Odd-skipped related 2 (Osr2) encodes a key intrinsic regulator of secondary palate growth and morphogenesis
Yu Lan,
Catherine E. Ovitt,
Eui-Sic Cho*,
Kathleen M. Maltby,
Qingru Wang and
Rulang Jiang
Center for Oral Biology and Department of Biomedical Genetics, Aab
Institute of Biomedical Sciences, University of Rochester School of Medicine
and Dentistry, Rochester, NY 14642, USA
Author for correspondence (e-mail:
rulang_jiang{at}urmc.rochester.edu)
Accepted 17 March 2004
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SUMMARY
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Development of the mammalian secondary palate involves multiple steps of
highly regulated morphogenetic processes that are frequently disturbed during
human development, resulting in the common birth defect of cleft palate.
Neither the molecular processes governing normal palatogenesis nor the causes
of cleft palate is well understood. In an expression screen to identify new
transcription factors regulating palate development, we previously isolated
the odd-skipped related 2 (Osr2) gene, encoding a zinc-finger protein
homologous to the Drosophila odd-skipped gene product, and showed
that Osr2 mRNA expression is specifically activated in the nascent
palatal mesenchyme at the onset of palatal outgrowth. We report that a
targeted null mutation in Osr2 impairs palatal shelf growth and
causes delay in palatal shelf elevation, resulting in cleft palate. Whereas
palatal outgrowth initiates normally in the Osr2 mutant embryos, a
significant reduction in palatal mesenchyme proliferation occurs specifically
in the medial halves of the downward growing palatal shelves at E13.5, which
results in retarded, mediolaterally symmetric palatal shelves before palatal
shelf elevation. The developmental timing of palatal growth retardation
correlates exactly with the spatiotemporal pattern of Osr1 gene
expression during palate development. Furthermore, we show that the
Osr2 mutants exhibit altered gene expression patterns, including
those of Osr1, Pax9 and Tgfb3, during palate
development. These data identify Osr2 as a key intrinsic regulator of palatal
growth and patterning.
Key words: Cleft palate, Odd-skipped, Osr2, Palate development
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Introduction
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The mammalian secondary palate arises from the medial sides of the
maxillary processes flanking the embryonic oral cavity as two bilateral
outgrowths that initially grow vertically down the sides of the developing
tongue. At a precise developmental stage, the bilateral palatal shelves
elevate to a horizontal position above the dorsum of the tongue and fuse with
each other at the midline to form the intact secondary palate that separates
the nasal cavity from the oral cavity
(Ferguson, 1988
). Any
disturbance of the growth, elevation or fusion of the palatal shelves could
result in cleft palate, one of the most common birth defects in humans.
Considerable efforts have gone into genetic and epidemiological studies of
human orofacial clefting (Murray,
2002
; Carinci et al.,
2003
). Several cleft-causing mutations have recently been
identified, including mutations in the IRF6, MSX1, PVRL1 and
TBX22 genes (van den Boogaard et
al., 2000
; Suzuki et al.,
2000
; Braybrook et al.,
2001
; Sozen et al.,
2001
; Kondo et al.,
2002
; Jezewski et al.,
2003
). In addition, studies of targeted mutations in mice have
shown that mutations in a growing number of genes each results in cleft palate
in nullizygous mutants (Thyagarajan et
al., 2003
). Most of these mutant mice also have gross craniofacial
developmental defects (e.g.
Gendron-Maguire et al., 1993
;
Rijli et al., 1993
;
Satokata and Maas, 1994
;
Martin et al., 1995
;
Sanford et al., 1997
;
Peters et al., 1998
),
indicating that cleft palate is a common secondary effect of other
craniofacial abnormalities. Mutations in Msx1 and Tgfb3,
which are normally expressed during palate development, have been shown to
cause cleft palate as a primary effect, because the gene products normally
regulate palatal mesenchyme proliferation and palatal shelf fusion,
respectively (Fitzpatrick et al.,
1990
; Pelton et al.,
1990
; Satokata and Maas,
1994
; Kaartinen et al.,
1995
; Proetzel et al.,
1995
; Taya et al.,
1999
; Zhang et al.,
2002
).
Despite the large number of genes associated with cleft palate formation,
strikingly little is known about the molecular processes governing normal
palatal growth and patterning. For example, there is little understanding of
what controls the initial phases of palatal outgrowth from the medial sides of
the maxillary processes, although this occurs at the developmental time when
many drugs are administered to experimentally induce cleft palate in animal
models (Salomon and Pratt,
1979
; Shah, 1984
;
Ferguson, 1988
;
Diehl and Erickson, 1997
). In
an attempt to identify genes regulating the initial phases of palate
development, we previously carried out an expression screen in mouse embryos
and showed that the odd-skipped related 2 (Osr2) gene is specifically
activated in the nascent palatal mesenchyme at the onset of palatal outgrowth
(Lan et al., 2001
). The
Osr2 gene encodes a zinc-finger protein with extensive sequence
similarity to the Drosophila Odd-skipped family of putative
transcription factors (Lan et al.,
2001
). The odd-skipped gene was initially identified as a
pair-rule gene because mutations at this locus cause loss of the odd-numbered
segments in the Drosophila embryo
(Nusslein-Volhard and Wieschaus,
1980
; Coulter et al.,
1990
). Gene expression and phenotypic analyses indicate that the
odd-skipped gene product functions to prevent inappropriate
expression of other segmentation genes
(Coulter and Wieschaus, 1988
;
DiNardo and O'Farrell, 1987; Mullen and
DiNardo, 1995
; Saulier-Le
Drean et al., 1998
). During mouse embryogenesis, Osr2
mRNA expression marks the medial maxillary regions where the initial palatal
outgrowths occur and persists strongly in the downward growing palatal
shelves. Upon palatal shelf elevation and fusion at the midline, Osr2
expression in the palate is downregulated
(Lan et al., 2001
). The
sequence homology to the Odd-skipped family of transcription factors and the
dynamic expression pattern suggest that Osr2 may play an important role in
palate development. Interestingly, recent annotation of the human genome
database assigned the orthologous human OSR2 gene to chromosome 8q23,
in a chromosomal region strongly associated with non-syndromic orofacial
clefting (Prescott et al.,
2000
). To analyze the function of Osr2 in palate
development, we have generated mutant mice carrying a targeted deletion of the
Osr2 coding region and found that Osr2 mutants have specific
defects in palatal shelf growth and morphogenesis.
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Materials and methods
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Targeted disruption of the mouse Osr2 gene
A BAC clone containing the Osr2 genomic region was isolated from
the RPCI-22 129/SvEvTac mouse BAC library (BACPAC Resources, Children's
Hospital of Oakland, Oakland, CA). A targeting vector was made with a 9.4 kb
HindIII fragment containing all three coding exons of the
Osr2 gene subcloned from the BAC clone
(Fig. 1A). The targeting vector
contains a 2 kb 5' homology arm containing the initial 15 codons of the
Osr2 open reading frame fused in-frame with a modified bacterial
lacZ gene that encodes a nuclearly localized ß-galactosidase, a
loxP-flanked PGK-neo expression cassette, a 3.3 kb 3' homology
arm and a PGK-DTA expression cassette. Correct targeting of the
Osr2 locus with this vector results in the replacement of 2.6 kb of
the Osr2-coding region with the lacZ gene and the neo
expression cassette, which is expected to produce a ß-galactosidase
fusion protein containing the N-terminal 15 amino acid residues of the Osr2
protein.

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Fig. 1. Targeted disruption of the mouse Osr2 gene. (A) The Osr2
gene consists of four exons spanning 8 kb of genomic DNA. Boxes indicate
exons, with the protein-coding region marked in black. The positions of the
translation start (ATG) and stop (TAG) codons are also indicated. Restriction
sites are: B, BamHI; E, EcoRI; H, HindIII; P,
PstI; X, XbaI. The targeting vector used the 2.2 kb
XbaI-PstI fragment containing the intron 1/exon 2 junction
as the 5' arm and the 3.3 kb XbaI-HindIII fragment
3' to the Osr2-coding region as the 3' arm. A modified
bacterial lacZ gene and a neo expression cassette were
inserted in between the arms and a diphtheria toxin A (DTA)
expression cassette was cloned 3' to the 3' arm for negative
selection against random integration. Correct targeting results in the
lacZ gene and the neo cassette replacing most of the
Osr2 coding region, from the sixteenth codon of the open reading
frame to the XbaI site in the 3' untranslated region.
Arrowheads above the wild-type and mutant genomic schematics indicate the
positions of PCR primers used for genotyping. (B) Southern hybridization
analysis of tail DNA samples from a litter of F1 progeny of a chimeric male
generated with a targeted ES clone. Tail DNA samples were digested with
BamHI, separated by electrophoresis through a 1% agarose gel,
transferred onto a Zetaprobe nylon membrane (BioRad), and hybridized with
random prime-labeled probes made from the 600 bp
HindIII-EcoRI fragment isolated from the Osr2
genomic region 5' to the targeted region. The 14 kb BamHI
fragment corresponding to the wild-type allele was detected in all F1 progeny,
while the 7.7 kb mutant allele-specific fragment was detected only in
heterozygotes. (C) PCR analysis of tail DNA samples from a litter of newborn
F2 progeny. The fragments amplified from wild-type and mutant alleles are 490
bp and 460 bp, respectively. Homozygous mutants were born at the expected
Mendelian frequency (25%). m, DNA fragment size markers; +/+, wild type; +/-,
heterozygote; -/-, homozygote.
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The targeting vector was linearized and electroporated into CJ7 ES cells as
previously described (Swiatek and Gridley,
1993
). G418-resistant ES colonies were screened by Southern
hybridization for homologous recombination. Two independently targeted ES cell
clones were injected into blastocysts from C57BL/6J mice and the resultant
chimeras bred with C57BL/6J females. F1 mice were genotyped by Southern
hybridization analysis of tail DNA. Mice and embryos from subsequent
generations were genotyped by PCR. PCR with primer 1 (5'-GAT ACG GGT AAG
ACA GAA ACT G-3') and primer 2 (5'-CTA CAA GGA TCT AGC ACA TGC
TG-3') amplified a product of 490 bp from the wild-type Osr2
allele. PCR with primer 2 and primer 3 (5'-CTT CTT GAC GAG TTC TTC TGA
GG-3') amplified a mutant allele-specific product of 460 bp.
Heterozygous F1 mice were backcrossed with C57BL/6J mice and N2 heterozygous
mice were intercrossed for analysis of homozygous phenotype.
Detection of ß-galactosidase and skeletal analysis
X-gal staining of whole-mount embryos and cryostat sections for
ß-galactosidase detection was performed as described previously
(Hogan et al., 1994
). Cryostat
sections were counterstained with eosin after X-gal staining. Skeletal
preparations were made from newborn mice as described previously
(Martin et al., 1995
).
Histology and in situ hybridization
For histology, embryos were fixed in Bouin's fixative, dehydrated through
graded alcohols, embedded in paraffin wax, sectioned at 7 µm and stained
with Hematoxylin and Eosin. For in situ hybridization, embryos were fixed
overnight at 4°C in 4% paraformaldehyde in PBS. In situ hybridization of
whole-mount embryos and tissue sections were performed as described previously
(Lan et al., 2001
).
Detection of cell proliferation and apoptosis
For detection of cell proliferation in the palatal shelves, pregnant
Osr2 heterozygous females were injected intraperitoneally on
gestational day 12.5 or 13.5 with BrdU (Roche) labeling reagent (45 µg/g
body weight). One hour after injection, embryos were dissected, fixed in
Carnoy's fixative, dehydrated through graded alcohols, embedded in paraffin
wax and sectioned in the coronal plane at 5 µm. Immunodetection of BrdU was
performed using the BrdU labeling and detection kit (Roche) according to
manufacturer's instructions and the sections were counterstained with Eosin.
The total number of mesenchymal cell nuclei as well as the number of
BrdU-labeled mesenchymal nuclei in a fixed area of 0.057 mm2
beginning at the distal tip and encompassing more than two-thirds of the
vertically-oriented palatal shelves were counted using an ocular scale grid.
Sections were selected from the middle of the anteroposterior axis of the
palatal shelves in comparable positions in the wild-type and mutant embryos
and cell counts were recorded for each of the bilateral palatal shelves from
five continuous sections of each embryo. The cell proliferation index was
calculated as percentage of the cell nuclei with BrdU labeling. Student's
t-test was used to analyze the significance of difference and a
P value less than 0.01 was considered statistically significant.
Apoptotic cell death in the palatal shelves was assessed by TUNEL labeling
of either paraffin wax or cryostat sections using the in situ cell death
detection kit (Roche) following the manufacturer's instructions.
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Results
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Generation of Osr2tm1Jian mutant mice
To examine the function of Osr2 in palate development, we have
generated mice carrying a targeted mutation replacing most of the
Osr2-coding region with a modified bacterial lacZ gene
through homologous recombination in ES cells
(Fig. 1A). The targeted allele,
Osr2tm1Jian, is expected to encode a functional
ß-galactosidase fusion protein containing the N-terminal 15 amino acid
residues of the Osr2 protein and to result in loss of Osr2 function.
Correctly targeted ES cell clones were used to generate chimeric animals and
germline transmission of the targeted mutation was confirmed by Southern
hybridization and PCR analysis of tail DNA samples of the F1 and F2 progeny of
the chimeric founders (Fig.
1B,C). Embryos heterozygous for the
Osr2tm1Jian allele were isolated and stained with X-gal to
reveal domains of ß-galactosidase activity. As shown in
Fig. 2, ß-galactosidase
staining was restricted to the mesonephros at E9.5
(Fig. 2A). By E10.5,
ß-galactosidase staining is also detected in the limb buds, in the
rostrolateral regions of the mandibular processes, in the mesenchyme posterior
to the optic placodes, and in the nascent palatal mesenchyme at the medial
sides of the maxillary processes (Fig.
2B,C). At E13.5, ß-galactosidase activity was detected
throughout the downward growing palatal mesenchyme, with lateral sides of the
palatal mesenchyme showing higher ß-galactosidase activity than the
medial sides (Fig. 2D), which
is similar to the Osr2 mRNA expression pattern reported previously
(Lan et al., 2001
). At E14.75,
the palatal shelves have elevated and ß-galactosidase expression is
downregulated in the palatal mesenchyme, while it is strongly expressed in the
tooth bud mesenchyme, the olfactory mesenchyme, the periocular mesenchyme and
the eyelids (Fig. 2E). These
data indicate that expression of the ß-galactosidase reporter fusion
protein recapitulates the endogenous Osr2 expression pattern reported
previously (Lan et al., 2001
),
further confirming correct integration of the lacZ gene into the
Osr2 locus.

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Fig. 2. Expression of ß-galactosidase in Osr2 heterozygous embryos.
(A) At E9.5, ß-galactosidase expression (blue) was detected specifically
in the mesonephric vesicles (arrow). (B) At E10.5, ß-galactosidase
activity was detected in the mesonephros (arrow), in the limb buds, in the
maxillary and mandibular processes, in the mesenchyme posterior to the eye and
in the mesenchyme adjacent to the first branchial cleft (arrowhead). (C)
Facial view of a stained E10.5 embryo showing ß-galactosidase expression
in the palatal primordia (arrowheads). (D,E) Frontal sections of E13.5 (D) and
E14.75 (E) heterozygous embryos showing ß-galactosidase expression in the
palatal mesenchyme, olfactory mesenchyme, tooth bud mesenchyme, and the
periocular mesenchyme and the eyelids. e, eye; fl, forelimb bud; hl, hindlimb
bud; mb, mandibular process; mx, maxillary process; oe, olfactory epithelia;
p, palatal shelf, t, tongue; tb, tooth bud.
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Osr2tm1Jian/tm1Jian homozygous mutants die at birth with open eyelids and cleft palate
Mice heterozygous for the Osr2tm1Jian allele are normal
and fertile. Heterozygous animals were intercrossed and genotypes of their
progeny were determined 2 weeks after birth. No mice homozygous for the
mutation were found. Careful examination of staged embryos and newborn mice
from heterozygous intercrosses revealed that the
Osr2tm1Jian/tm1Jian homozygous mutants could complete
embryogenesis but died within 24 hours after birth. All homozygous neonates
exhibited open eyelids and bilateral cleft of the secondary palate
(Fig. 3B,D), which correlate
with Osr2 mRNA expression during normal eyelid and palate development
(Lan et al., 2001
). As
Osr2 also exhibits a dynamic expression pattern during kidney and
limb development (Lan et al.,
2001
), we carefully examined development of these structures by
histological and skeletal preparations, but did not find any abnormalities in
them (data not shown). In addition to open eyelids and cleft palate, another
abnormality observed in the homozygous mutants is that they have thickened
tympanic rings (Fig. 3F), which
correlates with Osr2 expression in the mesenchymal cells that give
rise to the tympanic rings proximal to the first branchial clefts
(Fig. 2B). Other craniofacial
structures, including the middle ear ossicles that develop from proximal first
arch mesenchyme adjacent to the tympanic rings, are unaffected by the
Osr2tm1Jian mutation
(Fig. 3G,H).

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Fig. 3. Osr2tm1Jian/tm1Jian mutant mice are born with open
eyelids and cleft palate. (A,B) Dorsal view of heterozygous (A) and homozygous
(B) mutant newborn heads. (C,D) Frontal sections of heterozygous (C) and
homozygous (D) newborn mutant heads. (E,F) Ventral view of stained skeletal
preparations of heterozygous (E) and homozygous (F) mutant neonatal skulls.
Arrowheads indicate palatal processes of the palatine bones that have fused to
each other in the heterozygous mouse (E) but are absent in the homozygous
mutant, exposing the presphenoid bone (marked with an asterisk) underneath
(F). The tympanic rings are significantly thicker in the homozygous mutant
than in the heterozygous mouse (arrows). (G,H) Comparison of tympanic rings
with associated middle ear ossicles and the Meckel's cartilage dissected from
wild-type (G) and homozygous mutant (H). Whereas the mutant tympanic ring is
significantly thicker than that of the wild type, the associated Meckel's
cartilage and middle ear ossicles are similar in size in wild-type and mutant
newborns. e, eye; i, incus; m, malleus; mc, Meckel's cartilage; mm, manubrium
of the malleus; p, palate; t, tongue; tr, tympanic ring.
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Osr2tm1Jian/tm1Jian mutants exhibit impaired palatal shelf growth
To investigate which palatal developmental processes require Osr2
gene function, we carried out histological analyses of embryos throughout
palate development from E12 to birth. As shown in
Fig. 4, palatal outgrowth and
initial downward palatal growth were normal in the homozygous mutants
(Fig. 4A,B). Between E13.5 and
E14.5, the vertically oriented palatal shelves undergo rapid growth and
initiate reorientation to a horizontal position above the dorsum of the tongue
in the wild-type and heterozygous embryos
(Fig. 4C,E). By contrast, the
Osr2tm1Jian/tm1Jian homozygous mutant littermates exhibit
retarded palatal shelves that remain vertically oriented at this stage
(Fig. 4D,F). Palatal shelf
retardation is observed throughout the anteroposterior axis of the palatal
shelves in all homozygous mutants examined at E14.5 (data not shown). By
E15.5, the elevated palatal shelves have initiated fusion at the midline in
wild-type and heterozygous embryos, whereas the homozygous mutant palatal
shelves are elevated but failed to contact each other at the midline
(Fig. 4G,H). As the palatal
fusion process continues to generate intact secondary palates in wild-type and
heterozygous embryos (Fig. 4I),
the homozygous mutant palatal shelves are further separated from each other
(Fig. 4J), resulting in
bilateral cleft of the secondary palate at birth
(Fig. 3D).

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Fig. 4. Histological analysis of palate development in
Osr2tm1Jian/tm1Jian mutants. (A,B) At E13.5, wild-type (A)
and Osr2tm1Jian/tm1Jian homozygous mutant (B) embryos
exhibited similar palatal shelf size and shape. (C,D) At E14.5, palatal
shelves appeared retarded in the homozygous mutant (D) compared with the
heterozygous littermate (C). (E,F) At E15.0, the heterozygous mutant (E)
palatal shelves had elevated to the horizontal position above the tongue,
while the homozygous mutant (F) palatal shelves were still vertically
oriented. (G,H) At E15.5, the heterozygous mutant palatal shelves had made
contact and initiated fusion at the midline, but the palatal shelves remained
separated from each other in the homozygous mutant littermate (H). (I,J) At
E16.5, the heterozygous mutant (I) palatal shelves had completed fusion, but
the homozygous mutant (J) palatal shelves were retarded and separate from each
other. p, palatal shelf, t, tongue.
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To investigate whether palatal shelf retardation in the
Osr2tm1Jian/tm1Jian mutant embryos was due to impaired
cell proliferation, we analyzed BrdU incorporation in E12.5 and E13.5 embryos.
No difference in the cell proliferation index between wild-type and mutant
palatal shelves was observed in E12.5 embryos (data not shown). However,
whereas the wild-type and heterozygous embryos showed no significant
differences in palatal cell proliferation at E13.5, the homozygous mutants
exhibited a 26% reduction (P<0.01) in cell proliferation index in
the palatal mesenchyme (Fig.
5C,D). The wild-type and heterozygous palatal shelves exhibit
faster growth in the medial halves than the lateral halves
(Fig. 5A,B,D), which is
consistent with previous reports and results in the characteristic erectile
shape of the palatal shelves before elevation
(Ferguson, 1988
). Remarkably,
when the percentage of BrdU-labeled cells was recorded separately for the
medial and lateral halves of the palatal shelves, there was a 37% reduction
(P<0.01) in the cell proliferation index in the medial halves of
homozygous mutant palatal shelves, while the cell proliferation index was not
significantly different (P>0.05) in the lateral halves of the
palatal shelves in wild-type, heterozygous and homozygous mutant embryos
(Fig. 5D). This preferential
reduction in cell proliferation in the medial halves of the palatal shelves
results in the appearance of retarded, mediolaterally symmetric palatal
shelves in the homozygous mutants at E14.5
(Fig. 4D). We also examined
whether abnormal cell death contributed to the palatal shelf retardation by in
situ TUNEL assays, but did not find any differences in cell death between
wild-type and homozygous mutant palatal shelves (data not shown).

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Fig. 5. Analysis of cell proliferation in Osr2tm1Jian/tm1Jian
mutants at E13.5. (A-C) Frontal sections showing BrdU-labeled cell nuclei
(blue color) in wild type (A), heterozygous (B) and homozygous mutant (C)
palatal shelves at E13.5. Arrows indicate medial side of the palatal shelves.
(D) Comparison of the percentage of BrdU-labeled cells in a fixed area of
palatal mesenchyme in wild-type (+/+), heterozygous (+/-) and homozygous
mutant (-/-) embryos. Standard deviation values were used for the error
bars.
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The palatal mesenchyme proliferation defect in Osr2tm1Jian/tm1Jian mutants correlates with the spatiotemporal pattern of Osr1 mRNA expression
As Osr2 mRNA expression is activated at the onset of palatal
outgrowth and persists throughout the downward growing palatal shelves during
mouse embryogenesis (Lan et al.,
2001
), the normal initiation of palatal outgrowth and preferential
reduction in cell proliferation in the medial halves of the palatal shelves in
the Osr2tm1Jian/tm1Jian mutants are surprising
observations. One possible explanation for the mutant phenotype is that loss
of Osr2 function in the initial stage of palatal outgrowth is
compensated by expression of a related gene product. The Osr1 gene
encodes a protein with 65% overall amino acid sequence identity and 98%
sequence identity in the zinc-finger domain with the Osr2 protein
(So and Danielian, 1999
;
Lan et al., 2001
). No other
odd-skipped related gene has been found in the mouse or human
genomes. Thus, we examined Osr1 mRNA expression and compared that
with the expression pattern of Osr2 mRNA during palate development.
We also compared the expression patterns of Osr1 and Osr2
with that of Pax9, which encodes a paired-class
homeodomain-containing transcription factor required for proper palatal
patterning (Peters et al.,
1998
). From E12.5 to E13.5, Osr2 mRNA is expressed
abundantly throughout the palatal mesenchyme, with lateral regions expressing
higher levels than the medial regions (Fig.
6A-C). Osr1 mRNA is strongly expressed in the developing
tongue and at the maxillary-mandibular junction but is only weakly expressed
in the palatal mesenchyme at E12.5 and E13.0
(Fig. 6D,E). By E13.5,
Osr1 mRNA expression is significantly up-regulated in the lateral
halves and is completely downregulated in the medial halves of the palatal
shelves, forming a sharp boundary in the middle of the palatal shelves
(Fig. 6F). Pax9 mRNA
is expressed in a pattern similar to that of Osr2 mRNA in the palatal
mesenchyme at E12.5 and E13.0 (Fig.
6G,H). However, at E13.5, Pax9 mRNA expression is
upregulated in the mediodistal regions and downregulated in the lateral
regions of the palatal shelves (Fig.
6I). These data indicate that dynamic molecular changes occur
between E13.0 and E13.5 during palate development. Interestingly, the defect
in palate development in the Osr2tm1Jian/tm1Jian mutants
is first detectable at this stage and the specific reduction in cell
proliferation in the mutant medial palatal mesenchyme correlates with the
spatiotemporal downregulation of Osr1 mRNA expression in those
cells.

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Fig. 6. Comparison of the patterns of Osr2, Osr1 and Pax9 mRNA
expression during palate development. mRNA signals are shown in red in all
panels. (A-C) Osr2 mRNA is abundantly expressed throughout the
developing palatal shelves from E12.5 to E13.5. Osr2 mRNA is also
highly expressed in the developing tooth bud mesenchyme at E13.5 (arrows in
C). (D-F) Osr1 mRNA expression is weak in the developing palatal
shelves at E12.5 (D) and E13.0 (E) but it is highly abundant in the developing
tongue and several regions of the mandible. By E13.5, the lateral halves of
the palatal shelves express moderate levels of Osr1 mRNA, while the
medial halves of the palatal shelves completely lack Osr1 mRNA
expression (F). Arrowheads indicate the sharp boundaries between the lateral
Osr1-expressing and the medial Osr1-nonexpressing palatal
mesenchyme cells. In contrast to Osr2, Osr1 is not expressed in the
developing tooth bud mesenchyme (arrows in F). (G-I) Pax9 mRNA
exhibits a lateral to medial expression gradient in the downward growing
palatal shelves at E12.5 (G) and E13.0 (H), with higher levels in the lateral
regions. By E13.5, Pax9 expression is upregulated in the medial
regions of the palatal shelves and the Pax9 mRNA gradient is
reversed, with higher levels in the medial regions of the palatal shelves (I).
Pax9 mRNA is also expressed in the tooth bud mesenchyme at E13.5
(arrows in I). p, palatal shelf; t, tongue.
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Defects in palatal shelf molecular patterning and elevation in Osr2tm1Jian/tm1Jian mutants
To investigate the role of Osr2 in palate development further, we
examined the expression patterns of several genes implicated in regulating
palatal growth and/or patterning. Msx1, a homeobox gene required for
anterior palate mesenchyme proliferation, is expressed weakly in the anterior
but not posterior regions of the palatal mesenchyme and strongly in maxillary
and mandibular mesenchyme at E14.5 (Zhang
et al., 2002
). No differences in Msx1 expression were
found between wild-type and Osr2tm1Jian/tm1Jian mutants,
although the palatal shelves are retarded in the anterior region in the
homozygous mutants (Fig. 7A,B).
Bmp4 has recently been implicated in regulating palate mesenchyme
growth because a Bmp4 transgene driven by the Msx1 promoter
was able to rescue palate growth defects in the Msx1-/-
mutant mice (Zhang et al.,
2002
). However, Bmp4 expression appears unaltered in the
Osr2tm1Jian/tm1Jian mutant palatal shelves compared with
the wild-type embryos (Fig.
7C,D). Expression of Tbx22, the mouse ortholog of the
human X-linked cleft palate gene (Braybrook
et al., 2002
; Bush et al.,
2002
; Herr et al.,
2003
), is also unaltered in the mutant palatal shelves
(Fig. 7E,F). Pax9,
encoding a paired-class transcription factor required for proper palate
patterning (Peters et al.,
1998
), exhibits a dynamic expression pattern during palate
development (Fig. 6G-I). In the
Osr2tm1Jian/tm1Jian mutants, Pax9 expression is
similar to that in wild-type embryos during early palatal outgrowth (data not
shown). However, by E13.5 when the wild-type palatal shelves showed a strong
mediolateral gradient of Pax9 mRNA expression, the
Osr2tm1Jian/tm1Jian mutants exhibited a uniform lower
level of Pax9 mRNA expression in the palatal mesenchyme
(Fig. 8A,B). By E14.5, when
strong Pax9 expression is observed in the palatal shelves in
wild-type littermates (Fig.
8C), significantly reduced levels of Pax9 mRNA are found
throughout the retarded palatal shelves in
Osr2tm1Jian/tm1Jian mutants
(Fig. 8D). The alteration in
Pax9 mRNA expression in the Osr2tm1Jian/tm1Jian
mutants is specific to the palatal mesenchyme, as Pax9 mRNA levels in
the tooth bud mesenchyme and in other craniofacial regions are similar in
wild-type and mutant embryos (Fig.
8A-D). Tgfb3, which encodes a growth factor required for
normal palatal shelf fusion (Kaartinen et
al., 1995
; Proetzel et al.,
1995
), is expressed in the medial and distal epithelia of the
vertically oriented palatal shelves at E14.5 in wild-type embryos
(Fig. 8E). In the homozygous
Osr2tm1Jian/tm1Jian mutants, Tgfb3 expression in
the distal palatal epithelia is lost (Fig.
8F). Complementary to the Tgfb3 expression pattern in the
epithelia, Osr1 is expressed in the mesenchyme of the proximolateral
one-third of the palatal shelves in wild-type embryos at E14.5
(Fig. 8G). In the homozygous
mutant palatal shelves, the Osr1 expression domain extends to the
distal tip of the palatal mesenchyme (Fig.
8H), most probably as a result of the severe reduction in medial
palatal mesenchyme proliferation. By E15.0, the palatal shelves have elevated
to the horizontal position above the tongue and the Osr1 expression
domain is still restricted in the same regions of the palatal mesenchyme in
the wild-type and heterozygous embryos
(Fig. 8I). By contrast, the
homozygous mutant littermates exhibit a low level of Osr1 expression
throughout the retarded, still vertically oriented palatal shelves
(Fig. 8J). These data indicate,
in addition to the role in palatal shelf growth, that Osr2 function is
required for the normal mediolateral patterning and elevation of the palatal
shelves.

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|
Fig. 7. Expression of Msx1, Bmp4 and Tbx22 in the developing
palatal shelves is not altered in the Osr2tm1Jian/tm1Jian
homozygous mutant embryos. (A,B) At E14.5, Msx1 mRNA is abundantly
expressed in medial maxillary processes and the tooth mesenchyme and weakly
expressed in the anterior palatal shelves (arrows) in both wild-type (A) and
the homozygous (B) mutant embryos. Note the palatal shelves are retarded in
the homozygous mutant (arrows in B). (C,D) At E13.5, Bmp4 mRNA is
expressed at comparable levels in wild-type (C) and the homozygous mutant (D)
palatal shelves. (E,F) At E13.5, Tbx22 mRNA is abundantly expressed
in the palatal mesenchyme and the base of the tongue in both wild-type (E) and
the homozygous mutant (F) embryos. Tbx22 is also expressed similarly
in the periocular mesenchyme in wild-type and the mutant embryos. mx,
maxillary process; t, tongue.
|
|

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|
Fig. 8. Expression of Pax9, Tgfb3 and Osr1 is altered in the
Osr2tm1Jian/tm1Jian mutant palatal shelves. (A,B)
Pax9 mRNA expression in wild-type (A) and mutant (B) embryos at
E13.5. Pax9 mRNA exhibits a mediolateral gradient in the wild-type
palatal mesenchyme at this stage, with higher levels in the medial palatal
mesenchyme (A). By contrast, uniform lower levels of Pax9 mRNA
expression are observed in the mutant palatal mesenchyme (B), although similar
levels of Pax9 mRNA are observed in the tooth bud mesenchyme in
wild-type and mutant embryos. (C,D) At E14.5, Pax9 mRNA in the
palatal mesenchyme is significantly reduced in the mutant (D) compared with
that in the wild-type littermate (C). Pax9 mRNA levels in the tooth
bud mesenchyme remains similar in wild-type and mutant embryos. (E,F)
Tgfb3 mRNA is abundantly expressed in the medial and distal regions
of the palatal epithelium in wild-type embryos (E), but its expression domain
is shifted medially in the mutant palatal epithelium (F) at E14.5. Arrowheads
indicate the boundaries between Tgfb3-expressing and
Tgfb3-nonexpressing cells in the distal palatal regions. (G,H)
Osr1 mRNA expression in wild-type (G) and the mutant (H) palatal
shelves at E14.5. (I,J) Osr1 mRNA expression in wild-type (I) and the
mutant (J) palatal shelves at E15.0. p, palatal shelf; t, tongue; tb, tooth
bud.
|
|
 |
Discussion
|
---|
Osr2 is an intrinsic regulator of secondary palate development
Cleft palate is among the most common birth defects in humans and has been
observed in a number of mice carrying mutations in genes encoding
transcription factors (Gendron-Maguire et
al., 1993
; Rijli et al.,
1993
; Satokata and Maas,
1994
; Martin et al.,
1995
; Qiu et al.,
1997
; De Felice et al.,
1998
; Peters et al.,
1998
; Zhao et al.,
1999
), growth factors and their receptors
(Kaartinen et al., 1995
;
Proetzel et al., 1995
;
Sanford et al., 1997
;
Miettinen et al., 1999
), other
signaling molecules (Matzuk et al.,
1995
; Jiang et al.,
1998
), and extracellular matrix molecules
(Pace et al., 1997
;
Lavrin et al., 2001
). Whereas
the cleft palate phenotypes at birth may appear similar, the underlying
mechanisms in cleft pathogenesis are very different. Palate development occurs
at the time of significant growth and morphogenesis of the entire craniofacial
complex and is dependent on normal development of other craniofacial
structures (Ferguson, 1988
).
The cleft palate phenotype observed in many mutant mice, such as those with
mutations in Dlx1, Dlx2, Hoxa2, Mhox (Prrx1 Mouse
Genome Informatics) and
EF1 (Zfhx1a Mouse
Genome Informatics) genes (Gendron-Maguire
et al., 1993
; Rijli et al.,
1993
; Martin et al.,
1995
; Qiu et al.,
1997
; Takagi et al.,
1998
), is accompanied by gross craniofacial and skeletal
malformations and can be attributed, at least partly, to secondary effects of
those malformations. Several mutations cause mechanical hindrance to palatal
shelf elevation. For example, the palatal shelves fuse aberrantly to the
lateral sides of the tongue in the Jag2-/- mutant mice and
the tongue fails to descend during palatal shelf elevation in the
Foxf2-/- mutant mice, causing cleft palate
(Jiang et al., 1998
;
Wang et al., 2003
). Another
class of mutations specifically interferes with palatal shelf fusion. For
example, Titf2 and Tgfb3 are normally expressed in palatal
epithelium and mutations in these genes specifically disrupt palatal shelf
fusion (Kaartinen et al.,
1995
; Proetzel et al.,
1995
; De Felice et al.,
1998
).
The cleft palate phenotype in the Osr2tm1Jian/tm1Jian
mutant mice is clearly different from those of all previously reported mutant
mice. All Osr2tm1Jian/tm1Jian homozygous mutants examined
after E15.5 exhibited cleft palate. In addition to cleft palate,
Osr2tm1Jian/tm1Jian mutants exhibited open eyelids and
thickened tympanic rings, which correlated with Osr2 mRNA expression
during normal development of these structures. Open eyelids have been observed
in several mouse mutant strains without cleft palate
(Keeler, 1935
;
Bennett and Gresham, 1956
;
Jeriloff et al., 2000), indicating that it does not cause cleft palate. The
thickened tympanic ring phenotype is unique to the
Osr2tm1Jian/tm1Jian mutants, but it most probably reflects
a developmental role for Osr2 that is independent from that in palate
development as other craniofacial structures immediately adjacent to the
tympanic rings are unaffected by the mutation. Moreover, Osr2 mRNA is
normally expressed during palatal outgrowth and
Osr2tm1Jian/tm1Jian mutants exhibited specific impairment
of palatal mesenchymal cell proliferation, which resulted in retarded,
mediolaterally symmetric palatal shelves before palatal shelf elevation. Thus,
whereas Osr2 is required for the normal development of several craniofacial
structures, the cleft palate phenotype of the
Osr2tm1Jian/tm1Jian mutants resulted from a primary defect
in palatal shelf growth.
Impairment of palatal mesenchyme cell proliferation has been reported
previously in the Msx1-/- mutant mice and recently in mice
with neural crest-specific inactivation of the Tgfbr2 gene
(Zhang et al., 2002
;
Ito et al., 2003
).
Msx1 is strongly expressed in the maxillary processes and tooth buds,
but only very weakly expressed in the anterior region of the palatal shelves
(Zhang et al., 2002
).
Reduction in cell proliferation was found in the anterior but not in the
posterior regions of the palatal shelves in Msx1-/-
mutants (Zhang et al., 2002
).
In mice with neural crest-specific inactivation of the Tgfbr2 gene,
palatal shelves grow normally up to E13.5 but palatal mesenchyme growth is
reduced after palatal shelf elevation at E14.5
(Ito et al., 2003
). These
mutant mice also exhibited gross craniofacial skeletal growth defects
(Ito et al., 2003
), consistent
with expression of Tgfbr2 mRNA in migrating cranial neural crest
cells (Wang et al., 1995
). By
contrast, Osr2 mRNA expression is specifically activated in the
nascent palatal mesenchyme at the onset of palatal outgrowth and persists
strongly in the palatal mesenchyme during the downward palatal growth
(Lan et al., 2001
). Palatal
growth retardation is observed throughout the anteroposterior axis of the
palatal shelves before palatal shelf elevation in the
Osr2tm1Jian/tm1Jian mutants. Therefore, Osr2
expression is a specific marker of the early palatal mesenchyme and is
essential for the rapid downward growth of the palatal shelves before
elevation.
In the Msx1-/- mutant mice, impairment of mesenchyme
growth in the anterior palate was shown to be due to lack of Bmp4
expression and transgenic expression of Bmp4 under the control of the
Msx1 promoter was able to restore anterior palatal mesenchyme
proliferation in the Msx1-/- mutant mice
(Zhang et al., 2002
). We found
that Msx1 and Bmp4 are expressed normally in the developing
palatal shelves in Osr2tm1Jian/tm1Jian mutant mice
(Fig. 7). Furthermore, we
observed no differences in Shh and Bmp2 expression, which
are believed to function downstream of the Msx1-BMP4 pathway in the palatal
shelves (Zhang et al., 2002
),
between wild-type and Osr2tm1Jian/tm1Jian mutant palatal
shelves (data not shown). The differences in phenotype and molecular marker
expression patterns in Msx1-/- and
Osr2tm1Jian/tm1Jian mutants indicate that Osr2 and Msx1
function in distinct molecular pathways to regulate palate development. The
fact that impairment of palatal mesenchyme proliferation occurs earlier in the
Osr2tm1Jian/tm1Jian mutants than in mice lacking
Tgfbr2 in the neural crest derivatives suggests that Osr2 function in
the palatal mesenchyme is also independent of Tgfß signaling. The
identification of Osr2 as an essential regulator of palatal mesenchyme growth
warrants future investigation of how Osr2 expression is activated in
the nascent palatal mesenchyme and how it controls palatal mesenchyme
proliferation. Interestingly, the human OSR2 gene is located at
chromosome 8q23, in a region with strong association to non-syndromic
orofacial clefting (Prescott et al.,
2000
), suggesting that OSR2 is a new candidate gene for
human cleft palate formation.
The Osr genes and palatal patterning
Whereas the Osr2 gene is activated at the onset of palatal
outgrowth and is expressed throughout the downward growing palatal mesenchyme,
the Osr1 gene exhibits a unique, mediolaterally differentially
regulated pattern of expression during palate development. During early
palatal outgrowth, Osr1 mRNA is expressed weakly throughout the
palatal mesenchyme. By E13.5 of mouse development, Osr1 mRNA
expression is strongly upregulated in the lateral halves and completely
downregulated in the medial halves of the palatal shelves
(Fig. 6D-F). As the
mediolateral pattern is laid down, Pax9 mRNA expression is
upregulated in the medial palatal mesenchyme
(Fig. 6I). In the
Osr2tm1Jian/tm1Jian mutants, early Osr1 and
Pax9 expression patterns are unaltered during palatal outgrowth (data
not shown). However, by E13.5, the upregulation of Pax9 mRNA
expression seen in the medial regions of the wild-type palatal shelves is not
observed in the mutants (Fig.
8B), indicating a molecular patterning defect in the mutant
palatal mesenchyme. At the same developmental stage, a significant reduction
in BrdU incorporation is observed specifically in the medial palatal
mesenchyme in the Osr2tm1Jian/tm1Jian mutants
(Fig. 5). Although mice
deficient in the Pax9 gene have cleft palate
(Peters et al., 1998
), it is
not known whether Pax9 directly regulates palatal mesenchyme proliferation.
Thus, it remains to be investigated whether the reduction in Pax9
mRNA expression in the medial palatal mesenchyme is the cause of the
region-specific reduction in palatal cell proliferation in the
Osr2tm1Jian/tm1Jian mutants. Nevertheless, these data
indicate that Osr2 plays an important role in the mediolateral patterning of
the palatal mesenchyme. The differential regulation of Osr1 mRNA
expression in the medial and lateral halves of the palatal shelves suggests
that Osr1 may also have a role in mediolateral palatal patterning.
In addition to alterations in gene expression in the palatal mesenchyme, we
observed alteration of Tgfb3 mRNA expression in the palatal
epithelium at E14.5 in the Osr2tm1Jian/tm1Jian mutants.
Interestingly, the epithelial Tgfb3 expression domain complements
that of Osr1 mRNA expression in the palatal mesenchyme in both
wild-type and the Osr2tm1Jian/tm1Jian mutant embryos
(Fig. 8E-H). Because the
Osr1 and Tgfb3 mRNAs are expressed normally up to E13.5 in
the Osr2tm1Jian/tm1Jian mutants (data not shown), the
alterations in their expression in the palatal shelves at E14.5 are most
probably secondary effects of the reduction in medial palatal mesenchyme
proliferation. However, the corresponding shift in epithelial Tgfb3
and mesenchymal Osr1 expression domains suggest that Osr1
expression in the palatal mesenchyme may regulate mesenchymal-epithelial
interactions that in turn regulate Tgfb3 expression in the palatal
epithelium. Further characterization of the molecular pathways through which
Osr2 and Osr1 regulate palate development will provide novel insights into the
molecular mechanisms governing palate growth and patterning.
 |
ACKNOWLEDGMENTS
|
---|
We thank Bob Angerer and Lin Gan for comments on the manuscript; Tom
Gridley for the CJ7 ES cell line; YiPing Chen for cDNA probes; and the
University of Rochester Transgenic Mouse Facility for generation of chimeric
mice from targeted ES cells. This work was supported by a NIH/NIDCR grant
(DE13681) to R.J.
 |
Footnotes
|
---|
* Present address: Department of Oral Anatomy, Chonbuk National University
School of Dentistry, Chonju 561-756, Republic of Korea 
 |
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