Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development

Outgrowth and fusion of the lateral and medial nasal processes and of the maxillary process of the first branchial arch are integral to lip and primary palate development. Wnt9b mutations are associated with cleft lip and cleft palate in mice; however, the cause of these defects remains unknown. Here, we report that Wnt9b(-/-) mice show significantly retarded outgrowth of the nasal and maxillary processes due to reduced proliferation of mesenchymal cells, which subsequently results in a failure of physical contact between the facial processes that leads to cleft lip and cleft palate. These cellular defects in Wnt9b(-/-) mice are mainly caused by reduced FGF family gene expression and FGF signaling activity resulting from compromised canonical WNT/β-catenin signaling. Our study has identified a previously unknown regulatory link between WNT9B and FGF signaling during lip and upper jaw development.     

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.     

Tgf-beta3-induced palatal fusion is mediated by Alk-5/Smad pathway

Cleft palate is among the most common birth defects in humans, caused by a failure in the complex multistep developmental process of palatogenesis. It has been recently shown that transforming growth factor beta3 (Tgf-beta3) is an absolute requirement for successful palatal fusion, both in mice and humans. However, very little is known about the mechanisms of Tgf-beta3 signaling during this process. Here we show that putative Tgf-beta type I receptors, Alk-1, Alk-2, and Alk-5, are all endogenously expressed in the palatal epithelium. Activation of Alk-5 in the Tgf-beta3 (-/-) palatal epithelium is able to rescue palatal fusion, whereas inactivation of Alk-5 in the wild-type palatal epithelium prevents palatal fusion. The effect of Alk-2 is similar, but less pronounced. The induction of fusion by activation of Alk-5 or Alk-2 is stronger in the posterior parts of the palates at the embryonic day 14 (E14), while their activation at E13.5 also restores anterior fusion, reflecting the natural anterior-posterior direction of palate maturation in vivo. We also show that Smad2 is endogenously activated in the palatal midline epithelial seam (MES) during the fusion process. By using a mutant Alk-5 receptor that is an active kinase but is unable to activate Smads, we show that activation of Smad-independent Tgf-beta responses is not sufficient to induce fusion of shelves deficient in Tgf-beta3. Based on these observations, we conclude that the Smad2-dependent Alk-5 signaling pathway is dominant in palatal fusion driven by Tgf-beta3.     

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

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

Overexpression of Smad2 in Tgf-beta3-null mutant mice rescues cleft palate

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta3-/-), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3-/-/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion.     

Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion

Transforming growth factor beta (TGF-beta) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-beta action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-beta type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-beta type II receptor (TGFbetaRII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-beta, and (2) ALK5 acts also in conjunction with type II receptors other than TGFbetaRII.     

Tissue-specific expression of Cre recombinase from the Tgfb3 locus

Tgfb3, a member of the TGF-beta superfamily, is tightly regulated, both spatially and temporally, during embryogenesis. Previous mouse knockout studies have demonstrated that Tgfb3 is absolutely required for normal palatal fusion and pulmonary development. We have generated a novel tool to ablate genes in Tgfb3-expressing cells by targeting the promoterless Cre-pgk-Neo cassette into exon 1 of the mouse Tgfb3 gene, which generates a functionally null Tgfb3 allele. Using the Rosa26 reporter assay, we demonstrate that Cre-induced recombination was already induced at embryonal day 10 (E10) in the ventricular myocardium, limb buds, and otic vesicles. At E14, robust recombination was detected in the prefusion palatal epithelium. Deletion of the TGF-beta type I receptor Alk5 (Tgfbr1) specifically in Tgfb3 expressing cells using the Tgfb3-Cre driver line lead to a cleft palate phenotype similar to that seen in conventional Tgfb3 null mutants. In addition, Alk5/ Tgfb3-Cre mice displayed hydrocephalus, and severe intracranial bleeding due to germinal matrix hemorrhage.     

Growth status of children treated for unilateral cleft lip and palate

Cephalometric distances, angles, and proportions were evaluated for 32 children 5 to 8 years of age treated for unilateral cleft lip and palate. The children were age and sex matched with untreated controls with normal skeletal relationships. The unilateral cleft lip and palate sample was treated by the same surgeon and orthodontist using the same techniques and appliances. Measures of overall facial proportions, facial convexity, and prognathism were not significantly different between the two groups. The primary group differences pertain to the posterior aspect of the maxilla, which is vertically short in the unilateral cleft lip and palate sample. Horizontally, the maxilla of the unilateral cleft lip and palate children was significantly longer, producing a steeper palatal plane. In addition, the zygoma and orbits of unilateral cleft lip and palate children were somewhat retruded; the posterior cranial base and total mandibular length also were longer in the unilateral cleft lip and palate children.     

Modified technique of presurgical infant maxillary orthopedics for complete unilateral cleft lip and palate

This article introduces a modified device fabrication and facial taping method that increases the efficiency and efficacy of presurgical infant maxillary orthopedic therapy for babies born with complete cleft lip and palate. Interarch and intra-arch relationships of the maxillary and mandibular dental arches were evaluated on mounted stone models before and after treatment. The palatal plate device was custom-fabricated in a manner that bypassed the need for periodic acrylic addition and removal, thereby eliminating the risk of natural maxillary growth restriction during therapy. Elastic labial tapes were fabricated and applied in a configuration that mimicked normal function of the orbicularis oris muscle. A nasal stent wire was utilized from the initiation of therapy to enhance intraoral retention and stability of the device. Examples of infants undergoing a unilateral complete cleft lip and palate treatment protocol are presented. Treatment objectives were achieved within 7 to 8 weeks of therapy for patients who had an initial alveolar cleft size more than 10 to 12 mm. The modified protocol of presurgical infant maxillary orthopedic therapy is an effective and efficient treatment modality in reversing the pre-existing orofacial dysmorphism by redirecting the infant's natural growth.     

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.     

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.     

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.     

The inductive role of Wnt-β-Catenin signaling in the formation of oral apparatus

Proper patterning and growth of oral structures including teeth, tongue, and palate rely on epithelial–mesenchymal interactions involving coordinated regulation of signal transduction. Understanding molecular mechanisms underpinning oral–facial development will provide novel insights into the etiology of common congenital defects such as cleft palate. In this study, we report that ablating Wnt signaling in the oral epithelium blocks the formation of palatal rugae, which are a set of specialized ectodermal appendages serving as Shh signaling centers during development and niches for sensory cells and possibly neural crest related stem cells in adults. Lack of rugae is also associated with retarded anteroposterior extension of the hard palate and precocious mid-line fusion. These data implicate an obligatory role for canonical Wnt signaling in rugae development. Based on this complex phenotype, we propose that the sequential addition of rugae and its morphogen Shh, is intrinsically coupled to the elongation of the hard palate, and is critical for modulating the growth orientation of palatal shelves. In addition, we observe a unique cleft palate phenotype at the anterior end of the secondary palate, which is likely caused by the severely underdeveloped primary palate in these mutants. Last but not least, we also discover that both Wnt and Shh signalings are essential for tongue development. We provide genetic evidence that disruption of either signaling pathway results in severe microglossia. Altogether, we demonstrate a dynamic role for Wnt-β-Catenin signaling in the development of the oral apparatus.     

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.     

Avian Facial Morphogenesis Is Regulated by c-Jun N-terminal Kinase/Planar Cell Polarity (JNK/PCP) Wingless-related (WNT) Signaling

Wingless-related proteins (WNTs) regulate extension of the central axis of the vertebrate embryo (convergent extension) as well as morphogenesis of organs such as limbs and kidneys. Here, we asked whether WNT signaling directs facial morphogenesis using a targeted approach in chicken embryos. WNT11 is thought to mainly act via β-catenin-independent pathways, and little is known about its role in craniofacial development. RCAS::WNT11 retrovirus was injected into the maxillary prominence, and the majority of embryos developed notches in the upper beak or the equivalent of cleft lip. Three-dimensional morphometric analysis revealed that WNT11 prevented lengthening of the maxillary prominence, which was due in part to decreased proliferation. We next determined, using a series of luciferase reporters, that WNT11 strongly induced JNK/planar cell polarity signaling while repressing the β-catenin-mediated pathway. The activation of the JNK-ATF2 reporter was mediated by the DEP domain of Dishevelled. The impacts of altered signaling on the mesenchyme were assessed by implanted Wnt11- or Wnt3a-expressing cells (activates β-catenin pathway) into the maxillary prominence or by knocking down endogenous WNT11 with RNAi. Host cells were attracted to Wnt11 donor cells. In contrast, cells exposed to Wnt3a or the control cells did not migrate. Cells in which endogenous WNT11 was knocked down were more oriented and shorter than those exposed to exogenous WNT11. The data suggest that JNK/planar cell polarity WNT signaling operates in the face to regulate several morphogenetic events leading to lip fusion.     

Timing of Egf treatment differentially affects Tgf-beta2 induced cranial suture closure

Premature suture obliteration results in an inability of cranial and facial bones to grow, with craniofacial dysmorphology requiring surgical correction as a consequence. Understanding signaling pathways associated with suture morphogenesis might enable non-invasive treatment of patients with fused sutures. Tgf-beta 2 induces premature suture fusion associated with increased cell proliferation both in vitro and in vivo. Tgf-beta 2 and Egf signal transduction pathways use some signaling proteins in common to regulate proliferation and differentiation, leading to speculation that these two pathways converge to regulate normal suture development. It was therefore hypothesized that Egf could induce suture fusion, and that Tgf-beta 2-induced suture closure occurred via an Egf-dependent pathway. A well-established fetal calvarial organ culture system was used to expose developing E19.5 fetal rat coronal sutures to Egf, Tgf-beta 2 and SC-120, a blocker of Egf receptor activity. Co-culture experiments examined the effect of Egf on Tgf-beta 2-induced suture closure when Egf was given either prior to or after Tgf-beta 2 treatment. Histomorphometric measurement of suture width was done on sagittal sections through coronal sutures harvested after 5 days in culture. Western blotting using phospho-antibodies against Egf receptors was used to confirm Egf receptor activity. Suture width increased with increasing concentrations of Egf, demonstrating that Egf-induced cell activity alone was not sufficient to cause premature suture obliteration. Egf administered prior to Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced suture obliteration, demonstrating that pre-exposure of cells to this powerful mitogen prevented their response to signals induced by Tgf-beta 2. However, Egf added after Tgf-beta 2 treatment had no effect on Tgf-beta 2-induced suture closure. Blocking Egf activity after Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced obliteration, indicating that Tgf-beta 2 required Egf activity to induce suture obliteration. Appropriate timing of signal generation by Egf and Tgf-beta 2 is critical for normal suture development and maintenance of suture patency.     

Craniofacial defects in mice lacking BMP type I receptor Alk2 in neural crest cells

Neural crest cells (NCCs) are pluripotent migratory cells that contribute to the development of various craniofacial structures. Many signaling molecules have been implicated in the formation, migration and differentiation of NCCs including bone morphogenetic proteins (BMPs). BMPs signal through a receptor complex composed of type I and type II receptors. Type I receptors (Alk2, Alk3 and Alk6) are the primary determinants of signaling specificity and therefore understanding their function is important in revealing the developmental roles of molecular pathways regulated by BMPs. Here we used a Cre/loxP system for neural crest specific deletion of Alk2. Our results show that mice lacking Alk2 in the neural crest display multiple craniofacial defects including cleft palate and a hypotrophic mandible. Based on the present results we conclude that signaling via Alk2 receptors is non-redundant and regulates normal development of a restricted set of structures derived from the cranial neural crest.     

Developmental deficiencies of the upper facial skeleton due to partial elimination of mesencephalic neural crest cells in the chick embryo

With the aim to test the hypothesis that cells derived from the mesencephalic portion of the neural crest, are involved in the process of differentiation of various upper facial bones, in 41 chick embryos of the 6-somite stage (approx. 26 hours of incubation) the anterior and middle thirds of this part of the neural crest were partially eliminated by micro-laser irradiation, either unilaterally or bilaterally. Of the 14 embryos sacrificed at the age of 12 days, a number of 6 proved to have developed harelip and/or cleft palate conditions. In these embryos, in addition a reduction or absence of the maxillary, palatal, jugale and quadrato-jugale was observed. On the contrary, other facial bones as well as the first and second branchial arch cartilages proved to have developed normally. From these results the conclusion may be drawn that (a sufficient number of) cells from the anterior and middle thirds of the mesencephalic neural crest are indispensable for a normal differentiation of the maxillary, palatal, jugale and quadrato-jugale.     

Loss of distinct arterial and venous boundaries in mice lacking endoglin,a vascular-specific TGFbeta coreceptor

Several characteristic morphological and functional differences distinguish arteries from veins. It was thought that hemodynamic forces shaped these differences; however, increasing evidence suggests that morphogenetic programs play a central role in blood vessel differentiation. Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by the inappropriate fusion of arterioles with venules. The genes implicated in this disease, ALK1 and endoglin, may be involved in defining the fundamental boundaries between arteries and veins. We previously showed that mice lacking Alk1 lost structural, molecular, and functional distinctions between arteries and veins. Here, we report that mice lacking endoglin develop arterial-venous malformations and fail to confine intraembryonic hematopoiesis to arteries. In contrast to Alk1 mutants, endoglin mutants do not show profound vessel dilation or downregulation of arterial ephrinB2. Finally, our data indicate that a failure in cardiac cushion formation observed in both strains may be secondary to the peripheral vasculature defect. The phenotypic similarities, yet reduced severity, implicates endoglin as an accessory coreceptor that specifically modulates Alk1 signaling. We propose that endoglin and Alk1 are necessary for the maintenance of distinct arterial-venous vascular beds and that attenuation of the Alk1 signaling pathway is the precipitating event in the etiology of HHT.