Homozygosity for a Rare Plec Variant Suggests a Contributory Role in Congenital Insensitivity to Pain.

Congenital insensitivity to pain is a rare human condition in which affected individuals do not experience pain throughout their lives. This study aimed to identify the molecular etiology of congenital insensitivity to pain in two Thai patients. Clinical, radiographic, histopathologic, immunohistochemical, and molecular studies were performed. Patients were found to have congenital insensitivity to pain, self-mutilation, acro-osteolysis, cornea scars, reduced temperature sensation, tooth agenesis, root maldevelopment, and underdeveloped maxilla and mandible. The skin biopsies revealed fewer axons, decreased vimentin expression, and absent neurofilament expression, indicating lack of dermal nerves. Whole exome and Sanger sequencing identified a rare homozygous variant c.4039C>T; p.Arg1347Cys in the plakin domain of Plec, a cytolinker protein. This p.Arg1347Cys variant is in the spectrin repeat 9 region of the plakin domain, a region not previously found to harbor pathogenic missense variants in other plectinopathies. The substitution with a cysteine is expected to decrease the stability of the spectrin repeat 9 unit of the plakin domain. Whole mount in situ hybridization and an immunohistochemical study suggested that Plec is important for the development of maxilla and mandible, cornea, and distal phalanges. Additionally, the presence of dental anomalies in these patients further supports the potential involvement of Plec in tooth development. This is the first report showing the association between the Plec variant and congenital insensitivity to pain in humans.

Genetic Variants in KCTD1 Are Associated with Isolated Dental Anomalies.

KCTD1 plays crucial roles in regulating both the SHH and WNT/ß-catenin signaling pathways, which are essential for tooth development. The objective of this study was to investigate if genetic variants in KCTD1 might also be associated with isolated dental anomalies. We clinically and radiographically investigated 362 patients affected with isolated dental anomalies. Whole exome sequencing identified two unrelated families with rare (p.Arg241Gln) or novel (p.Pro243Ser) variants in KCTD1. The variants segregated with the dental anomalies in all nine patients from the two families. Clinical findings of the patients included taurodontism, unseparated roots, long roots, tooth agenesis, a supernumerary tooth, torus palatinus, and torus mandibularis. The role of Kctd1 in root development is supported by our immunohistochemical study showing high expression of Kctd1 in Hertwig epithelial root sheath. The KCTD1 variants in our patients are the first variants found to be located in the C-terminal domain, which might disrupt protein-protein interactions and/or SUMOylation and subsequently result in aberrant WNT-SHH-BMP signaling and isolated dental anomalies. Functional studies on the p.Arg241Gln variant are consistent with an impact on ß-catenin levels and canonical WNT signaling. This is the first report of the association of KCTD1 variants and isolated dental anomalies.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

MicroRNAs regulate distal region of mandibular development through Hh signaling.

Mandibular anomalies are often seen in various congenital diseases, indicating that mandibular development is under strict molecular control. Therefore, it is crucial to understand the molecular mechanisms involved in mandibular development. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating the level of gene expression. We found that the mesenchymal conditional deletion of miRNAs arising from a lack of Dicer (an essential molecule for miRNA processing, Dicerfl/fl ;Wnt1Cre), led to an abnormal groove formation at the distal end of developing mandibles. At E10.5, when the region forms, inhibitors of Hh signaling, Ptch1 and Hhip1 showed increased expression at the region in Dicer mutant mandibles, while Gli1 (a major mediator of Hh signaling) was significantly downregulated in mutant mandibles. These suggest that Hh signaling was downregulated at the distal end of Dicer mutant mandibles by increased inhibitors. To understand whether the abnormal groove formation inDicer mutant mandibles was caused by the downregulation of Hh signaling, mice with a mesenchymal deletion of Hh signaling activity arising from a lack of Smo (an essential molecule for Hh signaling activation, Smofl/fl ;Wnt1Cre) were examined. Smofl/fl ;Wnt1Cre mice showed a similar phenotype in the distal region of their mandibles to those in Dicerfl/fl ;Wnt1Cre mice. We also found that approximately 400 miRNAs were expressed in wild-type mandibular mesenchymes at E10.5, and six microRNAs were identified as miRNAs with binding potential against both Ptch1 and Hhip1. Their expressions at the distal end of the mandible were confirmed by in situ hybridization. This indicates that microRNAs regulate the distal part of mandibular formation at an early stage of development by involving Hh signaling activity through controlling its inhibitor expression level.

Juberg-Hayward syndrome is a cohesinopathy, caused by mutation in ESCO2.

BACKGROUND: Juberg-Hayward syndrome (JHS; MIM 216100) is a rare autosomal recessive malformation syndrome, characterized by cleft lip/palate, microcephaly, ptosis, short stature, hypoplasia or aplasia of thumbs, and dislocation of radial head and fusion of humerus and radius leading to elbow restriction. OBJECTIVE: To report for the first time the molecular aetiology of JHS. PATIENT AND METHODS: Clinical and radiographic examination, whole exome sequencing, Sanger sequencing, mutant protein model construction, and in situ hybridization of Esco2 expression in mouse embryos were performed. RESULTS: Clinical findings of the patient consisted of repaired cleft lip/palate, microcephaly, ptosis, short stature, delayed bone age, hypoplastic fingers and thumbs, clinodactyly of the fifth fingers, and humeroradial synostosis leading to elbow restriction. Intelligence is normal. Whole exome sequencing of the whole family showed a novel homozygous base substitution c.1654C>T in ESCO2 of the proband. The sister was homozygous for the wildtype variant. Parents were heterozygous for the mutation. The mutation is predicted to cause premature stop codon p.Arg552Ter. Mutations in ESCO2, a gene involved in cohesin complex formation, are known to cause Roberts/SC phocomelia syndrome. Roberts/SC phocomelia syndrome and JHS share similar clinical findings, including autosomal recessive inheritance, short stature, cleft lip/palate, severe upper limb anomalies, and hypoplastic digits. Esco2 expression during the early development of lip, palate, eyelid, digits, upper limb, and lower limb and truncated protein model are consistent with the defect. CONCLUSIONS: Our study showed that Roberts/SC phocomelia syndrome and JHS are allelic and distinct entities. This is the first report demonstrating that mutation in ESCO2 causes JHS, a cohesinopathy.

Ift88 is involved in mandibular development.

The mandible is a crucial organ in both clinical and biological fields due to the high frequency of congenital anomalies and the significant morphological changes during evolution. Primary cilia play a critical role in many biological processes, including the determination of left/right axis patterning, the regulation of signaling pathways, and the formation of bone and cartilage. Perturbations in the function of primary cilia are known to cause a wide spectrum of human diseases: the ciliopathies. Craniofacial dysmorphologies, including mandibular deformity, are often seen in patients with ciliopathies. Mandibular development is characterized by chondrogenesis and osteogenesis; however, the role of primary cilia in mandibular development is not fully understood. To address this question, we generated mice with mesenchymal deletions of the ciliary protein, Ift88 (Ift88fl/fl ;Wnt1Cre). Ift88fl/fl ;Wnt1Cre mice showed ectopic mandibular bone formation, whereas Ift88 mutant mandible was slightly shortened. Meckel's cartilage was modestly expanded in Ift88fl/fl ;Wnt1Cre mice. The downregulation of Hh signaling was found in most of the mesenchyme of Ift88 mutant mandible. However, mice with a mesenchymal deletion of an essential molecule for Hh signaling activity, Smo (Smofl/fl ;Wnt1Cre), showed only ectopic mandibular formation, whereas Smo mutant mandible was significantly shortened. Ift88 is thus involved in chondrogenesis and osteogenesis during mandibular development, partially through regulating Sonic hedgehog (Shh) signaling.

Overactivation of the NF-κB pathway impairs molar enamel formation.

OBJECTIVE: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal structures and functions of the ectoderm-derived organs, including teeth. HED patients exhibit a variety of dental symptoms, such as hypodontia. Although disruption of the EDA/EDAR/EDARADD/NF-κB pathway is known to be responsible for HED, it remains unclear whether this pathway is involved in the process of enamel formation. EXPERIMENTAL SUBJECTS AND METHODS: To address this question, we examined the mice overexpressing Ikkß (an essential component required for the activation of NF-κB pathway) under the keratin 5 promoter (K5-Ikkß). RESULTS: Upregulation of the NF-κB pathway was confirmed in the ameloblasts of K5-Ikkß mice. Premature abrasion was observed in the molars of K5-Ikkß mice, which was accompanied by less mineralized enamel. However, no significant changes were observed in the enamel thickness and the pattern of enamel rods in K5-Ikkß mice. Klk4 expression was significantly upregulated in the ameloblasts of K5-Ikkß mice at the maturation stage, and the expression of its substrate, amelogenin, was remarkably reduced. This suggests that abnormal enamel observed in K5-Ikkß mice was likely due to the compromised degradation of enamel protein at the maturation stage. CONCLUSION: Therefore, we could conclude that the overactivation of the NF-κB pathway impairs the process of amelogenesis.

MicroRNAs control eyelid development through regulating Wnt signaling.

BACKGROUND: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. RESULTS: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. CONCLUSIONS: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.

TFAP2B mutation and dental anomalies.

Mutations inTFAP2B has been reported in patients with isolated patent ductus arteriosus (PDA) and Char syndrome. We performed mutation analysis of TFAP2B in 43 patients with isolated PDA, 7 patients with PDA with other congenital heart defects and 286 patients with isolated tooth agenesis with or without other dental anomalies. The heterozygous c.1006G>A mutation was identified in 20 individuals. Those mutation carriers consisted of 1 patient with term PDA (1/43), 16 patients with isolated tooth agenesis with or without other dental anomalies (16/286; 5.6%), 1 patient with PDA and severe valvular aortic stenosis and tooth agenesis (1/4) and 2 normal controls (2/100; 1%). The mutation is predicted to cause an amino-acid substitution p.Val336Ile in the TFAP2B protein. Tfap2b expression during early mouse tooth development supports the association of TFAP2B mutation and dental anomalies. It is hypothesized that this incidence might have been the result of founder effect. Here we report for the first time that TFAP2B mutation is associated with tooth agenesis, microdontia, supernumerary tooth and root maldevelopment. In addition, we also found that TFAP2B mutations, the common causes of PDA in Caucasian, are not the common cause of PDA in Thai population.

Regional regulation of Filiform tongue papillae development by Ikkα/Irf6.

BACKGROUND: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood. RESULTS: We found Ikkα and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikkα and Irf6 in filiform papillae development. Ikkα and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue. CONCLUSIONS: Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikkα and Irf6. Developmental Dynamics 245:937-946, 2016. © 2016 Wiley Periodicals, Inc.

Multiple postnatal craniofacial anomalies are characterized by conditional loss of polycystic kidney disease 2 (Pkd2).

Polycystin 2 (Pkd2), which belongs to the transient receptor potential family, plays a critical role in development. Pkd2 is mainly localized in the primary cilia, which also function as mechanoreceptors in many cells that influence multiple biological processes including Ca(2+) influx, chemical activity and signalling pathways. Mutations in many cilia proteins result in craniofacial abnormalities. Orofacial tissues constantly receive mechanical forces and are known to develop and grow through intricate signalling pathways. Here we investigate the role of Pkd2, whose role remains unclear in craniofacial development and growth. In order to determine the role of Pkd2 in craniofacial development, we located expression in craniofacial tissues and analysed mice with conditional deletion of Pkd2 in neural crest-derived cells, using Wnt1Cre mice. Pkd2 mutants showed many signs of mechanical trauma such as fractured molar roots, distorted incisors, alveolar bone loss and compressed temporomandibular joints, in addition to abnormal skull shapes. Significantly, mutants showed no indication of any of these phenotypes at embryonic stages when heads perceive no significant mechanical stress in utero. The results suggest that Pkd2 is likely to play a critical role in craniofacial growth as a mechanoreceptor. Pkd2 is also identified as one of the genes responsible for autosomal dominant polycystic kidney disease (ADPKD). Since facial anomalies have never been identified in ADPKD patients, we carried out three-dimensional photography of patient faces and analysed these using dense surface modelling. This analysis revealed specific characteristics of ADPKD patient faces, some of which correlated with those of the mutant mice.

R-spondins/Lgrs expression in tooth development.

BACKGROUND: Tooth development is highly regulated in mammals and it is regulated by networks of signaling pathways (e. g. Tnf, Wnt, Shh, Fgf and Bmp) whose activities are controlled by the balance between ligands, activators, inhibitors and receptors. The members of the R-spondin family are known as activators of Wnt signaling, and Lgr4, Lgr5, and Lgr6 have been identified as receptors for R-spondins. The role of R-spondin/Lgr signaling in tooth development, however, remains unclear. RESULTS: We first carried out comparative in situ hybridization analysis of R-spondins and Lgrs, and identified their dynamic spatio-temporal expression in murine odontogenesis. R-spondin2 expression was found both in tooth germs and the tooth-less region, the diastema. We further examined tooth development in R-spondin2 mutant mice, and although molars and incisors exhibited no significant abnormalities, supernumerary teeth were observed in the diastema. CONCLUSIONS: R-spondin/Lgr signaling is thus involved in tooth development.

The role of Irf6 in tooth epithelial invagination.

Thickening and the subsequent invagination of the epithelium are an important initial step in ectodermal organ development. Ikkα has been shown to play a critical role in controlling epithelial growth, since Ikkα mutant mice show protrusions (evaginations) of incisor tooth, whisker and hair follicle epithelium rather than invagination. We show here that mutation of the Interferon regulatory factor (Irf) family, Irf6 also results in evagination of incisor epithelium. In common with Ikkα mutants, Irf6 mutant evagination occurs in a NF-κB-independent manner and shows the same molecular changes as those in Ikkα mutants. Irf6 thus also plays a critical role in regulating epithelial invagination. In addition, we also found that canonical Wnt signaling is upregulated in evaginated incisor epithelium of both Ikkα and Irf6 mutant embryos.

Distinct roles of microRNAs in epithelium and mesenchyme during tooth development.

BACKGROUND: Tooth development is known to be mediated by the cross-talk between signaling pathways, including Shh, Fgf, Bmp, and Wnt. MicroRNAs (miRNAs) are 19- to 25-nt noncoding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs, which is believed to be important for the fine-tuning signaling pathways in development. To investigate the role of miRNAs in tooth development, we examined mice with either mesenchymal (Wnt1Cre/Dicer(fl/fl)) or epithelial (ShhCre/Dicer(fl/fl)) conditional deletion of Dicer, which is essential for miRNA processing. RESULTS: By using a CD1 genetic background for Wnt1Cre/Dicer(fl/fl), we were able to examine tooth development, because the mutants retained mandible and maxilla primordia. Wnt1Cre/Dicer(fl/fl) mice showed an arrest or absence of teeth development, which varied in frequency between incisors and molars. Extra incisor tooth formation was found in ShhCre/Dicer(fl/fl) mice, whereas molars showed no significant anomalies. Microarray and in situ hybridization analysis identified several miRNAs that showed differential expression between incisors and molars. CONCLUSION: In tooth development, miRNAs thus play different roles in epithelium and mesenchyme, and in incisors and molars.

A Mutation in CACNA1S Is Associated with Multiple Supernumerary Cusps and Root Maldevelopment.

BACKGROUND: Enamel knots and Hertwig epithelial root sheath (HERS) regulate the growth and folding of the dental epithelium, which subsequently determines the final form of tooth crown and roots. We would like to investigate the genetic etiology of seven patients affected with unique clinical manifestations, including multiple supernumerary cusps, single prominent premolars, and single-rooted molars. METHODS: Oral and radiographic examination and whole-exome or Sanger sequencing were performed in seven patients. Immunohistochemical study during early tooth development in mice was performed. RESULTS: A heterozygous variant (c. 865A>G; p.Ile289Val) in CACNA1S was identified in all the patients, but not in an unaffected family member and control. Immunohistochemical study showed high expression of Cacna1s in the secondary enamel knot. CONCLUSIONS: This CACNA1S variant seemed to cause impaired dental epithelial folding; too much folding in the molars and less folding in the premolars; and delayed folding (invagination) of HERS, which resulted in single-rooted molars or taurodontism. Our observation suggests that the mutation in CACNA1S might disrupt calcium influx, resulting in impaired dental epithelium folding, and subsequent abnormal crown and root morphology.

Expression of R-spondins/Lgrs in development of movable craniofacial organs.

Wnt signaling plays a critical role in the development of many organs, including the major movable craniofacial organs tongue, lip, and eyelid. Four members of the R-spondin family (Rspo1-4) bind to Lgr4/5/6 to regulate the activation of Wnt signaling. However, it is not fully understood how Rspos/Lgrs regulate Wnt signaling during the development of movable craniofacial organs. To address this question, we examined the expression of Rspos, Lgrs, and Axin2 (major mediator of canonical Wnt signaling) during tongue, lip, and eyelid development. The expression of Axin2, Rspos and Lgrs was observed in many similar regions, suggesting that Rspos likely activate canonical Wnt signaling through the Lgr-dependent pathway in these regions. Lgr expression was not detected in regions where Axin2 and Rspos were expressed, suggesting that Rspos might activate canonical Wnt signaling through the Lgr-independent pathway in these regions. In addition, the expression of Rspos and Lgrs were observed in some other regions where Axin2 was not expressed, suggesting the possibility that Rspos and/or Lgrs are involved in non-canonical Wnt signaling or the Wnt-independent pathway. Thus, we identified a dynamic spatiotemporal expression pattern of Rspos and Lgrs during the development of the eyelid, tongue, and lip.

A novel P3H1 mutation is associated with osteogenesis imperfecta type VIII and dental anomalies.

OBJECTIVE: Our objective was to investigate the molecular etiology of osteogenesis imperfecta type VIII and dental anomalies in 4 siblings of a Karen tribe family. MATERIALS AND METHODS: Four patients and their unaffected parents were studied by clinical and radiographic examination. In situ hybridization of P3h1 during early murine tooth development, whole-exome sequencing, and Sanger direct sequencing were performed. RESULTS: A novel homozygous missense P3H1 mutation (NM_001243246.1; c.2141A>G; NP_001230175.1; p.Lys714Arg) was identified in all patients. Their unaffected parents were heterozygous for the mutation. The mutation is hypothesized to belong to isoform c of P3H1. Mutations in P3H1 are associated with autosomal recessive osteogenesis imperfecta type VIII. Hypodontia, a mesiodens, and single-rooted permanent second molars found in our patients have never been reported in patients with P3H1 mutations. Single-rooted second permanent molars or failure to form multiple roots implies effects of the P3H1 mutation on root development. CONCLUSIONS: We report a novel P3H1 mutation as the underlying cause of osteogenesis imperfecta type VIII with dental anomalies. Our study suggests that isoform c of P3H1 is also a functional isoform of P3H1. We report, for the first time, to our knowledge, the association of P3H1 mutation and osteogenesis imperfecta type VIII with dental anomalies.

Molecular mechanisms in palatal rugae development.

BACKGROUND: Periodic patterning of iterative structures is diverse across the animal kingdom. Clarifying the molecular mechanisms involved in the formation of these structures helps to elucidate the genetic commonality of developmental processes, as organs with these structures are believed to share the same molecular mechanisms and fundamental processes. Palatal rugae are periodic corrugated structures on the hard palate and are conserved in all mammals. Although the numbers and patterns of the palatal rugae are species specific, they are consistent in each mammalian species, except humans. HIGHLIGHT: Palatal rugae development is thus under strict genetic control in most mammals and is an excellent model to investigate the genetic commonality of developmental processes to form periodic patterning. CONCLUSION: This review highlights the current understanding of the molecular mechanisms of palatal rugae development.

Primary cilia in murine palatal rugae development.

Periodic patterning of iterative structures is a fundamental process during embryonic development, since these structures are diverse across the animal kingdom. Therefore, elucidating the molecular mechanisms in the formation of these structures promotes understanding of the process of organogenesis. Periodically patterned ridges, palatal rugae (situated on the hard palate of mammals), are an excellent experimental model to clarify the molecular mechanisms involved in the formation of periodic patterning of iterative structures. Primary cilia are involved in many biological events, including the regulation of signaling pathways such as Shh and non-canonical Wnt signaling. However, the role of primary cilia in the development of palatal rugae remains unclear. We found that primary cilia were localized to the oral cavity side of the interplacode epithelium of the palatal rugae, whereas restricted localization of primary cilia could not be detected in other regions. Next, we generated mice with a placodal conditional deletion of the primary cilia protein Ift88, using ShhCre mice (Ift88 fl/fl;ShhCre). Highly disorganized palatal rugae were observed in Ift88 fl/fl;ShhCre mice. Furthermore, by comparative in situ hybridization analysis, many Shh and non-canonical Wnt signaling-related molecules showed spatiotemporal expression patterns during palatal rugae development, including restricted expression in the epithelium (placodes and interplacodes) and mesenchyme. Some of these expression were found to be altered in Ift88 fl/fl;ShhCre mice. Primary cilia is thus involved in development of palatal rugae.

Ift88 limits bone formation in maxillary process through suppressing apoptosis.

OBJECTIVE: The development of the maxillary bone is under strict molecular control because of its complicated structure. Primary cilia play a critical role in craniofacial development, since defects in primary cilia are known to cause congenital craniofacial dysmorphologies as a wide spectrum of human diseases: the ciliopathies. The primary cilia also are known to regulate bone formation. However, the role of the primary cilia in maxillary bone development is not fully understood. DESIGN: To address this question, we generated mice with a mesenchymal conditional deletion ofIft88 using the Wnt1Cre mice (Ift88fl/fl;Wnt1Cre). The gene Ift88 encodes a protein that is required for the function and formation of primary cilia. RESULTS: It has been shown thatIft88fl/fl;Wnt1Cre mice exhibit cleft palate. Here, we additionally observed excess bone formation in the Ift88 mutant maxillary process. We also found ectopic apoptosis in the Ift88 mutant maxillary process at an early stage of development. To investigate whether the ectopic apoptosis is related to the Ift88 mouse maxillary phenotypes, we generated Ift88fl/fl;Wnt1Cre;p53-/- mutants to reduce apoptosis. The Ift88fl/fl;Wnt1Cre;p53-/- mice showed no excess bone formation, suggesting that the cells evading apoptosis by the presence of Ift88 in wild-type mice limit bone formation in maxillary development. On the other hand, the palatal cleft was retained in the Ift88fl/fl;Wnt1Cre;p53-/- mice, indicating that the excess bone formation or abnormal apoptosis was independent of the cleft palate phenotype in Ift88 mutant mice. CONCLUSIONS: Ift88 limits bone formation in the maxillary process by suppressing apoptosis.