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.

Fetal and early postnatal development of the porcine tonsils of the soft palate.

Tonsils are mucosa-associated lymphoid tissues located at the openings of the gastrointestinal and respiratory tracts, which play a key role in the surveillance of inhaled or ingested pathogens and can concurrently be reservoirs of infectious agents. Therefore, tonsils are important for the immunology and hygiene management of domestic animals, including pigs. However, the process of their fetal developmental has been poorly described, at least in part, because rodents lack tonsils. Therefore, we performed a histological analysis of porcine tonsils of the soft palate from 60 to 100 days of gestation (DG) and from 2 to 14 days post partum (DP). This analysis showed that lymphoid aggregations first appear at DG65, gradually develop during the fetal stage, and expand after birth. In addition, the mRNA expression of chemokine genes involved in lymphoid aggregation and localization was analyzed. CCL19 expression showed the most marked increase and a sharp peak after birth. CCL21 expression changed moderately but showed an interesting bimodal pattern. CXCL13 expression steadily increased throughout the study period. Thus, we demonstrated the mRNA expression of chemokine characteristically changed accompanying tonsillar 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.

Developmental mechanism of muscle-tendon-bone complex in the fetal soft palate.

OBJECTIVE: This study was performed to investigate how the palatine aponeurosis, medial pterygoid process (MPP) of the sphenoid bone, and tensor veli palatini (TVP) muscle form the pulley: muscle-tendon-bone complex. DESIGN: Mice at embryonic day (ED) 14-17 were used as sample in this study. Azan staining was performed to observe the morphology, and immunohistochemical staining of desmin was performed to closely observe the development of the myotendinous junction. To confirm the bone formation process, immunohistochemical staining of type II collagen (col II), tartrate-resistant acid phosphatase (TRAP), and alkaline phosphatase (ALP) staining were performed. Furthermore, to objectively evaluate bone formation, the major axis and width of the MPP were measured, and osteoclasts that appeared in the MPP were counted. RESULTS: At ED 14 and 14.5, ALP showed a reaction throughout the MPP. The col II-positive area expanded until ED 16.5, but it was markedly reduced at ED 17. The TVP initially contacted with the palatine aponeurosis at ED 16.5. The major axis and width of the MPP and the number of TRAP-positive osteoclasts were significantly increased as the TVP and palatine aponeurosis joined. CONCLUSIONS: Therefore, in addition to the tissue units: muscle, tendon, and bone, the interaction in organogenesis promotes rapid growth of the pulley: muscle-tendon-bone complex.

Implicaciones moleculares del Factor de crecimiento Transformante Beta (TGF-ß) en el desarrollo de las fisuras labiopalatinas / Molecular implications of transforming growth factor beta (TGF-ß) signaling pathway in the development of cleft lip and palate

Las fisuras labio palatinas se generan por la falta de fusión de los tejidos del labio o del paladar durante las primeras etapas del desarrollo fetal, estas se encuentran entre los defectos congénitos más comunes causados por el desarrollo facial anormal durante la gestación; su etiología no se encuentra totalmente aclarada, sin embargo se intenta explicar por medio del modelo de umbral multifactorial, planteándose que es producto de la interacción de factores endógenos y exógenos, entre los endógenos se han reportado alteraciones en la señalización del TGF-ß, el cual está involucrado en el desarrollo embrionario, diferenciación celular y en la regulación del desarrollo del paladar. En esta revisión se muestran los recientes avances sobre las implicaciones moleculares de la vía de señalización TGF-ß en el desarrollo de las fisuras labio palatinas (AU)

Cleft lip and palate are generated by the lack of fusion of the tissues of the lip or palate during early fetal development, these are among the most common birth defects caused by abnormal facial development during gestation. The etiology of these cracks is not fully elucidated, however attempts to explain by means of multifactorial threshold model, considering that is the product of the interaction of endogenous and exogenous factors, endogenous alterations have been reported in TGF-ß signaling, which is involved in embryonic development, cell differentiation and in the regulation of development of the palate. In this review, the recent advances implications of the molecular signaling pathway TGF-ß in the development of cleft lip and palate shown (AU)

Fetal Tendinous Connection Between the Tensor Tympani and Tensor Veli Palatini Muscles: A Single Digastric Muscle Acting for Morphogenesis of the Cranial Base.

Some researchers contend that in adults the tensor tympani muscle (TT) connects with the tensor veli palatini muscle (TVP) by an intermediate tendon, in disagreement with the other researchers. To resolve this controversy, we examined serial sections of 50 human embryos and fetuses at 6-17 weeks of development. At 6 weeks, in the first pharyngeal arch, a mesenchymal connection was found first to divide a single anlage into the TT and TVP. At and after 7 weeks, the TT was connected continuously with the TVP by a definite tendinous tissue mediolaterally crossing the pharyngotympanic tube. At 11 weeks another fascia was visible covering the cranial and lateral sides of the tube. This "gonial fascia" had two thickened borders: the superior one corresponded to a part of the connecting tendon between the TT and TVP; the inferior one was a fibrous band ending at the os goniale near the lateral end of the TVP. In association with the gonial fascia, the fetal TT and TVP seemed to provide a functional complex. The TT-TVP complex might first help elevate the palatal shelves in association with the developing tongue. Next, the tubal passage, maintained by contraction of the muscle complex, seems to facilitate the removal of loose mesenchymal tissues from the tympanic cavity. Third, the muscle complex most likely determined the final morphology of the pterygoid process. Consequently, despite the controversial morphologies in adults, the TT and TVP seemed to make a single digastric muscle acting for the morphogenesis of the cranial base.

A Comprehensive Study of Soft Palate Development in Mice.

Cleft palate is one of the most common congenital birth defects. Tremendous efforts have been made over the last decades towards understanding hard palate development. However, little is known about soft palate morphogenesis and myogenesis. Finding an appropriate surgical repair to restore physiological functions of the soft palate in patients with cleft palate is a major challenge for surgeons, and complete restoration is not always achievable. Here, we first analyzed the morphology, orientation and attachments of the four muscles of the murine soft palate and found that they are very similar to their counterparts in humans, validating the use of mus musculus as a model for future studies. Our data suggests that muscle differentiation extends from the lateral region to the midline following palatal fusion. We also detected an epithelial seam in the fusing soft palatal shelves, consistent with the process of fusion of the posterior palatal shelves, followed by degradation of the epithelial remnants. Innervation and vascularization are present mainly in the oral side of the soft palate, complementing the differentiated muscles. Cell lineage tracing using Wnt1-Cre;Zsgreenfl/fl mice indicated that all the tendons and mesenchyme embedding the soft palate muscles are neural crest-derived. We propose that the posterior attachment of the soft palate to the pharyngeal wall is an interface between the neural crest- and mesoderm-derived mesenchyme in the craniofacial region, and thus can serve as a potential model for the study of boundaries during development. Taken together, our study provides a comprehensive view of the development and morphology of the murine soft palate and serves as a reference for further molecular analyses.

Analysis of human soft palate morphogenesis supports regional regulation of palatal fusion.

It is essential to complete palate closure at the correct time during fetal development, otherwise a serious malformation, cleft palate, will ensue. The steps in palate formation in humans take place between the 7th and 12th week and consist of outgrowth of palatal shelves from the paired maxillary prominences, reorientation of the shelves from vertical to horizontal, apposition of the medial surfaces, formation of a bilayered seam, degradation of the seam and bridging of mesenchyme. However, in the soft palate, the mechanism of closure is unclear. In previous studies it is possible to find support for both fusion and the alternative mechanism of merging. Here we densely sample the late embryonic-early fetal period between 54 and 74 days post-conception to determine the timing and mechanism of soft palate closure. We found the epithelial seam extends throughout the soft palates of 57-day specimens. Cytokeratin antibody staining detected the medial edge epithelium and distinguished clearly that cells in the midline retained their epithelial character. Compared with the hard palate, the epithelium is more rapidly degraded in the soft palate and only persists in the most posterior regions at 64 days. Our results are consistent with the soft palate following a developmentally more rapid program of fusion than the hard palate. Importantly, the two regions of the palate appear to be independently regulated and have their own internal clocks regulating the timing of seam removal. Considering data from human genetic and mouse studies, distinct anterior-posterior signaling mechanisms are likely to be at play in the human fetal palate.

The formation of endoderm-derived taste sensory organs requires a Pax9-dependent expansion of embryonic taste bud progenitor cells.

In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

Expression of GAD67 and Dlx5 in the taste buds of mice genetically lacking Mash1.

It has been reported that a subset of type III taste cells express glutamate decarboxylase (GAD)67, which is a molecule that synthesizes gamma-aminobutyric acid (GABA), and that Mash1 could be a potential regulator of the development of GABAnergic neurons via Dlx transcription factors in the central nervous system. In this study, we investigated the expression of GAD67 and Dlx in the embryonic taste buds of the soft palate and circumvallate papilla using Mash1 knockout (KO)/GAD67-GFP knock-in mice. In the wild-type animal, a subset of type III taste cells contained GAD67 in the taste buds of the soft palate and the developing circumvallate papilla, whereas GAD67-expressing taste bud cells were missing from Mash1 KO mice. A subset of type III cells expressed mRNA for Dlx5 in the wild-type animals, whereas Dlx5-expressing cells were not evident in the apical part of the circumvallate papilla and taste buds in the soft palate of Mash1 KO mice. Our results suggest that Mash1 is required for the expression of GAD67 and Dlx5 in taste bud cells.

TGFß regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate.

Clefting of the soft palate occurs as a congenital defect in humans and adversely affects the physiological function of the palate. However, the molecular and cellular mechanism of clefting of the soft palate remains unclear because few animal models exhibit an isolated cleft in the soft palate. Using three-dimensional microCT images and histological reconstruction, we found that loss of TGFß signaling in the palatal epithelium led to soft palate muscle defects in Tgfbr2(fl/fl);K14-Cre mice. Specifically, muscle mass was decreased in the soft palates of Tgfbr2 mutant mice, following defects in cell proliferation and differentiation. Gene expression of Dickkopf (Dkk1 and Dkk4), negative regulators of WNT-ß-catenin signaling, is upregulated in the soft palate of Tgfbr2(fl/fl);K14-Cre mice, and WNT-ß-catenin signaling is disrupted in the palatal mesenchyme. Importantly, blocking the function of DKK1 and DKK4 rescued the cell proliferation and differentiation defects in the soft palate of Tgfbr2(fl/fl);K14-Cre mice. Thus, our findings indicate that loss of TGFß signaling in epithelial cells compromises activation of WNT signaling and proper muscle development in the soft palate through tissue-tissue interactions, resulting in a cleft soft palate. This information has important implications for prevention and non-surgical correction of cleft soft palate.

Down-regulation of Wnt10a by RNA interference inhibits proliferation and promotes apoptosis in mouse embryonic palatal mesenchymal cells through Wnt/Beta-catenin signaling pathway

Cleft palate is one of the most common birth defects. Both environmental and genetic factors are involved in this disorder. Here, we investigated the function of Wnt10a in proliferation and apoptosis of mouse embryonic palatal mesenchymal (MEPM) cells. Expression of Wnt10a was down-regulated at both the mRNA and protein levels in transfected MEPM cells containing Wnt10a-specific small hairpin RNA (shRNA) plasmid. Down-regulation of Wnt10a inhibited cell proliferation and induced cell cycle arrest in the S phase in MEPM cells. Moreover, apoptosis was significantly increased in MEPM cells of Wnt10a gene silencing. Finally, the expression of β-catenin was markedly reduced in MEPM cells transfected with shRNA plasmid, indicating that the canonical Wnt/β-catenin signaling pathway was involved in the alterations of cell proliferation and apoptosis induced by Wnt10a knockdown. Thus, our findings reveal that Wnt10a regulates proliferation and apoptosis of MEPM cells at least partially through the canonical Wnt/β-catenin signaling pathway

[Early morphogenesis of ciliated cells in human oral cavity].

Ciliated cells were found in the epithelium of the oral cavity of human embryos and fetuses starting from the seventh week of prenatal development. At the early stages of prenatal development (until the 13th week), cells with cilia cover most of the dorsal surface of the tongue and the soft palate, whereas they are found only near the gland ducts in the circumvallate and foliate lingual papillae after 17 weeks of development. The ultrastructure of the axoneme of cilia corresponds to the structure of motile cilia and is represented by nine microtubule doublets that surround the central pair of microtubule singlets. An immunohistochemical study performed on weeks 10-12 of development identified nerve endings associated with the ciliated cells. Until the 14th week of development, the cytoplasm of ciliated cells is immunopositive for NSE. The spatial distribution of ciliated cells in the tongue epithelium until the 13th week of development is not related to the morphogenesis of lingual papillae, and their role in the human oral cavity during the first trimester of pregnancy is unclear and requires further study.

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.

Development of the human tensor veli palatini: specimens measuring 13.6-137 mm greatest length; weeks 6-16 of development.

The present study seeks to determine the main events that occur in the development of the tensor veli palatini (TVP). A light microscope was used on serial sections of 60 human specimens from weeks 6 to 16 of development. The TVP becomes visible in an embryo of 14.5 mm greatest length (GL; week 6) from a common blastema with the medial pterygoid muscle. In embryos of Carnegie stage 20 (week 7), the TVP is differentiated and relates to the anlage of the pterygoid hamulus. At week 8 of development, when the palatal shelves become horizontal, the presence of the anlage of the palatine aponeurosisis distinguished and is reached by the TPV. In an embryo of 30 mm GL, the chondrification nucleus of the pterygoid hamulus and the synovial bursa of the TVP are identifiable. At week 9, the TVP is continuous with the palatine aponeurosis. At week 13, a connective tissue lamina appears between the TVP and the intramembranous ossification center for the anterior process of the malleus, which we know as the goniale and interpret as an attachment of the muscle to the primary vertebrate jaw or incudomalleal joint. The TVP from its origin, innervation and relation to the goniale appears to be a muscle of mastication that, at the end of the embryonic period, reaches the palatine aponeurosis anlage and the mesenchyme of the auditory tube and specializes in the movements of the soft palate and the auditory tube.

Developmental expression of Bdnf, Ntf4/5, and TrkB in the mouse peripheral taste system.

Brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4), and their TrkB receptor regulate taste system development. To determine where and when gustatory neurons come in contact with these important factors, temporospatial expression patterns of Bdnf, Ntf4/5, and TrkB in the peripheral taste system were examined using RT-PCR. In the lingual epithelium, Ntf4/5 mRNA expression was higher than that of Bdnf at embryonic day 12.5 (E12.5), and the expression of both factors decreased afterwards. However, Ntf4/5 expression decreased at an earlier age than Bdnf. Bdnf and Ntf4/5 are expressed in equal amounts at E12.5 in geniculate ganglion, but Bdnf expression increased from E14.5 to birth, whereas Ntf4/5 expression decreased. These findings indicate that NT4 functions at early embryonic stages and is derived from different sources than Bdnf. TrkB expression in the geniculate ganglion is robust throughout development and is not a limiting factor for neurotrophin function in this system.

Delayed osteoprogenitor differentiation in cleft-palate models.

Failure of palatal shelf fusion results in cleft palate (CP) and may lead to malformation of palatal bones and undergrowth of the maxilla. It is not known whether defects in bone formation may contribute to this phenotype. We tested the hypothesis that impaired fusion of developing palatal shelves affects palatal bone development using palate organotypic cultures. Using two different approaches, we show that induction of cleft results in increased expression of pre-osteoblast and early osteoblast markers, Twist1, Snai1 and Runx2, and decreased expression of more mature markers of bone differentiation, collagen-1 and osteopontin, indicating delayed osteoblast differentiation in CPs. This, together with the increase in immature osteoblasts and proliferation observed in non-fused palatal shelves, suggests that palatal osteoblast differentiation is at least partly modulated by shelf fusion. Delayed osteoblast differentiation may therefore contribute to defects in gross morphology and function of the maxilla in CP patients.

The 'equals sign': a novel marker in the diagnosis of fetal isolated cleft palate.

OBJECTIVES: To determine the feasibility of visualization of the uvula and the soft palate during routine two-dimensional (2D) ultrasound examination and to develop a sonographic procedure that facilitates prenatal detection of isolated fetal cleft palate. METHODS: We examined 667 consecutive patients with a singleton pregnancy between 20 and 25 weeks of gestation who were referred to our practice for a detailed fetal anomaly scan. The uvula was visualized either in a frontal section through the neck and pharynx or via a transverse section with subsequent slight tilting of the transducer. The soft palate was visualized in a median sagittal section through the head. To test whether the techniques described are suitable for inclusion in routine examinations, the time allowed for a normal ultrasound scan was not changed from 30 min. RESULTS: A normal uvula could be visualized with a typical echo pattern (the 'equals sign') in 90.7% of the cases and the soft palate could be completely visualized in a median sagittal section in 85.3% of the cases. Visualization of at least one of the two structures (either the uvula or the soft palate) was successful in 98.4% of the cases. In one case an isolated cleft palate (in an otherwise normal fetus) was diagnosed; in one case with a cleft lip and palate, the cleft palate and the completely split uvula were detected. In 1.2% of the cases the examination did not provide sufficient information on either the uvula or the soft palate. CONCLUSIONS: In a high percentage of cases, relevant soft palate structures can be visualized successfully with 2D ultrasound, permitting cleft palate to be ruled out in routine examinations. Visualization of the equals sign proves an intact palate. Absence of the equals sign indicates cleft palate and should prompt further examination of the soft palate in a median sagittal section. Cleft palate can be confirmed when the soft palate cannot be visualized. This technique also facilitates evaluation of the soft palate in the event of a cleft lip and palate.

Viewing of the soft and the hard palate on routine 3-D ultrasound sweep of the fetal face--a feasibility study.

OBJECTIVES: To report a technique to view the fetal soft and hard palates, utilizing acquired routine 3-D volumes of the fetal face. METHOD: The axial, sagittal and coronal planes in acquired volumes of 3-D surface rendering of 5 normal fetal faces obtained at 19, 21, 23, 26 and 28 weeks, respectively, were reviewed by focusing on the uvula. RESULTS: The surfaces of the soft and the hard palate could be viewed in oblique axial, oblique sagittal and targeted coronal planes in all fetuses. CONCLUSION: Rotating or tilting of the axial, sagittal and coronal planes of the fetal head allows the visualization of the various aspects of the soft and hard palates, with the uvula as a useful landmark.

Anatomy and growth of the fetal soft palate: a cadaveric study to improve its ultrasonographic observation.

OBJECTIVE: To determine the anatomy of the soft and hard palate during fetal growth in order to improve its ultrasonographic prenatal visualization. DESIGN: Anatomic study in human formalin-fixed fetus. METHODS: The heads of 18 second and third trimester fetuses were studied in the median sagittal plan. Measurements of the soft palate, the velopharynx, the root of the tongue, and the oral floor were taken. The hard palate/soft palate angle and the anterior cranial base/soft palate angle were measured. RESULTS: The growth of the hard palate was linear, and the growth of the soft palate was polynomial (second order) during the period studied. The hard palate/soft palate angle was 150.33 degrees +/- 7.62 and 150.20 degrees +/- 6.67 in the second and third trimester, respectively. The anterior position of the soft palate in relation to the anterior cranial base was 48.8 degrees +/- 3.13 in the second trimester and 52.26 degrees +/- 3.31 in the third trimester. Its posterior position was 89.66 degrees +/- 5.51 in the second trimester and 92.97 degrees +/- 4.01 in the third trimester. Throughout the fetal period, the soft palate moved downward relative to the clivus and cervical spine. CONCLUSIONS: Despite the nonlinear growth and downward displacement of the soft palate during fetal life, its position remains stable. These results may be useful to explore the fetal soft palate using 2D and 3D ultrasonography and to improve the prenatal diagnosis of isolated cleft palate.