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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., (© 2020 Wiley Periodicals LLC.)

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.

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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (© 2016 Wiley Periodicals, Inc.)

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.

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., (© 2015 Anatomical Society.)

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.

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., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 TGFβ 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-β-catenin signaling, is upregulated in the soft palate of Tgfbr2(fl/fl);K14-Cre mice, and WNT-β-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 TGFβ 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.

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.

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.

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., (Copyright © 2011 S. Karger AG, Basel.)

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.

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., (Copyright © 2010 ISUOG. Published by John Wiley & Sons, Ltd.)

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., (Copyright © 2010 S. Karger AG, Basel.)

Although embryonic expression of Shh in the fungiform papilla placodes has a critical role in fungiform papilla patterning, it remains unclear whether its appearance indicates the differentiation of the basal cells of taste buds. To examine the embryonic development of the basal cells, the expression of Shh, Prox1, and Mash1 was determined in the anterior tongue and soft palate in mouse embryos by in situ hybridization. In the anterior tongue, Prox1 was coexpressed with Shh from the beginning of Shh expression in the fungiform papilla placodes at E12.5. Shh was expressed in the soft palate in a band-like pattern in the anteriormost region and in a punctate pattern in the posterior region at E14.5. The number (21.4 +/- 4.3, at E14.5) of locations where Shh was observed (i.e., spots) rapidly increased and reached a peak level (54.8 +/- 4.0 at E15.5). Also in the soft palate, Prox1 was coexpressed with Shh from the beginning of Shh expression. These results suggest that basal cell differentiation occurs synchronously with the patterning of Shh spots both in the anterior tongue and in the soft palate. In contrast, Mash1 expression lagged behind the expression of Shh and Prox1 and began after the number of Shh spots had reached its peak level in the soft palate. Furthermore, immunohistochemistry of PGP9.5 and Shh revealed that epithelial innervation slightly preceded Mash1 expression both in the tongue and in the soft palate. This is the first report describing the time courses of the embryonic expression of basal cell markers of taste buds., ((c) 2008 Wiley-Liss, Inc.)

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.

Objectives: To describe a three-dimensional (3D) ultrasound technique for assessing the fetal soft palate., Methods: A prospective study conducted from April to December 2006 included 87 fetuses in a low-risk population. Fetal ultrasound scans were performed between 21 and 25 weeks of gestation to determine the normal 3D ultrasound view of the fetal soft tissues of the palate. The sonographers used a 30 degrees-inclined axial 3D view of the fetal palate. Ultrasound images obtained in this view were compared with fetopathological specimens of the same gestational age by two observers, both pediatric surgeons. Each observer indicated whether they thought that the uvula or the velum could be detected, and the differences in responses between the observers were assessed., Results: The frequencies of detection of the uvula and velum of each observer varied between 80% and 90%. The observers showed a significant difference in judging the visualization of the uvula, but not in judging the velum., Conclusions: A 30 degrees -inclined axial 3D ultrasound view seems to be effective in assessing the integrity of the fetal soft palate.

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., ((c) 2008 S. Karger AG, Basel.)

Members of the Tshz gene family encode putative zinc fingers transcription factors that are broadly expressed during mouse embryogenesis. Tshz1 is detected from E9.5 in the somites, the spinal cord, the limb buds and the branchial arches. In order to assess the function of Tshz1 during mouse development, we generated Tshz1-deficient mice. Tshz1 inactivation leads to neonatal lethality and causes multiple developmental defects. In the craniofacial region, loss of Tshz1 function leads to specific malformations of middle ear components, including the malleus and the tympanic ring. Tshz1(-/-) mice exhibited Hox-like vertebral malformations and homeotic transformations in the cervical and thoracic regions, suggesting that Tshz1 and Hox genes are involved in common pathways to control skeletal morphogenesis. Finally, we demonstrate that Tshz1 is required for the development of the soft palate.

Cleft palate is a common birth defect caused by disruptions in secondary palate development. Anterior-posterior (A-P) regional specification plays a critical role in palate development and fusion. Previous studies have shown that at the molecular level, the anterior palate can be defined by the expression of Shox-2 and the posterior palate by Meox-2 expression in certain mouse strains. Here, we have extended previous studies by performing a more detailed analysis of these genes during mouse palate development. We found that the expression patterns of Shox-2 and Meox-2 are dynamic during palate development. At embryonic day 12.5 (E12.5), Shox-2 expression is localized to the anterior end and its expression domain covers less than 25% of the length of the palate shelf. The Shox-2 expression domain then gradually expands towards the posterior end and ultimately occupies more than 60% of the palate shelf by E14.5. The expansion of the Shox-2 domain may involve induction of Shox2 expression in additional cells. Reciprocally, the Meox-2 expression domain at E12.5 covers a large portion of the palate shelf, a region more than 70% of the entire palate, but then regresses to the posterior 25% by E14.5. This regression is likely caused by the repression of Meox-2 expression in certain Meox2 expressing cells, rather than the cessation of cell proliferation. Therefore, certain Meox-2 positive "primitive posterior cells" are differentiated/converted into Shox-2 positive "definitive anterior cells" during A-P regional specification.

Background: SOX9 is a critical transcription factor for chondrogenesis and sex determination. Haploinsufficiency mutations of Sox9 in humans lead to campomelic dysplasia. Inactivation of Sox9 in the craniofacial region of mice results in an absence of endochondral bones and in malformation of other structures. This suggests that Sox9 plays multiple roles in craniofacial development and these remain to be elucidated. In order to study the functions of Sox9 in craniofacial development, a preliminary expression examination was performed., Material and Methods: To detect the expression of Sox9 mRNA, antisense riboprobe was synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagittal and coronal sections of mice head from organogenesis to the early postnatal stage., Results: It was found that Sox9 was expressed in multiple stages and distinct processes. Besides the expression in cartilage, it was seen in the fusing stage of palatogenesis. Sox9 was also present during differentiation and maturation of craniofacial muscles. In addition, it was observed in intramembranous skeletal elements at restricted sites and stage., Conclusions: The expression pattern suggests that Sox9 serves broad roles in craniofacial development.

Physiological atresia, or fetal occlusion, is an epithelial vegetation arising at a certain stage of embryonal fetal life in some organs of esophageal, respiratory and urogenital system as well as in natural openings of the head--the lid slit, external nasal openings, external and internal acoustic meatus. In parallel, tubal organs close to open later, i.e. recanalization takes place. In view of this, epithelial "plugs" in the larynx and external nasal openings have a positive biological role making a temporary obstruction for amniotic fluid penetration into the viscera. Plug-induced difference of pressure between closed nasal cavity and open oral cavity in reflexes of mouth opening helps horizontal adjustment of the processes of the palate bone. This is important for formation of the hard palate. Before appearance of functioning muscles of the soft palate, epithelial plugs in the external nasal openings block nasal cavity. If the plugs were absent, fetal swallowing movement would be impossible, as amniotic fluid would outpour through the nose. Epithelial adhesions in external nasal openings as well as in the larynx and esophagus are temporary structures contributing to formation of palatine shape and establishment of fetal functions.

Six to eight copies of a transgene integrated into mouse chromosome 15 resulting in a new transgene insertional mutant, Footless, presenting with malformations of the limbs, kidney, and soft palate. Homozygotes possess a unique asymmetric pattern of limb truncations. Posterior structures from the autopod and zeugopod of the hindlimbs are missing with left usually more severely affected than right. In contrast, anterior structures are missing from the right forelimbs. The left forelimb is usually normal except for the absence of the distal telephalanges and nails. These structures are absent on all formed digits. In situ hybridization assays examined the expression of Shh, dHand, Msx2, Fgf8, En1, and Lmx1b in mutant limb buds and indicated normal establishment of the anterior/posterior and dorsal/ventral axes of the developing limbs. However, dysmorphology of the apical ectodermal ridge was observed in the mutant limb buds.

Objective: To propose a new method, which allows for a complete description of primary and secondary cleft palates, incorporating elements that are related to the palate, lip, and nose that will also reflect the complexity of this problem., Method: To describe the type of cleft, two embryonic structures were considered: (1) the primary palate, formed by the prolabium, premaxilla, and columella and (2) the secondary palate, which begins at the incisive foramen and is formed by a horizontal portion of the maxilla, the horizontal portion of the palatine bones, and the soft palate. Anatomical characteristics to be considered were defined, and a new method is proposed to more fully describe any cleft., Results: A description of five cases was made using the method proposed in this work and compared with other published methods for the classification and description of clefts., Conclusions: A mathematical expression was developed to characterize clefts of the primary palate, including the magnitude of palatal segment separation and the added complexity of bilateral clefts, yielding a numerical score that reflects overall complexity of the cleft. Clefts of the secondary palate are also considered in a separate score. Using this method, it is possible to incorporate elements that are not considered in other approaches and to describe all possible clefts that may exist.

Palatal taste buds are intriguing partners in the mediation of taste behavior and their spatial distribution is functionally important for suckling behavior, especially in the neonatal life. Their prenatal development has not been previously elucidated in the rat, and the onset of their maturation remains rather controversial. We delineated the development and frequency distribution of the taste buds as well as the immunohistochemical expression of alpha-gustducin, a G protein closely related to the transduction of taste stimuli, in the nasoincisor papilla (NIP) and soft palate (SP) from the embryonic day 17 (E17) till the postnatal day 70 (PN70). The main findings in the present study were the development of a substantial number of taste pores in the SP of fetal rats (60.3 +/- 1.7 out of 122.8 +/- 5.5; mean +/- SD/animal at E19) and NIP of neonatal rats (9.8 +/- 1.0 out of 44.8 +/- 2.2 at PN4). alpha-gustducin-like immunoreactivity (-LI) was not expressed in the pored taste buds of either prenatal or newborn rats. The earliest expression of alpha-gustducin-LI was demonstrated at PN1 in the SP (1.5 +/- 0.5 cells/taste bud; mean +/- SD) and at PN4 in the NIP (1.4 +/- 0.5). By age the total counts of pored taste buds continuously increased and their morphological features became quite discernible. They became pear in shape, characterized by distinct pores, long subporal space, and longitudinally oriented cells. Around the second week, a remarkable transient decrease in the total number of taste buds was recorded in the oral epithelium of NIP and SP, which might be correlated with the changes of ingestive behaviors. The total counts of cells showing alpha-gustducin-LI per taste bud gradually increased till the end of our investigation (14.1 +/- 2.7 in NIP and 12.4 +/- 2.5 in SP at PN70). We conclude that substantial development of taste buds began prenatally in the SP, whereas most developed entirely postnatal in the NIP. The present study provides evidence that the existence of a taste pore which is considered an important criterion for the morphological maturation of taste buds is not enough for the onset of the taste transduction, which necessitates also mature taste cells. Moreover, the earlier maturation of palatal taste buds compared with the contiguous populations in the oral cavity evokes an evidence of their significant role in the transmission of gustatory information, especially in the early life of rat., (Copyright 2001 Wiley-Liss, Inc.)

We studied corresponding structures on the oral and basal surfaces of the oral epithelial layer, focusing on the microanatomy of gustatory papillae. Specimens for scanning electron microscopy were obtained from the rat soft palates and fixed with a mixture of 2% paraformaldehyde and 1% glutaraldehyde. They were first prepared without postfixation and conductive-staining to study the oral surface. After examination, the epithelium was reinforced by additional sputter coating and treated with 6 N NaOH at 60 degrees C to exfoliate the basal epithelial surfaces without any significant artifacts. The papillae, showing circular, elliptical or fusiform protuberances on the oral surface, were classified into two types: types I and II. The type I and type II papillae contained one and two taste pores, respectively. On the basal epithelial surface, the basal portions of the taste buds were associated with concentrically arranged nerve fibers and Schwann cells. Another characteristic finding on the basal epithelial surface was the presence of excretory ducts of minor salivary glands in a close spatial relationship to taste buds. It is suggested that saliva coming out through the duct is mixed with food, thus enabling intimate contact with the taste pores of the papillae., (Copyright 2001 S. Karger AG, Basel.)

The ultrastructural development of the surface belonging to the epithelium-connective tissue interface of the hard and soft palatine mucosa of mouse was studied using field emission scanning electron microscopy and high voltage transmission electron microscopy methods. The tissues were analysed at 18 days of gestation, and, 1, 3, 7, 14, 21, 30 and 90 days after birth. The three-dimensional architecture of the connective tissue in the lamina propria showed different shapes. The papillae began to form on days 7 and 14 but continued to growth until the adult period. Different stages of primary and secondary connective tissue papillae were recognized in the hard palatine mucosa whereas the soft palate showed erect connective tissue papillae, small round formations containing taste buds and several openings of the salivary glands ducts.

Objective: The purpose of this study was to investigate, in a normal human fetal sample, the development of the anterior area of the palate. Emphasis has been placed on the bilateral epithelial fusion lines between the soft-tissue palatal shelves and the premaxillary mucosa, and on the relationship between these fusion lines and the locations of the inclusive fissures and of the tooth buds. The goal was to evaluate how the location of clefts in cleft lip and palate (CLP) patients interferes with tooth buds that are often affected in clefting., Design: Nineteen maxillas of human fetuses underwent histologic examination after serial horizontal sectioning. Superimpositions of tracings from photographs of the histologic sections yielded information on the maxillary morphology at different levels., Results: After elevation and fusion of the soft-tissue palatal shelves, the palatal fusions were Y-shaped. During subsequent osseous palate development, including formation of the incisive fissure and intermaxillary suture, the Y-shaped epithelial fusions disintegrated. The locations of the anterior arms of the epithelial Y and of the incisive fissure did not coincide., Conclusions: The incisive fissure is likely not the location of clefts in CLP. The location of anterior palatal clefting in CLP patients follows the original course of the epithelial fusion between the palatal shelves and the premaxillary mucosa found in the region of the lateral incisors.

The biosynthesis and hydration of glycosaminoglycans (GAG) has been implicated in the generation of palatal shelf-elevating force(s) in mammals, although the nature of the palatal shelf extracellular matrices during cleft palate formation remains poorly understood. This study quantifies the GAG composition in the palatal shelves of Wistar rat fetuses at various periods of palatogenesis where clefts were induced experimentally using 5-fluoro-2-deoxyuridine (FUDR). For both normal and cleft palatal shelves, hyaluronan, heparan sulphate and chondroitin-4-sulphate were detected but not dermatan sulphate or chondroitin-6-sulphate. Throughout the period of cleft development studied, the total amount of GAG was significantly decreased (by approx. 30%) compared with normal development, this decrease being particularly marked at a time equivalent to post-elevation during normal development (approx. 75%). Furthermore, and unlike normal palatogenesis, no significant differences were recorded between the anterior and posterior parts of the palatal shelves during cleft formation. As for normal palatogenesis, however, the percentages of each GAG were not altered at any stage. The findings are consistent with the view that suppression of GAG biosynthesis is related to the development of cleft palate in FUDR-treated rat fetuses and can therefore be interpreted as providing evidence of a role for the mesenchymal glycoconjugates in shelf elevation during normal palatogenesis.

The two specific aims of this study were as follows: to evaluate the appropriateness of the A/Jax mouse model in the investigation of the key cellular stages in prenatal soft palate morphogenesis and myogenesis; and to describe structural differences in the histomorphology of the soft palate anatomy from cleft and noncleft mice prior to, during, and after palatogenesis. Cleft-induced and control groups of A/Jax mouse embryos from timed pregnancies were harvested sequentially on gestational days 15 to 19. Embryos were weighed and staged for external body morphology. The heads were removed and fixed for light microscopy, sectioned serially in the frontal plane at 10 microns and stained with hematoxylin-eosin to characterize and compare the soft palate musculature. All observations were made at the head depth of the trigeminal ganglion in both age- and stage-matched embryos. The following findings were made: (1) the A/Jax mouse is a suitable animal model for the study of soft palate myogenesis; (2) there were no discernible morphologic differences between the soft palate muscles in cleft and noncleft A/Jax mice when viewed under light microscopy; (3) the soft palate and related muscles were identifiable as muscle fields, in both the cleft and noncleft fetuses, as early as gestational day 15 and as specific muscles at gestational day 18; (4) in both the cleft and noncleft A/Jax fetuses, the soft palate muscles appeared in a sequential anatomic fashion (the palatine aponeurosis appeared first, next the tensor palatini, and then the levator palatini muscles); and (5) in the cleft palate fetuses, both pterygoid plates were angulated and displaced laterally.

Five pregnant rats were treated during organogenesis with sc injections of acyclovir (50 mg/kg body weight) on days 9, 10 and 11 of pregnancy. The fetuses (N=62) were evaluated on day 20 of gestation and presented decreased body weight as well as delayed differentiation of fetal rat palate epithelium, with increased nuclear volume, decreased cytoplasmic and cellular volumes, decreased epithelial and keratin thicknesses, and increased cellular numerical density.

Processes of the development and growth of the soft palate mucosas in ontogenesis and also proliferation, growth and differentiation of epithelial structures were studied in the experiment. The epithelium of the soft palate nasal surface was established to be more labile and to be characterized with polymorphism forming single-layer and pseudostratified structures. Under these conditions the oral surface epithelium keeps stratified structures firmly. On the basis of discovered differences of the epithelium biological qualities a suggestion is made on the different genetic origin of epithelial tissues of mucosas of the soft palate oral and pharyngeal surfaces.

To test the hypothesis that soft palate muscles are abnormal in cleft palate, we compared soft palate morphogenesis in fetuses with cleft palate (n = 4) to age-matched (n = 3) and nonmatched (n = 1) control specimens. The morphologic status of all soft palate and masticatory structures were classified into one of six stages based on the level of histogenesis. At 54 mm crown-rump length (CRL), the levator veli palatini (L), palatopharyngeus (PP), and palatoglossus (PG) in cleft subjects demonstrated mesenchymal condensation into myoblastic fields, lagging behind the control specimens (97 mm CRL), which displayed definitive fields of myoblasts and myotube formation. In the 175 mm and 225 mm cleft and the 170 mm and 192 mm control specimens, muscular morphology was similar and had reached its postnatal appearance for the tensor veli palatini (175 m only) and L, PP, PG (225 mm only). Muscle fiber directions were, however, disoriented and disorganized, especially close to the medial epithelial edge of the cleft. The levator veli palatini, could not be distinguished as a discrete muscle in the cleft specimens, and what we believed to be the PP and PG seemed "normal" at the level of light microscopy, but malpositioned in a superior direction. This preliminary study demonstrates for the first time that early myogenesis in cleft palates differs from normal.

The synthesis and hydration of glycosaminoglycans (GAG) has been implicated in the generation of a palatal shelf-elevating force in mammals. This study quantifies the GAG composition in the palatal shelves of Wistar rat fetuses at various stages of palatogenesis. Hyaluronan, heparin sulphate and chondroitin-4-sulphate were detected but not dermatan sulphate or chondroitin-6-sulphate. The distribution of the GAG differed in the anterior and posterior regions (i.e. the presumptive hard and soft palates) and with the stage of development. At the time of palatal-shelf reorientation, there were no significant differences for either the total amount of GAG or for the percentages of specific GAG types between anterior and posterior regions. Indeed, the most marked differences were detected at the stages of histogenesis after shelf elevation. Nevertheless, the results support the view that hyaluronan is involved in palatal-shelf reorientation, its percentage being initially high and decreasing after shelf elevation. No changes were detected for the other (sulphated) GAG during the time of elevation. The findings point to the need to correlate the events during histogenesis with changes in the ground substance of palatal-shelf mesenchyme and indicate that there are different developmental mechanisms within the presumptive hard and soft palates.

Surgical correction of clefts of the soft palate leads to varying degrees of normal function although the repair itself is successful. Explanations for this include structural abnormalities of the muscles. Previous studies have focused primarily on gross anatomical features of late fetal and postnatal cleft palate musculature; however, infrequent reference has been made to early prenatal morphologic patterns of soft-palate development, beginning with the embryo. Thus we evaluated the chronology of prenatal myogenesis of the soft palate from its early mesenchymal phase through the appearance of definitive palatal muscles and associated structures in a sample of 22 human fetuses that represented postfertilization weeks 6.5 to 20.5 (18- to 192-mm crown-rump length). Specimens were histologically prepared for descriptive and morphometric light microscopy. Data were collected on the earliest appearance times of identifiable soft palate and associated structures within the mesenchymal field and on their individual stages of myogenesis (e.g., for muscles, from mesenchyme to myoblasts to fascicles). Analyses showed that (1) palatal muscles and related bony structures emerge sequentially as densely staining mesenchymal subfields within the larger mesenchymal soft-palate field during the 6- to 9-week period, with the tensor veli palatini muscle appearing earliest, and the musculus uvulae latest; (2) further morphogenesis of the soft palate and associated structures follows a definite timeline; and (3) by 16 to 17 weeks the postnatal palatal morphology is in place.

To provide information concerning biosynthetic activities within the mesenchymal cells of rat palatal shelves, the numbers of nucleolar organiser regions (NORs) were detected using an Ag-NOR staining technique within tissues obtained from coronal sections of 30 fetal heads prepared for light microscopy. The study involved comparisons between stages of palatogenesis and between location along the palatal shelves, anterior and posterior regions being analysed to correspond to the presumptive hard and soft palates. For both anterior and posterior regions, the mean Ag-NOR count/cell was initially high (approximately 3.4 anteriorly and approximately 3.0 posteriorly). The count/cell decreased significantly immediately after elevation but increased again after shelf reorientation and complete fusion. When comparisons were made between anterior and posterior regions of the palatal shelves, no differences were discerned at a stage prior to elevation (day 15) and a stage after the shelves had elevated and completely fused (day 17). Immediately post-elevation (day 16.5), however, the mean count/cell was significantly higher anteriorly. Thus the degree of mesenchymal cell activity, as expressed in terms of protein biosynthesis, can change in the developing palate according to both site and stage of development.

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

Much attention has been focused on the mechanisms responsible for the elevation and fusion of the palatal processes during formation of the secondary palate. In a former publication this closure, defined as fusion of the palatal shelves along their length, was described as occurring at a period in osseous palatal development characterized by incipient ossification of the maxillae and palatine bones, and at a stage in early mandibular development when the mandibular condyle has not developed. The purpose of the present study was to examine the osseous maturation of the cranial base at the time of palatal closure. The investigation was based on 62 human embryos and fetuses, CRL 22-57 mm, and general skeletal hand and foot maturation CNO = 0-0 to CNO = 8-1 (CNO, Composite Number of Ossified bones in hand and foot). From the fetuses, cranial tissue was dissected out and skeletal maturity was determined radiographically according to formerly described skeletal maturity stages in the cranial base. The study revealed that the soft tissue palatal shelves always elevate and fuse along their length at a specific, well-defined stage of maturity in the cranial base. The cranial base development in this stage is characterized by unossified cartilage. This information is essential for understanding abnormal skeletal development of the oral and nasal cavities.

A new method of anatomic dissection and image reconstruction using computer techniques for better understanding of eustachian tube (ET) functioning is presented. Coronal sections of two noncleft fetal skulls were photographed and projected on a graphic tablet. Contours of the pertinent structures were digitized using a mouse. Coordinates of all digitized points were entered into a special computer program. The data were transformed into three-dimensional representations of anatomic structures. The levator veli palatini muscle (M-LVP) was found to have a close relationship with the ET running underneath it and passing at the inside of the pharyngeal edge of the medial cartilage before entering the soft palate. On M-LVP contraction, this part of the medial ET cartilage appears to be the sole point of impact for ET opening. The tensor veli palatini muscle (M-TVP) is connected with the lateral cartilage of the ET and leaves the ET, rounding the pterygoid hamulus before entering the palatal aponeurosis. On this anatomic basis, action of the muscle by isotonic contraction appears to be more likely than isometric contraction.

Some clefts of the lip and palate appear to result from the rupture after fusion has occurred. This possibility is suggested by (1) epithelial remnants found in the palatal shelves, (2) regenerating epithelium found in the palatal clefts of human fetuses older than 13 weeks, (3) immature epithelium visible on the margin of the cleft, (4) nasopalatine ducts cut off by clefts, (5) perforation type cleft found in the hard palate, (6) a Simonart band visible in six cleft mouse fetuses, (7) duplicated upper lateral incisors enclosing a lip-jaw cleft, and (8) rodent cavities in the lip or palate related to cleft formation.

Fetuses were obtained from CD-1 mice at a time estimated to be 12 h prior to vivo secondary palate closure. One of the palatal shelves of each partially dissected fetal head was lesioned in one of five ways, the other left intact to serve as control. Single transverse cuts extending the width of the shelf were made at one of three positions along the longitudinal axis of the shelf: one-third, one-half or two-thirds the shelf length estimated from the rostral edge. Some specimens were cut in two places, dividing the shelf into three equal segments. Another group received a lesion which separated the caudal third of the shelf from its maxillary connections. All specimens were cultured for 18 h. At the end of the culture period the heads were fixed, examined and the degree of elevation of each shelf piece assessed. Intact, control shelves of all preparations were elevated in the rostral two-thirds of the shelf, while the caudal third was partially elevated. Results seen in lesioned shelves depended upon both the size of the segment and the region of the shelf contained in the segment. The rostral two-thirds of the shelf, the presumptive hard palate, whether intact or in segments elevated without physical connections to neighboring shelf tissue. Thus, it is unlikely that this elevation requires a wave of contraction be transmitted from the caudal soft palate region. In contrast, the presumptive soft palate requires continuity with the rostral portions of the shelf both to maintain structural stability and to elevate.

Closure between the secondary palate and the primary palate and between the former and the nasal septum was studied in the Syrian golden hamster at light- and electron-microscopic level. Sequential changes in the epithelia before, during and after the closure of the primary and the secondary palate were described. A characteristic epithelial thickening was observed prior to epithelial fusion between the nasal septum and the secondary palatal shelf. Fusion of the opposing epithelia was characterized by formation of desmosomes. An unilateral or bilateral empty space was observed on each side of the epithelial seam, and it was suggested that this may represent the incisive foramen in the adult.

The intrinsic forces necessary for directing the reorientation of the secondary palate appear to reside in the anterior two thirds of the palate or presumptive hard palate. The hard palate could reorient regardless of whether it was intact or separated from the posterior third or presumptive soft palate. The soft palate could only reorient if the palate shelves are left intact. These intrinsic forces, within the hard palate, may be mediated by the mesenchymal cells, their extracellular matrix, or the epithelium surrounding the shelves. This latter possibly was tested by removing the epithelium, from either the presumptive oral or nasal surface followed by measurement of reorientation in vitro. Only after removal of the oral epithelium was a significant inhibition in reorientation observed. The treatment used to remove the epithelium, EDTA and scraping, was shown to remove 41% of the oral epithelium leaving the majority of the basement membrane intact. The observed inhibition of reorientation did not appear to be a consequence of wound healing. Creation of wounds twice the area that was observed after treatment with EDTA and scraping inhibited reorientation minimally. These results suggest that the epithelium and particularly the anterior oral epithelium plays a major role in the reorientation of the murine secondary palate.

The morphology of musculus levator veli palatini in the 15-week fetus was analyzed using 30-micrometer subserial sections. The sample included 26 specimens, of which 9 each were sectioned coronally and sagittally while 8 were sectioned in the transverse plane. At this stage of development m. levator veli palatini takes a general attachment to the precursor of the petrous part of the temporal bone, and, in some cases, auxiliary attachments to the auditory tube complex were also observed. At its origin, the muscle is located anterior to the tube. It then runs medially, passing beneath the auditory tube prior to entering the velum. As it nears the region of the lateral pharyngeal wall, a small fascicle trails posteriorly and inferiorly to the main muscle mass and occasionally runs into the upper margin of m. constrictor pharyngis superior. The levator muscle is more localized within the velum at this stage of development than it has been reported to be in the adult, being confined here to the central third of the soft palate. Most of the fibers of the muscle appear to form a sling within the central 20% of the velum, although some were seen to take attachment to loose connective tissue and the palatine raphe. Upon its entry into the velum, m. levator veli palatini is intersected vertically by bundles of both mm. palatoglossus and palatopharyngeus.

Cell release from the palatal shelves and the fusion line prior to and during closure of the secondary palate was studied in Sprague-Dawley rat fetuses of a precise, individually determined age. Fetuses with normal and with experimentally retarded or failing palatal closure were examined by light- and electronmicroscopy. Three different phenomena were observed involving either locally restricted areas or the entire medial shelf edge: (1) desquamation of superficial cells, (2) extrusion of cell blebs, and (3) surface accumulation of basal cells. It is suggested, that desquamation is a process facilitating primary contact and fusion between undifferentiated cells, while extrusion of cell blebs probably represents degradation of the epithelial seam by cellular escape. Basal cells were seen at the surface of unfused shelf edges in fetuses with partial or total palatal clefting, as well as in fetuses with a normal but timewise delayed palatogenesis. It was assumed that, under normal conditions, epithelial surface desquamation is followed or accompanied by basal cell migration and that this may provide an additional mechanism assuring shelf contact between deep-seated cells.

Responses of the epithelial and mesenchymal cells of the developing hamster secondary palate to hadacidin, 5-fluorouracil, and hydrocortisone were analysed. Correlation of data on cellular response with morphological and biochemical observations suggested that several aspects of differentiation of palatal cells needs to be analysed before pathogenesis of teratogen-induced cleft palate can be defined.