Genetics of Cortisone-Induced Cleft Palate in the Mouse—embryonic and Maternal Effects

Differences between mouse strains in frequency of embryonic, cortisone-induced cleft palate were examined. Probit analysis demonstrated a family of linear and parallel dose-response curves for different inbred and hybrid embryos. Since the differences between genotypes were not in the slopes of the response curves but rather in their location, it is proposed that the median effective dose (ED₅₀) of cortisone required to induce cleft palate (or the tolerance) provides a more appropriate definition of the response trait and its difference than a frequency statement. The tolerance of C57BL/6J is dominant to that of A/J. A maternal effect of A/J relative to C57BL/6J dams caused a two-fold reduction in the embryonic tolerance of cortisone. Cortisone-induced cleft palate and mortality were separate response traits.—In these and previous studies on cortisone- and other glucocorticoid-induced cleft palate in the mouse, the nature of the cleft-palate-response curve appeared to be the same for all glucocorticoids, and within-strain differences in tolerance could be used as measures of potency or bioassays for a particular effect of the glucocorticoids.     

Palatogenesis

Cleft palate represents the second most common birth defect and carries substantial physiologic and social challenges for affected patients, as they often require multiple surgical interventions during their lifetime. A number of genes have been identified to be associated with the cleft palate phenotype, but etiology in the majority of cases remains elusive. In order to better understand cleft palate and both surgical and potential tissue engineering approaches for repair, we have performed an in-depth literature review into cleft palate development in humans and mice, as well as into molecular pathways underlying these pathologic developments. We summarize the multitude of pathways underlying cleft palate development, with the transforming growth factor beta superfamily being the most commonly studied. Furthermore, while the majority of cleft palate studies are performed using a mouse model, studies focusing on tissue engineering have also focused heavily on mouse models. A paucity of human randomized controlled studies exists for cleft palate repair, and so far, tissue engineering approaches are limited. In this review, we discuss the development of the palate, explain the basic science behind normal and pathologic palate development in humans as well as mouse models and elaborate on how these studies may lead to future advances in palatal tissue engineering and cleft palate treatments.     

Palatogenesis: engineering, pathways and pathologies

Cleft palate represents the second most common birth defect and carries substantial physiologic and social challenges for affected patients, as they often require multiple surgical interventions during their lifetime. A number of genes have been identified to be associated with the cleft palate phenotype, but etiology in the majority of cases remains elusive. In order to better understand cleft palate and both surgical and potential tissue engineering approaches for repair, we have performed an in-depth literature review into cleft palate development in humans and mice, as well as into molecular pathways underlying these pathologic developments. We summarize the multitude of pathways underlying cleft palate development, with the transforming growth factor beta superfamily being the most commonly studied. Furthermore, while the majority of cleft palate studies are performed using a mouse model, studies focusing on tissue engineering have also focused heavily on mouse models. A paucity of human randomized controlled studies exists for cleft palate repair, and so far, tissue engineering approaches are limited. In this review, we discuss the development of the palate, explain the basic science behind normal and pathologic palate development in humans as well as mouse models and elaborate on how these studies may lead to future advances in palatal tissue engineering and cleft palate treatments.     

PDGF-C controls proliferation and is down-regulated by retinoic acid in mouse embryonic palatal mesenchymal cells

BACKGROUND: Platelet-derived growth factor C (PDGF-C) was recently identified as a member of the PDGF ligand family. Some observation suggests that PDGF-C could play an important role in palatogenesis highlighted by the Pdgfc(-/-) mouse with cleft palate, which led us to examine the mechanism of PDGF-C signaling in palatogenesis. It is well known that retinoic acid (RA) is a teratogen that can effectively induce cleft palate in the mouse. Due to the critical roles of PDGF-C and RA in cleft palate, the link between cleft palate induced by RA and loss of PDGF-C was investigated. METHODS: Retarded mesenchymal proliferation is an important cause for cleft palate. To clarify the mechanism of PDGF-C in palatogenesis, we evaluated the effects of PDGF-C and anti-PDGF-C neutralizing antibody on proliferation activity in mouse embryonic palatal mesenchymal (MEPM) cells. RESULTS: Briefly, our results show PDGF-C promotes proliferation, anti-PDGF-C antibody inhibits it in MEPM cells, and RA downregulates the PDGF-C expression both at the mRNA and protein levels. CONCLUSIONS: These demonstrate that PDGF-C is a potent mitogen for MEPM cells, implying that inactivated PDGF-C by gene-targeting or reduced PDGF-C by RA may both cause inhibition of proliferation in palatal shelves, which might account for the pathogenesis of cleft palate in Pdgfc(-/-) mouse or RA-treated mouse. In conclusion, our results suggest that PDGF-C signaling is a new mechanism of cleft palate induced by RA.     

Hydrocortisone-induced embryotoxicity and embryonic drug disposition in H-2 congenic mice

Congenic mouse strains C57BL/10Sn (B10) and B10.A/SgSn (B10A), genetically different only in the region of the H-2 complex, were compared for sensitivity to hydrocortisone-induced embryotoxicity and embryonic drug disposition. Pregnant B10A mice dosed intramuscularly with 0, 100, 150, and 200 mg hydrocortisone/kg body weight and B10 mice injected with 0, 200, 400, 600, and 800 mg/kg, both on gestational day (GD) 12, were evaluated on GD 18 for reproductive toxicity. The induction of cleft palate demonstrated a linear dose-response by probit analysis: The ED50s were 143.6 mg/kg and 512.0 mg/kg for B10A and B10 mice, respectively. Comparison of fetal weight revealed statistically significant intrauterine growth retardation at all doses administered to B10 mice. However, growth retardation was shown only in the high-dose group in the B10A strain. Embryonic drug concentrations were evaluated by administration of hydrocortisone to mice of both strains on GD 12, at the ED50 for cleft palate production in the B10A strain, with 3H-hydrocortisone (5 muci/mouse) added as a tracer. Maternal serum and embryos were analyzed for steroid content. Disposition and pharmacokinetics of 3H-hydrocortisone were similar in both strains, with the majority of serum radioactivity recovered as hydrocortisone and the major radioactive peak in embryos comigrating with cortisone. The results indicate that H-2 haplotype does not influence hydrocortisone-induced cleft palate sensitivity through an alteration of embryonic drug exposure.     

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.     

H-2 haplotype and heterochronic orofacial morphogenesis in congenic mice: consideration as a possible explanation for differential susceptibility to teratogenesis.

For over 40 years it has been known that genetically different inbred strains of mice have different degrees of susceptibility to corticosteroid-induced cleft palate. Gene(s) at or near the H-2 region on chromosome 17 have been implicated. One postulated explanation is that the strain difference in susceptibility is not related to differential corticosteroid action, but to differences in normal developmental pattern. Studies have demonstrated significant quantitative differences between inbred strains for a number of growth variables relative to palatal development. It is also known that there are genes at or near the H-2 complex that influence pre- and post-implantation development. Thus, we sought to determine the relationship in H-2 congenic mice between haplotype differences and variation in normal orofacial development. Morphometric analyses of the palatal region in serially sectioned E13 and E17 B10 and B10.A mice were completed. We were able to find some evidence for H-2 haplotype related phenotypic differences, but these differences are less than compelling as an explanation for haplotype-dependent susceptibility differences. A more likely explanation is GR-mediated differential corticosteroid responsiveness and its consequent effects on palatal shelf growth.     

A unique mouse strain expressing Cre recombinase for tissue-specific analysis of gene function in palate and kidney development

Mammalian palate development is a multistep process, involving initial bilateral downward outgrowth of the palatal shelves from the oral side of the maxillary processes, followed by stage-specific palatal shelf elevation to the horizontal position above the developing tongue and subsequent fusion of the bilateral palatal shelves at the midline to form the intact roof of the oral cavity. While mutations in many genes have been associated with cleft palate pathogenesis, the molecular mechanisms regulating palatal shelf growth, patterning, and elevation are not well understood. Genetic studies of the molecular mechanisms controlling palate development in mutant mouse models are often complicated by early embryonic lethality or gross craniofacial malformation. We report here the development of a mouse strain for tissue-specific analysis of gene function in palate development. We inserted an IresCre bicistronic expression cassette into the 3' untranslated region of the mouse Osr2 gene through gene targeting. We show, upon crossing to the R26R reporter mice, that Cre expression from the Osr2(IresCre) knockin allele activated beta-galactosidase expression specifically throughout the developing palatal mesenchyme from the onset of palatal shelf outgrowth. In addition, the Osr2(IresCre) mice display exclusive Cre-mediated recombination in the glomeruli tissues derived from the metanephric mesenchyme and complete absence of Cre activity in other epithelial and mesenchymal tissues in the developing metanephric kidney. These data indicate that the Osr2(IresCre) knockin mice provide a unique tool for tissue-specific studies of the molecular mechanisms regulating palate and kidney 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.     

Inhibitory effect of corticoids on the proliferative pattern in mouse palatal processes.

The formation of the secondary palate in mice is accompanied by intensive mitotic activity, which is mainly concentrated at the medial edges of the palatal processes. In control H-Velaz randombred fetuses the mitotic activity culminated approximately 24 h before palatal-shelf horizontalization, so that the period of intensive cell proliferation coincided with the period when cleft palate could be induced by cortisone administration. Effects of teratogenic doses of corticoids, injected directly into amniotic sac of embryos on day 13 (0.3 mg hydrocortisone) or im to pregnant females on day 12 (7.5 mg cortisone acetate), on the proliferative peak in palatal processes were studied using intraamniotic injection of colchicine. Counts of colchicine-blocked mitoses in histological serial sections revealed both a significant decrease in overall mitotic density and a posterior shift of the proliferative peak in the palatal processes of fetuses treated with doses of corticoids producing cleft palate.     

Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion

Palatal fusion is a complex, multi-step developmental process; the consequence of failure in this process is cleft palate, one of the most common birth defects in humans. Previous studies have shown that regression of the medial edge epithelium (MEE) upon palatal fusion is required for this process, and TGF-beta signaling plays an important role in regulating palatal fusion. However, the fate of the MEE and the mechanisms underlying its disappearance are still unclear. By using the Cre/lox system, we are able to label the MEE genetically and to ablate Tgfbr2 specifically in the palatal epithelial cells. Our results indicate that epithelial-mesenchymal transformation does not occur in the regression of MEE cells. Ablation of Tgfbr2 in the palatal epithelial cells causes soft palate cleft, submucosal cleft and failure of the primary palate to fuse with the secondary palate. Whereas wild-type MEE cells disappear, the mutant MEE cells continue to proliferate and form cysts and epithelial bridges in the midline of the palate. Our study provides for the first time an animal model for soft palate cleft and submucous cleft. At the molecular level, Tgfb3 and Irf6 have similar expression patterns in the MEE. Mutations in IRF6 disrupt orofacial development and cause cleft palate in humans. We show here that Irf6 expression is downregulated in the MEE of the Tgfbr2 mutant. As a recent study shows that heterozygous mutations in TGFBR1 or TGFBR2 cause multiple human congenital malformations, including soft palate cleft, we propose that TGF-beta mediated Irf6 expression plays an important, cell-autonomous role in regulating the fate of MEE cells during palatogenesis in both mice and humans.     

Cleft palate susceptibility linked to histocompatibility-2 (H-2) in the mouse

Data have been obtained indicating that cortisone-induced cleft palate in the mouse is linked to theH-2 ^a complex. Cortisone (2.5 mg) was administered to pregnant females on days 11 through 14 of pregnancy. On day 17 of pregnancy, the fetuses were inspected for cleft palates. Sham experiments were done by injecting sterile saline instead of cortisone. The inbred strains, A/J and C57BL/6, and the congenic strains C57BL/10ScSn and B10.A were tested for susceptibility to cleft palate. The clefting frequency was also observed in hybrids of the congenic strains. The A/J and B10.A strains showed a characteristic high susceptibility to cleft palate (i.e., 99% and 81% incidence of cleft palate, respectively) after teratogenic treatment. The C57BL/6 and C57BL/ 10ScSn demonstrated a significant resistance to the teratogen (i.e., 25% and 21 % incidence of clefting, respectively). The teratogenic treatment of congenic hybrids indicated that maternal influences significantly affected the incidence of cleft palate formation. The maternal influence appeared to depend upon the specificH-2 haplotype of the mother.     

Restriction fragment length polymorphisms, glucocorticoid receptors, and phenytoin-induced cleft palate in congenic strains of mice with steroid susceptibility differences.

A gene that affects susceptibility to cortisone-induced cleft palate maps between H-2S and H-2D on mouse chromosome 17. Congenic mouse strains that differ at this locus, designated Cps-1 (cleft palate susceptibility-1), have been tested for the presence of several closely linked markers. All data obtained so far are consistent with a gene order of H-2S-Cps-1-BAT-5-BAT-2-TNF-H-2D. The Cps-1 gene does not appear to affect the level of glucocorticoid receptors or the susceptibility of mice to phenytoin-induced cleft palate.     

A critical review of the developmental toxicity and teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: recent advances toward understanding the mechanism

A specific teratogenic response is elicited in the mouse as a result of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). The characteristic spectrum of structural malformations induced in mice following exposure to TCDD and structurally related congeners is highly reproducible and includes both hydronephrosis and cleft palate. In addition, prenatal exposure to TCDD has been shown to induce thymic hypoplasia. These three abnormalities occur at doses well below those producing maternal or embryo/fetal toxicity and are thus among the most sensitive indicators of dioxin toxicity. In all other laboratory species tested, TCDD causes maternal and embryo/fetal toxicity but does not induce a significant increase in the incidence of structural abnormalities even at toxic dose levels. Developmental toxicity occurs in a similar dose range across species; however, mice are particularly susceptible to development of TCDD-induced terata. Recent experiments using an organ culture were an attempt to address the issue of species and organ differences in sensitivity to TCDD. Human palatal shelves examined in this in vitro system were found to approximate the rat in terms of sensitivity for induction of cleft palate. Investigators have suggested that altered regulation of growth factors and their receptors may involve inappropriate proliferation and differentiation of target cells, ultimately producing TCDD-induced terata. Why the teratogenic effects of TCDD are so highly species and tissue specific, and which animal species most accurately predicts the response of the human embryo/fetus, at the levels of exposure experienced by humans, still remains to be clarified.     

Mouse genetic models of cleft lip with or without cleft palate

Nonsyndromic cleft lip and palate (CLP) is among the most common human birth defects. Transmission patterns suggest that the causes are "multifactorial" combinations of genetic and nongenetic factors, mostly distinct from those causing cleft secondary palate (CP). The major etiological factors are largely unknown, and the embryological mechanisms are not well understood. In contrast to CP or neural tube defects (NTD), CLP is uncommon in mouse mutants. Fourteen known mutants or strains express CLP, often as part of a severe syndrome, whereas nonsyndromic CLP is found in two conditional mutants and in two multifactorial models based on a hypomorphic variant with an epigenetic factor. This pattern suggests that human nonsyndromic CLP is likely caused by regulatory and hypomorphic gene variants, and may also involve epigenetics. The developmental pathogenic mechanism varies among mutants and includes deficiencies of growth of the medial, lateral or maxillary facial prominences, defects in the fusion process itself, and shifted midline position of the medial prominences. Several CLP mutants also have NTD, suggesting potential genetic overlap of the traits in humans. The mutants may reflect two interacting sets of genetic signaling pathways: Bmp4, Bmpr1a, Sp8, and Wnt9b may be in one set, and Tcfap2a and Sox11 may be in another. Combining the results of chromosomal linkage studies of unidentified human CLP genes with insights from the mouse models, the following previously unexamined genes are identified as strong candidate genes for causative roles in human nonsyndromic CLP: BMP4, BMPR1B, TFAP2A, SOX4, WNT9B, WNT3, and SP8.     

Further evidence for a role of arachidonic acid in glucocorticoid teratogenic action in the palate

Arachidonic acid produces a significant reversal of the production of cleft palate by cortisone in the offspring of sensitive strains of mice in vivo. Arachidonic acid in nanogram per milliliter concentrations also produces a significant reversal of the cortisol inhibition of the programmed cell death of the medial edge epithelium of palatal shelves in vitro. This corrective action of arachidonic acid in vitro is significantly blocked by indomethacin at a nanogram per milliliter concentration which selectively inhibits the conversion of arachidonic acid to prostaglandins and/or thromboxanes at the level of cyclooxygenase. These results support the hypothesis that the inhibition of arachidonic acid release and subsequent prostaglandin and/or thromboxane production by glucocorticoids is involved in the teratogenic action of glucocorticoids and demonstrate that one site of this action is the inhibition of epithelial loss.     

Immunohistochemical localization of TGF-B1 during morphogenetic movements of the developing mouse palate.

Transforming growth factor-beta (TGF-B1) may play an important role in developmentally active tissues in which it is found in high concentrations. We localized TGF-B1 in the developing fetal mouse palate immunohistochemically using a polyclonal antibody. Mouse fetal palates at 12-17 days (inclusive) of gestation were examined and specific focal concentrations of TGF-B1 identified regions undergoing active morphogenesis. The association of TGF-B1 with aggregates of mesenchymal cells in the palate and chondroblasts, rhabdomyocytes, and epithelia of the craniofacial complex strongly implicates its role in proliferation and differentiation in the developing mouse palate. We believe these findings have important bearing on the normal development of the palate as well as cleft anomalies.     

Spontaneous cleft palate in CF#1/Ohu mice]

We found a spontaneous cleft palate in a mouse of CF#1/Ohu (Ohu University, Japan). Further, the frequency of the spontaneous cleft palate in strains of CF#1/Jms (Institute of Medical Science, University of Tokyo, Japan) and CF#1/Jah (National Institute of Animal Health, Japan) were about 3%, respectively. The frequency and the types of spontaneous cleft palate in CF#1 were clearly different from those in A/J and CL/Fr strains which were used as model animals of lip and cleft palate. We think that CF#1 is the new animal models to analyze a cleft palate genetically and biochemically.     

GABA uptake in embryonic palate mesenchymal cells of two mouse strains

To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate (1). Palate cells were capable of accumulating [³H]GABA by saturable uptake mechanisms characteristic of a high and a low affinity active transport as indicated by temperature, Na⁺ ion and carrier dependence as well asK _m andV _max values that were comparable to other biological systems. TheV _max of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential resonses to GABA.