Runx3 expression during mouse tongue and palate development.

EXPRESSions
THE ANATOMICAL RECORD PART A 288A:695– 699 (2006)
Runx3 Expression During Mouse
Tongue and Palate Development
HIROMITSU YAMAMOTO,1* KOSEI ITO,2 MARIKO KAWAI,3
YOTA MURAKAMI,4 KAZUHISA BESSHO,1 AND YOSHIAKI ITO2
1
Department of Oral and Maxillofacial Surgery, Graduate School of Medicine,
Kyoto University, Kyoto, Japan
2
Institute of Molecular and Cell Biology and Oncology Research Institute, Singapore
3
Department of Oral Morphology, Graduate School of Medicine, Dentistry, and
Pharmaceutical Sciences, Okayama University, Okayama, Japan
4
Institute for Virus Research, Kyoto University, Kyoto, Japan
ABSTRACT
Studies of molecular mechanisms underlying the development of the
mammalian oral mucosa have revealed a major involvement of transforming growth factor ␤ (TGF-␤) and bone morphologic protein (BMP) signaling
pathways. Here, we examined the expression of a downstream target of
TGF-␤ and BMPs, Runx3, in oral mucosa. Runx3 is a runt-related transcription factor that acts as a gastric tumor suppressor and regulator of
growth and differentiation in mammalian gastric epithelial cells. Another
member of the Runx family in C. elegans, run, is involved in the development of a functional hypodermis and gut. In this report, we examined
Runx3 expression using reverse transcription-polymerase chain reaction,
immnunohistochemistry, and in situ hybridization and found that Runx3 is
expressed in the tongue and palate epithelium of mouse embryos from
embryonic day 12.5 to 16.5. The functional relationship between Runx3 and
TGF-␤/BMPs signaling in tongue and palate development is discussed.
Anat Rec Part A, 288A:695– 699, 2006. © 2006 Wiley-Liss, Inc.
Key words: Runx3; tongue epithelium; palate epithelium; TGF␤/BMP signaling
Tongue development in mice is initiated during embryonic day 11 (E11) by the appearance of two lateral lingual
swellings on branchial arch I. By E13, the general proportions of the tongue occupied by the body, root, and epiglottis are established (Paulson et al., 1985). On the other
hand, the palatal shelves first appear later on at approximately E12.5 and rapidly grow vertically to flank the
developing tongue. The shelves, consisting of proliferating
mesenchymal cells, undergo a sudden elevation that
brings them into a horizontal apposition above the flattening tongue. Cleft palate, the most common birth defect
in human, occurs when the palatal shelves fail to meet in
the horizontal plane (Stanier and Moore, 2004).
Previous studies have revealed that several developmental factors are expressed in the epithelial cells of the
tongue and palate. Signaling pathways, including those
comprising bone morphogenetic proteins (BMPs), transforming growth factor ␤ (TGF-␤), sonic hedgehog (Shh),
and fibrobrast growth factors (FGFs), are known to be
involved in vertebrate facial development (Francis-West
et al., 1998). Among these various factors, the expression
©
2006 WILEY-LISS, INC.
of ligands and specific receptors of TGF-␤/BMPs in tongue
and palate epithelial cells have been particularly well
demonstrated (see Results and Discussion). This is especially the case for TGF-␤3, which was shown to be essential for the fusion of palatal shelves through the study of
TGF-␤3-deficient mice (Kaartinen, 1995; Proetzel et al.,
1995). TGF-␤3 is expressed by medial edge epithelium
cells just prior to the fusion of palatal shelves and ceases
to be expressed shortly after the formation of the midline
epithelial seam (MES) (Fitzpatrick et al., 1990). There-
*Correspondence to: Hiromitsu Yamamoto, Department of Oral
and Maxillofacial Surgery, Graduate School of Medicine, Kyoto
University, 54 Shogoin-kawaharacho, Sakyo, Kyoto 606-8507, Japan. Fax: 81-75-761-9732. E-mail: hyamamot@kuhp.kyoto-u.ac.jp
Received 21 September 2005; Accepted 22 February 2006
DOI 10.1002/ar.a.20339
Published online 7 June 2006 in Wiley InterScience
(www.interscience.wiley.com).
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YAMAMOTO ET AL.
fore, TGF-␤3 is predicted to be involved in the removal of
the MES, a process essential for the confluence of the
palatal mesenchyme. Moreover, Msx, a factor acting
downstream of BMPs, is frequently mutated in people
with orofacial clefting, suggesting distinct functions for
BMP signaling. This was further corroborated by studies
using Msx-, type IA Bmp receptor (Alk3)-, and Bmp4deficient mouse models (Satokata and Maas, 1994; van
den Boogaard et al., 2000; Liu et al., 2005). Thus, TGF-␤/
BMPs signaling pathways are heavily involved in mammalian palatogenesis.
The Runt-related (RUNX) gene family includes three
members, which encode heterodimeric transcription factors (Ito, 2004). Runx1/AML1 plays a role in the regulation of hematopoiesis (Okuda et al., 1996), and its dysregulation leads to acute myeloid leukemia (Look, 1997).
Runx2/Cbfa1 is essential for osteoblast differentiation,
bone formation, and chondrocyte maturation (Komori et
al., 1997; Otto et al., 1997; Inada et al., 1999; Enomoto et
al., 2000), and haploinsufficiency of Runx2 causes cleidocranial dysplasia, a human skeletal disease (Mundlos et
al., 1997). Runx3/PEBP2␣C (Bae et al., 1995) has multiple
functions and was first correlated with the genesis and
progression of human gastric cancer as a tumor suppressor. The gastric mucosa of Runx3-null (Runx3⫺/⫺) mice
exhibits hyperplasia as a result of stimulated proliferation
and suppressed apoptosis of epithelial cells (Li et al.,
2002), and RUNX3 is inactivated in more than 80% of
human gastric cancer cells by gene silencing or protein
mislocalization (Ito et al., 2005). Runx3 has an essential
role in the development of dorsal root ganglia neurons
(Inoue et al., 2002; Levanon et al., 2002) and functions in
development of CD8-lineage T-lymphocytes (Taniuchi et
al., 2002; Woolf et al., 2003). Furthermore, together with
Runx2, Runx3 plays fundamental roles in skeletal development by regulating and coordinating chondrocyte maturation and proliferation to form limb bones of the appropriate size and shape (Yoshida et al., 2004).
Runx3 is expressed in the gastrointestinal tract of
mouse and human (Li et al., 2002), and run, a homolog of
the Runx family in C. elegans, is involved in the development of a functional epidermis and gut (Nam et al., 2001).
Furthermore, RUNX3 interacts with receptor-regulated
Smads (R-Smads), Smad1, 2, 3, and 5, and a commonpartner Smad (Co-Smad), Smad4, to regulate target gene
expression downstream of TGF-␤/BMPs (Hanai et al.,
1999; Chi et al., 2005). Thus, Runx3 possibly functions
downstream of TGF-␤/BMPs in tongue and palate development.
In order to address the involvement of Runx3 in oral
epithelial development, we examined the expression of
Runx3 in mouse embryonic oral mucosa using reverse
transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization techniques.
Our findings demonstrate that Runx3 is expressed in the
tongue and palate epithelium from E12.5 to E16.5, which
corresponds to the developmental stage involving TGF-␤/
BMPs.
MATERIALS AND METHODS
Generation of Runx3 Mutant Mice
Runx3⫹/⫺ and Runx3⫺/⫺ mice were generated as described previously (Li et al., 2002). The lac-Z reporter sequence was inserted in-frame into the Runx3 gene to detect
the expression of Runx3. The Runx3 genotype of each mouse
was determined by PCR using tail DNA and the following
primers: 5⬘-GACTGTGCATGCACCTTTCACCAA-3⬘ and 5⬘TAGGGCTCAGTAGCACTTACGTCG-3⬘, or 5⬘-GACTGTGCATGCACCTTTCACCAA-3⬘ and 5⬘-ATGAAACGCCGAGTTAACGCCATCA-3⬘. All experiments in this study were
approved by the Animal Research Committee, Graduate
School of Medicine, Kyoto University.
Tissue Dissection and RT-PCR
Whole tongues were dissected from E12.5 to 18.5 and
neonate wild-type mice. Five or six samples were collected
at each stage except at E12.5 and subjected to RT-PCR
independently. The amount of tissue from the E12.5 embryos was so small that two preparations were mixed for
each assay. Stomachs and left hind limb cartilages were
also dissected from E14.5 wild-type embryos. Total RNA
was isolated from these tissues using Isogen (Nippon
Gene, Tokyo, Japan). Total RNA (1 ␮g) was reverse-transcribed with reverse transcriptase and oligo-(dT) primers
at 42°C for 1 hr (Invitrogen, SuperScript kit). Runx3
cDNA was amplified by PCR with EX Taq DNA polymerase (TaKaRa) under the following conditions: 94°C for 3
min, followed by 28, 30, and 32 cycles of 94°C for 1 min,
60°C for 1 min, and 72°C for 1 min. The primers used
for amplification were 5⬘-ACCGCTTTGGAGACCTGCGCATG-3⬘ and 5⬘-CGCTGTAGGGGAAGGCGGCAGA-3⬘ for
Runx3, and 5⬘-CGTATTGGGCGCCTGGTCAC-3⬘ and 5⬘CCAGTGAGCTTCCCGTTCAC-3⬘ for GAPDH (G. Huang
et al., 2001).
␤-Galactosidase Staining and
Immunohistochemistry
␤-galactosidase activity was measured as described previously (Li et al., 2002). For whole-mount ␤-galactosidase
histochemical staining, Runx3⫹/⫺ E15.5 embryos were
fixed with 2% paraformaldehyde and 0.2% glutaraldehyde
in PBS, then ␤-galactosidase activity was detected by incubating the samples in an X-gal staining cocktail, which
contained nitroblue tetrazolium (NBT) and potassium ferrocyanide as described (Huang et al., 1998; Li et al., 2002).
For histological analysis, the embryos were fixed with 4%
paraformaldehyde, then embedded in paraffin and cut into
7 ␮m thick sections. To detect ␤-galactosidase, immunohistochemistry was performed using an anti-␤-galactosidase mouse monoclonal antibody (Promega) and the Dako
EnVision⫹ mouse Kit (Dako).
In Situ Hybridization
For in situ hybridization, digoxigenin-11-UTP-labeled
single-stranded RNA probes were prepared using a DIG
RNA-labeling kit (Roche Biochemica). A 1.1 kb fragment
of Runx3 cDNA was used to generate antisense and sense
probes. Sections prepared as described above were also
used for in situ hybridization as described previously
(Komori et al., 1997; Inada et al., 1999; Yoshida et al.,
2002). Hybridization was performed at 55°C overnight
and washed at the same temperature.
RESULTS AND DISCUSSION
Expression of Runx3 in the developing tongue from
E12.5 to newborn mice was detected by RT-PCR. Runx3
mRNA was expressed in mouse tongue at E12.5. Expression was strong between E13.5 and E15.5, slowly de-
Runx3 EXPRESSION
Fig. 1. Runx3 expression in mouse tongue revealed by RT-PCR. A:
Runx3 expression in the tongue from E12.5 to E18.5 wild-type mouse
embryos and newborn mice. The number of PCR cycles was 30. B:
Runx3 expression in the tongue, stomach, and hind limb cartilage at
E14.5 in the wild-type mouse embryo. PCR was performed for the
indicated number of cycles to detect expression. GAPDH expression
was used as a control.
creased after E16.5, and was barely detectable in newborn
mice (Fig. 1A). The level of Runx3 expression in the tongue
was comparable to that in the stomach and hind limb
cartilage at E14.5 (Fig. 1B), where Runx3 expression has
been previously observed (Li et al., 2002; Yoshida et al.,
2004).
The expression pattern of Runx3 in the oral mucosa was
examined using Runx3 heterozygous (Runx⫹/⫺) embryos
in which the lac-Z gene was integrated into exon3 of one
allele of Runx3 (Li et al., 2002). ␤-galactosidase activity
was measured in whole-mount embryos at various stages.
Expression of the lac-Z allele was detected in the developing tongue at E15.5 (Fig. 2A–C). Both the dorsal and the
ventral (sublingual) surfaces of the tongue showed ␤-galactosidase activity in Runx3⫹/⫺ embryos (Fig. 2B and C).
No staining was detected on either surface of wild-type
tongue (Fig. 2D and E).
To determine Runx3 expression at the cellular level, we
analyzed embryonic tissues histologically. In the oral region of the E12.5 embryos, Runx3 expression was observed in the epithelial lining of the dorsal mucosal surface of the tongue and throughout the epithelium of the
palate (Fig. 2F–H). ␤-galactosidase expression was detected predominantly in the epithelial cells of the tongue
and palate, and it was stronger on the dorsal than on the
ventral surface of the tongue (Fig. 2F–H). In E13.5 embryos, when the structure of the epithelial layer seemed
complete, the Runx3 expression pattern (Fig. 2I–K) was
697
Fig. 2. Runx3 expression in mouse tongue as revealed by ␤-galactosidase activity. A–E: Runx3 expression in Runx3⫹/⫺ (A–C) and wildtype (D and E) mice was detected by ␤-galactosidase staining of wholemount preparations from E15.5 embryos. The dorsal (B and D) and the
ventral (sublingual; C and E) surfaces of the tongues are shown. F–N:
Runx3 expression in the Runx3⫹/⫺ mouse oral region at E12.5 (F–H),
E13.5 (I–K), and E14.5 (L–N) was revealed by immunodetection of ␤-galactosidase in sagittal sections of embryos. P, palate; T, tongue; E,
epithelium; M, mesenchyme. Scale bars ⫽ 1 mm (A–E); 500 ␮m (F, I, and
L); 200 ␮m (G, J, and M); 100 ␮m (H, K, and N).
similar to that seen in the E12.5 embryos. In contrast,
Runx3 expression was observed in both the dorsal and the
ventral mucosal epithelial layers of the tongue and in the
palate in E14.5 embryos (Fig. 2L–N).
We confirmed the Runx3 mRNA expression in the oral
mucosa by in situ hybridization (Figs. 3 and 4). The expression pattern in the tongue and palate epithelium at
E13.5 and E14.5 was similar to that observed in the immunohistochemical analysis of ␤-galactosidase activity
(Fig. 3A–D). Runx3 was expressed in the mesenchyme as
well as in the epithelium of tongue as detected by increased ␤-galactosidase activity (Figs. 2L–M and 3C and
D). Additionally, a fall-off in expression was observed at
E16.5 as shown by the RT-PCR results in Figure 1A (Fig.
3G and H). In frontal sections of E13.5 and E15.5 embryos,
we also observed Runx3 expression in the tongue and
palate epithelium (Fig. 4) and intense expression in the
eyelid mesenchyme, Meckel’s cartilage, and the mandibular chondrocytes (data not shown; Yamashiro et al., 2002).
Runx3 expression was also observed in the nasal epithelium at E15.5 (data not shown).
We observed that Runx3 is expressed in mouse tongue
and palate epithelium from E12.5 to E16.5, suggesting
that it may play a role downstream of TGF-␤/BMP signaling in these stages of oral epithelial development. In fact,
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YAMAMOTO ET AL.
epithelium, which have been reported to express Tgf-␤3,
Bmp-2 and -4, and Alks (Zhang et al., 2002; Dudas et al.,
2004a, 2004b; Liu et al., 2005). This is the first comprehensive report to demonstrate Runx3 expression during
mouse tongue development and palatogenesis.
The gastric mucosa of Runx3-null mice exhibit hyperplasia due to the continued proliferation and low apoptosis
of epithelial cells (Fukamachi and Ito, 2004), and the loss
of Runx3 alters gastric differentiation (Fukamachi et al.,
2004). Therefore, we analyzed epithelial proliferation and
apoptosis in the oral region of these mutants. However,
there was no significant difference between Runx3⫺/⫺ and
wild-type mice in cell proliferation or apoptosis with no
morphological difference (data not shown). All RUNX family genes are important targets of TGF-␤ superfamily signaling (Ito and Miyazono, 2003). Runx1 shows similar
expression to TGF-␤3 in contact epithelium during palatal
fusion (Yamashiro et al., 2002). Therefore, it will be crucial to study the involvement of the other Runx genes with
or without Runx3 in tongue and palate development. In
addition, the differentiation of Runx3⫺/⫺ oral epithelium
using established markers remains to be studied.
ACKNOWLEDGMENTS
The authors thank T. Komori and C.A. Yoshida for their
advice on in situ hybridization techniques.
LITERATURE CITED
Fig. 3. Runx3 expression in the developing oral region from E13.5 to
E16.5 revealed by in situ hybridization. Runx3 expression was observed
in the tongue and palate epithelium (arrowhead). Sagittal sections of
embryos were stained antisense (AS) or sense (S) probes of Runx3.
Scale bars ⫽ 200 ␮m (A–D); 500 ␮m (E–H).
Fig. 4. Runx3 expression in tongue and palate development at E13.5
and E15.5 revealed by in situ hybridization. Frontal sections of embryos
were stained with antisense (AS) or sense (S) probes of Runx3. PS,
palatal shelf. Scale bars ⫽ 500 ␮m.
it was found that the location of Runx3 expression coincided with that of Bmp-4 and -7 expression in chick (R.
Huang et al., 2001) and occurred in the same place and
with the same timing as Bmp-2 and -4 in mouse (Jung et
al., 1999; Kim et al., 2003). Likewise, Runx3 expression
was observed in mouse embryonic palate and palatal shelf
Bae SC, Takahashi E, Zhang YW, Ogawa E, Shigesada K, Namba Y,
Satake M, Ito Y. 1995. Cloning, mapping and expression of PEBP2
alpha C, a third gene encoding the mammalian Runt domain. Gene
159:245–248.
Chi XZ, Yang JO, Lee KY, Ito K, Sakakura C, Li QL, Kim HR, Cha EJ,
Lee YH, Kaneda A, Ushijima T, Kim WJ, Ito Y, Bae SC. 2005.
RUNX3 suppresses gastric epithelial cell growth by inducing p21
(WAF1/Cip1) expression in cooperation with transforming growth
factor beta-activated SMAD. Mol Cell Biol 25:8097– 8107.
Dudas M, Nagy A, Laping NJ, Moustakas A, Kaartinen V. 2004a.
Tgf-beta3-induced palatal fusion is mediated by Alk-5/Smad pathway. Dev Biol 266:96 –108.
Dudas M, Sridurongrit S, Nagy A, Okazaki K, Kaartinen V. 2004b.
Craniofacial defects in mice lacking BMP type I receptor Alk2 in
neural crest cells. Mech Dev 121:173–182.
Enomoto H, Enomoto-Iwamoto M, Iwamoto M, Nomura S, Himeno M,
Kitamura Y, Kishimoto T, Komori T. 2000. Cbfa1 is a positive
regulatory factor in chondrocyte maturation. J Biol Chem 275:
8695– 8702.
Fitzpatrick DR, Denhez F, Kondaiah P, Akhurst RJ. 1990. Differential expression of TGF beta isoforms in murine palatogenesis. Development 109:585–595.
Francis-West P, Ladher R, Barlow A, Graveson A. 1998. Signalling
interactions during facial development. Mech Dev 75:3–28.
Fukamachi H, Ito K. 2004. Growth regulation of gastric epithelial
cells by Runx3. Oncogene 23:4330 – 4335.
Fukamachi H, Ito K, Ito Y. 2004. Runx3⫺/⫺ gastric epithelial cells
differentiate into intestinal type cells. Biochem Biophys Res Commun 321:58 – 64.
Hanai JI, Chen LF, Kanno T, Ohtani-Fujita N, Kim WY, Guo WH,
Imamura T, Ishidou Y, Fukuchi M, Shi MJ, Stavnezer J, Kawabata
M, Miyazono K, Ito Y. 1999. Interaction and functional cooperation
of PEBP2/CBF with Smads: synergistic induction of the immunoglobulin germline C␣ promoter. J Biol Chem 274:31577–31582.
Huang D, Chen SW, Langston AW, Gudas LJ. 1998. A conserved
retinoic acid responsive element in the murine Hoxb-1 gene is
required for expression in the developing gut. Development 125:
3235–3246.
Huang G, Shigesada K, Ito K, Wee HJ, Yokomizo T, Ito Y. 2001.
Dimerization with PEBP2␤ protects RUNX1/AML1 from ubiquitinproteasome-mediated degradation. EMBO J 20:723–733.
Runx3 EXPRESSION
Huang R, Lang ER, Otto WR, Christ B, Patel K. 2001. Molecular and
cellular analysis of embryonic avian tongue development. Anat
Embryol (Berl) 204:179 –187.
Inada M, Yasui T, Nomura S, Miyake S, Deguchi K, Himeno M, Sato
M, Yamagiwa H, Kimura T, Yasui N, Ochi T, Endo N, Kitamura Y,
Kishimoto T, Komori T. 1999. Maturational disturbance of chondrocytes in Cbfa1-deficient mice. Dev Dyn 214:279 –290.
Inoue K, Ozaki S, Shiga T, Ito K, Masuda T, Okado N, Iseda T,
Kawaguchi S, Ogawa M, Bae SC, Yamashita N, Itohara S, Kudo N,
Ito Y. 2002. Runx3 controls the axonal projection of proprioceptive
dorsal root ganglion neurons. Nat Neurosci 5:946 –954.
Ito Y, Miyazono K. 2003. RUNX transcription factors as key targets of
TGF-␤ superfamily signaling. Curr Opin Genet Dev 13:43– 47.
Ito Y. 2004. Oncogenic potential of the RUNX gene family: “overview.”
Oncogene 23:4198 – 4208.
Ito K, Liu Q, Salto-Tellez M, Yano T, Tada K, Ida H, Huang C, Shah
N, Inoue M, Rajnakova A, Hiong KC, Peh BK, Han HC, Ito T, Teh
M, Yeoh KG, Ito Y. 2005. RUNX3, a novel tumor suppressor, is
highly inactivated in gastric cancer by protein mislocalization. Cancer Res 65:7743–7750.
Jung HS, Oropeza V, Thesleff I. 1999. Shh, Bmp-2, Bmp-4 and Fgf-8
are associated with initiation and patterning of mouse tongue papillae. Mech Dev 81:179 –182.
Kaartinen V, Voncken JW, Schuler C, Warburton D, Bu D, Heisterkamp N, Groffen J. 1995. Abnormal lung development and cleft
palate in mice lacking TGF␤3 indicates defect of epithelial-mesenchymal interaction. Nat Genet 11:415– 421.
Kim JY, Mochizuki T, Akita K, Jung HS. 2003. Morphological evidence of the importance of epithelial tissue during mouse tongue
development. Exp Cell Res 290:217–226.
Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K,
Shimizu Y, Bronson RT, Gao Y, Inada M, Sato M, Okamoto R,
Kitamura Y, Yoshiki S, Kishimoto T. 1997. Targeted disruption of
Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764.
Levanon D, Bettoun D, Harris-Cerruti C, Woolf E, Negreanu V, Eilam
R, Bernstein Y, Goldenberg D, Xiao C, Fliegauf M, Kremer E, Otto
F, Brenner O, Lev-Tov A, Groner Y. 2002. The Runx3 transcription
factor regulates development and survival of TrkC dorsal root ganglia neurons. EMBO J 21:3454 –3463.
Li QL, Ito K, Sakakura C, Fukamachi H, Inoue K, Chi XZ, Lee KY,
Nomura S, Lee CW, Han SB, Kim HM, Kim WJ, Yamamoto H,
Yamashita N, Yano T, Ikeda T, Itohara S, Inazawa J, Abe T,
Hagiwara A, Yamagishi H, Ooe A, Kaneda A, Sugimura T, Ushijima
T, Bae SC, Ito Y. 2002. Causal relationship between the loss of
RUNX3 expression and gastric cancer. Cell 109:113–124.
Liu W, Sun X, Braut A, Mishina Y, Behringer RR, Mina M, Martin JF.
2005. Distinct functions for Bmp signaling in lip and palate fusion
in mice. Development 132:1453–1461.
Look AT. 1997. Oncogenic transcription factors in the human acute
leukemias. Science 278:1059 –1064.
Mundlos S, Otto F, Mundlos C, Mulliken JB, Aylsworth AS, Albright
S, Lidhout D, Cole WG, Henn W, Knoll JH, Owen MJ, Mertelsmann
R, Zabel BU, Olsen BR. 1997. Mutations involving the transcription
factor CBFA1 cause cleidocranial dysplasia. Cell 89:773–779.
699
Nam S, Jin YH, Li QL, Lee KY, Jeong GB, Ito Y, Lee J, Bae SC. 2001.
Expression pattern, regulation, and biological role of Runt domain
transcription factor, run, in Caenorhabditis elegans. Mol Cell Biol
22:547–554.
Okuda T, van Deursen J, Hiebert SW, Grosweld G, Downing JR. 1996.
AML1, the target of multiple chromosomal translocation in human
leukemia, is essential for normal fetal liver hematopoiesis. Cell
84:321–330.
Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR,
Stamp GWH, Beddington RSP, Mundlos S, Olsen BR, Selby PB,
Owen MJ. 1997. Cbfa1, a candidate gene for cleidocranial dysplasia
syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765–771.
Paulson RB, Hayes TG, Sucheston ME. 1985. Scanning electron microscope study of tongue development in the CD-1 mouse fetus. J
Craniofac Genet Dev Biol 5:59 –73.
Proetzel G, Pawlowski SA, Wiles MV, Yin M, Boivin GP, Howles PN,
Ding J, Ferguson MW, Doetschman T. 1995. Transforming growth
factor-beta 3 is required for secondary palate fusion. Nat Genet
11:409 – 414.
Satokata I, Maas R. 1994. Msx1 deficient mice exhibit cleft palate and
abnormalities of craniofacial and tooth development. Nat Genet
6:348 –356.
Stanier P, Moore GE. 2004. Genetic of cleft lip and palate: syndromic
genes contribute to the incidence of non-syndromic clefts. Hum Mol
Genet 13:R73–R81.
Taniuchi I, Osato M, Egawa T, Sunshine MJ, Bae SC, Komori T, Ito
Y, Littman DR. 2002. Differential requirements for Runx proteins
in CD4 repression and epigenetic silencing during T lymphocyte
development. Cell 111:621– 633.
van den Boogaard MJ, Dorland M, Beemer FA, van Amstel HK. 2000.
MSX1 mutation is associated with orofacial clefting and tooth agenesis in humans. Nat Genet 24:342–343.
Woolf E, Xiao C, Fainaru O, Lotem J, Rosen D, Negreanu V, Bernstein
Y, Goldenberg D, Brenner O, Berke G, Levanon D, Groner Y. 2003.
Runx3 and Runx1 are required for CD8 T cell development during
thymopoiesis. Proc Natl Acad Sci USA 100:7731–7736.
Yamashiro T, Aberg T, Levanon D, Groner Y, Thesleff I. 2002. Expression of Runx1, -2 and -3 during tooth, palate and craniofacial
bone development. Mech Dev 119S:S107–S110.
Yoshida CA, Furuichi T, Fujita T, Fukuyama R, Kanatani N, Kobayashi S, Satake M, Takada K, Komori T. 2002. Core-binding factor
␤ interacts with Runx2 and is required for skeletal development.
Nat Genet 32:633– 638.
Yoshida CA, Yamamoto H, Fujita T, Furuichi T, Ito K, Inoue K,
Yamana K, Zanma A, Takada K, Ito Y, Komori T. 2004. Runx2 and
Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog. Genes Dev
18:952–963.
Zhang Z, Song Y, Zhao X, Zhang X, Fermin C, 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.