Download The dorsal neural tube organizes the dermamyotome

Development 122, 231-241 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
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231
The dorsal neural tube organizes the dermamyotome and induces axial
myocytes in the avian embryo
Martha S. Spence1, Joseph Yip2 and Carol A. Erickson1,*
1Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
2Department of Neurobiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261,
USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
Somites, like all axial structures, display dorsoventral
polarity. The dorsal portion of the somite forms the dermamyotome, which gives rise to the dermis and axial musculature, whereas the ventromedial somite disperses to
generate the sclerotome, which later comprises the
vertebrae and intervertebral discs. Although the neural
tube and notochord are known to regulate some aspects of
this dorsoventral pattern, the precise tissues that initially
specify the dermamyotome, and later the myotome from it,
have been controversial. Indeed, dorsal and ventral neural
tube, notochord, ectoderm and neural crest cells have all
been proposed to influence dermamyotome formation or to
regulate myocyte differentiation. In this report we describe
a series of experimental manipulations in the chick embryo
to show that dermamyotome formation is regulated by
interactions with the dorsal neural tube. First, we demonstrate that when a neural tube is rotated 180° around its
dorsoventral axis, a secondary dermamyotome is induced
from what would normally have developed as sclerotome.
Second, if we ablate the dorsal neural tube, dermamyotomes are absent in the majority of embryos. Third, if we
graft pieces of dorsal neural tube into a ventral position
between the notochord and ventral somite, a dermamyotome develops from the sclerotome that is proximate
to the graft, and myocytes differentiate. In addition, we also
show that myogenesis can be regulated by the dorsal neural
tube because when pieces of dorsal neural tube and unsegmented paraxial mesoderm are combined in tissue culture,
myocytes differentiate, whereas mesoderm cultures alone
do not produce myocytes autonomously. In all of the experimental perturbations in vivo, the dorsal neural tube
induced dorsal structures from the mesoderm in the
presence of notochord and floorplate, which have been
reported previously to induce sclerotome. Thus, we have
demonstrated that in the context of the embryonic environment, a dorsalizing signal from the dorsal neural tube
can compete with the diffusible ventralizing signal from the
notochord.
In contrast to dorsal neural tube, pieces of ventral neural
tube, dorsal ectoderm or neural crest cells, all of which
have been postulated to control dermamyotome formation
or to induce myogenesis, either fail to do so or provoke only
minimal inductive responses in any of our assays. However,
complicating the issue, we find consistent with previous
studies that following ablation of the entire neural tube,
dermamyotome formation still proceeds adjacent to the
dorsal ectoderm. Together these results suggest that,
although dorsal ectoderm may be less potent than the
dorsal neural tube in inducing dermamyotome, it does
nonetheless possess some dermamyotomal-inducing
activity.
Based on our data and that of others, we propose a model
for somite dorsoventral patterning in which competing diffusible signals from the dorsal neural tube and from the
notochord/floorplate specify dermamyotome and sclerotome, respectively. In our model, the positioning of the
dermamyotome dorsally is due to the absence or reduced
levels of the notochord-derived ventralizing signals, as well
as to the presence of dominant dorsalizing signals. These
dorsal signals are possibly localized and amplified by
binding to the basal lamina of the ectoderm, where they can
signal the underlying somite, and may also be produced by
the ectoderm as well.
INTRODUCTION
lack obvious morphological polarity (Bellairs, 1963; Christ et
al., 1972; Mestres and Hinrichsen, 1976). Within a few hours,
however, morphogenetic movements generate regional histological differences along the dorsal-ventral axis of each somite
(Mestres and Hinrichsen, 1976; reviewed by Stern et al., 1988;
Ordahl, 1993). Cells comprising the ventromedial portion of
the somite undergo an epithelial-to-mesenchymal transforma-
The paraxial mesoderm first arises by ingressing through the
primitive streak to form a uniform band of tissue that flanks
the neural tube. Although initially unsegmented, the paraxial
mesoderm soon becomes partitioned in an anterior-to-posterior
wave to form the somites, which are epithelial in nature and
Key words: dermamyotome, myogenesis, neural tube, somites, avian
embryo
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M. S. Spence, J. Yip and C. A. Erickson
tion to form the sclerotome, which lies lateral to the neural tube
and notochord (Hay, 1968). In contrast, cells in the dorsal
portion of the somite maintain an epithelial organization and
constitute the transient dermamyotome. Cells at the cranial
edge of the dermamyotome later give rise to the more ventrally
situated myotome, while the remaining dermamyotome cells
become dermatome (Kaehn et al., 1988; Tosney et al., 1994).
Although the derivatives of the somites (i.e. dermatome,
myotome and sclerotome) are positioned with obvious dorsalventral polarity, such regionalization is not intrinsic to either
the unsegmented paraxial mesoderm or the most recently
formed somites. This absence of regional specification at early
stages is revealed in experiments in which 180° rotation of the
paraxial mesoderm or early somites has no effect on the subsequent dorsal-ventral patterning (Aoyama and Asamoto,
1988). Similarly, when the dorsal half of an early somite is
replaced with a ventral half, the grafted ventral tissue
undergoes normal myogenesis (Christ et al., 1992). These
studies suggest that dorsal-ventral pattern within each somite
is established through interactions with surrounding axial
structures during and subsequent to segmentation.
Several axial tissues could potentially contribute to somite
patterning. For example, the neural tube and notochord are
known to play a role in the development of the sclerotome. Cocultures of somite with neural tube and notochord produce an
abundance of cartilage, a sclerotome derivative (Lash et al.,
1957; Lash, 1968; Kosher and Lash, 1975). Moreover, when a
notochord is grafted ectopically to dorsal regions of the somite,
the sclerotome expands at the expense of the dermamyotome
(Pourquié et al., 1993; Bober et al., 1994; Goulding et al.,
1994). Finally, early removal of the notochord enhances the
development of the dermamyotome and results in the absence
of sclerotome (van Stratten and Hekking, 1991; Rong et al.,
1992; Goulding et al., 1993, 1994). Recent studies reveal that
Sonic hedgehog is produced by the notochord and floor plate
at the correct time to induce sclerotome (Johnson et al., 1994)
and, furthermore, that heterologous cells expressing Sonic
hedgehog can induce sclerotome formation, as assessed using
molecular markers, both in culture assays (Fan and TessierLavigne, 1994) and in vivo (Johnson et al., 1994).
Unlike the sclerotome, candidate molecules that regulate the
formation of dermamyotome or the myotome from it have not
been identified. Indeed, our understanding of how these tissues
develop is further complicated by persistent controversy concerning which neighboring tissues exert inductive influences
on the paraxial mesoderm. The neural tube has been demonstrated repeatedly to play a role in myogenesis (e.g. Watterson
et al., 1954; Vivarelli and Cossu, 1986; Kenny-Mobbs and
Thorogood, 1987; Christ et al., 1992; Rong et al., 1992;
Borman and Yorde, 1994; Buffinger and Stockdale, 1994;
Stern and Hauschka, 1995; Münsterberg and Lassar, 1995),
and there is some evidence that a signal is produced specifically by the dorsal neural tube (Fan and Tessier-Lavigne,
1994). Yet several studies also indicate that dermamyotomeand myocyte-inducing molecules are produced by other
tissues, including the surface ectoderm (Christ et al., 1972;
Rong et al., 1992; Fan and Tessier-Lavigne, 1994; Kuratani et
al., 1994), the notochord or ventral neural tube (Buffinger and
Stockdale, 1994; Stern and Hauschka, 1995; Münsterberg and
Lassar, 1995) and neural crest cells (Christ et al., 1992;
Borman and Yorde, 1994). Finally, some evidence suggests
that dorsal fate is a default pathway in the absence of a ventralizing signal from the neural tube and notochord (Pourquié
et al., 1993), and therefore that dorsal-specifying signals may
not be necessary at all.
To characterize the cellular interactions that signal
formation of the dermamyotome as well as specification of
myocytes, we have employed the techniques of experimental
embryology. Because most of our experiments attempt to
identify and characterize various signaling centers in the
context of the embryo, the environmental conditions and
spatial organization of inducing and responding tissues are
close to normal, in contrast with previously employed tissue
culture assays where these signals may be lost or altered.
Results from a series of manipulations including dorsoventral
rotation of the neural tube, extirpation of the dorsal neural tube
and grafting of putative inducing tissues ventrally adjacent to
the sclerotome together support a model in which the dorsal
neural tube induces the formation of the dermamyotome. In
contrast, surface ectoderm, ventral neural tube and neural crest
cells appear to have only modest, direct signaling roles, or do
not participate in the induction of the dermamyotome. Furthermore, our study suggests that the signal emanating from
the dorsal neural tube is a diffusible molecule that can compete,
in the embryo, with the ventralizing signal from the notochord.
MATERIALS AND METHODS
Embryo culture
White Leghorn chicken embryos were used for all in vivo manipulations (Avian Sciences Department, University of California-Davis or
Western Scientific, Sacramento, CA). Some embryos were treated and
maintained in ovo. On the day before the operation, a small hole was
cut into the egg shell above the embryo, the window sealed with cellophane tape and the egg returned to the incubator. Just prior to any
experimental manipulation, a drop of 0.02% neutral red in Locke’s
saline was applied to the vitelline membrane to stain the embryo and
reveal the vitelline membrane, which was subsequently slit with a
tungsten needle to access the embryo. After surgery, the eggs were
resealed and incubated at 38°C and 70% humidity until the embryos
were fixed.
Alternatively, some embryos were cultured in vitro after surgery.
Embryos were cut from the blastoderm and transferred to a 100 mm
Petri dish coated with a layer of 2% agar. The vitelline membrane was
teased away, the experimental manipulation performed (see below)
and the embryo cultured according to previously published methods
(Erickson and Goins, 1995). Briefly, the embryo was oriented ventral
side up, the saline removed so that the embryo was flat and stretched,
and a filter paper ring (Whatman #1) centered over the embryo to keep
the embryo fully spread. A Nuclepore filter (8 µm pore size, Costar
Corp., Pleasanton, CA) was then layered over the embryo and ring,
which served to support the embryo during later culture. The whole
assemblage was then turned so that the embryo was dorsal side up
and suspended over a well in an organ culture dish (Falcon #3037)
containing 1.5 ml Liebowitz L-15 medium supplemented with 10%
fetal calf serum, 2 mM glutamine and 1% penicillin-streptomycin (all
from GIBCO, Grand Island, NY). The moat surrounding the well was
filled with sterile double-distilled water and the embryo returned to
the egg incubator at 38°C and 70% humidity.
Neural tube rotations and ablations
To discern whether the neural tube influenced the polarity of adjacent
axial structures, the orientation of the neural tube was experimentally
altered. Segments of neural tubes were excised with tungsten needles,
Dorsal neural tube induces the dermamyotome
leaving the notochord in place, as described previously (Yip, 1990).
The neural tube was then rotated 180° around the dorsal-ventral axis
and immediately repositioned in the embryo (Fig. 1). Embryos were
incubated for another 9-26 hours and fixed.
The dorsal neural tube was ablated from some embryos to
determine if dermamyotome development was inhibited. The dorsal
portion of the neural tube of stage 12-14 embryos was suctioned off
using a mouth-controlled micropipette with a 0.1 mm tip diameter.
Embryos were reincubated until they reached stage 16-18 and then
fixed.
Grafting experiments
The ability of various embryonic tissues to induce an ectopic dermamyotome from the ventral somite was assessed by grafting these
tissues into the space between the notochord and segmental plate.
Embryos were cut from the blastoderm and transferred, ventral side
up, to an agar-coated Petri dish filled with warm Locke’s saline. A
small slit was made in the endoderm between the neural tube and
paraxial mesoderm just posterior to the last-formed somite. The
piece of donor tissue was lightly stained with neutral red and
maneuvered into the slit with a tungsten needle. The embryos were
then incubated dorsal side up for another 18 to 22 hours (until stage
16-18) and fixed.
Embryonic tissues tested for their ability to induce the formation
of a dermamyotome included dorsal, lateral and ventral neural tube,
as well as ectoderm and neural crest cells. To obtain these tissues,
trunks at the axial level of the unsegmented mesoderm of stage 1315 embryos were excised with tungsten needles and digested with
Pancreatin (GIBCO) at 37°C until the tissues began to separate. The
trunks were then transferred to cold Hank’s balanced salt solution
(HBSS) and the various tissues separated by manual dissection. At
this time, the neural tube was cut longitudinally into dorsal, ventral
and lateral segments. The isolated tissues were incubated in F12
medium supplemented with 10% fetal calf serum, 2 mM glutamine,
3% chick embryo extract and 1% penicillin-streptomycin solution
(complete medium) for 2 hours to allow the cells to re-express proteolytically digested surface proteins. Neural crest cells were obtained
from 24-hour-old neural tube cultures, as described previously (e.g.
Loring et al., 1981; Erickson and Goins, 1995). The tissues were
washed in HBSS and stained with neutral red prior to grafting. In
some cases, the tissues were labeled for 2 hours in a 50 µg/ml solution
of wheat germ lectin conjugated with fluorescein (Sigma, St Louis,
MO) to identify unequivocally the graft in tissue sections.
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Co-culture studies
To assay the ability of various embryonic tissues to induce myocytes
from unspecified paraxial mesoderm, the trunk regions (encompassing the last 9 somites plus segmental plate) of stage 13-15 quail
embryos (Coturnix coturnix japonica) were removed with tungsten
needles and subsequently cut into 3-somite-length pieces (300 µm in
length), including a piece comprising the entire segmental plate. The
pieces were digested briefly with full-strength Pancreatin at 37°C until
the tissues could be separated easily from each other using tungsten
needles. For each axial level, the neural tube, notochord, ectoderm
and mesoderm (either somites or segmental plate) were collected separately and stored in complete F12 medium until used.
A piece of segmental plate approximately 300 µm in length was
introduced into a small culture well (8-chamber culture slides, LabTek, Nunc, Naperville, IL) in complete F12 medium. A potential
inducing tissue was also added to each well and the tissues
maneuvered with a blunt tungsten needle until they touched. Cultures
were incubated at 37°C in 5% CO2 for 4 days and the medium was
replaced every other day. The presence of myocytes was determined
by immunocytochemistry (see below).
Because ectoderm does not readily spread on a planar substratum,
we also employed collagen gels to immobilize and culture the tissues.
Collagen solution was prepared as described previously (Tucker and
Erickson, 1984) and 50 µl droplets gelled in 35 mm plastic Petri
dishes. The mesoderm and potential inducers were pipetted onto this
base and covered with a second layer of collagen so that the tissues
were completely enveloped. Cultures were incubated for 4 days.
Immunocytochemistry
We used a variety of techniques to identify myocytes and neural crest
cells, both in embryo sections and in culture. For most of our studies,
we employed whole-mount labeling (see Erickson et al., 1992; Tosney
et al., 1994 for details). A polyclonal antibody against the intermediate filament desmin (1:200, Biogenex, San Ramon, CA) was used to
identify myocytes (Erickson et al., 1987; Kaehn et al., 1988; Tosney
et al., 1994). In some cases, operated embryos were labeled with the
HNK-1 antibody to visualize the distribution of neural crest cells.
HNK-1-producing hybridoma cells were obtained from the American
Type Culture Collection and culture supernatant produced in our facilities. After tissues were immunolabeled, they were embedded in
Spurr’s resin, sectioned at 3 µm on a Sorvall MT-2 microtome using
a diamond histoknife (Diatome) and viewed with a Leitz Diaplan
microscope equipped with epifluorescence. The color micrographs in
Somite
Neural crest cells
Ectoderm from
dorsal surface
Notochord
Fig. 1. Diagrammatic representation
of the neural tube rotation
experiments. A segment of the neural
tube from the level of somites 16 to
21 was excised, rotated dorsoventrally
and replaced. The embryo was
reincubated for another 9 to 26 hours.
Note that these rotations were
performed at an axial level where the
dorsal-ventral axis of the somite was
already apparent. Even so, a
secondary dermamyotome develops
from the sclerotome.
234
M. S. Spence, J. Yip and C. A. Erickson
Fig. 4 were captured using a cooled CCD C72 MTI camera and NIH
Image software and merged in Photoshop.
Cultures were labeled with the monoclonal antibody 13F4 to
identify myocytes (Rong et al., 1987). In our hands this antibody
produced less background fluorescence in cultured tissue than the
desmin antibody. Cultures were fixed in 4% paraformaldehyde for 20
minutes, washed in PBS, blocked in 0.1% BSA/PBS and incubated in
13F4 (1:100 dilution in 0.1% BSA/PBS; Developmental Studies
Hybridoma Bank) for 2 hours. The cultures were washed with PBS
(2×), blocked in 0.1% BSA/PBS and incubated in secondary antibody
(goat anti-mouse conjugated with FITC or RITC, 1:100; Cappel) for
one hour. The cultures were then washed, coverslipped and viewed
as described above. In some cases, cultures were doubled-labeled with
the HNK-1 antibody and the desmin antibody.
RESULTS
Nomenclature
In this study, we use somite pairs as a convenient marker for
identifying and reporting axial positions. Traditionally, the first
formed somite pair is designated as ‘1’, the next most posterior
pair as ‘2’, and so on. Our study focuses on the thoracic level,
which encompasses somite pairs 20-27.
Regardless of the stage of development, the relative maturity
of each somite is consistent from one embryo to the next at the
thoracic level when measured with reference to the most
recently formed somite (Loring and Erickson, 1987; Ordahl,
1993; Tosney et al., 1994). Consequently, we also use a
posterior-to-anterior staging scheme employed previously (see
Loring and Erickson, 1987; Tosney et al., 1994), in which the
most recently formed pair of somites is designated as ‘−1’, the
next most anterior pair as ‘−2’, etc.
Neural tube rotations
Since there is reason to believe that the dorsoventral pattern of
the somites is established through interactions with the neural
tube (summarized in INTRODUCTION), we rotated the neural
tube around its dorsoventral axis, leaving the notochord in its
normal position, to test whether this could produce a corresponding inversion of paraxial mesoderm pattern. The neural
tube was excised at the level of somites 16-21 in stage 17-20
chick embryos, rotated 180° and the embryos reincubated for
9-26 hours (Fig. 1). Prior to surgery, the ventral portion of the
somites at the axial level of rotation had begun to disperse to
form the sclerotome while the dermamyotome was morphologically distinct in the dorsal somite. After rotation of the
neural tube and further development, however, the ventromedial margin of the somite was no longer mesenchymal, but
rather was epithelial in character, resembling the dermamyotome that persisted dorsally (n=5; Fig. 2). This was particularly noteworthy since the sclerotome had already begun to
disperse at the time of the operation. Proximity to the dorsal
neural tube appears to be important for the induction of a
second dermamyotome, because in one embryo in which the
neural tube was oriented tangentially, the length of the ectopic
dermamyotome that formed from each somite pair corresponded to the extent of the dorsal neural tube with which the
somite was in contact (Fig. 2C).
To examine whether the secondary dermamyotome could
have developed from pieces of dorsal somite that may have
adhered to the neural tube and been subsequently displaced
ventrally, we fixed 6 embryos 7-10 hours after the rotation. No
obvious clusters of cells could be detected adhering to the
rotated neural tube, suggesting that the ectopic dermamyotome
was not derived from dorsal somite.
Dorsal neural tube ablations
To test further the hypothesis that the dorsal neural tube
induces dermamyotome formation, we ablated the dorsal
portion of the neural tube, along with the narrow strip of
ectoderm that adheres to it, in stage 12-14 embryos. Manipulated embryos were allowed to develop for an additional 1820 hours and subsequently analyzed for the presence of a dermamyotome at the level of ablation.
When the dorsal neural tube was removed at the level of
the unsegmented mesoderm (n=16), 87.5% of the embryos
displayed either a complete absence (10 out of 16; 62.5%) or
Fig. 2.
Histological
sections through
embryos whose
neural tubes (nt)
were rotated and
then fixed 26
hours later.
(A,B) Phase
contrast and
fluorescence
micrographs of an
HNK-1-labeled
embryo. HNK-1
immunoreactivity
identifies early
migratory neural
crest cells. The
notochord (n) was
left in its normal
position, although
a small piece
remains attached
to the former
ventral surface of
the neural tube. A
dermamyotome
(dm) developed in
a normal position.
Also an ectopic
dermamyotomelike epithelium
developed from
what would have
been sclerotome
(arrowheads). The
vesicle attached to
the dorsal neural
tube is probably a
remnant of
ectoderm (e). (C) Fluorescence micrograph of a section taken
through a region where the neural tube is tangential with respect to
the embryonic dorsal-ventral axis. The size of the ectopic
dermamyotome (limits marked by arrowheads) is proportional to the
extent of dorsal neural tube in direct contact with the somite,
suggesting that close contact is necessary for signaling to occur. s,
sensory ganglion. Scale bar, 50 µm.
Dorsal neural tube induces the dermamyotome
235
Fig. 3. Sections through 6 embryos whose
dorsal neural tubes were ablated at the level
of the segmental plate. The degree to which
dermamyotome formation was affected is
variable. (A,B) In most cases, there was a
complete absence of the dermamyotome.
Note that there are no neural crest cells in
these sections (as assayed by HNK-1
immunoreactivity), indicating that the dorsal
neural tube has been removed and has not
regenerated. (C,D) In a smaller number of
instances, remnants of the dermamyotome
develop (arrowheads). Generally these are
found in lateral positions in the embryo, as
seen in C. Rarely, small epithelial vesicles
are observed (D). (E) Although the dorsal
portion of the neural tube has been
successfully ablated in this embryo, as
evidenced by the lack of neural crest cells,
normal dermamyotomes are found. Possibly
enough neural tube with inducing ability
remains to stimulate dermamyotome
development. (F) In this instance, where the
entire neural tube was removed, one large
dermamyotome forms dorsally. nt, neural
tube; n, notochord; dm, dermamyotome.
Scale bar, 50 µm.
a significant loss (4 out of 16; 25%) of the dermamyotome at
the level of ablation (Fig. 3A-D). The remaining embryos (2
out of 16; 12.5%) revealed no apparent effect on dermamyotome morphology (Fig. 3E). These results suggest that
the dorsal neural tube, or perhaps the narrow strip of
ectoderm covering it, specifies dorsal somite structures. The
appearance of part or all of the dermamyotome in some cases
may be due to the variability in the amount of neural epithelium that was removed from each embryo. Alternatively,
several studies show that the lateral dermamyotome develops
independently of the cues that regulate formation of the
medial dermamyotome (Bober et al., 1994; Pourquié et al.,
1995), which may explain why when remnants of dermamyotome are found, they are positioned laterally (e.g. Fig. 2C).
Curiously, when the entire neural tube was removed in one
individual, leaving an intact notochord in place, a fused
somite with a dorsally positioned dermamyotome resulted
(Fig. 3F).
The paraxial mesoderm apparently responds to a dorsalizing
cue associated with the dorsal neural tube quite early and irreversibly, because when the dorsal neural tube is ablated at progressively more anterior levels (i.e. embryologically older
regions), dermamyotome formation is less affected. Specifically, when the dorsal neural tube is ablated at the more
anterior level of somites −1 to −3 (n=7), only 43% (3 out of 7)
of the embryos exhibit a loss of the dermamyotome. There is
no loss of the dermamyotome when the neural tube is removed
from the axial level of somites −4 to −6 (n=5).
Embryonic tissues grafted medial to the ventral
somite
The previous two experiments demonstrated a role for dorsal
axial tissues in the induction of the dermamyotome. However,
these could not rule out the possibility that dermamyotomeinducing signals are produced either by the medial ectoderm
adhering to the neural tube or by neural crest cells emigrating
from the dorsal neural tube. To test potential roles for these
tissues and further investigate the influence of the neural tube,
we used two additional experimental strategies.
First, pieces of isolated embryonic tissues were grafted
ventrally into the space between the notochord and paraxial
mesoderm at the axial level where somites have not yet formed
to determine what embryonic tissues are capable of stimulating
ectopic dermamyotome formation. The presence of a dermamyotome was assayed by morphological criteria. We also used
an antibody against the intermediate filament protein desmin in
order to immunocytochemically detect myocytes. Out of 37
embryos in which pieces of dorsal neural tube were grafted
ventrally (Fig. 4A,B), 49% displayed either the presence of a
morphologically distinct dermamyotome (6 out of 37) or
desmin-positive cells proximal to the graft (12 out of 37). In the
remainder of the embryos that showed no obvious ectopic dermamyotome, the graft was quite distant from the somite, suggesting that the grafted tissue must be close to the mesoderm to
exert an effect. Pieces of lateral neural tube also induced either
an organized epithelium in the ventral somite or desmin-positive
cells (Fig. 4C,D), although the response was less robust.
236
M. S. Spence, J. Yip and C. A. Erickson
Fig. 4. Pieces of isolated embryonic
tissues were labeled with fluoresceinconjugated lectin (green labeling),
grafted between the notochord and
somite, and the presence of myocytes
assessed by immunoreactivity with an
antibody to desmin (red labeling).
(A,C,E) Fluorescence images; (B,D,F)
corresponding phase images. (A,B) A
piece of dorsal neural tube was grafted
ventrally and the embryo fixed 24 hours
later. Myocytes are distributed ventrally
(arrowheads) proximate to the grafted
tissue. In a phase micrograph of the
same section (B), note also the epithelial
arrangement of the somitic cells
(indicated by arrowheads) associated
with the grafted piece of dorsal neural
tube. Normal myotome structure and
position is observed on the contralateral
side. (C,D) A piece of lateral neural tube
was grafted between the notochord and
somite. A few myocytes arise in contact
with the graft (arrowheads), but these
cells are not organized into an
epithelium. (E,F) A piece of ventral
neural tube was grafted into a ventral
position but, although it is in intimate
contact with the somite, no myocytes
have differentiated. Two small areas of
rhodamine fluorescence (arrowheads)
dorsal to the graft are blood vessels.
Scale bar, 50 µm.
In contrast, when ventral neural tube pieces were grafted in
an identical fashion (n=14), the ventral somite cells were not
organized in an epithelium in any of the embryos, and only 7%
of the embryos (1 out of 14) displayed desmin-positive cells
next to the graft (Fig. 4E,F). This difference between the dorsal
and ventral grafts is statistically significant (Chi-square
analysis, P=0.05).
When pieces of ectoderm were grafted adjacent to the
notochord (n=10), 90% of these embryos displayed either a
vague epithelial arrangement of somite cells (5 out of 10; Fig.
5A) or a few desmin-positive cells (4 out of 10). The response
was not nearly as dramatic as that generated by dorsal neural
tube pieces, however (Fig. 5B).
Finally, we tested the role of neural crest cells in dermamyotome formation by grafting clusters of neural crest cells
isolated from 24-hour-old cultures between the somite and
notochord. In 12 embryos, none showed any evidence of dermamyotome formation proximate to the graft (data not shown).
Co-culture studies
To further distinguish between the relative roles of the dorsal,
ventral and lateral neural tube in the induction of myogenesis,
we co-cultured portions of the neural tubes with paraxial
mesoderm in vitro. Such studies allowed us to better control
the size of tissue pieces used and quantitate the inductive
response.
Dissected neural tubes approximately 300 µm in length were
taken from the axial level of the segmental plate, and cut
longitudinally into 3/4 dorsal and 1/4 ventral pieces, or alternatively, into 3/4 ventral and 1/4 dorsal pieces (Fig. 6A). These
neural tube pieces were then cultured individually with a piece
of segmental plate (approximately 300 µm in length). The
extent of myogenesis was assessed by quantifying the number
of 13F4-positive cells per culture. Because the density of overlapping cells precluded accurate counting, we categorized the
cultures as: <10 13F4-positive cells; 10≥100 cells; and >100
cells (Fig. 7). Control cultures of paraxial mesoderm alone
produced no myocytes (n=29 cultures). The results of coculture treatments are summarized in Fig. 6B.
The greatest number of 13F4-immunoreactive cells was
found in the 1/4 dorsal neural tube cultures (n=39 cultures;
over 90% of all cultures contained more than 10 myocytes).
Fewer immunoreactive cells were found in cultures that
contained either 3/4 dorsal or 3/4 ventral neural tube pieces.
The smallest number of immunoreactive cultures resulted from
1/4 ventral neural tube explants (n=56 cultures; 55% of these
Dorsal neural tube induces the dermamyotome
237
Fig. 5. (A) When pieces of ectoderm
(e) are grafted ventrally, cells that
should have been mesenchymal, as on
the contralateral side, are organized
into a thin epithelium (indicated by
arrowheads). This is not as robust a
response as seen when pieces of dorsal
neural tube (dnt) are similarly grafted
(B). Note in B the epithelial
organization of the ventral somite
(arrowheads) despite its proximity to
the notochord (n). The grafted tissue is not acting as a physical barrier between the somite and notochord and thereby blocking ventralizing
signals. nt, neural tube. Scale bar, 50 µm.
contained no myocytes), demonstrating that the dorsal neural
tube has the greatest ability to induce myocyte formation under
these culture conditions.
Neural crest cells derived from 24-hour-old cultures and cocultured with paraxial mesoderm (n=11) resulted in no
myocytes in 55% (6 out of 11) of the cultures, or a moderate
level (10≥100 myocytes) of myogenesis in 45% (5 out of 11)
of the cultures.
We found it difficult to establish cultures of dorsal ectoderm
on planar substrata and therefore could not assess their ability
to induce myogenesis using this assay. However, we repeated
some of these experiments by culturing tissues in collagen gels,
where adhesion to the substratum was not essential and where
the tissues could be forced to stay together. Under these conditions, two or three pieces of ectoderm co-cultured with a
piece of unsegmented mesoderm (n=14) produced no response
in 43% (6 out of 14), a minimal response in 43% (6 out of 14),
and a substantial response in 14% (2 out of 14) of the cultures.
A
Co-culture of dorsal neural tube and paraxial mesoderm in
collagen gels still yielded a dramatic response (>100
myocytes/culture) in all cultures (n=4), as expected from the
planar culture assay.
We also assessed the relative ability of progressively older
neural tubes to induce myogenesis (Fig. 8). Neural tubes from
4 different axial levels – unsegmented mesoderm (n=11),
somite level −1 to −3 (n=11), −4 to −6 (n=10), and −7 to −9
(n=12) – were cut in half longitudinally and the dorsal halves
co-cultured with segmental plate. This experiment did not
reveal statistically significant differences in the inductive
effects of dorsal neural tube from different axial levels. Stern
and Hauschka (1995) also reported that rostral and caudal
neural tubes are equivalent in their capacity to promote myogenesis. This result is not surprising in light of the neural tube
rotation experiments described earlier, in which all levels of
the rotated piece could induce a secondary dermamyotome.
Our culture system also allowed us to evaluate at what axial
B
Neural Tube Pieces + SP
3/4 D
1/4 V
+
+
Percentage of Cultures
75
< 10 myocytes
10 ≥ 100 myocytes
> 100 myocytes
31
32
50
27
20 19
18
3/4 V
11
14
25
7
1/4 D
24
6
4
+
+
0
1/4 V
3/4 V
3/4 D
1/4 D
Neural Tube Pieces
Fig. 6. (A) Diagrammatic summary of co-culture experiments in which paraxial mesoderm derived from the axial level of the segmental plate
(SP; indicated by arrow) was combined with dorsal or ventral pieces of neural tube for 4 days and the differentiation of myocytes assessed by
immunoreactivity with the 13F4 monoclonal antibody, which is a marker for myocytes. Cultures were characterized as: containing <10
myocytes; 10≥100 myocytes or >100 myocytes. (B) Percentages of cultures in each category are summarized in the bar graph. Total number of
cultures for each category are indicated at the top of each bar.
238
M. S. Spence, J. Yip and C. A. Erickson
Fig. 7. Examples
of cultures
immunolabeled
with the 13F4
antibody, which
reveals myocytes,
and rated as >100
myocytes (A),
10≥100 myocytes
(B) and <10
myocytes (C).
Note in A how far
the myocytes are
distributed from
the piece of dorsal
neural tube. i,
putative inducer.
Scale bar, 100 µm.
level myocytes were specified. As noted above, unsegmented
mesoderm cultured alone produced no myocytes, as measured
by 13F4 immunoreactivity (n=29). In contrast, somites −1 to
−3 cultured alone (n=15) produced myocytes in 60% of the
cultures and somites −4 to −6 (n=10) and somites −7 to −9
(n=12) produced myocytes 100% of the time. These results are
identical to other studies that analyze myogenesis in stage 13
embryos using culture assays (Stern and Hauschka, 1995) or
in vivo experimental manipulations (Aoyama and Asamoto,
1988). Thus, myocyte specification occurs within a few hours
of somite segmentation.
DISCUSSION
Our results, derived from both in vivo and in vitro analysis,
suggest that factors from the dorsal neural tube induce dermamyotome formation and myocyte differentiation, that these
factors are diffusible over a short range and compete with ventralizing factors, and that specification of dorsal fate occurs
shortly after somite segmentation.
Dorsal neural tube is an inducer
Many different embryonic tissues have been proposed to
specify dorsal somite structures, but our results indicate that
the dorsal neural tube is the most potent of these. When the
neural tube is rotated so that the dorsal surface abuts the ventral
somite, sclerotome dispersion is inhibited locally and an
epithelial structure resembling the dermamyotome forms.
Also, when the dorsal neural tube is ablated at a posterior axial
level, no dermamyotome develops in the majority of cases.
Finally, when pieces of dorsal neural tube are co-cultured with
paraxial mesoderm either in tissue culture or in the embryo, an
ectopic dermamyotome forms and myocytes differentiate from
the unspecified mesoderm. Since no dermamyotome forms or
muscle cells differentiate in the absence of the dorsal neural
tube in all our experimental conditions, our results further
suggest that a dorsalizing signal is required to specify the dermamyotome, rather than dorsal structures simply arising by
default.
Although we demonstrate that the dorsal neural tube can
induce dermamyotome formation and myocyte differentiation, and is also the most potent inducer in all of our assays,
we have not ruled out the possibility that redundant or overlapping cues emanate from other tissues. For example,
ectoderm has been proposed to signal dermamyotome
formation directly (Christ et al., 1972; Fan and TessierLavigne, 1994; Kuratani et al., 1994). In support of a role for
ectoderm is the observation that in the absence of the entire
neural tube, but in the presence of a notochord, the somites
fuse in the midline and one large overarching dermamyotome
develops (our results; Christ et al., 1972; Bober et al., 1994).
Thus, in the absence of dorsal neural tube cues, a normal dermamyotome develops in association with the ectoderm and
suggests that redundant cues are produced by ectoderm and
neural tube. If the ectoderm does produce such a cue, it is
difficult to explain why dermamyotomes do not develop when
the dorsal neural tube is ablated but the ectoderm over the
somites remains intact. Moreover when pieces of ectoderm
are grafted ventrally in the embryo adjacent to the ventral
somite, only a very minimal response is observed (see also
Kenny-Mobbs and Thorogood, 1987; Fan and TessierLavigne, 1994) compared with the dramatic response to the
dorsal neural tube. Given that a putative inducing factor
produced by the dorsal neural tube appears to be diffusible
(Fan and Tessier-Lavigne, 1994; see below), another explanation for the weak inductive activity displayed by the
ectoderm is that such a factor could diffuse from the dorsal
neural tube and bind to the extracellular matrix associated
with the basal lamina of the ectoderm.
Another possibility that our study cannot rule out is that the
dorsal neural tube may signal the overlying ectoderm to
acquire dermamyotome-inducing ability. There is precedent
for such a mechanism being involved in ventral somite specification, since Sonic hedgehog produced by the notochord
induces formation of the neural tube floor plate, which in turn
produces more Sonic hedgehog and is apparently responsible
for both stimulating sclerotome formation as well as repressing dermamyotome formation (Johnson et al., 1994; Fan and
Tessier-Lavigne, 1994). Thus when the dorsal neural tube was
ablated in our studies, it is conceivable that we may have interfered with signaling to the ectoderm. Nevertheless, one additional observation argues against this possibility. Bober et al.
(1994) report that, when one lateral half of a neural tube is
removed, the medial portion of the dermamyotome on the
depleted side does not form, even though the other half of the
neural tube is still intact, including its dorsal component. The
remaining neural tube could presumably still signal the
overlying ectoderm just as effectively. Thus we suggest that if
Dorsal neural tube induces the dermamyotome
239
Dorsal neural tube
from different axial
levels co-cultured with
segmental plate (SP)
DNT
SP
DNT
-7 to -9
75% positive (n=12)
40% positive (n=10)
SP
-4 to -6
-1 to -3
DNT
64% positive (n=11)
Segmental
Plate
SP
DNT
SP
the ectoderm plays a role in dermamyotome development, it is
likely to be redundant.
Neural crest cells have also been proposed to induce the
formation of the myotome as well as stimulate myogenesis, but
we view this possibility to be unlikely. First, neural crest cells
grafted ventrally adjacent to the sclerotome fail to induce dermamyotome formation. Similarly in co-culture studies, few or
no myocytes arise in the presence of pure populations of neural
crest cells. The moderate number of myocytes observed in
some cultures can be explained by the fact that purified crest
cells must be harvested from neural tube cultures, which may
contaminate them with a dorsal neural tube-derived inducing
factor. Finally, myotome formation occurs concomitant with
the invasion of neural crest cells into the somite at the thoracic
level (e.g. Loring and Erickson, 1987; Tosney et al., 1994) but,
at more posterior levels, crest cells invade the somite many
hours prior to the appearance of the myotome (our unpublished
results). Thus, there is no strict correlation between contact
with neural crest cells and the appearance of the myotome.
There are several reports suggesting that the notochord may
induce dermamyotome formation and muscle cell differentiation. First, as already discussed, when the entire neural tube
is ablated but the notochord in left in place (Christ et al.,1972;
Bober et al., 1994; and this study), a single large dermamyotome spans the space where the neural tube would be
normally. Second, the notochord apparently has a myocyteinductive capability in the absence of the neural tube in the
embryo. For example, Rong and co-workers (1992) removed
the neural tube and notochord along the entire embryonic axis
and then replaced these axial structures with 2 or 3 exogenous
notochords. Under these circumstances muscle cells differentiate. Finally, when paraxial mesoderm is co-cultured with
notochord, muscle cells differentiate (Buffinger and Stockdale,
1994; Stern and Hauschka, 1995; Münsterberg and Lassar,
1995; our unreported results). It is difficult to reconcile these
observations with the fact that grafting a notochord into the
72% positive (n=11)
Fig. 8. Summary of co-culture experiments in
which the myocyte-inducing ability of the
dorsal neural tube (DNT) from increasingly
anterior axial levels was assessed. The dorsal
half of the neural tube was isolated from four
different axial levels and then combined with
pieces of paraxial mesoderm isolated from the
level of the segmental plate (SP). There is no
significant difference in the inducing ability of
the neural tube as it ages (i.e. from the more
anterior axial levels).
dorsal portion of a somite represses dermamyotome formation
(Pourquié et al., 1993; Goulding et al., 1994) and that
notochord-derived Sonic hedgehog competes with the dorsal
signal to turn off dermamyotome-specific gene expression
(Johnson et al., 1994; Fan and Tessier-Lavigne, 1994). In
addition, when the notochord is removed, resulting in the dorsalization of the ventral neural tube (van Straaten and Hekking,
1991; Goulding et al., 1993), dermatome-specific gene
expression and myotome formation is extended ventrally,
again supporting the model that the notochord and ventral
neural tube suppress the development of the dermamyotome.
A definitive explanation of these conflicting results must await
the identification of the inducing factors. However, one possibility is that the neural tube reciprocally signals the notochord
and that, in the absence of neural tube influence (as would be
the case in Rong et al., 1992; Buffinger and Stockdale, 1994;
Stern and Hauschka, 1995), the notochord no longer produces
factors that repress dermamyotome formation, thereby
revealing myogenic factors that it also generates. The observation that ectopically expressed Sonic hedgehog alone in the
dorsal somite results in the expansion of the myotome rather
than its repression is evidence of a notochord-derived
myogenic inducer (Johnson et al., 1994).
The dorsal neural tube product is diffusible
It is likely that the inducing signals derived from the dorsal
neural tube are diffusible, although the distance over which
they can diffuse is difficult to discern from our current observations. We noted from our co-cultures on planar substrata that
the dorsal neural tube had to be placed in close proximity to
the mesoderm (but not necessarily touching) to see strong
induction but, in this assay, we were not able to quantitate
precisely this distance. Stern and Hauschka (1995) also noted
the need for close positioning. Under our culture conditions, a
diffusible factor might fall to very low levels a short distance
from the dorsal neural tube. In fact, co-cultures embedded in
240
M. S. Spence, J. Yip and C. A. Erickson
collagen gels (Fan and Tessier-Lavigne, 1994), where the
diffusing molecule may be concentrated or even bound to the
gel matrix, suggest effects of over 100 µm. We also noted that,
although the inducing and responding tissues needed to be
close, myocytes appeared widely throughout the paraxial
mesoderm (>100 µm from the inducing source) and not just in
the contact zone (see Fig. 8A), suggesting that this factor can
diffuse through and perhaps be concentrated in the tissue environment of the paraxial mesoderm. We did not rule out directly
that an inducing signal in our system is propagated from one
myocyte to the next. Such propagation seems unlikely,
however, because we might have observed similar numbers of
myocytes arising in our cultures of somites at different ages
but, instead, we found that greater numbers of myocytes differentiated from progressively older somites.
Our in vivo studies also cannot reveal how far such a factor
may diffuse since tests of induction were always done in competition with the host notochord or floorplate, which complicates the interpretation. Nevertheless myocytes often appeared
>50 µm from the closest edge of the dorsal neural tube
fragments. Any further tests of how far such an inducer could
act must clearly be carried out in the embryo since rates and
distances of diffusion will depend upon the ability to move
through and bind to the extracellular matrix or through other
tissues.
Our studies, as well as those of others, suggest that dorsal
neural tube-derived factors can compete with ventralizing
signals. Fan and Tessier-Lavigne (1994) elegantly demonstrated such competition when they co-cultured dorsal neural
tube and Sonic hedgehog-producing heterologous cells with
unspecified mouse presomitic mesoderm. Similarly in our
study, when 3/4 dorsal neural tubes, which are likely to contain
ventralizing signals, are cultured with paraxial mesoderm,
fewer myocytes differentiate compared to 1/4 dorsal neural
tubes, again suggesting antagonizing signals. We have been
able to demonstrate directly that such a competition occurs in
the embryo as well. In two different experimental paradigms –
either when the neural tube was rotated 180° around its dorsalventral axis or when pieces of dorsal neural tube were grafted
ventrally – host notochord was always left in its normal
position. Under these circumstances, dorsal structures (either a
dermamyotome or myocytes) differentiated in response to a
dorsalizing cue despite their proximity to the host notochord.
This competition is not due simply to the grafted tissue physically blocking a ventralizing signal from the notochord, since
the dorsalizing effect of the grafted dorsal neural tube was
noted even when the notochord had unimpeded simultaneous
contact with the somite (Fig. 5B).
Timing of specification
Several recent studies where progressively older somites were
placed in culture suggest that, within only a few hours after
separation from the segmental plate, the myocytes are already
determined and will later differentiate, even in the absence of
the neural tube (Vivarelli and Cossu, 1986; Kenny-Mobbs and
Thorogood, 1987; Buffinger and Stockdale, 1994; Stern and
Hauschka, 1995). Our tissue culture data are consistent with
these observations; although myocytes do not differentiate
from unsegmented paraxial mesoderm, older somites give rise
to progressively more myocytes. The dorsal neural tube
ablation studies extended these observations to the embryonic
environment. A complete dermamyotome rarely forms when
the dorsal neural tube is ablated at the level of the segmental
plate, suggesting that specification has not yet occurred. In
contrast, dorsal neural tube ablation at the level of the three
last-formed somites results in dermamyotome development in
50% of the cases, while somites −4 to −6 apparently no longer
need the influence of the dorsal neural tube since ablation has
no effect on dermamyotome differentiation. Aoyama and
Asamoto (1988) also showed that myocytes are specified by
somite −3 in stage 13 embryos by rotating a somite around its
dorsoventral axis, which then produced an inverted dorsoventral pattern. Interestingly, the somites remain responsive to
signaling from the neural tube after segmentation (see also
Stern and Hauschka, 1995) and the dorsal neural tube itself
retains inducing properties at more anterior axial levels, presumably many hours after they are needed. Such precision in
identifying the timing of specification should be useful in elucidating the molecular basis for the various steps in the specification, determination and differentiation of myocytes and
dermal precursors.
Model for dorsal-ventral pattern
Our data, along with a wide array of previously published
studies, suggest the following model for the establishment of
dorsal-ventral pattern in the somite. Competing diffusible
signals from the dorsal neural tube and notochord/floorplate
specify dermamyotome and sclerotome, respectively. The
dorsal neural tube signal is proposed to diffuse broadly but in
ventral regions is out-competed by Sonic hedgehog and
perhaps other ventral signals. Thus, the spatial positioning of
the dermamyotome dorsally is due to the absence or low concentration of notochord-derived signals, as well as to the
presence of dorsal signals. It is possible that these latter
signals are localized and amplified by binding to the ectoderm
or that the ectoderm also produces dorsalizing factors. An
alternative possibility is that the dorsal neural tube signals
only the medial edge of the somite, and this dermamyotomespecifying signal is propagated laterally in the plane of the
somitic epithelium. This signal, if propagated ventrally,
would be inhibited by ventralizing signals from the floorplate
and notochord. Identification of the dorsal signaling
molecules awaits further experimentation, but particularly
attractive candidates are members of the Wnt family, which
are diffusible signaling factors produced in the neural tube
that act over both a short and long range (Parr et al., 1993).
Finally, it is likely that several dorsalizing signals are
necessary, including those that maintain the epithelial
structure of the dermamyotome, as well as additional factors
that may dictate specific cell fate.
We thank Charles Ordahl and David McClay for insightful
comments concerning somitogenesis and signaling mechanisms and
Mark Reedy, David Parichy and Tuan Duong for careful scrutiny of
the manuscript. Two anonymous reviewers contributed substantially
to the tightened focus and balance of the Discussion, for which we
thank them. This research was supported by NIH grants DE 05630 to
C. A. E. and NS 23916 to J. Y.
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(Accepted 27 September 1995)