|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 23 October 2008
doi: 10.1242/dev.025304
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Biomedical Genetics and Center for Oral Biology, University of
Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
2 Institute of Child Health, University College London, London WC1N 1EH,
UK.
3 Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam,
The Netherlands.
* Author for correspondence (e-mail: rulang_jiang{at}urmc.rochester.edu)
Accepted 6 October 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Cleft palate, Mn1, Palate development, Anterior-posterior patterning, Tbx22, Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). MN1 encodes a protein of 1319 amino acids, with no
homology to any known functional domains, but the gene is evolutionarily
conserved from Drosophila to human
(Lekanne Deprez et al., 1995).
Although its relation to meningioma remains unclear, as no mutations or
deletions of the MN1 gene have been found in other individuals with
meningioma, the MN1 gene has been shown to play important roles in
acute myeloid leukemia (AML) pathogenesis (reviewed by
Grosveld, 2007). MN1 is the
target of a recurrent chromosomal translocation, t(12;22)(p13;q12), which is
associated with human AML (Buijs et al.,
1995). The translocation fuses the MN1 gene with the
TEL gene that encodes an ETS family DNA-binding transcription factor.
Both in vitro and in vivo studies showed that the MN1-TEL fusion protein is
oncogenic and that the transforming activity of the fusion protein depended on
the N-terminal 500 amino acid residues of the MN1 protein
(Buijs et al., 2000;
Kawagoe and Grosveld, 2005a;
Kawagoe and Grosveld, 2005b;
Carella et al., 2006). In
addition, MN1 is overexpressed in many individuals with AML
associated with other chromosomal abnormalities and in some individuals with
AML without karyotype abnormalities (Ross
et al., 2004; Valk et al.,
2004; Du et al.,
2005; Heuser et al.,
2006) (reviewed by Grosveld,
2007). Moreover, overexpression of MN1 in the bone marrow
caused malignant myeloid disease in mice
(Carella et al., 2007).
Although the molecular mechanisms involving MN1 in AML pathogenesis
remains to be elucidated, several biochemical studies have showed that MN1 can
function as a transcriptional co-activator of the nuclear hormone receptors
for retinoic acid or vitamin D (van Wely
et al., 2003; Sutton et al.,
2005). MN1 has also been shown to interact with the
transcriptional co-activators p300/CBP and RAC3, and to mediate
transcriptional activation via CACCC-rich DNA sequences
(van Wely et al., 2003;
Meester-Smoor et al., 2007).
Thus, in addition to involvement in AML, MN1 may interact with other
transcription factors to regulate cell proliferation and cell differentiation
during mammalian development.
To further investigate the roles of MN1 in oncogenesis and development,
Meester-Smoor et al. (Meester-Smoor et
al., 2005) generated mice with a targeted deletion in the
orthologous Mn1 gene. Although the mutant mice did not exhibit any
increased incidence of tumor formation, all Mn1-/-
homozygous mutant mice died shortly after birth and exhibited severe
craniofacial developmental defects, including cleft palate, and some
Mn1+/- heterozygous mutant mice also had cleft palate
(Meester-Smoor et al., 2005).
In mice, as in humans, the secondary palate develops from bilateral outgrowth
on the oral side of the developing maxillary processes. The palatal processes
initially grow vertically flanking the developing tongue. At a specific
developmental time, the bilateral palatal shelves reorient to the horizontal
position above the tongue, grow toward and fuse with each other at the midline
to form the intact roof of the oral cavity
(Ferguson, 1988). Cleft palate
may result from disturbances in the growth, elevation or fusion of the palatal
shelves. Gene inactivation studies in mice have demonstrated that many genes
play essential roles in palatal shelf growth, including Bmp4, Bmpr1a,
Fgf10, Fgfr2b, Msx1, Osr2, Shox2 and Tgfbr2, indicating that
multiple molecular pathways interact to regulate palate development
(Zhang et al., 2002;
Han et al., 2003;
Ito et al., 2003;
Lan et al., 2004;
Rice et al., 2004;
Alappat et al., 2005;
Liu et al., 2005;
Yu et al., 2005). Moreover, as
palate development occurs concurrently with significant growth and
morphogenesis of the craniofacial complex, gross defects in structures outside
of the palatal shelves may sometime hinder palatal shelf elevation or contact,
resulting in cleft palate (reviewed by
Chai and Maxson, 2006). To
understand the roles of Mn1 in palate development, we have characterized its
expression patterns during normal palate development and have identified a
primary role for Mn1 in differential regulation of palatal growth along the
anteroposterior axis.
The mammalian secondary palate is divided anatomically into the anterior
bony region (hard palate) and the posterior muscular region (soft palate)
(Sperber, 2002). Cleft palate
defects affecting the entire palate (complete cleft of the secondary palate)
or either the anterior or posterior regions (incomplete cleft of the secondary
palate) have been well documented (reviewed by
Hilliard et al., 2005).
Consistent with the morphological and pathological differences in the anterior
and posterior palate, recent studies have clearly demonstrated that there is
molecular heterogeneity along the anteroposterior axis of the developing
secondary palate (reviewed by Hilliard et
al., 2005; Li and Ding,
2007). During early palate development, expression of several
crucial signaling molecules and transcription factors, including Bmp4, Fgf10,
Msx1 and Shox2, is highly restricted along the anteroposterior axis.
Expression of Bmp4 and Msx1 is restricted to the most
anterior 25%, whereas Fgf10 and Shox2 are expressed in the
anterior half of the developing palatal shelves, up to the level of the first
molar tooth buds, prior to palatal fusion
(Zhang et al., 2002;
Alappat et al., 2005;
Yu et al., 2005;
Hilliard et al., 2005;
Li and Ding, 2007). Fgf10
signals through the Fgfr2b receptor to regulate palatal epithelial cell
proliferation and survival (Rice et al.,
2004; Alappat et al.,
2005). Bmp4 and Msx1 appeared to function in a positive-feedback
loop to regulate mesenchymal proliferation in the anterior palate
(Zhang et al., 2002).
Interestingly, exogenous Bmp4 induced Msx1 expression and cell
proliferation in the anterior, but not in the posterior, palatal mesenchyme in
explant culture assays (Zhang et al.,
2002; Hilliard et al.,
2005). Shox2 is also required for growth of the anterior
palate, and mice that lack Shox2 exhibited incomplete cleft within
the anterior palate while the mutant posterior palate fused normally
(Yu et al., 2005). Although
the anterior and posterior palatal regions exhibit similar growth rates during
palatal outgrowth (Zhang et al.,
2002; Li and Ding,
2007), no factor has been reported to regulate preferentially the
growth of the posterior palate. The Meox2 homeobox gene has been
reported as being expressed in the posterior but not in the anterior palatal
shelves in certain strains of mice (Jin
and Ding, 2006; Li and Ding,
2007). Some, but not all, Meox2 mutant mice exhibited
cleft palate, but they did not have defects in palatal shelf growth and their
cleft palate defect appeared to result from postfusion rupture
(Jin and Ding, 2006). In this
report, we show that Mn1 preferentially regulates the growth of the posterior
palate in mice. In addition, we show that palatal expression of
Tbx22, mutations of which cause X-linked cleft palate and
ankyloglossia (CPX) in humans (Braybrook et
al., 2001; Braybrook et al.,
2002), is specifically regulated by Mn1. These data provide novel
insights into the molecular mechanisms that regulate the regional growth and
patterning of the secondary palate.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Mn1+/- mice were maintained in the FVB
congenic background and were intercrossed to generate homozygous mutant
embryos for experimental analysis. Wild-type C57BL/6J and CD-1 mice were also
used for in situ hybridization analysis of Mn1 and Tbx22
mRNA expression during palate development.
In situ hybridization and histological analyses
Embryos at different stages were dissected, fixed in 4% paraformaldehyde
(PFA) in PBS overnight at 4°C. Whole-mount in situ hybridization was
performed as described previously (Lan et
al., 2001). For section in situ hybridization, PFA-fixed embryos
were dehydrated through graded alcohols and embedded in paraffin, sectioned at
7 µm, followed by prehybridization processing and by hybridization with
digoxigenin-labeled cRNA probes as described previously
(Zhang et al., 1999).
For histology, embryos were collected at predetermined stages, fixed in either Bouin's fixative or 4% PFA overnight, dehydrated through graded ethanol, embedded in paraffin wax and sectioned at 7 µm, followed by staining with Hematoxylin and Eosin.
Analyses of cell proliferation and cell apoptosis
Cell proliferation was measured by BrdU incorporation assays or detection
of Ki67. For BrdU incorporation assays, timed mating was set up between
Mn1+/- heterozygous mice and pregnant female mice were
injected intraperitoneally with BrdU (5-bromo-2-deoxy-uridine, Roche) labeling
reagent at gestational day 12 or 13, with a dose of 15 µl/g body weight.
One hour after injection, embryos were dissected, fixed in Carnoy's fixative,
dehydrated through graded ethanol, embedded in paraffin wax and sectioned at 5
µm. Sections from anterior, middle or posterior regions of the developing
palatal shelves were selected for detection of BrdU-labeled cells by using the
BrdU labeling and detection kit (Roche) following the manufacturer's protocol.
Following BrdU detection, sections were counterstained with nuclear fast red
(Vector Laboratories) to label all cellular nuclei. The total number of cells
and the number of BrdU-positive cells in the palatal epithelium and mesenchyme
on each of five consecutive sections were counted. Cell proliferation index
was calculated as the percentage of the total cells being BrdU-positive. ANOVA
was applied for statistical analyses and a P value less than 0.01 was
considered statistically significant.
For detection of Ki67, paraffin sections from selected palatal regions of
staged mouse embryos were stained with an antibody against Ki67 as described
previously (Casey et al.,
2006). Cell apoptosis was detected by TUNEL assays. Paraffin
sections from selected palatal regions of staged mouse embryos were analyzed
by using the DeadEnd Fluorometric TUNEL System (Promega) following the
manufacturer's instructions.
Expression vectors and promoter-luciferase constructs
The Mn1 expression vector was constructed by subcloning an
Mn1 cDNA fragment containing the full-length protein-coding region
into the pcDNA3TOPO (Invitrogen) expression vector. The human TBX22
promoter-luciferase reporter vectors (pGL3-TBX22 hP0 and
pGL3-TBX22 hP1) have been described previously
(Andreou et al., 2007). The
mouse Tbx22 promoter-luciferase reporter vectors were similarly
constructed by PCR amplifying the mouse Tbx22 promoter regions (see
Fig. S3 in the supplementary material) followed by subcloning into the
pGL3-basic vector (Promega). All subcloned fragments were sequence
verified.
Cell culture, transfection, and luciferase reporter assays
NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For
luciferase reporter assays, cells were plated in 24-well tissue culture plates
(Corning) and co-transfected with 0.05 µg of a luciferase reporter vector,
0.05 µg of the pRL-Renilla luciferase expression vector (Promega) and
increasing amounts of the Mn1 expression vector. Transfections were
performed by using the Lipofectamine reagents (Invitrogen) in accordance with
the manufacturer's instructions. Cells were cultured for 48 hours after
transfection and then assayed using the Dual-Luciferase Assay Kit (Promega).
Firefly luciferase activity was normalized to Renilla luciferase activity. All
transfection experiments were carried out in triplicate and data were
summarized from three repeat experiments.
Real-time RT-PCR
For detection of the effects of Mn1 on endogenous Tbx22 gene
expression, NIH3T3 cells were plated in six-well tissue culture plates and
transfected with increasing amounts of the Mn1 expression vector.
Cells were cultured for 48 hours after transfection and total RNA was
extracted using Trizol reagents (Invitrogen). First-strand cDNA was
synthesized using SuperScript First-Strand Synthesis System (Invitrogen).
Quantitative PCR amplifications were performed in an iCycler real-time PCR
machine (Bio-Rad) using the SYBR GreenER qPCR Supermix (Invitrogen).
Mn1 gene-specific PCR primers are
5'-AGATCCAGCTGCAGAGACAA-3' and
5'-TACTCATGGCGCTCTTGACT-3'. Tbx22 gene-specific PCR
primers are 5'-GACCTGTCCCTGATTGAGTCC-3' and
5'-GCTGGTTTTGGTAAGCTGTCA-3'. Hprt gene-specific primers
are 5'-TGCTGGTGAAAAGGACCTCTCG-3' and
5'-CTGGCAACATCAACAGGACTCC-3'. For each sample, the relative levels
of Mn1 and Tbx22 mRNAs were normalized to that of HPRT using
the standard curve method.
|
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
The reported cleft palate phenotype of the Mn1 mutant mice
prompted us to carry out a detailed analysis of Mn1 mRNA expression
during palate development. Recent studies have demonstrated that several genes
exhibit differential expression along the anteroposterior axis of the
developing palatal shelves in mice (Zhang
et al., 2002; Alappat et al.,
2005; Yu et al.,
2005; Hilliard et al.,
2005; Li and Ding,
2007). The distinct gene expression patterns appear to divide the
developing palatal shelves into three regions along the anteroposterior axis:
(1) anterior palate that expresses high levels of Msx1 and
Shox2 mRNAs (Zhang et al.,
2002; Hilliard et al.,
2005; Yu et al.,
2005); (2) middle palate (roughly corresponding to the region
flanked by the upper first molar tooth germs)
(Alappat et al., 2005) that
lacks Msx1 expression and exhibits anterior-to-posterior expansion of
Shox2 mRNA expression from E12.5 to E14.5
(Li and Ding, 2007); and (3)
posterior palate that expresses high levels of Meox2 mRNA but lacks
Msx1 and Shox2 mRNA expression
(Li and Ding, 2007). In situ
hybridization of serial sections of the developing palatal shelves showed that
Mn1 mRNA is differentially expressed along the anteroposterior axis
of the developing palatal shelves, with high levels of expression in both
mesenchyme and epithelium in the middle and posterior regions and very low
levels in the anterior region of the palatal shelves during the vertical
growth period from E12.5 to E13.5 as well as after palatal shelf elevation at
E14.5 (Fig. 2).
Mn1-/- mutant mice exhibit palatal retardation and failure of palatal shelf elevation
To investigate which palatal developmental steps require Mn1 function, we
carried out detailed histological analyses of Mn1-/- mouse
embryos throughout palate development. Mn1-/- mutant
embryos displayed normal palatal shelf outgrowth at E12.5 (data not shown). At
E13.5, the palatal shelves of Mn1-/- mutant embryos
exhibited similar size and shape to those of wild-type embryos
(Fig. 3A,B). By E14.5, the
palatal shelves of wild-type embryos had already elevated to the horizontal
position and initiated fusion by forming the midline epithelial seam
(Fig. 3C), which was
disintegrated by E15.5 to form the fused secondary palate
(Fig. 3E). By contrast, the
bilateral palatal shelves of Mn1-/- mutant embryos failed
to elevate and remained at the vertical position at E14.5 and at E15.5
(Fig. 3D,F).
|
|
Mn1 is required for proper palatal shelf growth
Impairment of palatal shelf elevation is often accompanied by and partially
due to retarded palatal shelf growth, as has been reported in the
Osr2-/- mutant mice
(Lan et al., 2004). To
investigate the cellular mechanisms of palatal shelf retardation and elevation
failure in Mn1-/- mutant mice, we examined whether there
are alterations in cell proliferation and cell survival during palate
development in Mn1-/- mutant mice. No differences in cell
apoptosis were found in the palatal shelves between wild-type and
Mn1-/- mutant embryos at E12.5 and at E13.5 (data not
shown). No significant alterations in cell proliferation were observed in the
palatal shelves in Mn1-/- mutant embryos at E12.5 (data
not shown). At E13.5, we detected a 57% reduction (P<0.01) in the
posterior and a 49% reduction (P<0.01) in the middle regions of
the palatal mesenchyme in Mn1-/- mutant embryos in
comparison with their wild-type littermates
(Fig. 5G). By contrast, the
cell proliferation index was not significantly different in the anterior
palatal mesenchyme in the same Mn1-/- mutant and wild-type
embryos (Fig. 5G). Similarly,
palatal epithelial cell proliferation was also significantly reduced in the
middle and posterior palate but not in the anterior palate in
Mn1-/- mutant embryos, in comparison with the wild-type
littermates (Fig. 5H). The
selective reduction in palatal cell proliferation in the middle and posterior
regions of the palatal shelves in Mn1-/- mutant mice
correlates with the differential expression of Mn1 mRNA along the
anteroposterior axis during normal palate development.
To understand the dramatic retardation of the palatal shelves at later stages in Mn1-/- mutant embryos, we also examined cell proliferation and cell apoptosis in E14.5 and E15.5 embryos. The decrease in cell proliferation rate in the posterior and middle regions of the palatal shelves in the Mn1-/- mutant embryos continued through E15.5 (Fig. 6A-C and data not shown), indicating that Mn1 is an important regulator of palatal shelf growth before and after palatal shelf elevation. Moreover, by TUNEL assays, we detected increased apoptosis at E15.5 in the posterior regions of the palatal mesenchyme in Mn1-/- mutant embryos (Fig. 6D,E). These data indicate that the dramatic degeneration of the posterior palatal shelves observed by E18.5 in Mn1-/- mutant embryos resulted from decreased proliferation and increased apoptosis in the posterior palatal shelves, in combination with retraction of the freely projecting palatal shelves into the maxillary processes due to the morphogenetic expansion of the craniofacial width.
|
|
;
Xiong et al., 1991;
Motokura et al., 1991; Morgan
et al., 1997). Conditional inactivation of Tgfbr2 in cranial neural
crest (CNC) cells downregulated cyclin D1 expression and reduced CNC cell
proliferation in the palatal mesenchyme
(Ito et al., 2003). Msx1
regulates neural crest cell proliferation and differentiation also through
maintaining cyclin D1 expression (Hu et
al., 2001). To understand the cellular mechanism by which Mn1
regulates palatal growth in the middle and posterior palatal shelves, we
investigated possible alterations in the expression levels of D-type cyclins
in Mn1-/- mutant embryos. No change in the levels of
cyclin D1 (Ccnd1) expression was detected in either the epithelium or
mesenchyme of the developing palate in Mn1-/- embryos in
comparison with wild-type littermates (data not shown). By contrast, the
expression of cyclin D2 (Ccnd2) is reduced in the middle and
posterior regions of the palatal mesenchyme, as well as in the posterior
palatal epithelium in Mn1-/- embryos at E13.5
(Fig. 7). These data suggest
that Mn1 regulates palatal shelf growth, at least in part, through maintaining
Ccnd2 expression in the middle and posterior palatal shelves.
Effects of Mn1 deficiency on palatal gene expression
To investigate the molecular mechanisms involving Mn1 in palate
development, we examined the expression patterns of other genes known to play
important role in palate development, including Fgf10, Fgfr2, Osr2, Shh,
Patch1, Pax9, Shox2 and Tgfb3. No obvious differences in either
levels or patterns of expression of these genes were found in the developing
palatal shelves in wild-type and Mn1-/- mutant embryos
(Fig. 8; see Fig. S1 in the
supplementary material). In particular, the differential expression patterns
of Fgf10, Shox2 and Meox2 mRNAs along the anteroposterior
axis of the developing palatal shelves are maintained in the
Mn1-/- mutant embryos
(Fig. 8), indicating that there
is no gross anteroposterior patterning defects in the developing palate in
Mn1-/- mutant embryos.
Extensive expression analyses of other genes implicated in palate development showed that expression of Tbx22, which is homologous to the gene associated with CPX in humans, is specifically reduced in the developing palatal shelves in Mn1-/- mutant embryos (Fig. 9). Interestingly, expression of Tbx22 mRNA also exhibits a posterior preference in the developing palatal shelves, similar to the expression pattern of Mn1, during normal palate development in mice (Fig. 9A-C). Compared with wild-type embryos, the expression levels of Tbx22 are dramatically reduced in the middle and posterior palatal shelves of Mn1-/- mutant embryos (Fig. 9D-F), indicating that Mn1 and Tbx22 function in the same molecular pathway to regulate mammalian palate development.
|
|
10 kb apart
(Andreou et al., 2007).
Quantitative RT-PCR assays of human embryonic RNA samples indicated that
embryonic TBX22 mRNA expression was predominantly generated by the
hP0 promoter (data not shown). Analyses of 5' RACE (rapid
amplification of cDNA ends) products from E12.5 mouse embryonic craniofacial
RNA templates revealed that the mouse Tbx22 gene is also transcribed
from at least two distinct promoters: a distal promoter corresponding to the
human TBX22 hP0 and a proximal promoter corresponding to the human
TBX22 hP1 (see Fig. S2 in the supplementary material). Interestingly,
the previously determined core binding DNA sequence for the Mn1 transcription
factor, CACCC, is present in various locations within 2 kb of the
transcription start site for each of the mouse and human TBX22
promoters (see Fig. S3 in the supplementary material). To investigate whether
Mn1 can activate transcription from the mouse and human TBX22 gene
promoters, we constructed Tbx22 promoter-luciferase reporter plasmids
(see Materials and methods) and tested the effects of Mn1
co-transfection on luciferase reporter expression in NIH3T3 cells.
Transfection of each of the promoter-luciferase plasmids showed that the mouse
mP0 and mP1 as well as human hP0 promoters each had
promoter activity in NIH3T3 cells, with the hP0 promoter being
threefold stronger than the mP0 or mP1 promoter
(Fig. 10A). By contrast, the
hP1-luciferase construct did not show promoter activity in NIH3T3 and
three other cell lines tested (Fig.
10A) (Andreou et al.,
2007) (and data not shown). Co-transfection with the Mn1
expression vector resulted in a dose-dependent increase in luciferase
expression from each of the mP0-, mP1- and
hP0-luciferase constructs but not the hP1
promoter-luciferase construct (Fig.
10A). Moreover, transfection of NIH3T3 cells with the Mn1
expression vector resulted in a dose-dependent increase in endogenous
Tbx22 mRNA expression (Fig.
10B,C). These data indicate that Mn1 functions as a
transcriptional activator of Tbx22 gene expression. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Schutte and Murray,
1999; Gorlin et al.,
2001). Approximately 50% of cleft palate cases are non-syndromic,
although cleft palate has been associated with more than 300 syndromic
developmental disorders. Several genes underlying syndromic forms of cleft
palate have been identified, including IRF6 (Van der Woude and
popliteal pterygium syndromes) (Kondo et
al., 2002), MSX1 (cleft lip or palate with hypodontia)
(van den Boogaard et al.,
2000), P63 (ectodermal dysplasia and cleft lip or palate)
(Celli et al., 1999;
Ianakiev et al., 2000),
PVRL1 (autosomal recessive cleft lip/palate with ectodermal
dysplasia) (Suzuki et al.,
2000) and TBX22 (CPX)
(Braybrook et al., 2001). In
addition, genetic studies in mice have revealed that mutations in more than 60
different genes each resulted in cleft palate and have uncovered specific
molecular and cellular mechanisms controlling palate development (reviewed by
Gritli-Linde, 2007).
Interestingly, recent studies have demonstrated that palatal shelf growth is
differentially regulated along the anteroposterior axis (reviewed by
Hilliard et al., 2005). Zhang
et al. (Zhang et al., 2002)
first demonstrated that Msx1 and Bmp4 are both only
expressed in and required for the growth of the anterior region of the
developing palatal shelves. In palatal explant culture assays, exogenous Bmp4
induced Msx1 mRNA expression and increased cell proliferation in the
anterior but not in posterior palatal mesenchyme
(Zhang et al., 2002;
Hilliard et al., 2005). The
Shox2 transcription factor is also specifically expressed in the anterior
palate and mice lacking Shox2 exhibited growth defects in the
anterior but not posterior regions of the developing palatal shelves
(Yu et al., 2005). However,
previous studies have also showed that the anterior and posterior palatal
regions have similar cell proliferation rates during palatal outgrowth
(Zhang et al., 2002;
Li and Ding, 2007), suggesting
that there must be factors that preferentially regulate posterior palatal
growth. Our finding that Mn1 is differentially expressed along the
anteroposterior axis and preferentially regulates middle and posterior palatal
cell proliferation fills a longstanding gap in the understanding of the
molecular mechanisms of palate development.
|
|
;
Braybrook et al., 2002;
Chaabouni et al., 2005). In
addition, mutation-screening studies of individuals with isolated cleft palate
showed that mutations in TBX22 represent the most common single cause
of cleft palate known (Marcano et al.,
2004; Suphapeetiporn et al.,
2007). These data suggest that TBX22 plays primary and crucial
roles in palate development. Consistent with this hypothesis, Braybrook et al.
(Braybrook et al., 2002) showed
that TBX22 mRNA is strongly expressed in the developing human palatal
shelves prior to palatal elevation and fusion. TBX22 mRNA expression
was also detected in the base of the developing tongue, which correlated well
with the ankyloglossia phenotype in individuals with CPX. Several laboratories
independently isolated the mouse Tbx22 gene and reported
Tbx22 mRNA expression in the developing palatal shelves and at the
base of the developing tongue (Braybrook et
al., 2002; Bush et al.,
2002; Herr et al.,
2003). Although the exact roles of Tbx22 in palate
development remain to be elucidated, these studies showed that palatal
expression of Tbx22 mRNA was high during vertical palatal growth and
declined after palatal shelf elevation in both human and mice, suggesting that
Tbx22 may play a conserved role in palatal shelf growth. Welsh et al.
(Welsh et al., 2007) showed,
by whole-mount in situ hybridization, that Tbx22 mRNA is
preferentially expressed in the posterior palate in E13.5 and E14.5 mouse
embryos. We confirmed, by using section in situ hybridization, the
differential expression of Tbx22 mRNA along the anteroposterior axis
of the developing secondary palate (Fig.
9). Interestingly, Tbx22 mRNA expression was dramatically
downregulated in the Mn1-/- mutant palatal shelves. The
fact that palatal expression of many genes, including that of Meox2,
which is also preferentially expressed in the posterior palate during palatal
outgrowth (Li and Ding, 2007)
(Fig. 8), is not affected by
Mn1 deficiency indicates that Tbx22 is a specific downstream
target of Mn1. Our data that Mn1 activated, in a dose-dependent manner,
expression of endogenous Tbx22 mRNA and the luciferase reporter gene
driven by either the human or mouse Tbx22 promoter sequences in
transfected NIH3T3 cells provide further support that Mn1 activates
transcription of the Tbx22 gene. These data link two spatially
co-expressed transcription factors in a novel molecular pathway for the
regulation of posterior palate development.
|
). We found that Mn1 is differentially expressed along
the anteroposterior axis of the developing palatal shelves and required for
proper palatal growth in the middle and posterior regions but not in the
anterior region. The specific downregulation of Tbx22 mRNA expression
in the Mn1-/- mutant palatal shelves also identifies a
primary role for Mn1 in palate development. However,
Mn1-/- mutant mice exhibit complete cleft of the secondary
palate, although no significant growth deficiency was found in the anterior
palate at any stage of palate development. In addition, the palatal shelves
failed to elevate, in particular in the middle and posterior regions, in the
Mn1-/- mutant mice. Impairment of palatal shelf elevation
is often caused by mechanical hindrance, such as aberrant palatal-tongue and
palatal-mandible fusions in the Jag2-/- and
Fgf10-/- mutant mice, respectively
(Casey et al., 2006;
Alappat et al., 2005). Defects
in mandibular development or deformation of the tongue have also been
suggested as causes of failure of palatal shelf elevation in several mutant
mouse strains, including Ryk-/- and
Foxf2-/- mutant mice
(Halford et al., 2000;
Wang et al., 2003). In
addition, Pax9-/- mutant mice exhibited impairment of
palatal shelf elevation that was attributable to deformation of the palatal
shelves themselves (Peters et al.,
1998). Mn1-/- mutant mice exhibited normal
mandibular development (Meester-Smoor et
al., 2005). However, the developing tongue did not properly
descend to the floor of the mouth during the time of palatal shelf elevation
in the Mn1-/- mutant mice, in comparison with that in the
wild-type littermates (Fig. 3).
Whereas Mn1 mRNA is strongly expressed in the middle and posterior
palatal mesenchyme, little Mn1 expression was observed in the
developing tongue (Fig. 2).
Thus, the palatal elevation defect in the Mn1-/- mutant
mice may be due to an intrinsic defect in the palatal shelves or secondary to
as yet unidentified defects in a mandibular component involved in tongue
movement. Taken together, the cleft palate phenotype in
Mn1-/- mutant mice probably results from a combination of
the primary defects in palatal growth and secondary effects of other
craniofacial abnormalities.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/23/3959/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Alappat, S. R., Zhang, Z., Suzuki, K., Zhang, X., Liu, H.,
Jiang, R., Yamada, G. and Chen, Y. (2005). The cellular and
molecular etiology of the cleft secondary palate in Fgf10 mutant
mice. Dev. Biol. 277,102
-113.[CrossRef][Medline]
Andreou, A. M., Pauws, E., Jones, M. C., Singh, M. K., Bussen,
M., Doudney, K., Moore, G. E., Kispert, A., Brosens, J. J. and Stanier, P.
(2007). TBX22 missense mutations found in patients with
X-linked cleft palate affect DNA binding, sumoylation, and transcriptional
repression. Am. J. Hum. Genet.
81,700
-712.[CrossRef][Medline]
Braybrook, C., Doudney, K., Marçano, A. C., Arnason, A.,
Bjornsson, A., Patton, M. A., Goodfellow, P. J., Moore, G. E. and Stanier,
P. (2001). The T-box transcription factor gene TBX22
is mutated in X-linked cleft palate and ankyloglossia. Nat.
Genet. 29,179
-183.[CrossRef][Medline]
Braybrook, C., Lisgo, S., Doudney, K., Henderson, D.,
Marçano, A. C., Strachan, T., Patton, M. A., Villard, L., Moore, G. E.,
Stanier, P. et al. (2002). Craniofacial expression of human
and murine TBX22 correlates with the cleft palate and ankyloglossia
phenotype observed in CPX patients. Hum. Mol. Genet.
11,2793
-2804.
Buijs, A., Sherr, S., van Baal, S., van Bezouw, S., van der
Plas, D., Geurts van Kessel, A., Riegman, P., Lekanne Deprez, R., Zwarthoff,
E. and Hagemeijer, A. (1995). Translocation (12;22) (p13;q11)
in myeloproliferative disorders results in fusion of the ETS-like TEL
gene on 12p13 to the MN1 gene on 22q11. Oncogene
10,1511
-1519.[Medline]
Buijs, A., van Rompaey, L., Molijn, A. C., Davis, J. N.,
Vertegaal, A. C., Potter, M. D., Adams, C., van Baal, S., Zwarthoff, E. C.,
Roussel, M. F. et al. (2000). The MN1-TEL fusion protein.,
encoded by the translocation (12;22)(p13;q11) in myeloid leukemia, is a
transcription factor with transforming activity. Mol. Cell.
Biol. 20,9281
-9293.
Bush, J. O., Lan, Y., Maltby, K. M. and Jiang, R.
(2002). Isolation and developmental expression analysis of
Tbx22, the mouse homolog of the human X-linked cleft palate gene.
Dev. Dyn. 225,322
-326.[CrossRef][Medline]
Carella, C., Bonten, J., Rehg, J. and Grosveld, G. C.
(2006). MN1-TEL, the product of the t(12;22) in human myeloid
leukemia, immortalizes murine myeloid cells and causes myeloid malignancy in
mice. Leukemia 20,1582
-1592.[CrossRef][Medline]
Carella, C., Bonten, J., Sirma, S., Kranenburg, T. A.,
Terranova, S., Klein-Geltink, R., Shurtleff, S., Downing, J. R., Zwarthoff, E.
C., Liu, P. P. et al. (2007). MN1 overexpression is
an important step in the development of inv(16) AML.
Leukemia 21,1679
-1690.[CrossRef][Medline]
Casey, L. M., Lan, Y., Cho, E. S., Maltby, K. M., Gridley, T.
and Jiang, R. (2006). Jag2-Notch1 signaling regulates oral
epithelial differentiation and palate development. Dev.
Dyn. 235,1830
-1844.[CrossRef][Medline]
Celli, J., Duijf, P., Hamel, B. C., Bamshad, M., Kramer, B.,
Smits, A. P., Newbury-Ecob, R., Hennekam, R. C., Van Buggenhout, G., van
Haeringen, A. et al. (1999). Heterozygous germline mutations
in the p53 homolog p63 are the cause of EEC syndrome.
Cell 99,143
-153.[CrossRef][Medline]
Chaabouni, M., Smaoui, N., Benneji, N., M'rd, R., Jemaa, L. B.,
Hachicha, S. and Chaabouni, H. (2005). Mutation analysis of
TBX22 reveals new mutation in Tunisian CPX family. Clin.
Dysmorphol. 14,23
-25.[CrossRef][Medline]
Chai, Y. and Maxson, R. E., Jr (2006). Recent
advances in craniofacial morphogenesis. Dev. Dyn.
235,2353
-2375.[CrossRef][Medline]
Du, Y., Jenkins, N. A. and Copeland, N. G.
(2005). Insertional mutagenesis identifies genes that promote the
immortalization of primary bone marrow progenitor cells.
Blood 106,3932
-3939.
Ferguson, M. W. (1988). Palate development.
Dev. Suppl. 103,41
-60.
Gorlin, R. J., Cohen, M. M., Jr and Hennekam, R. C. M.
(2001). Syndromes of the Head and Neck.
New York: Oxford University Press.
Gritli-Linde, A. (2007). Molecular control of
secondary palate development. Dev. Biol.
301,309
-326.[CrossRef][Medline]
Grosveld, G. C. (2007). MN1, a novel player in
human AML. Blood Cells Mol. Dis.
39,336
-339.[CrossRef][Medline]
Halford, M. M., Armes, J., Buchert, M., Meskenaite, V., Grail,
D., Hibbs, M. L., Wilks, A. F., Farlie, P. G., Newgreen, D. F., Hovens, C. M.
et al. (2000). Ryk-deficient mice exhibit
craniofacial defects associated with perturbed Eph receptor crosstalk.
Nat. Genet. 25,414
-418.[CrossRef][Medline]
Han, J., Ito, Y., Yeo, J. Y., Sucov, H. M., Maas, R. and Chai,
Y. (2003). Cranial neural crest-derived mesenchymal
proliferation is regulated by Msx1-mediated p19(INK4d) expression during
odontogenesis. Dev. Biol.
261,183
-196.[CrossRef][Medline]
Herr, A., Meunier, D., Müller, I., Rump, A., Fundele, R.,
Ropers, H. H. and Nuber, U. A. (2003). Expression of mouse
Tbx22 supports its role in palatogenesis and glossogenesis.
Dev. Dyn. 226,579
-586.[CrossRef][Medline]
Heuser, M., Beutel, G., Krauter, J., Döhner, K., von
Neuhoff, N., Schlegelberger, B. and Ganser, A. (2006). High
meningioma 1 (MN1) expression as a predictor for poor outcome in acute myeloid
leukemia with normal cytogenetics. Blood
108,3898
-3905.
Hilliard, S. A., Yu, L., Gu, S., Zhang, Z. and Chen, Y. P.
(2005). Regional regulation of palatal growth and patterning
along the anterior-posterior axis in mice. J. Anat.
207,655
-667.[Medline]
Hu, G., Lee, H., Price, S. M., Shen, M. M. and Abate-Shen,
C. (2001). Msx homeobox genes inhibit
differentiation through upregulation of cyclin D1.
Development 128,2373
-2384.
Ianakiev, P., Kilpatrick, M. W., Toudjarska, I., Basel, D.,
Beighton, P. and Tsipouras, P. (2000). Split-hand/split-foot
malformation is caused by mutations in the p63 gene on 3q27. Am. J.
Hum. Genet. 67,59
-66.[Medline]
Ito, Y., Yeo, J. Y., Chytil, A., Han, J., Bringas, P., Jr,
Nakajima, A., Shuler, C. F., Moses, H. L. and Chai, Y.
(2003). Conditional inactivation of Tgfbr2 in cranial
neural crest causes cleft palate and calvaria defects.
Development 130,5269
-5280.
Jin, J. Z. and Ding, J. (2006). Analysis of
Meox2 mutant mice reveals a novel postfusion-based cleft palate.
Dev. Dyn. 235,539
-546.[CrossRef][Medline]
Kawagoe, H. and Grosveld, G. C. (2005a).
Conditional MN1-TEL knock-in mice develop acute myeloid leukemia in
conjunction with overexpression of HOXA9.
Blood 106,4269
-4277.
Kawagoe, H. and Grosveld, G. C. (2005b).
MN1-TEL myeloid oncoprotein expressed in multipotent progenitors perturbs both
myeloid and lymphoid growth and causes T-lymphoid tumors in mice.
Blood 106,4278
-4286.
Kondo, S., Schutte, B. C., Richardson, R. J., Bjork, B. C.,
Knight, A. S., Watanabe, Y., Howard, E., de Lima, R. L., Daack-Hirsch, S.,
Sander, A. et al. (2002). Mutations in IRF6 cause
Van der Woude and popliteal pterygium syndromes. Nat.
Genet. 32,285
-289.[CrossRef][Medline]
Lan, Y., Kingsley, P. D., Cho, E. S. and Jiang, R.
(2001). Osr2, a new mouse gene related to Drosophila
odd-skipped, exhibits dynamic expression patterns during craniofacial, limb,
and kidney development. Mech. Dev.
107,175
-179.[CrossRef][Medline]
Lan, Y., Ovitt, C. E., Cho, E. S., Maltby, K. M., Wang, Q. and
Jiang, R. (2004). Odd-skipped related 2
(Osr2) encodes a key intrinsic regulator of secondary palate growth
and morphogenesis. Development
131,3207
-3216.
Lekanne Deprez, R. H., Riegman, P. H., Groen, N. A., Warringa,
U. L., van Biezen, N. A., Molijn, A. C., Bootsma, D., de Jong, P. J., Menon,
A. G., Kley, N. A. et al. (1995). Cloning and
characterization of MN1, a gene from chromosome 22q11, which is
disrupted by a balanced translocation in a meningioma.
Oncogene 10,1521
-1528.[Medline]
Li, Q. and Ding, J. (2007). Gene expression
analysis reveals that formation of the mouse anterior secondary palate
involves recruitment of cells from the posterior side. Int. J. Dev.
Biol. 51,167
-172.[CrossRef][Medline]
Liu, W., Sun, X., Braut, A., Mishina, Y., Behringer, R. R.,
Mina, M. and Martin, J. F. (2005). Distinct functions for Bmp
signaling in lip and palate fusion in mice.
Development 132,1453
-1461.
Marcano, A. C. B., Doudney, K., Braybrook, C., Squires, R.,
Patton, M. A., Lees, M., Richieri-Costa, A., Lidral, A. C., Murray, J. C.,
Moore, G. E. et al. (2004). TBX22 mutations are a
frequent cause of cleft palate. J. Med. Genet.
41, 68-74.
Matsushime, H., Roussel, M. F., Ashmun, R. A. and Sherr, C.
J. (1991). Colony-stimulating factor 1 regulates novel
cyclins during the G1 phase of the cell cycle. Cell
65,701
-713.[CrossRef][Medline]
Meester-Smoor, M. A., Vermeij, M., van Helmond, M. J., Molijn,
A. C., van Wely, K. H., Hekman, A. C., Vermey-Keers, C., Riegman, P. H. and
Zwarthoff, E. C. (2005). Targeted disruption of the
Mn1 oncogene results in severe defects in development of membranous
bones of the cranial skeleton. Mol. Cell. Biol.
25,4229
-4236.
Meester-Smoor, M. A., Molijn, A. C., Zhao, Y., Groen, N. A.,
Groffen, C. A., Boogaard, M., van Dalsum-Verbiest, D., Grosveld, G. C. and
Zwarthoff, E. C. (2007). The MN1 oncoprotein activates
transcription of the IGFBP5 promoter through a CACCC-rich consensus
sequence. J. Mol. Endocrinol.
38,113
-125.
Morgan, D. O. (1997). Cyclin-dependent kinases:
engines, clocks, and microprocessors. Annu. Rev. Cell. Dev.
Biol. 13,261
-291.[CrossRef][Medline]
Motokura, T., Bloom, T., Kim, H. G., Jüppner, H., Ruderman,
J. V., Kronenberg, H. M. and Arnold, A. (1991). A novel
cyclin encoded by a bcl1-linked candidate oncogene.
Nature 350,512
-515.[CrossRef][Medline]
Peters, H., Neubüser, A., Kratochwil, K. and Balling,
R. (1998). Pax9-deficient mice lack pharyngeal pouch
derivatives and teeth and exhibit craniofacial and limb abnormalities.
Genes. Dev. 12,2735
-2747.
Rice, R., Spencer-Dene, B., Connor, E. C., Gritli-Linde, A.,
McMahon, A. P., Dickson, C., Thesleff, I. and Rice, D. P. C.
(2004). Disruption of Fgf10/Fgfr2b-coordinated
epithelial-mesenchymal interactions causes cleft palate. J. Clin.
Invest. 113,1692
-1700.[CrossRef][Medline]
Ross, M. E., Mahfouz, R., Onciu, M., Liu, H. C., Zhou, X., Song,
G., Shurtleff, S. A., Pounds, S., Cheng, C., Ma, J. et al.
(2004). Gene expression profiling of pediatric acute myelogenous
leukemia. Blood 104,3679
-3687.
Schutte, B. C. and Murray, J. C. (1999). The
many faces and factors of orofacial clefts. Hum. Mol.
Genet. 8,1853
-1859.
Sperber, G. H. (2002).Palatogenesis: closure of
the secondary palate. In Cleft Lip and Palate: From Origin To
Treatment (ed. D. F. Wyszynski), pp.14
-24. Oxford: Oxford University Press.
Suphapeetiporn, K., Tongkobpetch, S., Siriwan, P. and
Shotelersuk, V. (2007). TBX22 mutations are a frequent cause
of non-syndromic cleft palate in the Thai population. Clin.
Genet. 72,478
-483.[Medline]
Sutton, A. L., Zhang, X., Ellison, T. I. and Macdonald, P.
N. (2005). The 1,25(OH)2D3-regulated transcription factor MN1
stimulates vitamin D receptor-mediated transcription and inhibits osteoblastic
cell proliferation. Mol. Endocrinol.
19,2234
-2244.
Suzuki, K., Hu, D., Bustos, T., Zlotogora, J., Richieri-Costa,
A., Helms, J. A. and Spritz, R. A. (2000). Mutations of
PVRL1, encoding a cell-cell adhesion molecule/herpesvirus receptor,
in cleft lip/palate-ectodermal dysplasia. Nat. Genet.
25,427
-430.[CrossRef][Medline]
Valk, P. J., Delwel, R. and Löwenberg, B.
(2004). Gene expression profiling in acute myeloid leukemia.
Curr. Opin. Hematol. 12,76
-81.[CrossRef]
van den Boogaard, M. J., Dorland, M., Beemer, F. A. and van
Amstel, H. K. (2000). MSX1 mutation is associated
with orofacial clefting and tooth agenesis in humans. Nat.
Genet. 24,342
-343.[CrossRef][Medline]
van Wely, K. H., Molijn, A. C., Buijs, A., Meester-Smoor, M. A.,
Aarnoudse, A. J., Hellemons, A., den Besten, P., Grosveld, G. C. and
Zwarthoff, E. C. (2003). The MN1 oncoprotein synergizes with
coactivators RAC3 and p300 in RAR-RXR-mediated transcription.
Oncogene 22,699
-709.[CrossRef][Medline]
Vanderas, A. P. (1987). Incidence of cleft lip,
cleft palate, and cleft lip and palate among races: a review. Cleft
Palate J. 24,216
-225.[Medline]
Wang, T., Tamakoshi, T., Uezato, T., Shu, F., Kanzaki-Kato, N.,
Fu, Y., Koseki, H., Yoshida, N., Sugiyama, T. and Miura, N.
(2003). Forkhead transcription factor Foxf2 (LUN)-deficient mice
exhibit abnormal development of secondary palate. Dev.
Biol. 259,83
-94.[CrossRef][Medline]
Welsh, I. C., Hagge-Greenberg, A. and O'Brien, T. P.
(2007). A dosage-dependent role for Spry2 in growth and
patterning during palate development. Mech. Dev.
124,746
-761.[CrossRef][Medline]
Xiong, Y., Connolly, T., Futcher, B. and Beach, D.
(1991). Human D-type cyclin. Cell
65,691
-699.[CrossRef][Medline]
Yu, L., Gu, S., Alappat, S., Song, Y., Yan, M., Zhang, X.,
Zhang, G., Jiang, Y., Zhang, Z., Zhang, Y. et al. (2005).
Shox2-deficient mice exhibit a rare type of incomplete clefting of
the secondary palate. Development
132,4397
-4406.
Zhang, Y., Zhao, X., Hu, Y., St Amand, T., Zhang, M.,
Ramamurthy, R., Qiu, M. and Chen, Y. (1999). Msx1 is
required for the induction of Patched by Sonic hedgehog in the mammalian tooth
germ. Dev. Dyn. 215,45
-53.[CrossRef][Medline]
Zhang, Z., Song, Y., Zhao, X., Zhang, X., Fermin, C. and Chen,
Y. (2002). Rescue of cleft palate in Msx1-deficient
mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the
regulation of mammalian palatogenesis. Development
129,4135
-4146.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  E Pauws, G E Moore, and P Stanier A functional haplotype variant in the TBX22 promoter is associated with cleft palate and ankyloglossia J. Med. Genet., August 1, 2009; 46(8): 555 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhang, D. R. Dowd, M. C. Moore, T. A. Kranenburg, M. A. Meester-Smoor, E. C. Zwarthoff, and P. N. MacDonald Meningioma 1 Is Required for Appropriate Osteoblast Proliferation, Motility, Differentiation, and Function J. Biol. Chem., July 3, 2009; 284(27): 18174 - 18183. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
