BounTI (boundary-preserving threshold iteration): A user-friendly tool for automatic hard tissue segmentation.

X-ray Computed Tomography (CT) images are widely used in various fields of natural, physical, and biological sciences. 3D reconstruction of the images involves segmentation of the structures of interest. Manual segmentation has been widely used in the field of biological sciences for complex structures composed of several sub-parts and can be a time-consuming process. Many tools have been developed to automate the segmentation process, all with various limitations and advantages, however, multipart segmentation remains a largely manual process. The aim of this study was to develop an open-access and user-friendly tool for the automatic segmentation of calcified tissues, specifically focusing on craniofacial bones. Here we describe BounTI, a novel segmentation algorithm which preserves boundaries between separate segments through iterative thresholding. This study outlines the working principles behind this algorithm, investigates the effect of several input parameters on its outcome, and then tests its versatility on CT images of the craniofacial system from different species (e.g. a snake, a lizard, an amphibian, a mouse and a human skull) with various scan qualities. The case studies demonstrate that this algorithm can be effectively used to segment the craniofacial system of a range of species automatically. High-resolution microCT images resulted in more accurate boundary-preserved segmentation, nonetheless significantly lower-quality clinical images could still be segmented using the proposed algorithm. Methods for manual intervention are included in this tool when the scan quality is insufficient to achieve the desired segmentation results. While the focus here was on the craniofacial system, BounTI can be used to automatically segment any hard tissue. The tool presented here is available as an Avizo/Amira add-on, a stand-alone Windows executable, and a Python library. We believe this accessible and user-friendly segmentation tool can benefit the wider anatomical community.

Multiscale mechanical characterisation of the craniofacial system under external forces.

Premature fusion of craniofacial joints, i.e. sutures, is a major clinical condition. This condition affects children and often requires numerous invasive surgeries to correct. Minimally invasive external loading of the skull has shown some success in achieving therapeutic effects in a mouse model of this condition, promising a new non-invasive treatment approach. However, our fundamental understanding of the level of deformation that such loading has induced across the sutures, leading to the effects observed is severely limited, yet crucial for its scalability. We carried out a series of multiscale characterisations of the loading effects on normal and craniosynostotic mice, in a series of in vivo and ex vivo studies. This involved developing a custom loading setup as well as software for its control and a novel in situ CT strain estimation approach following the principles of digital volume correlation. Our findings highlight that this treatment may disrupt bone formation across the sutures through plastic deformation of the treated suture. The level of permanent deformations observed across the coronal suture after loading corresponded well with the apparent strain that was estimated. This work provides invaluable insight into the level of mechanical forces that may prevent early fusion of cranial joints during the minimally invasive treatment cycle and will help the clinical translation of the treatment approach to humans.

Synchondrosis fusion contributes to the progression of postnatal craniofacial dysmorphology in syndromic craniosynostosis.

Syndromic craniosynostosis (CS) patients exhibit early, bony fusion of calvarial sutures and cranial synchondroses, resulting in craniofacial dysmorphology. In this study, we chronologically evaluated skull morphology change after abnormal fusion of the sutures and synchondroses in mouse models of syndromic CS for further understanding of the disease. We found fusion of the inter-sphenoid synchondrosis (ISS) in Apert syndrome model mice (Fgfr2S252W/+ ) around 3 weeks old as seen in Crouzon syndrome model mice (Fgfr2cC342Y/+ ). We then examined ontogenic trajectories of CS mouse models after 3 weeks of age using geometric morphometrics analyses. Antero-ventral growth of the face was affected in Fgfr2S252W/+ and Fgfr2cC342Y/+ mice, while Saethre-Chotzen syndrome model mice (Twist1+/- ) did not show the ISS fusion and exhibited a similar growth pattern to that of control littermates. Further analysis revealed that the coronal suture synostosis in the CS mouse models induces only the brachycephalic phenotype as a shared morphological feature. Although previous studies suggest that the fusion of the facial sutures during neonatal period is associated with midface hypoplasia, the present study suggests that the progressive postnatal fusion of the cranial synchondrosis also contributes to craniofacial dysmorphology in mouse models of syndromic CS. These morphological trajectories increase our understanding of the progression of syndromic CS skull growth.

Mechanical loading of cranial joints minimizes the craniofacial phenotype in Crouzon syndrome.

Children with syndromic forms of craniosynostosis undergo a plethora of surgical interventions to resolve the clinical features caused by the premature fusion of cranial sutures. While surgical correction is reliable, the need for repeated rounds of invasive treatment puts a heavy burden on the child and their family. This study explores a non-surgical alternative using mechanical loading of the cranial joints to prevent or delay craniofacial phenotypes associated with Crouzon syndrome. We treated Crouzon syndrome mice before the onset of craniosynostosis by cyclical mechanical loading of cranial joints using a custom designed set-up. Cranial loading applied to the frontal bone partially restores normal skull morphology, significantly reducing the typical brachycephalic appearance. This is underpinned by the delayed closure of the coronal suture and of the intersphenoidal synchondrosis. This study provides a novel treatment alternative for syndromic craniosynostosis which has the potential to be an important step towards replacing, reducing or refining the surgical treatment of all craniosynostosis patients.

Familial Absent Uvula With Velopharyngeal Incompetence-A New Syndrome?

We present a family with a previously undescribed abnormality of the palate and oropharynx which involved the absence of the uvula and the anterior pillar of the fauces, rudimentary posterior pillar of the fauces, and hypernasality. Eight family members over 4 generations are affected in a pattern consistent with autosomal dominant inheritance. A causal role for the FOXF2 gene has been identified and previously reported. We describe the management of the proband, which involved attempting to lengthen the palate and to retroposition the abnormally anteriorly directed velar musculature, along with speech therapy.

Mouse Models of Syndromic Craniosynostosis.

Craniosynostosis is a common craniofacial birth defect. This review focusses on the advances that have been achieved through studying the pathogenesis of craniosynostosis using mouse models. Classic methods of gene targeting which generate individual gene knockout models have successfully identified numerous genes required for normal development of the skull bones and sutures. However, the study of syndromic craniosynostosis has largely benefited from the production of knockin models that precisely mimic human mutations. These have allowed the detailed investigation of downstream events at the cellular and molecular level following otherwise unpredictable gain-of-function effects. This has greatly enhanced our understanding of the pathogenesis of this disease and has the potential to translate into improvement of the clinical management of this condition in the future.

Overexpression of Fgfr2c causes craniofacial bone hypoplasia and ameliorates craniosynostosis in the Crouzon mouse.

FGFR2c regulates many aspects of craniofacial and skeletal development. Mutations in the FGFR2 gene are causative of multiple forms of syndromic craniosynostosis, including Crouzon syndrome. Paradoxically, mouse studies have shown that the activation (Fgfr2cC342Y; a mouse model for human Crouzon syndrome), as well as the removal (Fgfr2cnull), of the FGFR2c isoform can drive suture abolishment. This study aims to address the downstream effects of pathogenic FGFR2c signalling by studying the effects of Fgfr2c overexpression. Conditional overexpression of Fgfr2c (R26RFgfr2c;ßact) results in craniofacial hypoplasia as well as microtia and cleft palate. Contrary to Fgfr2cnull and Fgfr2cC342Y, Fgfr2c overexpression is insufficient to drive onset of craniosynostosis. Examination of the MAPK/ERK pathway in the embryonic sutures of Fgfr2cC342Y and R26RFgfr2c;ßact mice reveals that both mutants have increased pERK expression. The contrasting phenotypes between Fgfr2cC342Y and R26RFgfr2c;ßact mice prompted us to assess the impact of the Fgfr2c overexpression allele on the Crouzon mouse (Fgfr2cC342Y), in particular its effects on the coronal suture. Our results demonstrate that Fgfr2c overexpression is sufficient to partially rescue craniosynostosis through increased proliferation and reduced osteogenic activity in E18.5 Fgfr2cC342Y embryos. This study demonstrates the intricate balance of FGF signalling required for correct calvarial bone and suture morphogenesis, and that increasing the expression of the wild-type FGFR2c isoform could be a way to prevent or delay craniosynostosis progression.

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

Predicting calvarial growth in normal and craniosynostotic mice using a computational approach.

During postnatal calvarial growth the brain grows gradually and the overlying bones and sutures accommodate that growth until the later juvenile stages. The whole process is coordinated through a complex series of biological, chemical and perhaps mechanical signals between various elements of the craniofacial system. The aim of this study was to investigate to what extent a computational model can accurately predict the calvarial growth in wild-type (WT) and mutant type (MT) Fgfr2C342Y/+ mice displaying bicoronal suture fusion. A series of morphological studies were carried out to quantify the calvarial growth at P3, P10 and P20 in both mouse types. MicroCT images of a P3 specimen were used to develop a finite element model of skull growth to predict the calvarial shape of WT and MT mice at P10. Sensitivity tests were performed and the results compared with ex vivo P10 data. Although the models were sensitive to the choice of input parameters, they predicted the overall skull growth in the WT and MT mice. The models also captured the difference between the ex vivoWT and MT mice. This modelling approach has the potential to be translated to human skull growth and to enhance our understanding of the different reconstruction methods used to manage clinically the different forms of craniosynostosis, and in the long term possibly reduce the number of re-operations in children displaying this condition and thereby enhance their quality of life.

Sumoylation in Craniofacial Disorders.

Craniofacial development requires a complex series of coordinated and finely tuned events to take place, during a relatively short time frame. These events are set in motion by switching on and off transcriptional cascades that involve the use of numerous signalling pathways and a multitude of factors that act at the site of gene transcription. It is now well known that amidst the subtlety of this process lies the intricate world of protein modification, and the posttranslational addition of the small ubiquitin -like modifier, SUMO, is an example that has been implicated in this process. Many proteins that are required for formation of various structures in the embryonic head and face adapt specific functions with SUMO modification. Interestingly, the main clinical phenotype reported for a disruption of the SUMO1 locus is the common birth defect cleft lip and palate. In this chapter therefore, we discuss the role of SUMO1 in craniofacial development, with emphasis on orofacial clefts. We suggest that these defects can be a sensitive indication of down regulated SUMO modification at a critical stage during embryogenesis. As well as specific mutations affecting the ability of particular proteins to be sumoylated, non-genetic events may have the effect of down-regulating the SUMO pathway to give the same result. Enzymes regulating the SUMO pathway may become important therapeutic targets in the preventative and treatment therapies for craniofacial defects in the future.

Analysis of the Fgfr2C342Y mouse model shows condensation defects due to misregulation of Sox9 expression in prechondrocytic mesenchyme.

Syndromic craniosynostosis caused by mutations in FGFR2 is characterised by developmental pathology in both endochondral and membranous skeletogenesis. Detailed phenotypic characterisation of features in the membranous calvarium, the endochondral cranial base and other structures in the axial and appendicular skeleton has not been performed at embryonic stages. We investigated bone development in the Crouzon mouse model (Fgfr2C342Y) at pre- and post-ossification stages to improve understanding of the underlying pathogenesis. Phenotypic analysis was performed by whole-mount skeletal staining (Alcian Blue/Alizarin Red) and histological staining of sections of CD1 wild-type (WT), Fgfr2C342Y/+ heterozygous (HET) and Fgfr2C342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E)12.5-E17.5 stages. Gene expression (Sox9, Shh, Fgf10 and Runx2) was studied by in situ hybridisation and protein expression (COL2A1) by immunohistochemistry. Our analysis has identified severely decreased osteogenesis in parts of the craniofacial skeleton together with increased chondrogenesis in parts of the endochondral and cartilaginous skeleton in HOM embryos. The Sox9 expression domain in tracheal and basi-cranial chondrocytic precursors at E13.5 in HOM embryos is increased and expanded, correlating with the phenotypic observations which suggest FGFR2 signalling regulates Sox9 expression. Combined with abnormal staining of type II collagen in pre-chondrocytic mesenchyme, this is indicative of a mesenchymal condensation defect. An expanded spectrum of phenotypic features observed in the Fgfr2C342Y/C342Y mouse embryo paves the way towards better understanding the clinical attributes of human Crouzon-Pfeiffer syndrome. FGFR2 mutation results in impaired skeletogenesis; however, our findings suggest that many phenotypic aberrations stem from a primary failure of pre-chondrogenic/osteogenic mesenchymal condensation and link FGFR2 to SOX9, a principal regulator of skeletogenesis.

Gain-of-Function Mutations in ZIC1 Are Associated with Coronal Craniosynostosis and Learning Disability.

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5-12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded.

Mechanical properties of calvarial bones in a mouse model for craniosynostosis.

The mammalian cranial vault largely consists of five flat bones that are joined together along their edges by soft fibrous tissues called sutures. Premature closure of the cranial sutures, craniosynostosis, can lead to serious clinical pathology unless there is surgical intervention. Research into the genetic basis of the disease has led to the development of various animal models that display this condition, e.g. mutant type Fgfr2C342Y/+ mice which display early fusion of the coronal suture (joining the parietal and frontal bones). However, whether the biomechanical properties of the mutant and wild type bones are affected has not been investigated before. Therefore, nanoindentation was used to compare the elastic modulus of cranial bone and sutures in wild type (WT) and Fgfr2C342Y/+mutant type (MT) mice during their postnatal development. Further, the variations in properties with indentation position and plane were assessed. No difference was observed in the elastic modulus of parietal bone between the WT and MT mice at postnatal (P) day 10 and 20. However, the modulus of frontal bone in the MT group was lower than the WT group at both P10 (1.39±0.30 vs. 5.32±0.68 GPa; p<0.05) and P20 (5.57±0.33 vs. 7.14±0.79 GPa; p<0.05). A wide range of values was measured along the coronal sutures for both the WT and MT samples, with no significant difference between the two groups. Findings of this study suggest that the inherent mechanical properties of the frontal bone in the mutant mice were different to the wild type mice from the same genetic background. These differences may reflect variations in the degree of biomechanical adaptation during skull growth, which could have implications for the surgical management of craniosynostosis patients.

Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening.

Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data.

Development of the lip and palate: FGF signalling.

The fibroblast growth factor (FGF) signalling pathway is critically involved in several aspects of craniofacial development, including formation of the lip and the palate. FGF receptors are activated by extracellular FGF ligands in order to regulate cellular processes such as migration and morphogenesis through instruction of specific target gene expression. A key factor in the development of orofacial structures is the interaction between mesodermal- and neural crest-derived mesenchyme and ecto- and endodermal-derived epithelium. FGF signalling occurs in both cell types and promotes epithelial-mesenchymal communication through region-specific expression of receptor subtypes. Many FGF ligands and receptors are expressed at specific stages and at precise locations during normal palatogenesis and an absolute requirement of some has been demonstrated by their (conditional) inactivation resulting in a cleft palate phenotype. Other important signalling pathways involving SHH and SPRY are intricately involved in the interpretation of FGF signalling. As a cause of human pathology, functionally validated FGF pathway gene mutations have been exclusively associated with syndromic forms of cleft lip and palate. Most commonly, this includes patients with mutations in FGFR1 and FGFR2 (Kallmann, Pfeiffer, Apert and Crouzon syndromes) where cleft palate is part of a broad craniofacial phenotype, including craniosynostosis. Similarly, FGF8 mutations have been found in patients with Kallmann-like idiopathic hypogonadotropic hypogonadism, some also with cleft lip and palate. In this chapter, we will provide an overview of the relevant FGF ligands and receptors important for lip and palate morphogenesis, correlating their expression patterns with the effects of their perturbation that lead to a clefting pathogenesis.

Investigation of SUMO pathway genes in the etiology of nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: SUMO1 has been implicated as having a role in the causation of cleft lip with or without cleft palate (CLP), both directly and through association studies in humans and, perhaps more controversially, in transgenic mouse studies. METHODS: To screen for sequence variants that might be responsible for human CLP, we performed direct DNA sequence analysis in a well-characterized white European cohort of 192 patients. We screened the genes encoding SUMO1, SUMO2, and SUMO3, as well as the E3 ligases PIAS1 and PIAS2, which are required for sumoylation. Variants were analyzed in a cohort of 192 unaffected white European controls. RESULTS: Only two missense variants were identified, both within SUMO3, however, these were both present in multiple affected individuals and a similar number of controls. Other variants identified, apart from a single synonymous change in PIAS1, were all present within flanking intronic regions distant from splice consensus sites. Moreover, most other variants were previously reported in dbSNP and were shown to be present at a similar frequency in cases and controls. CONCLUSIONS: Our findings indicate that mutations identified in the SUMO-related genes tested, including three novel coding SNPs, do not directly contribute to the incidence of CLP.

Tbx22null mice have a submucous cleft palate due to reduced palatal bone formation and also display ankyloglossia and choanal atresia phenotypes.

Craniofacial defects involving the lip and/or palate are among the most common human birth defects. X-linked cleft palate and ankyloglossia results from loss-of-function mutations in the gene encoding the T-box transcription factor TBX22. Further studies show that TBX22 mutations are also found in around 5% of non-syndromic cleft palate patients. Although palate defects are obvious at birth, the underlying developmental pathogenesis remains unclear. Here, we report a Tbx22(null) mouse, which has a submucous cleft palate (SMCP) and ankyloglossia, similar to the human phenotype, with a small minority showing overt clefts. We also find persistent oro-nasal membranes or, in some mice a partial rupture, resulting in choanal atresia. Each of these defects can cause severe breathing and/or feeding difficulties in the newborn pups, which results in approximately 50% post-natal lethality. Analysis of the craniofacial skeleton demonstrates a marked reduction in bone formation in the posterior hard palate, resulting in the classic notch associated with SMCP. Our results suggest that Tbx22 plays an important role in the osteogenic patterning of the posterior hard palate. Ossification is severely reduced after condensation of the palatal mesenchyme, resulting from a delay in the maturation of osteoblasts. Rather than having a major role in palatal shelf closure, we show that Tbx22 is an important determinant for intramembranous bone formation in the posterior hard palate, which underpins normal palate development and function. These findings could have important implications for the molecular diagnosis in patients with isolated SMCP and/or unexplained choanal atresia.

The Mn1 transcription factor acts upstream of Tbx22 and preferentially regulates posterior palate growth in mice.

The mammalian secondary palate exhibits morphological, pathological and molecular heterogeneity along the anteroposterior axis. Although the cell proliferation rates are similar in the anterior and posterior regions during palatal outgrowth, previous studies have identified several signaling pathways and transcription factors that specifically regulate the growth of the anterior palate. By contrast, no factor has been shown to preferentially regulate posterior palatal growth. Here, we show that mice lacking the transcription factor Mn1 have defects in posterior but not anterior palatal growth. We show that Mn1 mRNA exhibits differential expression along the anteroposterior axis of the developing secondary palate, with preferential expression in the middle and posterior regions during palatal outgrowth. Extensive analyses of palatal gene expression in wild-type and Mn1(-/-) mutant mice identified Tbx22, the mouse homolog of the human X-linked cleft palate gene, as a putative downstream target of Mn1 transcriptional activation. Tbx22 exhibits a similar pattern of expression with that of Mn1 along the anteroposterior axis of the developing palatal shelves and its expression is specifically downregulated in Mn1(-/-) mutants. Moreover, we show that Mn1 activated reporter gene expression driven by either the human or mouse Tbx22 gene promoters in co-transfected NIH3T3 cells. Overexpression of Mn1 in NIH3T3 cells also increased endogenous Tbx22 mRNA expression in a dose-dependent manner. These data indicate that Mn1 and Tbx22 function in a novel molecular pathway regulating mammalian palate development.

FGF signalling and SUMO modification: new players in the aetiology of cleft lip and/or palate.

Owing to the complex aetiology and the variable penetrance of cleft lip and/or palate (CL/P), understanding the molecular basis has been challenging. Recent reports have identified two independent biochemical pathways that will help to elucidate the underlying pathology. Fibroblast growth factor signalling, previously known for its involvement in craniofacial development, is now implicated in the genetic basis of both syndromic and non-syndromic CL/P. At the same time, an important role in lip and palate development is beginning to emerge for small ubiquitin-like modifier modification, a widely used posttranslational regulatory mechanism. Both of these pathways might interact with environmental risk factors for CL/P. Here we review their contribution to normal and abnormal orofacial development.

TBX22 missense mutations found in patients with X-linked cleft palate affect DNA binding, sumoylation, and transcriptional repression.

The T-box transcription factor TBX22 is essential for normal craniofacial development, as demonstrated by the finding of nonsense, frameshift, splice-site, or missense mutations in patients with X-linked cleft palate (CPX) and ankyloglossia. To better understand the function of TBX22, we studied 10 different naturally occurring missense mutations that are phenotypically equivalent to loss-of-function alleles. Since all missense mutations are located in the DNA-binding T-box domain, we first investigated the preferred recognition sequence for TBX22. Typical of T-box proteins, the resulting sequence is a palindrome based around near-perfect copies of AGGTGTGA. DNA-binding assays indicate that missense mutations at or near predicted contact points with the DNA backbone compromise stable DNA-protein interactions. We show that TBX22 functions as a transcriptional repressor and that TBX22 missense mutations result in impaired repression activity. No effect on nuclear localization of TBX22 was observed. We find that TBX22 is a target for the small ubiquitin-like modifier SUMO-1 and that this modification is required for TBX22 repressor activity. Although the site of SUMO attachment at the lysine at position 63 is upstream of the T-box domain, loss of SUMO-1 modification is consistently found in all pathogenic CPX missense mutations. This implies a general mechanism linking the loss of SUMO conjugation to the loss of TBX22 function. Orofacial clefts are well known for their complex etiology and variable penetrance, involving both genetic and environmental risk factors. The sumoylation process is also subject to and profoundly affected by similar environmental stresses. Thus, we suggest that SUMO modification may represent a common pathway that regulates normal craniofacial development and is involved in the pathogenesis of both Mendelian and idiopathic forms of orofacial clefting.