Isolation and Developmental Expression Analysis of
Tbx22, the Mouse Homolog of the Human X-Linked Cleft
JEFFREY O. BUSH,1YU LAN,2KATHLEEN M. MALTBY,2
1Department of Biology, University of Rochester, Rochester, New York
2Center for Oral Biology, University of Rochester, Rochester, New York
AND RULANG JIANG1,2*
have been identified recently in patients with the
X-linked cleft palate and ankyloglossia syn-
drome, suggesting that the TBX22 transcription
factor plays an important role in palate develop-
ment. However, because ankyloglossia has been
reported in the majority of patients with TBX22
mutations, it has been speculated that the cleft
palate phenotype is secondary to defective fetal
tongue movement. To understand the role of
TBX22 in disease pathogenesis and in normal de-
velopment, it is necessary to carry out a detailed
temporal and spatial gene expression analysis.
We report here the isolation and developmental
expression analysis of the mouse homolog Tbx22.
The mouse Tbx22 gene encodes a putative pro-
tein of 517 amino acid residues, which shares 72%
overall amino acid sequence identity with the
human TBX22 protein. By using interspecific
backcross analysis, we have localized the Tbx22
gene to mouse chromosome X, in a region syn-
tenic to human chromosome Xq21, where the
TBX22 gene resides, indicating that Tbx22 is the
ortholog of human TBX22. Our in situ hybridiza-
tion analysis shows that Tbx22 is expressed in a
temporally and spatially highly restricted pat-
tern during mouse palate and tongue develop-
ment. Together with the mutant phenotypes in
human patients, our data indicate a primary role
© 2002 Wiley-Liss, Inc.
Mutations in the TBX22 gene
palate and tongue
Key words: T-box; Tbx; Tbx22; palate develop-
tongue tie; tongue development
The mammalian secondary palate develops initially
as two bilateral shelves that grow vertically down the
sides of the developing tongue. At a precise develop-
mental time, 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. Any disturbance of the growth, ele-
vation, and fusion of the palatal shelves causes cleft
palate, which occurs frequently and affects approxi-
mately 1 in 1,500 births in humans. The molecular
mechanisms underlying normal palate development
and cleft palate pathogenesis are not well understood.
Recently, mutations in the TBX22 gene have been
identified in patients with X-linked cleft palate and
ankyloglossia (Braybrook et al., 2001). TBX22 is a new
member of an evolutionarily conserved gene family in
which each gene encodes a transcription factor contain-
ing a conserved DNA binding domain, termed T-box
(Papaioannou and Silver, 1998; Papaioannou, 2001).
Mutations in three other members of the T-box gene
family have been associated with human developmen-
tal disorders, the ulnar-mammary syndrome (TBX3),
Holt-Oram syndrome (TBX5), and isolated adrenocor-
ticotropic hormone deficiency (TBX19) (Bamshad et al.,
1997; Basson et al., 1997; Lamolet et al., 2001). In
addition, the TBX1 gene is deleted in the majority of
the velo-cardio-facial/DiGeorge syndrome patients, and
mice carrying targeted mutations in the Tbx1 gene
show developmental abnormalities resembling defects
in DiGeorge syndrome patients (Chieffo et al., 1997;
Jerome and Papaioannou, 2001; Lindsay et al., 2001;
Merscher et al., 2001). Many DiGeorge syndrome pa-
tients have a cleft palate phenotype, and mice homozy-
gous for a targeted mutation in Tbx1 have cleft palate
(Jerome and Papaioannou, 2001). Interestingly, recent
studies show that Tbx1 is only expressed in the meso-
dermal core and the endoderm of the developing
branchial arches but not in the developing neural crest
derivatives (Garg et al., 2001; Vitelli et al., 2002), sug-
Grant sponsor: NIH; Grant number: DE13681; Grant number: T32
*Correspondence to: Rulang Jiang, Center for Oral Biology, Univer-
sity of Rochester, 601 Elmwood Avenue, Box 611, Rochester, NY
14642. E-mail: firstname.lastname@example.org
Received 26 June 2002; Accepted 5 August 2002
Published online 3 October 2002 in Wiley InterScience (www.
DEVELOPMENTAL DYNAMICS 225:322–326 (2002)
© 2002 WILEY-LISS, INC.
gesting that the cleft palate phenotype of Tbx1 mutant
mice is likely to be a secondary effect. It is not known
whether TBX22 is expressed during palate develop-
ment. Because the majority of the male patients with
TBX22 mutations exhibit both cleft palate and anky-
loglossia, it has been speculated that the cleft palate
phenotype may be secondary to defective fetal tongue
movement (Gorski et al., 1992; Stanier et al., 1993). To
understand the role of TBX22 in disease pathogenesis
and in normal development, it is necessary to carry out
a detailed temporal and spatial expression analysis of
the TBX22 gene. Of the only two reports on the TBX22
gene to date, one failed to detect TBX22 mRNA expres-
sion in any fetal and adult tissue, whereas the other
showed a reverse transcriptase-polymerase chain reac-
tion (RT-PCR) product of expected size in every fetal
tissue examined, which did not include the palatal
tissue (Laugier-Anfossi and Villard, 2000; Braybrook et
al., 2001). To better characterize the developmental
role of TBX22, we have isolated the mouse Tbx22 gene
and analyzed its expression pattern during mouse em-
RESULTS AND DISCUSSION
We isolated the mouse Tbx22 cDNA through a com-
bination of database mining and RT-PCR cloning (see
Experimental Procedures section). The mouse Tbx22
gene encodes a protein of 517 amino acid residues that
shares 72% overall amino acid sequence identity with
the human TBX22 protein (Fig. 1). Because a previous
study failed to detect the existence of a mouse homolog
of TBX22 by using Southern and Northern hybridiza-
tion methods (Laugier-Anfossi and Villard, 2000), we
mapped the mouse Tbx22 gene by using the Jackson
Laboratory BSS interspecific backcross panel to con-
firm orthology. The Tbx22 gene is localized to the cen-
tral part of mouse chromosome X and cosegregates
with the markers DXMit65 and DXMit214. This loca-
sequences. The mouse Tbx22 cDNA sequence has been submitted to
Genbank (accession no. AY125891). The human TBX22 protein se-
quence is from Genbank (Genbank accession no. NM_016954). De-
Comparison of the mouse Tbx22 and human TBX22 proteinduced amino acid sequences of mouse Tbx22 and human TBX22 are
compared. Identical amino acid residues are highlighted. The T-box in the
mouse Tbx22 protein contains amino acid residues 98-280.
EXPRESSION PATTERNS OF Tbx22
tion is in a region of known synteny to human chromo-
some Xq21, where TBX22 resides (Oeltjen et al., 1997;
Phippard et al., 2000; Braybrook et al., 2001). The
chromosomal localization, together with the high de-
gree of sequence identity in and outside of the T-box
region (Fig. 1), indicates that Tbx22 is the mouse or-
tholog of human TBX22.
We analyzed Tbx22 gene expression during mouse
embryonic development by using in situ hybridization
of both whole-mount and paraffin sections. Tbx22
mRNA expression is first detected at embryonic day (E)
9 during mouse embryogenesis in the developing
somites (Fig. 2A,B). At E10.5, in addition to the
somites, Tbx22 mRNA is detected in the frontonasal
processes and mandibular processes of the first
branchial arches (Fig. 2C). In situ hybridization of par-
affin sections of E10.5 and E11.5 embryos shows that
the mandibular Tbx22 expression domain is in the
primordial tissue of the developing tongue (Fig. 2C,D).
Tbx22 expression in embryos older than E11.5 is only
detected in the craniofacial regions by in situ hybrid-
ization of both whole-mount and sections. At E11.5,
Tbx22 is expressed in the developing tongue primordia
as well as in the oral sides of the maxillary and nasal
processes (Fig. 3A,B). By E12.0, the palatal shelves
have emerged from the oral sides of the maxillary
processes and started growing down the sides of the
developing tongue. Tbx22 expression in the maxillary
A: Tbx22 expression is detected at embryonic day (E) 9.25 by whole-
mount in situ hybridization in the nascent somites and the newly formed
somite (the mRNA signal is shown in purple/blue in A and B). B: Tbx22
expression is dynamic during somite development. Tbx22 mRNA expres-
sion is initiated in the nascent somite (white arrow) and is down-regulated
in the newly formed somites (s1, s2, and s3) but is turned back on in more
mature rostral somites (black arrows). C: A sagittal section of an E10.5
embryo hybridized with radiolabeled antisense RNA probes shows strong
Tbx22 expression in the frontonasal processes and mandibular arch
mesenchyme. (The mRNA signal detected by autoradiography is shown
in red in C and D.) D: Tbx22 expression in the mandibular processes is
restricted to the tongue primordial region. fl, forelimb bud; m, mandibular
process; n, nasal process; pm, presomitic mesoderm; s, somite.
Expression patterns of Tbx22 mRNA in early mouse embryos.
velopment. A: At embryonic day (E) 11.5, Tbx22 mRNA signals are
detected in medial nasal and maxillary processes. B: Tbx22 is expressed
in the tongue primordial swellings (arrowheads) and lateral mesenchyme
(arrows) of the mandibular processes at E11.5. C: At E12.5, Tbx22
expression in the maxillary processes is restricted to the palatal shelves
(arrows). Strong Tbx22 expression is also detected at the base of the
developing tongue. D: Frontal section of an E12.5 embryonic head shows
strong Tbx22 expression at the base of the tongue and in the lateral
regions of the mandible. Moderate levels of Tbx22 expression is detected
in the mesenchymal precursor cells of Meckel’s cartilage (arrows).
E: Strong Tbx22 expression persists in the palatal shelves and the base
of the tongue at E13.5. F: High-magnification view of a frontal section of
the palatal and tongue region at E13.5, showing that Tbx22 expression is
restricted to the mesenchyme in these tissues. G,H: Sagittal (G) and
frontal (H) sections of E13.5 embryonic heads, showing strong Tbx22
expression in the frontonasal processes and in the tissues surrounding
the developing eyes, in addition to expression in the palatal shelves and
the base of the tongue. I: At E14.5, the palatal shelves have elevated and
fused to each other at the midline. Tbx22 expression is down-regulated in
the palatal shelves and the tongue/mandible region but persists in the
tissues surrounding the eyes. e, eye; mb, mandible; mn, medial nasal
process; mx, maxillary process; n, nasal cavity; p, palatal shelf; t, tongue;
ul, upper lip.
Expression patterns of Tbx22 mRNA during craniofacial de-
BUSH ET AL.
processes becomes restricted to the palatal shelves
(Fig. 3C). Tbx22 expression in the mandibular pro-
cesses is detected at the base of the developing tongue,
in the mesenchymal precursors of Meckel’s cartilage,
as well as in two subsets of lateral mesenchymal cells
(Fig. 3C,D). High levels of Tbx22 expression persist in
mesenchyme of the palatal shelves and at the base of
the tongue through E13.5 (Fig. 3E–G). Tbx22 expres-
sion is also detected in the mesenchyme surrounding
the developing eyes at E13.5 (Fig. 3H). By E14.5, the
palatal shelves have elevated to a horizontal position
above the dorsum of the tongue and fused with each
other at the midline. At this stage, Tbx22 expression is
turned off in the palatal shelves and is significantly
down-regulated in the mesenchyme at the base of the
tongue, whereas high levels of Tbx22 mRNA persist in
the mesenchyme surrounding the developing eyes (Fig.
3I). The temporally and spatially highly restricted pat-
tern of expression during palate and tongue develop-
ment, together with the cleft palate and ankyloglossia
phenotypes in patients with mutations in the TBX22
gene, indicate a primary role for Tbx22 in both palate
and tongue development.
Isolation of Tbx22
Searching the mouse Expressed Sequence Tags
(EST) database (www.ncbi.nlm.nih.gov) by using the
human TBX22 protein sequence identified four mouse
ESTs with high sequence similarities (Genbank acces-
sion nos. BB606355,BB614519,
BB664980). Comparison of the assembled cDNA se-
quence with the human genomic and mouse genomic
trace sequences shows that the assembled Tbx22 cDNA
sequence is missing the 3? part of the last coding exon
(exon 8). RT-PCR using a 5? primer corresponding to
sequences in exon 5 and a 3? primer corresponding to
sequences downstream of the putative inframe stop
codon amplified a product of approximately 650 bp
from E12.5 mouse head RNA. Sequencing of the RT-
PCR product confirmed the predicted open reading
frame. Sequence analyses were performed by using
MacVector (Oxford Molecular, Ltd.).
A microsatellite sequence containing 24 (GA) repeats
was identified in the genomic sequence of the last exon
of the Tbx22 gene. PCR primers (primer 1, 5?-GACAT-
GCTATCAGTGATAATTGAGG-3? and primer 2, 5?-
(GA) repeat region were tested for polymorphisms be-
tween C57BL/6J and SPRET/Ei mouse strains and
were found to amplify unique products of 213 bp and
approximately 190 bp, respectively, from the two
strains. This microsatellite polymorphism was used to
map the chromosomal location of the Tbx22 gene by
using the Jackson Laboratory BSS interspecific back-
cross panel, which consists of genomic DNA from 94 N2
animals [(C57BL/6Jei ? SPRET/Ei)F1 ? SPRET/Ei].
No recombinants were detected between Tbx22 and the
markers DXMit65 and DXMit214, which places Tbx22
at approximately 49 centimorgans from the centromere
on mouse chromosome X. Raw mapping data for this
gene as well as thousands of markers typed by using
the Jackson Laboratory BSS backcross panel are avail-
able on the World Wide Web (www.jax.org/resources/
In Situ Hybridization
In situ hybridization of whole-mount and sectioned
embryos was carried out as previously described (Jiang
et al., 1998; Lan et al., 2001). Digoxigenin- and33P-
labeled antisense RNA probes were made by using the
subcloned 650-bp cDNA fragment as described above.
We thank Paul Kingsley for critical reading of the
manuscript and discussions. This work was supported
by a NIH grant (DE13681) to R.J. J.O.B. was supported
by a NIH training grant (J32 DE07202).
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