Institute of Neuroscience, 1254 University of Oregon, Eugene, OR
97403-1254, USA
Present address: Department of Developmental Biology, Beckman Center B300, 279
Campus Drive, Stanford University School of Medicine, Stanford, CA 94305-5329,
USA
* Author for correspondence (e-mail: kimmel{at}uoneuro.uoregon.edu)
Accepted 3 December 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Endothelin 1, Pharyngeal arch, Branchial arch, Operculum, Dermal bone, Morphogen, Zebrafish
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The differences along the DV axis may have had their origins in the
earliest osteichthyans (McAllister,
1968), the clade that includes all living bony fish and the basal
extinct paleoniscid shown in Fig.
1B. In an outgroup, acanthodians, the bones of the hyoid series
are all branchiostegal-like, similar in shape and size. Therefore, an
evolutionary scenario leading to the condition in a derived teleost such as
the zebrafish would include (1) the establishment of a DV size gradient within
the series, (2) further modification of the dorsal-most element that includes
new muscle attachments and its new highly functional joint, and (3) the
reduction in the number of ventral elements.
Here we report studies supporting the idea that changes in developmental
regulation involving a gene network controlled by a single conserved
intercellular signaling molecule, endothelin 1 (Edn1), may have been involved
in all three of these evolutionary changes. Edn1 is expressed in the embryonic
pharyngeal arches, where these bones develop. Reduced function of the
Edn1-controlled network produces a prominent skeletal phenotype shared among
mice, chickens and zebrafish namely the reduction or loss of specific
pharyngeal cartilages (Kurihara et al.,
1994; Clouthier et al.,
1998
; Clouthier et al.,
2000
; Yanagisawa et al.,
1998
; Kempf et al.,
1998
). Prominent among these affected elements is Meckel's
cartilage of the embryonic lower (ventral) jaw, the mandible, suggesting that
Edn1 plays a key role in pharyngeal skeletal patterning in all jawed
vertebrates, the gnathostomes (Miller et
al., 2000
; Kimmel et al.,
2001a
). The gene encoding the Edn1 ortholog in zebrafish was first
identified in a genetic screen: a single allele was recovered and named
sucker for the prominent facial phenotype of mutant larvae
(Piotrowski et al., 1996
).
Subsequent work establishes that the mutant phenotype is because of a severe
loss of function of the sucker (edn1) gene
(Miller et al., 2000
).
Phenotypic analyses show that the zebrafish Edn1 network regulates development
of ventral pharyngeal cartilages in both the mandibular (first) and hyoid
(second) arch, is required for aspects of dorsal cartilage development in the
same arches, and is also required for development of the joints normally made
between the dorsal and ventral cartilage in both arches
(Piotrowski et al., 1996
;
Kimmel et al., 1998
;
Miller et al., 2000
;
Miller and Kimmel, 2001
). Edn1
may function generally in gnathostomes to specify skeletal pattern along the
DV axis of the pharynx.
How Edn1 plays this DV patterning role, particularly the patterning of
ventral cartilage and joint development, is beginning to be unraveled
(Miller et al., 2000;
Miller et al., 2003
). The
expression of the edn1 gene is complex and dynamic. Prominent
expression domains located ventrally in the pharyngeal arch primordia
correlate with the prominent loss of function phenotype of ventral cartilage
reduction. Both ventral epithelium and the ventral mesodermal arch cores
express edn1; the secreted Edn1 protein probably acts directly on the
postmigratory neural crest-derived ectomesenchyme that differentiates into
ventral cartilage. Immediate responses in the ventral postmigratory crest to
Edn1 signaling include the initiation and/or maintenance of transcription of
several developmental regulatory genes, including genes encoding transcription
factors such as Goosecoid and Hand2 (dHAND). These genes are also Edn1 targets
in the mouse (Clouthier et al.,
1998
; Clouthier et al.,
2000
; Thomas et al.,
1998
; Charité et al.,
2001
), suggesting broad conservation of aspects of the entire
genetic regulatory system among gnathostomes. Expression studies in zebrafish,
particularly of the homeobox gene bapx1 in the mandibular arch, also
show that the joints develop, under control of Edn1, from an intermediate
region along the DV axis of neural crest-derived mesenchyme that appears
remote (but only slightly so) from the ventral Edn1 source. These findings
suggest that secreted Edn1 can act at a distance from its source
(Miller et al., 2003
).
In contrast to the functions of Edn1 in cartilage patterning, its role in development of pharyngeal bones in zebrafish was unknown. Here we describe how reducing Edn1 function affects development of these bones in the young larva, with special focus on the dermal bones of the hyoid arch. The severe loss of function phenotype is dramatic; every bone that is normally present in the pharynx of a 1-week old larva is missing or modified in edn1 mutants. Dermal bones developing in the mandibular arch are malformed and show polarity reversals in mutants, consistent with the cartilage phenotypes in this arch. Lowering Edn1 results in an outstanding variety of dermal bone phenotypes in the hyoid arch. The changes involve both the opercle and branchiostegal rays, and include bone loss, expansion, shape change, fusion and probable homeotic transformation. We provide evidence that these remarkably different bone phenotypes result from lowering edn1 gene function to different levels, and we interpret the results to mean that bone patterning is exquisitely sensitive to the strength of the Edn1 signal. Our data support the hypothesis that Edn1 acts as a morphogen, regulating bone development along the DV axis in a concentration-dependent manner. Changes in, or in responses to, the Edn1 gradient may have underlain evolution of the branchiostegal-opercle series in teleosts and their ancestors.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Larval skeletal and neuromast staining
Alcian Green was used for cartilage staining in whole fixed larvae as
described (Kimmel et al.,
1998; Miller et al.,
2000
), and cartilages dissected and prepared as flat mounts
(Kimmel et al., 1998
).
Developing bone matrix was usually labeled by overnight immersion of live or
fixed larvae in 50 µg/ml Calcein (Molecular Probes, Eugene, OR) made in
Embryo Medium (Westerfield, 1995), followed by five rinses in Embryo Medium to
remove excess dye. For double labeling of bones and neuromasts, the larvae
were first vitally stained overnight with 4 µg/ml of Alizarin Red S (Sigma,
St Louis, MO), and then rinsed and stained for 10 minutes with 40 µg/ml of
DASPEI (Molecular Probes), followed by five rinses. Immunocytochemistry with
the zns5 monoclonal antibody (see Johnson
and Weston, 1995
) was performed as described by Maves et al.
(Maves et al., 2002
).
Image acquisition and processing
Bone and neuromast phenotypes in the Calcein or Alizarin Red-DASPEI
preparations were scored at a magnification of 50x, with a Leica MZ
FLIII stereomicroscope equipped with epifluorescence optics. Higher
magnification images were obtained with a Zeiss Pascal confocal microscope:
z-series were captured through the pharyngeal regions of interest of larvae
mounted at orientations to provide optimal views of the bones. We studied the
bones in projection views made from the z-stacks (Zeiss software); generally
we processed the images with Adobe PhotoShop to enhance contrast and decrease
background. Several of the figures below (e.g.
Fig. 2) show black and white
negative images (to improve reproduction) of the fluorescent bones in such
projections. Alcian Green-labeled flat-mounted cartilages were photographed
with differential interference contrast (DIC) optics, using a Zeiss Axiophot
microscope (Kimmel et al.,
1998).
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We observed that all of the cartilage replacement bones of the anterior two arches are invariably absent in sucker(edn1)tf216b mutants (edn1-). The dermal bones are absent or malformed. Below we explore the condition of the dermal bones of the mandibular arch and especially the hyoid arch in some detail. Briefly, in the mutant mandibular arch, the maxilla and dentary are fused together, misoriented and misshapen. In the mutant hyoid arch, branchiostegal rays are absent (but see below), and the opercle is either absent or alternatively the opercle is present and enlarged (Fig. 2B).
The dermal jaw bones
Development of the dermal bones of the mandibular arch (first pharyngeal
arch, forming the upper and lower jaws) is severely perturbed when Edn1 is
lowered, either as in edn1 mutants
(Fig. 3), or by the
translation-blocking edn1 morpholino (edn1-MO). In wild-type
larvae, a dentary is positioned superficially to Meckel's cartilage in the
lower (ventral) jaw on each side of the midline. It is fan-shaped posteriorly
(Fig. 3A) and anteriorly curves
(Fig. 3B), deep to the lower
lip. At this stage the bilateral dentaries do not yet join together at the
anterior midline, but are well separated. The maxilla, anterior and
superficial to the pterygoid process of the palatoquadrate cartilage in the
upper (dorsal) jaw, is more dorsal-ventrally oriented than the dentary (best
seen in the side view in Fig.
3A).
In edn1 mutants, the mouth is wide open and is extremely
malformed. The lower jaw points in the wrong direction. It is reversed in
anterior-posterior orientation, such that the lower lip curves posteriorly
rather than anteriorly, and is located ventral to the eyes rather than
anterior to the eyes as in the wild type. In place of the two bilateral pairs
of mandibular arch dermal bones curving anteriorly in the wild type, in the
edn1 mutants or edn1-MO-injected larvae we frequently
observe a thin, bilateral wicket-shaped single element with a posterior
curvature that follows the outline of the mouth, just deep to the lips
(Fig. 3C). However, the
expressivity of the phenotype varies among individual mutants. Sometimes there
are gaps in the bone at the midline and approximately midway along the arms of
the wicket (arrows, Fig. 3D,E).
We infer from these patterns (and also from phenotypes in
edn1-MO-injected embryos; see legend to
Fig. 3) that the wicket
includes the rudiments of both dentaries and maxillas, fused together more or
less completely in different individual mutants. The dentary would represent
the posterior part of the wicket reversed in anterior-posterior polarity, as
suggested from the curvature of the bone and the way it follows the lower lip
(as in the wild type). We note that the cartilage phenotypes in the same arch
of edn1 mutants are interpreted similarly: the dorsal cartilages and
the remnants of the ventral cartilages are invariably fused to one another,
and the anterior-posterior polarity of the ventral cartilage is reversed
(Piotrowski et al., 1996;
Kimmel et al., 1998
) (see
Discussion).
The opercle loss-gain phenotypes
The second or hyoid arches of the larva 1-week after fertilization include
the paired rudiments of two cartilage replacement bones (hm, ch;
Fig. 2) and at least two
elements of the dermal branchiostegalopercle series (op, bsrp, and
variably at this stage bsrm,
Fig. 2). All of the bones are
missing in some edn1 mutants. In others, only a bone fragment is
present, located dorsally in the segment, approximately at the normal position
of the opercle but much smaller than the wild-type opercle and lacking its
characteristic fan shape. In yet other individuals, we could easily recognize
an opercle. Curiously and almost invariably in the latter class the opercle
was enlarged, usually markedly so (Fig.
4). Such contrasting phenotypes, ranging from complete absence to
marked expansion, resulting from the same genetic lesion as we observe for the
opercle in edn1 mutants is unusual. To emphasize the contrast we
refer to the opercle loss or reduction as the `loss' phenotype, and to the
opercle expansion as the `gain' phenotype.
|
The opercle normally makes an articulating joint with a dorsal hyoid
cartilage, the hyosymplectic, and the expansion of the gain opercles in
edn1 mutants invariably involves the bone around this joint region
(Fig. 4). The hyosymplectic
cartilage is present in edn1 mutants, although often considerably
changed in appearance (Kimmel et al.,
1998). DIC imaging of both bone and cartilage together in mutant
larvae revealed that the gain opercle makes an enlarged articulation with the
cartilage at approximately the correct location
(Fig. 4B,D). In contrast, in
opercle loss examples that include a bone fragment (i.e. as opposed to opercle
loss examples in which the bone is completely missing), the fragment is
considerably removed from the cartilage, located superficially in dermal
mesenchyme just beneath the epidermis (not shown). Another feature of the
opercle-gain phenotype revealed by DIC imaging is the frequent presence of
ectopic muscles attaching to the enlarged bone. For example, the arrow in
Fig. 4D shows a muscle
projecting to the blade of the opercle from an anterior location. Normally no
such muscle is present.
What accounts for such contrasting loss versus gain opercle phenotypes? The
phenotypes seem unlikely to be entirely because of difference in genetic
background, because in many cases we see a loss phenotype on one side, and a
gain phenotype on the other side of the same mutant larva. Furthermore, the
mutant phenotype is not limited to edn1 mutants. Mutations at three
other loci unlinked to edn1 are all grouped in the same phenotypic
class as edn1 mutants, termed the `anterior arch class', and some or
all of these genes, schmerle (she), sturgeon
(stu) and hoover (hoo), may function along the
edn1 genetic pathway (see Piowtroski et al., 1996;
Kimmel et al., 1998;
Miller and Kimmel, 2001
) (C.
T. M. and M. W., unpublished). We observed the opercle-gain phenotype in
mutants at each of these three other loci, and the opercle-loss phenotype in
two of them (Table 1;
branchiostegal ray phenotypes are discussed below). Hence, the opercle
loss-gain phenotypic pair is not locus-specific.
|
Study of histological sections (not shown) reveals that all bone in early
zebrafish (whether dermal or cartilage replacement) is acellular (reviewed by
Beresford, 1996) (see also
Parenti, 1986
), formed by
osteoblasts present along the surfaces of the bone matrix. Dyes such as
Calcein or Alizarin Red stain bone matrix but not the osteoblasts, which can
be labeled with the monoclonal antibody zns-5
(Johnson and Weston 1995
). In
early wild-type larvae a prominent cluster of osteoblasts delineates the
developing opercle (Fig. 5A,
arrow; a cluster of branchiostegal ray osteoblasts is also present at the
asterisk). Comparing immunoreactivity in wild type and stu (not
shown) or she mutants revealed two phenotypes, the probable cellular
correlates of the loss and gain opercle phenotypes shown by the matrix
labeling. Some mutants had a reduction of the size of the opercle osteoblast
population (Fig. 5B) and others
showed an expansion (Fig. 5C).
These findings suggest that the number of embryonic cells recruited as
osteoblasts ultimately determines whether a loss or gain opercle phenotype
will form.
|
The opercle-loss phenotype results from severe reduction of Edn1 and
the gain phenotype results from milder reduction
What determines how many opercle osteoblasts develop? The data in
Table 1 suggest an explanation
for the loss-gain opercle phenotypic pair. We propose that lowering Edn1
function to different levels results in the different phenotypes: we observed
only the opercle-gain phenotype in the clutch of hoo mutants scored
(Table 1) and, because
hoo- typically has the mildest cartilage and facial
phenotypes of all of the anterior arch mutants, this result suggests that a
mild reduction of Edn1 results in the gain phenotype and a more severe
reduction results in the opercle-loss phenotype. We tested this postulate
directly by reducing Edn1 function to different levels with
edn1-MO.
Injecting the edn1-MO at moderate or high levels reliably
phenocopies in detail the edn1 mutant defects in gene expression,
cartilages and facial appearance (Miller
and Kimmel, 2001). When injected at lower levels, the
edn1-MO phenocopies the facial and cartilage defects of the other
anterior arch mutants, resulting in a phenotypic series according to the level
of Edn1 reduction, namely hoo- (the mildest phenotype,
usually obtained with the lowest effective amount of edn1-MO) <
stu- < she- <
edn1- (the most severe, obtained with highest levels)
(Miller and Kimmel, 2001
).
In the present study, injecting the same morpholino over the same 30-fold
concentration range studied previously yielded both the opercle-gain and -loss
phenotypes, in proportion to the amount of edn1-MO. At the highest
useful level we examined (15 ng), edn1-MO specifically phenocopies
all defects in pharyngeal bones of the edn1 mutants, with the notable
change in several experiments that we observed a higher fraction of
opercle-loss phenotypes than we usually observe in edn1 mutants (85%
of injected larvae, n=163; compare with
Table 1). The increase might be
because of differences in genetic background, or might mean that the single
available mutant allele, even though severe, is not a null allele
(Miller et al., 2000). In the
experiment quantified in Fig.
6A-C the opercle-loss phenotype still predominated when the
morpholino was injected at 5 ng (A: opercle ranks 1 and 2, totaling 75%).
However, at 0.5 ng the opercle-loss phenotype becomes the minority class (C:
19%), and opercles of normal size and with the gain phenotype predominate
(respectively in Fig. 6C, wild
type; 44%, and opercle gain ranks 3 and 4; 35%). In other experiments with the
lower amounts of edn1-MO, as many as 50% of the injected larvae showed the
opercle-gain phenotype. Our findings suggest that as predicted, the
opercle-gain phenotype results when Edn1 is only mildly reduced, and the loss
phenotype results from reduction that is more substantial.
|
|
|
DIC observations during the course of this study revealed that opercular
neuromasts, sensory organs of the anterior lateral line system, were
frequently missing in anterior arch mutants and in edn1-MO-injected
larvae. We used two-color methods to score the bone and lateral line
phenotypes together in a set of edn1 mutants (data not shown) and in
the set of edn1-MO-injected animals used for
Fig. 6, and observed strong
correlation between the severities of the two phenotypes. Neuromasts occur at
stereotyped and largely invariant positions along this region of the pharynx,
as well described by Raible and Kruse
(Raible and Kruse, 2000). In
most uninjected larvae at 6 or 7 days postfertilization the operculum bears
two neuromasts located just superficially to the opercle (83%,
Table 2;
Fig. 7B, OP1, OP2). There is a
mild reduction in the number of opercular neuromasts in
edn1-MO-injected larvae that show the opercle-gain phenotype (74%
have both OP neuromasts, Table
2; Fig. 7C).
However, only 5% of opercle-loss arches have both opercular neuromasts; rather
they have one (60%) or none at all (35%;
Fig. 7D). Other neuromasts in
the vicinity of the operculum are also disrupted by compromising Edn1. These
other neuromast changes are less frequent than the loss of opercular
neuromasts, and include duplications as well as losses
(Table 2 includes the scores
for one of the more severely affected of these, neuromast M2 located just
dorsal and superficial to the opercle-hyosymplectic joint). Similar to the
losses of the neuromasts OP1 and OP2, we observed these losses in animals
injected with any of the three MO concentrations, and they occurred most
frequently in arches also exhibiting the opercle-loss phenotype (e.g.
Fig. 7D). Hence, as for the
cartilage-bone phenotypes, the severities of neuromast-bone phenotypes often
vary together within a single arch.
|
|
DV identity of hyoid bones; homeotic shift between branchiostegal ray
and opercle
Similar to the opercle, the hyoid arch branchiostegal ray phenotype also
changes according to how severely Edn1 is lowered. At 6 days
postfertilization, one and usually two branchiostegal rays are present ventral
to the opercle. The posterior and more dorsal ray (bsrp,
Fig. 2) appears earlier in
development (our work in progress). The branchiostegal rays are often missing
(or sometimes one is present but reduced in size) when Edn1 is severely
lowered as in edn1 mutants (Figs
2,
4;
Table 1). However, at the
minimum, the more dorsal branchiostegal ray develops after only milder Edn1
reduction, as apparently in she, stu and hoo mutants
(Table 1) and in larvae
injected with the lower amounts of edn1-MO (not shown). Formation of
a branchiostegal ray is apparently more sensitive to reduction of Edn1 than
the opercle; the fraction of branchiostegal loss is always higher than the
fraction of opercle loss, and furthermore we never observed the converse
situation, i.e. an individual hyoid arch in which the opercle is absent and a
branchiostegal ray is present. Rather, in general, the presence of a
branchiostegal ray is associated with the opercle-gain phenotype. As for the
opercle phenotype, our data suggest that presence or absence of staining of
branchiostegal ray bone matrix (e.g. with Calcein) correlates with presence or
absence of branchiostegal osteoblasts labeled with zns-5
(Fig. 5).
In cases in which a branchiostegal ray is present, it is abnormally close to the opercle, and is sometimes malformed (Fig. 8). Normally the branchiostegal rays are saber-shaped (Fig. 8A). In she and stu mutants and in edn1-MO-injected larvae the branchiostegal ray is often sickle- or scimitar-shaped (Fig. 8G, and extreme case in Fig. 8H), curved with the convexity toward the opercle. Further, in what we term the `walking stick' phenotype, the opercle and dorsal branchiostegal ray are fused together (Fig. 8B,D,F). Fig. 8K shows a dramatic example of fusion, in which a thin continuous sheet of bone connects the opercle and branchiostegal ray.
The bone fusions suggest that after a mild reduction of Edn1 the opercle and branchiostegal ray are becoming more similar to one another in character i.e. that we are observing homeosis. Antibody labeling seems to support this interpretation; for example, the osteoblast labeling in the she mutant shown in Fig. 5C is suggestive that branchiostegal and opercle osteoblast populations are fused together at the region where the bones attach to the cartilage. Furthermore, the walking stick-branchiostegal ray makes a differentiated joint with the underlying ceratohyal cartilage resembling the joint the opercle makes with the hyosymplectic; a particularly clear example of the branchiostegal joint region bone is shown in Fig. 8F. Normally a branchiostegal ray does not make such a distinctive structure; a blunt end of the bone (uppermost end in Fig. 8A) is simply bound to the ceratohyal by connective tissue.
A rare phenotype in hoo mutants is that along with making a joint, the branchiostegal ray enlarges (in 4% of the mutants, Table 1) and strikingly, can take on a fan shape (Fig. 9). Here the dorsal branchiostegal ray might be homeotically transformed, rather completely, into an opercle.
These branchiostegal ray phenotypes, similar to those of the opercle
described above, appear to be because of change in Edn1 level, and not because
of some other changes associated with mutations at these several loci. To
demonstrate this we added back Edn1 into edn1 mutants, either as
human EDN1 protein or as a wild-type zebrafish edn1 DNA
(Miller et al., 2000). Again,
at low frequency we observed both the walking stick phenotype and the
branchiostegal-opercle transformation phenotype
(Fig. 10). As indicated by the
absence of joints between the palatoquadrate and Meckel's cartilage
(Fig. 10, *), only partial
rescue was obtained in both cases, suggesting that [Edn1] was lower than in
the wild type.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dermal bone fusions in the mandibular and hyoid arches
The two anterior pharyngeal arches, mandibular and hyoid, are understood to
be serially homologous pharyngeal segments, supported by the role of Edn1 in
specifying DV pattern in both (reviewed by
Kimmel et al., 2001a). Among
the phenotypes caused by lowering Edn1 are fusions between ventral and dorsal
cartilages in both arches. The dermal bone phenotypes extend this evidence: we
suggest that both the `wicket' phenotype in the mandibular arch and the
`walking stick' phenotype in the hyoid arch represent abnormal fusions between
dorsal and ventral dermal bones, reminiscent of the DV cartilage fusions.
However, there are interesting differences. The cartilages that fuse normally
articulate with one another; the fusions represent missing joints. This is
true for neither of the bone fusions. Whereas the DV cartilage fusions occur
with mild Edn1 loss in both arches (Miller
and Kimmel, 2001
), dermal bone fusions resulting from mild Edn1
loss occur only in the hyoid arch, not in the mandibular arch. Furthermore,
the way the bone wicket follows the line of the lips in the mandibular arch
has no counterpart in the hyoid arch. Grafting studies have suggested a role
of the oral epithelium in skeletal patterning in the chick
(Tyler and Hall, 1977
).
Similarly, in zebrafish the lip epithelium may be involved in patterning
mandibular dermal bone in a way that is not mirrored in the hyoid arch.
A morphogen gradient model of bone patterning
Our loss of function analyses strongly suggest that the hyoid arch bone
phenotype is exquisitely sensitive to the level of the edn1 gene
product, a secreted peptide. The findings extend previous understanding of
edn1 function derived from analyses of pharyngeal cartilages. The
gene is expressed segmentally by ventrally located arch epithelia and mesoderm
in the embryonic pharyngeal walls (Miller
et al., 2000). The Edn1 peptide is secreted, such that it can
serve as an extracellular signal acting on postmigratory skeletogenic neural
crest cells. As a consequence, these cells transcriptionally upregulate target
genes, such as the bHLH transcription factor-encoding gene hand2. In
edn1 mutants and in other anterior arch mutants the ventral
cartilages are variably reduced and misoriented
(Piotrowski et al., 1996
;
Kimmel et al., 1998
). As shown
previously (Miller and Kimmel,
2001
) and also by this work, more severe ventral cartilage
reductions correlate with more severe loss of Edn1, implying a developmental
role of signal strength.
In common among all of the anterior arch mutants is the loss of joints
between the dorsal and ventral cartilages of the first two pharyngeal arches,
resulting in cartilage fusions. Edn1 appears to pattern joint development in a
domain that lies either entirely or mostly more dorsal and not contiguous with
the ventral signal source in the embryonic pharyngeal walls
(Miller et al., 2003). This
geometry also implies a role of signal strength, for we suppose that a ventral
signal locally patterns crest cells to make ventral cartilage, and represses
cartilage formation in slightly more dorsal cells, such that they make a joint
rather than cartilage. In this model, the ventral cells respond to a higher
level of the signal than the more dorsal ones.
Our new findings also support a model in which the bone phenotype depends not only on the amount of signal released, but also on the DV position within an arch of the responding cells. For example, extreme reduction of Edn1, as in injections of the highest levels of the edn1-MO, usually leads to the complete loss of both the ventral and dorsal bones in the hyoid arch (the ventral branchiostegal rays and the dorsal opercle). With only moderate loss of signal, the dorsal bone frequently persists and indeed, is enlarged, but ventral branchiostegal rays are usually missing. With mildest reduction of Edn1, both ventral and dorsal bones persist although their shapes and sizes are abnormal. Dorsal structures respond to a ventral signal, suggesting that the signal acts at a location remote from its source. Ventral structures are more sensitive than dorsal ones to reduced signal, suggesting that higher levels of signal normally pattern the ventral bones and lower levels pattern the dorsal ones. These findings are consistent with the hypothesis that Edn1 functions as a morphogen in pharyngeal skeletal development: diffusion (or transport) of the peptide away from its ventral source sets up a concentration gradient to which the postmigratory neural crest cells differentially respond according to their DV positions in the arches. Fig. 11 depicts how the gradient model works to explain normal DV positioning of the branchiostegal ray and opercle (A), and how lowering the gradient to a more moderate level predicts both loss of the ventral bone and expansion of the dorsal one (B). Lowering the gradient still further predicts the observed loss of both bones (not shown).
|
The gradient model makes a prediction that we do not observe; namely loss of DV joints between cartilages in the anterior arches is more sensitive to Edn1 reduction than is loss of more ventral cartilage. By the model just outlined we expect the opposite, the more ventral tissue should be the more sensitive one (just as we observe for hyoid bone development). To explain this discrepancy, one can postulate that DV joint position is determined by an Edn1 gradient, but that joint differentiation requires additional Edn1-sensitive factors.
A morphogen gradient hypothesis provides a simple explanation for much of
the phenotypic variation we observe, yet many factors must determine the
cellular response to the gradient. For example, the hyoid dermal bone fusions,
the formation of joints by branchiostegal rays, and, in hoo mutants,
branchiostegal ray shape transformation toward opercle (ventral toward
dorsal), suggests that Edn1 is playing some role not only in positioning where
bone develops, but also in specifying the character or `identity' of the bone.
Reduced signal can be interpreted by ventral bone-forming cells (in the sense
of the French Flag model of Wolpert)
(Wolpert, 1971) to mean that
they are developing at a dorsal position more remote from a ventral
source, hence accounting for the transformation toward the shape of the dorsal
bone.
Patterning how much bone develops: negative regulation and the
opercle-gain phenotype
Development of the opercle depends on Edn1, as revealed by its loss when
the signal is severely lowered. In contrast, in the opercle-gain phenotype, a
dramatically enlarged opercle develops. By the morphogen hypothesis it is the
gradient of Edn1 in the arch that determines the mutant phenotype (i.e. the
level at the ventral source and the slope, as in
Fig. 11). An alternative
scenario, that the signal is expressed locally to the bone, is suggested by
studies in the mouse showing Edn1 expression within bone-forming primordia
(Sasaki and Hong, 1993;
Kitano et al., 1998
). However,
this explanation seems unlikely in zebrafish: the only arch mesenchyme in
which we have observed edn1 RNA expression is ventral mesoderm, and
although our fate mapping of the embryonic pharyngeal arches is preliminary,
the opercle appears to arise from postmigratory neural crest located dorsally
in the arch primordia, distant from the Edn1 source (J. G. C., unpublished
observations). Hence, in zebrafish the dermal bone primordia may not express
the gene. Furthermore, we observed that the severities of both the cartilage
and lateral line neuromast phenotypes correlate with the severities of opercle
phenotypes (gain versus loss) in the same hyoid arch. These correlations might
not be expected if only local expression of the edn1 gene within the
bone primordia determined the bone phenotype.
Correlated bone-cartilage and bone-neuromast phenotypes might also be
indicating the presence of patterning interactions between these tissues, even
if an Edn1 gradient is providing the positional information for where these
interactions occur. The correlations are strong but they are not absolute,
which might argue against local tissue interaction. Nevertheless, it is
reasonable to suppose that interactions occur between the two tissues
contributing to a joint, such as the hyosymplectic cartilage and the opercle
bone. Ablation or transplantation experiments could reveal such interactions.
Interactions between neuromasts and underlying dermal bone have been
postulated for many years; e.g. in positioning lateral line canals that run
through dermal bones (Allis,
1889; Parrington,
1948
; Webb, 1989
).
Mutations are available that block zebrafish neuromast development
(Whitfield et al., 1996
), but
it is not known if hyoid bones are affected in these mutants.
Regardless of the patterning mechanism, a key finding from our study is
that with reduction of the Edn1 signal the size of the opercle expands,
meaning that Edn1 negatively regulates dermal bone size. A role of Edn1 as a
negative upstream regulator in zebrafish was previously suggested
(Piotrowski et al., 1996),
from an observation that dorsal cartilages are expanded in the loss of
function edn1 mutant. A more recent example is in muscle patterning.
Dorsal muscles in the mandibular arch specifically express an Engrailed
homeobox gene, eng2. In edn1 mutants the eng2
expression domain is expanded ventrally, showing that the ventral signal
directly or indirectly is functioning as a repressor, and probably at some
distance from its source (Miller et al.,
2003
).
Hyoid dermal bone evolution and homeosis
Negative regulation is a well-known phenomenon in developmental genetics,
but is generally not considered in discussions of how evolution of development
works. Mutation toward loss of function is of course much more probable than
mutation toward gain of function, and when the mutated gene is a negative
regulator, the loss of function mutation results in an expanded rather than a
reduced phenotype, here with respect to bone size. More bone comes from less
genetic function, and this certainly has important implications for
evolutionary change.
The highly specialized dermal bone pattern in the zebrafish hyoid arch
might derive evolutionarily from a long DV series of uniform branchiostegal
ray-like elements (McAllister,
1968) (Fig. 1).
Changes in Edn1 regulation might underlie the evolutionary changes. We
discovered that Edn1 negatively regulates the size of the opercle, the
dorsal-most element of the branchiostegal-opercle series in zebrafish. Hence a
ventral to dorsal gradient of Edn1 accounts for a dorsal to ventral gradient
of bone size that apparently evolved in early osteichthyans. The evolving
opercle would have gained new muscle attachments and a more efficient hinged
joint. We observed that when Edn1 is mildly reduced the opercle-gain phenotype
includes ectopic muscle attachments (Fig.
4), that a branchiostegal ray can develop a joint region
resembling that of the opercle and develop the opercle's fan shape. Finally,
when Edn1 is severely reduced (as in edn1 mutants), branchiostegal
rays are absent, correlating with the evolutionary loss of branchiostegal rays
that occurred in parallel in several teleost lineages
(McAllister, 1968
). Of the two
branchiostegal rays generally developing in the young wild-type larvae, the
more ventral one (bsrm, closer to the putative signal source) is
almost never present in mutants. Therefore, Edn1 positively regulates
formation of branchiostegal rays, and possibly in a concentration- and
position-dependent manner. Evolution of a lower number of them may also have
involved changes in Edn1 level or in the sensitivities of the responding
cells.
We interpret the branchiostegal-opercle transformation as homeosis;
dissimilar meristic elements become similar
(Bateson, 1894). Hubbs
(Hubbs, 1920
) previously
placed the branchiostegal rays and opercle into a single meristic series,
which is in accord with a hypothesis of homeotic change between the elements.
However, there are caveats concerning our interpretation. We do not have
specific genetic markers for bone identity that we can use to test for
homeosis. Furthermore, even if the transformation is indeed one of bone
identity, and can be induced by just manipulating the level of Edn1 (as
suggested by the rescue experiments in
Fig. 10), it is important to
note that hoo might be playing other roles in skeletal patterning
than its putative role in the Edn1 signaling pathway. Similar to she
and stu, the molecular nature of the hoo gene has not yet
been described. Furthermore, the low penetrance of the transformation
phenotype (expansion of the branchiostegal ray occurs in only 4% of
hoo mutants) clearly indicates that unknown factors (other than the
proposed Edn1 gradient) are playing critical roles in specifying bone
identity.
Homeosis is well-known along the anterior-posterior axis of the embryo. The
transformations are between segmental homologs. The homeotic genes are usually
either Hox genes or genes that interact with Hox genes. In
contrast, the axis of patterning along the hyoid arch is the DV axis, and
there is no well-understood role of Hox genes in DV pharyngeal
patterning (but see Davenne et al.,
1999). Other candidates are available: Dlx homeobox genes
regulate DV patterning differences (maxillary versus mandibular) in the
mammalian first arch (Depew et al.,
2002
) (reviewed by Panganiban
and Rubenstein, 2002
), and members of the TGFß superfamily
regulate the formation of molars versus incisors along the mammalian jaw bones
(Tucker et al., 1998
;
Ferguson et al., 2001
).
Msx homeobox genes regulate bone development (e.g.
Dodig et al., 1999
), including
a negative regulation of bone size through interaction with the
osteoblast-specifying homeobox gene Runx2
(Shirakabe et al., 2001
).
Msx and Dlx genes, targets of Edn1 signaling in the
zebrafish pharyngeal arches (Miller et
al., 2000
), also control bone matrix development by reciprocal
regulation of Osteocalcin, a bone matrix protein
(Newberry et al., 1998
).
Learning the nature of target genes controlled by Edn1 in the zebrafish hyoid
arch that cell-autonomously control dermal bone size and DV identity would be
a fruitful avenue for future work.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allis, E. P. (1889). The anatomy and development of the lateral line system in Amia calva. J. Morphol. 2,463 -567.
Bateson, W. (1894). Materials for the Study of Variation. London: Macmillan & Co.
Beresford, W. A. (1996). Cranial skeletal tissues: diversity and evolutionary trends. In The Skull, vol. 2 (ed. J. Hanken and B. K. Hall), pp. 69-130. Chicago and London: The University of Chicago Press.
Carroll, R. L. (1988). Vertebrate Paleontology and Evolution. New York: W. H. Freeman.
Charité, J., McFadden, D. G., Merlo, G., Levi, G.,
Clouthier, D. E., Yanagisawa, M., Richardson, J. A. and Olson, E. N.
(2001). Role of Dlx6 in regulation of an endothelin-1-dependent,
dHAND branchial arch enhancer. Genes Dev.
15,3039
-3049.
Clouthier, D. E., Hosoda, K., Richardson, J. A., Williams, S.
C., Yanagisawa, H., Kuwaki, T., Kumada, M., Hammer, R. E. and Yanagisawa,
M. (1998). Cranial and cardiac neural crest defects in
endothelin-A receptor-deficient mice. Development
125,813
-824.
Clouthier, D. E., Williams, S. C., Yanagisawa, H. Wieduwilt, M., Richardson, J. A. and Yanigisawa, M. (2000). Signaling pathways crucial for craniofacial development revealed by endothelin-A receptor-deficient mice. Dev. Biol. 217, 10-24.[CrossRef][Medline]
Cubbage, C. C. and Mabee, P. M. (1996). Development of the cranium and paired fins in the zebrafish Danio rerio (Ostariophysi, cyprinidae). J. Morphol. 229,121 -160.[CrossRef]
Davenne, M., Maconochie, M. K., Neun, R., Pattyn, A., Chambon, P., Krumlauf, R. and Rijli, F. M. (1999). Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain. Neuron 22,677 -691.[Medline]
Depew, M. J., Lufkin, T. and Rubenstein, J. L. R.
(2002). Specification of jaw subdivisions by Dlx genes.
Science 298,381
-385.
Dodig, M., Tadic, T., Kronenberg, M. S., Dacic, S., Liu, Y. H., Maxson, R., Rowe, D. W. and Lichtler, A. C. (1999). Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation. Dev. Biol. 209,298 -307.[CrossRef][Medline]
Ferguson, C. A., Tucker, A. S., Heikinheimo, K., Nomura, M., Oh,
P., Li, E. and Sharpe, P. T. (2001). The role of effectors of
the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in
patterning of tooth development. Development
128,4605
-4613.
Hubbs, C. L. (1920). A comparative study of the bones forming the opercular series of fishes. J. Morphol. 33,61 -71.
Hubbs, C. L. and Hubbs, L. C. (1945). Bilateral asymmetry and bilateral variation in fishes. Papers of the Michigan Academy of Sciences, Arts and Letters 30,229 -310.
Janvier, P. (1996). Early Vertebrates. Oxford: Clarendon Press.
Jarvik, E. (1980). Basic Structure and Evolution of Vertebrates. London, New York, Toronto, Sydney, San Francisco: Academic Press.
Johnson, S. L. and Weston, J. A. (1995).
Temperature-sensitive mutations that cause stage-specific defects in zebrafish
fin regeneration. Genetics
141,1583
-1595.
Jollie, M. (1962). Chordate Morphology. New York: Rheinhold.
Kempf, H., Linares, C., Corvol, P. and Gasc, J.-M.
(1998). Pharmacological inactivation of the endothelin type A
receptor in the early chick embryo: a model of mispatterning of the branchial
arch derivatives. Development
125,4931
-4941.
Kimmel, C. B., Miller, C. T. and Keynes, R. J. (2001a). Neural crest patterning and the evolution of the jaw. J. Anat. 199,105 -119.[CrossRef][Medline]
Kimmel, C. B., Miller, C. T., Kruse, G., Ullmann, B., BreMiller, R. A., Larison, K. D. and Snyder, H. C. (1998). The shaping of pharyngeal cartilages during early development of the zebrafish. Dev. Biol. 203,245 -263.[CrossRef][Medline]
Kimmel, C. B., Miller, C. T. and Moens, C. B. (2001b). Specification and morphogenesis of the zebrafish larval head skeleton. Dev. Biol. 233,239 -257.[CrossRef][Medline]
Kitano, Y., Kurihara, H., Kurihara, Y., Maemura, K., Ryo, Y., Yazaki, Y. and Harii, K. (1998). Gene expression of bone matrix proteins and endothelin receptors in endothelin-1-deficient mice revealed by in situ hybridization. J. Bone Miner. Res. 13,237 -244.[Medline]
Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagal, R., Oda, H., Kuwaki, T., Cao, W.-H., Kamada, N. et al. (1994). Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368,703 -710.[CrossRef][Medline]
Maisey, J. G. (1986). Heads and tails: a chordate phylogeny. Cladistics 2, 201-256.
Maves, L., Jackman, W. and Kimmel, C. B.
(2002). FGF3 and FGF8 mediate a rhombomere 4 signaling activity
in zebrafish hindbrain. Development
129,3825
-3837.
McAllister, D. E. (1968). The evolution of branchiostegals and associated opercular, gular, and hyoid bones. Bull. Natl. Mus. Canada 221, 1-239.
Miles, R. S. (1973). Articulated acanthodian fishes from the Old Red Sandstone of England, with a review of the structure and evolution of the acanthodian shoulder-girdle. Bull. Br. Mus. Nat. His. Geol. 24,115 -213.
Miller, C. T. and Kimmel, C. B. (2001). Morpholino phenocopies of endothelin 1 (sucker) and other anterior arch class mutations. Genesis 30,186 -187.[CrossRef][Medline]
Miller, C. T., Schilling, T. F., Lee, K.-H., Parker, J. and Kimmel, C. B. (2000). sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development. Development 127,3825 -3828.
Miller, C. T., Yelon, D., Stainier, D. Y. R, and Kimmel, C.
B. (2003). Two endothelin 1 effectors,
hand2 and bapx1, pattern ventral pharyngeal cartilage and
the jaw joint. Development
130,1353
-1365.
Nelson, G. J. (1970). The hyobranchial apparatus of teleostean fishes of the families Engraulidae and Chirocentridae. Am. Mus. Nov. 2410,1 -30.
Newberry, E. P., Latifi, T. and Towler, D. A. (1998). Reciprocal regulation of osteocalcin transcription by the homeodomain proteins Msx2 and Dlx5. Biochemistry 37,16360 -16368.[CrossRef][Medline]
Nquyen, V. H., Schmid, B., Trout, J., Connors, S. A., Ekker, M. and Mullins, M. C. (2003). Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes. Dev. Biol. 199,93 -110.[CrossRef]
Panganiban, G. and Rubenstein, J. L. (2002). Developmental functions of the Distal-less/Dlx homeobox genes. Development 129,4372 -4386.
Parenti, L. R. (1986). The phylogenetic significance of bone types in euteleost fishes. Zool. J. Linnean Soc. 87,37 -51.
Parrington, F. R. (1948). A theory of the relations of lateral lines to dermal bones. Proc. Zool. Soc. Lond. 119,65 -78.
Piotrowski, T., Schilling, T. F., Brand, M., Jiang, Y. J.,
Heisenberg, C. P., Beuchle, D., Grandel, H., Van Eeden, F. J. M.,
Furutani-Seiki, M., Granato, M. et al. (1996). Jaw and
branchial arch mutants in zebrafish. 2. Anterior arches and cartilage
differentiation. Development
123,345
-356.
Raible, D. W. and Kruse, G. J. (2000). Organization of the lateral line system in embryonic zebrafish. J. Comp. Neurol. 421,189 -198.[CrossRef][Medline]
Russell, E. S. (1916). Form and Function: a Contribution to the History of Animal Morphology. London: J. Murray.
Sasaki, T. and Hong M. H. (1993). Endothelin-1 localization in bone cells and vascular endothelial cells in rat bone marrow. Anat. Rec. 237,332 -337.[Medline]
Schultze, H.-P. (1993). Patterns of diversity in the skulls of jawed fishes. In The Skull, Vol.2 (ed. J. Hanken and B. K. Hall), pp.189 -254. Chicago and London: The University of Chicago Press.
Shirakabe, K., Terasawa, K., Miyama, K., Shibuya, H. and
Nishida, E. (2001). Regulation of the activity of the
transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5.
Genes Cells 6,851
-856.
Thomas, T., Kurihara, H., Yamagishi, H., Kurihara, Y., Yazaki,
Y., Olson, E. N. and Srivastava, D. (1998). A signaling
cascade involving endothelin-1, dHAND and Msx1 regulates development of
neural-crest-derived branchial arch mesenchyme.
Development 125,3005
-3014.
Tucker, A. S., Matthews, K. L. and Sharpe, P. T.
(1998). Transformation of tooth type induced by inhibition of BMP
signaling. Science 282,1136
-1138.
Tyler, M. S. and Hall, B. K. (1977). Epithelial influences on skeletogenesis in the mandible of the embryonic chick. Anat. Rec. 188,229 -240.[Medline]
Verraes, W. (1977). Postembryonic ontogeny and functional anatomy of the ligamentum mandibulo-hyoideum and the ligamentum interoperculo-mandibulare, with notes on the opercular bones and some other cranial elements in Salmo gairdneri Richardson, 1836 (Teleostei: Salmonidae). J. Morphol. 151,111 -120.
Webb, J. F. (1989). Developmental constraints and evolution of the lateral line system in teleost fishes. In The Mechanosensory Lateral Line (ed. S. Coombs, P. Görner and H. Münz), pp. 79-97. New York: Springer-Verlag.
Whitfield, T. T., Granato, M., Van Eeden, F. J. M., Schach, U.,
Brand, M., Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C.
P., Jiang, Y. J. et al. (1996). Mutations affecting
development of the zebrafish inner ear and lateral line.
Development 123,241
-254.
Wolpert, L. (1971). Positional information and pattern formation. Curr. Top. Dev. Biol. 6, 183-224.[Medline]
Yanagisawa, H., Yanagisawa, M., Kapur, R. P., Richardson, J. A.,
Williams, S. C., Clouthier, D. E., de Wit, D., Emoto, N. and Hammer, R. E.
(1998). Dual genetic pathways of endothelin-mediated
intercellular signaling revealed by targeted disruption of endothelin
converting enzyme-1 gene. Development
125,825
-836.