GATA3 is essential for separating patterning domains during facial morphogenesis.

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

Assessment of the maxilla-mandible-nasion angle in normal and aneuploid foetuses in the first trimester of pregnancy.

PURPOSE: This retrospective study aims to determine whether the maxilla-mandible-nasion (MMN) angle can be reliably measured in the first trimester, to describe normal ranges, and to determine if significant changes occur in foetuses with aneuploidies. METHODS: The MMN angle was measured in stored 2D-ultrasound images of 200 normal fetal profiles between 11+0 and 13+6 weeks of gestation. Each image was analyzed by two observers at two independent time points. Bland-Altmann analysis was performed to evaluate the reliability of the measurements. Additionally, the MMN angle was measured on sonograms from 140 aneuploid foetuses. RESULTS: The mean MMN angle in normal foetuses from 11 to 14 weeks of gestation was 15.4°. Reliability of the measurement was high when repeatedly measured by the same observer (ICC = 0.92 and 0.82) and between two observers (ICC = 0.77 and 0.63). Average MMN values in foetuses with trisomy 21, 13, and Turner syndrome were significantly higher than those measured in normal foetuses. The highest differences were observed in foetuses with trisomy 13. Among those, 62% had an MMN angle above the 95th percentile and 92% above the normal mean. CONCLUSION: The MMN angle can be reliably measured in early pregnancy and is abnormal in about 60% of foetuses with trisomy 13.

Shox2 regulates osteogenic differentiation and pattern formation during hard palate development in mice.

During mammalian palatogenesis, cranial neural crest-derived mesenchymal cells undergo osteogenic differentiation and form the hard palate, which is divided into palatine process of the maxilla and the palatine. However, it remains unknown whether these bony structures originate from the same cell lineage and how the hard palate is patterned at the molecular level. Using mice, here we report that deficiency in Shox2 (short stature homeobox 2), a transcriptional regulator whose expression is restricted to the anterior palatal mesenchyme, leads to a defective palatine process of the maxilla but does not affect the palatine. Shox2 overexpression in palatal mesenchyme resulted in a hyperplastic palatine process of the maxilla and a hypoplastic palatine. RNA sequencing and assay for transposase-accessible chromatin-sequencing analyses revealed that Shox2 controls the expression of pattern specification and skeletogenic genes associated with accessible chromatin in the anterior palate. This highlighted a lineage-autonomous function of Shox2 in patterning and osteogenesis of the hard palate. H3K27ac ChIP-Seq and transient transgenic enhancer assays revealed that Shox2 binds distal-acting cis-regulatory elements in an anterior palate-specific manner. Our results suggest that the palatine process of the maxilla and palatine arise from different cell lineages and differ in ossification mechanisms. Shox2 evidently controls osteogenesis of a cell lineage and contributes to the palatine process of the maxilla by interacting with distal cis-regulatory elements to regulate skeletogenic gene expression and to pattern the hard palate. Genome-wide Shox2 occupancy in the developing palate may provide a marker for identifying active anterior palate-specific gene enhancers.

Negative regulation of endothelin signaling by SIX1 is required for proper maxillary development.

Jaw morphogenesis is a complex event mediated by inductive signals that establish and maintain the distinct developmental domains required for formation of hinged jaws, the defining feature of gnathostomes. The mandibular portion of pharyngeal arch 1 is patterned dorsally by Jagged-Notch signaling and ventrally by endothelin receptor A (EDNRA) signaling. Loss of EDNRA signaling disrupts normal ventral gene expression, the result of which is homeotic transformation of the mandible into a maxilla-like structure. However, loss of Jagged-Notch signaling does not result in significant changes in maxillary development. Here we show in mouse that the transcription factor SIX1 regulates dorsal arch development not only by inducing dorsal Jag1 expression but also by inhibiting endothelin 1 (Edn1) expression in the pharyngeal endoderm of the dorsal arch, thus preventing dorsal EDNRA signaling. In the absence of SIX1, but not JAG1, aberrant EDNRA signaling in the dorsal domain results in partial duplication of the mandible. Together, our results illustrate that SIX1 is the central mediator of dorsal mandibular arch identity, thus ensuring separation of bone development between the upper and lower jaws.

Absent 'superimposed-line' sign: novel marker in early diagnosis of cleft of fetal secondary palate.

OBJECTIVES: To describe a novel sign, the 'superimposed-line' sign, for early diagnosis of cleft of the fetal secondary palate on two-dimensional imaging of the vomeromaxillary junction in the midsagittal view. METHODS: This was a prospective evaluation of the superimposed-line sign using two-dimensional sonography (midsagittal view) in 9576 singleton fetuses referred for routine screening between 12 and 20 weeks of gestation. In this view, the vomer bone appears as a line superimposed on the distal two-thirds of the maxillary line, as the vomer fuses with the secondary palate in the midline. If there is a midline cleft of the secondary palate, the line formed by the palate is absent and hence only the vomer bone is visualized, creating a single line instead of the normal superimposed double line. Multiplanar three-dimensional (3D) views were assessed in cases in which the superimposed-line sign was absent. RESULTS: The superimposed line was absent in 17 fetuses with a cleft of the secondary palate that was confirmed by 3D evaluation. Of these, 13 had defects involving the premaxilla and four had an isolated cleft of the secondary palate. Postnatal confirmation was available in all cases. The sign was useful in ruling out cleft of the fetal secondary palate in 32 high-risk cases with a family history of cleft palate. The superimposed-line sign had a sensitivity of 89.5% in detecting cleft of the secondary palate. CONCLUSIONS: The superimposed-line sign is a new sonographic marker for evaluation of cleft of the fetal secondary palate; documentation of this sign proves the presence of both the palate and vomer in the midline. This marker can be demonstrated clearly in the late first trimester, allowing early diagnosis of secondary palatine cleft. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Cis-control of Six1 expression in neural crest cells during craniofacial development.

BACKGROUND: Six1 is a transcriptional factor that plays an important role in embryonic development. Mouse and chick embryos deficient for Six1 have multiple craniofacial anomalies in the facial bones and cartilages. Multiple Six1 enhancers have been identified, but none of them has been reported to be active in the maxillary and mandibular process. RESULTS: We studied two Six1 enhancers in the chick neural crest tissues during craniofacial development. We showed that two evolutionarily conserved enhancers, Six1E1 and Six1E2, act synergistically. Neither Six1E1 nor Six1E2 alone can drive enhancer reporter signal in the maxillary or mandibular processes. However, their combination, Six1E, showed robust enhancer activity in these tissues. Similar reporter signal can also be driven by the mouse homolog of Six1E. Mutations of multiple conserved transcriptional factor binding sites altered the enhancer activity of Six1E, especially mutation of the LIM homeobox binding site, dramatically reduced the enhancer activity, implying that the Lhx protein family be an important regulator of Six1 expression. CONCLUSION: This study, for the first time, described the synergistic activation of two Six1 enhancers in the maxillary and mandibular processes and will facilitate more detailed studies of the regulation of Six1 in craniofacial development.

Critical Growth Processes for the Midfacial Morphogenesis in the Early Prenatal Period.

BACKGROUND: Congenital midfacial hypoplasia often requires intensive treatments and is a typical condition for the Binder phenotype and syndromic craniosynostosis. The growth trait of the midfacial skeleton during the early fetal period has been assumed to be critical for such an anomaly. However, previous embryological studies using 2-dimensional analyses and specimens during the late fetal period have not been sufficient to reveal it. OBJECTIVE: To understand the morphogenesis of the midfacial skeleton in the early fetal period via 3-dimensional quantification of the growth trait and investigation of the developmental association between the growth centers and midface. METHODS: Magnetic resonance images were obtained from 60 human fetuses during the early fetal period. Three-dimensional shape changes in the craniofacial skeleton along growth were quantified and visualized using geometric morphometrics. Subsequently, the degree of development was computed. Furthermore, the developmental association between the growth centers and the midfacial skeleton was statistically investigated and visualized. RESULTS: The zygoma expanded drastically in the anterolateral dimension, and the lateral part of the maxilla developed forward until approximately 13 weeks of gestation. The growth centers such as the nasal septum and anterior portion of the sphenoid were highly associated with the forward growth of the midfacial skeleton (RV = 0.589; P < .001). CONCLUSIONS: The development of the midface, especially of the zygoma, before 13 weeks of gestation played an essential role in the midfacial development. Moreover, the growth centers had a strong association with midfacial forward growth before birth.

Upper jaw development in the absence of teeth: New insights for craniodental evo-devo integration.

In p63-null mice (p63-/- ), teeth fail to form but the mandible forms normally; conversely, the upper jaw skeleton is malformed. Here we explored whether lack of dental tissues contributed to midfacial dysmorphologies in p63-/- mice by testing if facial prominence defects appeared before odontogenesis failed. We also investigated gene dose effects by testing if one wild type (WT) p63 allele (p63+/- ) was sufficient for normal upper jaw skeleton formation. We micro-CT scanned PFA-fixed p63-/- , p63+/- , and WT (p63+/+ ) adult and embryonic mice aged E10-E14. Next, we landmarked mandibular (MdP), maxillary (MxP) and nasal prominences (NPs), and facial bones. 3D landmark data were assessed using Principal Component, Canonical Variate, Partial Least Squares, and other statistical analyses. The p63-/- embryos showed MxP and NP malformations by E12, despite the presence of dental tissues. MdP shape was comparable among p63-/- , p63+/- , and p63+/+ embryos. Upper jaw shape was comparable between p63+/+ and p63+/- adults. The upper jaw and its dentition both require p63 signaling, but not each other's presence, to form properly. One WT p63 allele enables normal midfacial morphogenesis; gene dose may be a target for jaw macroevolution. Jaw-specific genetic mechanisms likely integrate the evo-devo of dentitions with upper versus lower jaws.

Craniofacial structures' development in prenatal period: An MRI study.

INTRODUCTION: The development of skeletal structures (cranial base, upper and lower) and upper airways spaces (oropharyngeal and nasopharyngeal) of the skull has always been an issue of great interest in orthodontics. Foetal MRI images obtained as screening exam during pregnancy can help to understand the development of these structures using a sample cephalometric analysis. MATERIAL AND METHODS: A total of 28 MRI images in sagittal section of foetuses from 20th to 32th weeks of gestation were obtained to dispel doubts about the presence of skeletal malformations. Cephalometric measurements were performed on MRI T2-dependent images acquired with a 1.5 T scanner. The Software Osirix 5 permits to study sagittal and vertical dimensions of the skull analysing linear measurements, angles and areas of the skeletal structures. RESULTS: Vertical and sagittal dimension of cranial base, maxilla and mandible grow significantly (P < .01) between the second and third trimester of gestational period as well as nasopharyngeal and oropharyngeal spaces (P < .05). High correlation between the development of anterior cranial base and functional areas devoted to speech and swallow is demonstrated (r: .97). CONCLUSIONS: The development of craniofacial structures during foetal period seems to show a close correlation between skeletal features and functional spaces with a peak between the second and third trimester of gestation. MRI images result helpful for the clinician to detect with a sample cephalometric analysis anomalies of skeletal and functional structures during prenatal period.

Separate development of the maxilla and mandible is controlled by regional signaling of the maxillomandibular junction during avian development.

BACKGROUND: Syngnathia is a congenital craniofacial disorder characterized by bony or soft tissue fusion of upper and lower jaws. Previous studies suggested some causative signals, such as Foxc1 or Bmp4, cause the disruption of maxillomandibular identity, but their location and the interactive signals involved remain unexplored. We wanted to examine the embryonic origin of syngnathia based on the assumption that it may be located at the separation between the maxillary and mandibular processes. This region, known as the maxillomandibular junction (MMJ), is involved in segregation of cranial neural crest-derived mesenchyme into the presumptive upper and lower jaws. RESULTS: Here we investigated the role of Fgf, Bmp, and retinoid signaling during development of MMJ in chicken embryos. By changing the levels of these signals with bead implants, we induced syngnathia with microstomia on the treated side, which showed increased Barx1 and neural cell adhesion molecule (NCAM) expression. Redistribution of proliferating cells was also observed at the proximal region to maxillary and mandibular arch around MMJ. CONCLUSIONS: We propose that interactive molecular signaling by Fgfs, Bmps, and retinoids around MMJ is required for normal separation of the maxilla and mandible, as well as the proper positioning of beak commissure during early facial morphogenesis. Developmental Dynamics 246:28-40, 2017. © 2016 Wiley Periodicals, Inc.

Foxf2 is required for secondary palate development and Tgfß signaling in palatal shelf mesenchyme.

The secondary palate separates the oral from the nasal cavity and its closure during embryonic development is sensitive to genetic perturbations. Mice with deleted Foxf2, encoding a forkhead transcription factor, are born with cleft palate, and an abnormal tongue morphology has been proposed as the underlying cause. Here, we show that Foxf2(-/-) maxillary explants cultured in vitro, in the absence of tongue and mandible, failed to close the secondary palate. Proliferation and collagen content were decreased in Foxf2(-/-) palatal shelf mesenchyme. Phosphorylation of Smad2/3 was reduced in mutant palatal shelf, diagnostic of attenuated canonical Tgfß signaling, whereas phosphorylation of p38 was increased. The amount of Tgfß2 protein was diminished, whereas the Tgfb2 mRNA level was unaltered. Expression of several genes encoding extracellular proteins important for Tgfß signaling were reduced in Foxf2(-)(/)(-) palatal shelves: a fibronectin splice-isoform essential for formation of extracellular Tgfß latency complexes; Tgfbr3 - or betaglycan - which acts as a co-receptor and an extracellular reservoir of Tgfß; and integrins αV and ß1, which are both Tgfß targets and required for activation of latent Tgfß. Decreased proliferation and reduced extracellular matrix content are consistent with diminished Tgfß signaling. We therefore propose that gene expression changes in palatal shelf mesenchyme that lead to reduced Tgfß signaling contribute to cleft palate in Foxf2(-)(/)(-) mice.

Lhx6 and Lhx8 promote palate development through negative regulation of a cell cycle inhibitor gene, p57Kip2.

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57(Kip2) (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6(-/-);Lhx8(-/-) mutants. p57(Kip2) has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57(Kip2) by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57(Kip2) via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57(Kip2) expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.

Molecular mechanisms of midfacial developmental defects.

The morphogenesis of midfacial processes requires the coordination of a variety of cellular functions of both mesenchymal and epithelial cells to develop complex structures. Any failure or delay in midfacial development as well as any abnormal fusion of the medial and lateral nasal and maxillary prominences will result in developmental defects in the midface with a varying degree of severity, including cleft, hypoplasia, and midline expansion. Despite the advances in human genome sequencing technology, the causes of nearly 70% of all birth defects, which include midfacial development defects, remain unknown. Recent studies in animal models have highlighted the importance of specific signaling cascades and genetic-environmental interactions in the development of the midfacial region. This review will summarize the current understanding of the morphogenetic processes and molecular mechanisms underlying midfacial birth defects based on mouse models with midfacial developmental abnormalities.

The Hox gene Dfd controls organogenesis by shaping territorial border through regulation of basal DE-Cadherin distribution.

Hox genes are highly conserved selector genes controlling tissue identity and organogenesis. Recent work indicates that Hox genes also controls cell segregation and segmental boundary in various species, however the underlying cellular mechanisms involved in this function are poorly understood. In Drosophila melanogaster, the Hox gene Deformed (Dfd) is required for specification and organogenesis of the adult Maxillary (Mx) palp. Here, we demonstrate that differential Dfd expression control Mx morphogenesis through the formation of a physical boundary separating the Mx field and the Peripodial Epithelium (PE). We show that this boundary relies on DE-cadherin (DE-cad) basal accumulation in Mx cells controlled by differential Dfd expression. Indeed, Dfd controls boundary formation through cell autonomous basal redistribution of DE-cad which leads to subsequent fold at the Dfd expression border. Finally, the loss of Mx DE-cad basal accumulation and hence of Mx-PE folding is sufficient to prevent Mx organogenesis thus revealing the crucial role of boundaries in organ differentiation. Altogether, these results reveal that Hox coordination of tissue morphogenesis relies on boundary fold formation through the modulation of DE-cad positioning.

Identification and functional analysis of novel facial patterning genes in the duplicated beak chicken embryo.

Cranial neural crest cells form the majority of the facial skeleton. However exactly when the pattering information and hence jaw identity is established is not clear. We know that premigratory neural crest cells contain a limited amount of information about the lower jaw but the upper jaw and facial midline are specified later by local tissue interactions. The environmental signals leading to frontonasal identity have been explored by our group in the past. Altering the levels of two signaling pathways (Bone Morphogenetic Protein) and retinoic acid (RA) in the chicken embryo creates a duplicated midline on the side of the upper beak complete with egg tooth in place of maxillary derivatives (Lee et al., 2001). Here we analyze the transcriptome 16 h after bead placement in order to identify potential mediators of the identity change in the maxillary prominence. The gene list included RA, BMP and WNT signaling pathway genes as well as transcription factors expressed in craniofacial development. There was also cross talk between Noggin and RA such that Noggin activated the RA pathway. We also observed expression changes in several poorly characterized genes including the upregulation of Peptidase Inhibitor-15 (PI15). We tested the functional effects of PI15 overexpression with a retroviral misexpression strategy. PI15 virus induced a cleft beak analogous to human cleft lip. We next asked whether PI15 effects were mediated by changes in expression of major clefting genes and genes in the retinoid signaling pathway. Expression of TP63, TBX22, BMP4 and FOXE1, all human clefting genes, were upregulated. In addition, ALDH1A2, ALDH1A3 and RA target, RARß were increased while the degradation enzyme CYP26A1 was decreased. Together these changes were consistent with activation of the RA pathway. Furthermore, PI15 retrovirus injected into the face was able to replace RA and synergize with Noggin to induce beak transformations. We conclude that the microarrays have generated a rich dataset containing genes with important roles in facial morphogenesis. Moreover, one of these facial genes, PI15 is a putative clefting gene and is in a positive feedback loop with RA.

ADAM10 is essential for cranial neural crest-derived maxillofacial bone development.

Growth disorders of the craniofacial bones may lead to craniofacial deformities. The majority of maxillofacial bones are derived from cranial neural crest cells via intramembranous bone formation. Any interruption of the craniofacial skeleton development process might lead to craniofacial malformation. A disintegrin and metalloprotease (ADAM)10 plays an essential role in organ development and tissue integrity in different organs. However, little is known about its function in craniofacial bone formation. Therefore, we investigated the role of ADAM10 in the developing craniofacial skeleton, particularly during typical mandibular bone development. First, we showed that ADAM10 was expressed in a specific area of the craniofacial bone and that the expression pattern dynamically changed during normal mouse craniofacial development. Then, we crossed wnt1-cre transgenic mice with adam10-flox mice to generate ADAM10 conditional knockout mice. The stereomicroscopic, radiographic, and von Kossa staining results showed that conditional knockout of ADAM10 in cranial neural crest cells led to embryonic death, craniofacial dysmorphia and bone defects. Furthermore, we demonstrated that impaired mineralization could be triggered by decreased osteoblast differentiation, increased cell death. Overall, these findings show that ADAM10 plays an essential role in craniofacial bone development.

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.

Identification of a face enhancer reveals direct regulation of LIM homeobox 8 (Lhx8) by wingless-int (WNT)/ß-catenin signaling.

Development of the mammalian face requires a large number of genes that are expressed with spatio-temporal specificity, and transcriptional regulation mediated by enhancers plays a key role in the precise control of gene expression. Using chromatin immunoprecipitation for a histone marker of active enhancers, we generated a genome-wide map of candidate enhancers from the maxillary arch (primordium for the upper jaw) of mouse embryos. Furthermore, we confirmed multiple novel craniofacial enhancers near the genes implicated in human palate defects through functional assays. We characterized in detail one of the enhancers (Lhx8_enh1) located upstream of Lhx8, a key regulatory gene for craniofacial development. Lhx8_enh1 contained an evolutionarily conserved binding site for lymphoid enhancer factor/T-cell factor family proteins, which mediate the transcriptional regulation by the WNT/ß-catenin signaling pathway. We demonstrated in vitro that WNT/ß-catenin signaling was indeed essential for the expression of Lhx8 in the maxillary arch cells and that Lhx8_enh1 was a direct target of the WNT/ß-catenin pathway. Together, we uncovered a molecular mechanism for the regulation of Lhx8, and we provided valuable resources for further investigation into the gene regulatory network of craniofacial development.

Patterning of mammalian heterodont dentition within the upper and lower jaws.

Mammalian heterodont dentition is differentiated into incisors, canines, premolars, and molars in the mesial-distal direction, in both the upper and lower jaws. Although all the lower teeth are rooted in the mandible, the upper incisors are rooted in the premaxilla and the upper canine and the teeth behind it are in the maxilla. The present study uncovers ontogenetic backgrounds to these shared and differing mesiodistal patterns of the upper and lower dentition. To this end, we examined the dentition development of the house shrew, Suncus murinus, instead of the rodent model animals because the dentition of this primitive eutherian species includes all the tooth classes, and no toothless diastema region. In the shrew, the upper incisor-forming region extended over the medial nasal prominence and the mesial part of the maxillary prominence. Consequently, the maxillary and mandibular prominences were in a mirror-image relationship in terms of the mesiodistally differentiated tooth-forming regions and of the complementary gene expression pattern, with Bmp4 in the mesial and Fgf8 in the distal regions. This suggests shared molecular mechanisms regulating tooth class differentiation between the upper and lower jaws. However, the premaxillary bone appeared within the mesenchyme of the medial nasal prominence, but grew distally beyond the former epithelial boundary with the maxillary prominence to form, finally, the incisive (premaxillary-maxillary) suture just mesial to the canine. Therefore, the developmental locations of the upper incisors are not inconsistent with the classical osteological criterion of the upper canine by comparative odontologists.

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/ß-catenin signaling. Our study has identified a previously unknown regulatory link between WNT9B and FGF signaling during lip and upper jaw development.