A new two-dimensional sonographic approach to the assessment of the fetal hard and soft palates.

Facial clefts are among the most common congenital defects. Ultrasound (US) imaging of secondary fetal palate, especially the detection of isolated defects, remains challenging. Currently described two-dimensional (2D) and three-dimensional methods are technically demanding and impractical for application during routine fetal anatomy evaluation. As an adjunct method, magnetic resonance imaging can provide additional information but has its limitations. We present a novel 2D US approach using axial and sagittal planes to evaluate the fetal palate and demonstrate the main differences between an intact palate, isolated cleft palate, and a cleft lip with cleft palate.

Shox2 regulates osteogenic differentiation and pattern formation during hard palate development in mice.

During mammalian palatogenesis, cranial neural crest-derived mesenchymal cells undergo osteogenic differentiation and form the hard palate, which is divided into palatine process of the maxilla and the palatine. However, it remains unknown whether these bony structures originate from the same cell lineage and how the hard palate is patterned at the molecular level. Using mice, here we report that deficiency in Shox2 (short stature homeobox 2), a transcriptional regulator whose expression is restricted to the anterior palatal mesenchyme, leads to a defective palatine process of the maxilla but does not affect the palatine. Shox2 overexpression in palatal mesenchyme resulted in a hyperplastic palatine process of the maxilla and a hypoplastic palatine. RNA sequencing and assay for transposase-accessible chromatin-sequencing analyses revealed that Shox2 controls the expression of pattern specification and skeletogenic genes associated with accessible chromatin in the anterior palate. This highlighted a lineage-autonomous function of Shox2 in patterning and osteogenesis of the hard palate. H3K27ac ChIP-Seq and transient transgenic enhancer assays revealed that Shox2 binds distal-acting cis-regulatory elements in an anterior palate-specific manner. Our results suggest that the palatine process of the maxilla and palatine arise from different cell lineages and differ in ossification mechanisms. Shox2 evidently controls osteogenesis of a cell lineage and contributes to the palatine process of the maxilla by interacting with distal cis-regulatory elements to regulate skeletogenic gene expression and to pattern the hard palate. Genome-wide Shox2 occupancy in the developing palate may provide a marker for identifying active anterior palate-specific gene enhancers.

IRF6 and TAK1 coordinately promote the activation of HIPK2 to stimulate apoptosis during palate fusion.

Cleft palate is a common craniofacial defect caused by a failure in palate fusion. The palatal shelves migrate toward one another and meet at the embryonic midline, creating a seam. Transforming growth factor-ß3 (TGF-ß3)-induced apoptosis of the medial edge epithelium (MEE), the cells located along the seam, is required for completion of palate fusion. The transcription factor interferon regulatory factor 6 (IRF6) promotes TGF-ß3-induced MEE cell apoptosis by stimulating the degradation of the transcription factor ΔNp63 and promoting the expression of the gene encoding the cyclin-dependent kinase inhibitor p21. Because homeodomain-interacting protein kinase 2 (HIPK2) functions downstream of IRF6 in human cancer cells and is required for ΔNp63 protein degradation in keratinocytes, we investigated whether HIPK2 played a role in IRF6-induced ΔNp63 degradation in palate fusion. HIPK2 was present in the MEE cells of mouse palatal shelves during seam formation in vivo, and ectopic expression of IRF6 in palatal shelves cultured ex vivo stimulated the expression of Hipk2 and the accumulation of phosphorylated HIPK2. Knockdown and ectopic expression experiments in organ culture demonstrated that p21 was required for HIPK2- and IRF6-dependent activation of caspase 3, MEE apoptosis, and palate fusion. Contact between palatal shelves enhanced the phosphorylation of TGF-ß-activated kinase 1 (TAK1), which promoted the phosphorylation of HIPK2 and palate fusion. Our findings demonstrate that HIPK2 promotes seam cell apoptosis and palate fusion downstream of IRF6 and that IRF6 and TAK1 appear to coordinately enhance the abundance and activation of HIPK2 during palate fusion.

The correlative hypotheses between Pitchfork and Kif3a in palate development.

It is well known that dysfunction of primary cilia during embryonic development causes a range of developmental disorders such as cleft lip and palate, lung, kidney and heart disease. Both Pitchfork and Kinesin family member 3a (Kif3a) are associating with primary cilia, but whether there is a correlation between them are still inconclusive. Our research confirmed that Pitchfork over-expression induced lateral cleft palate and primary cilia disassembly during palate development. We also demonstrated that Sonic hedgehog (Shh) and Patched1 (Ptc1) expression levels were altering in the over-expressed Pitchfork group during palate development. Then we observed by consulting a vast amount of literature that specific knockout of the Kif3a also induced lateral cleft palate and expended the expression domains of Shh and Gli1 during palate development. Furthermore, loss of the Kif3a results in disassembly of the primary cilia and eventually leads to abnormal palatal development. Finally, we found that both Pitchfork and Kif3a are accumulating at the basal body and ciliary necklace during the early phase of cilia assembly and disassembly and both of them are involved in ciliary transport. Based on the above evidence, we hypotheses that there may be a potential correlation between Pitchfork and Kif3a, that could regulate primary cilia disassembly during palate development.

Development of Hard and Soft Palate During the Fetal Period and Hard Palate Asymmetry.

In the present study, it was aimed to perform the morphometric analysis of the hard and soft palate in fetal cadavers and evaluate hard palate asymmetry during the fetal development. The development of the palate was investigated in 40 (21 males, 19 females) fetal materials aged between the 17th and 40th gestational week. In this study, distances between the incisive papilla-staurion (Ip-Sr), staurion-posterior nasal spine (Sr-Pns), incisive papilla-greater palatine foramen (Ip-Gpf) on the right and left sides, Sr-Gpf, and Pns-Gpf were measured. In cases with asymmetry, the ratio of asymmetry was determined in percentage using the asymmetry index. Moreover, angular values between Ip-Sr-Gpf and Ip-Pns-Gpf on the right and left sides were measured, and the right and left side values were compared with each other. The hard and soft palate lengths were measured on the planum medianum. Upon comparing the measured parameters between males and females, the mean values of male fetuses were higher in all parameters, but a significant difference was found only in the Sr-Pns distance among these parameters. Upon comparing the angular data and asymmetry index data on the hard palate between the trimester groups, a significant difference was found only in the Ip-Sr-Gpf (left) parameter. The mean ratio of the hard palate to the soft palate was found as 1.90. It is believed that the obtained data will contribute to studies to be conducted in fields such as plastic surgery, maxillofacial surgery, intrauterine surgery, fetopathology, embryology, anatomy, and obstetrics.

Lrp4/Wise regulates palatal rugae development through Turing-type reaction-diffusion mechanisms.

Periodic patterning of iterative structures is diverse across the animal kingdom. Clarifying the molecular mechanisms involved in the formation of these structure helps to elucidate the process of organogenesis. Turing-type reaction-diffusion mechanisms have been shown to play a critical role in regulating periodic patterning in organogenesis. Palatal rugae are periodically patterned ridges situated on the hard palate of mammals. We have previously shown that the palatal rugae develop by a Turing-type reaction-diffusion mechanism, which is reliant upon Shh (as an inhibitor) and Fgf (as an activator) signaling for appropriate organization of these structures. The disturbance of Shh and Fgf signaling lead to disorganized palatal rugae. However, the mechanism itself is not fully understood. Here we found that Lrp4 (transmembrane protein) was expressed in a complementary pattern to Wise (a secreted BMP antagonist and Wnt modulator) expression in palatal rugae development, representing Lrp4 expression in developing rugae and Wise in the inter-rugal epithelium. Highly disorganized palatal rugae was observed in both Wise and Lrp4 mutant mice, and these mutants also showed the downregulation of Shh signaling, which was accompanied with upregulation of Fgf signaling. Wise and Lrp4 are thus likely to control palatal rugae development by regulating reaction-diffusion mechanisms through Shh and Fgf signaling. We also found that Bmp and Wnt signaling were partially involved in this mechanism.

A score-based method for quality control of fetal hard palate assessment during routine second-trimester ultrasound examination.

INTRODUCTION: When an orofacial cleft lip is discovered, precise characterization of this malformation is necessary, especially the extension of this cleft to the secondary palate. We aimed to develop and evaluate the feasibility/reproducibility of a score-based quality control for the visualization of the fetal hard palate during the second-trimester scan. MATERIAL AND METHODS: All ultrasound images of fetal hard palate assessed routinely during second-trimester scan were retrospectively retrieved for a 6-month period. One hundred of these images were randomly selected and analyzed by two blinded reviewers, according to a scoring system (0-6 points). Criteria retained in the score were complete palate bone horizontal plate, presence of two pterygoid processes, visible alveolar ridge, and horizontal axis of insonation. A score ≥4 defined images of good quality. Inter- and intra-reviewer reproducibility was assessed. RESULTS: Inter-reviewer reproducibility was excellent with significant correlation (Pearson coefficient 0.953; P < .0001), global adjusted κ coefficient (0.86, 95% CI 0.79-0.94) and individual criteria adjusted κ coefficient always > 0.8. Rates of images of good quality (score ≥ 4) were 75%-77%, also with excellent agreement (κ coefficient 0.89, 95% CI 0.79-0.99). Intra-reviewer reproducibility retrieved the same results (excellent agreement) except for the axis of insonation (satisfactory agreement). CONCLUSIONS: This simple image scoring system for the fetal palate is easy, has excellent inter- and intra-reviewer reproducibility and could also help sonographers to correctly identify the palate structure.

Diversity in primary palate ontogeny of amniotes revealed with 3D imaging.

The amniote primary palate encompasses the upper lip and the nasal cavities. During embryonic development, the primary palate forms from the fusion of the maxillary, medial nasal and lateral nasal prominences. In mammals, as the primary palate fuses, the nasal and oral cavities become completely separated. Subsequently, the tissue demarcating the future internal nares (choanae) thins and becomes the bucconasal membrane, which eventually ruptures and allows for the essential connection of the oral and nasal cavities to form. In reptiles (including birds), the other major amniote group, primary palate ontogeny is poorly studied with respect to prominence fusion, especially the formation of a bucconasal membrane. Using 3D optical projection tomography, we found that the prominences that initiate primary palate formation are similar between mammals and crocodilians but distinct from turtles and lizards, which are in turn similar to each other. Chickens are distinct from all non-avian lineages and instead resemble human embryos in this aspect. The majority of reptiles maintain a communication between the oral and nasal cavities via the choanae during primary palate formation. However, crocodiles appear to have a transient separation between the oral and nasal cavities. Furthermore, the three lizard species examined here, exhibit temporary closure of their external nares via fusion of the lateral nasal prominences with the frontonasal mass, subsequently reopening them just before hatching. The mechanism of the persistent choanal opening was examined in chicken embryos. The mesenchyme posterior/dorsal to the choana had a significant decline in proliferation index, whereas the mesenchyme of the facial processes remained high. This differential proliferation allows the choana to form a channel between the oral and nasal cavities as the facial prominences grow and fuse around it. Our data show that primary palate ontogeny has been modified extensively to support the array of morphological diversity that has evolved among amniotes.

Type III transforming growth factor beta receptor regulates vascular and osteoblast development during palatogenesis.

BACKGROUND: Cleft palate occurs in up to 1:1,000 live births and is associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palatal shelf elongation, elevation, and fusion. Transforming growth factor ß (TGFß) and bone morphogenetic protein 2 (BMP2) canonical signaling is required during each stage of palate development. The type III TGFß receptor (TGFßR3) binds all three TGFß ligands and BMP2, but its contribution to palatogenesis is unknown. RESULTS: The role of TGFßR3 during palate formation was found to be during palatal shelf elongation and elevation. Tgfbr3(-) (/) (-) embryos displayed reduced palatal shelf width and height, changes in proliferation and apoptosis, and reduced vascular and osteoblast differentiation. Abnormal vascular plexus organization as well as aberrant expression of arterial (Notch1, Alk1), venous (EphB4), and lymphatic (Lyve1) markers was also observed. Decreased osteoblast differentiation factors (Runx2, alk phos, osteocalcin, col1A1, and col1A2) demonstrated poor mesenchymal cell commitment to the osteoblast lineage within the maxilla and palatal shelves in Tgfbr3(-) (/) (-) embryos. Additionally, in vitro bone mineralization induced by osteogenic medium (OM+BMP2) was insufficient in Tgfbr3(-) (/) (-) palatal mesenchyme, but mineralization was rescued by overexpression of TGFßR3. CONCLUSIONS: These data reveal a critical, previously unrecognized role for TGFßR3 in vascular and osteoblast development during palatogenesis.

TCDD disrupts posterior palatogenesis and causes cleft palate.

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion.

Vitamin B-complex application promotes secondary palate development in a palate organ model of the A/WySnJ mouse.

PURPOSE: This study analyzed the direct influence of vitamin B-complex supplements (Polybion N, Merck Pharma GmbH, Germany) in medium on secondary palatal development in palatal organ cultures of A/WySnJ mice. Because of positive clinical experiences with prophylactic vitamin B substitution in mothers of cleft-related families, the direct influence of the vitamin B-complex on palatal tissue was analyzed. MATERIALS AND METHODS: The inbred A/WySnJ mouse strain shows a highly spontaneous, genetically determined clefting rate of 20% to 44%. One hundred seventy-seven A/WySnJ fetuses were microdissected on gestational day 14.3 before the occurrence of palatal fusion. Palatal organ cultures were prepared and incubated in chemically defined serum-free medium with different concentrations (0.1% and 1.0%) of the vitamin B-complex Polybion N for 72 hours. Palatal development was analyzed microscopically according to the 6-step visual scale that describes the approximation of palatal shelves during development. RESULTS: At the beginning of the experiment (gestational day 14.3), the palatal development of all specimens used for in vitro organ culture showed a clear approach of the palatal shelves at stage II (2.25±0.78). Seventy-two hours after in vitro cultivation, the palatal shelves of the organ cultures supplemented with the vitamin B-complex showed significant growth (0.1%, P=.00017; 1.0%, P=.00078), whereas the untreated control group remained at initial developmental stage II (P=.291). CONCLUSIONS: The results of this in vitro study suggest a significant positive influence of vitamin B supplementation on palatal shelf development in organ culture. Further studies will focus on the vitamin B concentration in the amniotic fluid of dams with or without cleft in their offspring.

Association of AXIN2 with non-syndromic oral clefts in multiple populations.

We have previously shown the association of AXIN2 with oral clefts in a US population. Here, we expanded our study to explore the association of 11 AXIN2 markers in 682 cleft families from multiple populations. Alleles for each AXIN2 marker were tested for transmission distortion with clefts by means of the Family-based Association Test. We observed an association with SNP rs7224837 and all clefts in the combined populations (p = 0.001), and with SNP rs3923086 and cleft lip and palate in Asian populations (p = 0.004). We confirmed our association findings in an additional 528 cleft families from the United States (p < 0.009). We tested for gene-gene interaction between AXIN2 and additional cleft susceptibility loci. We assessed and detected Axin2 mRNA and protein expression during murine palatogenesis. In addition, we also observed co-localization of Axin2 with Irf6 proteins, particularly in the epithelium. Our results continue to support a role for AXIN2 in the etiology of human clefting. Additional studies should be performed to improve our understanding of the biological mechanisms linking AXIN2 to oral clefts.

OmniView algorithm: a novel 3-dimensional sonographic technique in the study of the fetal hard and soft palates.

The purpose of this pictorial essay is to report on the application of OmniView (GE Healthcare, Zipf, Austria), new 3-dimensional sonographic software, and its application in the prenatal sonographic study of the fetal hard and soft palates. We will show that this novel technique is easy and feasible, requires a limited learning curve, and provides correct volume interrogation of the region of interest. The OmniView algorithm may be useful in training programs, and volume data sets can be interpreted by experts in remote sites. Future prospective studies with consecutive patients will be necessary to evaluate whether the routine application of OmniView will increase the prenatal diagnosis of facial clefting, especially those with isolated palate defects.

Roles of collagen and periostin expression by cranial neural crest cells during soft palate development.

The tissue in the palatal region can be divided into the hard and the soft palates, each having a specialized function such as occlusion, speech, or swallowing. Therefore, an understanding of the mechanism of palatogenesis in relation to the function of each region is important. However, in comparison with the hard palate, there is still a lack of information about the mechanisms of soft palate development. In this study, the authors investigated the contribution of cranial neural crest (CNC) cells to development of both hard and soft palates. They also demonstrated a unique pattern of periostin expression during soft palate development, which was closely related to that of collagen type I (Col I) in palatine aponeurosis. Furthermore, organ culture analysis showed that exogenous transforming growth factor-ß (TGF-ß) induced the expression of both periostin and Col I. These novel patterns of expression in the extracellular matrix (ECM) induced by CNC cells suggest that these cells may help to determine the character of both the hard and soft palates through ECM induction. TGF-ß signaling appears to be one of the mediators of Col I and periostin expression in the formation of functional structures during soft palate development.

Accuracy of prenatal three-dimensional ultrasound in the diagnosis of cleft hard palate when cleft lip is present.

OBJECTIVE: To investigate the accuracy of prenatal axial three-dimensional (3D) ultrasound in predicting the absence or presence of cleft palate in the presence of cleft lip. METHODS: Between March 2005 and January 2009, there were 81 cases with a prenatal two-dimensional (2D) ultrasound screening diagnosis of unilateral or bilateral cleft lip at 22-25 weeks of gestation referred to our tertiary care center. Of these, 79 fetuses were included in this prospective study and two were excluded. Axial 3D ultrasound imaging of the fetal palate was performed and the diagnoses were compared with clinical findings at delivery. The frequencies of intact and cleft palate, the degree of association between the prenatal predictions and postnatal findings and the probability of detection of cleft lip and palate were determined. RESULTS: Of 79 prenatal predictions, 77 (97%) were correct and the association between the prenatal predictions and postnatal findings was strong. The sensitivity for detection of cleft lip and palate within this high-risk population was 100% and the specificity was 90%. In one of the excluded cases, the palate could not be visualized due to a fetal prone position. There were chromosomal anomalies in 4% of cases and associated structural or growth anomalies in 23%, termination of pregnancy was carried out in 4% and intrauterine fetal demise occurred in 3%. CONCLUSION: Axial 3D ultrasound of the fetal palate has high accuracy in identifying prenatal cleft palate when cleft lip is diagnosed at mid-trimester 2D ultrasound screening.

The genetic basis of craniofacial and dental abnormalities.

The embryonic head development, including the formation of dental structures, is a complex and delicate process guided by specific genetic programs. Genetic changes and environmental factors can disturb the execution of these programs and result in abnormalities in orofacial and dental structures. Orofacial clefts and hypodontia/ oligodontia are examples of such abnormalities frequently seen in dental clinics. An insight into the mechanisms and genes involved in the formation of orofacial and dental structures has been gradually gained by genetic analysis of families and by the use of experimental vertebrate models such as the mouse and chick models. The development of novel clinical therapies for orofacial and dental pathological conditions depends very much on a detailed knowledge of the molecular and cellular processes that are involved in head formation.

Tgf-beta-mediated FasL-Fas-Caspase pathway is crucial during palatogenesis.

Programmed cell death, or apoptosis, is one of the fates of the medial edge epithelium (MEE) during palatal fusion. Transforming growth factor ß (Tgf-ß) signaling (such as Tgf-ß3) is required for the disappearance of the MEE, but the relationship between Tgf-ß3 and apoptosis remains unclear. Here we show that the Fas ligand (FasL)-Fas-Caspase extrinsic apoptosis pathway functions during palatal fusion in wild-type mice, but is not detectable in mice lacking Tgf-ß3 (Tgf-ß3 (-/-) ) or Tgfßr2 in the MEE (K14-Cre;Tgfbr2 (fl/fl)). Inhibition of the FasL-Fas system results in persistence of the midline epithelial seam (MES) and inhibition of caspase activity during palatal organ culture. Moreover, ectopic FasL protein induces apoptosis in MES of K14-Cre;Tgfbr2 (fl/fl) mice. Thus, we conclude that the FasL-Fas-caspase extrinsic apoptosis pathway is regulated by the Tgf-ß3 signaling cascade and is essential for palatal fusion during craniofacial development.