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J. Embryol. exp. Morph. 90, 415-436 (1985)
415
Printed in Great Britain © The Company of Biologists Limited 1985
Myogenic differentiation in early chick wing
mesenchyme in the absence of the brachial somites
TERRY KENNY-MOBBS
Department of Biology, Dalhousie University, Halifax, Nova Scotia,
Canada, B3H 4J1
SUMMARY
A controversy exists in the literature over the ability of wing mesenchyme of somatopleural
origin to form skeletal muscle. Experimental approaches used in such studies leave open the
possibility of postoperative accessibility of the experimental wings to somitic cell invasion. In the
present study wing somatopleural tissue was isolated from HH stage-12 to -21 chick embryos and
grown either in organ culture (OC) or on the chorioallantoic membrane (CAM) of host chicks,
conditions under which postoperative entry of somitic cells is impossible. In the presence of axial
and somitic tissues of the brachial region, the wing territories from all four stages underwent
comparable growth and tissue differentiation. However, isolated wing regions showed a stagedependency in the differentiation of skeletal muscle but not of other limb tissues. The incidence
and amount of skeletal muscle was markedly reduced in HH stage-12 and -15 isolated wing
regions while myogenesis in HH stage-18 and -21 wing buds was not affected by the absence of
the somitic tissues. These results are consistent with reported stages of somitic cell migration
into wings with the exception of HH stage-12 explants which should have been muscleless if
somitic cells are the sole source of wing myofibres. The possibility that somitic cells had been
included in these explants was investigated by 1) testing the myogenic potential of lateral plate
tissue adjacent to the wing, 2) altering the dissection procedure for isolating wing territories and
3) using antibodies to skeletal muscle myosin and actin to detect myotubes. The results from this
series of experiments illustrate the need for extraordinary care in the isolation of wing regions
when investigating limb-somite relationships and suggest that the myogenic capacity attributed
to wing somatopleural cells in the past can be accounted for by either postoperative entry of
somitic cells into experimental wings or inadvertent inclusion of somitic cells in the primordia
when dissected. Overall, the results show that somitic cells are the sole source of wing myofibres
for, in their absence, somatopleural cells form all mesodermally-derived wing cell types except
skeletal myofibres.
INTRODUCTION
It has been clearly established from the chick/quail recombination experiments
of Christ, Jacob & Jacob (1974, 1977, 1979) and Chevallier, Kieny & Mauger
(1976, 1977; Chevallier, 1978; Kieny & Chevallier, 1980; Mauger, Kieny &
Chevallier, 1980) that cells from the somites adjacent to the limb territories of
chick and quail embryos migrate into the somatopleural mesoderm of the limb and
there differentiate exclusively into skeletal myofibres. Morphological studies of
Present address: Medical Oncology Unit, Centre Block, Southampton General Hospital,
Southampton, SO9 4XY, U.K.
Key words: limb-somite, myogenesis, wing bud, mesenchyme, somatopleural, chick embryo.
416
T. KENNY-MOBBS
normal embryos (Fischel, 1895; Grim, 1970; Christ et al. 1977; Chevallier, 1978;
Jacob etal. 1978) and other experimental approaches (Chevallier etal. 1911,1978;
Chevallier, 1979; Christ etal. 1977,1979; McLachlan & Hornbruch, 1979; Mauger
etal. 1980) have corroborated this conclusion. All other mesenchymal limb tissues,
including the connective tissues of the skeletal muscles, develop from
somatopleurally-derived cells.
Taken as a whole, these studies show that there are at least two distinct
populations of cells within the undifferentiated limb mesenchyme - one that gives
rise to skeletal muscle and one that forms the rest of the mesenchymally-derived
limb tissues. While all evidence indicates that the somitically-derived cells will give
rise only to skeletal myofibres, the developmental fates of the somatopleurallyderived cells are not so clearly established. When the brachial somites were
extirpated and replaced by a piece of 9-day gut tissue or destroyed by X-irradiation
(Chevallier etal. 1977, 1978; Chevallier, 1979), 100% and 3 3 % , respectively, of
the experimental wings contained muscle. Replacement of chick brachial somites
with those of quail resulted in the presence of quail nuclei in all skeletal myofibres
of the wing on the operated side (Christ et al. 1977; Chevallier et al. 1977, 1978).
However, when quail brachial somites were replaced by chick somites, chick
nuclei were found only in skeletal myofibres but some myofibres were of quail
origin and some of both chick and quail origin. These various experimental results
suggest that somatopleurally-derived limb mesenchyme may also have skeletal
myogenic potential, at least under the experimental conditions that prevailed in
these studies.
More direct tests of the developmental potentials of somatopleurally-derived
limb cells have been made by isolating wing regions and grafting them to the
coelomic cavities of chick hosts (Christ etal. 1979; McLachlan & Hornbruch, 1979;
Mauger et al. 1980) or into other embryonic sites (Mauger et al. 1980), but these
experiments, too, gave contradictory results. Christ et al. (1979) maintained that
the somatopleural cells did not have the ability to form skeletal muscle while both
McLachlan & Hornbruch (1979) and Mauger et al. (1980) concluded that the
somatopleural cells were developmentally labile and capable of expressing a
myogenic phenotype under certain experimental conditions.
Before one can ascribe a myogenic potential to the somatopleurally-derived
cells of the limb mesenchyme, it is essential that inclusion or postoperative entry of
somitic cells into the wing territory be obviated. Removal of the brachial somites
or their destruction by X-irradiation are not infallible methods for ensuring
complete removal or destruction of all somitic cells (Mauger, 1970; Kieny et al.
1972; Chevallier etal. 1978). Furthermore, it is important that the sites in which the
isolated wing tissues are grown are not accessible to somitic cell invasion. For
many of the intraembryonic graft sites (e.g., the coelom) this cannot be
guaranteed, as shown by the results of Mauger et al. (1980).
The experiments described in this paper attempt to resolve the question of the
possible myogenic potential of the wing mesenchyme of somatopleural origin by
isolating wing territories at early stages and growing them either in organ culture
Myogenic potential of early wing mesenchyme
All
or grafted to the chorioallantoic membrane of host chicks, conditions under which
postoperative entry of somitic cells into the wing region is impossible. The results
show a stage-related dependence of limb myogenesis on the presence of the
brachial somites and also show that somatopleurally-derived limb mesenchymal
cells do not have skeletal myogenic potential - thai: the somitic cells that migrate
into the limb regions are the sole source of limb myofibres.
Some of these results were presented at the Third International Conference on
Limb Development and Regeneration (Kenny-Mobbs & Hall, 1983).
MATERIALS AND METHODS
Fertile eggs of the White Leghorn variety of Gallus domesticus were obtained from a local
hatchery. The eggs were incubated to the required stage of development at 37°C and a relative
humidity of 50-60 %. Embryonic development was staged according to Hamburger & Hamilton
(1951) using the number of somites as the main criterion.
Tissues were dissected from embryos of four specific stages of development; HH stages 12,15,
18 and 21. Wing primordia from these embryos should have contained different numbers of
somitic cells. Migration into wing primordia had not yet begun in the HH stage-12 embryos; HH
stage-15 wing primordia should have contained a few somitic cells since migration would have
been under way for a few hours before isolation; HH stage-18 wing buds should have contained
nearly a full complement of somitic cells since migration was nearing completion (Christ et al.
1977; Chevallier, 1978) and, in HH stage-21 wing buds, the onset of histological differentiation
was imminent.
(i) Dissections
Embryos were removed from the yolk with the aid of filter paper discs and transferred to
saline. Wing regions were then dissected free of surrounding tissues as illustrated in Fig. 1A. For
the two younger stages this resulted in a rectangular piece of tissue that included the three germ
layers of the lateral plate opposite somites 12/13 to'20/21 (Fig. IB) and sometimes included
portions of the intermediate mesoderm. In the older embryos only the wing buds were removed
(lateral plate somatopleure only), although occasionally some intermediate mesoderm was
included (Fig. 1C).
Wing territories that included the somitic and axial tissues were also dissected from embryos
of each of the four stages. In these cases, the embryo was cut up the midline of the neural tube
(Fig. 1A) so that each dissected piece contained the wing territory and the tissues medial to it,
viz., the intermediate mesoderm, half of the neural tube and portions of the notochord (Fig.
ID). The covering epithelial layers were also present.
In a second series of experiments, two areas of lateral plate tissue were dissected from HH
stage-12 embryos (Fig. 2A). The first of these (diagonally-crossed in Fig. 2A) was wing lateral
plate and included all three germ layers adjacent to somites 14/15 to presumptive somites 20/21.
Care was taken to ensure that the medial cut of this block of tissue was such that intermediate
mesoderm was excluded from the dissected tissues. The second piece of tissue (stippled in Fig.
2A) included the lateral plate tissue (all three layers) adjacent to somites 10/11 to somites 13/14.
In both cases the distal boundary of the dissected tissues was at the margin of the areas pellucida
and opaca. At both levels some dissections were made that included medial tissues - the
intermediate mesoderm, somitic mesoderm and axial tissues.
Lateral plate tissues of the wing region and that posterior to the wing level were dissected
from embryos of HH stage-15 embryos. In HH stage-12 embryos the posterior boundary of the
wing territory can be established by estimating where the remaining brachial somites will
segment; however, it is difficult to determine the exact location of more posterior tissues, in this
418
T. KENNY-MOBBS
1A
Myogenic potential of early wing mesenchyme
419
case the flank region of the embryo (at the level of somites 21-24). Consequently, the ability of
lateral plate tissue at this level to form skeletal muscle was assessed in slightly older embryos.
The dissected area is shown stippled in Fig. 2B. The anterior cut was made at the level of somites
21/22 and the posterior at the level of somites 24/25. Again some tissues were dissected to
include the more medial tissues.
(ii) Organ cultures
Each wing territory was mounted on a small square of Millipore filter and placed, filter side
down, on wire mesh supports in 35 mm plastic dishes (Falcon Type 3001). Three tofivepieces of
tissue were placed in each dish and 1-5 ml of culture medium was added. The culture medium
consisted of BGJb with 15 % horse serum and 5 % embryo extract. All components of the
medium were purchased from GIBCO. The cultures were maintained at 37 °C in an atmosphere
of 5 % carbon dioxide in air. The medium was changed every other day over the culture period
which varied from 4 to 9 days. At the end of the culture period the explants were rinsed in saline
and fixed as described below.
Fig. 2. A schematic diagram of (A) an HH stage-12 embryo and (B) an HH stage-15
embryo illustrating the lateral plate tissues dissected from the level of the wing (wlp),
the region anterior to the wing (alp) and that posterior to the wing (pip). Somite 15 is
shaded. Bar = 1 mm.
Fig. 1. (A) A schematic diagram of an HH stage-12 embryo illustrating the boundaries
of areas dissected when (a) the wing territory was isolated with the adjacent somitic
and axial tissues and (b) only wing region lateral plate was isolated. The 15th somite
pair (cross-hatched) marks the beginning of the wing (or brachial) region of the
embryo. Bar = lmm. (B-D) Paraffin sections of the isolated tissues before explantation. d, distal; p, proximal. Bars = 50/an. (B) Lateral plate tissue of the brachial
region of an HH stage-12 embryo. Both somato- and splanchnopleural layers of the
lateral plate were included in the dissected wing regions from HH stage-12 and -15
embryos. (C) Isolated wing bud of an HH stage-18 embryo. Only the wing buds (lateral
plate somatopleure) were isolated from HH stage-18 and -21 embryos. (D) Wing
region of an HH stage-15 embryo that was dissected with the adjacent somitic and axial
tissues. /, intermediate mesoderm; nt, neural tube; ,?, somitic mesoderm; wlp, wing
region lateral plate.
420
T. KENNY-MOBBS
(iii) Chorioallantoic grafts
Host embryos for chorioallantoic (CAM) grafting were incubated for 8 to 10 days. The host
eggs were windowed using a fine-toothed hacksaw blade and were then returned to the
incubator until required for grafting. Tissues to be CAM-grafted were dissected and set up for
organ culture as described in the previous sections. They were held in the CO2 incubator until
grafted.
The tissue to be grafted was positioned on the CAM at a bifurcation of the blood vessels, with
either the filter or the tissues abutting the CAM. The window was replaced and sealed in
position with Scotch tape. The hosts were then returned to the incubator for a further 4 to 7
days.
(iv) Histological techniques
All cultures, grafts and normal tissues prepared for light-microscopic examination were fixed
in one of three fixatives; neutral buffered formalin (Drury & Wallington, 1967), half-strength
Karnovsky's (Karnovsky, 1965) or phosphate-buffered 4 % formaldehyde, 1 % glutaraldehyde
(McDowell & Trump, 1976). Fixed tissues were processed for paraffin embedding through an
ascending series of alcohol and xylene and were infiltrated and embedded with Paraplast Plus
(m.p. 56-57°C; Canlab.).
6 jum serial sections were prepared and stained by one of three staining procedures; Mallory's
triple stain (modification of Pantin, 1960), haematoxylin/alcian blue/chlorantine fast red
(modified from Lison, 1954), or haematoxylin/eosin/alcian blue, and were mounted in DPX
synthetic mountant (BDH Chemicals).
Photomicrographs were taken on Kodak Panatomic X film (ASA 32) with a Zeiss
Photomicroscope II.
(v) Indirect immunofluorescence
Antibodies to skeletal muscle myosin and actin were generously supplied by Dr B. G.
Atkinson, Department of Zoology, The University of Western Ontario. The antibodies were
raised in rabbits to skeletal muscle proteins isolated from frog hindlimb muscles and have been
found to cross-react with their antigens in various species, including chick (Atkinson, pers.
comm.). The procedures for the isolation and purification of these proteins and for the raising
and purification of the antibodies have been described by Dhanarajan (1979) and Dhanarajan &
Atkinson (1980).
Grafts or cultures of HH stage-12 wing regions were rinsed in saline and immediately frozen in
O.C.T. Compound (Tissue-Tek II, Canlab). 10/zm sections were cut and serially mounted on
precleaned microscope slides. The sections were air-dried for 5-15 min and then fixed for 30 min
in 3 % paraformaldehyde in phosphate-buffered saline (PBS). They were then rinsed in PBS and
permeabilized in absolute acetone at —20°C. Sections were exposed to the skeletal muscle
antibodies, diluted 1:1 with PBS, for 1 h at 37°C, rinsed in PBS, and exposed to goat anti-rabbit
globulin conjugated tofluoresceinisothiocyanate (FITC-GaR-IgG; GIBCO) for 30min at 37°C.
After several rinses in PBS and distilled water, the sections were mounted in 6 % polyvinyl
alcohol. As controls the skeletal muscle antibody was replaced by serum from pre-immune
rabbits, also diluted 1:1 with PBS, or the first layer was omitted altogether and the sections
exposed only to the second layer - the FITC-GaR-IgG.
The sections were examined with a Zeiss Universal fluorescence microscope, equipped for
epifluorescence, using an HBO 200 W/4 light source, exciter filter BG12/4, barrier filters 44 and
50 and Neofluar objectives. Photographs were taken on Kodak Tri-X film.
RESULTS
(i) Explants of wing territories and adjacent somitic and axial tissues
In the first series of experiments, wing territories, along with the somites and
portions of the neural tube and notochord of the brachial region (e.g., Fig. ID)
Myogenic potential of early wing mesenchyme
All
were isolated from HH stage-12 to -21 embryos. 40 such explants were cultured
and 42 were grafted onto the CAM.
Little stage-related difference was found in the growth and differentiation of
explants grown in culture or on the CAM. Generally though, growth was considerably greater on the CAM than in culture and some grafts from all four stages
exhibited some evidence of a wing morphology (Fig. 3A) while none of the
cultures did so. Feather germs, kidney tubules and gut were common features of
both cultures and grafts, with the exception of HH stage-21 explants in which no
gut tissue differentiated. Neural tissue was present in all and notochord in most of
the explants. In the CAM grafts, in which vertebral cartilages underwent considerable morphogenesis, ganglia were observed on the neural side of these cartilages
and nerve bundles and blood vessels were seen to extend into the surrounding
tissues through gaps in the vertebral cartilage (Fig. 4A).
Cartilage differentiated in all cultures and grafts (Fig. 3). At least three distinct
cartilage elements were observed in all cultures and 65-75 % contained four or
more elements. Cartilage morphogenesis was greater in the CAM grafts and,
when the grafts formed identifiable wings, elements could be identified as intrinsic
limb cartilages, including autopodial elements. Membranous bone of the clavicle
was found in 23 % of HH stage-18 grafts (Fig. 4B). Discrete muscle bundles with
readily identified myotubes were present in both vertebral and limb regions of the
explants (Fig. 4A,C). In some sections, especially of more distal wing regions,
early stages of tendon formation could be seen (Fig. 4C).
From these results it is clear that explants of wing primordia from all four stages,
when grown in the presence of the brachial somites and portions of the neural tube
and notochord, exhibited a similar pattern of growth and differentiation of the
wing region. The incidence of a recognizable wing morphology increased with
increasing age of the explant but tissue differentiation was the same.
(ii) Explants of isolated wing territories
(a) In this series of experiments, wing primordia were dissected free of adjacent
tissues and grown in culture or on the CAM. Growth on the CAM was more
substantial than in culture and, like explants that included the axial tissues, grafts
with a wing-like morphology were more commonly obtained from the older
explants (Fig. 5). Differentiation in isolated wing primordia differed from the
explants described above in that there was a decrease in the number of cartilage
elements that formed and in the incidence of kidney differentiation for all four
stages (Tables 1 and 2). Grafts of HH stage-18 and -21 wing buds contained more
cartilage elements than cultures of the same age.
Differences in the presence of skeletal muscle in these explants were found to be
stage-related; while all explants of HH stage-18 and -21 wing buds contained welldeveloped skeletal muscle bundles (Tables 1 and 2; Figs 5,6), HH stage-12 and -15
explants showed a marked decrease in the incidence of skeletal muscle formation,
although a few explants from both stages contained one or two muscle bundles
(Tables 1 and 2; Fig. 7). Most of the HH stage-12 and -15 explants contained
422
T. KENNY-MOBBS
Myogenic potential of early wing mesenchyme
423
Table 1. Differentiation in explants of HH stage-12 to -21 wing primordia grown in
culture for 4 to 9 days*
Cartilage elements
Stage
1-2
3-4
HH12
HH15
HH18
HH21
69%
47%
0%
18%
31%
53%
62%
77%
Muscle
Condensations Myotubes
0%
0%
38%
5%
75%
65%
25%
35%
100%
100%
Gut
94%
82%
15%
0%
Kidney n
94 %
59 %
23 %
13%
16
17
13
44
* Differentiation expressed as a percentage of samples (n) containing the tissue.
Table 2. Differentiation in grafts of HH stage-12 to- 21 wing primordia grown on the
CAM for 4 to 7 days*
Cartilage elements
Stage
1-2
3-4
HH12
HH15
HH18
HH21
73%
73%
0%
0%
27%
27%
19%
12%
Muscle
Condensations Myotubes
0%
0%
81%
88%
72%
82%
15%
18%
100%
100%
Gut
91%
91%
52%
0%
Kidney n
48 %
55 %
57 %
12%
33
11
21
16
* Differentiation expressed as a percentage of samples (n) containing the tissue.
condensations in 'myogenic' areas (Tables 1 and 2; Fig. 8A,B), but no myotubes
could be found within these condensations.
The low incidence of skeletal myogenesis in the explants from HH stage-12 and
-15 embryos prompted a careful examination of the differentiation of other
mesodermally-derived tissues in these explants. It was found that differentiation of
kidney tubules occurred in 94 % of cultured and 48 % of CAM-grafted explants
from HH stage-12 embryos (see Tables 1 and 2). Myotubes were present in four of
the cultures and in five of the grafts and all nine of these explants contained kidney
tubules. This coincidence of kidney tubule differentiation with the presence of
Fig. 3. Survey sections of CAM-grafts of wing regions explanted with the adjacent
somitic and axial tissues. (A) Wing region of a graft from an HH stage-12 embryo
grown on the CAM for 6 days showing a wing-like morphology and the presence of
cartilage (c) and skeletal muscle (m) bundles. Bar = 200jum. (B) An amorphouslyshaped graft of an HH stage-18 wing bud grown on the CAM for 6 days.
Differentiation of axial and somitic tissues (top) and lateral plate tissues (bottom),
including cartilage (c) and muscle (m) can be seen, nt, neural tube; k, kidney.
Bar = 300/an.
Fig. 4. Paraffin sections of wing regions CAM-grafted with adjacent somitic and axial
tissues. Bars = 100 jum. (A) The axial region of an HH stage-15 graft grown for 6 days
on the CAM showing the differentiation of vertebral cartilage (c) and muscle (m) and
spinal ganglia (g). (B) A graft from an HH stage-18 embryo, grown for 6 days on the
CAM, in which the clavicle (b) had formed, c, cartilage. (C) A graft from an HH stage12 embryo left on the CAM for 10 days showing tendon formation (arrow), c, cartilage;
m, muscle.
424
T. KENNY-MOBBS
Myogenic potential of early wing mesenchyme
425
Fig. 8. Paraffin sections through an HH stage-12 isolated wing region grown on the
CAM for 5-5 days. (A) Cartilage (c) and feather germs (f) are seen and, in the loose
connective tissue between these two, there is an area of condensation. Bar = 100 /mi.
(B) A higher magnification of the condensed area. No myotubes could be identified in
this region. The asterisk (*) marks the area indicated at the top of the arrow in Fig. 8A.
Bar = 25
skeletal muscle raised the possibility that somitic cells had been inadvertently
included in the initial dissections. Such 'contaminating' somitic cells might have
been included i) by making the medial cut of the dissection of the wing regions too
close to the intermediate mesoderm and thereby accidentally including some
somitic cells which lie medial to the intermediate mesoderm, or ii) by including
lateral plate tissue anterior and/or posterior to the wing territories in the initial
dissections. Such tissues already may have contained cells capable of myogenic
Fig. 5. A survey section of an isolated HH stage-18 wing bud that had been grown on
the CAM for 6 days. Note the wing-like morphology and the differentiation of cartilage
elements (c), muscle (m) and bone (b). Bar = 1000ym.
Fig. 6. A section through an HH stage-18 isolated wing bud grown in culture for 6
days, c, cartilage; m, muscle. Bar = 200 jum.
Fig. 7. A survey section of an HH stage-12 isolated wing region grown on the CAM for
6-5 days. Cartilage elements (c), kidney tubules (k) and one small muscle bundle (m)
formed in this graft. Bar = 200 urn.
426
T. KENNY-MOBBS
Table 3. Differentiation in CAM grafts of HH stage-12 and -15 wing territories
explantedfor 6 days*
Stage
12
15
Cartilage elements
3-4
5
1-2
41%
23%
30%
27 %
30%
50 %
Muscle
Condensations Myotubes
96%
82%
0%
9%
Gut
Kidney
n
85%
95%
4%
5%
27
22
'Differentiation expressed as a percentage of samples (n) containing the tissue.
expression. Two series of experiments were carried out to check these
possibilities.
(b) Lateral plate tissue of the wing territory was dissected from HH stage-12 and
-15 embryos (Fig. 2) and grafted to the CAM. These explants differed from those
described in section (a) above in that the region dissected was restricted to the
lateral plate tissue opposite somites 15-20 and the medial cut for the dissection
avoided not only the somites but also the intermediate mesoderm.
Tissue differentiation in these explants was not appreciably different from that
observed in the HH stage-12 and -15 CAM grafts described before (cf. Tables 2
and 3) with the notable exceptions of a marked decrease in the incidence of kidney
tubule formation and a reduction in the incidence of skeletal muscle differentiation in both HH stage-15 explants (9 % compared with 18 % in the previous
dissections) and HH stage-12 grafts, in which no myotubes could be found (Table
3; Figs 9,10).
Areas of cellular condensation in the loose connective tissues adjacent to
cartilage elements were common in grafts from both stages (Figs 9, 10). No
myotubes could be found within these condensations in HH stage-12 grafts but
because they were such a common feature of the areas in which muscle would
normally form, they were investigated further with antibodies to skeletal muscle
myosin and actin using indirect immunofluorescence. Eleven explants of HH
stage-12 wing regions (seven grafts and four cultures) and five explants of wing
regions with the adjacent axial and somitic tissues (three grafts and two cultures)
were studied using these antibodies, the latter serving as positive controls.
All grafts and cultures that included the somitic and axial tissues in the initial
dissection were strongly positive for the presence of skeletal muscle myosin
(Fig. 11) and gave a fainter, though positive, reaction with skeletal muscle actin
antibodies. In the absence of somitic tissue, all cultures and grafts gave a negative
reaction for both skeletal muscle myosin (Fig. 12A) and actin (Fig. 12B). Smooth
Figs 9, 10. Sections through CAM grafts showing tissue differentiation in explants of
wing lateral plate from HH stage-12 (Fig. 9) and HH stage-15 (Fig. 10) embryos.
Cartilage (c), feather germs (/), loose connective tissue and areas of condensation
(arrows) were common features of these grafts. Within the condensations (Figs 9B,
10B), cells were more densely packed but no myotubes could be identified.
Bars = 100(im (A); bars = 50/an (B).
Myogenic potential of early wing mesenchyme
\v
427
428
T. KENNY-MOBBS
muscle cells of the gut wall did not react with either antibody. The controls with
pre-immune serum (Fig. 13A) and the application of only the second layer (FITCGaR-IgG) (Fig. 13B) were negative.
(iii) Grafts of lateral plate tissue anterior and posterior to the wing regions
In the final series of experiments in this study, lateral plate tissues anterior to the
wing region (in HH stage-12 embryos) and posterior to the wing region (in HH
stage-15 embryos) were analysed for their ability to form skeletal muscle when
isolated from the adjacent somites and grafted to the CAM.
In the majority of anterior lateral plate grafts (aLP) one or two cartilage
elements formed (Table 4, Fig. 14). Membranous bone of the clavicle, and gut,
liver, and kidney tissues also differentiated in many of the grafts. However, none
of the grafts of aLP contained myotubes or condensations in myogenic areas.
Grafts of posterior lateral plate (pLP) formed large mounds of tissue consisting
mainly of a vascularized loose connective tissue in which one S-shaped cartilage
element was frequently seen (Table 4; Fig. 15A). Feather germs and gut were the
only other tissues identified in these grafts. In two samples an area of condensation
adjacent to the cartilage element was seen (Fig. 15B) but no myotubes were found.
In grafts of aLP and pLP in which the axial and somitic tissues were included in
the initial dissection, neural tissue, notochord, vertebral cartilage and kidney
tubules differentiated and all explants contained well-developed skeletal muscle
bundles in both vertebral and lateral plate regions of the grafts (Fig. 16).
DISCUSSION
The results of this study demonstrate a stage-related dependence of limb
myogenesis on the presence of somites in the brachial region consistent with the
reported stages of somitic cell migration into wing primordia (Chevallier, 1978;
Christ et al. 1977). They not only support a somitic origin for wing skeletal muscle
but also show that these somitic cells are the sole source of such muscle for, in their
absence, cells of somatopleural origin form all mesodermally-derived cell types of
the wing except skeletal myofibres.
There are two critical requirements for an ectopic site in these limb-somite
studies, namely, i) that the site supports the growth and differentiation of the
explanted limbs and ii) that it is isolated from the somitic tissues. Another obvious
criterion is that no somitic cells are included in the initially dissected tissues. What
Figs 11-13. Immunofluorescence in frozen sections of HH stage-12 CAM grafts of
wing regions explanted with (controls) and without adjacent somitic tissues. All
negatives were given equal exposure on printing. Bars = 200 /im.
Fig. 11. Positive reaction to skeletal muscle myosin antibodies in a control graft.
Fig. 12. In the absence of somitic tissue, explants of wing regions gave negative
reactions to antibodies both to skeletal muscle myosin (12A) and actin (12B).
Fig. 13. Controls in which explants containing somitic tissues were treated with preimmune serum (13A) instead of antibody or treated only with FITC-GaR-IgG (13B).
c, cartilage; m, muscle.
429
Myogenic potential of early wing mesenchyme
<7
.1'"
12A
—-
12 ft
<*
13 :
430
T. KENNY-MOBBS
5ȣ
Myogenic potential of early wing mesenchyme
431
Table 4. Differentiation in grafts ofHHstage-12 lateral plate opposite somites 10/11 to
13/14 (aLP) and HH stage-15 lateral plate opposite somites 21-25 (pLP) grown on the
CAM for 6 and 5 days, respectively
Stage
Cartilage elements
1-2
3
12 (aLP)
15 (pLP)
73%
92%
7%
0%
Clavicle
Muscle
Gut
Liver
Kidney
n
40%
0%
0%
0%
87%
85%
73%
0%
27%
0%
15
13
is not so obvious is the special care that must be taken to ensure that this does not
occur. Each of these points will now be considered in detail.
(i) Differentiation in explanted wing regions
None of the ectopic sites used in the different limb-somite studies appears to be
appreciably better than any other in permitting normal myogenesis to occur.
McLachlan & Hornbruch suggested that the CAM and the coelom may not
be equivalent 'in their ability to evoke differentiation of cell types' (p. 216,
McLachlan & Hornbruch, 1979), implying that the CAM, while fostering cartilage
differentiation, might not be conducive for muscle differentiation. This is clearly
not the case as large, healthy muscle bundles were found in CAM grafts of wing
buds isolated from older embryos (HH 18 and 21), as well as in wing regions from
all four stages when adjacent axial and somitic tissues were included.
Differentiation of skeletal muscle in explanted limbs might also be affected by
the lack of innervation of the explants since skeletal muscle eventually degenerates
without innervation, although initial differentiation of skeletal muscle is not nervedependent (e.g., Hunt, 1932; Eastlick, 1943). It has been suggested that Christ
et al. observed no skeletal muscle in their coelomic grafts because it had
degenerated (McLachlan & Hornbruch, 1979; Mauger et al. 1980). The lack of
skeletal muscle in the explants of the present study cannot be attributed to
degenerative breakdown; although the explants were not innervated, there was no
evidence of degeneration over the periods of explantation used.
(ii) Exclusion of somitic cells from explanted wing regions
As already stressed in the Introduction, to assign a myogenic capacity to wing
somatopleural cells, one must be sure that there are no somitic cells present in the
experimental limbs as they develop. An alternative explanation for the presence of
Figs 14-16. Paraffin sections of 5- to 6-day-old CAM grafts of lateral plate tissue
anterior (aLP) and posterior (pLP) to the wing region.
Fig. 14. An HH stage-12 aLP explant showing cartilage (c), bone (b) and a feather
germ(/). Bar = 200 ^m.
Fig. 15. Grafts of HH stage-15 pLP in which (A) an S-shaped cartilage element (c)
and loose connective tissue formed. Bar = 200jum. (B) A condensation (*) in which
myotubes could not be identified, c, cartilage. Bar = 200 ^m.
Fig. 16. An explant of HH stage-12 aLP that included axial and somitic tissues.
Neural tissue (nt), cartilage (c) and muscle (m) differentiated in these grafts.
Bar =100 jan.
432
T. KENNY-MOBBS
skeletal muscle in the explants of earlier studies and the first series of experiments
in this study lies in the assumption that somitic cells were present in the
experimental limbs. This could have happened if a) the explanted wing regions
were .accessible to somitic cell invasion after grafting or b) somitic cells were
included inadvertently in the initially dissected tissues.
(a) Access to postoperative invasion by somitic cells
The first of these possibilities has no bearing on the results of the present study
since explants grown in culture or on the CAM of host chicks are completely
isolated from somitic regions. However, it does have a bearing on past studies for
all explants to different ectopic sites within host chicks were made to regions in and
around the limb territories and in hosts in which somitic cell migration was under
way, i.e., between HH stages 13 and 17 (Christ et al. 1979; McLachlan &
Hornbruch, 1979; Mauger et al. 1980). Several observations suggest that these
graft sites are less than ideal for studies on limb-somite relationships because they
do not ensure isolation of the grafted primordium from host somitic cells.
In order to survive, grafts must attach to the host tissue and become vascularized
(Hamburger, 1938; Rudnick, 1945). The location and extent of this attachment
varies but in many cases it forms in the somatopleural layer and sometimes very
close to the lateral edges of the somites (e.g., fig. 1, Dhouailly & Kieny, 1972).
Such continuity clearly makes the graft susceptible to invasion by host cells. That
such an invasion occurs has been shown in grafts to the flank (Dhouailly & Kieny,
1970, 1972; Mauger et al. 1980) and in grafts in place of the neural tube (Mauger
etal. 1980). Dhouailly & Kieny (1970,1972) assumed that flank somatopleural cells
had invaded the grafts; however, evidence suggests that somitic cells also
contributed to the ectopic limbs. First, the distribution of host cells in the ectopic
limbs (fig. 1, Dhouailly & Kieny, 1970) was exactly that seen for somitic cells in
limbs formed after chick somites had been replaced by those of quail (e.g.,
Chevallier, 1978). Second, somitic cells from all cephalocaudal levels tested were
found to be capable of entering limb territories and giving rise to normal limb
musculature when placed adjacent to limb regions (Chevallier et al. 1977; Christ
et al. 1978). Third, limb territories seem to elicit the migration of adjacent somitic
cells (Mauger & Kieny, 1980). Fourth, myotubes of host origin were found in
ectopic limbs after grafting to the flank and in place of the neural tube (Mauger
et al. 1980). Considering these observations, and the fact that somitic cell
migration is under way in the hosts, it must be assumed that host somitic cells will
invade limb primordia grafted to these sites. McLachlan & Hornbruch (1979) did
not describe the distribution of host cells in their grafts of quail wing primordia to
the chick coelom but showed host cells in muscle bundles in the recovered grafts.
Likewise, Christ etal. (1979) made no mention of the presence of host cells in their
coelomic grafts but later stated that such invasion probably occurs (Christ &
Jacob, 1980). Grafts between the folds of the neural tube, the third site used by
Mauger et al. (1980), can be considered impervious to somitic cell invasion and
such grafts were found to be muscleless when recovered.
Myogenic potential of early wing mesenchyme
433
There is an important difference in the grafting procedure used by these three
groups of workers that should be noted. Both McLachlan & Hornbruch (1979) and
Christ et al. (1979) grafted intact limb primordia. On the other hand, Mauger et al.
(1980) stripped the ectoderm from the primordia before grafting them. In the
latter grafts the limb somatopleure induced the formation of an ectopic limb that
incorporated both host and graft mesodermal cells and was covered by host
ectoderm (Dhouailly & Kieny, 1970, 1972). These explants may have been more
susceptible than the intact primordia to invasion by host somitic cells since the
absence of the graft ectoderm may have eliminated any physical barrier to such
invasion.
In itself, the presence of host somitic cells does nor negate the fact that graft cells
formed myotubes in wing primordia explanted before somitic migration had begun
in the donor (McLachlan & Hornbruch, 1979; Mauger et al. 1980) but the findings
of Mauger et al. (1980) suggest that this potential can only be expressed in the
presence of somitic myogenic cells since no graft-only myotubes were found. The
majority of myotubes (>69%) were of host origin only; 26% of the myotubes
contained graft-originated nuclei, but always in association with host nuclei
(Mauger etal. 1980, Table 1). Again, however, the critical point is the origin of the
graft cells that form muscle. These observations do not support the suggestion of
McLachlan & Hornbruch (1979) that the presence of somitic myogenic cells in
normal limbs suppresses skeletal myogenesis in limb somatopleural cells.
(b) Inclusion of somitic cells in dissected wing regions
The presence of muscle in the first series of HH stage-12 grafts in this study was
not immediately interpreted as evidence that somatopleurally-derived cells have
myogenic potential because both the amount and incidence were so low, especially
considering that the grafts had otherwise differentiated normally (compared with
controls). In attempting to explain these results, one was led to the conclusion that
the routine dissection procedure for the isolation of wing primordia used by
workers studying limb-somite relationships may not be sufficient to ensure
complete exclusion of somitic cells. There are two possible ways that somitic cells
might be included in the isolated wing regions; by including adjacent lateral plate
tissue that had myogenic potential (already invaded by somitic cells) or by
including parts of the somites themselves.
In the first case one must consider the timing of the onset of somitic migration
into lateral plate tissues. Studies by Chevallier (1978), Christ et al (1977) and
Mauger et al. (1980) show that it is unlikely that migration into wing lateral plate
begins before all brachial somites have segmented, so wing regions from embryos
with 14 to 19 somite pairs (HH stages 11 to 13") can be considered devoid of
somitic cells. However, migration of somitic cells into the lateral plate occurs in a
cephalocaudal sequence, for example, reaching the level of somite 12 at HH stage
12 (Mauger et al. 1980), somite 19 at HH stage 14 (Pinot, 1969) and somite 26 by
HH stage 16 (Jacob et al. 1979). Given this sequential initiation of somitic cell
migration it seemed possible that lateral plate tissue anterior or, less likely,
434
T. KENNY-MOBBS
posterior to the wing region might already contain myogenic cells. In the first
experiments of the present study portions of this adjacent tissue were included in
the dissected pieces to ensure that the wing primordium was dissected intact. The
results from two different types of grafts showed that inclusion of this adjacent
tissue does not account for the differentiation of skeletal muscle in the initial HH
stage-12 cultures and grafts. CAM grafts of HH stage-12 lateral plate anterior to
the wing (aLP) and that posterior to the wing (pLP) from HH stage-15 embryos
showed that these regions do not have myogenic potential at these stages.
Obviously, in limb-somite studies one takes special care not to include somitic
tissue in the dissected wing regions, but the results of the present study suggest that
it is necessary to exclude tissue even more lateral to the somites, namely, the
intermediate mesoderm, to be absolutely sure of not including some somitic cells.
In the first series of experiments, the nine explants that contained skeletal muscle
also contained kidney tubules (e.g., Fig. 7). However, in the second series, in
which not only the somitic tissue but also the intermediate mesoderm was
excluded from the dissected pieces, kidney differentiated in one of 27 explants and
none contained skeletal muscle.
Whether accidental inclusion could be the source of somitic cells in the grafts of
McLachlan & Hornbruch (1979) and Mauger et al. (1980) cannot be determined
from their papers; however, both groups of workers explanted wing primordia as
young as HH stages 10/11 and Pinot (1969) has shown that the boundaries
between the somitic, intermediate and lateral plate mesoderms at the wing level
cannot be distinguished until HH stage 12. Without some indication of these
boundaries, there is a strong possibility that some somitic cells were included in the
dissected primordia. Since the differentiation of other tissue types in the
experimental wings was not described in these papers, a coincidence of kidney
tubule differentiation with the presence of skeletal muscle would not have been
noticed and the possibility of inclusion of somitic cells in the explant not
recognized.
To summarize, of the four possible explanations for the presence of somitic cells
in experimental wings that contained skeletal muscle, namely, postoperative
entry, early migration, derivation from adjacent lateral plate and inadvertent
inclusion during dissection of the explants, the last seems the most likely for the
explants in the first series of experiments in this study and, albeit on circumstantial
evidence, for the explants of McLachlan & Hornbruch (1979) and Mauger et al.
(1980). In the experiments involving the destruction or replacement of brachial
somites (e.g., Chevallier et al. 1911,1978), a combination of incomplete removal of
host somitic cells, regulation, and developmental asynchrony between chick and
quail cells could reasonably account for the presence of somitic cells and, hence,
skeletal muscle in the experimental wings.
In conclusion, the results of this study argue against the possibility that limb cells
of somatopleural origin have skeletal myogenic potential. In all cases in which
skeletal myotubes were found, there was evidence that somitic cells entered or
were included in the experimental wings. When these possibilities were excluded,
Myogenic potential of early wing mesenchyme
435
as in the second series of HH stage-12 grafts in this study, no skeletal muscle
differentiated in the explants. Therefore, limb cells of somatopleural origin have
the potential to form all mesodermal derivatives of the limb except the skeletal
myofibres, which differentiate from somitic cells that invade the limb as it begins
its outgrowth from the body wall.
The author gratefully acknowledges scholarship support from the Natural Sciences and
Engineering Research Council (NSERC) of Canada and from the I.W. Killam Memorial Fund
at Dalhousie University. The work was done in the laboratory of Dr Brian Hall and was funded
by an NSERC grant to Dr Hall. My thanks to Drs Brian Hall, Ian Mobbs and Peter Thorogood
for comments on the manuscript.
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