Download Combinatorial signals from the neural tube, floor plate and

Development 121, 651-660 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
651
Combinatorial signals from the neural tube, floor plate and notochord induce
myogenic bHLH gene expression in the somite
Andrea E. Münsterberg and Andrew B. Lassar
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA
02115, USA
SUMMARY
The neural tube, floor plate and notochord are axial tissues
in the vertebrate embryo which have been demonstrated to
play a role in somite morphogenesis. Using in vitro coculture of tissue explants, we have monitored inductive
interactions of these axial tissues with the adjacent somitic
mesoderm in chick embryos. We have found that signals
from the neural tube and floor plate/notochord are
necessary for expression of the myogenic bHLH regulators
MyoD, Myf5 and myogenin in the somite. Eventually
somitic expression of the myogenic bHLH genes is maintained in the absence of the axial tissues. In organ culture,
at early developmental stages (HH 11−), induction of myogenesis in the three most recently formed somites can be
mediated by the neural tube together with the floor
plate/notochord, while in more rostral somites (stages IVIX) the neural tube without the floor plate/notochord is sufficient. By recombining somites and neural tubes from
different axial levels of the embryo, we have found that a
second signal is necessary to promote competence of the
somite to respond to inducing signals from the neural tube.
Thus, we propose that at least two signals from axial tissues
work in combination to induce myogenic bHLH gene
expression; one signal derives from the floor
plate/notochord and the other signal derives from regions
of the neural tube other than the floor plate.
INTRODUCTION
opmental stages somitic cells are committed to particular cell
fates and differentiate irrespective of their location within the
embryo (Aoyama and Asamoto, 1988; Christ et al., 1992;
Ordahl and LeDouarin, 1992). Efforts to identify the source of
the cues that dictate somitic cell fate have focused attention on
the axial tissues of the vertebrate embryo: the developing
neural tube and the underlying notochord. It was noted that
somites that form in the absence of a neural tube do not
progress beyond a hollow epithelial sphere morphology reminiscent of immature somites, and therefore seem to be arrested
in their maturation (Packard and Jacobson, 1976). Recently, it
was demonstrated that excision of the neural tube and
notochord from early chick embryos results in the striking
absence of axial skeletal muscle (Christ et al., 1992; Rong et
al., 1992). In vitro experiments have provided evidence that the
neural tube directly promotes skeletal muscle differentiation
(Avery et al., 1956; Vivarelli and Cossu, 1986; Kenny-Mobbs
and Thorogood, 1987; Rong et al., 1992; Buffinger and
Stockdale, 1994; Stern and Hauschka, 1994). In these assays,
developmentally immature somites were explanted and
cultured under various conditions; muscle differentiation
markers (i.e. myoblast fusion or myosin heavy chain synthesis)
were not activated unless the somites were co-cultivated with
cells from the adjacent neural tube. In contrast, developmentally more mature somites could differentiate into muscle when
explanted and cultured in the absence of neural tube.
The initiation of the myogenic program is thought to be con-
In all vertebrate embryos, the paraxial mesoderm gives rise to
transient structures called somites. The somites are repeated
units which are progressively generated during development
along the anteroposterior axis on either side of the neural tube.
Somite morphogenesis is outlined in Fig. 1. Initially, somites
form as epithelial spheres from the unsegmented paraxial
mesoderm. [Throughout this paper, we refer to the most
recently formed somite as somite I, the next youngest somite
as somite II, etc., (as proposed in Ordahl, 1993).] As development proceeds, a columnar epithelial sheet develops in the
dorsal somite (dermomyotome), while cells in the ventral
somite loose their epithelial morphology and give rise to a
loosely connected mesenchyme (sclerotome). The sclerotome
gives rise to precursor cells of the ribs, vertebrae and intervertebral discs (discussed in Christ and Wilting, 1992). Subsequently, cells of the dermomyotome that lie adjacent to the
neural tube form an intermediate sheet of cells in the middle
of the somite (myotome) that gives rise to axial skeletal muscle
(i.e. vertebral and back muscle). Cells at the lateral edge of the
dermomyotome migrate to give rise to the body wall/limb musculature (reviewed in Wachtler and Christ, 1992). Cells from
the dorsalmost epithelial sheet (dermatome) give rise to
dermis.
At an early developmental stage, cell fate within the somite
is plastic and responsive to extrinsic cues, while at later devel-
Key words: neural tube, floor plate, notochord, myogenic bHLH,
somitogenesis
652
A. E. Münsterberg and A. B. Lassar
trolled by a set of basic-Helix-Loop-Helix (bHLH) transcription factors, which include MyoD, myogenin, Myf-5 and
MRF-4 (reviewed in Buckingham, 1992; Emerson, 1993;
Sassoon, 1993; Weintraub, 1993; Lassar and Münsterberg,
1994; Olson and Klein, 1994). Gene knockout by homologous
recombination in mice has demonstrated that either MyoD or
Myf-5 is necessary for the determination or survival of
myogenic precursor cells and that myogenin is necessary to
execute the differentiation program (Hasty et al., 1993;
Nabeshima et al., 1993; Rudnicki et al., 1993). Detailed in situ
hybridization studies have shown that the myogenic bHLH
genes are expressed early during embryogenesis (Hopwood et
al., 1989a; Sassoon et al., 1989; Bober et al., 1991; Charles de
la Brousse and Emerson, 1990; Hinterberger et al., 1991; Ott
et al., 1991; Pownall and Emerson, 1992) and, thus, they
provide molecular markers for myogenic precursor cells in
vivo. In different species, the order in which the myogenic
bHLH factors are expressed differs; however, in all species
examined, at least one member of this gene family is expressed
in the early somite prior to its morphological segregation into
dermatome, myotome and sclerotome (discussed in Buckingham, 1992). Interestingly, in both mouse and quail, myogenic
bHLH transcripts first accumulate in the medial portion of the
somite adjacent to the neural tube (Ott et al., 1991; Pownall
and Emerson, 1992, Barth and Ivarie, 1994).
In this report, we focus on the inductive interactions that are
necessary to activate high level expression of the myogenic
bHLH genes and induce skeletal muscle differentiation in
somites. To study these interactions, we have dissected and
cultured embryonic somites in the absence
or presence of various axial tissues (neural
tube, floor plate and notochord) and
monitored the expression of skeletal musclespecific genes by RT-PCR. This approach
has revealed that myogenesis of epithelial
XI
somites (stages I-III), isolated from stage
11− or younger chick embryos, requires
interaction with both neural tube and floor
plate in vitro. In contrast, myogenesis of
more rostral somites (stages IV-IX), from
V
similar stage embryos, requires interaction
only with the neural tube in the absence of
floor plate. This finding suggests that two III
II
signals are necessary for somite myogenesis
I
in vitro. We propose a model in which interaction with the floor plate/notochord
psm
mediates the competence of the somite to
respond to a muscle-promoting signal from
the neural tube.
MATERIALS AND METHODS
Chick embryo dissections and culture of
tissues
Fertilized chick eggs (Spafas) were incubated at
38.5°C in a humidified incubator (Petersime).
Embryos were staged according to Hamburger
and Hamilton, 1951, collected in silicon (Dow
Corning)-coated Petri dishes containing Tyrodes
buffer (10× stock: NaCl 80 g/l, KCl 2 g/l, CaCl2
2 g/l NaH2PO4 0.5 g/l, MgCl2 2 g/l, Glucose 10
g/l) and pinned down with their ventral side facing up using insect
pins. The endodermal epithelium was removed from the region to be
dissected, and the following tissues were explanted: somites alone;
somites with the adjacent neural tube, floor plate and notochord;
somites with the adjacent neural tube excluding floor plate; or somites
with floor plate and notochord (see figure legends for details and
stages of embryos). Following dissection with micro feather scalpels
(Oasis, CA) the tissues were transferred into Tyrodes buffer containing Dispase (Boehringer Mannheim, 1 mg/ml) and incubated for 5
minutes at room temperature to facilitate the removal of the ectodermal epithelia. After washing in medium, the tissues were cultured on
gelatin-coated 24-well dishes. Tissues that had been separated during
the dissection were aggregated on the tissue culture dish. The cultures
were maintained in α-MEM (Gibco) supplemented with 15% heatinactivated horse serum (Gibco), 2.5% chick embryo extract and 1%
penicillin/Streptomycin solution, in vitro for 5 days in 5% CO2 at
37°C.
Chick embryo extract was prepared as follows: 9- to 11-day chick
embryos were rinsed in PBS and then forced through a 60 ml syringe.
An equal volume of α-MEM was added and incubated for 1 hour on
ice. After removing the debris by centrifugation at 25,000
revs/minute, the supernatant was sterile filtered and kept frozen at
−20°C until used.
RNA isolation and reverse transcription
At the end of the culture period, medium was aspirated and RNA was
prepared from the explants using the method described by Chomczynski and Sacchi (1987). Tissues were lysed in 100 µl lysis buffer
(25 g guanidinium thiocyanate, 1.76 ml 0.75 M NaCitrate, pH 7, 2.64
ml 10% Sarkosyl, 38 µl β-mercaptoethanol, 29.3 ml H2O) and transferred to a 1.5 ml centrifuge tube, 10 µl 2 M sodium acetate, pH 4
and 100 µl water saturated phenol were added and mixed, then 20 µl
Nt
Nc
Dermatome
Myotome
Sclerotome
Dermomyotome
Sclerotome
Epithelial
Somite
5 day culture;
RT-PCR analysis
Fig. 1. Somite maturation and experimental scheme. The diagram on the left depicts the
axis of a stage 11+ chick embryo with rostral at the top and caudal at the bottom. The
roman numerals indicate the somite stage according to Ordahl, 1993; the last somite
formed from the presegmented mesoderm (=psm) being somite I. The diagrams in the
middle of the figure depict transverse sections through different axial levels of the embryo.
Somites are immature in caudal regions of the embryo, and are progressively more mature
in rostral regions. Somites I-III consist of a columnar epithelia and are referred to as
epithelial or immature somites within this text. Somite V contains two morphologically
distinct groups of cells; dermomyotome and sclerotome. Somite XI contains three
morphologically distinct cell types; dermatome, myotome and sclerotome. The
experimental design is outlined at the bottom right; unless otherwise stated somites I-III
were dissected and cultured for 5 days either alone or in the presence of axial tissues. The
induction of skeletal muscle-specific genes was examined by RT-PCR.
Axial tissues induce myogenic bHLH genes
chloroform/iso-amylalcohol (49:1) was added, mixed and incubated
on ice for 15 minutes. Following centrifugation at 4°C, the supernatant was precipitated with 240 µl ethanol in a new tube. Glycogen
was included as a carrier (1 µl RNAase-free glycogen, 20 µg/µl,
Boehringer Mannheim). After precipitation at −80°C for at least 1
hour, the RNA was pelleted by centrifugation at 4°C for 20 minutes,
washed with 80% ethanol, air dried and resuspended in 50 µl buffer
(40 mM Tris, ph 8.0; 10 mM NaCl; 6 mM MgCl2) containing 0.5 µl
DNAase (RNAase-free DNAase, Boehringer Mannheim, 40 U/ml)
and 0.1 µl RNAase inhibitor (USB, 120 U/µl). This was incubated for
1 hour at 37°C, then extracted with Tris-buffered phenol/chloroform
and precipitated by adding 1/10 volumes of 3 M sodium acetate, pH
4, and 2.5 volumes ethanol at −80°C. After centrifugation, the RNA
pellet was washed twice with 80% ethanol, air dried, dissolved in 10
µl DEPC-treated H2O and kept at −80°C.
Before cDNA synthesis, the RNA was incubated at 65°C for 3
minutes then immediately cooled on ice, 5 µl were added to 25 µl
Reverse Transcription cocktail, containing 1× transcription buffer (as
provided by BRL), 0.5 mM of each dNTP, 200 ng of a random
hexamer primer, 0.1 µl RNAase inhibitor, 3.3 mM DTT and 200 U
MoMuLV-Reverse Transcriptase (BRL) and incubated at 42°C for 1
hour. The cDNA was kept at −20°C until used for PCR analysis.
PCR amplification
1 µl of cDNA was used as template for PCR amplification. Each 50
µl reaction contained 50 mM KCl; 10 mM Tris/HCl, pH 7; 1.5 mM
MgCl2, 0.01% gelatin; 200 mM of each dNTP; 0.1 µCi α32P-dCTP
(3000 Ci/mMole); 500 ng of the appropriate primers and 1 U Taq
polymerase (Boehringer Mannheim). Myf-5 and SHH cDNAs were
amplified in the presence of 5% formamide. After an initial denaturation step of 93°C for 3 minutes, the reactions were cycled between
93°C (30 seconds), 60°C (30 sec) and 72°C (1 minute) in a MJ
Research thermocycler. The reaction products for MyoD, Myf-5,
Myogenin, Myosin Heavy Chain, Twist, choline acetyl-transferase
and Sonic hedgehog were amplified in 30 cycles while the product of
GAPDH was amplified in 23 cycles. It was determined that at 23
cycles the PCR for GAPDH was in the linear range of amplification.
We chose 30 cycles of amplification for the other cDNAs to detect
trace amounts of gene products which were induced during in vitro
culture. Depending on the amount of template in the culture, 30 cycles
amplification of the myogenic bHLH gene products were usually just
within the linear range. 5 µl of each PCR reaction was analyzed on a
6% polyacrylamide gel, which was dried and exposed to X-ray film
at −20°C for 16 hours. The following primers were used: MyoD (Lin
et al., 1989, nt 620-639) 5′-CGTGAGCAGGAGGATGCATA-3′, (nt
864-883)
5′-GGGACATGTGGAGTTGTCTG-3′;
Myogenin
(Fujisawa-Sehara et al., 1990, nt 435-453) 5′ AGCCTCAACCAGCAGGAG-3′,
(nt
694-713)
5′
TGCGCCAGCTCAGTTTTGGA-3′; Myf-5 (Saitoh et al., 1993, nt
283-302) 5′-CTGAGGAAGAGGAACACGTC-3′, (nt 453-434) 5′AGGTCTCGAATGCTTGGTTC-3′; Myosin Heavy Chain (Kavinsky
et al., 1983, nt 392-411) 5′-GATCCAGCTGAGCCATGCCA-3′, (nt
1008-989) 5′-GCTTCTGCTCAGCATCAACC-3′; Twist (Doug
Spicer, unpublished) 5′-TGTTATCTAGGGCTCTTGCCGG-3′, 5′GCAGAGCGACGAGCTGGACTC-3′; Sonic hedgehog (Riddle et
al.,
1993)
5′-TGGAAGATATGAAGGGAAGA-3′,
5′CTGAGTTTTCTGCTTTGACG-3′; GAPDH (Dugaiczyk et al.,
1983, nt 680-699) 5′-AGTCATCCCTGAGCTGAATG-3′, (nt 9901009) 5′-AGGATCAAGTCCACAACACG-3′. The expected product
sizes are as follows: MyoD, 280 nt; Myf-5, 174 nt; Myogenin, 284
nt; MHC, 616 nt; Twist, 150 nt; Sonic hedgehog, 395 nt; GAPDH,
330 nt. In all cases, PCR amplification was shown to be dependent on
reverse transcription of RNA templates and on the presence of both
forward and reverse primers in the PCR reaction. The identity of each
of the PCR products was confirmed by restriction enzyme digestion
and, in each case, the expected size fragments were obtained.
653
RESULTS
We have employed an in vitro assay to study cell specification
in the developing somite with the aim of characterizing
inducers of skeletal muscle differentiation. Several studies
have demonstrated that somitic cells express immunohistochemically detectable muscle actin and myosin in vitro if cocultured with neural tube (Kenny-Mobbs and Thorogood,
1987; Vivarelli and Cossu, 1986; Buffinger and Stockdale,
1994; Stern and Hauschka, 1994). However, it is not clear from
these studies whether signals from the neural tube are
necessary for the initial specification of myogenic cells or
rather for the differentiation of already determined muscle
progenitor cells. Because MyoD and Myf-5 are known to be
expressed in proliferating myoblasts prior to their differentiation into skeletal muscle (Tapscott et al., 1988; Braun et al.
1989), these myogenic transcription factors are the earliest
available markers of myogenic precursor cells. Therefore, we
monitored the expression of MyoD and Myf-5 to assay for the
presence of myogenic cells, and myogenin and myosin heavy
chain (MHC, embryonic fast form) to assay for muscle differentiation in chick somites co-cultivated with axial tissues
(neural tube, floor plate and notochord). If signals from axial
tissues were necessary to either induce myogenic precursor
cells or maintain their viability, then we expected that no
myogenic markers would be expressed in somites cultured in
the absence of axial tissues. If, however, axial tissues were
necessary solely for differentiation of myogenic precursors,
then somites cultured in the absence of axial tissues would
express MyoD and/or Myf-5, but fail to express muscle differentiation genes.
The neural tube/floor plate/notochord complex
promotes myogenic bHLH and myosin heavy chain
gene expression
The three most caudal somites (somites I-III) of a HamburgerHamilton (HH) stage 12+ chick embryo (containing 17
somites, Hamburger and Hamilton, 1951) were isolated and
cultured in the absence or presence of the adjacent neural
tube/floor plate/notochord complex. When these somites were
isolated and cultured for 5 days in the absence of axial tissues,
MyoD, Myf-5, myogenin, and MHC transcripts were not
detectable (Fig. 2, lane 1). In contrast, if somites I-III from the
contralateral side of the same embryo were cultured in the
presence of the adjacent neural tube/floor plate/notochord
complex, expression of both myogenic lineage markers (MyoD
and Myf-5) and differentiation markers (myogenin and MHC)
was observed (Fig. 2, lane 2). No skeletal muscle gene
expression was detected in the neural tube or the notochord
(data not shown), indicating that skeletal muscle formation in
these cultures required the presence of somitic tissue. Furthermore, co-culture of quail somites (I-III) with chick axial tissues
resulted in the induction of quail but not chick skeletal muscle
(data not shown), indicating that all skeletal muscle in these
cultures was somite-derived.
These findings indicate that signals from axial tissues are
necessary to promote the expression of both myogenic lineage
markers (MyoD and Myf-5) and differentiation markers
(myogenin and myosin heavy chain). Interestingly, expression
of MyoD can first be detected in somite II of stage 11-12 quail
embryos by in situ hybridization (Pownall and Emerson, 1992;
654
A. E. Münsterberg and A. B. Lassar
cultures, the gene Twist is expressed mainly in somitic
mesoderm and very weakly in notochord (data not shown).
Readily detectable levels of both Twist and GAPDH in somites
cultured alone (Fig. 2, lane 1) attest to the viability of somitic
cells cultured in the absence of axial tissues. However, this data
does not rule out the possibility that some somitic lineages may
selectively die in the absence of axial tissues. The increased
amount of Twist expression in somites cultivated with axial
tissues (Fig. 2, lane 2) could be due to either proliferation of
somitic cells or up-regulation of Twist expression in sclerotomal cells (Hopwood et at., 1989b; Wolf et al., 1991).
Signals from both the neural tube and the ventral
midline tissues (floor plate and notochord) are
necessary for somite myogenesis in vitro
To investigate which axial tissues are required for somitic
muscle differentiation, we dissected the neural tube away from
the floor plate/notochord complex. We subsequently co-cultivated immature somites of stage 10+/11 embryos with either
neural tube lacking floor plate, neural tube plus floor
Fig. 2. The neural tube/ floor plate/ notochord complex is necessary
for myogenic bHLH gene expression. Somites I-III of a stage 12+
(17 somite) chick embryo were dissected either alone or together
with the neighboring neural tube/floor plate/notochord complex as
diagrammed above each lane. The endodermal and ectodermal
epithelia were removed, and the tissues were cultured for 5 days in
the absence (lane 1) or the presence (lane 2) of the neural tube/floor
plate/notochord complex. At the end of the culture period, the tissues
were harvested and assayed for the expression levels of MyoD, Myf5, Myogenin, Myosin Heavy Chain (MHC), the bHLH gene Twist,
and GAPDH by Reverse Transcriptase-PCR analysis (RT-PCR). The
lower amounts of GAPDH in the culture containing somites only
(lane 1) reflect a lower amount of tissue in this culture, where neural
tube and notochord are absent. Neural tube and notochord do not
express detectable levels of MyoD, Myf-5, Myogenin or Myosin
Heavy Chain (data not shown). Similar results were obtained with 34
embryos ranging from stage 10 to stage 13.
Barth and Ivarie, 1994), and is detectable by RT-PCR at
extremely low levels in chick somites I-III which have not been
cultured in vitro (data not shown). The absence of detectable
MyoD in chick somites I-III cultured alone indicates that the
initial expression of MyoD in these somites is not maintained
in the absence of axial tissues (see also Bober et al. 1994).
Taken together, these results suggest that, in the presence of
axial tissues, the expression of MyoD is both maintained and
significantly up-regulated.
Previous studies have suggested that either the stability of
the somite, or the viability of somitic cells may require the
presence of adjacent axial tissues (Lipton and Jacobson, 1974;
Teillet and Le Douarin, 1983; Rong et al. 1992). To monitor
the viability of somites cultured in vitro, we assayed the
expression of GAPDH and Twist in somites cultured either
alone or in the presence of axial tissues. In these explant
Fig. 3. Signals from both dorsal neural tube and floor plate/
notochord are necessary for myogenesis of somites I-III in stage 11
embryos. Somites I-III, dissected from a stage 10+ embryo were
cultured in the presence of neural tube alone (lane 1), or in the
presence of neural tube, floor plate and notochord (lane 2). Somites
IV-VI, dissected from a stage 10+ embryo were cultured with floor
plate and notochord (lane 3). Somites I-III, dissected from a stage 11
embryo were cultured either in the presence of neural tube alone
(lane 4), or in the presence of neural tube and floor plate (lane 5).
The tissues present in each culture are diagrammed above each lane.
Note that in these and in subsequent dissections the neural tube was
transected along its dorsal-ventral axis such that ipsilateral and
contralateral somites were cultured with comparable neural tissue.
Expression levels of MyoD, Myf-5, Myosin Heavy Chain (MHC),
Sonic hedgehog (SHH) and GAPDH were assayed by RT-PCR
analysis. In these explant cultures, SHH is a specific marker for floor
plate and notochord. Similar results were obtained with eight (lanes 1
and 2), three (lane 3), or four independent embryos (lanes 4 and 5).
Axial tissues induce myogenic bHLH genes
plate/notochord, or floor plate/notochord in the absence of
neural tube (Fig. 3). As shown in the previous experiment,
robust activation of myogenic bHLH genes and skeletal muscle
differentiation was observed in somites that had been cultured
with neural tube plus floor plate and notochord (Fig. 3, lane 2).
If, however, the somites were cultured either with neural tube
alone (Fig. 3, lane 1), or with the floor plate/notochord
complex, alone (Fig. 3, lane 3), only trace levels of skeletal
muscle gene expression were observed. In these tissue
explants, Sonic hedgehog (SHH) is a specific marker for both
floor plate and notochord (Echelard et al., 1993; Krauss et al.,
1993; Riddle et al., 1993; Roelink et al., 1994). Thus SHH
expression was monitored to assay for the presence of these
ventral midline tissues in the various cultures. This experiment
indicates that, under the culture conditions employed, neither
the neural tube nor the ventral midline tissues (floor plate and
notochord) were sufficient to support myogenesis of immature
somites from stage 10+/11 embryos. However, the presence of
both neural tube and ventral midline tissues was able to
promote skeletal muscle differentiation in vitro of similar stage
somites.
The notochord has been demonstrated to induce the floor
plate in the ventral neural tube (van Straaten et al. 1988; Smith
and Schoenwolf, 1989; Placzek et al., 1990, 1993; Yamada et
al., 1991). Furthermore, the notochord shares several inducing
capacities with the floor plate (Yamada et al., 1991, 1993;
Pourquié et al., 1993; Placzek et al. 1993). Thus, we reasoned
that signals from the floor plate/notochord complex could be
emitted by one or both of these tissues. To clarify if the floor
655
plate itself was able to promote somitic myogenesis, somites
I-III from stage 11 embryos were cultured with neural tube,
after removal of the underlying notochord. After 5 days in
culture, this neural tube, which contained floor plate as shown
by high level expression of SHH, was capable of inducing
skeletal muscle in somitic tissue (Fig. 3, lane 5). Thus somites
I-III from a stage 11 embryo can be induced to form skeletal
muscle by a combination of signals derived from the neural
tube and floor plate.
The requirements for somite myogenesis in vitro
differ along the anteroposterior axis of the embryo
In vertebrate embryos, a developmental gradient exists, with
rostral regions of the axis being developmentally more
advanced than caudal regions. Skeletal muscle differentiation
of somites, independent of axial tissues, is observed first in
rostral regions of the embryo and later in caudal regions (Rong
et al., 1992; Borman et al., 1994; Buffinger and Stockdale,
1994; Stern and Hauschka, 1994). We therefore analyzed
whether the tissue requirements for somite myogenesis in vitro
differ in somites taken from different axial levels.
Paraxial mesoderm was explanted in blocks of three somites
from a stage 11− embryo (containing 12 somites), starting
caudally with the presegmented mesoderm and going successively more rostral. The somites were cultured either alone
(Fig. 4A, lanes 1-4), or in the presence of neural tube excluding
the floor plate (Fig. 4A, lanes 5-8). In the absence of neural
tube, the somites at all axial levels examined did not differentiate into skeletal muscle; MyoD was either undetectable or
Fig. 4. (A) Signals from neural tube
alone are sufficient to induce
myogenesis in rostral but not caudal
regions of a stage 11− embryo. Somites
were dissected along the rostrocaudal
axis of a stage 11− embryo (12 somite)
in blocks of 3 (presegmented
mesoderm, lanes 1 and 5; somites I-III,
lanes 2 and 6; somites IV-VI, lanes 3
and 7; somites VII-IX, lanes 4 and 8)
and somites were either cultured alone
(lanes 1-4) or in the presence of
adjacent neural tube lacking floor plate
(lanes 5-8). The tissues present in each
culture are diagrammed above each
lane. Expression levels of MyoD, Myf5, Myosin heavy chain (MHC), the
bHLH gene Twist, Sonic Hedgehog
(SHH) and GAPDH were assayed by
RT-PCR analysis. In these explant
cultures SHH is a specific marker for
floor plate and notochord. Similar
results were obtained in four
independent experiments. (B) Axial
tissues are necessary to initiate but not
to maintain the myogenic program
during somite maturation. Somites XIIXIV were dissected from a stage 16
(28 somite) embryo and either
harvested for RNA analysis without
culture (lane 1), or cultured in the absence (lane 2) or presence (lane 3) of the neural tube/floor plate/notochord complex. The tissues present in
each culture are diagrammed above each lane. Expression levels of MyoD, Myogenin, Myosin Heavy Chain (MHC) and GAPDH were assayed
by RT-PCR analysis.
656
A. E. Münsterberg and A. B. Lassar
expressed at very low levels, and Myf-5 and MHC transcripts
were not detected (Fig. 4A, lanes 1-4). In caudal regions of the
embryo, the presence of the neural tube alone was not sufficient for somitic myogenesis (Fig. 4A, lanes 5 and 6). In
contrast, in more rostral regions of the embryo the presence of
neural tube resulted in robust induction of skeletal muscle (Fig.
4A, lanes 7 and 8). The extremely low levels of Sonic
hedgehog expression in all the above explants suggest that
myogenesis in cultures containing neural tube from rostral
regions of the embryo was not due to the presence of floor plate
in these samples. These findings suggest that, as somites
mature, myogenesis of the somite (in vitro) becomes independent of the ventral midline tissues, but still requires interaction
with the neural tube.
As somites mature further, myogenesis in vitro becomes
independent of axial tissues (Kenny-Mobbs and Thorogood,
1987; Vivarelli and Cossu, 1986; Buffinger and Stockdale,
1994; Stern and Hauschka, 1994). We observed equivalent
levels of myogenesis in somites XII-XIV isolated from a stage
16 embryo cultured in the absence or presence of axial tissues
(Fig. 4B, lanes 2 and 3, respectively). Therefore, these somites
can operationally be defined as specified for myogenesis. Interestingly, the myogenic regulators MyoD and myogenin were
expressed in these somites at low levels at the time of dissection. Transcripts for MHC were below levels of detection (Fig.
4B, lane 1). Thus, somites that are specified for myogenesis
have received sufficient signals to allow up-regulation of
myogenic bHLH genes and the expression of muscle differentiation markers in the absence of further induction.
Taken together, these observations indicate that immature
paraxial mesoderm (i.e., presegmented plate and somites I-III
in stage 11− embryos) requires at least two signals for myogenesis. In vitro these signals can be provided by the floor plate
and more dorsal regions of the neural tube, respectively (and
perhaps by other tissues in the embryo). Slightly more mature
somites (somite IV-IX in stage 11− embryos) only require
signals from the neural tube for myogenesis in vitro. In still
more mature somites, myogenesis becomes independent of the
adjacent axial tissues (somites XII-XIV in stage 16 embryos).
The competence of somites to initiate myogenesis
differs along the axis of the embryo
The ability of neural tube lacking floor plate to induce myogenesis in rostral (somites IV-IX) but not caudal (presegmented mesoderm - somite III) paraxial mesoderm is consistent with a number of possibilities. Either somites IV-IX (from
a stage 11− embryo) have different intrinsic signaling requirements than more caudal paraxial mesoderm or they have progressed in their maturation such that they can respond to a
signal(s) from neural tube alone. In addition, it is conceivable
that neural tube adjacent to somites IV-IX has different
signaling properties than neural tube adjacent to more caudal
paraxial mesoderm, allowing the former to induce skeletal
muscle in the absence of floor plate and notochord. To address
whether it is the mesoderm or the neural tube that differs in its
capacity to support myogenesis in caudal versus rostral
paraxial mesoderm, we recombined paraxial mesoderm and
neural tube from these different axial levels.
Robust myogenesis was observed in somites VII-IX (taken
from a stage 10+ embryo) that had been cultured with either
adjacent neural tube or neural tube that had been adjacent to
presegmented mesoderm – somite I (Fig. 5, lanes 3 and 4). In
contrast, extremely low levels of myogenesis were observed in
caudal paraxial mesoderm (presegmented mesoderm – somite
I from the same embryo) that had been cultured with either
adjacent neural tube or neural tube from axial levels adjacent
to somites VII-IX (Fig. 5, lanes 1 and 2). The trace levels of
MyoD expression observed in these latter cultures can
probably be attributed to the presence of a few floor plate cells
in these cultures, as evidenced by the low level of Sonic
hedgehog expression. Importantly, paraxial mesoderm from
either of these axial levels (from this stage embryo) did not differentiate into skeletal muscle in the absence of axial tissues
(data not shown; Rong et al., 1992; Borman et. al., 1994;
Buffinger and Stockdale, 1994; Stern and Hauschka, 1994).
Thus somites VII-IX taken from a stage 10+ embryo were
competent to differentiate into skeletal muscle in vitro in
response to a signal from the neural tube, whereas more caudal
Fig. 5. The competence of somites to initiate myogenesis in response
to a neural tube signal changes along the axis of a stage 10+ embryo.
Presegmented mesoderm and somites I were dissected from a stage
10+ embryo and either cultured with the adjacent neural tube (lane 1)
or with more rostral neural tube (adjacent to somites VII-IX) from
the same embryo (lane 2). Somites VII-IX were dissected from the
same embryo and either cultured with the adjacent neural tube (lane
4) or with neural tube from more caudal regions (adjacent to
presegmented mesoderm-somite I, lane 3). The origin of somitic
mesoderm and neural tubes in each co-culture is diagrammed above
each lane (tissues from caudal axial levels are depicted as white,
tissues from rostral axial levels are depicted as stippled). Expression
levels of MyoD, Myosin Heavy Chain (MHC), Sonic hedgehog
(SHH), and GAPDH were assayed by RT-PCR analysis. In these
explant cultures, Sonic hedgehog (SHH) is a marker for both
notochord and floor plate. The absence of SHH expression in lanes 3
and 4 indicates that these explants contained no floor plate cells. The
low amount of SHH PCR product in lanes 1 and 2 indicates the
presence of a few floor plate cells in these explants. Identical results
were obtained in four independent experiments (lanes 1 and 4) or in
duplicate (lanes 2 and 3).
Axial tissues induce myogenic bHLH genes
paraxial mesoderm lacked this ability; neural tube from both
axial levels could provide this signal.
A
Series
Signaling
NT
3
657
M
DISCUSSION
In this study, we demonstrate that signals from both the neural
tube and floor plate together support the expression of the
myogenic bHLH regulators in the somite in vitro. This finding
is consistent with a requirement for signals from axial tissues
in either mediating the initial induction of somitic cells to
become myoblasts or in supporting the proliferation and
survival of such determined cells. Prior embryological manipulations have shown that differentiation of the axial musculature in the chicken embryo requires proximity between the
somite and the neural tube (Christ et al., 1992; Rong et al.,
1992). Our work has confirmed and extended this analysis to
demonstrate that stable expression of the myogenic bHLH regulatory family of transcription factors requires at least two
signals. In our in vitro co-culture system, these signals can be
provided by the floor plate and regions of the neural tube dorsal
to the floor plate. We provide evidence to suggest that the competence of somites to respond to the signal from the neural tube
(in the absence of floor plate) changes as somites mature.
Therefore, we propose that signals from neural tube and floor
plate act directly upon the somite and together induce skeletal
muscle formation. Previous studies have documented the
importance of notochord and floor plate in the induction of
sclerotome (Grobstein and Holtzer 1955; Lash et al. 1957; Hall
1977, Brand-Saberi et al. 1993; Dietrich et al., 1993; Koseki
et al. 1993; Pourquié et al., 1993). This study, together with
other recent reports (Rong et al. 1993; Buffinger and Stockdale
1994; Stern and Hauschka 1994), suggests that ventral midline
tissues (floor plate and notochord) may also play a role in the
formation of somitic skeletal muscle.
Parallel signals from the neural tube and floor
plate/notochord synergistically activate the
myogenic program
Skeletal myogenesis in either presegmented plate or somites IIII (isolated from a stage 11− or younger chick embryo)
required co-cultivation of the paraxial mesoderm with both
neural tube dorsal to the floor plate and the floor plate itself.
The requirement for two different regions of the neural tube
for somitic myogenesis in vitro is consistent with three possible
signaling schemes. Either signals from the floor plate induce a
secondary signal in more dorsal regions of the neural tube,
which in turn activates somite myogenesis (Series Signaling;
see Fig. 6A) or signals from the floor plate act together with
signals from the neural tube to synergistically activate somite
myogenesis (Parallel Signaling; see Fig. 6B). In the former, the
signals act in a dependent sequential cascade; in the latter the
signals are unlinked and work independently. Lastly, a combination of the above two models is possible; in this case, signals
from the floor plate induce a secondary signal in the neural
tube, which then acts in parallel with other signals from the
floor plate to induce somite myogenesis (Series + Parallel
Signaling; see Fig. 6C).
In contrast to presegmented mesoderm and somites I-III from
stage 11− embryos, which require neural tube plus ventral midline tissues to form skeletal muscle, somites IV-IX from the
2
1
B
Parallel
Signaling
NT
1
C
Series + Parallel
Signaling
NT
Fp
Nc
3
Fp
2
Nc
3
2
1
M
Fp
M
4
Nc
Fig. 6. Multiple signals are involved in somitic myogenesis. (A)
Series signaling. Signal 1 from the notochord induces floor plate in
the ventral neural tube. Signal 2 from the floor plate travels within
the neural tube to affect the production of Signal 3. Signal 3 from the
neural tube induces skeletal muscle formation in the adjacent somite.
(B) Parallel signaling. Signal 1 from the notochord induces floor
plate in the ventral neural tube. Signal 2 from the floor plate travels
directly to the somite. Signal 3 from the neural tube travels directly
to the somite. The convergence of signals 2 and 3 within the somite
induces skeletal muscle formation. (C) Series + parallel signaling.
Signal 1 from the notochord induces floor plate in the ventral neural
tube. Signal 2 from the floor plate travels within the neural tube to
affect the production of signal 3. Signal 4 from the floor plate travels
directly to the somite. The convergence of signals 3 and 4 within the
somite induces skeletal muscle formation. Our experiments do not
rule out the possibility that signals 2 and 4 could also be emitted
directly by the notochord.
same stage embryos form skeletal muscle if co-cultured solely
with neural tube lacking floor plate. We analyzed whether the
paraxial mesoderm or the neural tube differs in its capacity to
support myogenesis at different axial levels (Fig. 5). Our data
suggest that the competence of paraxial mesoderm to respond to
a signal from the neural tube is different at varying axial levels.
It is unclear what signals in the embryo have promoted the com-
658
A. E. Münsterberg and A. B. Lassar
petence of somites IV-IX to respond to a signal from the neural
tube. However, we have found that the floor plate/notochord
can provide this signal to somites I-III from a similar stage
embryo (Fig. 3). Taken together, these findings imply that signals from both the neural tube and ventral midline tissues are
required for somitic myogenesis. In addition, the data are most
consistent with a parallel signaling scenario (see Fig. 6B) in
which a signal from the floor plate and notochord (see below) is
transmitted directly to the somite; this signal renders these cells
responsive to muscle-inducing signals from the neural tube.
Therefore, we propose that a combination of independent signals from the neural tube and the floor plate/notochord act
directly on the paraxial mesoderm to induce myogenesis. As a
result of these interactions, the myogenic bHLH genes are
stably activated. Stable activation of myogenic bHLH regulators correlates with somitic muscle differentiation that is independent of the axial tissues, suggesting that inductive signals
from axial tissues are necessary for the initiation but not the
maintenance of somite myogenesis.
Is the notochord involved in skeletal myogenesis?
Two groups have recently shown that notochord can induce a
small but detectable level of myogenesis in somites II-III, but
not in presegmented mesoderm or somite I from stage 11-13
chick embryos (Buffinger and Stockdale, 1994; Stern and
Hauschka, 1994). These observations, together with previous
observations (Rong et al. 1992), indicate that notochord can
induce skeletal muscle in some paraxial mesoderm (somite II
or older). This is in contrast to our own finding where somites
I-III of stage 11 or younger embryos required two signals (from
the neural tube and the ventral midline tissues) for robust myogenesis (Fig. 3). It is not clear whether this difference is due
to different culture conditions, or whether a small amount of
notochord-dependent myogenesis was below levels of
detection by RT-PCR. However, the inability of notochord to
induce myogenesis in presegmented plate (Buffinger and
Stockdale, 1994; Stern and Hauschka, 1994) is consistent with
our findings that two signals are required for somite myogenesis, and suggests that induction of muscle by notochord may
only occur in somites that have received another signal.
In our in vitro culture system, somitic skeletal muscle is
induced by factors present in the neural tube and the floor plate.
It seems that notochord is not required (Fig. 3). However,
numerous studies have documented that the differentiation of
floor plate depends on signals from the notochord (van Straaten
et al., 1989; Placzek et al., 1990, 1993; Yamada et al., 1991,
1993). Therefore, the apparent requirement of floor plate signals
for somitic muscle formation in vitro implies that notochord may
also be involved in myotome formation in vivo. Furthermore,
the documented muscle-inducing capacity of either floor plate
(this study) or notochord (Rong et al., 1992: Buffinger and
Stockdale, 1994; Stern and Hauschka, 1994) suggest that both
these tissues may play a direct role in promoting somite myogenesis in the embryo. However, it is not clear whether muscleinducing signals from either one or both of these ventral midline
tissues are necessary for somite myogenesis. In light of the many
signaling properties common to both notochord and floor plate
(Yamada et al., 1991, 1993; Echelard et al., 1993; Pourquié et
al., 1993; Placzek et al. 1993; Riddle et al., 1993; Roelink et al.,
1994), it seems plausible that the muscle-inducing activities of
these tissues may be similar, if not identical.
A prediction of all the models schematized in Fig. 6 is that
removal of notochord, prior to induction of floor plate, should
inhibit the formation of myotome in vivo. Whereas it was
demonstrated that sclerotome formation in the somite is
disrupted following notochord removal (Dietrich et al., 1993;
Koseki et al., 1993; Goulding et al., 1994), myotome formation
was reported to occur in somites that had fused beneath the
remaining neural tube (van Straaten and Hekking 1991;
Goulding et al., 1994). Interestingly, Goulding and colleagues
noted that MyoD expression did not occur in somites that were
positioned at a distance from the residual notochord, suggesting that signals from the notochord may be necessary for
MyoD induction in vivo (Goulding et al., 1994). Myotome
formation in vivo, in the absence of notochord, is consistent
with a number of explanations: (1) possible signaling by the
notochord to paraxial mesoderm prior to removal of the
notochord, (2) differentiation of floor plate in the absence of
underlying notochord (described in Artinger and BronnerFraser, 1993) possibly mediated by homeogenetic induction of
floor plate within the plane of the neural tube (Placzek et al.,
1993), (3) signaling of floor plate and notochord to axial levels
that are anterior and posterior to the transected ends of the
notochord, and (4) another signaling source in the embryo that
shares the muscle promoting properties of the floor
plate/notochord complex such that these signals would be
redundant in the embryo but unique in explant cultures containing only somite and axial tissues.
The relative position of somite cells with respect to
neural tube, floor plate and notochord may
determine cell fate
There is a wealth of experimental evidence indicating that sclerotome formation in the somite is induced by signals from the
axial tissues (Grobstein and Holtzer, 1955; Lash et al., 1957;
Hall, 1977, Brand-Saberi et al., 1993). These classical studies,
together with more recent grafting experiments in vivo
(Pourquié et al., 1993), and the analysis of gene expression
patterns in mutant mouse embryos (Dietrich et al., 1993)
surgically manipulated chick embryos (Brand-Saberi et al.,
1993; Koseki et al., 1993) and explant culture (Fan and
Tessier-Lavigne, 1994), have implied a role for the floor plate
and notochord in sclerotome formation. Our own experiments
indicate that specification of somitic cells into myotome results
from the combined action of at least two signals; from the floor
plate/notochord and regions dorsal to the floor plate in the
neural tube. Dermatome formation may either result as a
default pathway for somitic cells in the absence of signals from
axial tissues, or may be actively induced by interaction with
the overlying epidermis. Thus specification of somitic cell fate
into either dermatome, myotome or sclerotome may in part be
dictated by the position of somitic cells relative to the neural
tube, floor plate and notochord (see also Pourquié et al., 1993;
Fan and Tessier-Lavigne, 1994).
Implantation of either an ectopic floor plate or notochord
between the neural tube and the paraxial mesoderm induces
ectopic sclerotome while inhibiting the formation of myotome
(Pourquié et al. 1993). These authors suggested that signals
from the floor plate and notochord prevented myotome
formation by inducing ventral cell types (i.e. sclerotome), and
therefore it seems paradoxical that the ventral midline tissues
would play a positive role in myotome differentiation (this
Axial tissues induce myogenic bHLH genes
report, Buffinger and Stockdale, 1994; Stern and Hauschka,
1994). A possible resolution to this paradox lies in the fact that
the ectopic notochord/floor plate in these experiments was
implanted between the neural tube and the paraxial mesoderm
and therefore may have interfered with signaling between these
two tissues.
It is not clear whether different signaling molecules or a
gradient of the same single signaling molecule emanating from
the floor plate/ notochord complex promotes formation of the
myotome versus the sclerotome. Furthermore, it is possible
that myotome formation requires cell-cell signaling within the
somite, for example from induced sclerotomal cells. In
summary, it seems well established that somitic cell fate is
dictated by cues from the neural tube, floor plate and
notochord. However, clarification of whether these signals are
expressed in an interdependent manner in the axial tissues,
and/or whether they induce a further cascade of signals in
somitic tissue must await characterization of the molecules
involved.
We thank Hazel Sive, Cliff Tabin, Tom Schultheiss and Mark
Mercola for their comments on previous versions of this manuscript;
the anonymous reviewers of this manuscript for their insightful suggestions; Tom Jessell, Sam Pfaff, Toshiya Yamada and Doug Spicer
for sharing reagents and unpublished sequence information with us,
and Nicholas Buffinger, Charlie Emerson, Steve Hauschka, Howard
Stern and Frank Stockdale for sharing unpublished information with
us. This work was supported by grants to A. B. L. from the National
Science Foundation, the Lucille P. Markey Charitable Trust, the
Muscular Dystrophy Association, The Council for Tobacco Research
USA, and a Basil O’Connor Award no. 1 FY93-0848 from the March
of Dimes Birth Defects Foundation. A. B. L. was a Lucille P. Markey
Scholar. A. E. M. was supported by fellowships from the Human
Frontiers Science Program Organization and the Muscular Dystrophy
Association.
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(Accepted 6 December 1994)