Bmpr1a signaling plays critical roles in palatal shelf growth and palatal bone formation

Cleft palate, including submucous cleft palate, is among the most common birth defects in humans. While overt cleft palate results from defects in growth or fusion of the developing palatal shelves, submucous cleft palate is characterized by defects in palatal bones. In this report, we show that the Bmpr1a gene, encoding a type I receptor for bone morphogenetic proteins (Bmp), is preferentially expressed in the primary palate and anterior secondary palate during palatal outgrowth. Following palatal fusion, Bmpr1a mRNA expression was upregulated in the condensed mesenchyme progenitors of palatal bone. Tissue-specific inactivation of Bmpr1a in the developing palatal mesenchyme in mice caused reduced cell proliferation in the primary and anterior secondary palate, resulting in partial cleft of the anterior palate at birth. Expression of Msx1 and Fgf10 was downregulated in the anterior palate mesenchyme and expression of Shh was downregulated in the anterior palatal epithelium in the Bmpr1a conditional mutant embryos, indicating that Bmp signaling regulates mesenchymal-epithelial interactions during palatal outgrowth. In addition, formation of the palatal processes of the maxilla was blocked while formation of the palatal processes of the palatine was significantly delayed, resulting in submucous cleft of the hard palate in the mutant mice. Our data indicate that Bmp signaling plays critical roles in the regulation of palatal mesenchyme condensation and osteoblast differentiation during palatal bone formation.     

Signaling through Tgf-beta type I receptor Alk5 is required for upper lip fusion

Cleft lip with or without cleft palate is one of the most common congenital malformations in newborns. While numerous studies on secondary palatogenesis exist, data regarding normal upper lip formation and cleft lip is limited. We previously showed that conditional inactivation of Tgf-beta type I receptor Alk5 in the ectomesenchyme resulted in total facial clefting. While the role of Tgf-beta signaling in palatal fusion is relatively well understood, its role in upper lip fusion remains unknown. In order to investigate a role for Tgf-beta signaling in upper lip formation, we used the Nes-Cre transgenic mouse line to delete the Alk5 gene in developing facial prominences. We show that Alk5/Nes-Cre mutants display incompletely penetrant unilateral or bilateral cleft lip. Increased cell death seen in the medial nasal process and the maxillary process may explain the hypoplastic maxillary process observed in mutants. The resultant reduced contact is insufficient for normal lip fusion leading to cleft lip. These mice also display retarded development of palatal shelves and die at E15. Our findings support a role for Alk5 in normal upper lip formation not previously reported.     

The Etiology of Cleft Palate Formation in BMP7-Deficient Mice

Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.     

Correlation of susceptibility to 6-aminonicotinamide and hydrocortisone-induced cleft palate

Our previous genome-wide Quantitative Trait Locus (QTL) mapping study using mouse A/J by C57BL/6J recombinant inbred (RI) lines suggested several chromosomal regions contain genes influencing susceptibility to phenytoin (PT)-induced cleft lip with or without cleft palate [CL(P)] and 6-aminonicotinamide (6-AN)-induced isolated cleft palate (CP). Importantly, the same chromosomal regions but different RI parental strain alleles were sometimes implicated in susceptibility to these different kinds of orofacial clefting. Here we report the susceptibility to hydrocortisone (HC)-induced CP in these RI lines. We treated pregnant females with HC and studied the incidence of CP in day 17 fetuses. RI lines showed highly correlated responses to HC and 6-AN. The A/J parental line and five RI lines showed very high levels of clefting in response to both of these teratogens. The C57BL/6J parental line and five other RI lines exhibited low incidence of CP for these teratogens. In contrast, there was no significant correlation between incidence of PT-induced CL(P) and HC-induced CP.     

Report of two cases with Van der Woude syndrome: a child and her mother

Report of two cases with Van der Woude syndrome: a child and her mother: Congenital pits of the lower lip are rare malformations. They are closely associated with cleft lip (CL), cleft lip/palate (CL/CP) or isolated cleft palate (CP) and if so this condition is known as Van der Woude syndrome, which is inherited in an autosomal dominant fashion with high penetrance. Two individuals, one with lower lip pits and cleft palate and the other with isolated lower lip pit from the same family are described. Autosomal dominant pattern of inheritance was observed in this family and treatment consisted of complete removal of sinus tracts in one patient. Pathological features of sinus tracts consisted of stratified nonkeratinized squamous epithelium and a lamina propria of dense connective tissue. Importance of genetic counseling is emphasized as at least half of gene carriers have some kind of clefting.     

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2): structure,gene mapping,polymorphisms, and candidate evaluation for human orofacial clefts

BACKGROUND: Nonsyndromic orofacial clefts have an estimated incidence of 1/1000 live births. Population genetic and embryologic studies suggest that cleft palate only (CPO) may be a distinct clinical entity from cleft lip with or without cleft palate (CL/P). Both CPO and CL/P are thought to be multifactorial in etiology, with evidence indicating that genetic, environmental, and developmental determinants may all play a role. The ARNT2 gene localizes to a conserved linkage group on mouse chromosome 7 that is syntenic with human chromosome 15q23-25. This chromosomal region was previously identified as a teratogen-induced clefting susceptibility locus in a genome-wide scan of AXB and BXA recombinant inbred mice. Arnt2 is expressed in the first branchial arch in mice. The teratogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts through the aryl hydrocarbon receptor (Ahr) pathway to produce dose-dependent CPO and thymic wasting in mice exposed in utero. Arnt2 and Ahr proteins dimerize in vitro. TCDD exposure is also associated with orofacial clefting in children of parents involved in agricultural work. METHODS: To determine whether ARNT2 influences human craniofacial development, we identified the human ARNT2 gene and conducted genomic structural analysis. Mutational screening was performed in infants with nonsyndromic CPO or CL/P who were identified by the Iowa Birth Defects Registry. RESULTS: A common amino acid polymorphism was detected but, no obvious disease-causing mutations were detected by SSCP analysis. The microsatellite marker, GATA89D04 (D15S823) was identified within intron 11 of the human ARNT2 gene, and linkage disequilibrium of nonsyndromic CPO and CL/P parent-infant trios was conducted. CONCLUSIONS: No association was demonstrated with CPO (n = 45) and CL/P (n = 37). Teratology 66:85-90, 2002.     

Confirmation of the role of N-acetyltransferase 2 in teratogen-induced cleft palate using transgenics and knockouts

Previous work on Dilantin- and hydrocortisone-induced cleft palate and cleft lip with or without cleft palate using congenics for the N-acetyltransferase loci (Nat1 and Nat2 are closely linked) and recombinant inbred lines implicated the Nat1,2 region in susceptibility to teratogen-induced orofacial clefting. Since Nat1 does not differ between the two strains, Nat2 appeared to be responsible. We have now tested this conclusion using transgenics and knockouts. Transgenics for human NAT1 (equivalent to mouse Nat2) and knockouts for Nat2 were tested for susceptibility to Dilantin, hydrocortisone, and 6-aminonicotinamide-induced orofacial clefting. We found that Nat2 greatly influences teratogen-induced orofacial clefting on the A/J background but not on the C57BL/6J background. The magnitude and direction of the effects depended on which teratogen was used. The Nat2 knockout did not make C57BL/6J susceptible or A/J (already with very low activity) more susceptible but significantly decreased sporadic clefting in the A/J strain. We conclude that only the A/J strain, with several loci affecting orofacial clefting, is influenced by Nat2.     

Distinct developmental programs require different levels of Bmp signaling during mouse retinal development

The Bmp family of secreted signaling molecules is implicated in multiple aspects of embryonic development. However, the cell-type-specific requirements for this signaling pathway are often obscure in the context of complex embryonic tissue interactions. To define the cell-autonomous requirements for Bmp signaling, we have used a Cre-loxP strategy to delete Bmp receptor function specifically within the developing mouse retina. Disruption of a Bmp type I receptor gene, Bmpr1a, leads to no detectable eye abnormality. Further reduction of Bmp receptor activity by removing one functional copy of another Bmp type I receptor gene, Bmpr1b, in the retina-specific Bmpr1a mutant background, results in abnormal retinal dorsoventral patterning. Double mutants completely lacking both of these genes exhibit severe eye defects characterized by reduced growth of embryonic retina and failure of retinal neurogenesis. These studies provide direct genetic evidence that Bmpr1a and Bmpr1b play redundant roles during retinal development, and that different threshold levels of Bmp signaling regulate distinct developmental programs such as patterning, growth and differentiation of the retina.     

Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. We have used Msx1-deficient mice as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. We found that Msx1 expression was restricted to the anterior of the first upper molar site in the palatal mesenchyme and that Msx1 was required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicated that the cleft palate seen in Msx1 mutants resulted from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1(-/-) palatal mesenchyme rescued the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate was a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicated that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. Our findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations.     

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.     

Few associations of candidate genes with nonsyndromic orofacial clefts in the population of Lithuania

Nonsyndromic orofacial clefting (NS-OFC) is a common complex multifactorial trait with a considerable genetic component and a number of candidate genes suggested by various approaches. Twenty biallelic and microsatellite DNA markers in the strong candidate loci TGFA, TGFB3, GABRB3, RARA, and BCL3 were analysed for allelic association with the NS-OFC phenotype in 112 nuclear families (proband + both parents) from Lithuania by using the transmission disequilibrium test (TDT). Associations were found between the TGFA gene marker rs2166975 and nonsyndromic cleft palate only (CPO) phenotype (p = 0.045, df 1) as well as between the D2S292 marker and the cleft lip with or without cleft palate (CL/CP) phenotype in allele-wise TDT (P = 0.005, df 9) and genotype-wise TDT (P = 0.021, df 24). A weak association (P = 0.085, df 3) of the BCL3 marker (BCL3 gene) with the risk of CPO was also found. Thus our initial results support the contribution of allelic variation in the TGFA locus to the aetiology of CL/CP in the population of Lithuania but they do not point to TGFA as a major causal gene. Different roles of the TGFA and BCL3 genes in the susceptibility to NS-OFC phenotypes are suggested.     

The effect of teratogens on maternal corticosterone levels and cleft incidence in A/J mice.

It is unknown whether orofacial clefting, one consequence of teratogenic exposure, results from a direct interaction between the teratogen and the embryonic palate, or indirectly from maternal alterations caused by the teratogen. In the current study pregnant A/J mice were exposed to one of three cleft-inducing agents in order to examine the relationship between drug-induced clefting and the response of maternal plasma corticosterone to drug administration. The agents used, haloperidol (HAL), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or phenytoin (PHT), were administered in teratogenic doses between 0800 and 0930 on gestational day 10 (GD 10). For corticosterone determinations, mice were dosed on GD 10, and blood was collected at 1, 4, 24, or 48 hr after dosing. For fetal evaluation of cleft lip and/or cleft palate, mice were dosed on GD 10 and killed on GD 18. Phenytoin was the most potent inducer of cleft lip and palate and induced a sustained elevation of plasma corticosterone in maternal animals. The other treatments, in order of decreasing potency to induce clefting and/or cause an elevation of corticosterone in plasma were 2,4,5-T > HAL > controls. Correlations between maternal corticosterone levels and clefting incidence were very high at all time points examined; total exposure (area under the curve) was also highly correlated. A linear relationship between drug-induced increases in maternal corticosterone levels and the incidence of clefting in A/J mice was evident. Based on these findings, we believe that increased maternal corticosterone levels may play a role in orofacial clefting in A/J mice.     

Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly,Cleft Lip,and Cleft Palate

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15^th to 24^th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in “non-syndromic” orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.     

Orofacial clefts, parental cigarette smoking, and transforming growth factor-alpha gene variants.

Results of studies to determine whether women who smoke during early pregnancy are at increased risk of delivering infants with orofacial clefts have been mixed, and recently a gene-environment interaction between maternal smoking, transforming growth factor-alpha (TGFa), and clefting has been reported. Using a large population-based case-control study, we investigated whether parental periconceptional cigarette smoking was associated with an increased risk for having offspring with orofacial clefts. We also investigated the influence of genetic variation of the TGFa locus on the relation between smoking and clefting. Parental smoking information was obtained from telephone interviews with mothers of 731 (84.7% of eligible) orofacial cleft case infants and with mothers of 734 (78.2%) nonmalformed control infants. DNA was obtained from newborn screening blood spots and genotyped for the allelic variants of TGFa. We found that risks associated with maternal smoking were most elevated for isolated cleft lip with or without cleft palate, (odds ratio 2.1 [95% confidence interval 1.3-3.6]) and for isolated cleft palate (odds ratio 2.2 [1.1-4.5]) when mothers smoked > or =20 cigarettes/d. Analyses controlling for the potential influence of other variables did not reveal substantially different results. Clefting risks were even greater for infants with the TGFa allele previously associated with clefting whose mothers smoked > or =20 cigarettes/d. These risks for white infants ranged from 3-fold to 11-fold across phenotypic groups. Paternal smoking was not associated with clefting among the offspring of nonsmoking mothers, and passive smoke exposures were associated with at most slightly increased risks. This study offers evidence that the risk for orofacial clefting in infants may be influenced by maternal smoke exposures alone as well as in combination (gene-environment interaction) with the presence of the uncommon TGFa allele.     

Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate.

Complex segregation analysis of pedigrees having nonsyndromic cleft lip with or without cleft palate (CL/P) (Chung et al. 1986; Marazita et al. 1986) has shown that a major-locus model best explains the observed recurrence of CL/P in Caucasian families. To identify this major gene, we compared the frequencies of 12 RFLPs at five loci-epidermal growth factor, transforming growth factor-alpha, epidermal growth factor receptor, glucocorticoid receptor, and estrogen receptor-in both a group of 80 subjects with nonsyndromic CL/P and 102 controls. These candidate genes were selected because studies in rodents had suggested their possible involvement in palatogenesis. A significant association was observed between two RFLPs at the transforming-growth-factor-alpha (TGFA) locus and the occurrence of clefting (P = .0047 and P = .0052). This suggests that either the TGFA gene itself or DNA sequences in an adjacent region contribute to the development of a portion of cases of CL/P in humans and provides an opportunity to begin to examine the molecular events underlying lip and palate formation.     

Medical sequencing of candidate genes for nonsyndromic cleft lip and palate

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.     

Reduced epithelial surface activity is related to a higher incidence of facial clefting in A/WySn mice

The epithelial surfaces of the facial primordia were evaluated by scanning electron microscopy (SEM) during primary palatogenesis in two genetically related mouse strains, the A/J and the A/WySn strains. These two strains were selected because the reported frequency of spontaneous cleft lip with or without cleft palate [CL(P)] in the A/J strain approximates 0%, whereas the spontaneous frequency of CL(P) in the A/WySn strain is 20-30%. The embryos were examined prior to (two to six tail somites), during (seven to ten tail somites), and after (ten to 14 tail somites) primary palate fusion. During fusion, epithelial surface activity (characterized as cellular debris, dissociated cells, cellular projections, and epithelial bridging) was more pronounced in A/J embryos. A/WySn embryos with spontaneous cleft lip exhibited a marked deficiency in epithelial activity when compared to their normal littermates. No discernible differences were detected in the facial morphology, with the exception of the distal end of the medial nasal prominence, which appeared longer in the A/J strain. This study suggests that the degree of epithelial surface activity at the putative site of fusion and the relative length of the medial nasal prominence may account for the observed differences in facial clefting of the two strains. Face shape, related to prominence divergence, was similar in the two strains and could not explain the higher incidence of clefting observed in the A/WySn strain.     

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.