities of
the somitic mesoderm are not regionalized. The final differentiation of th&
somite-emigrated cells occurs 'ortsgemäss' (Chevallier et al. 1977).
(4) The muscle cells can originate from the somatopleure. This possibility,
although doubtful in these replacement experiments, cannot be discarded.
The latter hypothesis becomes more likely in the case of large somitic
elimination through radiodestructions. In this type of experiment which in
addition involved the somitic mesoderm ahead of and/or behind the wing level,,
the majority of the wings were muscleless and only one third of the cases
compensated their adjacently-somite-originating muscle deficiencies, mainly in
an incomplete way. Anyhow we have no argument to decide in favour of a
somitic or somatopleural origin of this poor musculature. But it is worth
278
A. CHEVALLIER, M. KIENY AND A. MAUGER
mentioning here that the somatopleure can give rise to muscle fibres. Indeed, in
preliminary experiments muscle fibres of convincing somatopleural origin have
been obtained in the case of ectopic wings resulting from the translocation of
quail wing somatopleural mesoderm into chick neural tube, at stages before the
somatopleural graft had become invaded by somitic cells. Thus in these
conditions, the somatopleure seems able to compensate for somitic deficiency.
In conclusion, the elimination of a portion of somitic mesoderm including
that of the wing level and extending ahead of and behind the wing level leads in
two thirds of the cases to muscleless wings. This confirms that the somitic
contribution is required for the development of a normal wing musculature.
Le mémoire représente une partie de la thèse qui sera soutenue par A. Chevallier derant l'Université scientifique et médicale de Grenoble pour l'obtention du grade de docteur
d'Etat es Sciences.
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CAPLAN,
(Received 24 June 1977)