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First published online 14 June 2006
doi: 10.1242/dev.02426
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King's College London, Department of Craniofacial Development, Floor 27 Guy's Tower, Guy's Hospital, London Bridge, London SE1 9RT, UK.
Author for correspondence (e-mail:
susanne.dietrich{at}kcl.ac.uk)
Accepted 3 May 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Chick, Quail, Embryo, Head mesoderm, Skeletal muscles, Eye muscles, Lateral rectus, Branchiomeric muscles, Mandibular arch, Jaw muscles, Lbx1, Paraxis, En2, Myf5, MyoR, Fgf8, Bmp7
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Muscles in the head develop from two sources: the unsegmented para- and
pre-otic head mesoderm, and post-otically, from segmented paraxial mesoderm,
namely the occipital somites (Couly et
al., 1992; Jacob et al.,
1984; Noden,
1983a; Wachtler and Jacob,
1986). The occipital somites deliver the posterior pharyngeal and
laryngeal muscles and the muscles of the tongue. They have been secondarily
incorporated into the head during vertebrate evolution and largely develop
like trunk somites (Gans and Northcutt,
1983). The unsegmented paraxial head mesoderm provides the muscles
of the first three branchial arches, including the jaw closure muscles [first
or mandibular arch muscles (MAM), innervated by the trigeminal nerve], the jaw
opening and facial muscles (second or hyoid arch muscles, innervated by the
facial nerve) and the anterior pharyngeal and laryngeal muscles (third
branchial arch muscles, innervated by the glossopharyngeal nerve)
(Couly et al., 1992;
Jacob et al., 1984;
Noden, 1983a;
Wachtler and Jacob, 1986).
Moreover, this mesoderm is the source of two of the extrinsic eye muscles
[lateral rectus extraocular muscle (EOM), innervated by the abducens nerve,
and dorsal oblique EOM, innervated by the trochlear nerve], while the
remaining four EOM originate from prechordal head mesoderm (dorsal rectus,
ventral rectus, medial rectus, ventral oblique EOM, all innervated by the
oculomotor nerve). Most of the prechordal mesoderm joins the paraxial head
mesoderm before muscle development, such that EOM and the muscles of the
anterior branchial arches develop from a morphologically continuous strip of
mesenchyme (Jacob et al.,
1984; Wachtler and Jacob,
1986). Moreover, this mesenchyme has the same myogenic potential
(this study). We thus will collectively refer to it as head mesoderm.
It was established only recently that the head mesoderm employs a distinct
programme of myogenesis (Mootoosamy and
Dietrich, 2002). Key regulators of somitic myogenesis such as
Pax3 are absent (Hacker and
Guthrie, 1998; Mootoosamy and
Dietrich, 2002). Moreover, signals that stimulate trunk muscle
development suppress myogenic differentiation in the head
(Tzahor et al., 2003). Myf5,
the first muscle determining factor to be expressed in the embryo, employs
distinct promoter and enhancer elements in the head and in the trunk
(Hadchouel et al., 2000;
Summerbell et al., 2000),
while knockout mice for the two basic helix-loop-helix transcription factors
MyoR and Capsulin (Lu et al.,
2002), mice lacking the T-box gene Tbx1
(Kelly et al., 2004), or mice
lacking the homeobox gene Pitx2
(Gage et al., 1999;
Kitamura et al., 1999),
display distinct craniofacial, but not trunk muscle, defects. However, neither
the cascades that trigger the myogenic differentiation of the head mesoderm,
nor the cascades that specify the individual head muscles, are known.
Classically, neural crest cells are thought to control the developing head
muscles (Noden, 1983b). Neural
crest cells provide the majority of skull bones, and in addition the
connective tissue, tendons and muscle attachment points for muscles in the
head (Couly et al., 1993;
Köntges and Lumsden,
1996; Noden,
1983a). Moreover, they carry positional information, the
alteration of which leads to patterning defects of both bone and muscle
(Grammatopoulos et al., 2000;
Noden, 1983b;
Pasqualetti et al., 2000). In
zebrafish neural crest cells in mutants such as chinless, and in
human DiGeorge patients, head muscle is severely impaired
(Kelly et al., 2004;
Schilling et al., 1996).
However, the candidate gene for DiGeorge syndrome, Tbx1, affects
neural crest cells indirectly (Vitelli et
al., 2002a; Vitelli et al.,
2002b). Moreover, the gene is expressed in branchial arch muscle,
and hence may influence muscle development directly
(Kelly et al., 2004). In
addition, in-vivo and in-vitro studies led to conflicting results regarding
the requirement of neural crest cells for muscle differentiation; thus the
role of these cells remains unclear
(Tzahor et al., 2003).
Besides neural crest cells, the structures targeted by head muscles are
also suspected to control craniofacial myogenesis. For example, in
ZRDCT-An anophthalmic mouse mutants lacking eyes, eye muscles are
severely reduced (Paterson and
Kaiserman-Abramof, 1981). Likewise, in eyeless hagfishes, no eye
muscles are found, a remarkable exception to the otherwise stereotype
arrangement of eye muscles in vertebrates
(http://tolweb.org/tree?group=Hyperotreti&contgroup=Craniata)
(Brodal and Ragnar, 1963).
However, hagfishes, which constitute a sister group to lampreys, have highly
specialised life styles and may have secondarily lost their eyes and eye
muscles. Thus, it remains open whether head muscle development requires the
presence of muscle targets.
Finally, the innervating nerves have been thought to control muscle
development in the head. In patients suffering from Duane's syndrome, the
abducens nerve fails to innervate the lateral rectus EOM, which either
degenerates or survives when misinnervated by the oculomotor nerve
(Engle, 2002). In a similar
vein, in patients suffering from fibrosis of extraocular muscles (FEOM) type2,
trochlear and oculomotor-innervated muscles fail, as these nerves are absent
(Nakano et al., 2001).
However, in animal models for Duane's and FEOM type2, muscle anlagen first
develop, suggesting that innervation may not be required for the initial steps
of muscle development (Engle,
2002).
Conceptually, muscles that develop from head mesoderm can be organised into
two groups, the EOM and the branchiomeric muscles. EOM develop from the more
anterior and medial aspect of the head mesoderm and remain outside the
branchial arches at all times (Couly et
al., 1992; Noden,
1983a; Wachtler and Jacob,
1986). Motor neurons innervating these muscles resemble somatic
motor neurons in the trunk (Jacob et al.,
2001). The branchiomeric muscles arise from the more posterior and
lateral aspect of the head mesoderm and develop within the branchial arches
(Couly et al., 1992;
Noden, 1983a;
Wachtler and Jacob, 1986).
Their motoneurons resemble visceral motoneurons
(Jacob et al., 2001). Notably,
branchiomeric muscle precursors express MyoR and Capsulin
before Myf5 (von Scheven et al.,
2006).
At the level of the anterior hindbrain, precursors for prospective eye and
branchiomeric muscles sit side by side as the anlage of the lateral rectus EOM
is medially adjacent to developing MAM
(Couly et al., 1992;
Noden, 1983a;
Wachtler and Jacob, 1986).
Here, muscles express additional markers, with Paraxis and
Lbx1 characterising the EOM
(Mootoosamy and Dietrich,
2002) and En2 labelling the MAM
(Gardner and Barald, 1992).
Thus, focusing on this territory, we have the tools to simultaneously address
the differentiation and specification of eye versus branchiomeric muscles.
Using heterotopic transplantation in the chick embryo, we first established that the head mesoderm develops into muscle according to localised, extrinsic cues. Ablation experiments then revealed that neural crest cells, the eye as the ultimate EOM target, or the innervating nerves do not control the onset of muscle differentiation or muscle specification. However, the neural tube provides a signal that specifies the lateral rectus EOM. This signal is anteroposteriorly unrestricted, soluble and acts in conjuction with a further, unidentified cue. In its absence, while muscle differentiation continued, the muscle anlage erroneously expressed the MAM marker En2. Moreover, the abducens nerve fell short of its target. This suggests that the neural tube supports the specification of EOM and suppresses the specification of arch muscles, in order to aid eye-muscle innervation.
The upregulation of En2 upon separation of head mesoderm and neural tube suggested that branchial arch derived signals might stimulate MAM and suppress EOM development. Implanting beads loaded with signalling molecules expressed in the brachial arches, we found that Bmp7 had a generalised, negative effect on head muscle development. Fgf8, however, displayed a dual function: it upregulated En2 expression and suppressed Paraxis expression, indicating that it antagonises the neural tube derived signals and supports MAM against EOM development. In addition, Fgf8 upregulated MyoR, a marker for proliferative, undifferentiated arch muscle precursors, and suppressed Myf5, indicating that Fgf8 controls muscle precursor proliferation versus differentiation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Protein-loaded beads
Recombinant human Fgf4 or Fgf8 (R&D) at 500 µg/ml or 100 µg/ml
was loaded onto Heparin-Acrylic beads (Sigma) as described by Alvares et al.
(Alvares et al., 2003).
Recombinant human Bmp2, Bmp4 or Bmp7 (R&D) at 500 µg/ml or 100 µg/ml
and Shh (R&D) at 100 µg/ml was loaded onto Affi-Gel blue agarose beads
(Biorad) as described in Dietrich et al.
(Dietrich et al., 1998).
Tissue culture cell implants
Rat B1 control cells and cells expressing Wnt1 were raised, labelled with
Celltracker Orange (Molecular Probes), aggregated and implanted as described
in Cheng et al. (Cheng et al.,
2004).
Microsurgery
In ovo microsurgery was carried out with flame-sharpened 100-µm tungsten
needles as described previously (Alvares et
al., 2003; Dietrich et al.,
1998; Dietrich et al.,
1997; Mootoosamy and Dietrich,
2002). Cranial operations were performed at HH10, before head
muscle development and innervation (Noden
et al., 1999). The exceptions were neural crest ablations and the
heterotopic transplantation of head mesoderm fragments, which were carried out
at HH8-9-, before the emigration of cranial neural crest cells
(Lumsden et al., 1991).
Barrier experiments were performed using 7.5-µm-thick tantalum foil
[Goodfellow; (Dietrich et al.,
1998; Dietrich et al.,
1997)], filter experiments using 25-µm-thick polycarbonate
filters with a pore size of 0.05 µm [Costar;
(Fan and Tessier-Lavigne,
1994)]. Operations at flank levels were performed at HH16 as
described by Lours and Dietrich (Lours and
Dietrich, 2005); the mid-hindbrain neural tube grafts were derived
from HH10 donors. After surgery, the eggs were incubated for 48 hours, then
harvested and fixed as outlined above.
Electroporation
The pCAß-IRES-eGFP vector described in Alvares et al.
(Alvares et al., 2003) or this
vector harbouring the open reading frame of mouse Hoxb1
(Bell et al., 1999) was
injected into the HH10 neural tube at midbrainhindbrain levels and
electroporated as described in Schubert and Lumsden
(Schubert and Lumsden,
2005).
In-situ hybridisation
Whole-mount in-situ hybridisation was carried out as previously described
(Dietrich et al., 1998;
Dietrich et al., 1997;
Mootoosamy and Dietrich,
2002). Probes and their expression patterns are detailed in the
following references: Bmp7 (Begbie
et al., 1999); Dlx5
(Ferrari et al., 1995);
En2 (Logan et al.,
1992); Fgf8 (Mahmood
et al., 1995); Lbx1
(Dietrich et al., 1998);
Myf5 (Saitoh et al.,
1993); MyoR (von
Scheven et al., 2006); Paraxis
(Sosic et al., 1997);
Shh (Johnson et al.,
1994).
Immunohistochemistry
Upon in-situ hybridisation, whole-mount immunohistochemistry was carried
out according to Guthrie and Lumsden
(Guthrie and Lumsden, 1992).
Axonal staining was performed using the RMO270 antibody (Zymed; dilution
1:3000), which recognises the 155-kDa intermediate neurofilament subunit.
Quail tissues were identified using the QCPN antibody (Developmental Studies
Hybridoma Bank, University of Iowa, IA; dilution 1:200), the developing eye
using a Pax6 antibody (Developmental Studies Hybridoma Bank, University of
Iowa, IA; dilution 1:200), and eGFP protein using an anti-eGFP antibody
(Molecular probes; dilution 1:2000). In all cases, signals were detected using
anti-mouse IgG conjugated with horseradish peroxidase (Dako; dilution 1:100)
and a peroxidase substrate kit (Vector Laboratories).
Sectioning
Embryos were embedded in 20% gelatin (Sigma) in PBS at 50°C, then
cooled to 4°C. Subsequently, blocks were trimmed and fixed in 4% PFA for
up to 2 days, then rinsed in PBS and sectioned to 50 µm on a Pelco 1000
Vibratome. Sections were collected on gelatinised slides and mounted in either
80% glycerol/PBS, or Aquamount (BDH).
Photomicroscopy
After in-situ hybridisation and/or immunohistochemistry, embryos were
cleared in 80% glycerol/PBS. Whole-mounted embryos were split midsagitally
before analysis. Embryos and sections were photographed on a Zeiss Axiophot,
using Nomarski optics.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Fate
mapping experiments for the head mesoderm have established that EOM and the
muscles of the first three branchial arches develop from distinct regions
(Couly et al., 1992;
Noden, 1983a;
Wachtler and Jacob, 1986).
Moreover, the head mesoderm can superimpose its identity on individuated
neural crest cells (Trainor and Krumlauf,
2000). We thus investigated whether the localised formation of
muscle anlagen in the head depends on intrinsic or extrinsic cues.
Heterotopic grafting of head mesoderm fragments was carried out on the
right side of embryos as indicated in Fig.
1A, using quail donors and stage-matched chicken hosts. The
operations were performed before neural crest cell emigration at
HH8-9-, as neural crest cells have been suggested to provide
positional information and patterning cues for the developing muscles
(Noden, 1983b). As a control,
head mesoderm at the level of the anterior hindbrain was grafted
orthotopically.
The grafted tissue was traced using the QCPN antibody
(Fig. 1B-H, brown staining).
The specification of the lateral rectus EOM was investigated using
Paraxis (not shown) and Lbx1
(Fig. 1B-F, blue staining) as
markers; both markers gave identical results. The position of all muscle
anlagen was examined, assaying for the expression of the muscle-determining
factor Myf5 (Fig.
1G,H, blue staining). The embryos were analysed 48 hours
post-surgery at HH19, as all markers are robustly expressed at this stage
(Mootoosamy and Dietrich,
2002).
|
;
Noden, 1983a;
Wachtler and Jacob, 1986)
(n=4, Fig. 1B). The
expression of lateral rectus EOM markers was confined to the territory
adjacent to rhombomere 2, i.e. at the correct position
(Mootoosamy and Dietrich,
2002). When the same tissue was grafted anteriorly next to the
anterior midbrain (n=4; Fig.
1C) or posteriorly to otic levels (n=2;
Fig. 1D), the lateral rectus
markers were not expressed in the graft. However, normal expression was seen
at the usual site of the developing lateral rectus, next to rhombomere 2
(arrows). This was also the case when head mesoderm from anterior midbrain
levels (n=6; Fig. 1E)
or from otic levels (n=6; Fig.
1F) was heterotopically grafted to anterior hindbrain levels
(arrows). The normal development of muscle anlagen upon the heterotopic
grafting of anterior or posterior head mesoderm fragments to rhombomere 2
levels was confirmed by Myf5 staining
(Fig. 1G,H). Moreover, operated
and unoperated sides of the embryos looked identical (not shown). This
suggests that head muscles develop in tune with their local environment
(ortsgemäss). This furthermore suggests that extrinsic cues govern the
development of head muscles.
|
), they were the
first candidates in our quest to identify signals positively regulating head
muscle differentiation and specification. To investigate their role in vivo,
the neural folds and the dorsal third of the neural plate were ablated
bilaterally from diencephalic to otic levels at HH8-9-
(n=34), as indicated in Fig.
2A. This operation eliminates the source of neural crest cells and
prevents crest regeneration (Begbie et al.,
1999; Lumsden et al.,
1991; Veitch et al.,
1999). To confirm that this was the case, embryos at HH19 were
analysed for the expression of Dlx5, a marker for branchial arch
neural crest (Ferrari et al.,
1995) (Fig. 2B-E).
Indeed, operated embryos lacked neural-crest-associated Dlx5
expression (Fig. 2D,E, open
arrowheads). Moreover, tracing the nervous system with the RMO antibody, we
found the typical neuronal phenotype of neural-crest-deficient embryos, namely
a separation of the ophthalmic and the mandibulomaxillary aspect of the
trigeminal ganglion (Begbie et al.,
1999; Veitch et al.,
1999) (Fig. 2D,E,
oph, mmx). Next, we investigated the muscular phenotypes of the operated
embryos, judging the neural-crest ablated state of the embryos on the basis of
their neuronal phenotype. We found that the lateral rectus EOM markers were
appropriately expressed at the level of rhombomere 2 (Paraxis:
Fig. 2F-I, Lbx1 not
shown). Likewise, En2 correctly labelled the MAM
(Gardner and Barald, 1992)
(Fig. 2J-M). The Myf5
expression pattern indicated that in the absence of neural crest cells, the
head mesoderm occupied all the available space in the branchial arches.
Importantly, all differentiating muscle anlagen present in the wild type were
also present in the operated embryos (Fig.
2N-Q). Finally, MyoR, a marker for proliferative,
undifferentiated arch muscle precursors
(von Scheven et al., 2006) was
present, although expression domains remained proximally unsegregated
(Fig. 2R-U, small arrows).
Thus, neural crest cells seem to play a role in the spacing and separation of
muscle anlagen, but not in the initiation of muscle differentiation or EOM-MAM
specification.
Ablation of the eye
The eye is the target of the developing EOM, and in ZRDCT-An
anophthalmic mouse mutants lacking eyes, EOM are strongly reduced
(Paterson and Kaiserman-Abramof,
1981). To investigate whether the eye is responsible for EOM
differentiation and specification, at HH10 we unilaterally ablated the optic
placode and the part of the forebrain providing the optic cup
(Fig. 3A; n=11). After
reincubation for 48 hours, the embryos were assayed for the expression of the
lateral rectus markers and, using a Myf5 probe, for the presence of
head muscles in general. The ablation was controlled using the Pax6
antibody (Fig. 3B-E, brown
colouring). Upon staining, the embryos were bisected mid-sagittally, and both
halves were photographed and compared. We found that on the operated side only
a small remnant of the optic stalk was present, while the eye was lacking
(Fig. 3C,E, open arrowheads).
Significantly, Paraxis (not shown), Lbx1
(Fig. 3C, blue staining) and
Myf5 (Fig. 3E, blue
staining) were correctly expressed. Thus, early muscle differentiation and
specification does not rely on the presence of the muscles' target.
|
). To test
this hypothesis, lateral rectus innervation was prohibited by surgically
removing rhombomeres 5 and 6 at HH10 (n=10;
Fig. 4B). At HH19-20, the
nervous system was traced with the RMO antibody (brown staining), and the
lateral rectus EOM using a probe for Lbx1 (not shown) or
Paraxis (blue staining). The embryos were then mid-sagittally
bisected and viewed from the inside. On the unoperated side, the rootlets of
the abducens nerve were clearly visible in the ventral region of rhombomeres 5
and 6 (Fig. 4A, arrowheads),
with axons extending anteriorly, innervating the lateral rectus anlage (arrow)
(Wahl et al., 1994). On the
operated side, the abducens nerve was missing. However, Paraxis
expression was identical to the unoperated side, indicating that for the onset
of muscle development the nerve was dispensable.
|
The embryos were analysed 48 hours after the operation for the expression of lateral rectus EOM markers, for MAM markers and for muscle differentiation markers. Moreover, the nervous system was traced with the RMO antibody except in the case of notochord/floor plate separation experiments, in which the notochord and floor plate were stained for the expression of Shh. To examine marker gene expression in detail, transverse vibratome sectioning at the level of rhombomere 2 and the mandibular arch was performed. However, only the experiments separating neural tube and head mesoderm led to significant changes in gene expression of the markers and are shown here (Fig. 5).
Separation of head mesoderm and neural tube-marker gene expression
When neural tube and head mesoderm were separated at HH10, as shown in
Fig. 5A, Ai, then 48 hours
later, the lateral rectus EOM was well established on the control side,
expressing Lbx1 (Fig.
5B,D, arrows) and Paraxis
(Fig. 5E,G, arrows). On the
operated side however, both markers were strongly reduced or absent
(Fig. 5C,D,F,G, open
arrowheads). This loss of EOM-marker expression was not due to the loss of the
muscle anlage, as Myf5 was expressed on the unoperated, as well as
the operated, side (Fig. 5H-J,
arrows). Likewise, the balance between muscle proliferation and
differentiation was normal, as evidenced by the wild-type expression of
MyoR (Fig. 5K-M,
arrows). Thus, muscle differentiation commenced correctly, but the
specification as lateral rectus EOM had failed.
To investigate whether the muscle in the position normally occupied by the lateral rectus may have adopted the fate of the laterally adjacent MAM, we stained the embryos for En2 expression. On the unoperated side, En2 signals were confined to the prospective jaw closure muscles developing in the mandibular arch, staying clear of the more medially located anlage of the lateral rectus EOM (Fig. 5N, arrows). On the operated side however, En2 staining encompassed this muscle anlage (Fig. 5O,P, arrowheads). Thus, in absence of the neural tube derived signal, muscle in the position of the lateral rectus anlage differentiates, but lacks the lateral rectus markers and, instead, expresses markers for the mandibular arch muscles.
Separation of head mesoderm and neural tubeinnervation phenotype
The specification of abducens motoneurons depends on positional information
and patterning cues within rhombomeres 5 and 6
(Hernandez et al., 2004;
Moens et al., 1998). However,
the axons have to sample their environment to extend along the ventral surface
of the neural tube in an anterior direction and to identify the lateral rectus
muscle anlage, disregarding the laterally adjacent MAM and the more anteriorly
located remaining five EOM (Wahl et al.,
1994). It thus seems conceivable that lateral rectus specification
and the target recognition of the abducens nerve are linked. We therefore
blocked the neural tube derived signal for the lateral rectus muscle as
before, cultivated the embryos to HH19-20, double stained for
Paraxis-RMO and mid-sagittally opened the embryos to reveal the
innervation patterns on the unoperated
(Fig. 6A) and operated sides
(Fig. 6B,C; n=17). We
found that on the left, unoperated side, the Paraxis-expressing
muscle anlage (arrowhead) was innervated by the abducens nerve (arrow). On the
right, operated side that lacked Paraxis, the nerve showed a
defasciculated appearance typical for axons not readily finding a target
(Tosney and Landmesser, 1985)
(arrowhead). This suggests that the communication between the respecified
lateral rectus anlage and the abducens nerve was defective.
Properties of the signal specifying the lateral rectus EOM
Transmission of the neural tube derived signal
The lateral rectus eye muscle anlage resides in close proximity to the
neural tube before becoming engaged with the developing eye. Thus, its
specification may be achieved either via a soluble signal or through cell-cell
contact. To investigate this, we used the same strategy as for the earlier
barrier experiments; this time, however, inserting a 25-mm-thick filter with a
pore size of 0.05 µm between the anterior hindbrain and the head mesoderm.
This filter excludes cellular extensions but admits soluble factors
(Fan and Tessier-Lavigne,
1994) (n=16, Fig.
6D-F). The embryos were analysed for the expression of
Paraxis and double stained with the RMO antibody for their
innervation phenotype. Notably, on the operated side, both Paraxis
expression and innervation of the lateral rectus by the abducens nerve was
restored (Fig. 6E,F,
arrows).
|
). With
the lateral rectus EOM developing next to rhombomere 2, we asked whether the
neural-tube-derived signal required for the development of this muscle would
be rhombomere 2-specific. For this, two sets of experiments were carried out:
(1) heterotopic grafting of neural tissues
(Fig. 7); and (2)
transformation of rhombomere 2 via Hoxb1 misexpression
(Fig. 8).
Heterotopic neural tube grafting
Rhombomeres, when grafted to a posterior position, lose their positional
values unless the isthmus is included
(Irving and Mason, 2000).
Therefore, we transplanted at HH10 rhombomere 2 orthotopically as control
(Fig. 7B, n=3), but
heterotopically grafted rhombomere 2 plus the anteriorly adjacent r1, isthmus
and posterior midbrain to anterior midbrain-diencephalic levels
(Fig. 7C, n=3) or otic
levels (Fig. 7D, n=5),
and we grafted the posterior diencephalon-anterior mesencephalon
(Fig. 7E, n=4) or the
posterior hindbrain (Fig. 7F,
n=7) to midbrain-rhombomere 2 levels. The scheme of transplantations
is shown in Fig. 7A. In all
cases, the lateral rectus marker Lbx1 was expressed at the normal
site, indicating that the neural tube derived signal is not rhombomere
2-specific, and furthermore, not sufficient to trigger lateral rectus EOM
development at an ectopic location.
Hoxb1 misexpression
Hoxb1 controls the identity of rhombomere 4 and, when misexpressed
in rhombomere 2, homeotically transforms rhombomere 2 into rhombomere 4
(Bell et al., 1999). We
electroporated at HH10 either the pCAß-IRES-eGPF control vector
(Alvares et al., 2003);
(Fig. 8B-D, n=5) or
this vector expressing mouse Hoxb1
(Fig. 8E-G, n=11) into
the posterior midbrain-anterior hindbrain, using the conditions established by
Schubert and Lumsden (Schubert and
Lumsden, 2005) (shown schematically in
Fig. 8A). However, neither the
lateral rectus EOM marker Paraxis
(Fig. 8C,F) nor the muscle
marker Myf5 (Fig.
8D,G) were affected. Thus, consistent with our heterotopic
grafting experiments, the signal supporting lateral rectus EOM development is
not confined to rhombomere 2.
Test for neural tube derived signalling molecules
Our analysis suggested that a soluble, anteroposteriorly unrestricted
signal from the neural tube acts in lateral rectus EOM specification. Shh
derived from the floor plate and Wnt1 derived from the dorsal neural tube fit
this description (Brent and Tabin,
2002). We therefore inserted beads loaded with 100 µg/ml
recombinant Shh protein (n=6) or RatB1 cells engineered to release
Wnt1 (Cheng et al., 2004;
Fan et al., 1997;
Münsterberg et al., 1995)
(n=13) into the head mesoderm at the level of the future lateral
rectus EOM (Fig. 9A). As
controls, beads loaded with bovine serum albumin (BSA) (n=12) and
RatB1 fibroblast carrying the empty cloning vector (n=2) were used.
We expected to find an upregulation of the lateral rectus markers in response
to Shh or Wnt1. Instead, Wnt1 slightly, Shh strongly suppressed
Paraxis (see Fig. S1 in the supplementary material), in line with
their repressive function for head myogenesis described in
(Tzahor et al., 2003).
|
Signalling molecules known to influence branchial arch development are Fgf
and Bmp molecules (Helms et al.,
2005). Importantly, receptors for these molecules are expressed in
the head mesoderm and developing muscles (S.D., unpublished). Moreover, Fgf
and Bmp molecules are expressed in the pharyngeal endoderm at HH10, the time
MyoR expression commences (von
Scheven et al., 2006) (Fig.
9Ai,iii). Subsequently, elevated signals are found in the
pharyngeal pouches (endoderm plus ectoderm) and the oral ectoderm (shown for
HH19, Fig. 9ii,iv). Thus,
recombinant Fgf4, Fgf8, Bmp2, Bmp4 or Bmp7 protein was loaded onto beads at
500 µg/ml or 100 µg/ml. Subsequently, the beads were implanted into the
head mesoderm at the level of rhombomere 2
(Fig. 9A). As a control,
BSA-soaked beads were used (Fig.
9B-E). Beads loaded with Fgf molecules gave identical results;
here only the Fgf8 experiments at 100 µg/ml are shown (n=30,
Fig. 9F-O). Also all Bmp
molecules led to the same phenotypes. However, at 500 µg/ml, we observed
strong bilateral effects. Thus, we focus here on the phenotype obtained with
Bmp7 at 100 µg/ml, which left the control side intact (n=18,
Fig. 9P-W).
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). Although our heterotopic neural tube grafting and
Hoxb1 misxepression experiments deemed this unlikely, we tested
whether Fgf8 beads implanted into the head mesoderm might also re-pattern the
neural tube, thereby indirectly influencing muscle development. However, the
operation did not affect positional values in the neural tube, as monitored by
Hoxa2 expression (Fig.
9F,G, n=7). Yet Fgf8 had a strong effect on the
craniofacial muscle markers (Fig.
9H-O, n=39). The molecule downregulated the expression of
lateral rectus EOM markers (shown for Paraxis,
Fig. 9H,I, open arrowhead) and
upregulated the expression of the MAM marker En2
(Fig. 9L,M, arrowhead), thus
evoking a phenotype similar to that obtained by the neural tube-head mesoderm
separation experiments. Unlike those, however, Myf5 expression was
downregulated (Fig. 9J,K, open
arrowhead), while MyoR was upregulated
(Fig. 9N,O, arrowhead). Thus,
Fgf8 supported the MAM over the EOM fate, but at the same time kept the cells
specified as MAM in an immature, proliferative state.
The isthmus is a prominent source of Fgf8, but in the neighbouring EOM,
neither En2 nor MyoR expression are found. We therefore
tested the ability of the isthmus to signal to adjacent mesoderm, employing
limb induction from the flank lateral mesoderm as a well-established Fgf8
response assay (Cohn et al.,
1995). Fgf8-loaded beads (n=3) or HH10 posterior
midbrain-anterior hindbrain (n=6) were implanted at HH16 as described
by Lours and Dietrich (Lours and Dietrich,
2005). Forty-eight hours later, we found that the Fgf8 beads had
induced ectopic limbs carrying Fgf8-expressing apical ectodermal
ridges, while the isthmus, though maintaining its own Fgf8
expression, had not (see Fig. S2 in the supplementary material).
Effect of Bmp7
Bmp7 beads (Fig. 9P-W,
n=23) downregulated the lateral rectus markers (shown for
Paraxis, Fig. 9P,Q,
open arrowhead), but also reduced En2 expression in the distal area
of the mandibular arch (Fig.
9T,U, open arrowheads). Moreover, Myf5 expression was
eliminated, both in the area of the lateral rectus and in the mandibular arch
(Fig. 9R,S, arrowheads).
MyoR expression was slightly downregulated
(Fig. 9T,U, open arrowheads).
Thus, Bmp7 has a generalised effect on the developing head muscles, halting
their progress of differentiation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Despite
their importance, their development is still a mystery. In this study, we
aimed at shedding light onto the control of early muscle differentiation and
specification of head muscles derived from the non-somitic head mesoderm. We
focused on the region of the anterior hindbrain, because here eye muscles
forming outside the arches and jaw closure muscles developing within the
mandibular arch reside side by side. Moreover, besides Myf5, a
muscle-determining factor indicating the onset of muscle differentiation
(Noden et al., 1999), these
muscles differentially express a set of markers with Lbx1 and
Paraxis labelling the lateral rectus EOM
(Mootoosamy and Dietrich,
2002), En2 labelling the MAM
(Gardner and Barald, 1992) and
MyoR labelling the proliferative, undifferentiated MAM precursors
(von Scheven et al., 2006).
Thus, we are in the fortunate position of being able to simultaneously
investigate muscle differentiation and specification.
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). Moreover, the various head muscles develop at defined,
stereotype positions, and eventually, the lateral rectus EOM and the MAM
express distinct markers (Couly et al.,
1992; Gardner and Barald,
1992; Mootoosamy and Dietrich,
2002; Noden,
1983a; Wachtler and Jacob,
1986). In addition, the head mesoderm possesses positional
information, which it can superimpose onto stray neural crest cells
(Trainor and Krumlauf, 2000).
Thus, it was equally possible that head myogenesis is controlled by intrinsic
or extrinsic cues. Grafting fragments of head mesoderm into heterotopic
locations showed, however, that head muscle differentiation and specification
is controlled by signals from the local environment. Head mesoderm from
midbrain or otic levels grafted to the level of the anterior hindbrain
initiated myogenesis and expressed the lateral rectus markers in the same
fashion as the endogenous muscle anlage on the control side. Conversely, when
head mesoderm normally providing this muscle was moved to the anterior
midbrain or to otic levels, then muscle development proceeded in accordance
with the new position and the lateral rectus markers were not expressed. Thus,
while the head mesoderm is bound to employ a myogenic programme distinct from
myogenic programmes in the trunk, the onset of this programme and the
specification of individual muscles rely on instructive cues from the
environment.
Neural crest cells, target tissues and nerves are dispensable for early head muscle development
Classically, head myogenesis was thought to depend on neural crest cells
(Grammatopoulos et al., 2000;
Noden, 1983b;
Pasqualetti et al., 2000),
target tissues (Paterson and
Kaiserman-Abramof, 1981) and innervating nerves
(Engle, 2002). However, when
the head mesoderm was separated from these tissues through ablation
experiments then muscle differentiation and specification commenced correctly.
Thus, neural crest cells, target tissues and innervating nerves are not
necessary for early head muscle development in vivo. Our findings contrast
with recent in-vitro experiments showing that cranial neural crest cells
promote head muscle differentiation
(Tzahor et al., 2003). It has
to be noted, however, that in the trunk, somitic myogenesis is controlled by
various surrounding tissues, of which the dorsal neural tube and the surface
ectoderm show a degree of redundancy
(Dietrich et al., 1997). If
signal redundancy also occurs in the head, then the in-vivo role of the neural
crest cells may become masked by other tissues fulfilling a similar function.
However, in vitro the head mesoderm expressed a number of cardiac markers
(Tzahor et al., 2003). Thus,
it is possible that under the chosen culture conditions, the neural crest
cells were required to override the signals recruiting mesoderm for cardiac
development.
As our work suggests that neural crest cells and innervation are
dispensable for early head muscle development, this implies that the reported
role of neural crests cells and nerves affects head muscle development after
the onset of differentiation and specification. Remarkably, studies on somitic
myogenesis established that in the trunk, muscle, connective tissues and
nerves begin their development independently. However, after the completion of
this initial phase, these tissues become interdependent
(Hippenmeyer et al., 2004;
Kardon, 1998;
Pun et al., 2002). Thus, it is
possible that although the onset of myogenesis in head and trunk is controlled
by distinct programmes, eventually these programmes may converge. In the
trunk, connective tissue is mesoderm-derived, while in the head it stems from
neural crest cells (Couly et al.,
1993; Noden,
1983a). Moreover, in the trunk skeletal muscle is innervated by
somatic motoneurons, while in the head, the branchial arch muscles are
supplied by neurons resembling visceral motoneurons
(Jacob et al., 2001). It thus
will be crucial to investigate whether the distinct tissues serving the
similar functions employ related or different molecular cascades.
A soluble signal from the neural tube specifies the lateral rectus versus mandibular arch muscles
Searching for the extrinsic cues that might control the early events in
head muscle formation, we found that most tissues had no effect (or the effect
was masked through signal redundancy). However, the neural tube provided a
signal that promoted the expression of the lateral rectus EOM markers. In the
absence of the neural tube derived signal, while all muscle anlagen
differentiated at the appropriate position, Paraxis and Lbx1
expression was lost. Instead, the muscle anlage normally providing the lateral
rectus expressed En2, a marker for MAM
(Gardner and Barald, 1992).
This suggests that the neural tube specifies the lateral rectus eye muscle
versus the MAM.
Given the proximity of the neural tube to the lateral rectus, it was
conceivable that the neural tube might transmit its signal through cell-cell
contact. However, when a filter with a pore size of 0.05 µm was inserted
between neural tube and head mesoderm, thereby preventing cell-cell contact
but admitting soluble molecules (Fan and
Tessier-Lavigne, 1994), then the expression of the lateral rectus
markers was restored. This suggests that the neural tube releases a soluble
factor.
To make inroads into the identification of the neural tube derived signal, we performed heterotopic grafting of neural tissues and changed the identity of the neural tube next to the lateral rectus EOM. These experiments showed that the neural tube derived signal is not sufficient to trigger ectopic lateral rectus development. Moreover, the signal is not confined to rhombomere 2, which neighbours the lateral rectus. This suggests that the neural-tube-derived signal might act in a permissive, rather than instructive, fashion.
Shh provided by the floor plate and Wnt1 by the dorsal neural tube are
soluble signalling molecules that operate at all axial levels
(Brent and Tabin, 2002); they
hence fit the phenomenological description of the elusive neural tube derived
signal. However, in gain-of-function experiments, these molecules, instead of
promoting the expression of the lateral rectus markers, suppressed them. This
observation is in line with our earlier collaborative study showing a
repressive role for Shh and Wnt signals in craniofacial myogenesis
(Tzahor et al., 2003).
Branchial-arch-based Fgf8 antagonises the neural tube signal and specifies MAM
It is established that during trunk muscle development, signals from the
neural tube and lateral mesoderm act antagonistically to specify epaxial and
hypaxial muscle precursors (Parkyn et al.,
2002). As in the absence of the neural tube, MAM markers were
ectopically expressed, we asked whether signals from the branchial arches
might specify MAM but suppress EOM development.
Fgf8 and Bmp7 are known regulators of branchial arch pattern and
development; they are expressed in the pharyngeal endoderm, pharyngeal pouches
(endo- and ectoderm) and oral ectoderm and act in epithelial-mesenchymal
interactions (Helms et al.,
2005). Performing gain-of-function experiments, we found that
Fgf8, but not Bmp7, had a specific effect on EOM and MAM markers. When Fgf8
beads were implanted into the mesoderm destined to form the lateral rectus,
the EOM markers were lost and En2 was ectopically expressed. This
effect was not an indirect consequence of neural tube re-patterning, as
Hoxa2 expression was unaltered. The head mesoderm harbours Fgf
receptors (S.D., unpublished), and in Fgf8 hypomorph mouse mutants that
succeed in gastrulation specific responses of the head mesoderm to the loss of
the epithelial Fgf signal have been observed
(Frank et al., 2002). This
suggests that Fgf8 signals to the head mesoderm directly, simultaneously
supporting branchial arch muscle development and opposing the neural tube/EOM
system.
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).
However, the mesoderm next to the isthmus does not express En2.
Employing limb induction as well-established Fgf8 response assay, we found
that the ability of the isthmus to signal to neighbouring mesoderm is limited.
Thus, isthmic Fgf8 may not be available to the head mesoderm. It cannot be
excluded, however, that further factors found in the mandibular arch
environment but not at the isthmus are required to upregulate
En2.
Role of Fgf8 and Bmp7 in the control of head muscle differentiation
When head mesoderm and neural tube were separated, muscle differentiation
remained on course. However, Fgf8, in addition to its patterning role also
influenced muscle differentiation: MyoR, a marker for proliferative,
undifferentiated MAM muscle precursors
(von Scheven et al., 2006) was
upregulated by Fgf8 while Myf5 was downregulated. Thus suggests that
Fgf8, similar to its role in the trunk
(Itoh et al., 1996;
Kahane et al., 2001), may
expand the pool of muscle precursor in the head and may underpin muscle
growth.
In contrast to Fgf8, Bmp7 strongly downregulated Myf5, and more
mildly, MyoR. Thus, Bmp7 may inhibit the entry of head muscle
precursors into any myogenic programmes. It has to be taken into account,
however, that branchial arch mesoderm not only contributes to skeletal muscle
but also to the anterior heart field and subsequently to the outflow tract of
the heart (Kelly, 2005). Bmp
molecules are important regulators of heart development
(Brand, 2003). Hence it is
possible that Bmp7 acted on the branchial arch mesoderm, diverting it from a
skeletal muscle to a cardiac fate.
Eye muscle specification may be a prerequisite for the target recognition of the cognate nerves
When the head mesoderm was separated from the neural tube, leading to
erroneous expression of En2 in the muscle normally expressing
Paraxis and Lbx1, then the abducens nerve destined for this
muscle fell short of its target. Moreover, the nerve defasciculated,
indicating that the axons struggled to identify their target and hence were
actively sampling the environment (Tosney
and Landmesser, 1985). When instead of the impermeable foil
barrier, the filter was used, then marker gene expression and innervation of
the lateral rectus was restored. This indicates that the progress of axonal
outgrowth and target recognition was not hindered by the obstacle in its path.
Rather, the innervation phenotype was linked to the presence of
Paraxis/Lbx1 or the absence of En2 or both. This
suggests that the neural tube specifies the eye muscle to aid its innervation.
However, as Paraxis, Lbx1 and En2 are all transcription
factors, we can expect that ultimately, the specification of the lateral
rectus EOM leads to the production of cell surface or soluble axon guidance
cues.
The abducens-lateral rectus pair is one of the rare examples of nerve and
muscle not originating from the same axial level: the abducens nerve is born
in rhombomeres 5 and 6; the lateral rectus muscle develops next to rhombomere
2 (Wahl et al., 1994).
Moreover, it is only at rhombomere 2 levels that eye and branchiomeric muscles
develop side by side (Couly et al.,
1992; Noden,
1983a; Wachtler and Jacob,
1986). Furthermore, this region is traversed by a number of
cranial nerves, with the abducens nerve arriving from posterior regions, the
oculomotor nerve arriving from anterior regions and the maxillo-mandibular
branch of the trigeminal projecting laterally into the mandibular arch
(Chilton and Guthrie, 2004;
Wahl et al., 1994). Thus, is
it possible that signalling events and marker gene expression serve a unique
function in head muscle development in this region. However, Fgf8 as signal to
specify MAM is expressed in all branchial arches, and in hypomorph Fgf8 mouse
mutants, severe pharyngeal and aortic arch defects were observed
(Abu-Issa et al., 2002;
Frank et al., 2002) (reviewed
in Helms et al., 2005). Thus,
it is likely that the neural tube-Fgf8/arch antagonism is a global regulator
of eye versus branchiomeric muscle development.
Model
Summarising our results (Fig.
10), we propose that at a soluble signal from the neural tube
(green), together with a further factor, specifies the neighbouring muscle
anlage as Paraxis/Lbx1-positive lateral rectus EOM (yellow).
Simultaneously these signals prevent the expression of the MAM marker
En2. The correct specification of the lateral rectus then facilitates
the innervation by the abducens nerve. Within the branchial arches, however,
the presence of Fgf8 (blue) ensures that EOM markers are repressed while MAM
markers (magenta) are supported. Thus, the neural tube derived signal and Fgf8
act antagonistically in head muscle specification. Yet, Fgf8 has an additional
role in preventing MAM differentiation, maintaining the cells in a
proliferative state.
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