Alkek Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Blvd, Houston, TX 77030, USA
* Author for correspondence (e-mail: jmartin{at}ibt.tamushsc.edu)
Accepted 1 September 2003
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SUMMARY |
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Key words: Homeobox, Craniofacial morphogenesis, Haploinsufficiency
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Introduction |
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Investigation of Pitx2 function using loss-of-function approaches
in mice has shown that Pitx2 plays an important role in early stages
of tooth development (Gage et al.,
1999; Kitamura et al.,
1999
; Lin et al.,
1999
; Lu et al.,
1999
). Pitx2-null mutant embryos had arrested tooth
development at placode or bud stage. Consistent with a haploinsufficient
mechanism, tooth phenotypes were observed in Pitx2 null
+/ mice (Gage et al.,
1999
). Early epithelial-mesenchymal signaling was intact in
Pitx2-null embryos as suggested by the presence of a condensed dental
mesenchyme (Lin et al., 1999
;
Lu et al., 1999
). Expression
of markers such as Shh and mesenchymal Bmp4 and
Msx1 also supported the idea that tooth initiation and specification
occurred but tooth germ expansion failed in Pitx2-null embryos
(Lin et al., 1999
;
Lu et al., 1999
). In situ also
showed that Bmp4 expression was expanded, while Fgf8 failed
to be expressed or was downregulated in oral epithelium of Pitx2-null
embryos (Lin et al., 1999
;
Lu et al., 1999
). Taken
together, these data suggest that the initial events in tooth development
occurred in the absence of Pitx2, subsequent signaling events were
deranged resulting in a premature extinction of Fgf8 expression and
failure of demarcation of Bmp4 expression to dental epithelium. These
experiments uncovered an early function for Pitx2 in tooth
morphogenesis but failed to address any later role for Pitx2 in
craniofacial development.
The Pitx2 gene encodes three isoforms, Pitx2a, Pitx2b and
Pitx2c in mice and a fourth Pitx2 isoform, Pitx2d,
has been identified in humans (Cox et al.,
2002). The different isoforms are generated by both alternative
splicing and alternative promoter usage
(Shiratori et al., 2001
)
(Fig. 1A,B) and have both
overlapping and distinct expression patterns. All Pitx2 isoforms have
a common C terminus and distinct N termini
(Fig. 1A). Pitx2c is
the asymmetrically expressed isoform while Pitx2a, Pitx2b and
Pitx2c isoforms are co-expressed in head mesoderm, oral ectoderm,
eye, body wall and central nervous system
(Kitamura et al., 1999
;
Liu et al., 2001
;
Schweickert et al., 2000
;
Smidt et al., 2000
).
Pitx2c, but not Pitx2a or Pitx2b, is expressed in
hematopoietic stem cells (Degar et al.,
2001
). Co-expression of Pitx2 isoforms is found in the
three developmental fields that are most frequently affected in individuals
with RGS I: eyes, teeth and anterior abdominal wall.
|
We investigated Pitx2 isoform function in craniofacial
morphogenesis by analyzing craniofacial phenotypes of isoform-specific
deletions. We used Pitx2 alleles that encode differing levels of
Pitx2 to investigate the requirements for total Pitx2 dose
in craniofacial morphogenesis (Liu et al.,
2001). Our results show that Pitx2 isoforms have
interchangeable function in craniofacial development and that signaling
pathways that are regulated by Pitx2 respond differently to changes
in total Pitx2 dose. The Fgf8 maintenance pathway uses low
Pitx2 doses, while Bmp4 repression requires high
Pitx2 doses. Our findings uncovered downstream functions for
Pitx2 in tooth development and fate mapping experiments with a
Pitx2 cre recombinase knock-in allele revealed that Pitx2
daughter cells are migratory. Movement of Pitx2 daughters was
aberrant in Pitx2 mutants, suggesting that Pitx2 regulates
cell movement in craniofacial primordia.
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Materials and methods |
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lacZ staining and histology
Mouse embryos were fixed in Bouin's, dehydrated and embedded in paraffin
wax. Sections were cut (7-10 µm) and stained with Hematoylin and Eosin.
lacZ staining was as previously described
(Lu et al., 1999).
Generation of the Pitx2 alleles
The Pitx2 abcnull,
abhypoc,
ab and
c
alleles have been described previously
(Liu et al., 2001
;
Liu et al., 2002
;
Lu et al., 1999
). For
Pitx2
abccreneo allele, a targeting vector
was constructed that introduced cre recombinase neofrt into
PvuII and Nru1 sites in Pitx2 fifth exon. Crosses
to a rosa26 eFlp deletor strain resulted in neomycin removal
(Farley et al., 2000
). Crosses
to Pitx2
abcnull allele confirmed that
Pitx2
abccreneo was a null allele and in
situ hybridization experiments showed cre expression recapitulated
endogenous Pitx2.
RT-PCR
Total mRNA was extracted using SV total RNA isolation system (Promega) and
cDNA produced with M-MLV reverse transcriptase (Invitrogen). Four
Pitx2 primers detected Pitx2 isoform expression: exon 2
(5'-attgtcgcaaactagtgtcgg-3'), exon 3
(5'-ccgtgaactcgacctttttga-3'), exon 4
(5'-tcctgggactcctccaaacat-3') and exon 5
(5'-gtttctctggaaagtggctcc-3'). A 104 bp Pitx2b fragment
was amplified with exon 2 and exon 3 primers, 159 bp Pitx2a fragment
with exon 2 and exon 5 primers and a 207 bp Pitx2c fragment with exon
4 and exon 5 primers.
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Results |
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We studied Pitx2a and Pitx2b isoform expression using the
abhypoc and
ab alleles that contain
a lacZ knock-in into Pitx2 exon 2 and deletes Pitx2
exon 3 (Fig. 1C-F). As
lacZ was introduced into exon 2, this analysis provides information
about Pitx2a and Pitx2b specific expression but does not
distinguish between these two isoforms because Pitx2a uses exon 2 and
Pitx2b uses both exon 2 and exon3
(Fig. 1A). We used RT-PCR to
distinguish between Pitx2a and Pitx2b expression (see
below). We also performed in situ analysis using a Pitx2c probe. At
10.5 dpc, lacZ was expressed uniformly throughout the oral ectoderm,
while at 14.5 dpc, lacZ expression was found in dental epithelium and
primary enamel knot of cap stage tooth
(Fig. 1D-F). Using a
Pitx2c probe for in situ, we detected Pitx2c expression
throughout the 10.5 dpc oral ectoderm (Fig.
1G). At 14.5 dpc, Pitx2c was expressed in dental
epithelium similarly to Pitx2a and Pitx2b
(Fig. 1H,I). To distinguish
between Pitx2a and Pitx2b isoform expression in oral
ectoderm, we performed RT-PCR with a primer set that distinguished between
Pitx2a, Pitx2b and Pitx2c. We identified all three isoforms
in the mandibular arch epithelium at 10.5 and 12.5 dpc
(Fig. 1J). These data suggest
that the Pitx2a, Pitx2b and the Pitx2c isoforms are
coexpressed in oral ectoderm and, at later stages, within tooth epithelial
structures.
Pitx2 isoforms have interchangeable functions in tooth
development
Co-expression of Pitx2 isoforms suggests a number of possibilities
for the regulation of target pathways by Pitx2. It is possible that
Pitx2 isoforms would regulate distinct target genes in tooth
formation or Pitx2 isoforms may have redundant functions. Isoform
co-expression also supports the idea that some Pitx2 target genes
have a requirement for Pitx2 heterodimers
(Cox et al., 2002). To address
these ideas, we analyzed forming teeth of
ab/ and
c/ embryos.
As a control, we analyzed teeth of ab;
c
mutant embryos. We reasoned that this allelic combination should encode near
normal levels of all Pitx2 isoforms, albeit from different
chromosomes, and should be functionally similar to
abcnull heterozygous embryos. Analysis of coronal
and sagittal sections through the teeth of
ab;
c embryos at 14.5 and16.5 dpc revealed that tooth development
was normal (Fig. 2A-D). From
this, we conclude that the
ab and
c alleles
encode adequate levels of Pitx2 isoforms to support normal tooth
development.
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We noted that Dlx2 was still expressed in the caudal aspect of the
Pitx2 mutant mandibular mesenchyme
(Fig. 3I,J). As Pitx2
expression is restricted to the rostral mandibular arch ectoderm, continued
expression of Dlx2 in caudal mandibular mesenchyme suggested that
Fgf8 signaling from the caudal aspect of the mandibular ectoderm was
intact in the Pitx2 abcnull mutant embryos
and that patterning of the mandibular process was disrupted in the
Pitx2
abcnull mutants. Goosecoid
(Gsc), an Fgf8 responsive homeobox gene, is normally
expressed in the caudal mandibular arch mesenchyme. Caudal Gsc
expression is normally maintained via a Fgf8 repressive pathway that
inhibits Gsc expression in the rostral mandibular process
(Tucker et al., 1999
). We
reasoned that if maintenance of Fgf8 signaling was disrupted in
Pitx2
abcnull mutants, then Gsc
expression should be expanded rostrally. We found that Gsc expression
was weakly expanded in a subset of Pitx2
abcnull mutants embryos
(Fig. 3M,N), while in the
remainder of mutant embryos Gsc expression was caudally restricted
(data not shown). The incomplete penetrance of expanded Gsc
expression suggests that in the subpopulation of Pitx2 mutant embryos
with correct Gsc expression, the early Fgf8 expression was
sufficient to specify the correct Gsc expression domain.
Correct patterning of the mandibular mesenchyme is necessary for formation
of Meckel's cartilage (Tucker et al.,
1999). Based on the weak expansion of Gsc expression, we
expected that Pitx2-null mutants would have a weak Meckel's cartilage
phenotype. To assess this, we performed whole-mount cartilage staining on
Pitx2
abcnull mutants and control
wild-type littermate embryos. The Pitx2
abcnull mutants had a variable deficiency of
Meckel's cartilage supporting the notion that rostral caudal polarity of the
mandibular process was weakly affected by loss of Pitx2 function
(Fig. 3O,P). Taken together,
these data suggest that in the absence of Pitx2, Fgf8 expression in
oral ectoderm fails to be maintained. In the absence adequate Fgf8
signaling, Fgf8-dependent signaling to underlying mesenchyme is
reduced leading to defective mandibular arch rostral caudal polarity.
Differential sensitivity of Pitx2 target pathways to changes
in total Pitx2 dose
To address the idea that Pitx2 target pathways have distinct
requirements for total Pitx2 dose, we examined Fgf8 and
Bmp-signaling pathways in Pitx2 allelic combinations that
encode differing levels of Pitx2 activity
(Liu et al., 2001). We used
the
abcnull allele, in conjunction with the
ab and
abhypoc alleles that encode
reduced levels of Pitx2c in the absence of Pitx2a and
Pitx2b to generate Pitx2 allelic combinations with
intermediate levels of Pitx2 activity. Previously, we showed that the
abcnull+/ embryos expressed
58% of
homozygous wild-type Pitx2c mRNA levels while the
abcnull;
ab and
abcnull;
abhypoc allelic
combinations expressed
50% and 38% of wild-type Pitx2c mRNA
levels respectively (Liu et al.,
2001
).
At 10.5 dpc, Fgf8 expression was not detectable in the
Pitx2 abcnull homozygous mutant oral
ectoderm, supporting the idea that Pitx2 was required for maintenance
of Fgf8 expression in the oral ectoderm
(Fig. 3Q,R)
(Lin et al., 1999
;
Lu et al., 1999
). In the
rostral mandibular process of Pitx2
abcnull mutant embryos, Barx1 and
Pax9, mesenchymal targets of Fgf8 signaling pathways
(Neubuser et al., 1997
;
Tucker et al., 1998
), were not
expressed or had greatly diminished expression
(Fig. 3T,U,W,X). Caudal
mandibular arch expression of Barx1 was maintained in Pitx2
abcnull mutant embryos as this expression is
probably dependent on Fgf8 and Edn1 signaling from the
caudal aspect of the mandibular process that does not express Pitx2
(Fig. 3R,S). By contrast, the
abcnull;
ab and
abcnull;
abhypoc allelic
combinations, that encode reduced levels of Pitx2c mRNA and lack
Pitx2a and Pitx2b (Liu
et al., 2001
) (Fig.
1C), expressed Fgf8 in the oral ectoderm of 10.5 dpc
embryos (Fig. 3Q-S and data not
sown). Barx1 and Pax9 were expressed in the
abcnull;
abhypoc embryos
that encode low levels of Pitx2
(Fig. 3T,W).
We investigated whether repression of Bmp signaling by Pitx2 was
also rescued in the
abcnull;
abhypoc allelic
combination that encodes low levels of Pitx2 function. To assess
expansion of Bmp signaling, we examined Bmp4 expression in oral
ectoderm of 10.5 dpc Pitx2 mutant embryos. In contrast to the
Fgf8 signaling pathway, Bmp repression required high levels of
Pitx2 function. In Pitx2
abcnull/ embryos Bmp4
expression was expanded laterally in mandibular process ectoderm
(Fig. 4A,B)
(Lu et al., 1999
). In
wild-type embryos, Bmp4 expression is found in the medial mandibular
process and the distal aspect of the ectoderm of the maxillary process at 10.5
dpc (Fig. 4B,E). In
Pitx2
abcnull;
abhypoc and
abcnull;
ab allelic combinations,
Bmp4 expression in the mandibular process was weakly expanded.
Moreover, in the maxillary process ectoderm of Pitx2
abcnull;
abhypoc and
abcnull;
ab mutants, Bmp4
expression failed to be distally restricted and was detected all the way to
the junction with the mandibular process
(Fig. 4C-G).
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Pitx2 regulates tooth orientation and cap formation
We investigated the tooth morphology of the
abcnull;
abhypoc and
abcnull;
ab allelic combinations
using histological analysis. Sections through 18.5 dpc wild-type, and
abcnull;
ab mutant embryos revealed
well-formed molars. We found that in the
abcnull;
ab embryos, the orientation of the molar tooth was abnormal
(Fig. 5A,C,E,G). In
abcnull;
abhypoc 18.5 dpc
mutant embryos, analysis of serial sections revealed that molar teeth were
absent (Fig. 5B,F). As
lacZ marks cells fated to express Pitx2a and
Pitx2b, serving as a marker of dental epithelium, we performed
lacZ staining on serial cryosections from heads of 14.5 dpc
Pitx2 allelic combinations. In
ab+/
and
abcnull;
ab embryos,
well-formed cap stage molar teeth were clearly evident with lacZ
staining (Fig. 5I,J). In
abcnull;
abhypoc mutant
embryos, the dental lamina invaginated but failed to form the dental cap
(Fig. 5K). In Pitx2
abcnull homozygous mutant embryos, tooth
development arrested at the placode or bud stage. The molar phenotype in
abcnull;
abhypoc
embryos, with a more developed dental lamina, suggests that tooth development
progressed further than in
abcnull mutant embryos.
These data show that as the dose of Pitx2 decreases there is evidence
of increasingly severe defects in tooth morphogenesis.
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Pitx2 regulates cell movement from the oral ectoderm into
oral cavity and facial ectoderm
Our previous data revealed that Pitx2 functioned to regulate local
cell movement in heart development (Liu et
al., 2002). To determine if a similar mechanism was at work in
craniofacial development, we used the
abccre knock
in allele and the Gtrosa 26 reporter mouse to follow the movement of
Pitx2 daughter cells within the first branchial arch. At 9.5-11.0
dpc, cre expression was detected in the oral ectoderm in both
abccre+/ and
abccre;
abcnull embryos,
although by 11.0 dpc cre expression was diminished in the
abccre;
abcnull embryos
(Fig. 6A,B and not shown).
Cre expression was restricted to oral ectoderm and was not found in
facial ectoderm or epithelium lining the oral cavity
(Fig. 6C-E). Fate mapping with
the GtRosa26 reporter showed that Pitx2 daughters were
detected in the oral ectoderm, periocular mesenchyme, guts, heart and body
wall (Fig. 6F,G).
|
Pitx2 daughters extensively populated the floor and roof inside the forming mouth (Fig. 6L-O). In Pitx2 mutants, fewer daughter cells populated the oral cavity roof as compared with wild type (Fig. 6N-Q). Pitx2 daughters contributed to Rathke's pouch and dental epithelium, of both the wild type and mutant although in the Pitx2 mutant tooth morphogenesis was arrested (Fig. 6N-S and not shown). These data reveal that Pitx2 daughter cells exit the oral ectoderm and contribute to both facial ectoderm and the ectoderm lining the oral cavity and Pitx2 function is necessary for correct deployment and expansion of daughter cells.
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Discussion |
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Pitx2 regulates mandibular morphogenesis by maintaining
Fgf8 and repressing Bmp4 expression
Deletion of Fgf8 in oral ectoderm revealed a role for
Fgf8 in survival and outgrowth of mandibular mesenchyme
(Trumpp et al., 1999), while
pharmacological suppression of Fgf signaling in explants suggested that Fgf
functioned primarily by signaling to the underlying mesenchyme
(Mandler and Neubuser, 2001
).
Bead implantation also suggested an early role for Fgf8 in
establishing the maxillo-mandibular region of the chick embryo
(Shigetani et al., 2000
).
Importantly, antagonistic interactions between Fgf and Bmp signaling has been
implicated in proximodistal mandibular arch patterning, placement of tooth
organ formation and determination of the maxillo-mandibular region of the
early embryo (Neubuser et al.,
1997
; Shigetani et al.,
2000
; Tucker et al.,
1998
).
Our data reveal that Pitx2 maintains Fgf8 expression in
branchial arch ectoderm. Expression of prospective Fgf8 target genes,
such as Barx1 and Pitx1, was severely reduced in
Pitx2 abcnull homozygous mutant embryos.
Consistent with a role of Fgf8 signaling in mandibular rostral caudal
polarity, expression of Gsc was expanded rostrally in the mandibular
process of Pitx2
abcnull mutants. In
addition, as Pitx2 is normally expressed in rostral mandibular arch
ectoderm that contributes to oral ectoderm, Pitx2
abcnull homozygous mutants lose Barx1
expression in the rostral but not caudal mandibular arch. The Pitx2
abcnull;
abhypoc and
abcnull;
ab mutant embryos express
Fgf8 and Fgf8 target genes, suggesting that maintenance of
this pathway requires only low doses of Pitx2.
In contrast to Fgf8, high doses of Pitx2 are required for
repression of Bmp signaling. In the
abcnull;
abhypoc and
abcnull;
ab mutants, expression of
Bmp4 was expanded in maxillary ectoderm while Msx1 and
Msx2 expression was expanded in mesenchyme of both maxillary and
mandibular processes. Thus, expression of the Bmp target genes was more
significantly expanded than expression of Bmp4 ligand. This may
reflect the induction of a signal relay cascade in the mandibular process. It
is also interesting to note that Dpp has been shown to act as a
classical morphogen in the wing imaginal disc of Drosophila
(Entchev et al., 2000
;
Teleman and Cohen, 2000
).
We found that in abcnull;
abhypoc and
abcnull;
ab Pitx2 mutants components of Bmp4 and Fgf8
signaling pathways, such as Msx1 and Barx1, are co-expressed
in mandibular mesenchyme. Previous work suggested an antagonistic interaction
between these two signaling pathways
(Neubuser et al., 1997
;
Tucker et al., 1998
). It is
likely that in the Pitx2 mutant allelic combinations, Bmp signaling
is only weakly expanded and this is insufficient to antagonize expression of
Barx1 in mandibular mesenchyme.
These data provide insight into the normal function of Pitx2 in
regulating gene expression. The Fgf8 pathway and the Bmp
suppression pathway have different requirements for total Pitx2 dose.
As Pitx2, Fgf8 and Bmp4 are co-expressed in many cells of
the oral ectoderm, one can envision a mechanism where Pitx2 would
directly regulate Fgf8 and Bmp4 expression. In this model,
one idea to explain the different requirements for Pitx2 dose in
regulating Bmp4 and Fgf8 would be that the regulatory
regions of Bmp4 and Fgf8 contain different numbers of
high-affinity Pitx2-binding sites, a mechanism suggested to underlie
the haploinsufficiency of individuals with Holt-Oram syndrome that are
heterozygous for tbx5 (Bruneau et
al., 2001). Thus, Pitx2 target genes with more
Pitx2-binding sites would require higher doses of Pitx2 for
correct levels of gene expression. However, this model is complicated by in
vitro observations showing that Pitx2 can cooperatively bind DNA
(Dave et al., 2000
;
Wilson et al., 1993
),
suggesting that low levels of Pitx2 can form higher order complexes
on DNA. It is likely that there are other mechanisms, such as interaction with
co-factors, to constrain or augment the ability of Pitx2 to activate
target genes. Further experiments are necessary to rule out the possibility
that Pitx2 indirectly regulates the Fgf8 and Bmp4
pathways.
Pitx2 in tooth morphogenesis and cell movement in
craniofacial development
Pitx2-null embryos have arrest of tooth development at the placode
or bud stage (Gage et al.,
1999; Lin et al.,
1999
; Lu et al.,
1999
). In the Pitx2
abcnull;
abhypoc and
abcnull;
ab embryos, molar tooth morphogenesis was partially rescued in
that an invaginated dental lamina formed without a cap or the orientation of
the dental cap was abnormal. Our in situ studies showed that Fgf8 was
expressed in the oral ectoderm of
abcnull;
abhypoc and
abcnull;
ab embryos. Moreover, expression of Pax9 was also
detected in the prospective dental mesenchyme and Barx1 was expressed
in proximal mandibular mesenchyme of these embryos revealing that Fgf
signaling to mandibular mesenchyme is intact in the Pitx2 hypomorphic
embryos. Although expanded Bmp signaling could account for tooth
defects in the
abcnull;
abhypoc and
abcnull;
ab embryos, the abnormal tooth morphology was not suppressed
by reducing Bmp4 dose using a Bmp4-null allele (W.L. and
J.F.M., unpublished). Based on these data, we favor the notion that
Pitx2 regulates tooth morphogenesis through a pathway that is
distinct from Fgf8 and Bmp4 signaling, although further
experiments are required to investigate these ideas.
Our fate-mapping studies show that Pitx2 daughter cells move from
oral ectoderm to populate facial and inner oral cavity ectoderm.
Pitx2-expressing cells make a decision to extinguish Pitx2
and become motile. It may be that Pitx2 expression promotes cell
compaction or inhibits cell motility. It is notable that one of the phenotypes
of the Pitx2-null embryos was failure of compaction and
differentiation of the periocular mesenchyme
(Lu et al., 1999).
Fgf8 signaling was implicated in cell movement as Fgf8-null
embryos had defects in cell migration through the primitive streak. Analysis
of Xenopus sprouty2, an inhibitor of Fgf signaling, revealed that Fgf
signaling in Xenopus regulated both mesoderm induction and convergent
extension movements (Nutt et al.,
2001
). Thus, it is plausible that Pitx2 regulates cell
movement in the craniofacial primordia through an Fgf8-mediated
pathway.
A direct connection of Pitx2 to cytoskeleton and morphogenetic
movement has been made by the observation that Pitx2 controls Rho
GTPase activity by regulating expression of the guanine nucleotide
exchange factor, Trio (Wei and
Adelstein, 2002). It has recently been proposed that
Pitx2 is a target of canonical Wnt ß-catenin signaling pathway
in pituitary and cardiac development
(Kioussi et al., 2002
). This
work uncovered a genetic interaction between Pitx2 and dishevelled 2,
a Wnt pathway branchpoint, in the heart. Other studies showed that
Rho family GTPases are downstream components of non-canonical planar
cell polarity (PCP) pathway (Habas et al.,
2003
; Strutt et al.,
1997
; Winter et al.,
2001
). Although further experiments are required, our data showing
that Pitx2 daughters are migratory supports the idea that
Pitx2 may be a component of a non-canonical Wnt pathway in
craniofacial development.
Pitx2 and the phenotypic heterogeneity of Rieger syndrome
I
The phenotypes in individuals with Rieger syndrome with PITX2
mutations are heterogeneous. Our data reveal that slight changes in
Pitx2 dose can have a large influence on resulting phenotypes. This
is illustrated most clearly by comparing the
abcnull;
abhypoc and
abcnull;
ab mutants that have only
slight changes in Pitx2 activity but dramatic differences in tooth
morphogenesis (Liu et al.,
2001
). Many organ systems, such as heart and lungs, cannot
distinguish between these small differences in Pitx2 activity
(Liu et al., 2001
).
The isoform deletions of Pitx2 reveal functional redundancy
between isoforms in tooth development. These data are consistent with the
observation that all Pitx2 mutations detected in individuals with
Rieger syndrome are in regions common to all isoforms
(Alward, 2000;
Kozlowski and Walter, 2000
;
Priston et al., 2001
;
Saadi et al., 2001
). Our data
suggest that the Pitx2 N terminus does not have a significant
function in tooth morphogenesis because this region is not conserved between
Pitx2a, Pitx2b and Pitx2c. This differs from pituitary and
skeletal muscle where the N terminus has an influence on Pitx2
function (Kioussi et al.,
2002
; Suh et al.,
2002
). It is also clear that Pitx1 functions
cooperatively with Pitx2 in pituitary organogenesis and limb
development (Marcil et al.,
2003
). As Pitx1 is co-expressed with Pitx2 in
developing teeth, it will be interesting to investigate potential cooperative
functions of Pitx1 and Pitx2 in oral and dental
epithelium.
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ACKNOWLEDGMENTS |
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