Genetic aspects of the effects of methylmercury in mice: the incidence of cleft palate and concentrations of adenosine 3':5' cyclic monophosphate in tongue and palatal shelf

Concentrations of adenosine 3':5' cyclic monophosphate (cAMP) were measured in the tongues and palates of 14.5-day-old fetuses from control and methylmercury-treated mothers of four inbred lines of mice which represent the four possible combinations of two H-2 alleles and two residual genetic backgrounds. The incidence of cleft palate in fetuses from control and methylmercury-treated mothers was also examined. The H-2 alleles significantly affected the degree of reduction of cAMP concentration in palates seen in fetuses from mothers treated with methylmercury. Neither the H-2 allele nor the residual genetic background played a role in the effect of methylmercury on cAMP concentrations in fetal tongues. The magnitude of increase in the incidence of cleft palate with methylmercury treatment was approximately the same for all lines. Thus, methylmercury-induced cleft palate may not be mediated by the reduction of cAMP. Finally, fetuses with cleft lip had increased palatal cAMP levels, whether or not they were from control or methylmercury treated mothers.     

Study of the mechanisms of BUdR-induced cleft palate in the mouse

This study was designed to examine the pathogenesis of bromodeoxyuridine (BUdR)-induced clefts of the secondary palate in the LACA mouse. Intraperitoneal injections of BUdR (500 mg/kg body weight) were given at various days and combinations of days between E11 and E15 (plug day = E1). Treatment on E11 alone resulted in approximately 22% of fetuses with cleft palate when the latter were examined either on E16 or E19. Treatment on E11 and E12 approximately doubled the above incidence, and treatment on E11, 12 and 13 raised it to 100%. However, no treatment, either single or multiple, caused cleft palate when given later than E11. This suggests that the cellular changes caused by BUdR that lead to cleft palate must be inflicted during E11 and that such damage can be repaired in about 80% of embryos. All fetuses with cleft palate had severe micrognathia on E16 and E19, which skeletal staining showed to be the result of a bilateral sigmoid buckling of Meckel's cartilage. Studies with the scanning electron microscope (SEM) on E15, 16, and 19 suggested strongly that the micrognathia caused a relative macroglossia and hence mechanical interference with palatal shelf reorientation. Histological studies with the light microscope showed that BUdR caused cellular necrosis in many embryonic tissues during the 24 hours after its administration. This necrosis was strikingly more severe in the mandibular rudiment of the first branchial arch than in the maxillary. The latter observation accords well with findings by other workers that cell proliferation is more rapid in the mandibular blastema than in the maxillary. Transmission electron microscope (TEM) studies of the buckled region of Meckel's cartilage failed to reveal any ultrastructural differences from control Meckel's cartilage. Hence BUdR had only interfered with the shape of the cartilage but not with its histiogenesis. We conclude that BUdR, by its cytotoxicity or antidifferentiative effects, interfered with the formation of the anterior end of Meckel's cartilage, initiating a chain of events leading through micrognathia and relative macroglossia to failure of palatal shelf reorientation and 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.     

Tissue phosphatase changes following triamcinolone associated with cleft palate in rats.

One theory of the development of cleft palate in rats involves the action of lysosomal enzymes secreted by epithelial cells at the time of fusion of the palatal shelves. To test this theory we studied the biochemistry of the palates of fetal rats daily between days 14 and 19 (from 3 days before to 3 days after palate closure). Triamcinolone was administered once im on gestation day 14 to Wistar rats; 0.5 mg/kg body weight produced approximately 50% cleft palates. Pooled control palatal tissue was compared with pooled experimental tissue; that from fetuses with clefts being pooled separately from those not affected. Acid phosphatase and beta-glucuronidase were assayed. Concentration vs. time curves for both enzymes were very similar. Prior to the time of palate closure both enzymes were present in low concentration. Between days 16 and 17, the normal time of closure, there was an abrupt increased in enzyme concentration, with experimental tissue showing a significant elevation over control tissue on days 17 and 18. Alkaline phosphatase was also present in small amounts before closure and significantly higher in control tissue on day 17. Protein was depressed in palates having clefts on day 17; thus the ratio of enzyme activities to protein synthesis was significantly elevated at a critical time. Unaffected experimental palates had a normal ratio. These results suggest imbalanced acid phosphatase, beta-glucuronidase, and alkaline phosphatase activity compared with protein synthesis at the time of palate closure following triamcinolone in rats.     

Amniotic fluid induces rapid epithelialization in the experimentally ruptured fetal mouse palate--implications for fetal wound healing

Cleft of the secondary palate is one of the most common congenital birth defects in humans. The primary cause of cleft palate formation is a failure of fusion of bilateral palatal shelves, but rupture of the once fused palate has also been suggested to take place in utero. The possibility of post-fusion rupture of the palate in humans has hardly been accepted, mainly because in all the cleft palate cases, the cleft palatal edge is always covered with intact epithelium. To verify whether the intrauterine environment of the fetus plays roles in wound healing when the once fused palate is torn apart, we artificially tore apart fetal mouse palates after fusion and cultivated them in culture medium with or without mouse or human amniotic fluid. We thereby found that the wounded palatal edge became completely covered with flattened epithelium after 36 hours in culture with amniotic fluid, but not in culture without amniotic fluid. Using histological and scanning electron microscopic analyses of the healing process, it was revealed that the epithelium covering the wound was almost exclusively derived from the adjacent nasal epithelium, but not from the oral epithelium. Such actions of amniotic fluid on the fetal wound were never simulated by exogenous epidermal growth factor (EGF), albumin, or both. In addition, the rapid epithelialization induced by amniotic fluid was not prevented by either PD168393 (an inhibitor of the EGF receptor-specific tyrosine kinase) or SB431542 (a specific inhibitor of TGFbeta receptor type I/ALK5). The present study provides new insights into the unique biological actions of amniotic fluid in the repair of injured fetal palate.     

The fetal cleft palate: II. Scarless healing after in utero repair of a congenital model.

The role of fetal surgery in the treatment of non-life-threatening congenital anomalies remains a source of much debate. Before such undertakings can be justified, models must be established that closely resemble the respective human anomalies, and the feasibility and safety of these in utero procedures must be demonstrated. The authors recently described and characterized a congenital model of cleft palate in the goat. The present work demonstrates the methodology they developed to successfully repair these congenital cleft palates in utero, and it shows palatal healing and development after repair. A surgically created cleft model was developed for comparative purposes. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Six pregnant goats were gavaged twice daily during gestational days 32 to 41 (term, 145 days) with a plant slurry of Nicotiana glauca containing the piperidine alkaloid anabasine; the 12 fetuses had complete congenital clefts of the secondary palate. Repair of the congenital clefts was performed at 85 days of gestation using a modified von Langenbeck technique employing lateral relaxing incisions with elevation and midline approximation of full-thickness, bilateral, mucoperiosteal palatal flaps followed by single-layer closure. Six congenitally clefted fetuses underwent in utero repair, six remained as unrepaired controls. Twelve normal fetuses underwent surgical cleft creation by excision of a 20 x 3 mm full-thickness midline section of the secondary palate extending from the alveolus to the uvula, at 85 days of gestation. Six surgically clefted fetuses underwent concurrent repair of the cleft at that time; six clefted fetuses remained as unrepaired controls. At 2 weeks of age, no congenitally or surgically created clefts repaired in utero demonstrated gross or histologic evidence of scar formation. A slight indentation at the site of repair was the only remaining evidence of a cleft. At 6 months of age, normal palatal architecture, including that of mucosal, muscular, and glandular elements, was seen grossly and histologically. Cross-section through the mid-portion of the repaired congenitally clefted palates demonstrated reconstitution of a bilaminar palate, with distinct oral and nasal mucosal layers, after single-layer repair. In utero cleft palate repair is technically feasible and results in scarless healing of the mucoperiosteum and velum. The present work represents the first in utero repair of a congenital cleft palate model in any species. The use of a congenital cleft palate model that can be consistently reproduced with high predictability and little variation represents the ideal experimental situation. It provides an opportunity to manipulate specific variables, assess the influence of each change on the outcome and, subsequently, extrapolate such findings to the clinical arena with a greater degree of relevance.     

The influence of experimentally produced oligohydramnios on lung growth and pulmonary surfactant content in fetal rabbits.

To study the effect of oligohydramnios on lung growth and biochemical lung development in fetal rabbits, amniotic fluid was drained through a tube inserted into the maternal peritoneal cavity on the 23 day of gestation. Littermate fetuses without an amniotic shunt were used as controls. The fetuses were delivered abdominally on the 28 day of gestation. In a total of 8 pregnant does, 17 fetuses underwent amniotic shunting and 22 fetuses were used as controls. The amniotic shunt produced a significant reduction in the amniotic fluid volume. There were no differences in the wet weights of the fetal body, liver or brain between the two groups. However, the amniotic shunt significantly decreased the wet weight of the fetal lung, fetal lung wet weight/body weight ratio, and protein concentration per lung as compared to the control fetuses. In the fetal liver and brain tissues, no changes were found in the concentrations of total phospholipids, phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC, the main component of lung surfactant) per g of wet tissue and per mg of protein. However, the lungs of the fetuses with amniotic shunts contained significantly more PC and DSPC, and the L/S ratio was higher than in the control fetuses. These results suggest that the oligohydramnios produced by an amniotic shunt causes pulmonary hypoplasia, but raises the pulmonary surfactant content of fetal rabbit lung.     

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.     

The effect of prolonged oligohydramnios on fetal lung development, maturation and ventilatory patterns in the newborn guinea pig

We drained the amniotic fluid surrounding guinea pig fetuses between days 45 and 65 of gestation (term is 67 days). The fetuses were delivered by Cesarean section and the impact of prolonged oligohydramnios on lung growth, maturation and postnatal ventilatory pattern was measured. Untouched littermate fetuses served as controls. Neither fetal body, liver nor brain weights were significantly affected by the experimental situation. When expressed in percent of control values, lung weight (63%), lung/body weight ratio (70%), lung volume (67%), total lung DNA content (63%) and lung DNA per gram of fetal weight (71%) were all significantly less following amniotic fluid drainage, confirming the diagnosis of lung hypoplasia. Disaturated phosphatidylcholine content per gram of lung tissue and total lung glycogen content were not affected by the procedure, indicating that the maturity of the hypoplastic lungs was not delayed. When measured 4 to 6 hours after birth, tidal volume was significantly less (62%) and respiratory frequency was significantly more (137%); however, minute ventilation per unit of body weight was not significantly changed. This animal model of sublethal lung hypoplasia could become useful to study the potential for, and the kinetics of, postnatal catch-up lung growth about which little is known.     

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.     

Morphological characteristics of monosomy X in spontaneous abortions

From a morphologic and cytogenetic study of 160 spontaneous abortion specimens with 45,X, as well as data from the literature, we conclude that monosomy X has characteristics which can suggest the diagnosis in prenatal life in the absence of a karyotype. Specimens in our study could be classified into four groups: (I) 49% consisted of a chorionic and amniotic sac, usually ruptured, containing a well-defined umbilical cord with a fragment of embryonic tissue at its end. (II) 25% consisted of small macerated embryos of 40-44 days developmental age, with pronounced retrognathia and lack of nasofrontal angle. Two specimens had a neural tube defect, one had cleft lip and palate, and two had an isolated cleft palate. The developmental age of the embryos was 5-6 weeks behind their gestational age. (III) 5% were very macerated second trimester fetuses with cystic hygromata, lymph-edema of the hands and feet, ascites, hydrothorax, and hypoplastic lungs. Four had coarctation of the aorta, three had single umbilical artery, and two had persistent left cardinal vein. Horseshoe kidney, ventricular septal defect, and anomalous subclavian artery were found once. (IV) The remaining 21% consisted only of fragments of ruptured sacs without an umbilical cord insertion site, pieces of fetal membranes or chorionic villi only. The spectrum of anomalies observed suggests that the pathogenetic mechanism for early death in 45,X embryos and fetuses may be related to vascular abnormalities or to abnormal fluid balance, leading to disturbed embryo-placental circulation, and excess fluid volume in the fetus. The 45,X karyotype is compatible with quite normal morphological development up to the fetal stage, and no specimens were found where development had ceased at a very early stage. The usual lethality of monosomy X may be explained by the presence of genes, essential for survival, on the pseudoautosomal segment of the X and Y. Lethality could result from recessive lethal mutations in this region, or from the necessity for two copies of this regions for normal development.     

Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses

We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on 22-day gestation (term) fetuses. Controls were nitrofen-exposed fetuses without CDH. Absorption of distal air space fluid was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. In controls, distal air space fluid absorption was rapid and mediated by beta-adrenoceptors as demonstrated by reversal to fluid secretion after propranolol. Normal lung fluid absorption was also partially inhibited by amiloride. In contrast, CDH fetuses continued to show lung fluid secretion, and this secretion was not affected by either propranolol or amiloride. CDH lungs showed a 67% reduction in alpha-ENaC and beta-ENaC expression, but no change in alpha1-Na-K-ATPase expression. These studies demonstrate: 1) CDH delays lung maturation with impaired distal air space fluid absorption secondary to inadequate Na+ uptake by the distal lung epithelium that results in fluid-filled lungs at birth with reduced capacity to establish postnatal breathing, and 2) the main stimulus to lung fluid absorption in near-term control fetuses, elevated endogenous epinephrine levels, is not functional in CDH fetuses.     

Comparison of cleft palate induction by Nicotiana glauca in goats and sheep

The induction of cleft palate by Nicotiana glauca (wild tree tobacco) during the first trimester of pregnancy was compared between Spanish-type goats and crossbred western-type sheep. Cleft palate was induced in 100% of the embryonic/fetal goats when their pregnant mothers were gavaged with N. glauca plant material or with anabasine-rich extracts from the latter, during gestation days 32-41. Seventy-five percent of newborn goats had cleft palate after maternal dosing with N. glauca during gestation days 35-41, while no cleft palates were induced when dosing periods included days 36-40, 37-39, or day 38 only. The induced cleft palates were bilateral, involving the entire secondary palates with complete detachment of the vomer. Eleven percent of the newborn goats from does gavaged during gestation days 32-41 had extracranial abnormalities, most often contractures of the metacarpal joints. Most of these contractures resolved spontaneously by 4-6 weeks postpartum. One newborn kid also had an asymmetric skull due to apparent fetal positioning. No cleft palates were induced in lambs whose mothers were gavaged with N. glauca plant or anabasine-rich extracts during gestation days 34-41, 35-40, 35-41, 36-41, 35-51, or 37-50. Only one of five lambs born to three ewes gavaged with N. glauca plant material during gestation days 34-55 had a cleft palate, but all five of these lambs had moderate to severe contractures in the metacarpal joints. The slight to moderate contracture defects resolved spontaneously by 4-6 weeks postpartum, but the severe contractures resolved only partially. Embryonic/fetal death and resorption (determined by ultrasound) occurred in 25% of pregnant goats fed N. glauca compared to only 4% of pregnant sheep. Nicotiana glauca plant material contained the teratogenic alkaloid anabasine at 0.175% to 0.23%, dry weight, demonstrating that Spanish-type goats are susceptible to cleft palate induction by the natural toxin anabasine, while crossbred western-type sheep are resistant. However, clinical signs of toxicity were equally severe in goats and sheep, even though maternal alkaloid tolerance was generally lower in sheep. We postulate that an alkaloid-induced reduction in fetal movement during the period of normal palate closure is the cause of the cleft palate and multiple flexion contractures. Teratology 61:203-210, 2000. Published 2000 Wiley-Liss, Inc.     

Evaluation of velopharyngeal closure by CT scan and endoscopy

Computerized tomography (CT) and endoscopy were used for the objective evaluation of velopharyngeal closure. In 19 patients with cleft palates and 9 normal subjects, CT scans of the velopharynx were made both at rest and during vowel phonation with a scanning time of 3.0 seconds and slicing width of 3 mm. At the same time, endoscopic observations of the velopharynx through the nose were carried out both at rest and during phonation. CT scan during phonation clearly demonstrated the mobility of the velopharynx, i.e., elevation of the soft palate, inward movement of the lateral pharyngeal walls, and protrusion of the posterior pharyngeal wall, in a single picture. Its disadvantage is exposure to x-rays and a rather complicated procedure. However, endoscopy is simple with no exposure to x-rays, but its disadvantage is occasional incomplete visualization because of the dead angle created by the elevated soft palate. Thus the combined use of CT and endoscopy can help to determine a rational choice of treatment for cleft palates.     

Genesis of hadacidin-induced cleft palate in hamster: morphogenesis, electron microscopy, and determination of DNA synthesis, cAMP, and enzyme acid phosphatase.

A morphological, electron microscopic, and biochemical study was undertaken to analyze the genesis of hadacidin-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that hadacidin affected the growth of vertical palatal shelves to induce cleft palate. Electron microscopic observations showed that initial hadacidin-induced changes were seen in the mesenchymal cells. Within 12 hr of drug administration, the perinuclear space was swollen and a lysosomal response injury was evident in the mesenchymal cells. Subsequently, 24 hr after hadacidin treatment, lysosomes appeared in the epithelial cells; changes were also seen in the basal lamina which included separation of the lamina densa from the basal cells, duplication of lamina densa, and complete loss of basal lamina. Between 36 and 42 hr post-treatment, the cellular and basal lamina changes subsided, and the epithelium of vertical shelves underwent stratification. Biochemical determination of enzyme acid phosphatase indicated that the levels of enzyme activity in both the control and treated palatal tissues corresponded to the appearance of lysosomes. Measurement of cAMP levels suggested that the peak activity of cAMP corresponded to that of enzyme acid phosphatase and cell injury. The cAMP activity in hadacidin-injured cells, however, was significantly lower in comparison to that of the dying cells of control palates. Hadacidin treatment also affected DNA synthesis in the developing primordia of the palate. It was suggested that hadacidin injures the precursor cells of the palate prior to the appearance of the primordia, and subsequently affects their proliferative behavior, stunting the vertical growth of the palatal shelves and inducing a cleft palate.     

Morphological study of the removed fetus after therapeutic abortion for echographic anomalies (apropos of 42 cases)

In 42 cases, fetal abnormalities were diagnosed by obstetric ultrasonography and the pregnancy was terminated. The malformations included: anencephaly (22), severe hydrocephaly (4, one with a spina bifida), encephalocele and meningocele (2) amniotic band syndrome (4; a correct prenatal diagnosis was performed during the second trimester in two cases), major anterior abdominal wall defects (2), Pena-Shokeir syndrome type I? (I), severe renal abnormalities (2), conjoined twins, dicephalus type (2), cystic hygroma and hydrops fetalis (2), osteogenesis imperfecta, type II (I). Thus, there were 23 fetuses with a polygenetically determined status; five fetuses could be affected by an autosomal recessive disorder.     

Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.     

Pulmonary hypoplasia in chondrodystrophic mice

Lungs of day-18 fetal mice with hereditary chondrodysplasia (cho) were examined histologically and biochemically for pulmonary hypoplasia. Compared with normal littermate controls, the mutant's lungs were smaller by 37 (wet weight) and 22% (dry weight). Total DNA and protein per whole lung were decreased by 13 and 19%, respectively. The significantly smaller-than-normal terminal sacs observed in histological sections of the mutant's lungs corresponded with the greater difference (37%) in lung wet weight. The developmental mechanism for this disorder was further explored by examining the volumes of thoracic cavity and amniotic fluid. The volume of the thoracic cavity of newborn mutants was less than half that of controls, suggesting that the pathogenetic mechanism for the hypoplastic lungs in chondrodysplastic mice includes thoracic dystrophy. Measurement of the amniotic fluid volume revealed polyhydramnios in the mutant, thereby ruling out oligohydramnios as a mechanism. The relevance of this study to human pulmonary hypoplasia in short-limb chondrodystrophy is discussed.     

Changes in lung liquid dynamics induced by prolonged fetal hypoxemia

Our aim was to determine the effect of prolonged fetal hypoxemia, induced by reduced maternal uterine blood flow (RUBF), on fetal lung liquid secretion, flow, and volume. In chronically catheterized fetal sheep, lung liquid volume (VL) and the secretion rate of lung liquid (Vs) were measured before and after a 24-h period of either RUBF or normoxemia. Tracheal fluid flow and the incidence of fetal breathing movements (FBM) were measured before, during, and after the 24-h period. In normoxic control fetuses Vs was not significantly altered. After 24 h of RUBF, Vs was significantly (P less than 0.005) reduced compared with pre-RUBF values. During 24 h of RUBF the incidence of FBM declined initially but returned to control values after 12-16 h. In seven of eight fetuses, over the 12- to 24-h period of RUBF, large amounts of liquid (22.7-62.6 ml) were drawn into the lungs during FBM, resulting in a net movement of amniotic fluid into the lungs. During the 18- to 24-h period of RUBF, changes in the incidence of FBM were found to be significantly and positively correlated (r = 0.86, P less than 0.005) with the changes in VL that occurred over the 24-h period. Thus, prolonged RUBF can result in the inhalation of large volumes of amniotic fluid by the fetus, which could be a cause of in utero meconium aspiration.