1 Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403,
USA
2 Department of Biochemistry and Biophysics and Programs in Developmental
Biology, Genetics and Human Genetics, University of California, San Francisco,
CA 94143, USA
* Present address: Department of Developmental Biology, Beckman Center B300, 279
Campus Drive, Stanford University School of Medicine, Stanford, CA 94305-5329,
USA
Present address: Developmental Genetics Program, Skirball Institute, NYU
School of Medicine, New York, NY, USA
Author for correspondence (e-mail:
ctm{at}stanford.edu)
Accepted 10 December 2002
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SUMMARY |
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First we show that edn1-expressing cells in the first (mandibular) and second (hyoid) pharyngeal arch primordia are located most ventrally and surrounded by hand2-expressing cells. Next we show that along the DV axis of the early first arch primordia, bapx1 is expressed in an intermediate domain, which later marks the jaw joint, and this expression requires edn1 function. bapx1 function is required for formation of the jaw joint, the joint-associated retroarticular process of Meckel's cartilage, and the retroarticular bone. Jaw joint expression of chd and gdf5 also requires bapx1 function.
Similar to edn1, hand2 is required for ventral pharyngeal cartilage formation. However, the early ventral arch edn1-dependent expression of five genes (dlx3, EphA3, gsc, msxe and msxb) are all present in hand2 mutants. Further, msxe and msxb are upregulated in hand2 mutant ventral arches. Slightly later, an edn1-dependent ventral first arch expression domain of gsc is absent in hand2 mutants, providing a common downstream target of edn1 and hand2. In hand2 mutants, bapx1 expression is present at the joint region, and expanded ventrally. In addition, expression of eng2, normally restricted to first arch dorsal mesoderm, expands ventrally in hand2 and edn1 mutants. Thus, ventral pharyngeal specification involves repression of dorsal and intermediate (joint region) fates. Together our results reveal two critical edn1 effectors that pattern the vertebrate jaw: hand2 specifies ventral pharyngeal cartilage of the lower jaw and bapx1 specifies the jaw joint.
Key words: Zebrafish, endothelin 1, hand2, bapx1, Joints, Jaw, Pharynx, Pharyngeal arch, Branchial arch
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INTRODUCTION |
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GDF5 and another signalling molecule of the TGFß superfamily, BMP5,
are required for partially overlapping subsets of joints in the mouse axial
skeleton, suggesting the skeleton is assembled piecemeal by partially
redundant TGFß signalling molecules
(Storm et al., 1994;
Storm and Kingsley, 1996
;
Storm and Kingsley, 1999
).
Gdf5 negatively regulates its own expression, and thus refines where
the joint is positioned (Storm and
Kingsley, 1999
). Despite being required for certain joints, GDF5
is not sufficient to induce ectopic joints
(Storm and Kingsley, 1999
;
Merino et al., 1999
;
Francis-West et al., 1999a
).
In chick embryos, another secreted molecule, Wnt14, is sufficient to induce
ectopic joints, and in addition inhibits nearby joints
(Hartmann and Tabin, 2001
).
Little is known about upstream factors that control the expression of these
joint-promoting signaling molecules.
The jaw joint forms in the first, or mandibular, pharyngeal arch,
articulating the upper and lower jaw. Work in mice, chicks and zebrafish has
begun to unravel molecular mechanisms responsible for jaw development. In both
mice and zebrafish, the secreted peptide endothelin 1 (encoded by the
edn1 or sucker gene in zebrafish) is required for
development of the jaw, as well as skeletal elements of the second, or hyoid,
pharyngeal arch (Kurihara et al.,
1994; Miller et al.,
2000
; Miller and Kimmel,
2001
). In mice, targeted inactivation of the Edn1 receptor, EdnrA,
produces a similar phenotype as Edn1 inactivation, namely loss of the mandible
and severe malformations of other pharyngeal skeletal elements
(Clouthier et al., 1998
).
Pharmacological inactivation of EdnrA in chick embryos results in a similar
disruption of lower jaw formation (Kempf
et al., 1998
). Thus, Edn1-EdnrA signaling is required for lower
jaw formation in chicks as well as mice and fish. Within the early pharyngeal
arch primordia, secretion of Edn1 from paraxial mesodermal cores and
surrounding epithelia, both surface ectoderm and pharyngeal endoderm, is
received by the EdnrA receptor, which is broadly expressed in the
postmigratory cranial neural crest (CNC) cylinder. Edn1 signaling sets up a
dorsoventral prepattern and promotes the specification of ventral fates within
this cylinder of postmigratory CNC (reviewed by
Kimmel et al., 2001a
). In
zebrafish, graded reduction of Edn1 function with edn1 antisense
morpholino oligonucleotides (Edn1-MOs) results in graded reduction in ventral
pharyngeal cartilage formation. The pharyngeal joints also require Edn1 and
are more sensitive to Edn1 reduction, because ventral cartilage, but not
joints, form in animals injected with lower doses of Edn1-MOs
(Miller and Kimmel, 2001
).
Thus, in zebrafish Edn1-signaling is required for both joint and ventral
pharyngeal fates, with joint fates being more sensitive to Edn1 reduction.
The requirement for Edn1 in activating expression of hand2 (also
known as dHAND) in the ventral arch primordia is conserved between
mice and zebrafish (Thomas et al.,
1998; Miller et al.,
2000
). hand2 mutant mice die before skeletal
differentiation occurs with severe heart and circulatory defects
(Srivastava et al., 1997
;
Thomas et al., 1998
;
Yamagishi et al., 2000
). In
the hand2 mutant mouse pharyngeal arch primordia, CNC fails to adopt
ventral fates and undergoes apoptosis
(Thomas et al., 1998
). Thus,
hand2 is an excellent candidate effector of Edn1-mediated pharyngeal
arch patterning.
Here we present functional analysis of two edn1-dependent genes, bapx1 and hand2, during zebrafish pharyngeal arch development. edn1 expression is complementary to and surrounded by hand2-expressing ventral arch CNC, whereas bapx1 expression defines an intermediate presumptive joint domain, ventral to some dlx2 expression and dorsal to hand2 expression. The loss of the jaw joint in edn1 mutants can in part be explained by a failure to upregulate expression of bapx1, whose function is required for multiple aspects of skeletal development of the jaw joint region including the joint itself, the retroarticular process of Meckel's cartilage, and the retroarticular bone. In the developing jaw joint, bapx1 is required for expression of chordin and gdf5. The loss of ventral pharyngeal cartilage in edn1 mutants can in part be explained by a second edn1 target gene, hand2. Similar to edn1, hand2 is required for formation of almost all ventral pharyngeal cartilage. Despite this phenotypic similarity to edn1 mutants, the early ventrally restricted edn1-dependent expression of dlx3, EphA3, gsc, msxe and msxb in cartilage precursors are all present in hand2 mutants. Further, msxe and msxb are upregulated in the ventral arches of hand2 mutants. However, similar to edn1 mutants, hand2 mutants lack late first arch expression of gsc. bapx1 expression in hand2 mutants is ectopically expanded ventrally, suggesting that hand2 helps position the jaw joint by repressing expression of bapx1. Finally, we show that both hand2 and edn1 restrict eng2 expression to dorsal mesoderm. Thus the specification of ventral pharyngeal arch fates involves the repression of other ventral, joint and dorsal arch fates. Collectively, our results identify bapx1 and hand2 as critical effectors of Edn1 in patterning the jaw joint and ventral pharyngeal cartilage.
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MATERIALS AND METHODS |
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Cloning bapx1
Degenerate PCR primers designed against the Nk3/Bap homeobox region
5'-TGGARMGNMGYTTYAAYCAYCA-3' and 5'-TTRTAN-
CKNCKRTTYTGRAACCA-3' were used with zebrafish genomic DNA as a template
and 48 cycles of: 94°C for 30 seconds, 48°C for 30 seconds and
72°C for 30 seconds, which generated a 117-bp band. This PCR product was
cloned using a TOPO TA kit (Invitrogen) and sequencing revealed a Bapx1-like
homeodomain. These primers and conditions were then used to screen DNA pools
from an arrayed genomic DNA PAC library
(Amemiya and Zon, 1999), which
identified a single positive PAC, 91M18. This PAC was isolated and subcloned,
and sequencing subclones yielded the sequence of the second exon and the
3' end of the intron. Using gene-specific primers
5'-GATCTTGACCTGCGTCTCG-3' and 5'-GCGTTATCTCTCCGG-
ACCG-3' from this PAC sequence to amplify a 72 bp band, phage dilution
pools of a 15-19 hpf zebrafish cDNA library
(Appel and Eisen, 1998
) were
screened by PCR. A single phage was isolated, which contained a 1357 bp
insert, containing the first exon and predicted full-length ORF of a
bapx1 gene (Accession Number, AY225416).
Tissue labeling procedures
Alcian staining and in situ hybridizations were performed as described
(Miller et al., 2000). After
Alcian staining, some wholemounts were treated with a solution of 3% hydrogen
peroxide and 1% potassium hydroxide for 10 minutes to remove pigmentation.
Bone labeling using calcein was performed as described
(Yan et al., 2002
;
Kimmel et al., 2003
). For
sectioning, embryos were embedded in Epon and sectioned at 5 microns.
A 1.2 kB PCR fragment of bapx1 genomic DNA, containing the entire
second exon and part of the 3' UTR, was used for all in situs.
chd and gdf5 probes are described in Schulte-Merker et al.
(Schulte-Merker et al., 1997)
and Bruneau et al. (Bruneau et al.,
1997
), respectively. All other riboprobes are described or
referenced in Miller et al. (Miller et
al., 2000
).
Morpholino oligo injections
edn1 morpholino oligo (edn1-MO) 5'-GTAGTATGCAAGTCCCGTATTCCAG-3'
(31 to 7 nucleotides 5' to predicted translation start site), (see
Miller and Kimmel, 2001),
bapx1 morpholino oligo (bapx1-MO1)
5'-GCGCACAGCCATGTCGAGCAGCACT-3' (ATG start
complementary sequence underlined) and bapx1-MO2
(5'-GCGGAGCATTAGGGTTAAGATTACG-3', complementary to 52 to 28
nucleotides 5' of the predicted ATG start codon) were purchased from
Gene Tools, Inc., and diluted to 25 mg/ml in 1 x Danieau buffer.
Subsequent dilutions were made in 0.2 M KCl and 0.2% Phenol Red. These
dilutions were injected into the yolk of 1-8 cell zebrafish embryos,
approximately 5 nL per embryo. bapx1-MO1, which seemed less toxic and gave
cleaner phenotypes (see below), was used for all phenotypic analyses. The
inbred *AB line was used for all MO-injections into wild types.
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RESULTS |
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We analyzed the pharyngeal arch expression domains of hand2, edn1,
and the more broadly expressed postmigratory CNC marker, dlx2, in
serial sections of 32 hpf embryos stained for the expression of each of these
genes (Fig. 1). These serial
section studies strongly support our previous findings in wholemounts
(Miller et al., 2000), and add
cellular resolution to the expression patterns. hand2-expressing
cells are a ventral subset of dlx2-expressing CNC cells, and are
closely apposed to cells of three different tissues expressing edn1:
ventral surface ectoderm, ventral mesodermal cores and pharyngeal endodermal
epithelia. In the first two arches, hand2-expressing cells cover
dorsally the edn1-expressing arch cores, which are in extreme
proximity to the yolk. edn1 expression is not appreciably detected in
the first pharyngeal pouch at this stage. The ventral surface ectodermal
domain of edn1 expression seems to extend to, but not beyond, the
dorsal extent of the adjacent hand2 expression domain
(Fig. 1E,F,H,I). Thus, in the
first two arches at this stage, edn1 expression is restricted to the
ventralmost tissues of the arches, appearing slightly more ventrally localized
than hand2 expression.
|
A zebrafish bapx1 gene is expressed at the first arch joint,
and this expression domain requires edn1 function
In this DV postmigratory CNC prepattern, approximately the ventral third of
the dlx2-expressing CNC cylinder expresses hand2, and
probably includes precursors of the ventral cartilages. We hypothesized that
joint-forming cells, although slightly farther away from the Edn1 source,
respond to Edn1 signaling, given the requirement of edn1 for
pharyngeal joint primordia (Miller and
Kimmel, 2001). We thus began a search for markers of pharyngeal
joint primordia. In Xenopus embryos, the
bapx1/bagpipe-related NK3 superfamily homeobox gene Xbap is
expressed in a large region in the intermediate first arch, encompassing the
jaw joint region (Newman et al.,
1997
). Using degenerate PCR with a zebrafish genomic DNA library,
followed by gene-specific PCR with a 15-19 hpf zebrafish cDNA library, we
cloned a zebrafish bapx1 gene. Phylogenetic analyses of this
zebrafish and other bagpipe-related genes reveals that this zebrafish
gene is orthologous to Xbap and other vertebrate bapx1
(nkx3.2) genes (data not shown).
Similar to expression of the Xenopus Xbap gene
(Newman et al., 1997),
expression of zebrafish bapx1 is present in mesenchyme of the
mandibular arch primordia. Mandibular expression begins at approximately 30
hpf (see Fig. 2H), and at 32
hpf a large patch of expression is present in the posterior intermediate first
arch postmigratory CNC cylinder (Fig.
2A,B). This domain persists through 54 hpf. By this stage, these
intermediate bapx1 domains clearly mark the jaw joints because the
upper and lower jaw cartilages have begun to chondrify, and bapx1
expression is present in cells within and surrounding the jaw joint
(Fig. 2C,D). By 54 hpf,
additional bapx1 expression domains are present in the midline of the
first two arches (Fig. 2C).
Expression is also detected in pharyngeal endodermal epithelia at 32 through
54 hpf (Fig. 2A,B,F). Other
bapx1 expression domains include putative sclerotomal derivatives at
48 hpf, the pectoral fin at 54 hpf, and cells closely apposed to the eye at 36
and 42 hpf (data not shown).
|
We next asked how bapx1 expression in the putative joint region primordium relates to the dlx2/hand2 DV prepattern in the zebrafish pharyngeal arch primordia. Double-labeling experiments show that bapx1 expression is ventral to a large dorsal domain of dlx2-expressing CNC cells and dorsal to hand2-expressing CNC cells (Fig. 2G,H). Thus at this early stage, from 30-32 hpf, in the first arch primordia, bapx1 marks an intermediate or presumptive joint region.
bapx1 expression in the developing jaw joint primordium
requires edn1 function
Because the jaw joint region is particularly affected in 4-day old animals
with reduced edn1 function
(Miller and Kimmel, 2001), we
next asked whether bapx1 downregulation prefigures the edn1
mutant phenotype. At 36 hpf, first arch mesenchymal bapx1 expression
is undetectable in edn1 mutants
(Fig. 3A,B). These defects are
not simply because of developmental delay, because by 54 hpf, the midline
domains of bapx1 expression in both arch one and two are present in
edn1 mutants, whereas the jaw joint domains of bapx1
expression remain undetectable (Fig.
3C,D).
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bapx1 is required for the jaw joint
Injecting morpholino antisense oligos (MOs) into embryos has been shown to
be highly successful at reducing gene function in vivo (reviewed by
Heasman, 2002). This technique
efficiently works to downregulate genes involved in zebrafish head skeletal
development, as shown by highly penetrant phenocopy of the edn1
mutant phenotype upon injection of edn1-morpholinos
(Miller and Kimmel, 2001
). To
assess bapx1 function in pharyngeal skeletal patterning, morpholino
oligos complementary to the region around the predicted translation start site
of bapx1 were injected. Animals injected with bapx1-MOs display
dose-dependent loss of the jaw joint (Fig.
4, Table 1).
Injection of 1 mg/ml (total of 5 ng) of bapx1-MO1 resulted in 51% of injected
animals lacking jaw joints, whereas injection of 3 mg/ml (total of 15 ng)
raised this frequency to 80% (Table
1). The loss of features of the jaw joint region in
bapx1-MO1-injected animals was graded in severity. Frequently the
cartilaginous retroarticular process (RAP), which projects ventrally from the
posterior end of Meckel's cartilage, was present, but just dorsally, the jaw
joint itself was lost with the dorsal and ventral cartilages locally fused
(Table 1,
Fig. 4A-H). In more severely
affected animals RAP was missing, and the joint was completely missing and
filled in with ectopic cartilage (Fig.
4C,D). Unlike animals with mild edn1 reduction, and
consistent with the absence of second arch joint bapx1 expression,
the second arch joint is unaffected. However, the second arch midline skeletal
element, the basihyal, which is prefigured by bapx1-expressing cells
(see Fig. 3C), was
characteristically reduced in bapx1-MO1-injected animals
(Table 1).
|
|
To confirm the specificity of this morpholino, we injected a second non-overlapping bapx1-MO (bapx1-MO2). Although injection of MO2 caused other possibly non-specific phenotypes including loss of the branchial cartilages (data not shown, see Discussion), this MO also caused highly penetrant, dose-dependent loss of the jaw joint (Table 1). To further confirm the specificity of these jaw joint region MO phenotypes, we injected these two bapx1-MOs together, at relatively lower concentrations. These combinatorial injections caused more frequent jaw joint loss at concentrations lower than that of the singles alone. Although injection of 5 ng of MO1 and MO2 alone resulted in 51% and 2% loss of the jaw joint, respectively, injecting half as much of each MO combinatorially enhanced the penetrance of this phenotype to 77%. Coinjection of MO1 and MO2 also enhanced the basihyal phenotype, resulting in frequent deletion of this element (Table 1). These phenotypic enhancements further suggest that these non-overlapping MOs are specifically reducing function of bapx1.
Slightly later in development, at around 6 dpf, the retroarticular bone
(RAB) begins ossifying perichondrally on RAP of Meckel's cartilage
(Fig. 4I)
(Cubbage and Mabee, 1996) (C.
B. K., unpublished). Because RAP is often missing in bapx1-MO-injected fish
(see above), we asked if skeletal defects in bapx1-MO-injected animals also
included defects in RAB. To examine potential defects in pharyngeal bones of
bapx1-MO-injected animals, we stained bones in uninjected and injected larvae
with the fluorescent dye Calcein (Kimmel
et al., 2003
; Yan et al.,
2002
). In bapx1-MO-injected animals, we observed a highly
penetrant RAB loss (Fig. 4J;
111/117, or 95% of injected animals lacking RAB vs. 14/51 or 28% of uninjected
siblings lacking RAB). Thus, bapx1 is required for at least three
aspects of skeletal development in and around the jaw joint: (1) local
inhibition of chondrogenesis at the jaw joint, (2) specification of the RAP of
Meckel's cartilage, and (3) ossification of the RAB.
To provide a potential genetic mechanism for bapx1's role in jaw
joint development, we asked whether markers of tetrapod limb joints also mark
the zebrafish jaw joint, and whether such domains require bapx1
function. In tetrapods, Gdf5, which encodes a TGFß-related
signaling molecule, is expressed in early cartilage condensations and later in
developing joints (Chang et al.,
1994 Storm et al.,
1994
; Storm and Kingsey, 1996;
Merino et al., 1999
;
Francis-West et al., 1999a
). A
subset of mouse appendicular and axial joints requires Gdf5 function
(Storm et al., 1994
;
Storm and Kingsley, 1996
;
Merino et al., 1999
). Another
marker of developing tetrapod limb joints is the BMP-antagonist
chordin (chd)
(Francis-West et al., 1999b
;
Scott et al., 2000
). In 56 hpf
zebrafish, chd expression, which is localized to the jaw joint in
wild types (Fig. 5A), is absent
in bapx1-MO-injected animals (Fig.
5B). At 78 hpf, gdf5 expression is detected in cells
within the jaw joint, and these expression domains are severely reduced in
bapx1-MO-injected animals. gdf5 expression was also detected in a
triangular cluster of cells at the second arch ventral midline, seemingly
prefiguring the unpaired ventral midline basihyal cartilage, and similar to
the second arch midline domain of bapx1 expression at 54 hours
(Fig. 5C,
Fig. 2C). This second arch
midline domain of gdf5 expression was also downregulated in
bapx1-MO-injected animals (Fig.
5D), correlating with the reduced basihyal cartilage phenotype
(Table 1). These expression
defects in bapx1-MO-injected animals were specific to the joint region,
because other expression domains of both genes, including cells around the
second arch joint for both genes, the heart and ears for chd, and
ceratohyal and palatoquadrate perichondrial cells for gdf5, were not
affected by bapx1-MO injection. Thus, bapx1 is required for
development of the jaw joint region: RAP, RAB and the jaw joint itself all
require bapx1 function, as does the early expression of two genes,
chd and gdf5, within the developing jaw joint.
|
hand2 is required for ventral pharyngeal cartilage
development
Although bapx1 is a required edn1 effector of joint
patterning, ventral pharyngeal cartilage formation outside of the joint region
is largely unaffected in bapx1-MO-injected animals. Thus, we next sought an
edn1 effector of ventral cartilage patterning. We focused on
hand2, an excellent candidate for three reasons. First, in the mouse
embryo, hand2 expression requires Edn1 function and
hand2 is required for ventral pharyngeal arch patterning
(Thomas et al., 1998). Second,
hand2 expression requires edn1 function in zebrafish,
correlating with the edn1 mutant ventral cartilage loss
(Miller et al., 2000
). Third,
as shown above, hand2 expression exquisitely complements
edn1 expression in the ventral pharyngeal arch primordia.
hand2 mutant zebrafish were found in a screen for mutations
affecting heart development, including a null allele completely deleting the
hand2 locus (hans6)
(Yelon et al., 2000). Despite
lacking a heart and circulating blood, hans6 mutants (for
clarity hereafter referred to as hand2 mutants) live for at least
four days and make differentiated pharyngeal cartilage in the mandibular and
hyoid arches (Fig. 6A,B).
Unlike mutants of the anterior arch class (e.g. edn1 mutants)
cartilages of the more posterior pharyngeal arches never develop. Dorsal
anterior arch cartilages of reduced size, but relatively normal shape, form in
hand2 mutants, whereas only a tiny amount of ventral cartilage forms
(Fig. 6C-I). Mutant dorsal
cartilages have readily recognizable components, including the pterygoid
process and parallel stacks of chondrocytes forming the lateral plate of the
palatoquadrate cartilage in arch one, and the symplectic (SY) and
hyomandibular regions of the hyosymplectic cartilage in arch two. Mutant
ventral cartilages, in contrast, are almost absent in the anterior arches
(Fig. 6C-I). Although similar
to edn1 mutants, hand2 mutants lack jaw joints, less
cartilage is present ventrally in hand2 mutants, and the two upper
jaws typically are connected by a small disorganized cartilage bridge spanning
the midline (Fig. 6C-E;
Table 2). In contrast and also
unlike edn1 mutants, the hand2 mutant second arch has
well-formed morphological joints between the dorsal and severely reduced
ventral second arch cartilages (Fig.
6C-I). Hence the differential requirement of hand2 for
joint formation reveals a key patterning difference between arch one and
two.
|
|
A complex role of hand2 in ventral pharyngeal arch
patterning
We previously showed that edn1 function is required for the
ventral arch expression of hand2 and four other genes: dlx3,
EphA3, gsc and msxe (Miller
et al., 2000; Miller and
Kimmel; 2001
). Homologs of dlx3, gsc and msxe
are all required for proper mammalian craniofacial development
(Kula et al., 1996
;
Price et al., 1998
;
Rivera-Perez et al., 1995
;
Yamada et al., 1995
;
Jabs et al., 1993
;
Satokata and Maas, 1994
;
Satokata et al., 2000
).
EphA3, the Ephrin transmembrane receptor tyrosine kinase, is
expressed in Meckel's cartilage of rats
(Kilpatrick et al., 1996
).
Thus, all four of these genes have potentially conserved pharyngeal arch
domains transducing the Edn1 signal.
Because absence of ventral cartilage is shared between hand2 and
edn1 mutants (Piotrowski et al.,
1996; Kimmel et al., 1996;
Miller et al., 2000
) and
because in the mouse pharyngeal arch msx1 expression requires
hand2, we expected that ventral arch expression of some or all of
these edn1 target genes would also require hand2. Instead,
early ventral arch expression of dlx3, EphA3, gsc and msxe
are robustly present in hand2 mutants
(Fig. 7). We see three classes
of effects on these genes in hand2 mutants: no effect or mild
upregulation, arch-specific requirement and clear upregulation. In the first
class, the ventrally restricted pharyngeal arch expression domains of
dlx3 and EphA3 expression are present in hand2
mutants, and appear slightly upregulated
(Fig. 7A-D).
|
In the second class, gsc expression requires hand2
function in ventral arch one, but not ventral arch two. At 32 hpf, dorsal and
ventral domains of gsc expression are present in the second arch, and
both domains are present in hand2 mutants at 32 hpf
(Fig. 7E,F). Later at 38 hpf, a
ventral arch one domain of gsc expression is present, and this domain
of gsc expression is absent in hand2 mutants
(Fig. 7G,H). Both the early
second arch ventral domain and the late first arch ventral domain of
gsc expression are missing in edn1 mutants
(Miller et al., 2000) (data
not shown).
In the third class, two msx genes are upregulated in hand2 mutants. Expression of msxe in ventral first and second arch CNC at 30 hpf is present in hand2 mutants and appears upregulated, possibly in other cells, but seemingly in the same cells that express msxe at lower levels in wild types (Fig. 7I,J). A second zebrafish msx gene, msxb, is more sparsely and weakly expressed in wild-type ventral arch CNC at 30 hpf (Fig. 7K). This expression, similar to that of msxe, requires edn1 but not hand2 function (Fig. 7L,M). Similar to msxe, but more dramatically so, msxb expression appears upregulated in hand2 mutants. This msxb upregulation in hand2 mutants appears, similar to the msxe upregulation, to involve upregulation of transcription in cells that normally express msxb at lower levels, and in addition seems to involve the ectopic expression of msxb in ventral CNC cells in which msxb expression is normally not detectable by in situ hybridization (i.e. in cells in the anterior ventral first and second arch, compare Fig. 7K,L). Thus, in stark contrast to edn1 mutants, and despite ventral cartilage being almost absent later, early expression of dlx3, EphA3, gsc, msxe and msxb are all present in the ventral arches of hand2 mutants. However, similar to edn1 mutants, hand2 mutants lack later first arch ventral gsc expression. These findings reveal complexity in the genetic network controlling ventral pharyngeal cartilage development. Based on the genes we have examined, we suggest that most edn1-dependent signaling in the early pharyngeal arch primordia occurs independently of hand2 function.
hand2 represses joint and dorsal arch fates
Next we asked whether hand2 mutants, similar to edn1
mutants that also lack jaw joints, have early bapx1 expression
defects. Despite lacking a jaw joint, hand2 mutants have expanded
intermediate first arch bapx1 expression
(Fig. 8B). However and
remarkably, bapx1 expression ectopically expands into the ventral
first arch of hand2 mutants, where hand2 is normally
expressed (Fig. 8B). To
determine whether this ectopic bapx1 expression in hand2
mutants requires Edn1 function, we injected Edn1 morpholinos (Edn1-MOs)
(Miller and Kimmel, 2001) into
clutches of hand2 mutants to obtain animals lacking both
hand2 and edn1 function. The expansion of bapx1
expression in hand2 mutants is edn1-dependent, because
hand2 mutants injected with Edn1-MOs, similar to edn1
mutants, completely lack first arch mesenchymal expression of bapx1
(Fig. 8C).
|
The expanded domain of bapx1 expression in hand2 mutants
suggests hand2 functions to repress the specification of joint fates.
Because the joint forms at an intermediate DV location (see above), we finally
asked whether even more dorsal fates are also repressed by hand2.
Although we currently know of no marker restricted to dorsal arch
postmigratory CNC in zebrafish, engrailed2 (eng2) expression
is restricted to dorsal first arch myoblasts (see
Hatta et al., 1990;
Ekker et al., 1992
;
Kimmel et al., 2001b
). In both
hand2 and edn1 mutants, eng2 expression expands
ventrally, apparently revealing an unsubdivided arch mesodermal core
(Fig. 8D-F). Thus,
edn1 and hand2 specify ventral fates at least in part by
repressing dorsal arch fates.
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DISCUSSION |
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Gene expression defines three domains within postmigratory pharyngeal
arch CNC
The serial section analyses we present here place edn1-expressing
cells as ventrally confined at a postmigratory stage. The ventral surface
ectoderm, paraxial mesodermal arch cores, and pharyngeal endodermal epithelia
expression domains of edn1 are all complementarily contained within
the hand2-expressing ventral region of the arch primordia. Because
the Edn1 signal is thought to be secreted and Edn1 function is required for
patterning throughout the DV extent of the pharyngeal arch, perhaps Edn1 acts
as a morphogen in patterning DV fates in the pharynx.
Our finding that a day before chondrogenesis, bapx1 is expressed in an intermediate region of the first arch, ventral to some dlx2 expression and dorsal to hand2 expression, raises the possibility that at least three domains of the resultant skeleton (dorsal, joint and ventral) fate map to stereotypical domains of the pharyngeal arch primordia. Preliminary experiments with uncaged fluorescein support this idea, because uncaged dorsal and ventral spots in the arch primordia gave rise to labeled cells in dorsal and ventral cartilages, respectively (C. T. M., C. B. K., S. Cheesman and S. Hutchinson, unpublished). Powerful new tools including green fluorescent protein (GFP) lines and 4D confocal microscopy promise to allow high resolution fate mapping of the postmigratory CNC cylinders (J. G. Crump and C. B. K., unpublished), and will test the proposals that the DV prepattern prefigures dorsal and ventral cartilages, and that the early intermediate bapx1 expression domain at 30 hpf prefigures the jaw joint.
An edn1 target, bapx1, is required for patterning
the intermediate jaw joint region of the first arch
Our bapx1-morpholino experiments reveal multiple requirements for
bapx1 in formation of the jaw joint region. Morphologically, the jaw
joint itself, the nearby RAP of Meckel's cartilage, and the RAB all require
bapx1 function. These phenotypes, all associated with the jaw joint
region, suggest bapx1 functions to specify multiple fates within the
intermediate first arch primordia. Because all three of these skeletal
phenotypes are seen in edn1 mutants
(Miller et al., 2000;
Kimmel et al., 2003
), and
bapx1 first arch expression requires edn1 signaling,
bapx1 is a critical effector of edn1 in patterning the
intermediate first arch.
bapx1 expression was detected at the ventral midline of the second
arch, i.e. in the position of the later basihyal cartilage, consistent with
the report of Xenopus Xbap expression
(Newman et al., 1997). Midline
expression of gdf5 in the second arch was downregulated in
bapx1-MO-injected animals, providing a potential earlier molecular correlate
of the basihyal reduction phenotype. Thus, in zebrafish, bapx1 is
required for jaw joints and a specific ventral midline cartilage, the
basihyal.
Although mammalian Bapx1 is expressed in mandibular arch
mesenchyme and joints in the appendicular skeleton, no defects have been
reported in these tissues in Bapx1 mutant mice. Bapx1 mutant
mice have defective vertebrae and basal skulls, and are asplenic (Tribioli et
al., 1999; Lettice et al.,
1999; Akazawa et al.,
2000
). Calcein labeling revealed no detectable differences in
early stages of vertebral formation in bapx1-MO injected-animals (data not
shown, but see below) and we did not examine spleen development in
bapx1-MO-injected zebrafish.
Although our morphological and molecular analysis of bapx1-MO-injected animals demonstrate that bapx1 is required for the jaw joint, we cannot be certain that MO injections completely eliminate bapx1 function, especially at later larval stages when the MO is probably significantly diluted. Thus, the lack of detectable differences in early vertebral development in bapx1-MO-injected animals could reflect the late stage this event occurs and the dilution of the injected MO. Once true genetic nulls of zebrafish bapx1 are discovered, the role of bapx1 in patterning the axial skeleton and spleen in zebrafish can be examined.
Morphologically, synovial joints are associated with a complex array of
cell types, including articular cartilage, tendons and ligaments (see
Kingston, 2000). Once markers
are discovered for these tissue types in zebrafish, their formation can be
assayed in anterior arch mutants and bapx1-MO-injected animals. Although GDF5
is insufficient to induce ectopic joints, GDF5 is sufficient to induce tendons
and ligaments (Wolfman et al.,
1997
). Perhaps gdf5 and/or bapx1 in the
developing jaw joint region also function to pattern connective tissues.
In chicks, RAP is derived from CNC emanating from the r4 level
(Kontges and Lumsden, 1996).
Thus, it will be particularly interesting to determine the axial level of
origin of RAP in zebrafish. Genetic null alleles of bapx1 will allow
mosaic analyses to determine which phenotypes (e.g. RAP loss) are
cell-autonomous.
A second edn1 target, hand2, is required for
ventral pharyngeal cartilage formation
In zebrafish, the hand2 mutant cartilage phenotype resembles the
edn1 mutant phenotype (i.e. ventral cartilage in the first two arches
is severely reduced, displaced ventrally and posteriorly, and the jaw joint is
missing). However, one notable difference is that the dorsal cartilages in
hand2 mutants are less affected than in edn1 mutants.
Organized stacks of palatoquadrate and SY chondrocytes are seen in
hand2 mutants, but not in edn1 mutants, which typically lack
the SY cartilage altogether (Kimmel et
al., 1998; Miller et al.,
2000
). Thus, edn1 affects a broader pharyngeal arch
domain than hand2 (see below).
Early edn1-dependent expression of dlx3, EphA3, gsc, msxe and msxb occurs in ventral postmigratory CNC of hand2 zebrafish mutants, showing that none of these genes are sufficient for ventral cartilage formation. Late ventral arch one gsc expression fails to be initiated in hand2 mutants (Fig. 2H,I). This defect, shared with edn1 mutants, may contribute to the shared loss of ventral cartilage and/or the jaw joint in hand2 and edn1 mutants.
hand2 represses joint, dorsal and ventral fates
Given the phenotypic similarity of ventral cartilage reduction in
edn1 and hand2 mutants and that in the mouse pharyngeal arch
expression of msx1 requires hand2 function
(Thomas et al., 1998), we
expected that a subset of the edn1-dependent ventral genes would also
require hand2. Although late ventral arch one gsc expression
failing to be initiated in hand2 mutants meets this prediction, it is
perhaps surprising that not only are the early edn1-dependent genes
expressed, but that msxe and msxb are upregulated.
Precedents exist for Hand2 functioning as a repressor, because in the mouse
limb bud, Hand2 represses expression of Gli3 and Alx4
(te Welscher et al., 2002
). We
suggest that in the zebrafish pharyngeal arches, the repression of
msxe by hand2 is largely in the same ventral arch cells that
in wild types express both genes. msxb, however, appears to be
upregulated in hand2 mutants both in cells that in wild types express
msxb, and in cells that in wild types do not contain detectable
msxb transcript by in situ hybridization (e.g. cells in the anterior
ventral first two arches, see Fig.
7). Once antibodies specific to zebrafish Hand2, MsxB and MsxE are
available, double-labeling experiments in sections could reveal the exact
cellular relationship of these expression domains in pharyngeal arches of wild
types and hand2 mutants. The difficulty in establishing orthology of
the zebrafish msx genes with mammalian msx genes, two of
which are required for craniofacial development, and one of which is
downstream to Edn1 signaling, complicates predicting the head skeletal
consequences of altered msxb and msxe expression in
zebrafish (Ekker et al., 1997
;
Satokata and Maas, 1994
;
Satokata et al., 2000
;
Thomas et al., 1998
).
Functional analyses of the zebrafish msx genes in head skeletal
patterning could reveal specific roles, if any, for msxb and
msxe. Because msx1 and msx2 in mice have redundant
roles in chondrogenesis and osteogenesis
(Satokata et al., 2000
),
perhaps the zebrafish msx genes do also.
Hu et al. (Hu et al., 2001)
show that msx genes maintain cells in a proliferative state by
blocking exit from the cell cycle, thus inhibiting differentiation. These
findings could provide an explanation for why the same phenotype (ventral
cartilage loss) is seen in edn1 and hand2 mutants, despite
opposite effects on msx expression. Perhaps in edn1 mutants,
the early failure to specify the entire ventral arch domain, including
expression of msxe and msxb, results in the almost complete
absence of tissues derived from this domain because of lack of proliferation.
Conversely, in hand2 mutants, perhaps excess and ectopic msx
expression prevents ventral arch CNC from differentiating into
chondrocytes.
The ventrally expanded expression domain of bapx1 in hand2 mutants indicate that the ventral first arch in hand2 mutants has partially adopted a joint fate. However, because the jaw joint later fails to form in hand2 mutants, bapx1 appears to be insufficient for formation of a differentiated jaw joint in this context. Because the expanded ventral domain of bapx1 in hand2 mutants also requires edn1 function, the positioning of bapx1 to the intermediate first arch is accomplished at least in part through the positive regulation of edn1 and the repression ventrally by hand2.
In wild-type zebrafish embryos, eng2 expression in the head
periphery is restricted to a dorsal first arch paraxial mesodermal core,
constrictor dorsalis (reviewed by Kimmel
et al., 2001b). Expression of eng2 expands ventrally in
both hand2 and edn1 mutants, showing that mandibular
mesoderm is dorsalized in edn1 and hand2 mutants, and
indicating that in the early pharyngeal arch primordia, these ventral
specifiers repress dorsal arch fates. Testing the initial proposal of
Piotrowski et al. (Piotrowski et al.,
1996
) that dorsal skeletal identity is expanded ventrally in
anterior arch mutants, awaits the identification of zebrafish dorsal-specific
postmigratory CNC markers. Excitingly, a dorsal second arch dermal bone, the
opercle, expands in animals with reduced edn1 function, providing
another line of evidence that Edn1 signaling represses dorsal pharyngeal arch
fates (Kimmel et al.,
2003
).
Model for DV pharyngeal arch patterning
The downregulation of bapx1 expression in the jaw joint primordium
and of dlx3, EphA3, gsc, msxe and msxb expression ventrally
(this work) (Miller et al.,
2000) in edn1 mutants reveals that Edn1 patterns both
ventral and joint fates. edn1 expression appears contained within the
ventral arch and at least some bapx1 expression does not overlap with
hand2 expression. This raises the hypothesis that Edn1 functions as a
morphogen in patterning the arch primordia, with the ventrally localized
secreted Edn1 signal specifying ventral and joint fates at high and
intermediate thresholds, respectively. An Edn1 gradient model, combined with
the repression of bapx1 by hand2, provides an attractive
mechanism for the positioning of the jaw joint. Analysis of dermal bone
phenotypes in edn1 mutants is consistent with a gradient model
(Kimmel et al., 2003
).
Embryological studies involving focal misexpression of Edn1 would directly
test the gradient model.
Based on our results, we propose the following genetic model for DV pharyngeal arch patterning (Fig. 9). Specification of ventral is in part performed by the edn1-dependent activation of hand2. Specification of the jaw joint is performed by the positive regulation of bapx1 by edn1, acting at a distance from the ventral edn1, source. In the first arch, hand2 restricts the jaw joint by repressing bapx1 in the ventral cartilage-forming domain, hence delimiting the position of the jaw joint. bapx1 positively regulates jaw joint expression of chd and gdf5, which might play roles in pharyngeal joint development.
|
Potential evolutionary implications of bapx1 expression
The localized expression of bapx1, chd and gdf5 to the
zebrafish jaw joint (this work) and tetrapod appendicular joints
(Tribioli et al., 1997;
Francis-West et al., 1999a
;
Francis-West et al., 1999b
;
Scott et al., 2000
;
Storm and Kingsley, 1996
;
Merino et al., 1999
) combined
with the requirements of bapx1 (this work) and gdf5 for
formation of particular joints (Storm and
Kingsley, 1996
) suggests that a conserved genetic network controls
joint formation in both the pharyngeal and appendicular skeleton. Whether
pharyngeal or appendicular joints arose first during evolution is currently
not clear, but some early vertebrates such as placoderms and acanthodians had
a clearly functional jaw joint (Janvier,
1996
).
Because bapx1 in vertebrates and invertebrates is expressed in
gut-associated mesenchyme (Tribioli et
al., 1997; Tribioli and
Lufkin, 1999
; Azpiazu and
Frasch, 1993
), this is probably an ancient role for
bapx1, one present before the jaw or skeletal joints evolved. The
localized expression of bapx1 in the jaw joint is particularly
fascinating given the transformation this region underwent during vertebrate
evolution. The expression and function of zebrafish bapx1 suggests a
specific genetic network exists for jaw joint formation and immediately raises
the question of whether agnathan lampreys have a first arch mesenchymal
bapx1 expression domain. If so, perhaps modification of
bapx1 downstream targets, or modification of Bapx1 function,
facilitated evolution of the jaw. If not, perhaps co-opting bapx1
expression in the first arch of agnathans played a role in the appearance of
jaws and joints during gnathostome evolution.
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ACKNOWLEDGMENTS |
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