Bmp4 gene is expressed at the putative site of fusion in the midfacial region

The molecular mechanisms by which the primordia of the midface grow and fuse to form the primary palate portion of the craniofacial region are not well characterized. This is in spite of the fact that failure of growth and/or fusion of these primordia leads to the most common craniofacial birth defect in humans (i.e. clefts of the lip and/or palate). Bmp4 plays a critical role during early embryonic development and has previously been shown to play a role in epithelial-mesenchymal interactions in the craniofacial region of chicks. We analyze the expression of bmp4 in mouse as the midfacial processes undergo fusion to form the primary palate. We show that bmp4 is expressed in a very distinct manner in the three midfacial processes (lateral nasal, LNP, medial nasal, MNP, and maxillary processes, MxP) that ultimately fuse to form the midface. Prior to fusion of the midfacial processes, bmp4 is expressed in the ectoderm of the LNP, MNP, and MxP in a distinct spatial and temporal manner near and at the site of fusion of the midface. Bmp4 appears to demarcate the cells in the LNP and MNP that will eventually contact and fuse with each other. As fusion of the three prominences proceeds, some bmp4 expressing cells are trapped in the fusion line. Later, the expression of bmp4 switches to the mesenchyme of the midface underlying its initial expression in the ectoderm. The switch occurs soon after fusion of the three processes. The pattern of expression in the midfacial region implicates the important role of bmp4 in mediating the fusion process, possibly through apoptosis of cells in the putative site of fusion, during midfacial morphogenesis.     

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.     

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.     

Microarray analysis of murine palatogenesis: temporal expression of genes during normal palate development

The mammalian face is assembled in utero in a series of complex and interdependent molecular, cell and tissue processes. The orofacial complex appears to be exquisitely sensitive to genetic and environmental influence and this explains why clefts of the lip and palate are the most common congenital anomaly in humans (one in 700 live births). In this study, microarray technology was used to identify genes that may play pivotal roles in normal murine palatogenesis. mRNA was isolated from murine embryonic palatal shelves oriented vertically (before elevation), horizontally (following elevation, before contact), and following fusion. Changes in gene expression between the three different stages were analyzed with GeneChip microarrays. A number of genes were upregulated or downregulated, and large changes were seen in the expression of loricrin, glutamate decarboxylase, gamma-amino butyric acid type A receptor beta3 subunit, frizzled, Wnt-5a, metallothionein, annexin VIII, LIM proteins, Sox1, plakophilin1, cathepsin K and creatine kinase. In this paper, the changes in genetic profile of the developing murine palate are presented, and the possible role individual genes/proteins may play during normal palate development are discussed. Candidate genes with a putative role in cleft palate are also highlighted.     

Distinct functions for Bmp signaling in lip and palate fusion in mice

Previous work suggested that cleft lip with or without cleft palate (CL/P) is genetically distinct from isolated cleft secondary palate (CP). Mutations in the Bmp target gene Msx1 in families with both forms of orofacial clefting has implicated Bmp signaling in both pathways. To dissect the function of Bmp signaling in orofacial clefting, we conditionally inactivated the type 1 Bmp receptor Bmpr1a in the facial primordia, using the Nestin cre transgenic line. Nestin cre; Bmpr1a mutants had completely penetrant, bilateral CL/P with arrested tooth formation. The cleft secondary palate of Nestin cre; Bmpr1a mutant embryos was associated with diminished cell proliferation in maxillary process mesenchyme and defective anterior posterior patterning. By contrast, we observed elevated apoptosis in the fusing region of the Nestin cre; Bmpr1a mutant medial nasal process. Moreover, conditional inactivation of the Bmp4 gene using the Nestin cre transgenic line resulted in isolated cleft lip. Our data uncover a Bmp4-Bmpr1a genetic pathway that functions in lip fusion, and reveal that Bmp signaling has distinct roles in lip and palate fusion.     

Flrt2 and Flrt3 have overlapping and non-overlapping expression during craniofacial development

Craniofacial morphogenesis is a complex multi-step process that involves numerous biological processes to coordinate the growth, proliferation, migration, and subsequent differentiation of the cranial neural crest cells. Members of the Fibronectin Leucine-Rich Transmembrane (Flrt) gene family have been previously reported to be widely expressed in the developing embryo. We mapped the expression of Flrt2 and Flrt3 at critical stages of craniofacial development and found that, during early craniofacial development, Flrt2 was highly expressed initially in the cranial neural crest cells and Flrt3 in the midbrain. Later both genes were expressed in the developing pharyngeal region. Flrt2 expression predominated in the neural crest-derived mesenchyme in the medial aspect of the developing frontonasal region in close relationships with the expression of Fgfr2, Shh, and Msx1, three genes shown previously to play critical roles in craniofacial development. Flrt2 was also present in the vomero-nasal organ, mandibular primodia, and the posterior aspects of the unfused and fused secondary palatal shelves. Flrt3, however, had a more restrictive expression, being present in the mesenchyme underlying the ectoderm of the medial nasal process and in the mandibular primordium and in regions undergoing outgrowth, in a pattern that overlapped with Bmp4 expression. Both Flrt2 and Flrt3 were later found to be present at sites of epithelial–mesenchymal interactions such as the developing tooth buds, hair follicles, and eye. Together the data suggested important roles for Flrt2 and Flrt3 in mediating events such as NCC migration, chondrogenesis and epithelial–mesenchymal interactions during craniofacial development.     

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.     

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.     

Characterizing expression pattern of Six2Cre during mouse craniofacial development

Craniofacial development is a complex process involving diverse cell populations. Various transgenic Cre lines have been developed to facilitate studying gene function in specific tissues. In this study, we have characterized the expression pattern of Six2Cre mice at multiple stages during craniofacial development. Our data revealed that Six2Cre lineage cells are predominantly present in frontal bone, mandible, and secondary palate. Using immunostaining method, we found that Six2Cre triggered reporter is co-expressed with Runx2. In summary, our data showed Six2Cre can be used to study gene function during palate development and osteogenesis in mouse models.     

From genotype to phenotype: the differential expression of FGF, FGFR, and TGFbeta genes characterizes human cranioskeletal development and reflects clinical presentation in FGFR syndromes.

Mutations in the fibroblast growth factor receptor (FGFR) genes 1, 2, and 3 are causal in a number of craniofacial dysostosis syndromes featuring craniosynostosis with basicranial and midfacial deformity. Great clinical variability is displayed in the pathologic phenotypes encountered. To investigate the influence of developmental genetics on clinical diversity in these syndromes, the expression of several genes implicated in their pathology was studied at sequential stages of normal human embryo-fetal cranial base and facial ossification (n = 6). At 8 weeks of gestation, FGFR1, FGFR2, and FGFR3 are equally expressed throughout the predifferentiated mesenchyme of the cranium, the endochondral skull base, and midfacial mesenchyme. Both clinically significant isoforms of FGFR2, IgIIIa/c and IgIIIa/b, are coexpressed in maxillary and basicranial ossification. By 10 to 13 weeks, FGFR1 and FGFR2 are broadly expressed in epithelia, osteogenic, and chondrogenic cell lineages. FGFR3, however, is maximally expressed in dental epithelia and proliferating chondrocytes of the skull base, but poorly expressed in the osteogenic tissues of the midface. FGF2 and FGF4, but not FGF7, and TGFbeta1 and TGFbeta3 are expressed throughout both osteogenic and chondrogenic tissues in early human craniofacial skeletogenesis. Maximal FGFR expression in the skull base proposes a pivotal role for syndromic growth dysplasia at this site. Paucity of FGFR3 expression in human midfacial development correlates with the relatively benign human mutant FGFR3 midfacial phenotypes. The regulation of FGFR expression in human craniofacial skeletogenesis against background excess ligand and selected cofactors may therefore play a profound role in the pathologic craniofacial development of children bearing FGFR mutations.     

Evidence for an early role for BMP4 signaling in thymus and parathyroid morphogenesis

The thymus and parathyroids are pharyngeal endoderm-derived organs that develop from common organ primordia, which undergo a series of morphological events resulting in separate organs in distinct locations in the embryo. Previous gene expression and functional analyses have suggested a role for BMP4 signaling in early thymus organogenesis. We have used conditional deletion of Bmp4 or Alk3 from the pharyngeal endoderm and/or the surrounding mesenchyme using Foxg1-Cre, Wnt1-Cre or Foxn1-Cre. Deleting Bmp4 from both neural crest cells (NCC) and early endoderm-derived epithelial cells in Foxg1-Cre;Bmp4 conditional mutants resulted in defects in thymus-parathyroid morphogenesis. Defects included reduced condensation of mesenchymal cells around the epithelium, partial absence of the thymic capsule, a delay in thymus and parathyroid separation, and failed or dramatically reduced organ migration. Patterning of the primordia and initial organ differentiation were not affected in any of the mutants. Deleting Bmp4 from NCC-derived mesenchyme or differentiating thymic epithelial cells (TECs) had no effects on thymus-parathyroid development, while loss of Alk3 from either neural crest cells or TECs resulted in only a mild thymic hypoplasia. these results show that the processes of cell specification and morphogenesis during thymus-parathyroid development are independently controlled, and suggest a specific temporal and spatial role for BMP4-mediated epithelial-mesenchymal interactions during early thymus and parathyroid morphogenesis.     

Directed Bmp4 expression in neural crest cells generates a genetic model for the rare human bony syngnathia birth defect

Congenital bony syngnathia, a rare but severe human birth defect, is characterized by bony fusion of the mandible to the maxilla. However, the genetic mechanisms underlying this birth defect are poorly understood, largely due to limitation of available animal models. Here we present evidence that transgenic expression of Bmp4 in neural crest cells causes a series of craniofacial malformations in mice, including a bony fusion between the maxilla and hypoplastic mandible, resembling the bony syngnathia syndrome in humans. In addition, the anterior portion of the palatal shelves emerged from the mandibular arch instead of the maxilla in the mutants. Gene expression assays showed an altered expression of several facial patterning genes, including Hand2, Dlx2, Msx1, Barx1, Foxc2 and Fgf8, in the maxillary and mandibular processes of the mutants, indicating mis-patterned cranial neural crest (CNC) derived cells in the facial region. However, despite of formation of cleft palate and ectopic cartilage, forced expression of a constitutively active form of BMP receptor-Ia (caBmprIa) in CNC lineage did not produce the syngnathia phenotype, suggesting a non-cell autonomous effect of the augmented BMP4 signaling. Our studies demonstrate that aberrant BMP4-mediated signaling in CNC cells leads to mis-patterned facial skeleton and congenital bony syngnathia, and suggest an implication of mutations in BMP signaling pathway in human bony syngnathia.     

Modulation of BMP signaling by Noggin is required for the maintenance of palatal epithelial integrity during palatogenesis

BMP signaling plays many important roles during organ development, including palatogenesis. Loss of BMP signaling leads to cleft palate formation. During development, BMP activities are finely tuned by a number of modulators at the extracellular and intracellular levels. Among the extracellular BMP antagonists is Noggin, which preferentialy binds to BMP2, BMP4 and BMP7, all of which are expressed in the developing palatal shelves. Here we use targeted Noggin mutant mice as a model for gain of BMP signaling function to investigate the role of BMP signaling in palate development. We find prominent Noggin expression in the palatal epithelium along the anterior-posterior axis during early palate development. Loss of Noggin function leads to overactive BMP signaling, particularly in the palatal epithelium. This results in disregulation of cell proliferation, excessive cell death, and changes in gene expression, leading to formation of complete palatal cleft. The excessive cell death in the epithelium disrupts the palatal epithelium integrity, which in turn leads to an abnormal palate-mandible fusion and prevents palatal shelf elevation. This phenotype is recapitulated by ectopic expression of a constitutively active form of BMPR-IA but not BMPR-IB in the epithelium of the developing palate; this suggests a role for BMPR-IA in mediating overactive BMP signaling in the absence of Noggin. Together with the evidence that overexpression of Noggin in the palatal epithelium does not cause a cleft palate defect, we conclude from our results that Noggin mediated modulation of BMP signaling is essential for palatal epithelium integrity and for normal palate development.     

Fgfr2b mediated epithelial-mesenchymal interactions coordinate tooth morphogenesis and dental trigeminal axon patterning

Dental trigeminal nerve fiber growth and patterning are strictly integrated with tooth morphogenesis, but it is still unknown, how these two developmental processes are coordinated. Here we show that targeted inactivation of the dental epithelium expressed Fgfr2b results in cessation of the mouse mandibular first molar development at the degenerated cap stage and the failure of the trigeminal molar nerve to establish the lingual branch at E13.5 stage while the buccal branch develops properly. This axon patterning defect correlates to the histological absence of the mesenchymal dental follicle and adjacent Semaphorin3A-free dental follicle target field as well as appearance of ectopic Sema3A expression domain in the lingual side of the epithelial bud. Although the mesenchymal ligands for Fgfr2b, Fgf3 and -10 were present in the Fgfr2b(-/)(-) dental mesenchyme, mutant dental epithelium showed dramatically reduced proliferation and the lack of Fgf3. Tgfbeta1, which controls Sema3A was absent from the Fgfr2b(-/-) tooth germ, and Sema3A was specifically downregulated in the dental mesenchyme at the bud and cap stage. In addition, the epithelial primary enamel knot signaling center although being molecularly present neither was histologically detectable nor expressed Bmp4 and Fgf3 as well as Fgf4, which is essential for tooth morphogenesis and stimulates mesenchymal Fgf3 and Tgfbeta1. Fgf4 beads rescued Tgfbeta1 in the Fgfr2b(-/-) dental mesenchyme explants and Tgfbeta1 induced de novo Sema3A expression in the dental mesenchyme. Collectively these results demonstrate that epithelial Fgfr2b controls tooth morphogenesis and dental axon patterning, and suggests that Fgfr2b, by mediating local epithelial-mesenchymal interactions, integrates these two distinct developmental processes during odontogenesis.     

Neural and orofacial defects in Folp1 knockout mice [corrected

BACKGROUND

Folic acid is essential for the development of the nervous system and other associated structures. Mice deficient in the folic acid‐binding protein one (Folbp1) gene display multiple developmental abnormalities, including neural and craniofacial defects. To better understand potential interactions between Folbp1 gene and selected genes involved in neural and craniofacial morphogenesis, we evaluated the expression patterns of a panel of crucial differentiation markers (Pax‐3, En‐2, Hox‐a1, Shh, Bmp‐4, Wnt‐1, and Pax‐1).

METHODS

Folbp1 mice were supplemented with low dosages of folinic acid to rescue nullizygotes from dying in utero before gestational day 10. The gene marker analyses were carried out by in situ hybridization.

RESULTS

In nullizygote embryos with open cranial neural tube defects, the downregulation of Pax‐3 and En‐2 in the impaired midbrain, along with an observed upregulation of the ventralizing marker Shh in the expanded floor plate, suggested an important regulatory interaction among these three genes. Moreover, the nullizygotes also exhibit craniofacial abnormalities, such as cleft lip and palate. Pax‐3 signals in the impaired medial nasal primordia were significantly increased, whereas Pax‐1 showed no expression in the undeveloped lateral nasal processes. Although Shh was downregulated, Bmp‐4 was strongly expressed in the medial and lateral nasal processes, highlighting the antagonistic activities of these molecules.

CONCLUSIONS

Impairment of Folbp1 gene function adversely impacts the expression of several critical signaling molecules. Mis‐expression of these molecules, perhaps mediated by Shh, may potentially contribute to the observed failure of neural tube closure and the development of craniofacial defects in the mutant mice. Birth Defects Research (Part A) 67:209–218, 2003. © 2003 Wiley‐Liss, Inc.

     

Cleft palate: A clinical review

Orofacial clefts, including cleft palates (CP), are one of the most common birth defects. CP have a multiplicity of effects on the individual and society in terms of economic costs, loss of productivity, psychosocial effects, and increased morbidity and mortality at all stages of life. Embryological development of the palate is well delineated, with developments in the last decade regarding the biomolecular processes involved. Etiology is complex, involving a number of genetic and environmental factors. Various techniques can be employed for the repair of CP, depending on whether the cleft is of the primary or secondary palate, the width of the cleft, whether lengthening of the palate is necessary, and with regard to concerns of disruption of midfacial growth. All surgical techniques have the goals of restoring functional speech, swallowing, and aesthetics. A multidisciplinary team is necessary for the long‐term pre‐ and postoperative care of CP patients to handle complications, associated anomalies, and to optimize function and quality of life. Birth Defects Research (Part C) 102:333–342, 2014. © 2014 Wiley Periodicals, Inc.