Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403, USA
Author for correspondence
(ctm{at}stanford.edu)
Accepted 17 February 2004
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SUMMARY |
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Although we find early patterning of the moz mutant hindbrain to be normal, we find a late defect in facial motoneuron migration in moz mutants. Pharyngeal musculature is transformed late, but not early, in moz mutants. We detect relatively minor defects in arch epithelia of moz mutants. Vital labeling of arch development reveals no detectable changes in CNC generation in moz mutants, but later prechondrogenic condensations are mispositioned and misshapen.
Mirror-image hox2-dependent gene expression changes in postmigratory CNC prefigure the homeotic phenotype in moz mutants. Early second arch ventral expression of goosecoid (gsc) in moz mutants and in animals injected with hox2-MOs shifts from lateral to medial, mirroring the first arch pattern. bapx1, which is normally expressed in first arch postmigratory CNC prefiguring the jaw joint, is ectopically expressed in second arch CNC of moz mutants and hox2-MO injected animals. Reduction of bapx1 function in wild types causes loss of the jaw joint. Reduction of bapx1 function in moz mutants causes loss of both first and second arch joints, providing functional genetic evidence that bapx1 contributes to the moz-deficient homeotic pattern. Together, our results reveal an essential embryonic role and a crucial histone acetyltransferase activity for Moz in regulating Hox expression and segmental identity, and provide two early targets, bapx1 and gsc, of moz and hox2 signaling in the second pharyngeal arch.
Key words: moz, Hox, hoxa2, Zebrafish, Cranial neural crest, Bapx1, Goosecoid, Homeosis, Pharynx
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Introduction |
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Although it is still not clear why Hox2 dysfunction results in
homeotic transformations, analyses in the mouse, chick and Xenopus
have begun to unravel the Hox2-responsive genetic circuitry. A
subtractive screen in mice discovered that Pitx1 is ectopically
expressed in Hoxa2 mutant second arch primordia, and inactivating
Pitx1 in a Hoxa2 mutant partially rescues the homeosis
(Bobola et al., 2003).
Expression analyses in mice reveal that at late stages Hoxa2
represses expression of the chondrogenic factor Sox9 and the
osteogenic factor Runx2 (Cbfa1)
(Kanzler et al., 1998
). Two
other known Hoxa2 target genes are the homeobox genes bapx1
and goosecoid (gsc). In Xenopus, ectopic expression
of Hoxa2 in the first arch represses expression of Bapx1
(Pasqualetti et al., 2000
). In
chicks, ectopic expression of Hoxa2 induces Gsc expression
(Grammatopoulos et al., 2000
),
and in zebrafish early gsc expression is reported to be downregulated
in hox2-MO injected animals (Hunter and
Prince, 2002
). Both Gsc and bapx1 are essential
for craniofacial development, although reducing function of either gene does
not result in homeosis (Rivera-Perez et
al., 1995
; Yamada et al.,
1995
; Miller et al.,
2003
).
Hox gene expression is maintained by trithorax group
(trxG) activity, which involves chromatin remodeling, including
histone acetylation (Simon and Tamkun, 1998). In humans, mutations in
trxG members cause leukemia
(Look, 1997;
Ernst et al., 2002
). The MYST
family histone acetyltransferase MOZ (monocytic leukemia zinc finger protein;
MYST3 - Human Gene Nomenclature Database) is mutated in human leukemias
(Borrow et al., 1996
). Human
MOZ is a large protein of 2004 amino acids and biochemical analyses reveal MOZ
to possess both histone acetyltransferase (HAT) and transcriptional activation
activity (Champagne et al.,
2001
; Kitabayashi et al.,
2001a
). Targets of either of these activities in vivo are unknown
and the function of MOZ during embryonic development has not been
reported.
Previous screens in zebrafish have identified a large number of mutations
causing craniofacial defects (Schilling
et al., 1996a; Piotrowski et
al., 1996
; Neuhauss et al.,
1996
). The cloning of several of these mutations
[endothelin1 (edn1 or sucker)
(Miller et al., 2000
);
tbx1 (van gogh)
(Piotrowski et al., 2003
);
tf2ap2a (lockjaw)
(Knight et al., 2003
)] reveals
remarkable conservation in the genetic control of vertebrate craniofacial
development, as each of these molecules is also required for patterning the
mammalian pharyngeal arches (Kurihara et
al., 1994
; Jerome and
Papaioannou, 2001
; Schorle et
al., 1996
; Zhang et al.,
1996
). To identify genes required for segmental identity in the
pharyngeal arches, we directly screened for mutations affecting cartilage
patterning in zebrafish.
We present the molecular identification and phenotypic characterization of
a zebrafish homeotic mutant discovered in this screen. Fine mapping,
positional cloning, sequencing and morpholino phenocopy experiments reveal
this homeotic locus to encode a zebrafish ortholog of the human oncogene MOZ,
a MYST family HAT. Severely reduced hox2 expression in moz
mutant zebrafish contributes to a mirror-image duplication of jaw cartilages
in place of second arch cartilages. moz is also more broadly required
for maintenance of most hox1-4 expression domains, probably resulting
in the homeotic transformation of the third and fourth arch gill support
cartilages. In the hindbrain, moz is required for maintenance, but
not initiation, of Hox gene expression, and moz mutants display
aberrant facial motoneuron migration. Inhibition of histone deacetylase
activity with Trichostatin A rescues Hox maintenance defects and homeotic
cartilage transformations in moz mutants, indicating that HAT
activity is essential for moz function. Pharyngeal musculature
appears transformed late but not early in moz mutants. We find little
evidence for patterning defects in arch epithelia of moz mutants.
However, striking gene expression changes in moz mutant postmigratory
hyoid CNC are apparent. Expression of bapx1, which is normally
restricted to the jaw joint (Miller et
al., 2003) is robustly duplicated in second arch CNC of
moz mutants. Although reduction of bapx1 function in
wild-type embryos results in absence of the jaw joint
(Miller et al., 2003
),
reduction of bapx1 function results in absence of both the first and
second arch joints in moz mutants. Expression of gsc is
profoundly reorganized in the moz mutant second arch, with lateral
CNC expression shifting to medial, mirroring the wild-type first arch pattern.
Together our results reveal that a zebrafish ortholog of the human oncogene
MOZ regulates Hox gene expression and segmental identity in the
vertebrate pharynx.
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Materials and methods |
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Mapping and positional cloning
Initial mapping was performed with mozb719 on an
outbred wik background. Fine mapping was performed with
mozb719 crossed onto the Islet1:GFP background
(Higashijima et al., 2000),
which was found to be highly polymorphic relative to AB within the z6371-z7351
interval. In these fish, primers 1 and 2
(Table 1) were used to amplify
the microsatellite z6371. Primers and enzymes were used to reveal co-dominant
polymorphisms in the 5' and 3' UTRs, respectively, of fc32e05
(3+4, MnlI) and fc15g12 (5+6, XmnI). All size polymorphisms
were resolved on 1-4% agarose gels using standard techniques. The 3' end
of mki67l was not present on PAC74G4. The SP6 end of PAC 14P16 begins
with the ninth nucleotide of the fc15g12 ORF. PAC ends were sequenced and the
following primers and enzymes used to reveal codominant polymorphisms: 4T (T7
end of PAC 4O19, 7+8, BclI); 14T (T7 end of PAC 14P16, 9+10,
ScfI); 114T (T7 end of PAC 114E16, 11+12, DraI). Accession
numbers are: AY600370 (moz cDNA) and CL525848-CL525855 (PAC end
sequences).
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Morpholino oligo injections
MOs were purchased from Gene Tools (Philomath, OR), and sequences are
listed in Table 1. MOs were
diluted to 25 mg/ml in 1x Danieau buffer. Subsequent dilutions were made
in 0.2 M KCl and 0.2% Phenol Red. These dilutions were injected into the yolk
of one- to four-cell zebrafish embryos, roughly 5 nl per embryo. hoxa2b-MO and
hoxb2a-MO were each injected at 3 mg/ml, and bapx1-MO was injected at 3
mg/ml.
Trichostatin A treatment
Trichostatin A (TSA; Sigma) was dissolved in DMSO to make a 3 mM stock
solution, which was stored at -20°C. For embryo incubations, this stock
solution was diluted to 0.1 M in Embryo Medium (with 0.003% PTU and
penicillin/streptomycin). This concentration of TSA has been shown to cause
increased H4 acetylation in zebrafish
(Collas et al., 1999);
treatments at higher concentrations cause severe edema and severely reduced
cartilage development in wild-type embryos (L.M., unpublished). Embryos in
their chorions were incubated in 0.1 M TSA, 0.003% DMSO beginning at 15 hours
postfertilization and were maintained in this treatment until fixation for in
situ hybridization or Alcian staining. Control sibling embryos were incubated
in 0.003% DMSO.
BODIPY labeling
Vital imaging with the fluorescent dye BODIPY-ceramide was performed as
described (Yan et al., 2002).
Briefly, clutches were soaked in dye continually from late gastrulation
onwards. Although animals continue to develop normally in this dye, it does
slightly retard development, as does keeping the fish at room temperature,
which we did while viewing repeatedly under a Zeiss LSM confocal microscope.
Therefore stages given are the corresponding stages at 28°C, based on the
head-trunk angle and other morphological criteria
(Kimmel et al., 1995
). A total
of 31 fish were examined, eight mutants and 23 wild-type siblings. One side of
the head was imaged from the outer surface to the midline with optical
sections 3 µm apart.
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Results |
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Positional cloning reveals the homeotic locus to encode moz
We mapped b719 using bulk segregant analysis and subsequent fine
mapping to an 4 cM interval between z7351 and z6371 on LG5
(Fig. 1A, data not shown). Two
zebrafish ESTs, fc15g12 and fc32e05, mapped to this region
(http://wwwmap.tuebingen.mpg.de;
http://134.174.23.167/zonrhmapper/Maps.htm)
were found to closely flank the b719 locus, by 0.11 cM and 0.05 cM,
respectively (Fig. 1A). Two
PACs for each of these two ESTs were isolated by PCR from DNA pools of an
arrayed zebrafish PAC library (Amemiya and
Zon, 1999
). Mapping polymorphisms derived from the PAC ends
revealed that ends of two of the PACs had crossed the recombinants
(Fig. 1A). Sequencing the T7
end of PAC4O19 revealed an exon highly homologous to the human histone
acetyltransferase MOZ, positioning moz as within the
non-recombinant interval (Fig.
1A). By aligning vertebrate Moz sequences and using degenerate
PCR, the rest of the predicted zebrafish moz ORF was isolated and is
predicted to encode a 2246 amino acid protein. The first five exons were found
to reside on PAC 114E16, whereas exons 8-16 were contained on PAC4O19.
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Expression of zebrafish moz at 28-48 hours postfertilization (hpf) appears ubiquitous in the head but expression is undetectable in the trunk and tail (Fig. 1C-E). The diffuse posterior boundary of moz expression during this time frame roughly coincides with the boundary between the hindbrain and spinal cord (Fig. 1C-E). Analyzing moz expression at 24, 28, 36 and 48 hpf in clutches of embryos from mozb719 heterozygotes yielded three clear classes of animals based on moz expression: strong, intermediate and faint. PCR genotyping revealed the strong class to be homozygous wild types, the intermediate class to be heterozygous for the mozb719 mutation, and the faint class to be mozb719 homozygous mutants. The reduction of moz expression in both classes of moz mutants appeared to globally affect moz expression levels in all cranial tissues (data not shown).
Reducing moz function results in pharyngeal cartilage homeosis
In moz mutants, second arch cartilages adopt a mirror-image first
arch pattern, forming an ectopic jaw (Fig.
2A,B). A novel large opening to the pharynx is present on either
side of the head, resembling an ectopic mouth
(Fig. 2B, but see below).
Especially ventrally, this homeotic phenotype resembles the phenotype seen
upon reducing function of both hoxa2b and hoxb2a (C. T.
Miller, PhD Thesis, University of Oregon, 2001)
(Hunter and Prince, 2002).
Flat-mounting dissected cartilages from moz mutants reveals that the
second arch cartilages adopt shapes characteristic of first arch cartilages
(Fig. 2D,E). In the
moz mutant second arch, the hyomandibular region of the dorsal second
arch cartilage that normally articulates with the otic capsule is missing
(Fig. 2D,E;
Table 2), presenting a more
complete homeotic transformation than observed in the earlier work (C. T.
Miller, PhD Thesis, University of Oregon, 2001)
(Hunter and Prince, 2002
). The
moz mutant dorsal second arch cartilage, in the more ventral position
of the thin symplectic cartilage (Kimmel
et al., 1998
) is thicker than its wild-type counterpart,
resembling the wild-type first arch dorsal (upper) jaw cartilage, the
palatoquadrate. The moz mutant ventral second arch cartilage is
shorter, thinner, contains fewer rows of chondrocytes, and forms a knob on its
lateral end, resembling the wild-type first arch ventral (lower) jaw
cartilage, Meckel's (Fig. 2;
and see below). Furthermore, in the first two arches of moz mutants,
the dorsal cartilages fuse to one another and the ventral cartilages fuse to
one another. by contrast, dorsal/ventral fusions within either arch are only
rarely seen (see below).
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|
To confirm that these homeotic phenotypes in moz mutants are due
to reduction of Moz function, we injected moz morpholino antisense
oligonucleotides (MOs). We have previously shown that MO injections can
efficiently phenocopy severe phenotypes of larval head skeletal mutants, as
well as reveal hypomorphic phenotypes at lower doses
(Miller and Kimmel, 2001).
Animals injected with any of three different moz MOs display
dose-dependent homeotic phenotypes seen in moz mutants
(Fig. 2C,F,I;
Table 2), strongly supporting
our conclusion that reduction of moz function causes the
b719 and b999 homeotic phenotypes.
Injection of lower doses of each morpholino, as well as analyses of the slightly variable b719 and hypomorphic b999 phenotypes (Table 2), show that the several homeotic phenotypes described above are separable, and that homeosis is not an all-or-nothing phenomenon. Some mutant animals display shape changes of the dorsal hyoid cartilage (the HM cartilage) without having deletions of HM (Table 2), showing moz controls at least two processes, positioning and shaping, of dorsal second arch cartilage formation. Likewise, some mutant animals display homeotic shape changes of the ventral hyoid cartilage without displaying the inversion (Table 2), similarly arguing that these two processes are separable. Interestingly, shape changes are more frequently seen than fusions, regardless of MO or dose (Table 2). The dorsal deletion of the hyomandibular portion of the hyosymplectic was the least penetrant for both mutant alleles and with both doses of all three MOs (Table 2). Thus, sensitivity to reduction of moz function ranges from high for shape changes to intermediate for fusions to low for dorsal deletions.
moz is required for most Hox group 1-4 expression domains
As anterior transformation of arch two to one resembles the mouse
Hoxa2 and zebrafish hoxa2b;hoxb2a loss-of-function
phenotypes (Gendron-Maguire et al.,
1993; Rijli et al.,
1993
; Hunter and Prince,
2002
) (C. T. Miller, PhD Thesis, University of Oregon, 2001), we
asked if moz functions upstream of hox2 genes. Although
zebrafish have at least seven Hox clusters, only two hox2 genes are
retained, hoxa2b and hoxb2a
(Amores et al., 1998
).
Expression of both hoxa2b and hoxb2a is broadly and severely
downregulated in the moz mutant second arch primordia by 33 hpf
(Fig. 3A-D, see below). Like
the second pharyngeal arch expression and despite the separable regulation of
pharyngeal arch and CNS Hox gene expression domains
(Prince and Lumsden, 1994
;
Maconochie et al., 1999
),
hindbrain expression of hoxa2b is also severely reduced in
moz mutants at 33 hpf (Fig.
3E,F). All rhombomeres that express hoxa2b (rhombomeres
2-5 or r2-r5) appear to do so at a lower level in moz mutants,
although r2 appears to be the most strongly affected. Expression of
hoxa2b is strikingly reduced in medial r2 and lateral r4
(Fig. 3E,F). To determine if
hoxa2b expression was simply delayed in moz mutants, we
assayed a later time point, 48 hpf. Even with overdeveloped in situ
hybridization and similar to expression at 33 hpf, arch expression of
hoxa2b is undetectable (Fig.
3G,H), and hindbrain expression is still drastically reduced (data
not shown).
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We next asked if moz is required for expression of other Hox
genes. hoxa1a expression is normally not present in a typical Hox
domain spanning one or multiple segments but instead is in clusters of cells
in the ventral forebrain and midbrain and in scattered cells in the anterior
hindbrain (McClintock et al.,
2001; McClintock et al.,
2003
). In contrast to the moz requirement for later
expression of hoxa2b and hoxb2a, hoxa1a expression is not
appreciably affected in moz mutants
(Fig. 3K,L).
For the hoxba cluster, expression of group 1-4 genes are affected
in a graded fashion in moz mutants, with hoxb1a being the
most severely affected and hoxb4a the most mildly affected. Severe
Hox expression defects in moz mutants are also present in the
embryonic hindbrain. At 36 hpf, the r4-restricted hindbrain expression of
hoxb1a is nearly abolished in moz mutants
(Fig. 3M,N). In addition to the
missing second arch domain (see above), hindbrain expression of
hoxb2a is reduced in moz mutants
(Fig. 3O,P). hoxb3a
expression is reduced in the hindbrain and in the third to fifth pharyngeal
arch primordia in moz mutants
(Fig. 3Q,R), perhaps
contributing to the third arch homeotic phenotype presented above. Expression
of hoxb4a is mildly reduced in both the hindbrain and pharyngeal
arches four through six of moz mutants
(Fig. 3S,T). Expression of the
other hox3-4 genes is similarly affected as their respective
hoxb3a and hoxb4a paralogs (data not shown). Initiation of
Hox genes b1a-b4a, like hoxa2b, appears unaffected
in the hindbrain of moz mutants. Hence, similar to trithorax
group (trxG) genes (Simon and
Tamkun, 2002; Ernst et al.,
2002
), moz appears to be required for the maintenance,
but not initiation, of expression of particular Hox genes in the
hindbrain.
However, hox5-6 gene expression appears unaffected in moz mutants (Fig. 3U,V; data not shown). Thus, moz expression, present throughout the embryonic head (see Fig. 1C-E), is specifically required for most hox1-4 expression domains in the hindbrain and pharyngeal arches.
Inhibition of histone deacetylase activity partially rescues the moz mutant phenotype
Because human MOZ has been shown to have histone acetyltransferase (HAT)
activity (Champagne et al.,
2001), and because trxG factors that maintain Hox gene
expression are associated with HAT activity
(Petruk et al., 2001
;
Milne et al., 2002
), we
wondered whether the inability of moz mutants to maintain Hox gene
expression was due to hypoacetylation. The histone deacetylase inhibitor
trichostatin A (TSA) has been shown to rescue defects caused by trxG
mutations in Drosophila (Sollars
et al., 2003
) and human cells
(Milne et al., 2002
). We
therefore asked whether TSA treatment could rescue the moz mutant
phenotype. moz mutant embryos that are incubated in 0.1 M TSA
starting at about 15 hpf show striking rescue of arch cartilage homeosis
(Table 3;
Fig. 4A-D) and rescue of Hox
gene expression (Fig.
4E-H).
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|
moz mutants display late hindbrain neuronal phenotypes
Given the broad expression defects of group 1-4 Hox genes, we analyzed
hindbrain neuronal development in moz mutants. Facial motoneurons
differentiate in r4 and begin to migrate posteriorly towards r5-6 around 15
hpf (Chandrasekhar et al.,
1997; Maves et al.,
2002
). At 48 hpf, some mutants display mispositioned facial
motoneurons (Fig. 5A,B),
resembling hoxb1 loss-of-function phenotypes seen in zebrafish and
mice (McClintock et al., 2002
;
Studer et al., 1996
;
Goddard et al., 1996
;
Gavalas et al., 2003
). In
contrast to Mll/Trithorax mutant mice, which also fail to maintain
Hox expression (Yu et al.,
1998
), cranial ganglia appear to innervate each pharyngeal arch
(Fig. 5C,D). The early
reticulospinal neurons, some of which are born by 10 hpf
(Mendelson, 1986
) and show
anterior transformations upon reduced function of hoxb1
(McClintock et al., 2002
),
display no detectable alterations in moz mutants (data not shown).
Thus, early segmentation and neuronal specification of the moz mutant
hindbrain occurs relatively normally, while later hindbrain phenotypes in
moz mutants are consistent with a defect in maintenance, but not
initiation, of Hox gene expression.
|
Pharyngeal arch muscles are derived from paraxial mesoderm, which initially
occupies central locations (arch `cores') in the pharyngeal arch, ensheathed
by postmigratory CNC (reviewed by Kimmel
et al., 2001b). Each arch mesodermal core subdivides into a
discrete pattern of identified myogenic cores. The first and second arches
display different sequences of mesodermal core subdivision. Although at
intermediate stages in fish the first arch contains three myogenic cores
[constrictor dorsalis (CD), adductor mandibulae (AM) and intermandibularis
(IM)], the second arch contains only two [constrictor hyoideus dorsalis and
ventralis (CHD and CHV)]. These myogenic cores subsequently subdivide into
primordia for individual muscles
(Edgeworth, 1935
) (reviewed by
Kimmel et al., 2001b
). This
early difference in the wild-type arch one and two intermediate myogenic core
pattern precludes assigning segmental homology to subsequently-forming dorsal
and intermediate muscles. However, this intermediate pattern serves as a
segmental character distinguishing the first two arches. Thus, we wondered
whether this aspect of segmental identity was transformed in moz
mutants.
eng2 expression marks the dorsal first arch myogenic condensation,
constrictor dorsalis (Hatta et al.,
1990; Ekker et al.,
1992
) (reviewed by Kimmel et
al., 2001b
). Expression of eng2 in moz mutants
at 28 hpf is not seen homeotically duplicated in the second arch
(Fig. 6A,B). myod
expression marks all pharyngeal arch myogenic condensations
(Schilling and Kimmel, 1997
).
We examined myod expression in moz mutants at 44 hpf, a
stage soon after myod expression labels the first and second arch
myogenic condensations (Fig.
6C,D). The arrangement of myod-expressing cores in
moz mutants at this stage appears grossly indistinguishable from the
wild-type pattern (Fig. 6C,D).
Slightly later in development at 54 hpf, subtle defects are observed in
moz mutant myod-expressing myogenic condensations. Ectopic
patches of myod expression are seen in the intermediate second arch
of moz mutants (Fig.
6E,F).
|
Defects in pharyngeal epithelia are not detected in moz mutants
Pharyngeal endoderm is required for many aspects of CNC patterning
(Piotrowski and Nüsslein-Volhard,
2000; Piotrowski et al.,
2003
; Couly et al.,
2002
) and chondrification of CNC requires contact with pharyngeal
endoderm (Epperlein, 1974
).
Given that the hyomandibular (HM) region of the dorsal second arch cartilage
almost never chondrifies in moz mutants
(Table 2), we wondered whether
missing, mispositioned or mis-specified pharyngeal pouches contribute to the
moz mutant phenotype.
We directly assayed developing pharyngeal pouch morphology and
specification by following the expression of the FGF target gene pea3
(Roehl and Nüsslein-Volhard,
2001) at 24, 34 and 54 hpf. Each pharyngeal pouch, which separates
the arch primordia, consists of an epithelial bilayer with an AP polarity:
expression of pea3 and the secreted ligand edn1 are both
expressed in posterior, but not anterior, pharyngeal endodermal epithelia
(Fig. 7A,B)
(Miller et al., 2000
). The
first pharyngeal pouch, which abuts dorsal second arch CNC, is present in
moz mutants and appears similarly patterned as its wild-type
counterpart at all three stages examined
(Fig. 7A,B; data not shown).
The same was true for more posterior pharyngeal pouches
(Fig. 7A,B; data not shown). We
wondered if the mirror-image duplication of the first arch pattern in the
moz mutant second arch could be due to defects or possibly even
reversals in pharyngeal pouch polarity. However, expression of pea3
(Fig. 7A,B) and edn1
(data not shown) in moz mutants revealed no defects in pouch
polarity.
|
Between the inverted ectopic jaw and the enlarged third arch in
moz mutants, large bilateral openings in the pharynx resemble ectopic
mouths (see Fig. 2B). To
determine if gene expression data support this interpretation, we examined
expression of a stomadeal marker, pitx2c
(Essner et al., 2000;
Schweickert et al., 2001
), in
moz mutants. pitx2c expression in wild-type embryos at 54
hpf strongly labels the mouth, presumably the ectodermal derivatives of the
stomodeum (Fig. 7E,F).
Expression of pitx2c in moz mutants was not detected
ectopically in these enlarged pharyngeal openings, providing no evidence for
stomadeal identity.
In chicks, the thymus forms largely from the third and fourth endodermal
pharyngeal pouches, which attract blood-borne lymphocyte precursors
(LeDouarin and Jotereau,
1975). In mice, hox3 genes regulate thymus formation
(Manley and Capecchi, 1995
;
Manley and Capecchi, 1998
). In
zebrafish, rag1 expression in lymphocytes marks the early thymus
(Willett et al., 1997
). In
pbx4(lzr) mutants, which have reduced hox3
expression, the thymus fails to form as assayed by rag1 expression
(Popperl et al., 2000
). Thus,
we similarly asked whether moz mutants form a thymus by examining
rag1 expression at 4 days. rag1 expression in the thymus is
present in moz mutants, although reduced
(Fig. 7G,H). Taken together,
these results reveal relatively minor defects in arch epithelial tissues in
moz mutants and are consistent with the idea that many aspects of the
arch environment are set up independent of the CNC
(Veitch et al., 1999
;
Gavalas et al., 2001
).
Early CNC generation appears normal in moz mutants but mispositioned and misshapen condensations form
Finding evidence suggesting that early patterning of non-CNC arch tissues
is normal in moz mutants, we next analyzed the CNC. In mice, Hox
genes not only control segmental identity, but also control the generation of
CNC (Gavalas et al., 2001). To
determine whether the broad Hox expression defects result in a defect in CNC
generation, we examined early pharyngeal arch primordia in living embryos with
the fluorescent dye BODIPY ceramide. This vital labeling offers nice
histological resolution of all major differentiated cell types
(Kimmel et al., 2001b
;
Yan et al., 2002
). Examining
arch primordia in labeled embryos from clutches of moz mutants
revealed mutants to be morphologically indistinguishable from their wild-type
siblings around 28 hpf when postmigratory CNC has populated the arch and
surrounded the mesodermal cores. No deficit in hyoid CNC was apparent
(Fig. 8A,B). Consistent with
this, expression of the broadly expressed CNC marker dlx2 at 28 hpf
appears unaffected in moz mutants (data not shown). Slightly later,
around a 34 hpf stage, the moz mutant hyoid arch appears slightly
hypoplastic (Fig8C,D), although
gross arch morphology appears relatively normal. These same optical sections
confirm our in situ results that no gross changes are apparent in the early
pharyngeal pouches in moz mutants (see above).
|
moz and hox2 genes repress early second arch expression of bapx1, which is required for aspects of moz-mediated homeosis
Reduced hox2 expression can at least in part account for the
anterior transformation homeotic skeletal and muscular phenotypes observed in
the second arch of moz mutants. To investigate the molecular
consequences of hox2 downregulation in the early second arch
primordium, we analyzed embryonic expression of a known hox2 target
gene, bapx1 (Pasqualetti et al.,
2000), in moz mutants and in embryos injected with
morpholinos to reduce function of hoxa2b and hoxb2a.
In embryos and larvae, bapx1 is expressed in a patch of
intermediate first arch, but not second arch, mesenchyme
(Fig. 9A,D)
(Miller et al., 2003). In
moz mutants at 33 hpf, first arch bapx1 expression is
present while an ectopic bapx1 domain is seen in the second arch
(Fig. 9B,E), providing
molecular confirmation of an anterior transformation in second arch CNC of
moz mutants. Ectopic second arch bapx1 expression is also
observed in embryos injected with hoxa2b and hoxb2a
morpholinos (95%, 37/39; Fig.
9C), demonstrating that hox2 dysfunction is sufficient to
result in these homeotic molecular changes. Ectopic bapx1 expression
is present in moz mutants and hox2-MO injected animals when
bapx1 expression first initiates arch expression, around 30 hpf (data
not shown). This molecular homeosis is stable, as bapx1 expression is
maintained in the second arch at 54 hpf
(Fig. 9F,G) and 4 days
(Fig. 9H,I).
|
|
Viewing early embryonic gsc expression from a ventral aspect reveals gsc expression to consist of a thin medial crescent in the ventral first arch and a broad lateral crescent in the ventral second arch (Fig. 10A). Examination of gsc expression in moz mutants from this ventral aspect reveals a startling patterning change. In moz mutants, ventral second arch expression of gsc appears as a thin medial crescent, mirroring the first arch pattern (Fig. 10B). Like the bapx1 expression change, this shifting of gsc expression is also observed in embryos injected with hoxa2b and hoxb2a morpholinos (92%, 23/25; Fig. 10C), demonstrating that hox2 dysfunction is also sufficient to result in this homeotic molecular change of gsc expression. At 41 hpf, dorsal arch two expression of gsc is reduced (Fig. 10D,F). However, lateral views do not reveal maintenance of the patterning change that ventral views do: the shifting of lateral gsc expression to the medial second arch (Fig. 10E,G). The pattern at 41 hpf is slightly different than the 33 hpf pattern (Fig. 10H), suggesting gsc expression is either dynamic, and/or that movements of gsc-expressing cells occur. Together our results suggest moz not only controls maintenance of an early pattern, but also specification of subsequent dynamic changes in patterning in the second arch CNC well before differentiation begins.
|
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Discussion |
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Although no other in vivo functional data have been reported for
moz in other vertebrates, biochemistry on human MOZ has revealed
multiple functional domains. These domains include a founding HAT domain of
the MYST family, an N-terminal transcriptional repression domain, and a
C-terminal transactivation domain
(Champagne et al., 2001;
Kitabayashi et al., 2001a
).
MOZ additionally contains two C4HC3 zinc fingers and a C2HC nucleosome
recognition motif (Borrow et al.,
1996
). This composite structure suggests MOZ can bind other
proteins and chromatin, acetylate histones, and modulate transcription. Our
moz alleles are predicted to truncate the C terminus, causing loss of
a transcriptional activation (TA) domain, but leaving the HAT domain intact.
However, the severely reduced moz mRNA levels detected by in situ
hybridization in mozb719 mutants makes it likely that the
overall activity of the remaining protein would be greatly reduced. This
reduced expression of moz in mozb719 mutants
suggests that mozb719 mutant transcripts are unstable or
that moz directly or indirectly regulates its own transcription.
Both moz lesions we present are loss of function lesions, as
mutant phenotypes are phenocopied by morpholino injections. However, we cannot
rule out the possibility that both alleles and morpholino phenotypes are all
hypomorphic until deletion alleles are found. Although we have no evidence of
a fish-specific duplication of moz, vertebrates do have a closely
related gene, Morf (monocytic leukemia zinc finger protein related
factor; Myst4 - Mouse Genome Informatics), which is also mutated in human
leukemias (Champagne et al.,
1999; Panagopoulos et al.,
2001
). An embryonic function has been reported for Morf
(named Querkopf) in mice (Thomas et al.,
2000
). An insertion in the 5'UTR of mouse MORF causes skull
and forebrain defects, but hindbrain or homeotic pharyngeal arch defects were
not reported (Thomas et al.,
2000
). Whether MORF and MOZ have overlapping functions is
unknown.
Biochemical analyses of human MOZ have also revealed multiple
MOZ-interacting partners. MOZ physically interacts with RUNX1 (AML1) and RUNX2
(AML3 or CBFA1) (Kitabayashi et al.,
2001b; Pelletier et al.,
2002
). The RUNX2 interaction is particularly interesting, as this
is the osteogenic gene shown in mice to be repressed by Hoxa2
(Kanzler et al., 1998
). Thus,
in our analyses of segmental identity in the pharyngeal arches of moz
mutants, we examined pharyngeal bones expecting to see ectopic bone formation.
However, the hyoid bone pattern (Kimmel et
al., 2003
) was undetectable and duplicated mandibular bones were
not seen in moz mutants (C.M., unpublished). Although we did not
examine bone patterning in hox2-MO injected fish, one possibility is that MOZ
functionally interacts with RUNX2 during pharyngeal development in
zebrafish.
Whether mammalian MOZ regulates Hox gene expression, as we predict, awaits
generation of moz mutant mice. The regulation of Hoxa2 by
the transcription factor AP2 is conserved from mammals to fish
(Maconochie et al., 1999;
Knight et al., 2003
). However,
no reduction in AP2 expression was observed in moz mutants at 28 hpf
(C.M., unpublished results). Thus, the regulation of hox2 genes by
moz appears to act through another mechanism, possibly by directly
transactivating Hox genes.
moz regulates segmental identity of pharyngeal cartilages
Our results extend the understanding that Hox genes specify segmental
identity in the vertebrate pharynx, as the transformed pharyngeal segments in
moz mutants correlate with reduced Hox expression in pharyngeal arch
primordia. A mirror-image duplicated jaw replaces the hyoid cartilages in
moz mutants, resembling the phenotype seen in animals co-injected
with hoxa2b and hoxb2a morpholinos (C. T. Miller, PhD
Thesis, University of Oregon, 2001)
(Hunter and Prince, 2002).
However, the moz mutant transformation is more complete, as dorsal
transformations are more severe and dorsal fusions more common than in hox2-MO
injected animals. It is likely that morpholinos cause incomplete
loss-of-function at later developmental timepoints when the injected
morpholino is significantly diluted. Alternatively, moz might
regulate other genes that are also expressed in hyoid CNC and contribute to
segmental identity.
Despite this stronger phenotype, the homeotic transformation in the hyoid
arch of moz mutants is still not complete in that the pterygoid
process of the palatoquadrate (PTP) is not seen duplicated. Perhaps in
moz mutants, as has been proposed for mouse Hoxa2 mutants,
only certain axial levels of CNC are transformed, i.e. perhaps PTP is derived
from midbrain crest whose derivatives are not seen duplicated in the second
arch of Hoxa2 mutants
(Köntges and Lumsden,
1996). de Beer (de Beer,
1937
) proposed that PTP was a premandibular element.
The pharyngeal arches are more sensitive to partial reduction of
Hoxa2 function in the mouse
(Ohnemus et al., 2001). The
partial transformations observed in these hypomorphic mouse mutants led these
authors to propose that homeosis was not an `all-or-nothing' phenomenon, as
the second arch did not act as a developmental unit as a whole. Our results,
in which a hypomorphic allele and low-level injections of Moz-MOs also
separate particular homeotic phenotypes from others, strongly support this
conclusion.
Experiments in Xenopus with an inducible Hoxa2 construct
revealed that the time of Hoxa2 overexpression affected the resultant
phenotype: overexpressing Hoxa2 early during CNC migration resulted
in `segmentation' phenotypes, where arch derivatives were fused, while
overexpressing late in postmigratory CNC resulted in `homeotic' phenotypes,
where cartilage shapes were altered
(Pasqualetti et al., 2000). As
interarch fusions could also be interpreted as homeosis (i.e. loss of
individual arch identity), this distinction is debatable. However, we note
that the moz mutant phenotype contains more frequent shape changes
(`homeotic') than fusions (`segmentation'), both dorsally and ventrally for
both mutant alleles and for all three morpholinos at two different doses each.
pbx4(lzr) mutants might display the converse phenotype, i.e.
segmentation appears more affected than homeosis. Interarch fusions seen in
moz mutants resemble the pbx4(lzr) mutant
phenotype, although fusions are more severe in pbx4(lzr)
mutants (Pöpperl et al.,
2000
). The more severe phenotype of pbx4(lzr)
mutants might reflect differences in the set of affected target genes and/or
temporal differences of target gene regulation (e.g. initiation versus
maintenance).
moz mutants also present with mild anterior homeotic
transformations of pharyngeal arches three and four (branchial or gill-bearing
arches one and two). Arch three and four cartilages in moz mutants
are slightly thicker, and typically contain an enlarged process on their
lateral end, resembling the retroarticular process of Meckel's (the lower jaw)
cartilage. Especially in the third arch of moz mutants at 5 days, an
ectopic dorsal cartilage is also frequently seen. These transformations in the
moz mutant anterior branchial arches are not seen in hox2-MO injected
animals (Hunter and Prince,
2002). These phenotypes probably result from additional Hox genes
(e.g. Hox3 and Hox4 genes) that moz regulates (see
above). Once genetic alleles of these zebrafish Hox genes are isolated, their
function in specifying pharyngeal segmental identity can be assessed.
This third arch cartilage phenotype in moz mutants somewhat
resembles the phenotype of valentino (val) mutants
(Moens et al., 1998) (reviewed
by Kimmel et al., 2001a
),
which was interpreted to be an ectopic interhyal cartilage based on ectopic
hoxb2a expression in the third arch of val mutants. As
neither hoxa2 nor hoxb2 is expressed in the moz
mutant third arch, we propose that the moz mutant third arch has
adopted mandibular fate. We did not detect ectopic bapx1 expression
in third or fourth arch CNC of moz mutants. However, given the subtle
nature of the skeletal change, the causative gene expression changes would
probably be subtle as well.
Moz is required for Hox maintenance and behaves like a trithorax group factor
Consistent with the homeotic pharyngeal arch phenotype, we observe defects
in hox1-4 gene expression in moz mutants. Three pieces of
evidence suggest that Moz functions similarly to trithrorax (trxG) factors in
regulating maintenance of Hox gene expression, as we discuss below.
First, we do not detect changes in initiation of Hox gene expression in the
hindbrain of moz mutants. By later stages, graded reduction of most
hox1-4 expression domains in the CNS domains is apparent. Defects in
Hox gene maintenance, but not initiation, are hallmarks of trx
mutants in Drosophila (Breen and
Harte, 1993) and mouse (Yu et
al., 1998
). Loss of Hox group 1 or 2 gene function in zebrafish or
in mice can cause severe homeotic neuronal transformations in the hindbrain
and motor axon pathfinding defects
(McClintock et al., 2002
;
Cooper et al., 2003
;
Studer et al., 1996
;
Gavalas et al., 1997
;
Gavalas et al., 1998
;
Gavalas et al., 2003
;
Rossel and Capecchi, 1999
;
Gendron-Maguire et al., 1993
;
Rijli et al., 1993
).
Supporting a role for moz in maintenance of Hox expression in the
hindbrain, we find that early neuronal specification in the hindbrain and
axonal trajectories in the head periphery are approximately normal in
moz mutants. The only consistent neuronal defect we are able to
detect in moz mutants is the disruption of facial motor neuron
migration. This phenotype may be consistent with the defect in maintenance of
hoxb1a expression in moz mutants, as loss of hoxb1a
in zebrafish or Hoxb1 in mice causes a similar defect
(McClintock et al., 2002
;
Studer et al., 1996
). In the
mouse Mll (trx) mutant, cranial ganglia are condensed and
fail to innervate the pharyngeal arches
(Yu et al., 1998
), but more
specific neuronal defects have not been reported and MLL mutant zebrafish have
not been described.
Second, Moz has a HAT domain, which for human MOZ has been demonstrated to
have HAT activity (Champagne et al.,
2001), and HAT activity has been associated with trxG factors
(Petruk et al., 2001
;
Milne et al., 2002
).
Furthermore, HAT activity is required for Moz function, as treatment with a
histone deacetylase inhibitor rescues many aspects of the moz mutant
phenotype.
Third, we find that moz mutant homeosis and Hox maintenance
defects are rescued by TSA, and TSA has been shown to rescue defects caused by
trxG mutations in Drosophila
(Sollars et al., 2003) and
human cells (Milne et al.,
2002
). Why does inhibition of histone deacetylase activity rescue
a putative decrease of histone acetylation? It seems likely that the
transcriptional on or off state of Hox genes is maintained through a balance
of chromatin modification activities, including histone acetylation by
trxG factors and histone deacetylation by Polycomb group
(PcG) factors (Milne et al.,
2002
) (reviewed by Francis and
Kingston, 2001
; Simon and
Tamkun, 2002
). In support of this idea, trxG and
PcG factors are antagonistic for proper Hox expression. For example,
homeotic axial transformations and Hox expression defects of
Mll-deficient mice and Bmi1-deficient mice are rescued when
function of both genes is removed (Hanson
et al., 1999
; Yu et al.,
1995
; Yu et al.,
1998
). We do not yet know whether Moz directly acetylates histones
associated with Hox regulatory regions. Western analyses show no detectable
decrease of acetylated histone H4 levels in moz mutants compared with
wild-type siblings (L.M., unpublished). We might expect to see rhombomere- or
arch-specific defects in acetylated histone H4 levels at specific Hox genes,
but at the present time this is very difficult to test.
Taken together, these findings implicate Moz as a trxG factor.
trxG genes have been genetically defined as suppressors of
PcG mutant phenotypes (reviewed by
Kennison, 1995). Further
studies demonstrating genetic interactions between moz and
PcG genes would provide firm support for moz as a
trxG gene.
One interesting aspect of Hox regulation that has emerged from our analysis
of moz mutants is that in general, there appears to be a gradient
effect of Moz activity within a Hox complex. We find that the hox1-4
requirement for Moz activity ranges from strong for group 1 to weak for group
4, while group 5 and 6 Hox genes show no Moz requirement. We see this gradient
effect on similar paralogs, where they have segmental domains in hindbrain and
CNC, but we do not see a moz requirement for hoxa1a
expression, even though moz appears to be expressed ubiquitously
throughout the head. Our findings suggest that Moz activity plays a global
role in Hox locus regulation, possibly through HAT activity. MYST family HAT
activity has been shown to have a chromosomal gradient of transcription
control in yeast (Kimura et al.,
2002). Whether a Moz-mediated gradient of histone acetylation
exists across group 1-5 genes in Hox clusters remains to be determined.
moz is required for late but not early patterning of head musculature
Our data suggest that at times when severe hox2, bapx1 and
gsc expression defects are present in postmigratory hyoid CNC of
moz mutants, head mesodermal patterning appears unaffected.
eng2, with eng3 the only segmentally restricted head
mesodermal marker that we know of (Ekker
et al., 1992; Hatta et al.,
1990
), appears appropriately confined to the first arch dorsal
muscle core (constrictor dorsalis) of moz mutants. Likewise, the
early myod expression pattern, which labels all proposed arch
myogenic cores (see Kimmel et al.,
2001b
), appears normal in moz mutants. The absence of
eng2 duplication or myod pattern disruption could be due to
residual hox2 activity in moz mutants. Alternatively,
moz and hox2 genes could play no role in restricting
eng expression to the first arch or setting up the pattern of
myod-expressing myogenic cores. The homeotic late muscle pattern seen
in moz mutants perhaps results from transformed CNC-derived
connective tissue, which has been shown to pattern paraxial-mesodermally
derived and somitic-derived myocytes
(Noden, 1983a
;
Noden, 1983b
;
Noden, 1986
).
Dramatic changes in CNC expression of bapx1 and gsc prefigure the moz mutant phenotype
In stark contrast to the apparently normal early patterning of moz
mutant mesoderm, endoderm and surface ectoderm, expression of two known
hox2 target genes, bapx1 and goosecoid
(gsc), is radically perturbed in postmigratory CNC of moz
mutant second arch primordia.
Within postmigratory CNC, bapx1 expression is confined to a patch
of intermediate first arch mesenchyme which appears to prefigure the jaw joint
(Miller et al., 2003). We
previously identified edn1 and hand2 (dHAND) as
positive and negative regulators, respectively, of bapx1 expression.
bapx1 expression spreads ventrally in hand2 mutants
(Miller et al., 2003
). We
report that moz and hox2 genes also contribute to
positioning bapx1 to the jaw joint, although these genes prevent
bapx1 from being expressed in an intermediate domain of the hyoid
arch. Thus, bapx1 integrates positional information from both the DV
(edn1, hand2) and AP axes (moz, hox2) to achieve its
jaw-joint-restricted expression. Furthermore, as an aspect of the moz
mutant homeotic pattern (the jaw joint) requires bapx1, these results
identify bapx1 as a crucial downstream effector contributing to the
homeotic transformation.
Microarray comparisons of gene expression in the second arches of wild-type
and Hoxa2 mutant mice revealed Pitx1 to be upregulated in
the Hoxa2 mutant second pharyngeal arch primordial
(Bobola et al., 2003), similar
to what we report here for bapx1. These authors report finding no
confirmed gene that is downregulated in Hoxa2 mutant second arches,
and suggest that Hoxa2-mediated segmental identity in the second arch
might largely involve repression of the first arch program. Although
microarray analyses promise to provide a global view of overall changes in
gene expression in Hoxa2 mutant arches, our demonstration of
spatially shifted gsc expression highlights the need to also analyze
potential spatial reorganization of affected genes.
In the mouse, gsc expression is spatially restricted within first
and second arch CNC (Gaunt et al.,
1993). Although gsc is required for specific aspects of
mouse craniofacial development, defects in first, but not second, arch
derivatives were reported (Rivera-Perez et
al., 1995
; Yamada et al.,
1995
). In both fruitflies and vertebrates, gsc functions
as a transcriptional repressor (Danilov et
al., 1998
; Ferreiro et al.,
1998
; Mailhos et al.,
1998
; Latinkic and Smith,
1999
; Yao and Kessler,
2001
), although precedent exists for gsc positively
regulating target genes (frzb)
(Yasuo and Lemaire, 2001
).
The identity of gsc target genes and the nature of their regulation
in the pharyngeal arches remains to be determined. bapx1 and
gsc are expressed in strikingly complementary patterns in the first
two arches (compare Fig. 9A-C with Fig. 10A-C), suggesting
one might repress expression of the other. Our previous report that in
hand2 mutant zebrafish ventral first arch expression of gsc
is lost, while bapx1 expression expands ectopically into this domain
(Miller et al., 2003
), is
consistent with gsc repressing bapx1 arch expression.
The inverted gsc expression domain in the early second arch
primordia of moz mutants suggests reorganization of the fate map at
an early prechondrogenic stage has occurred. This model is consistent with our
finding that moz mutant prechondrogenic condensations are
mispositioned and the finding that in the mouse Hoxa2 mutant,
chondrogenesis is induced in different regions of the arch than in wild types
(Kanzler et al., 1998). This
latter study additionally showed that transgenically driving Sox9
expression in the Hoxa2 domain partially phenocopied the
Hoxa2 mutant phenotype. Furthermore, transgenically driving
Hoxa2 with an Msx2 promoter resulted in loss of cranial
bones, suggesting Hoxa2 represses both cartilage and bone formation
(Kanzler et al., 1998
). Our
bapx1 and gsc expression data suggests that moz and
hox2 affect patterning within the second arch primordia long before
cartilage or bone differentiation occurs.
Perhaps gsc expression labels chondrogenic cells and these cells
shift medial in the early CNC cylinder. Alternatively, gsc expression
could label non-chondrogenic cells. Although the first model is more
consistent with the demonstrated cell-autonomous function of gsc in
mice (Rivera-Perez et al.,
1999), the latter is more consistent with the moz mutant
phenotype, in which the lateral end of the duplicated lower jaw cartilage
fuses laterally with the lower jaw near the jaw joint. In mice, although
gsc was found to be cell autonomous, gsc-null cells in the
presence of wild-type cells could contribute to the condensation of the
tympanic bone, a bone that never forms in gsc mutants
(Rivera-Perez et al., 1995
;
Rivera-Perez et al., 1999
;
Yamada et al., 1995
).
However, these gsc-null cells were not maintained
(Rivera-Perez et al., 1999
).
Exogenous gsc can induce neighboring cells to form a secondary axis,
suggesting in some contexts, Gsc can have non cell-autonomous functions
(Cho et al., 1991
;
Niehrs et al., 1993
).
A central mystery remaining is why the duplication in moz or
hox2-deficient animals is mirror image. At the time the gsc
expression defect appears, hoxa2 expression appears to mark all hyoid
postmigratory CNC. Thus, the spatially complex gsc defect in
hox2-injected animals is hard to reconcile with a model in which hox2
genes simply positively regulate gsc. The gsc expression
defect is also hard to reconcile with a model in which hox2 genes
modify responsiveness of second arch CNC to a single cue emanating from the
arch 1/2 boundary (Rijli et al.,
1993). We propose a modified version of the model of Rijli et al.,
in which hox2 modifies the responsiveness of hyoid CNC to multiple
environmental signals. The mediolateral inversion of gsc could be
explained if hox2 genes conferred responsiveness of second arch CNC
to a lateral surface ectodermal signal cue to activate gsc while
repressing responsiveness to a medial endodermal cue that normally repressed
gsc expression. Continued forward genetic screens in zebrafish could
reveal components of these putative signaling pathways that underlie segmental
identity in the pharyngeal arches.
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ACKNOWLEDGMENTS |
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Footnotes |
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