Development of the lung in mice with bromodeoxyuridine-induced cleft palate

Clinical and laboratory observations show that denial of free communication between the amniotic fluid and lung fluid results in pulmonary hypoplasia. Thus, cleft palate resulting from tongue obstruction to palatal shelf elevation might be associated with disturbed lung development. This association exists in the Pena-Shokeir phenotype. The goal of these experiments was to see what effect bromodeoxyuridine (BUdR)-induced cleft palate had on lung development. LACA mice were injected with 500 mg/kg BUdR on E11 or E11 and E12 of gestation, a treatment known to produce a 25% and 50% incidence of cleft palate, respectively. BUdR had a direct retarding effect on lung growth but, when cleft palate occurred as well, the lungs were more severely affected. Morphometry showed that lungs from fetuses with cleft palate had only one-half the saccular volume of controls or of treated fetuses with normal palates. Although hypoplastic, lungs associated with cleft palate had type I and type II pneumocytes, and the latter were shown by electron microscopy to be capable of producing surfactant. Hence, cellular differentiation had not been affected by the treatment. Fetuses with cleft palate had less amniotic fluid than controls but significantly more than those with normal palates after treatment. Thus, the pattern of abnormalities in this animal model bears some resemblance to that of the human Pena-Shokeir phenotype.     

Altered mandibular development precedes the time of palate closure in mice homozygous for disproportionate micromelia: an oral clefting model supporting the Pierre-Robin sequence

BACKGROUND: Development of the human craniofacial anatomy involves a number of interrelated, genetically controlled components. The complexity of the interactions between these components suggests that interference with the spaciotemporal interaction of the expanding tongue and elongating Meckel's cartilage correlates with the appearance of cleft palate. Mice homozygous for the semi-dominant Col2a1 mutation Disproportionate micromelia (Dmm), presenting at birth with both cleft palate and micrognathia, provide the opportunity to test the hypothesis that mandibular growth retardation coincides with formation of the secondary palate as predicted from our understanding of the Pierre Robin sequence. The present study was conducted in embryonic day 14 (E14) mice, 1 day before palate closure, to describe the relationship between growth of the lower jaw/tongue complex versus genotype of the embryo. METHODS: Whole heads, isolated from E14.25, E14.5 and E14.75 wild-type and homozygous mutant embryos, were fixed in Bouin's solution, embedded in paraffin, and serially sectioned. Mid-sagittal sections, stained with toluidine blue, were used to estimate growth of both tongue and lower jaw (Meckel's cartilage length) during a 12-hr period preceding palate closure. RESULTS: In control embryos, the largest increase in Meckel's cartilage length occurred between E14.5 and E14.75. Compared to control, the mean Meckel's cartilage length in the mutant was similar at E14.25, but was significantly less at E14.5 and E14.75. Absolute tongue size in control embryos increased linearly during this period of E14.25 to E14.75. Relative to the rapidly growing Meckel's cartilage, however, relative tongue size in control embryos actually decreased over time. Absolute tongue size in the mutant was not significantly different from that of control at any of the embryonic stages examined, however, relative tongue size in the mutant was significantly greater at E14.75 compared to control. CONCLUSION: Mandibular growth retardation, coupled with relative macroglossia in E14 Dmm/Dmm mice, suggests that the concerted development of the palate and lower jaw complex in the mutant is aberrant. Detection of micrognathia and pseudomacroglossia in homozygotes, before the time of palate closure, supports the hypothesis that a relationship exists between growth retardation of Meckel's cartilage and malformation of the secondary palate, as predicted by the Pierre-Robin sequence.     

A morphometric analysis of craniofacial growth and changes in spatial relations during secondary palatal development in human embryos and fetuses

Staged human embryos and fetuses in the Carnegie Embryological Collection were morphometrically analyzed to show craniofacial dimensions and changes in spatial relations, and to identify patterns that would reflect normal developmental events during palatal formation. Normal embryos aged 7-8 weeks postconception (Streeter-O'Rahilly stages 19-23) and fetuses aged 9-10 weeks postconception, in eight groups with mean crown-rump (CR) lengths of 18-49 mm, were studied with cephalometric methods developed for histologic sections. In the 4-week period studied, facial dimensions increased predominantly in the sagittal plane with extensive changes in length (depth) and height, but limited changes in width. Growth of the mandible was more rapid than the nasomaxillary complex, and the length of Meckel's cartilage exceeded the length of the oronasal cavity at the time of horizontal movement of the shelves during stage 23. Simultaneously with shelf elevation, the upper craniofacial complex lifted, and the tongue and Meckel's cartilage extended forward beneath the primary palate. Analysis of spatial relations in the oronasal cavity showed that the palatomaxillary processes became separated from the tongue--mandibular complex as the head extended, and the tongue became positioned forward with growth of Meckel's cartilage. As the head position extended by 35 degrees, the cranial base angulation was unchanged and the primary palate maintained a 90 degrees position to the posterior cranial base. However, the sagittal position of the maxilla relative to the anterior cranial base increased by 20 degrees between stages 19 and 23. In the late embryonic and early fetal periods, the mean cranial base angulation of approximately 128 degrees and the mean maxillary position angulation of approximately 84 degrees were similar to the angulations previously shown to be present later prenatally and post-natally. The results suggest that human patterns of cranial base angulation and maxillary position to the cranial base develop during the late embryonic period when the chondrocranium and Meckel's cartilage form the primary skeleton.     

Abnormal head posture associated with induction of cleft palate by methylmercury in C57BL/6J mice

Maternal treatment with methylmercury (MeHg) has been shown to induce a high frequency of cleft palate and produce growth retardation in rat and mouse fetuses, but the relation between these effects is unknown. The objective of this study was to determine if mandibular growth retardation was a factor that contributed to induction of cleft palate in C57BL/6J mice. Two doses of MeHg (10 mg/kg maternal body weight) were given subcutaneously on days 10 and 11 of gestation, and the fetuses were morphometrically studied on days 14, 15, and 18. Full clefts of the secondary palate were present in approximately half of the treated day 15 and 18 fetuses; therefore, the cleft palate (CP) and noncleft palate (NCP) groups were analyzed separately to facilitate identification of morphologic changes associated with the clefting. The results showed that, compared with controls, the day 14 MeHg-treated fetuses had significantly smaller placental weights, but only half of the fetuses had delayed palatal shelf elevation, reduced body weight, and delayed morphological development. However on day 15, the CP and the NCP groups had similar reductions in body weight and placental weight. A striking downward and forward positioning of the head was present in the MeHg-treated fetuses with the CP group more severely affected than the NCP group. Significant differences between the three groups (control, NCP, and CP) were present with mean head-to-body angles of 67 degrees, 60 degrees and 51 degrees, respectively. The absence of normal head lifting resulted in a relative mandibular retrognathia that when combined with a decrease in mandibular length produced alterations in spatial relations that were most severe in the CP fetuses. The results suggest that after exposure to MeHg, palatal closure is affected by altered tongue posture associated with the abnormal head positioning and shortening of the mandible that develop following placental and embryonic growth retardation.     

Effect of diazepam on the embryonic development of the palate in the rat

The results of previous studies on the effect of diazepam on palate formation in animals have been inconclusive. Teratogen-induced cleft palate is usually caused by a delay in palatal shelf elevation. The present study investigated the effect of diazepam on palate formation in the Sprague-Dawley rat. Five groups of dams received subcutaneous doses of either 10, 20, 30, 40, or 50 mg/kg body weight of diazepam. Control dams received propylene glycol (vehicle). Dams in each dosage group were killed at 16.9 (16 d 9 h); 16.16, and 17.9 days of gestation, respectively, to assess delay in palatal shelf elevation. Crown rump length (CRL) of 1,283 fetuses collected from 105 dams was measured. Fetuses in each time/dosage group showed a reduction in CRL (P less than .01). With increasing dosage the number of fetuses showing delayed palatal shelf elevation was significantly increased (P less than .01). These results demonstrate that with an increase in dose there is an increased delay in palatal shelf elevation and a decrease in CRL. However, in this strain there seems to be a rapid prenatal recovery, resulting in a marked reduction in the incidence of delayed palatal shelf elevation.     

Human prenatal palatal closure related to skeletal maturity of the jaws

The purpose of the present study was to elucidate the stages of skeletal maturation of the maxilla and the mandible at the time of soft tissue palatal closure. Similar studies were not found in the literature. This investigation was based on maxillae and mandibles from 19 human embryos/fetuses selected by visual inspection of palatal structures, eight fetuses "just before palatal closure," and 11 fetuses "just after palatal closure." The findings are related to formerly described skeletal developmental stages in the maxilla, to stages in the symphysis menti region, and to development in the mandibular condylar region. The present study revealed that elevation of the palatal shelves takes place at a specific stage of maxillary skeletal maturity (stage Max III), at a time of mandibular development characterized by absence of condylar cartilage and by constancy in symphysis menti maturity (stage SM I). Knowledge of the normal sequence of prenatal skeletal development is considered essential for understanding the abnormal sequence of skeletal development.     

Epidermal growth factor potentiates cortisone-induced cleft palate in the mouse.

Epidermal growth factor (EGF) injected into pregnant mice increased the frequency of cleft palate (CP) in cortisone-treated mouse fetuses. EGF alone produced proliferation and thickening of the epithelium of the palatal processes, but CP was not significantly increased over saline injected controls. Cortisone alone produced thinning of the palatal epithelium and caused CP in 61 percent of formed fetuses. The combination of EGF and cortisone treatment induced CP in 100 percent of formed fetuses; epithelial thickening still occurred with the combination treatment. Thus, EGF may be teratogenic under special circumstances. These observations suggest that the relative thickness of the palatal shelf epithelium may not be a critical factor in the fusion of the palatal shelves.     

Pathogenesis of bromodeoxyuridine-induced cleft palate in hamster

In the present study, the morphological, histochemical, biochemical, and cellular aspects of the pathogenesis of bromodeoxyuridine (BrdU)-induced cleft palate in hamster fetuses were analyzed. Morphological observations indicated that BrdU interferes with the growth of the vertical shelves and thus induces cleft palate. At an ultrastructural level, BrdU-induced changes were first seen in the mesenchymal cells. Eighteen hours after drug administration, the initial alterations were characterized by swelling of the nuclear membrane and the appearance of lysosomes in the mesenchymal cells of the roof of the oronasal cavity. During the next 6 hr, as the palatal primordia developed, lysosomes were also seen in the overlying epithelial cells. The appearance of lysosomal activity, which was verified by acid phosphatase histochemistry, was temporally abnormal and was interpreted as a sublethal response to BrdU treatment. Later the cellular alterations subsided; 48 hr after BrdU treatment, they were absent in both the epithelial and mesenchymal cells of the vertically developing palatal shelves. Subsequently, unlike controls (in which the palatal shelves undergo reorientation and fusion), the BrdU-treated shelves remained vertical until term. Biochemical determination of DNA synthesis indicated that although there was an inhibition of DNA synthesis at the time of appearance of palatal primordia, a catch-up growth during the ensuing 12 hr may have restored the number of cells available for the formation of a vertical palatal shelf. It was suggested that BrdU affected cytodifferentiation in the palatal tissues during the critical phase of early vertical development to induce a cleft palate.     

Radiographic analysis of the midface of a stillborn infant with a unilateral cleft lip and palate

The midface of a full-term stillborn infant with a right complete unilateral cleft lip and palate was studied with plain-film radiography and tomography, xeroradiography, and computerized axial tomography. Gross skeletal and soft-tissue deficiencies on the cleft side were evident as compared to the noncleft side and involved the entire bony maxillary complex and antrum, the orbit, and the nasal pyramid and intranasal structures. The area on the cleft side was 19 percent less than the noncleft side, and the maximal anteroposterior dimension was 16 percent less. The cleft bony palatal shelf was 12.5 mm wide compared to 20 mm on the noncleft side. The findings demonstrate the deficient and abnormal functional matrix inherent in the cleft condition.     

Experimental induction of palate shelf elevation in glutamate decarboxylase 67-deficient mice with cleft palate due to vertically oriented palatal shelf

BACKGROUND: Gamma-aminobutyric acid is an inhibitory neurotransmitter, synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65 and -67. Unexpectedly, inactivation of GAD67 induces cleft palate in mice. Reduction of spontaneous tongue movement resulting from decreased motor nerve activity has been related to the development of cleft palate in GAD67(-/-) fetuses. In the present study, development of cleft palate was examined histologically and manipulated with culture of the maxilla and partial resection of fetal tongue. METHODS: GAD67(-/-) mice and their littermates were used. Histological examination and immunohistochemistry were performed conventionally. Organ culture of the maxilla was carried out as reported previously. Fetuses were maintained alive under anesthesia and tips of their tongues were resected. RESULTS: Elevation of palatal shelves, the second step of palate formation, was not observed in GAD67(-/-) mice. In wild-type mice, GAD67 and gamma-aminobutyric acid were not expressed in the palatal shelves, except in the medial edge epithelium. During 2 days of culture of maxillae dissected from E13.5-E14.0 GAD67(-/-) fetuses, elevation and fusion of the palatal shelves were induced. When E13.5-15.5 mutant fetuses underwent partial tongue resection, the palatal shelves became elevated within 30 min. CONCLUSIONS: These results suggest that the potential for palate formation is maintained in the palatal shelves of GAD67(-/-) fetuses, but it is obstructed by other, probably neural, factors, resulting in cleft palate.     

Chondrodystrophic mice with coincidental agnathia: evidence for the tongue obstruction hypothesis in cleft palate

Mice homozygous for either of two mutations, chondrodysplasia (cho) or cartilage matrix deficiency (cmd), have short-limbed chondrodystrophy. This phenotype includes retrognathia, relative macroglossia, and cleft palate. It has been postulated that the cleft palate in these mice is the result of tongue obstruction during palatogenesis. Agnathia associated with microglossia is an independent spontaneously occurring defect in the strains bearing these mutations. The coincidental occurrence of agnathia-microglossia with chondrodystrophy lends itself to the study of the mechanism of cleft palate formation. We examined approximate midsagittal histological sections of normal and chondrodystrophic newborn mice, both with and without agnathia. Mandibular measurements and examinations of palate closure and tongue structure were made from photographic prints. Typical chondrodystrophic mutants with cleft palates had a mean mandibular length that was 66% of normal and a tongue that appeared large relative to the shortened mandible. Chondrodystrophic mutants with agnathia and microglossia had a mean mandibular length that was further reduced to 30% of normal, yet had a closed palate. We also observed two nonagnathic chondrodystrophic mutants that had slightly decreased mandibular lengths, microglossia, and closed palates. These observations suggest that tongue obstruction during palatogenesis is the pathogenetic mechanism of cleft palate in chondrodystrophic mice. A similar tongue obstruction hypothesis has been proposed as the mechanism of cleft palate formation in the human Pierre Robin sequence, which consists of retrognathia, glossoptosis, and cleft palate. This mechanistic hypothesis has been challenged, but our findings support the tongue obstruction hypothesis in the Robin cleft.     

Temporal and Spatial Expression of Hoxa-2 During Murine Palatogenesis

1. Mice homozygous for a targeted mutation of the Hoxa-2 gene are born with a bilateral cleft of the secondary palate associated with multiple head and cranial anomalies and these animals die within 24 hr of birth (Gendron-Maguire et al., 1993; Rijli et al., 1993; Mallo and Gridley, 1996). We have determined the spatial and temporal expression of the Hoxa-2 homeobox protein in the developing mouse palate at embryonic stages E12, E13, E13.5, E14, E14.5, and E15.2. Hoxa-2 is expressed in the mesenchyme and epithelial cells of the palate at E12, but is progressively restricted to the tips of the growing palatal shelves at E13.3. By the E13.5 stage of development, Hoxa-2 protein was found to be expressed throughout the palatal shelf. These observations correlate with palatal shelf orientation and Hoxa-2 protein may play a direct or indirect role in guiding the palatal shelves vertically along side the tongue, starting with the tips of the palatal shelves at E13, followed by the entire palatal shelf at E13.5.4. As development progresses to E14, the stage at which shelf elevation occurs, Hoxa-2 protein is downregulated in the palatal mesenchyme but remains in the medial edge epithelium. Expression of Hoxa-2 continues in the medial edge epithelium until the fusion of opposing palatal shelves.5. By the E15 stage of development, Hoxa-2 is downregulated in the palate and expression is localized in the nasal and oral epithelia.6. In an animal model of phenytoin-induced cleft palate, we report that Hoxa-2 mRNA and protein expression were significantly decreased, implicating a possible functional role of the Hoxa-2 gene in the development of phenytoin-induced cleft palate.7. A recent report by Barrow and Capecchi (1999), has illustrated the importance of tongue posture during palatal shelf closure in Hoxa-2 mutant mice. This along with our new findings of the expression of the Hoxa-2 protein during palatogenesis has shed some light on the putative role of this gene in palate development.     

D-penicillamine-induced cleft palate in mice

The teratogenic potential of the lathyrogen, D-penicillamine (DP), was assessed in pregnant mice, especially with respect to its ability to produce cleft palate. The dosage and the duration of treatment as they relate to the induction of cleft palate were also studied. Two different doses of DP were administered orally for either 5 or 4 consecutive days during the critical period of palatal closure. D-penicillamine (DP) at a dose level which does not have any apparent maternal toxic effects produced cleft palate in the offspring, and this teratogenic effect depended more upon the duration of treatment than the dosage administered. Inhibitory effects on the formation of bone matrix were observed at the base of the palatal shelf. It is suggested that DP is potentially an osteolathyrogenic agent. The mechanism of induction of cleft palate in DP-treated mice was explored by histological studies using light microscopy. Delayed elevation of the palatal shelves was observed and is considered to be the cause of the induction of cleft palate. No other external malformations could be detected in DP-treated fetuses.     

Developmental anomalies induced by all-trans retinoic acid in fetal mice: I. Macroscopic findings

The administration of a single dose of all-trans retinoic acid on day 8 of gestation to pregnant mice, ICR strain, led to malformed fetuses in all of the litters. All-trans retinoic acid (RA) was dissolved in olive oil and given in doses of 60 or 40 mg/kg of body weight. The control mice were given vehicle alone. Examination on day 18 of gestation of the fetuses exposed to 60 mg/kg showed various malformations, such as exencephaly, exophthalmus, micrognathia, agnathia, cleft palate, cleft lower lip, spina bifida, atresia ani, tail anomalies, agenesis of the kidneys, or hydronephrosis. In the fetuses exposed to 40 mg/kg, isolated cleft palate was much more common than in those exposed to 60 mg/kg. Double-stained preparations of bone and cartilage showed cranio-facial anomalies and axial skeletal anomalies: a- or hypogenesis of palatine or maxillary bones, tympanic ring, squamosal temporal bone or otic ossicles in cartilage, and fusion of basioccipital to basisphenoid and maxilla, zygomatic and mandibular bones; a- or hypogenesis of caudal vertebrae and supernumerary thoracic and lumbar vertebrae. These results indicate that anomalies comparable to those seen in the infants of mothers treated with isotretinoin, 13-cis retinoic acid, during pregnancy can also be induced in mice and suggest that the site affected by RA may be neural crest cells, including those in the cephalic and caudal regions, and cells committed to somitic mesoderm in the trunk region.     

Gross and cellular analysis of 6-mercaptopurine-induced cleft palate in hamster

The present study analyzes the morphological, histochemical, and ultrastructural aspects of the pathogenesis of 6-mercaptopurine (6MP)-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that 6MP stunts the growth of vertical palatal shelves and thus induces cleft palate. Ultrastructural analysis showed that, in contrast to controls, 6MP-induced alterations were first seen in the mesenchymal cells 24 hr after drug administration. The initial alterations were characterized by swelling of the nuclear membrane. During the next 12 hr, lysosomes were seen first in the mesenchymal cells and then in the cells of the medial edge epithelium (MEE) of the developing palatal primordia. The appearance of lysosomes was temporally abnormal and was interpreted as a sublethal response to 6MP treatment. Subsequently, the nuclear alterations and the lysosomes diminished; and 48 hr after 6MP administration, they were absent from the palatal tissues. Ninety hours after 6MP administration, unlike the controls (in which the palatal shelves were already fused), changes were seen at the epithelial-mesenchymal interface in the developing cleft palatal shelves. These changes were characterized by breakdown of the basal lamina and epithelial-mesenchymal contacts. Eventually, at term, the MEE of the vertical shelf stratified. It was suggested that 6MP affected cytodifferentiation in the palatal tissues during the critical phase of early vertical shelf development and thereby induced cleft palate.     

Morphological observations in normal primary palate and cleft lip embryos in the Kyoto collection

Normal developmental events during human primary palate formation and alterations associated with cleft lip remain poorly defined. The purpose of this study was to analyze serially sectioned human embryos to identify morphological changes during normal palatal closure and alterations associated with failure of palatal formation. Normal and cleft embryos from the histological collection at the Congenital Anomaly Research Center at the University of Kyoto were studied and photographed for detailed evaluation. Seven serially sectioned cleft lip embryos of stages shortly after primary palate formation (Streeter-O'Rahilly stages 19, 20, and 22) with unilateral or bilateral clefts with varying degrees of clefting were studied. In the normal Kyoto embryos, initial nasal fin (epithelial seam) formation was observed between the medial nasal process and the lateral nasal and maxillary processes at stage 17. During stages 18 and 19, the nasal fin epithelium was replaced by an enlarging mesenchymal bridge, as the maxillary processes united with the medial nasal processes to form the primary palate. The most prominent features observed in the cleft embryos were a reduced thickness of mesenchymal bridging between the medial nasal and maxillary processes, with an excessive amount of epithelium at the junctions between these processes. With ingrowth of the maxillary processes, greater cell dispersion and apparent extracellular matrix accumulation were observed in the medial nasal region. During closure of the primary palate, terminal branches of the maxillary nerve crossed the mesenchymal bridge to the medial nasal region. The partial clefts had reduced maxillary ingrowth and smaller union areas with the medial nasal process. Detailed studies of experimental animal models are required to identify regional growth required for contact between the facial prominences, to clarify the mechanisms of mesenchymal ingrowth and epithelial displacement during palatal formation, and to identify local and/or general factors causing alterations that lead to primary palatal clefting.     

Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFβ3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.     

Palate development after fetal tongue removal in cortisone-treated mice

Morphological studies of cortisone-induced cleft palate have shown retardation in the rotation of palatine shelves from a sagittal to a transverse plane. Cortisone also reduces fetal muscular movements, which may explain why displacement of the tongue from between the palatine shelves is delayed. Previous work with extrauterine development of control fetuses demonstrated that fetal membranes and tongue were major obstacles to shelf rotation. Thus, removal of these obstacles might permit rotation and fusion of palatine shelves in cortisone-treated fetuses. In the present experiment, fetuses from cortisone-treated strain CD-1 mice were released from uterus and membranes and allowed to develop for eight hours in a fluid medium with the umbilical cord left intact. Compared to 4% fusion in utero, there was palatal fusion in 20% of fetuses released from membranes. When the fetal tongue was removed during extrauterine development, the frequency of fusions increased to 61%. Fusion appeared normal by the criteria applicable through light microscopy. Thus, cortisone induces cleft palate primarily through interference with shelf rotation. The palatine shelves of treated fetuses retain their ability to fuse when they can come in contact during the normal time for palate closure.     

Prenatal repair of experimentally induced clefts in the secondary palate of the rat

A refined technique of amniotic sac puncturing at day 16.2 (i.e., 16 + 2/10 days) of gestation was employed in order to produce a series of total clefts and rare forms of partial clefts in Sprague-Dawley rat fetuses. From a total of 410 fetuses of a precise, individually determined age, 95 upper jaws were examined in the scanning electron microscope and, in part, in serial Epon sections. All fetal heads were examined macroscopically. Total clefts were found in 48.9% of a total of 184 viable rat fetuses examined at day 17.8 of smear age and in 21.8% of a total of 211 fetuses examined at day 19.3. Partial clefts were observed in 14.1% and 18.5% of fetuses at days 17.8 and 19.3 of smear age, respectively. At day 19.3, 16.1% of the viable fetuses showed a very inconspicuous, small abnormality (with residual clefting and incomplete fusion with the nasal septum) in the region of the palatine foraminae. Morphological observations suggested that under conditions of detained palatal closure (1) fusion of the soft palatal shelves commences independently from and prior to fusion of the hard palate, (2) delayed palatal shelf fusion proceeding in the anterior direction may occur with or without remaining sickle-shaped clefts in the anterior hard palate, and (3) in fetuses with small sickle-shaped clefts, fusion of the palatal shelves with the nasal septum does not occur. The present data imply that an almost total prenatal repair and delayed closure of the secondary palate may occur in rats that, at day 16.2 of multiple analysis age, most certainly had a total palatal cleft resulting from tongue resistance.