Download Myogenic specification in somites: induction by axial

1443
Development 120, 1443-1452 (1994)
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Myogenic specification in somites: induction by axial structures
Nicholas Buffinger and Frank E. Stockdale*
Stanford University, School of Medicine, Room M211, Stanford CA 94305-5306, USA
*Corresponding author
SUMMARY
Specification of the myogenic phenotype in somites was
examined in the early chick embryo using organotypic
explant cultures stained with monoclonal antibodies to
myosin heavy chain. It was found that myogenic specification (formation of muscle fibers in explants of somites or
segmental plates cultured alone) does not occur until
Hamburger and Hamilton stage 11 (12-14 somites). At this
stage, only the somites in the rostral half of the embryo are
myogenically specified. By Hamburger and Hamilton stage
12 (15-17 somites), the three most caudal somites were not
specified to be myogenic while most or all of the more
rostral somites are specified to myogenesis. Somites from
older embryos (stage 13-15, 18-26 somites) showed the
same pattern of myogenic specification — all but the three
most caudal somites were specified. We investigated the
effects of the axial structures, the notochord and neural
tube, on myogenic specification. Both the notochord and
neural tube were able to induce myogenesis in unspecified
somites. In contrast, the neural tube, but not the notochord,
was able to induce myogenesis in explants of segmental
plate, a structure which is not myogenic when cultured
alone. When explants of specified somites were stained with
antibodies to slow or fast MyHC, it was found that
myofiber diversity (fast and fast slow fibers) was established very early in development (as early as Hamburger
and Hamilton stage 11). We also found fiber diversity in
explants of unspecified somites (the three most caudal
somites from stage 11 to 15) when they were recombined
with notochord or neural tube. We conclude that myogenic
specification in the embryo results in diverse fiber types
and is an inductive process which is mediated by factors
produced by the neural tube and notochord.
INTRODUCTION
(Teillet and Le Douarin, 1983; Rong et al., 1992), though the
subsequent development of the musculature of the appendages
seems unaffected. Interestingly, the notochord appears able to
substitute for the neural tube in that it will support maturation
of somites (Rong et al., 1992), but the implantation of ectopic
notochord is reported to inhibit myogenesis in somites
(Pourquie et al., 1993). Using an in vitro model, Kenny-Mobbs
and Thorogood (1987) showed that the neural tube/notochord
complex can induce muscle fiber formation in explants of
brachial somites.
Understanding the molecular events leading to myogenic
differentiation has been advanced in recent years by the
discovery of the MyoD family of myogenic regulatory factors
(MRFs) (Davis et al., 1987; Wright et al., 1989; Edmondson
and Olson, 1989; Braun et al., 1989; Rhodes and Konieczny,
1989). Any of the members of this family (MyoD, myogenin,
myf5 and MRF4) can convert many non-myogenic cells to a
myogenic phenotype in vitro. Studies of early embryos show
that MRFs are expressed in somites long before there are overt
signs of myogenic differentiation (Sassoon et al., 1989; Charles
de la Brousse and Emerson, 1990; Piette et al., 1992).
However, the exact role these genes play in myogenesis in vivo
is unclear. Gene knock out experiments in transgenic mice
have yielded mixed results. Mice lacking the MyoD gene have
a normal phenotype, and the major defect in myf5 knock out
Inductive interactions play key roles throughout early development of vertebrate embryos. Indeed, the formation of one of
the three germ layers in amphibia, the mesoderm, is the result
of an inductive interaction between the animal and vegetal
hemispheres of the embryo (Nieuwkoop, 1969). Later in development, inductive events are required for the formation of
many of the tissues and organs of the embryo. The formation
of the heart, optic lens, kidney and vertebral cartilage are all
dependent on inductive events (Bacon, 1945; Spemann, 1901;
Grobstein, 1955; Holtzer and Detwiler, 1953). The axial structures of the embryo, the neural tube and notochord, are
important elements in several of these inductive interactions.
The neural tube is involved in the induction of kidney and
vertebral cartilage while the notochord also plays a role in
vertebral cartilage induction, as well as in specification of cell
fate in the neural tube (Yamada et al., 1993).
The axial structures have also been implicated in the
induction of somitic myogenesis. The somites, which lie
directly adjacent to the neural tube and notochord, are the
source of all of the skeletal muscle precursor cells of the trunk
and limbs of the developing vertebrate embryo (Chevallier et
al., 1977). Extirpation of the neural tube and notochord results
in the absence of myotomal structures in the adjacent somites
Key words: mesoderm, neural tube, notochord, muscle, somite,
induction, myogenesis
1444 N. Buffinger and F. E. Stockdale
mice is confined to abnormalities of the skeleton (Rudnicki et
al., 1992; Braun et al., 1992), although at least one of the two
genes is required, as mice lacking both genes have a complete
absence of muscle formation (Rudnicki et al., 1993). The
myogenin gene also appears to be essential for normal myogenesis, as two groups have shown that transgenic mice lacking
the myogenin gene form muscle fibers, but exhibit gross
defects in muscle formation and die as embryos (Hasty et al.,
1993; Nabeshima et al., 1993). In contrast, it has been reported
that there is a population of myoblasts in the somites of early
mouse which, when cultured, differentiate even though they do
not express MyoD or myogenin proteins (Cusella-De Angelis
et al., 1992).
To better define the earliest events of myogenesis, we have
used an in vitro organ culture system to examine when somitic
cells are first specified to myogenesis and which tissues are
involved in inducing the myogenic phenotype in somitic
mesoderm. We found that myogenic cells first appear in
explants formed from somites from the 13-somite embryo
(Hamburger and Hamilton stage 11), and that the neural tube
or notochord are able to induce myogenesis in somites. We
have also found that the segmental plates and the three most
newly formed somites, which do not autonomously form
muscle fibers in vitro (stages 10-15), make an excellent test
system for further characterization of myogenic induction by
the neural tube or notochord.
MATERIALS AND METHODS
Nomenclature
Somites are numbered utilizing a system that refers to the age of a
somite rather than the position of a somite. Somites are numbered,
using roman numerals, from the caudal end of the embryo to the
rostral end, with the most newly formed somite designated as somite
I, regardless of the stage of the embryo. Somites are designated as two
types, depending on whether muscle fibers form within them when
explanted in vitro. Somites are designated as specified somites if when
incubated alone as explants, they form muscle fibers. When somites
form muscle fibers only when recombined with other tissues in vitro,
such as the notochord or neural tube, they are designated as unspecified somites.
Explant culture
Fertile chicken eggs (white Leghorn) were obtained from a local
supplier (Western Scientific, Sacramento, CA) and incubated at 38°C
for the time appropriate for the desired stage. Embryos were staged
according to Hamburger and Hamilton (1951).
Eggs were cracked into a dish containing Pannett and Compton
saline (Pannett and Compton, 1924) and embryos were freed from the
yolk, transferred to a dish containing Tyrode’s solution and staged.
Embryos of the appropriate stages were transferred to a second dish
and held in place with the aid of a glass cloning ring. Several drops
of a 2.5% solution of trypsin (Difco, 1:250) in saline G (Gibco) were
added to the surface of the embryo. After a brief period of digestion,
the trypsin was diluted by the addition of a large volume of Pannett
and Compton saline. Segmental plates, somites and portions of the
neural tube and notochord were dissected using finely sharpened
stainless steel insect pins. The dissected structures were transferred to
dishes containing medium (10% horse serum (Gibco), 5% chick
embryo extract, with penicillin and streptomycin in DMEM) until the
explant cultures could be formed. Explant cultures were made in 12well tissue culture plates (Falcon or Costar) which had been lined with
a 1:1 mixture of medium and 0.5% agarose. Several types of explant
cultures were performed: individual somites were recombined in
clusters of 10 somites (Fig. 1A); 4 somites were recombined with a
small segment of either notochord (Fig. 1B) or neural tube (Fig. 1C)
from the same staged embryo; 4 individual segmental plates were
cultured alone (Fig. 1D); or 2 segmental plates were recombined with
notochord (Fig. 1E) or neural tube (Fig. 1F) from the same stage
embryo. Unless otherwise stated, the notochord and neural tube used
in the explants was from the same stage embryo as the somites. The
segments of neural tube and notochord used in the explants were from
portions adjacent to the somites used in the explants. The individual
tissue elements in an explant fused together into a single structure
within 2 hours of being placed in culture. At this time sufficient
medium to cover the explant was added. Cultures were incubated at
37°C for 4 days, with feeding every day.
Immunohistochemistry
At the end of the culture period, the explants were transferred to small
glass vials for whole-mount immunohistochemical staining. The
explants were washed once with Pannett and Compton saline and
fixed for 30 minutes at room temperature in 70% ethanol. The
explants were washed twice with TBS, then incubated in a blocking
solution of 20% horse serum in TBS for 2 to 3 hours. Hybridoma
supernatants containing monoclonal antibodies (mAb) for fast (mAb
F59) and/or slow (mAb S58) myosin heavy chain (Crow and
Stockdale, 1986) were added directly to the blocking solution to a
final concentration of 10% each. Explants were incubated overnight
at 4°C in primary antibodies, then washed 5 to 6 times with TBS.
Explants were incubated with secondary antibodies (fluorescein-conjugated anti-mouse IgA and Texas red-conjugated anti-mouse IgG,
Vector Labs and Zymed), diluted 1:100 or 1:50 in 20% horse serum,
overnight at 4°C. Explants were washed 5 to 6 times with TBS, and
mounted under a coverslip with 2.5% DABCO in PBS/glycerol 1:9.
Explants were surveyed under epifluorescent optics, and images were
collected with a laser scanning confocal microscope.
RESULTS
Myogenic specification in paraxial mesoderm
Evidence of specification of mesodermal cells to a myogenic
fate was sought in explant cultures of early stage somites or
segmental plates. Explants of paraxial mesoderm tissues
(somites or segmental plate) were isolated from the early chick
embryo and cultured in isolation in an organ culture system
that preserved tissue integrity. Explants of somites (the source
of skeletal muscle precursor cells of the limb and trunk in the
embryo) and segmental plates (the source of somites) were
assayed (Fig. 1). In each experiment, somites or segmental
plates were dissected from 5-10 embryos of a specific stage
and were held as separate pools of segmental plates or
numbered somites in separate dishes containing culture
medium until use. Explants of 4 segmental plates or 10 somites
of the same numbered group were assembled in random orientation. It should be noted that individual somites or
segmental plates were dissected free from other somites before
recombination into explants. The explants were incubated on
agarose pads that did not permit outgrowth and were assayed
for myogenesis at day 4 by staining as whole mounts for
myosin heavy chains using monoclonal antibodies directed
against fast (mAb F59) and slow (mAb S58) isoforms of
myosin heavy chain (MyHC).
Myogenesis was first detected in explanted somites from
stage 11 embryos, whereas myogenesis did not occur in
explants of segmental plate from any aged embryo (Fig. 2). No
Myogenic induction in somites 1445
A
B
C
D
E
F
Fig. 1. Somite nomenclature and types of explant cultures. Somites
are numbered from the caudal to the rostral end of the embryo. Only
the 12 most caudal somites are used except for stage 10 which has
only 9-11 somites. (A) Somites alone; (D) segmental plate alone;
(B) somites plus notochord; (E) segmental plate plus notochord;
(C) somites plus neural tube; (B) somites plus notochord.
explant of somites from the stage 10 embryo, nor explants of
segmental plates from stage 10-15 embryos formed muscle
fibers when cultured alone. Only one segmental plate explant
of the 50 studied was positive for myosin heavy chain (MyHC)
at the end of the culture period; this one was most likely due
to the inadvertent inclusion of a small amount of the neural
tube with segmental plate during dissection. Because somites
form in a rostral-caudal fashion, there exists a gradient of ages
in the somites of an embryo. We divided somites into four
groups of three somites each and tested each group of three
somites from stage 10 (9-11 somite stage) through stage 15
(24-26 somite stage). The groups were chosen with regard to
the time elapsed since the somites had formed from the
segmental plate. We have used a numbering system for the
somites which reflects this, in which the most newly formed
somite at any given stage is designated somite I, the second
youngest somite is somite II, and so on, counting from the
caudal to the rostral end of the series of somites. The groups
were: somites I-III, somites IV-VI, somites VII-IX, and
somites X-XII (for stage 10 embryos, the latter group consisted
of somites X-XI only). Ten somites from a particular group
were recombined in explant culture. A total of 26 explants were
assayed from stage 10 embryos, none of which stained for
MyHC. Stage 11 (12-14 somite stage) was the first stage at
which somite cells specified to a myogenic fate were detected
in explant culture. Only explants formed from stage 11 somites
VII-IX and X-XII were positive for MyHC after the 4 day
culture period. By stage 12, there was an expansion in the
groups of somites which formed myogenic cells when placed
Cumulative percentage of
explants that express MyHC
0
100
Somites X-XII
Somites VII-IX
Somites IV-VI
Somites I-III
Segmental Plate
Stage
10 36
11
12
13
14
15
200
300
400
500
51
36
41
24
29
10
11
12
13
14
15
Alone
29
35
45
28
38
32
With
notochord
17
22
21
11
5
5
10
11
12
13
14
15
0
100
200
300
400
500
With
neural tube
Fig. 2. Myogenesis in explants of
paraxial mesoderm from stages 1015. Myogenesis was assayed by
whole-mount staining with mAb
F59, which stains fast MyHC(s). All
four groups of somites and the
segmental plate were assayed at
each stage for each type of explant.
Note that addition of notochord to
the explants induces myogenesis in
somites I-III from stages 11-15, and
in the three rostral groups of somites
from stage 10. Neural tube induces
myogenesis in all unspecified
somites as well as the segmental
plate. The number at the top of each
column indicates the total number of
explants assayed.
1446 N. Buffinger and F. E. Stockdale
in explant culture. At this stage, explants of somites IV-VI, as
bined with segments of the notochord. Also, explants of stage
well as VII-IX and X-XII, were now positive for MyHC when
10 somites I-III recombined with notochord did not produce
cultured alone (Fig. 3). The results of explant cultures for
muscle fibers. Nevertheless, notochord induced myogenesis in
stages 13, 14, and 15 were similar to those of stage 12 (Fig. 2,
all other groups of somites that did not exhibit myogenesis
Alone).
when cultured alone. These included somite groups VI-VI,
We refer to groups of somites as either ‘specified’ or
VII-IX, and X-XI from stage 10 and all unspecified somites
‘unspecified’. Those groups of somites that did not form
from stages 11-15.
myogenic cells when cultured alone (all somites from stage 10,
Somites formed in isolation from the neural tube can be
somites I-VI from stage 11, and somites I-III from stages 12induced to be myogenic by the notochord. To determine if mat15) are unspecified, and groups of somites that did form
uration of the segmental plate must occur in the presence of
myogenic cells (somites VII-XII from stage 11 and somites IVthe neural tube for the resulting somites to be responsive to a
XII from stage 12-15) are specified. It should be noted that
myogenic signal from the notochord, the segmental plate was
somites I-III at stages 10-15 did not autonomously form
allowed to form somites in isolation from the neural tube and
myogenic cells when recombined in explant culture.
Only one of 43 explants of somites I-III from stage 1015 embryos was found to be positive for MyHC at the
end of the culture period. Thus only after somites
segmented from the segmental plate and matured beyond
Somites
the third somite position did specification to myogenesis
X-XII
occur.
Myogenic induction by axial structures
To determine the role of tissue-tissue interactions in
myogenic specification, we made recombinant explant
cultures, recombining isolated segmental plates or
somites with isolated segments of neural tube or
notochord from the same stage embryo (Fig. 1). Cells in
explants containing neural tube in combination with
segmental plate or with any group of somites from all
stages tested were always myogenic. Somites from any
of the groups of unspecified somites at stage 11-15 and
including all somites at stage 10, formed muscle fibers
when recombined with neural tube. Segmental plate from
stage 10-15 also formed myogenic cells when recombined in explant culture with neural tube (Fig. 2, with
Neural Tube; Fig. 4 C,F). The segments of neural tube
used for the explants were isolated from levels adjacent
to the somites. These observations suggest that the neural
tube induces myogenic specification in segmental plate
and unspecified somites.
To determine if only paraxial mesoderm can be
specified to a myogenic fate by interacting with the neural
tube, we cultured recombined isolated lateral plate
mesoderm (including both somatopleure and splanchnopleure) with segments of the neural tube. Only three of
20 such explants were positive for MyHC (data not
shown). The staining for MyHC in these three positive
explants was confined to single small patches of cells,
which we believe are the result of contamination of the
lateral plate explants with small numbers of somitic cells.
This observation suggests that only the paraxial
mesoderm, rather than all mesoderm, is responsive to the
neural tube-derived myogenic signal.
We found that the notochord was also able to initiate
myogenesis when combined with unspecified somites
(Fig. 2, with Notochord; Fig. 4B). However, the
myogenic-inducing effect of the notochord differed from
that of the neural tube, in that the notochord did not
induce myogenesis in explants of segmental plates (Fig.
4E) or explants of one group of somites. Segmental plates
from all stages did not form muscle cells when recom-
A
B
C
D
E
F
G
H
I
J
Somites
VII-IX
Somites
IV-VI
Somites
I-III
Segmental
Plate
Fig. 3. Myogenic specification in explants of somites or segmental plates of
stage 12 embryos. Differential interference contrast (A,C,E,G,I)) or
confocal fluorescence (B,D,F,H,J)) microscopy of explants double stained
with monoclonal antibodies to myosin heavy chain. (I,J) Segmental plate;
(G,H) somites I-III; (E,F) somites IV-VI; (C,D) somites VII-IX;
(A,B) somites X-XII. Explants of specified somites (B,D,F) all stained with
an antibody to fast MyHC (mAb F59), as indicated by red fluorescence.
Note that staining for slow MyHC (mAb S58, green fluorescence) is found
principally in explants with a large number of fibers (B,D). Images were
collected on a laser scanning confocal microscope with a 20× objective.
Myogenic induction in somites 1447
With
Notochord
Alone
A
B
With
Neural Tube
C
A
B
C
D
D
EE
FF
Somites
I-III
Segmental
Plate
Fig. 4. Induction of myogenesis in unspecified somites by notochord and neural tube. Somites I-III (A) and the segmental plate (D) are
unspecified, as shown by the absence of staining for MyHC. The notochord induces myogenesis in somites I-III (B), but not the segmental plate
(E). The neural tube induces myogenesis in somites I-III (C) and the segmental plate (F). Explants were formed with the neural tube or
notochord flanked by 4 somites (2 on each side) and incubated for 4 days. The explants were whole mounted double stained with antibodies to
fast and slow MyHC (mAb F59 and mAb S58), and detected with Texas Red- and FITC-labeled secondary antibodies, respectively. Images
were collected on a laser scanning confocal microscope with a 20× objective.
notochord. In these experiments, portions of embryos containing the segmental plate and lateral plate mesoderm, and
the over- and underlying ectoderm and endoderm, but
lacking the neural tube and notochord, were isolated and
permitted to segment in vitro (Packard and Jacobson,
1976). The somites formed in these cultures were then
dissected free from the ectoderm, endoderm and lateral
plate mesenchyme and were recombined with segments of
notochord. These somites became myogenic when they
were recombined with the notochord in explant culture (7
of 9 explants). The remaining unsegmented segmental
plates were also recombined with the notochord and none
of these explants formed muscle fibers (n=5) (data not
shown). These observations suggest that association of the
Fig. 5. Muscle fiber diversity in an explant of unspecified
somites recombined with neural tube. Somites of group I-III
from a stage 12 embryo were recombined with neural tube in
organ culture for 4 days, then whole-mount stained with
antibodies to fast and slow MyHC (mAb F59 and mAb S58).
Fluorescein indicates slow MyHC and Texas Red indicates fast
MyHC(s). Note the presence of a fast fiber (carets), a fast/slow
fiber (arrows) and a slow fiber (arrowheads). This image was
collected on a laser scanning confocal microscope with a 60×
objective.
1448 N. Buffinger and F. E. Stockdale
segmental plate with neural tube during segmentation of
somites is not required for somites to acquire responsiveness
to myogenic specification by notochord, although we could not
determine if it is segmentation per se, or a maturation event,
that permits segmental plates that undergo segmentation in
vitro to respond to the notochord.
Muscle fiber diversity in somite and segmental plate
explants
It is proposed that diversity among muscle fibers is an early
event in myogenesis and this early diversity has its origins in
the diversity of myoblasts (Stockdale, 1992). It has been shown
in both birds and mammals that myoblasts within the early
embryo have different fates because one can isolate clonal populations of myoblasts that form colonies of muscle fibers in cell
culture that express fast or fast and slow isoforms of MyHC
(Miller and Stockdale, 1986a,b; Stockdale and Miller, 1987;
Vivarelli et al., 1988). To determine whether the muscle fibers
formed within explants (autonomously or in response to neural
tube or notochord) were of diverse types, specified somites
cultured alone, and unspecified somites cultured with neural
tube or notochord, were stained with antibodies to fast and
slow isoforms of MyHC.
The muscle fibers that formed autonomously in specified
somites in explant culture always contained fast isoforms of
MyHC, and most, but not all explants also contained muscle
fibers that expressed slow MyHC isoforms (Fig. 6, Alone).
Explants of specified somites from stage 12-15 embryos
expressed fast isoforms of MyHC in 100% of the explants and
slow MyHC in about 88% of the explants (42 of 48 expressed
slow MyHCs). However, explants of specified somites X-XII
from the earlier stage 11 embryo, and explants of specified
somites that formed very small, rather than large numbers of
fibers, generally did not express slow MyHC (Fig. 3F). It
appears that explants which contained many fibers were more
likely to contain fibers that express slow MyHC (Fig. 3B,D).
Thus when specified somites undergo myogenesis in organ
culture, they autonomously form fibers of more than one type.
When unspecified somites or segmental plates were recombined with segments of neural tube, they always expressed
slow and fast MyHCs (80 of 80 explants) (Fig. 5, 6). However,
when explants of unspecified somites were recombined with
notochord (somites I-III from stage 11-15), fewer muscle fibers
formed than with neural tube, and the percentage of explants
expressing slow MyHC was lower (24 of 36 explants, 66%).
Thus, in cultures of somites that have been specified in vivo,
or segmental plate or unspecified somites that become specified
in vitro in response to neural tube or notochord, both fast and
fast/slow fibers appear. The frequency of differing types of
fibers appears to correlate, in general, with the number of fibers
that form in the explants.
DISCUSSION
These studies show that specification to myogenesis of unspecified somites from the early chick embryo can be induced by
the neural tube or the notochord. Axial mesenchyme need not
be segmented into somites for it to become specified to a
myogenic fate by interaction with the neural tube, although
Cumulative percentage of Explants
that express slow MyHC
0
100 200
300
400
500
Somites X-XII
Somites VII-IX
Somites IV-VI
Somites I-III
Segmental Plate
Stage
10
11
12
13
14
15
Alone
10
11
12
13
14
15
With
notochord
10
11
12
13
14
15
With
neural tube
0
100 200
300
400
500
Fig. 6. Muscle fiber diversity in
explants of paraxial mesoderm.
Muscle fiber diversity is
indicated by expression of slow
MyHC, which is detected by
staining with mAb S58. Note that
in explants of somites alone,
muscle fiber diversity is found
principally in explants of the
more rostral somites — explants
which produce a large number of
fibers. Explants with notochord
have a higher incidence of
muscle fiber diversity. Explants
with the neural tube showed
muscle fiber diversity and larger
numbers of fibers in almost all
cases.
Myogenic induction in somites 1449
only after segmentation of the segmental plate does the
notochord induce myogenesis. The muscle fibers that form in
segmental plate or somite explants in response to the neural
tube and notochord express fast or fast and slow isoforms of
myosin heavy chain.
None of the somites of the stage 10 embryo will form muscle
fibers when placed in explant culture. Somites that form
muscle fibers autonomously in explant culture are first found
in the rostral somites at stage 11 of development. We designate
these somites as specified somites, and those that do not form
muscle fibers when placed in explant culture as unspecified
somites. None of the somites of the stage 10 embryo are
specified when cultured alone, but the rostral somites (somite
group VII-IX and X-XII) of stage 11 embryos are. At later
stages (stages 12-15), the pattern of specification expands, and
somite groups IV-VI become specified. Because the somites
are pooled into groups, we cannot categorize a specific somite
within a somite group which lies at the unspecified/specified
somite boundary (the boundary between somites I-III and
somites IV, V or VI at stages 12-15). Although as a group
somites IV-VI are specified, it could be that the only somite
VI or somites V and VI are specified, as might be inferred by
the small number of fibers observed in explants of this group.
The three most newly formed somites are not specified at any
stage tested (stage 10 through 15). We also found that the
segmental plate is not specified at any of the stages tested. Thus
from stage 11 through 15 only the segmental plate and the three
youngest somites are unspecified — all other somites
autonomously form muscle fibers in explant culture. Thus
beyond stage 11, there do not appear to be stage-specific events
that lead to specification of a somite, but only events associated with the ‘age’ of a somite. On the other hand, before stage
11 all somites tested were unspecified. Therefore, there are two
phases in myogenic specification of somites, that in which the
embryo matures past stage 10, and that in which the somite
matures past somite III. That we find unspecified somites at
any stage indicates that myogenesis is not a ‘default’ fate for
somitic cells as has been recently suggested (Pourquie et al.,
1993).
The results reported here are in good agreement with the
work of Kenny-Mobbs and Thorogood (1987) and Rong and
co-workers (1992). Kenny-Mobbs and Thorogood used several
explant culture systems to investigate the myogenic specification of the six brachial somites (somites number 15-21) in
stages 12, 15, and 18 embryos. They found that the brachial
somites at stage 12 (which would be somite group I-III) were
not myogenic when cultured alone, and that the brachial
somites from stage 15 (which would be included in somite
groups IV-VI, VII-IX, and X-XII) were myogenic. Rong and
co-workers (1992) used a monolayer tissue culture system to
investigate myogenic specification of somites. Their results for
somites from stage 10-13 embryos are virtually identical to
ours. However, they report slightly different findings on
somites from older embryos. While we do not see myogenesis
in somites I-III from stage 14 and 15 embryos, Rong et al.
(1992) find small numbers of cells which stain with a monoclonal antibody to an unidentified protein expressed in smooth
as well as striated muscle. The basis for this difference is
unclear, but may be due to the markers of myogenesis used in
the different experiments.
Explants of segmental plate and the first three somites of
early embryos thus become an excellent test system to
determine the importance of tissue interactions in specification
of somites to myogenesis. The neural tube and the notochord
can induce myogenesis in unspecified somites of the early
embryo. However, there are differences in the inductive effects
of the neural tube and notochord. The neural tube can induce
myogenesis in all unspecified somites and segmental plates
tested from stage 10-15 embryos. The notochord can induce
myogenesis in all somites tested from stage 11-15 embryos,
but only in the rostral somites (somite groups IV-VI, VII-IX
and X-XI) of stage 10 embryos and not in segmental plate of
any staged embryo. It is not clear if this indicates a quantitative (where the neural tube produces more of the myogenic
factor(s) than the notochord) or a qualitative (where the neural
tube and notochord produce different myogenic signals) difference in the myogenic signals from the neural tube and
notochord. The differences between the effects of the neural
tube and notochord highlight another distinction between the
results reported here and those reported by Rong and coworkers (1992). While Rong et al. (1992) find that the
notochord and neural tube are equivalent in initiating myogenesis, we find that the myogenic effects of the neural tube have
a broader potency compared to the effects of the notochord.
However, both the notochord and neural tube appear to have a
positive effect on somitic myogenesis.
Jessell’s laboratory (Basler et al., 1993; Yamada et al., 1993)
has described signals involved in specification of cell fate in
the developing neural tube that occur in the same developmental time frame as those described here. These signals, some
diffusible and others cell-cell contact-mediated, are produced
by both the notochord and neural tube. Specification of the
dorsal-ventral pattern within the neural tube may be analogous,
if not related to, the system of signals involved in the specification of myogenesis in somites.
It has been shown in the quail embryo that somites II and
III express mRNA for qmf1 (the quail homologue of MyoD)
as early as stage 12 (Pownall and Emerson, 1992). However,
none of the explants formed from somites I-III from stage 12
embryos, or any staged embryo tested, formed muscle fibers
when cultured alone. This suggests that expression of MyoD,
at least at the RNA level, is not sufficient for myogenic specification in vitro. It may be that the signals from the neural tube
and notochord sustain the continuous expression of MyoD in
developing somites, suggesting that the function of the
notochord and neural tube is to maintain myogenic regulatory
gene expression. If this is the case, then after their removal
from the embryo, somites I-III would stop expressing MyoD
resulting in their subsequent failure to undergo myogenesis.
This implies that either the MyoD mRNA is not translated in
the early somites or that the cross- and auto-activation by
MyoD shown to operate in tissue culture model systems
(Thayer et al., 1989) may not be operative in the animal, or
that certain thresholds of myogenic regulatory factor protein
are required to initiate cross and auto-activation (reviewed in
Weintraub, 1993). Another possibility is that continued
induction by the neural tube and notochord is required to either
turn off an inhibitory factor, such as helix-loop-helix protein
inhibitor Id (Benezra et al., 1990) or to turn on a second
required myogenic factor, for example myogenin (Hollenberg
et al., 1993). While the results described here can neither
support nor refute any of these possibilities, results from John
1450 N. Buffinger and F. E. Stockdale
Gurdon’s laboratory (Hopwood et al., 1989, 1991; Hopwood
and Gurdon, 1990) suggest that this phenomenon is not
restricted to avian embryos. In early Xenopus embryos, both
MyoD and myf5 are expressed even before somites form, and
forced expression of either or both of these genes in mesoderm
is not sufficient to induce myogenesis.
Our data suggest that myogenic specification in paraxial
mesoderm by neural tube does not require segmentation. Cells
from segmental plates undergo myogenesis when recombined
with the neural tube even though they have not segmented into
somites. It seems unlikely that segmentation is occurring in the
paraxial mesoderm of these explants, as segmental plates
isolated from ectoderm and endoderm do not segment under
our culture conditions (Buffinger, unpublished data). However,
it appears that either a maturational event or segmentation of
the segmental plate is required for specification of myogenesis
by the notochord. The notochord does not induce myogenesis
when recombined with segmental plates, but will induce myogenesis in most somites from stage 10 and all somites from
stage 11-15. In addition, somites formed when segmental
plates are incubated in isolation from the neural tube and
notochord are induced to form muscle by the notochord in
explant culture. These data also demonstrate a difference
between the sensitivity of segmental plate and somites to the
myogenic factor(s) coming from the neural tube and
notochord. It is currently unclear whether these differences
between somite and segmental plate response are due to differences in the myogenic factors produced by notochord and
neural tube, or due to differences in sensitivity of somites and
segmental plate to the same factor.
We found muscle fiber type diversity in explants of specified
somites as well as in somites combined with neural tube or
notochord. Muscle fibers that formed in explants were almost
always of different types — those that expressed only fast and
those that expressed both fast and slow MyHC isoforms. Using
explants of specified somites cultured alone, slow MyHC (as
indicated by staining by mAb S58) was detected as early as
stage 11, the stage at which myogenic specification is first
observed. We found that, when large number of fibers were
formed in explants of specified somites cultured alone, there
usually were muscle fibers that contained slow MyHCs. When
unspecified somites from stage 10 embryos were cultured with
notochord or neural tube, muscle fibers appeared that stained
with mAb S58, indicating that induction produces fibers of
diverse types. Even in explants of segmental plate from stages
as early as stage 10 recombined with neural tube, muscle fibers
were produced that expressed slow MyHC.
Models of myogenic induction
One model for the induction of myotomal muscle implicates
neural crest cells in myogenic induction (Christ et al., 1992).
These authors found that ablation of the neural tube (and thus
the neural crest) at early stages led to the absence of a myotome
in adjacent somites. They also noted that myotome formation
is preceded by invasion of neural crest cells (Bronner-Fraser,
1986) and that neural crest cells migrate through the cranial
half of the somite (Rickmann et al., 1985), which coincides
with the portion of the somite in which the myotome begins to
form (Christ et al., 1992).
More recently, Pourquie and colleagues (1993) have implicated the notochord in dorsal/ventral patterning of the somite.
Using segments of notochord grafted ectopically into early
chick embryos, they found that myogenesis in the myotome
was blocked, and that the entire somite became chondrogenic.
They found the same effects when ectopic floor plate was
grafted to the lateral or dorsal aspect of the neural tube.
Combining these observations with those of van Straaten and
Hekking (1991), which showed that ablation of the notochord
had no effect on the differentiation of the myotome, Pourquie
et al. (1993) concluded that ‘dorsal’ structures of the somite
(the myotome and dermatome) form by default, that is that the
notochord, by inducing the floor plate, controls normal morphogenesis in the somite by inducing structures that are
‘ventral’ in character (i.e. sclerotome).
Neither model is sufficient to explain the specification of
myogenesis in somites reported here. Our experiments show
that myogenesis can occur in somites which do not contain
neural crest cells. It has been demonstrated by several techniques that neural crest cells begin to emigrate from the neural
tube into somites that are rostral to the most newly formed
somites (i.e. they migrate in at somite IV) (Weston and Butler,
1966; Bronner-Fraser, 1986; Serbedzija et al., 1989). However,
we have shown that notochord can induce myogenesis in
explants of both somites I-III, as well as somites that were
allowed to form from the segmental plate in isolation from the
neural tube (and thus neural crest cells), demonstrating somitic
myogenesis in the absence of neural crest cells. This, however,
does not rule out a role for neural crest cells in the morphogenesis of the myotome.
The observations reported here support a positive role for
the notochord in initiating myogenesis in somites. Our findings
that neither somites I-III nor the segmental plate form muscle
fibers when cultured in isolation from the notochord, suggest
that the myotomal muscle does not form as a default fate for
cells of the somite. In addition, the notochord is able to induce
muscle fiber formation in these unspecified somites. These
findings indicate that the notochord is able to act on its own in
somite cell specification, and is not restricted solely to the
induction of somites cells to ‘ventral’ type fates.
We think that a complex pattern of signals is produced by
the neural tube and notochord which compete to specify cell
fates in the somites. We have formulated a model, based on
our findings as well as those of Pourquie et al. (1993) and van
Straaten and Hekking (1991). The results of Pourquie and colleagues show that induction of an additional floor plate leads
to the induction of a wholly cartilaginous somite, suggesting
that the floor plate produces (or causes to be produced) a
cartilage-inducing factor that can induce cartilage (sclerotome)
at the expense of muscle (myotome). Extirpation of the
notochord leads to abnormal somites in the area of the ablation.
The somites that form in the absence of the notochord and floor
plate, lack a sclerotome, but do form a myotome (van Straaten
and Hekking, 1991), suggesting that, while induction of the
sclerotome requires the presence of the notochord and/or floor
plate, myogenic induction of the myotome does not. However,
we have shown that both the notochord and neural tube are
able to induce myogenesis in explants of somites that would
not form muscle if cultured alone. These observations lead us
to propose a model in which factors that induce cartilage and
factors that induce myogenesis compete to specify the fates of
cells in the somites. Cells that lie near the notochord and neural
tube are induced to form cartilage by non-diffusible factors
Myogenic induction in somites 1451
from the notochord and factors that require close proximity for
transmission from the neural tube (Lash et al., 1957). Somitic
cells more distant from the notochord and ventral neural tube
are specified to myogenesis by diffusible factors produced by
the neural tube and notochord.
Our experimental results confirm and extend the previously
reported work of Kenny-Mobbs and Thorogood (1987) and
Rong et al. (1992). We have shown that either the neural tube
or notochord can induce myogenesis in somites that would not
otherwise undergo myogenesis when cultured alone. Both the
neural tube and notochord are also able to induce diverse types
of muscle fibers. Moreover, the neural tube is able to induce
myogenesis in paraxial mesoderm in the absence of segmentation of the paraxial mesoderm. These observations indicate
that myogenic-inducing signals are communicated from neural
tube and notochord to somite cells.
We thank Sandra Conlon for technical assistance, and Drs W.
Nikovits and J. DiMario for helpful discussions. This investigation
was supported by PHS training grant number 5T32CA09302 awarded
by the National Cancer Institute, DHHS and a research grant from the
DHHS.
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(Accepted 16 March 1994)