Human Molecular Genetics, 2002, Vol. 11, No. 22 2793-2804
© 2002 Oxford University Press
Craniofacial expression of human and murine TBX22 correlates with the cleft palate and ankyloglossia phenotype observed in CPX patients
1Institute of Reproductive and Developmental Biology, Imperial College Faculty of Medicine–Hammersmith Campus, Du Cane Road, London W12 ONN, UK, 2Institute of Human Genetics, University of Newcastle, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK, 3Medical Genetics Unit, St George's Hospital Medical School, London SW17 0RE, UK and 4INSERM U491, Faculté de Médecine La Timone, Marseille, France
Received July 5, 2002; Accepted August 8, 2002
DDBJ/EMBL/GenBank accession no. AF516208
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cleft palate with ankyloglossia (CPX; MIM 303400) is inherited as a Mendelian, semidominant X-linked disorder and has been described in several large families from different ethnic origins. It is a useful genetic model for non-syndromic cleft palate, a common congenital disorder. Recently, the underlying genetic defect in CPX was identified, where unique mutations were found in the T-box-containing transcription factor TBX22. Here we report two new familial cases with novel missense and insertion mutations, each occurring within the T-box domain and highlighting the functional significance of this DNA-binding motif. We describe TBX22 expression in early human development, where expression is found in the palatal shelves and is highest prior to elevation to a horizontal position above the tongue. mRNA is also detected in the base of the tongue in the region of the frenulum that corresponds to the ankyloglossia seen in CPX patients. Other sites of expression include the inferior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop, and the tooth buds themselves. We have also identified the orthologous mouse Tbx22 gene and performed expression analysis in E12.5–E17.5 mouse embryos. The location of mRNA expression closely correlates between mouse and human, while at later stages of development, we also detected expression in mouse lung and whisker follicles. We conclude that expression of TBX22 is entirely consistent with the CPX phenotype and that the mouse should provide a useful model for elucidating its role in craniofacial development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Orofacial clefts are common congenital birth defects in humans, with an incidence of
1/800 live births. The embryology of cleft lip (CL) and cleft palate (CP) are largely distinct, with the upper lip and jaw forming between the 3rd and 5th week, and the palate between the 5th and 12th weeks of human development. The secondary palate arises from the maxillary processes that develop paired swellings, initially called lateral palatal processes. These processes grow downwards, as the lateral palatal shelves, on either side of the tongue before becoming elevated to a horizontal position above the tongue. The medial edge epithelia (MEE) of the two opposing palatal shelves then fuse to form the midline seam, which subsequently degenerates through a combination of epithelial–mesenchymal transformation (EMT), cell migration and apoptosis to create an intact palate separating the oral and nasal cavities (1). The palatal shelves also fuse with the nasal septum dorsally and the primary palate anteriorly. In the human, primary palate development occurs mainly in the 5th and 6th weeks of development, while secondary palate formation begins in the 6th week of development. This continues through to palatal shelf fusion, which occurs in the 8th and 9th weeks of development, and then on to ossification and development of associated structures (uvula, musculature, glands, etc.) at later stages of fetal development. Disruptions to the complex processes involved in palate development, such as growth, elevation or fusion of the palatal shelves, can result in clefting defects.
In the mouse, a variety of loss of function mutations that result in isolated cleft lip and or cleft palate have been described (2). The situation, however, is not as clear in humans. Whilst an inherited component for clefts was recognized by the studies of Fogh-Anderson (3), the vast majority of cases occur sporadically and display a multifactorial mode of inheritance (4). It is this complex interaction between genes and the environment that has hampered efforts to identify the underlying genetic predisposition to clefts. Numerous candidate genes have been studied, but few mutations have been detected in patients. Genetic causes of CL and/or CP have been uncovered when associated with syndromes such as deletions of 22q11 (DiGeorge syndrome), ectodermal dysplasia (5,6) or mutations in various collagen genes causing skeletal dysplasias (7–11). However, the genetic basis of isolated clefts remain poorly understood, with the recent exceptions of mutation in the cell adhesion molecule PVRL1 causing CL/P in a restricted population in Venezuela (12), and mutations in MSX1, which were identified in a small family with both clefts and tooth agenesis (13) as well as in 1–2% of patients with non-syndromic CL and/or CP (14).
The genetic basis for non-syndromic cleft palate has proved elusive, and one approach has been to study rare familial cases with X-linked inheritance (15). Linkage to Xq21 was obtained in a number of families (16–19), and causative mutations were recently identified in the gene encoding the T-box transcription factor TBX22 (20). X-linked cleft palate (CPX) is characterized by cleft of the secondary palate and ankyloglossia (tongue-tie). Phenotypic variation is seen within families, including bifid uvula, high arched palate and occasionally males with ankyloglossia only. Female carriers range from the full phenotype (CP+ankyloglossia) to completely asymptomatic, with 70–80% exhibiting ankyloglossia only (15,17). All of the carrier females and affected males in the different families carry mutations that are predicted to cause a loss of function for the transcription factor, either by premature introduction of a stop codon or by interfering with DNA binding to target sequences (20).
T-box genes are characterized by an 180-amino-acid DNA-binding domain homologous to that originally identified in the murine Brachyury (T) gene product. Many T-box genes have been identified throughout the animal kingdom, with at least 13 present in humans. T-box genes play important roles in the regulation of various aspects of embryonic development, in particular cell type specification and regulation of morphogenetic movements (21). Levels of T-box gene expression are critical to these developmental processes, as demonstrated by haploinsufficiency for several of the genes causing developmental abnormalities. Haploinsufficiency for TBX3 results in a pleiotropic disorder affecting limb, apocrine gland, tooth and genital development (22), and haploinsufficiency for TBX5 is associated with Holt–Oram syndrome affecting the heart and upper limbs (23,24). A null mutation in murine Tbx1 results in a range of phenotypic effects encompassing most of the common DiGeorge/velocardiofacial (DGS/VCFS) syndrome malformations, including cardiac outflow tract abnormalities, hypoplasia of the thymus and parathyroid glands, cleft palate, and facial dysmorphogenesis (25,26). The range of TBX22 mutations found in CPX patients (nonsense, frameshift, missense and splice site), indicate that the phenotype in affected males is the result of complete loss of TBX22 protein function, and in females a consequence of haploinsufficiency.
Here we report a further two novel mutations, both affecting the T-box domain and identified in familial cases of cleft palate and ankyloglossia where X-linkage had not previously been noted. Previously, we used RT–PCR to show that TBX22 is transcribed in a range of human fetal tissues at the appropriate developmental stage for palate formation (20). To better understand the role of TBX22 in palatogenesis, we have now studied the temporal and spatial distribution in more detail. Using in situ hybridization, both human and mouse embryos show a similar expression pattern consistent with the CPX phenotype, involving the palatal shelves, nasal septum and tongue during the period in which the secondary palate forms.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TBX22 mutation screening in familial cases with cleft palate and ankyloglossia
Six different mutations have previously been identified in familial cases of cleft palate and ankyloglossia (20). Here we investigate two new families that present with CPX-like phenotypes for TBX22 mutations. For each family, DNA samples were obtained from the proband and the mother and screened for TBX22 mutations by individually sequencing all eight exons as described in Braybrook et al. (20). In family 1, a single-base T>C transition was detected at nucleotide 641 (641T>C) with respect to GenBank sequence AY035371, within exon 5 (Fig. 1A). This results in a leucine-to-proline amino acid change at position 214 in the protein (L214P). The location of this missense mutation is at a highly conserved residue within the T-box gene family and throughout evolution. In family 2, a 3 bp insertion was detected within exon 4 between nucleotides 582 and 586: CAGCT>CAGCAGCT (Fig. 1B). At the amino acid level, this insertion results in addition of a serine residue (S195–F196 ins S), adjacent to highly conserved residues that form one of seven ß-barrel structures in the T domain (27). The absence of each mutation was confirmed by DNA sequencing in 100 normal chromosomes from unaffected and unrelated individuals of white European origins.
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Expression analysis of TBX22 during early human development
For the following section, we recommend reference to the website http://www.med.unc.edu/embryo_images/unit-hednk/hednk_htms/hednk033.htm developed by Drs Kathleen K. Sulik and Peter R. Bream Jr for a guided tour through normal palatogenesis. In the human, TBX22 expression is first detected at CS16 [
37 days post ovulation (dpo)], where there is a weak signal in the first pharyngeal arch (data not shown). By CS17 (41 dpo), expression is strong in the mesenchyme of the lateral and medial nasal processes, the lateral palatal processes and at the base of the tongue (Fig. 2A and B). There is also expression more generally in the mesenchyme of the developing face, including a band of signal underlying the base of the brain (arrowhead in Fig. 2B). The expression in the developing nose and primary palate (lateral and medial nasal processes; Fig. 2J) is also seen at CS19 (48 dpo), as is expression at the base of the tongue (arrow in Fig. 2F) and in the developing facial mesenchyme (Fig. 2F, H and J). At CS19, strong expression is detected in the oronasal membrane, which will rupture around this time to form the anterior connection between the nasal and pharyngeal cavities (Fig. 2D). Clear expression is still detected at CS19 in the developing lateral palatal processes, which have not as yet begun to grow downwards as lateral palatal shelves (Fig. 2F and H). Vertically directed palatal shelves are seen at CS20 and CS21, where there is strong expression of TBX22 (Fig. 2L and 3B). Expression in the palatal shelves appears stronger medially than laterally (Figs 2L and 3B), an observation that is supported by the expression pattern in the developing mouse (see below).
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By CS23, the palatal shelves have elevated to a horizontal position but have not yet fused (Fig. 3C). TBX22 is now barely detectable in the palatal shelves (Fig. 3D). TBX22 expression is still observed at the base of the tongue and also at the base of the developing nasal septum (Fig. 3D). There is also expression in the mesenchyme surrounding the developing nostrils and in the mesenchyme surrounding the developing eye, but it appears more restricted than at CS17 and CS19. In the 9-week fetus, at different anterior to posterior positions, the palatal shelves have made contact and are at different stages of fusion. TBX22 is not detectable at any level in the palatal shelves (Fig. 3H and data not shown). Expression is still detectable in the mesenchyme surrounding the developing nostrils and in the nasal cartilage that has now formed (Fig. 3F) but is no longer in the base of the nasal septum (Fig. 3H). There is still expression in the tongue, although it has become more diffuse (Fig. 3H). Strong expression is also seen in the odontogenic mesenchyme [Fig. 3F; first seen at CS23 (data not shown)] and in the developing tooth buds. At each stage, no signal was detected when sense TBX22 probes were hybridized to adjacent sections (data not shown).
Identification and characterization of the mouse orthologue of TBX22
Mouse Tbx22 sequences were first identified by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)-searching the mouse BAC end sequence library (http://www.tigr.org/tdb/bac_ends/mouse/bac_end_intro.html). This identified two adjacent clones (RP23-290M17 and RP23-275H10) that contained exons 5 and 6 respectively. Analysis of the BAC fingerprint database (http://www.bcgsc.bc.ca/projects/mouse_mapping/) identified further clones, including RP23-166N12, that appeared to span most of the two clones described above. This formed part of a contig containing a known X-chromosome marker, suggesting that this was likely to be the orthologous sequence for TBX22. We generated the genomic sequence of RP23-166N12 via the Sanger Institute as part of the MRC mouse sequencing initiative (http://www.hgmp.mrc.ac.uk/). The genomic structure of mouse Tbx22 was determined by comparison of human and mouse cDNA sequences with the overlapping mouse genomic sequence from the BAC clones RP23-18K13 (GenBank accession no. AL669851) and RP23-166N12 (AL663048). Alignment of mouse sequences in dbEST with the genomic sequence indicates that two alternative polyadenylation signals are used. The longer of the 3'-UTR regions extends for 3070 bp from the TAA stop codon. The human TBX22 3'-UTR extends for 642 bp from the TAG stop codon, but with no evidence of the use of an alternative poly adenylation signal. Comparison of the mouse (AF516208) and human (AY035371) cDNA sequences across the coding regions shows an 82% identity at the nucleotide level and 72% identity/80% similarity at the amino acid level (Fig. 4).
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At the 5' end of the Tbx22 cDNA, three EST sequences extend past the putative start codon. The longest of these, EST BB614519, is derived from adult male testis mRNA. The 5' sequence of this clone represents a novel exon mapping
8.5 kb upstream of the location of human exon 1. A full-length sequence containing this upstream exon and the longer 3'-UTR has been submitted to GenBank with the accession number AF516208. There is evidence for use of an alternative 5' exon from the EST D23002M15 (BB657920). This cDNA sequence utilizes a different 5' exon, only 307 bp upstream from the putative human exon 1. Genomic sequence analysis, EST database screening, RT–PCR and 5'-RACE using human tongue and palate mRNA (data not shown) indicate that while the GT 5' intronic splice site sequence for exon 1b is conserved, there is no evidence of the use of either exon 1a or 1b in human TBX22 transcripts. Comparing human and mouse genomic sequence by percentage identify plot (PIP) analysis (Fig. 5), there is no significant homology to the alternative mouse exon 1a, while mouse exon 1b retains 77% identity over 200 bp. The genomic sequence of the region corresponding to exon 1b is predicted to be a putative promoter region using neural network promoter prediction software (http://searchlauncher.bcm.tmc.edu/) in both human and mouse sequences. Within this region of homology, the human sequence includes a tandem (AC)19 and an imperfect (GTTTT)8 repeat both of which are absent in the mouse. There are several other conserved regions of sequence >100 bp and displaying >60% homology upstream of human exon 1. These do not have any homology to dbEST sequences by BLAST analysis, but could represent additional alternate exons or regulatory elements, and will be the subject of further investigation.
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Expression analysis of Tbx22 in the developing mouse embryo
To evaluate the temporal expression profile for Tbx22 before, during and after palatogenesis, total RNA was extracted from whole embryos from embryonic day (E)7.5 to E16.5. Using 35 cycles of RT–PCR, low levels of expression were detected at E9.5, E10.5, E12.5, E15.5 and E16.5, including the period of palatogenesis (data not shown). Using the same cycling conditions, no Tbx22 expression could be detected at or before E8.5. Although quantitative RT–PCR was not undertaken, Tbx22 transcripts could not be detected using <35 cycles of PCR following gel electrophoresis and ethidium bromide staining of PCR products, reflecting the relative low abundance of Tbx22 transcripts in mouse whole-embryo RNA. To perform a more detailed spatial analysis, in situ hybridization of E12.5–E17.5 craniofacial sections was used. Expression was first noted in the swelling palatal shelves at E12.5 (Fig. 6A). By E13.5, there is marked expression in the palatal mesenchyme (Fig. 6B). This appears as a gradient, with the strongest expression in the cells immediately subjacent to the MEE, particularly in the region where Tgfb3 staining is most intense (Fig. 6C). Thus, as in the human, expression in the palatal shelves appears stronger medially than laterally (Fig. 6B).
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There is some Tbx22 staining in epithelial cells, but this is not restricted to the palatal shelves and includes the cells lining the entire oral cavity. Another site of expression is the anterior region of the tongue, which arises from neural crest cells originating from the first branchial arch. In particular, expression is observed in the mesenchyme of the ventral region of the tongue (the ‘root’), which attaches the inferior surface to the floor of the mouth and corresponds to the frenulum (Fig. 6D). At E14.5, immediately prior to the palatal shelves elevating to a horizontal position above the tongue, expression in the palatal shelves has decreased (Fig. 6D). Expression in the base of the tongue is still strong, and expression in the odontogenic mesenchyme is evident. At E15.5, expression is still evident at the base of the nasal septum (Fig. 6E), and is elevated around the site of palatal shelf fusion and the disrupted epithelial seam (Fig. 6F). There is also more intense expression in the oral epithelial cells. This palatal expression disappears by E17.5, although, similar to the human, expression is now detectable in the developing nasal cartilage, the base of the nasal septum, and in glandular structures within the nose and facial structures (Fig. 6G). Expression is also observed in the odontogenic mesenchyme and the developing tooth buds (Fig. 6H and I), in the epithelial tubules of the lung and in the developing whisker follicles (Figs. 6J and K, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TBX22 is a major genetic determinant for familial cleft palate, particularly where ankyloglossia is also present. Previously, we have identified six different mutations that include nonsense, splice site, frameshift and missense alterations (20). Of these, five were present in extensive families in which linkage and/or haplotype analysis had been used to demonstrate that the cleft palate and ankyloglossia phenotype was X-linked and mapped to the approximate interval of CPX in Xq21 (16–19,28,29). A further mutation was also detected in a small family screened on the basis of a CPX-like phenotype alone (20). In this study, we have identified two additional mutations in familial cases who have the classic CPX phenotype, but where the pedigrees were insufficient to confirm X-linked inheritance. The mutation in family 1 is the third missense mutation to be described, occurring at highly conserved positions in the T-box domain. In this family, the mutation results in the substitution of a proline residue for a leucine (L214P) located within the e'f loop that forms contact with the major groove of the target DNA backbone (27). The other mutation in family 2 is a novel insertion that does not alter the reading frame but adds an extra serine residue, presumably altering the conformation of the ß-barrel structure and abolishing appropriate contact to the target DNA sequence. The TBX22 missense mutation in family 1, along with the seven previously described for TBX22, TBX3 and TBX5, highlight the importance of conserved residues within the DNA-binding domain either for protein folding or for direct contact with the DNA target sequence (20,22–24). It is likely that the mutations described here result in complete abolition of TBX22 function, since similar phenotypes are observed in families with mutations that result in premature introduction of a termination codon (20). It is notable, however, that a range of phenotypic variation is observed within individual CPX families, and this is likely to be a consequence of environmental and/or genotypic background, including variable non-random X inactivation, rather than differences between individual mutations.
In order to determine whether the expression of TBX22 correlates with the observed phenotype in CPX patients, we conducted an extensive analysis across the period of palatogenesis in human. Previously, we have demonstrated that TBX22 is expressed during the developmental period in which the palate forms, using RT–PCR (20). In this study, in situ hybridization was used to look at the spatial and temporal expression pattern of TBX22 during craniofacial development in the human. Expression was detected at CS16, around the time that the lateral palatal processes are beginning to form as swellings from the maxillary processes. By CS17, the lateral palatal processes are clearly identifiable and there is strong TBX22 expression. By CS20, the lateral palatal processes have extended downwards to become the vertically directed palatal shelves. Expression is most intense medially in the mesenchyme of the shelves, just subjacent to the epithelial cells, but declines from the time of shelf elevation, suggesting a role in cell proliferation and elongation of the shelves. No expression is detectable in the palatal shelves at 9 weeks of development at the stage when contact has been made and fusion is occurring. Strong expression of TBX22 is also observed in the developing primary palate (mesenchyme of the medial nasal process) and the developing nose: the mesenchyme of the lateral and medial nasal processes at CS17 and CS19, and the nasal septum at CS21, CS23 and 9 weeks of development. By this later time, expression is evident in the nasal cartilage. From CS17, there is also expression in facial mesenchyme, which is progressively restricted to mesenchyme surrounding the eye. There is also a high level of mRNA expression in the root of the tongue, corresponding to the frenulum that attaches the tongue to the floor of the mouth. This correlates very well with the CPX phenotype, in which ankyloglossia is one of the most characteristic features. In these patients, the frenulum develops abnormally, extending to the anterior tip of the tongue and severely restricting tongue movement and protrusion.
T-box genes are generally highly conserved through metazoan evolution, and it was therefore surprising that the first report of TBX22 suggested that it was unique to the human (30). However, using an in silico approach, we successfully identified mouse Tbx22 genomic and cDNA sequences, which were confirmed experimentally. Tbx22 expression was studied in the mouse in order to assess the potential validity of a mouse knockout model for the human disorder. This was considered important because of the increasing number of highly conserved genes that differ extensively in their human–mouse expression—and presumably therefore in their role in development (31). For example, Tbx5 is preferentially expressed in the murine and chick left cardiac ventricle (32), while asymmetry has not been noted in humans (24). There are also species differences of expression in other cardiac locations, such as the atrioventricular valves (32). However, expression of Tbx22 in the developing nose and palatal shelves and at the base of the tongue is very similar to that seen in the human from CS19 onwards (Fig. 6A and D) except that expression continues at the base of the nasal septum after fusion with the palatal shelves (Fig. 6E). At E15.5, there is intense expression in the region where contact between the palatal shelves has been made and fusion is occurring. As the epithelial seam has largely broken down this is probably a later stage than we have looked at in the human (Figs 6F and 3G, respectively). There is strong expression in both species in the odontogenic mesenchyme, and then more specifically in the developing tooth buds (Fig. 6H and I). In the mouse, we were able to study expression at later time points and to show that Tbx22 is also expressed in the developing lung epithelium and in the developing whiskers (Fig. 6J and K, respectively).
Whilst in situ data show that TBX22/Tbx22 has a restricted expression pattern during the time of palatogenesis, a much broader, low-level expression profile (20) was detected by RT–PCR. To date, the only consistent findings identified in CPX patients affect the tongue and secondary palate, with no reports of abnormal tooth or eye development. Loss of TBX22 function in other organs or tissues may be compensated for by other T-box proteins with overlapping function. Similar to Tbx22, Tbx1 is expressed in the lung epithelium, in contrast to Tbx2, 3, 4 and 5, which are predominantly expressed within the interacting lung mesenchyme (33). It is also interesting to note that Tbx1 is also expressed in the developing tongue of the mouse; however, in general, there are insufficient data available to compare overlapping expression domains of the various T-box gene family members.
In order to understand the role of TBX22 during palatogenesis, it will be important to identify its downstream target genes. The original T-box gene (T/Brachyury) binds the palindromic target sequence (T[G/C]ACACCTAGGTGTGAAATT) as a dimer (34), and can also bind a half-palindromic sequence (T/C)TTCACACCT) (35,36). All of the T-box proteins tested to date can also bind the Brachyury target sequence (36–38), although Brachyury does not necessarily bind to the target sequences of other T-box proteins (39). The regulatory elements of downstream target genes also contain variations of the Brachyury-binding site sequence, including members of the transforming growth factor ß (TGFB) and epidermal growth factor (EGF) families, paraxial protocadherin (Papc), and Brachyury-induced homeobox (Bix) genes (21). One possible target of Tbx22 is Tgfb3. Gene knockout of Tgfb3 results in a cleft palate phenotype in addition to delayed pulmonary development (40,41). Expression data (42,43) and the observation that exogenous TGFB3 induces palate fusion using a chicken model system, in which the palate is normally cleft (44), also support an important role for this growth factor during palate development. Tgfb3 and its receptor Tbr-II are expressed throughout the oral epithelial cells, but most strongly in the MEE cells of the palatal shelves, which undergo epithelial–mesenchymal transformation (EMT) (45). During lung development, in situ hybridization localized TGFB3 to both airway epithelium and pulmonary vascular smooth muscle cells (46,47). Whilst the expression patterns of Tbx22 and Tgfb3 overlap in certain tissues, there is a clear distinction in the palatal shelves. Here Tgfb3 is expressed very strongly in the MEE cells while Tbx22 expression is found in the oral epithelial cells, with the highest intensity in the mesenchyme immediately underlying the MEE. However, it has been proposed that the process of EMT involved in both palatal and lung development is controlled by interactive signalling between the epithelium and the underlying mesenchyme (48). Another member of the transforming growth factor ß family, TGFB2, is expressed in the mesenchymal component of several tissues, including bone and the secondary palate (49,50). Knockout mice generated for Tgfb2 have multiple developmental defects, including clefting of the secondary palate in 23% of the Tgfb2-null animals (51). Owing to the location of TBX22 expression, it may be that loss of function causes clefting due to a defect in mesoderm proliferation, preventing contact of the palatal shelves. This may be more similar to the phenotype observed in the Msx1 knockout mouse (52), rather than to the lack of MEE fusion as seen in the Tgfb3-null mouse (40,41) and it will be interesting to test whether Msx1 and Tbx22 interact at some level. Previous speculation has involved the role of the tongue as the site of the primary defect in CPX, whereby the ankyloglossia affects the tongue such that it creates a physical barrier for palatine shelf elevation (18,28,52). However, expression in the palatal shelves and nasal septum as well as the frenulum argues against the tongue being the sole cause of the clefting.
Clearly, TBX22 plays an important role in palatogenesis, and mutations express a phenotype in a predictable Mendelian fashion. Analyses in both human and mouse show that the gene is specifically expressed in the craniofacial tissues that are affected in CPX patients. The correlation of human and mouse TBX22/Tbx22 expression suggests that a gene knockout in the mouse will provide a useful model for cleft palate. In addition to elucidating the role of TBX22 in normal and disturbed palatogenesis, it will be important to identify the downstream target genes through which TBX22 is having an effect in order to facilitate the identification of novel risk factors for orofacial clefting.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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CPX families and mutation screening
Two families were investigated for TBX22 mutations on the basis of similarity to the CPX phenotype, which includes the presence of cleft palate in the proband and ankyloglossia (tongue-tie) either in the proband or elsewhere in the family. Both pedigrees were consistent with but too small to be conclusive for X-linkage. Family 1 was of white South African origin, and the proband was a male with cleft palate and ankyloglossia. The mother had ankyloglossia, as did her brother and mother. There was also a report of a further relative on the maternal side who had a cleft-palate-only phenotype. Family 2 is of white Brazilian origin. The proband has a cleft of the secondary palate, but the presence of ankyloglossia has not yet been confirmed. However, ankyloglossia is present in the mother and several distant maternal relatives.
Mutation screening was carried out by direct sequencing of individual exons amplified by PCR from genomic DNA, as described by Braybrook et al. (20). Mutations identified in the probands were then confirmed by sequencing in the reverse orientation and sequenced in the heterozygous mothers. More than 100 control chromosomes from mixed European origins were also examined for the presence of each mutation. Informed consent was obtained from all of the patients or their parents documented in this study.
In situ hybridization to human embryo sections
The collection and use of human embryos was carried out with ethical permission from the Joint Ethics Committee of the Newcastle Health Authority and with appropriate consent. Following either surgical or medically induced termination of pregnancy (53) embryos were staged, fixed in 4% paraformaldehyde in phosphate-buffered saline and then wax-embedded. Sense and antisense probes were synthesized by transcribing linearized plasmids containing a 336 bp fragment (nucleotides 618–954 of GenBank accession no. AY035371) with SP6 or T7 RNA polymerases, respectively, using 35S-UTP as a label. The probes were hybridized to the tissue-section slides at 52°C, and, following stringent washes, the slides were coated in Ilford K5 emulsion and exposed for 10 days following previously described protocols (54).
Identification of the mouse Tbx22 genomic and cDNA sequence
The sequence of the human TBX22 cDNA (AY035371) was used to BLAST-search (55; http://www.ncbi.nlm.nih.gov/BLAST/) the mouse RPCI23 BAC end clone sequences (http://www.tigr.org/tdb/bac_ends/mouse/bac_end_intro.html). The clones that correspond to positive matches were identified in the fingerprint database of the same RPCI23 library (http://www.bcgsc.bc.ca/projects/mouse_mapping/). Relevant clones were chosen for genomic sequencing at the Sanger Centre (Cambridge, UK) as part of the MRC mouse genome sequencing initiative. Alignment of the mouse genomic sequence (AL669851 and AL663048) with the human cDNA (AY035371) and BLAST analysis with mouse ESTs allowed the full-length mouse cDNA sequence (AF516208) to be predicted and the exon/intron boundaries to be determined.
Comparative analysis of human and mouse TBX22/Tbx22
PipMaker compares DNA sequences from different species to identify regions of sequence conservation (56). Genomic sequence generated from overlapping mouse BAC clones RP23-18K13 (AL669851) and RP23-166N12 (AL663048) containing Tbx22 was used as the first sequence and compared with sequence from the PAC clone RP4-795G23 (AL031000) containing human TBX22. The mouse sequence was screened for repeat elements using RepeatMasker (Smit and Green, unpublished data; http://ftp.genome.washington.edu/cgi-bin/ repeatmasker). Genomic sequences and exon locations were then submitted to the PipMaker program (http://nog.cse.psu.edu/pipmaker/). The resulting alignments are summarized as a percentage identity plot (PIP).
RT–PCR for Tbx22
Total RNA was extracted from mouse embryos between E7.5 and E16.5 using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. Reverse transcription was performed using 1–2 µg of total RNA, with 0.2 µg of random hexamers (Life Technologies) and MMLV RT (Life Technologies), following the manufacturer's instructions. PCR was performed on first-strand cDNA as previously described (57) using Tbx22-specific primers mT22e5F2 (5'-GATTGACCTGTCCCTGATT-3') and mT22e6R2 (5'-CTCCCAGGATCTCTAAATCC-3'), which generate a 162 bp PCR product using cDNA as a template. The gene-specific primers were designed to flank an intron so that products from cDNA could be distinguished from possible genomic DNA contamination. PCR was performed for 35 cycles using an annealing temperature of 58°C. RT–PCR control reactions were performed using primers specific for the housekeeping gene Hprt, as previously described (58).
Mouse in situ hybridization
To generate sense and antisense probes for Tbx22, a product of 465 bp spanning exons 4–8 incorporating the T-box domain was amplified from mouse cDNA using primers mT22e4F2 (5'-GCTCTGGAGAAAACTGGATG-3') and e8RTR2 (5'-GTAAGGAGTTCAAAGGAGAGG-3'). RT–PCR products were cloned into the pGEMT-Easy vector (Promega) and propagated in Escherichia coli JM109 cells. Recombinant clones were sequenced to determine orientation of the inserts. Following linearization of the plasmid DNA using either ApaI or SalI, antisense and sense digoxigenin-labelled riboprobes were synthesised with Sp6 or T7 RNA polymerase, respectively, using a DIG RNA labeling kit (Roche). For Tgfb3, a 609 bp cDNA product (HL Moses) was similarly labelled. CD1 strain mouse embryos from E12.5, E13.5, E14.5, E15.5 and E17.5 were fixed for 2 days in 4% paraformaldehyde in phosphate-buffered saline and embedded in paraffin wax. In situ hybridization was performed as previously described (59) using 8 µm sections.
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ACKNOWLEDGEMENTS |
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We are grateful to Antonio Richieri-Costa of Hopital de Reabilitação de Anomalias Craniofaciais, Universidade de São Paulo, Simon Gregory, Mark Ross and the Sanger Institute human and mouse chromosome sequencing teams, also for the services provided by the UK HGMP Resource Centre, to Andy Davies in the IRDB sequencing facility and to the MRC–Wellcome Human Developmental Biology Resource for provision of human developmental material. We wish to thank the Birth Defects Foundation, the Dunhill Medical Trust, the British Heart Foundation and the EU for their support of this work.
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FOOTNOTES |
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* To whom correspondence should be addressed Tel: +;44 2075942124; Fax: +;44 2075942129; Email: pstanier{at}ic.ac.uk
The authors wish it to be known what, in their opinion, the first two authors should be regarded as joint First Authors.
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