Indian Journal of Plastic Surgery Medknow Publications on behalf of Indian Journal of Plastic Surgery ISSN: 0970-0358 EISSN: 1998-376x Vol. 42, Num. S1, 2009, pp. 35-50

Indian Journal of Plastic Surgery, Vol. 42, Supp. 1 , 2009, pp. 35-50

Review Article

Cleft lip and palate genetics and application in early embryological development

Wenli Yu, Maria Serrano, Symone San Miguel, L. Bruno Ruest, Kathy K. H. Svoboda

Department of Biomedical Sciences, Texas A&M Health Science Center, Baylor College of Dentistry, Dallas, TX 75246 USA

Correspondence Address: Dr. Kathy K. H. Svoboda, Biomedical Sciences Department, Baylor College of Dentistry, 3302 Gaston Ave., Dallas, TX 75246 USA, Ksvoboda@bcd.tamhsc.edu

Code Number: pl09077

PMID: 19884679

DOI: 10.4103/0970-0358.57185

Abstract

Keywords: Cleft lip/palate; Human/murine palate genetics; Palate development

Introduction

Disturbances at any stage during palate development, e.g., defective palatal shelf growth, failed or delayed elevation, and blocked fusion, can result in cleft palate (CP) ^[1],[2] with or without cleft lip (CL/P). As one of the most common congenital cranio-facial defects, CL/P occurs in approximately 1 per 750 live births in the United States ^[2],[3] Clefts occur more frequently among Asians (about 1:400) and certain American Indians than Europeans or European descendants. Clefts are relatively less common among Africans and African Americans (about 1:1500). ^[4] Cleft lip and palate may not be life-threatening but many functions such as feeding, digestion, speech, middle-ear ventilation, hearing, respiration and facial and dental development can be disturbed because of the structures involved. These problems can also cause emotional, psycho-social, and educational difficulties. In addition, CP is an economic burden.

The aetiology of cleft lip with or without palate (CL/P) is theorized to be a combination of factors associated with genes and environment. ^[5],[6] The advent of gene targeting technology and basic conventional techniques using animal models has led to the identification of genes associated with known and unknown etiologic factors. Characterization of the genomic sequences will greatly impact regulation of gene networks and pinpoint any variations in the different stages of craniofacial morphogenesis. In this article, emphasis is placed on different genes associated with the classifications of CL/P into syndromic [Table - 1] and nonsyndromic [Table - 2]. Each classification plays a significant role in understanding the molecular and genetic mechanisms affecting these types of craniofacial defects. ^[7],[8],[9] In addition to known genes there is strong evidence that several environmental factors (e.g., alcohol consumption, tobacco, and anti-convulsants) increase the risk of CL/P. ^[10],[11] In contrast, several studies have shown that folic acid may have a protective effect on CL/P and neural tube defects. ^{[12],[13],[14],[15],[16]} Recent data from the National Birth Defect Prevention Network have indicated a decrease in neural tube defects from 5/10,000 to less than 2/10,000 after the fortification of the food supply with folic acid, indicating that this vitamin and the proteins that facilitate the uptake and metabolism of folic acid may be candidate genes in cranio-facial development. ^{[14],[17],[18],[19],[20]}

This review will concentrate on genetic contributions to facial clefts with/without cleft palate. We will begin with an overview of early palatal development, concentrate on muscle and bone development, and incorporate the effects of environmental insults and known genetic mutations that impact human palatal development.

Embryonic Palate Development

The palatal structures are composed of the cranial neural crest (CNC)-derived mesenchyme and pharyngeal ectoderm. ^[21-24] Epithelia that cover the palatal shelves are regionally divided into oral, nasal and medial edge epithelia (MEE). The nasal and oral epithelia differentiate into pseudo-stratified and squamous epithelia, whereas MEE is removed from the fusion line [Figure - 1].

The secondary palate originates as an outgrowth of the maxillary prominences at approximately embryonic day 11.5 in the mouse (E11.5-m) [Figure - 1] and post coital six weeks in humans (p.c.6wk-h). The palate shelves initially grow vertically along the sides of the tongue (E13.5-m; p.c.7wk-h) and then rise above the tongue as the latter drops in the oral cavity due to the forward and downward growth of the mandible (E14.0-m; p.c.8wks-h). With continued growth, the shelves appose at the midline (E14.5-m; p.c.10wks-h) and eventually fuse (E15.5-m; p.c.13wk-h). ^[25] Numerous genes similar in mice ^[26] and humans ^{[25],[27],[28]} are expressed [Table - 1] during palatal development.

During fusion the epithelium covering the tip of the opposing palatal shelves, adheres, intercalates and thins into a single-layer midline epithelial seam (MES). ^[23] The disintegration of this seam results in the confluence of the palatal mesenchyme. Tremendous interest has arisen in cellular mechanisms underlying MES degradation. Epithelial-mesenchymal transition (EMT) is one of the proposed models that regulates medial edge epithelial (MEE) cell fate. ^{[23],[29],[30],[31],[32],[33],[34],[35],[36]} However, other mechanisms have been proposed, such as apoptosis, ^{[37],[38],[39],[40]} in which all MEE cells are theorized to die during fusion. Alternatively, it is hypothesized by some researchers that MES cells disappear by migrating from the midline towards the nasal and oral epithelia. ^[41],[42] Other investigators postulate that all events, including apoptosis, migration and EMT, may occur. ^{[23],[39],[43]} Interestingly, the fusion of the external surface of the bilateral maxillary processes with the naso-frontal prominence in the chick is similar to palatal fusion [Figure - 2]. ^[44] The outer periderm layer dies through apoptosis, and the lateral edge epithelium of the inter-maxillary segment of the naso-frontal process fuses with the medial edge epithelium of the external maxillary process to form a seam that transitions to a confluent mesenchyme [Figure - 2]. ^[44] Evidence supporting these theories, especially those involving EMT and apoptosis, will be presented and further discussed.

Molecular Signalling Events in Embryonic Palatal Development

As stated above, cleft palate with or without cleft lip is a complex trait caused by a combination of multiple genes and environmental factors. ^[5] Palatal shelf development defects will be divided into five categories for the purpose of this review:

Failure of palatal shelf formation

The failure of the palatal shelf formation is a rare severe defect. Recent studies have identified several molecular networks operating between the palatal shelf epithelium and mesenchyme during different steps of palatogenesis. These networks include signalling molecules and growth factors such as sonic hedgehog (Shh), members of the transforming growth factor β (TGfβ) super family, including bone morphogenetic proteins (Bmps) and Tgfβs, fibroblast growth factors (Fgfs) and their receptors (FgfR), effectors and targets. ^[25] Studies addressing the role of Fgf signalling during early palatal development by analyzing Fgf10 and FgfR2b mutants found altered cell proliferation within both mesenchyme and epithelium in the palatal shelves and increased apoptosis within the epithelium. It was reported that Fgf10 and FgfR2b mutations affected the initial development of palatal shelves, and the mouse pups had complete CP. ^[45] By signalling via its receptor, FgfR2b, in the palatal shelf epithelium, the mesenchymal derived Fgf10 supports epithelial proliferation and survival and also induces the expression of Shh within the epithelium. Shh, in turn, signals to the mesenchyme and stimulates cell proliferation.

In general, signalling activities are subject to tight spatio-temporal control, and, in many instances, too much or too little control is detrimental to the developing organ. This situation is well illustrated in anomalies caused by de-regulated hedgehog (hh) and Fgf signalling. ^[46],[47] While Fgf10/FgfR2b activity plays a crucial role during palatogenesis, it appears to be subject to the tight spatio-temporal regulation shown in mice lacking Shox2. Shox2 mutant mice develop a very rare type of CP that may also be found in humans ^[48] the soft palate is intact, whereas the hard palate has a cleft. Abnormal proliferation and apoptosis are theorized to be the cause of the cleft. Surprisingly, a number of protagonists implicated in palatogenesis, including Msx1, Bmp4, Pax9, Lhx8, Osr2, Tgfβ3 and Jag 2, were expressed normally. ^[48] In contrast, Fgf10 and Fgfr2b were expressed at ectopic sites within the mesenchyme of the Shox2 mutant mice. ^[49] These studies emphasize the importance of the precise timing and determination of sites of signalling activities necessary for normal development. Mutation of activin-bA causes a severe facial primordial development defect, which may be responsible for the retardation of palatal shelf development and complete cleft palate. In addition, other genes, including Msx1, Lhx8, Shox2 and Osr2, assume important roles in the palatal shelf growth. The targeted mutation of these genes in mice generates CP, indicating the intrinsic requirement of these factors during palatogenesis. ^[49]

Fusion of the palatal shelf with the tongue or mandible

Under normal conditions, palatal shelves do not fuse with other oral structures. However, in mice that do not express Fgf10, the palatal shelf epithelium fuses with the tongue and mandible. ^[45] The loss of function mutations of Fgf10 results in anterior palatal shelf fusion with the tongue, whereas the middle and posterior palatal shelf regions adhere to the mandible, thus preventing the elevation of the palatal shelf. ^[50] There is a severe reduction of the expression of Jagged 2 (Jag2), thereby encoding a ligand for the Notch family receptors and ectopic Tgfβ3 production in the nasal epithelia of these mice. The analysis of Jag2 mutant embryos indicates that Jag2-Notch signalling prevents inappropriate palatal shelf adhesion to other oral epithelia through the control of oral epithelial differentiation. Another gene has also been associated with inappropriate adhesions. Mutations in TBX22 have been reported in families with X-linked cleft palate and ankyloglossia. ^{[51],[52],[53]} Tbx22 is expressed in the developing palate and tongue in mice, suggesting an important role in regulating tongue and palate development.

Failure of palatal elevation

Palatal shelf elevation is a rapid movement triggered by both intrinsic forces within the palatal shelves proper and by influences from other craniofacial and oral structures, including the movement of the tongue, and growth of the cranium and mandible. ^[1],[54] The role of the extra-cellular matrix in palatal shelf elevation has been supported by some studies and is presently accepted as an important determinant of palatal shelf elevation. ^[55],[56] Those studies ^[1] suggested that a progressive differential accumulation of glycosaminoglycans, primarily hyaluran in the palatal shelves, plays a role in their elevation. ^[55],[56] Hyaluronan is a highly charged glycosaminoglycan that retains high amounts of water, forming hydrated gels leading to the expansion of the extracellular matrix. Other constituents of the palatal shelves including collagen fibers, vascularization, and the epithelial covering; the polarized alignment of the mesenchyme cells may also contribute to the intrinsic elevation force of the PS. Mutations of Pax9, Pitx1 or Osr2 can lead to failed palatal shelf elevation and cleft palate defect. ^{[57],[58],[59],[60]} The cellular defect is associated with the CNC-derived palatal mesenchyme, suggesting the important functions of these transcription factors in regulating the fate of the CNC cells during palatogenesis.

Early studies attributed a role to neuro-transmitters during palatal shelf elevation. ^[1] At present, it is widely accepted that neuro-transmitter γ-aminobutyric acid (GABA) regulates not only neuronal activities but also cell migration, survival, proliferation and differentiation of neuronal and non-neuronal cells. ^{[61],[62],[63]} Terratological studies in rodents showed that GABA or GABA agonists generate CP by inhibiting palatal shelf elevation, whereas GABA antagonists stimulate the process. ^[64] The implication of GABA in palate development was demonstrated by genetic studies of mice lacking the β3 subunit of the GABA receptor that developed CP without other craniofacial malformations. ^[65]

Failure of palatal shelves to meet after elevation

Fusion of the opposing palatal shelves is an important step taking place through a sequence of events that includes the removal of the flat peridermal cells, contact and adhesion of the opposing MEE, which creates the MES, and the degeneration of the MES. The mesenchymal confluence thus forms at the midline. ^{[22],[23],[34]} Failure of shelf fusion is the most common type of cleft palate defect documented in animal studies. Mutations in Msx1 and Lhx8 and conditional inactivation of Tgfbr2 in CNC cells or Shh in the epithelium all result in retarded palatal shelf development. ^[45]

In many transgenic animals, the palatal shelves fail to meet at the midline because of hindrance by the tongue. This is usually associated with cases when the lower jaw does not move forward and downward during development, keeping the tongue between the palatal shelves. These secondary defects were evident in the Hand2 mutant mice in which the enhancer driving the expression of the gene in the pharyngeal arches was inactivated by targeted mutagenesis. ^[66] In these mice, the mandible did not grow properly, blocking the descent of the tongue, thus hindering palate fusion. ^[66],[67]

Persistence of middle edge epithelium

Adhesion of the opposing MEE is an important event in both human and mouse embryos. ^{[21],[27],[34],[44],[68]} E-cadherin is expressed in the epithelia covering the fronto-nasal and medial nasal processes as well as during the different stages of palate development, including the epithelial islands, remnants of the MES. ^{[69],[70],[71]} Mutations of CDH1/E cadherin, which deletes the extracellular cadherin repeat domains required for cell-cell adhesion, have recently been associated with CL/P in families with hereditary diffuse cancer. ^[72] E-cadherins are known to form dimers, indicating that the mutant proteins may have trans-dominant negative effects over the normal proteins. ^[72]

Extensive efforts have been made to elucidate the role of Tgfβ3 during palatal fusion. ^{[73],[74],[75],[76]} Adhesion of the MEE upon palatal shelf contact is a necessary step for fusion. Tgfβ3 is expressed in the MEE before and during fusion, and mediates MEE adhesion of the opposing palatal shelves through filopodia. E-cadherin is required for fusion, whereas filopodia seem to be crucial for proper alignment and guidance of cell sheets that are fated to fuse, but not for fusion itself. ^[77] Tgfβ3 is implicated in controlling the re-modelling of the extracellular matrix through regulation of the expression of the matrix metaloproteinases (Mmps) Mmp13, Mmp2 and the tissue inhibitor of metaloproteinase-2 (Timp). ^[78] Tgfβ3 signalling functions in the MEE by mediating the epithelial-masenchymal interactions leading to tissue changes that regulate palatal fusion. For example, EMT of the MES has been proposed as the major mechanism underlying the disappearance of the MES to generate mesenchyme continuity, thus preventing palatal clefts. ^[34] The establishment of the concept of EMT as the prevailing mechanism of MES disappearance led to studies attributing roles to different molecules, including Tgfβ3, Lef1, Smad, RhoA, phosphatidylinositol 3-kinase (PI-3 kinase), Mmps Twist and Snail. ^{[22],[33],[79]} In Tgfβ3 or Egfr mutant mice, there is an alteration of the fate of MEE cells. ^[80],[81] In Tgfβ3 null mutant mice, MEE cells fail to undergo apoptosis and remain along the midline, preventing normal fusion.

Ossification of the Palate

Palatal fusion signals the start of the ossification process in the anterior two-thirds of the palate to form the hard palatal tissues. This process entails the successful fusion of the three embryonic structures - lateral edges of the primary palate with the two anterior edges of the secondary palate. This process requires the synchronization of shelf movements together with the growth and withdrawal of the tongue and growth of the mandible and head. ^[82] Any form of disruption during the formative stages results in a pathological cleft. The same is true when ossification occurs too early. Sox9 is a gene controlling cartilage development and blocking the expression of Runx2, a transcription factor essential for osteoblast differentiation and bone formation associated with cleidocranial dysplasia. In Sox9 mutant, Runx2 expression is not repressed and ossification begins prematurely. ^[83] Since the palatal shelves are prematurely ossified, they cannot grow toward the midline and fail to fuse.

A wide range of studies on cranio-facial skeletal maturation has shown that the fusion of the palatal shelves along their length to form the mid-palatal (MP) suture occurs during the ossification of the maxillae and palatine bones before the mandibular condyle develops. ^{[48],[84],[85]} Ossification is observed where mesenchymal cells condense, the surrounding tissue vascularizes and the cells differentiate into osteoblasts that will form bone by mineral deposition. In this process, several growth and differentiation factors such as Bmps, core binding proteins (Cbf), Fgfs, and hedgehog (hh) proteins that interact with various signalling pathways to regulate the patterning of the undifferentiated mesenchyme, are involved. The Bmp-6 and the transcription factor Gli1 are also expressed during intra-membranous bone formation. ^[86],[87] As in cranio-facial sutures, the MP and trans-palatal (TP) suture osteoblasts express Tgfβ1, 2 and 3, while the suture cells express primarily Tgfβ3. ^[88],[89]

It has been established that cranial sutures are the growth sites for the neuro-cranium and that the dura mater provides the signalling molecules to regulate suture patency. ^[90] The MP and TP sutures have different morphology, so they are not in contact with the dura mater. Opperman's group hypothesized that these facial sutures are growth centres ^[88],[89] and that the nasal capsular cartilage produces signalling molecules to regulate the fusion of MP and TP sutures [Figure - 3]. ^[89] They found that the nasal cartilage maintained the TP sutures as growth sites in experiments on rat palatal organ cultures (E20) with or without nasal cartilage. They theorized that the nasal cartilage may regulate mid-facial growth. ^[89] Animal models have been developed to understand the aetiology and pathogenesis of orofacial clefts and the mechanisms of normal palatal ossification. The application of cyclic forces is an effective mechanical stimulus for the regulation of osteogenesis and osteoclastogenesis in the sutural growth of neonatal rats. ^[91] The process of tissue response and regeneration in the palato-maxillary suture under tensile forces was examined histologically and with fluorescence. A cyst-like zone appeared in the conjuncture of the bony front and the sutural connective tissue at the early stage of sutural expansion with increased proliferating osteoblasts and fibroblasts. New bone was deposited along the nasal septum and the front of the cyst until the new bone front formed and the suture restored its original morphology. ^[92]

The approach of utilizing MP suture expansion in mice has provided new insights into mechanical stress modulation as an important factor for the skeletal remodelling of bones and cartilage. The expansive force across the MP suture promotes both bone resorption through the activation of osteoclasts and bone formation through the increased proliferation and differentiation of the periosteal cells. ^[93] Similarly, the use of orthodontic wire expansion in growing rats showed that secondary cartilage can undergo chondrogenic and osteogenic differentiation in the maxillary arch. Interestingly, these induced changes were attributed to the alteration of the differentiation pathway of progenitor cells from chondroblastic to osteoblastic, in which many sutures temporarily form secondary cartilage during early development. Histological observations at days 7, 10, and 14 indicated that intra-membranous bone formation, which is partially recognized as mature bone, ^[94] occurred at the boundary between the pre-cartilaginous and cartilaginous cell layers where the calcified matrix was positive for osteocalcin antibody. The cellular events taking place at the MP suture cartilage in rat models as a result of expansion force have been observed as endochondral bone formation at the boundary between the maxillary bone and cartilage, whereas intra-membranous osteogenesis has appeared at the internal side of the cartilaginous layer. ^[95] To stimulate new bone formation in defective tissues, rat organ cultures with distracted palatal sutures were treated with Bmp-7 and Nell-1 for 8 days in vitro. The presence of Nell-1 increased chondrocyte hypertrophy and endochondral bone formation while Bmp-7 enhanced both chondrocyte proliferation and differentiation in the distracted palates of four-week-old male rats. This study indicates that Nell-1 is involved in the rapid osteoblast differentiation in palate sutures. ^[96] In another study, the application of TGF-β1 during the early stages of rat MP expansion induced rapid bone formation at the suture site. ^[97]

Oral and Palatal Musculature and Related Deformities

Overt CL/P encompasses a broad spectrum of defects, ranging from so-called microform clefts to complete unilateral or bilateral clefts of the lip and palate. The orbicularis oris (OO) muscle consists of numerous differently oriented strata of muscular fibres that surround the orifice of the mouth. At approximately seven weeks post-conception (p.c.) in humans, the two maxillary prominences fuse with the medial nasal prominence; however, lip fusion is not complete until the epithelial seam disappears through EMT and/or apoptosis ^[82] [Figure - 2]A-C. By eight weeks p.c., a dense, continuous band of mesenchymal cells corresponding to the future OO muscle can be seen, with discernible OO muscle fibers present by 12 weeks. ^[98],[104] The complete OO muscle architecture forms by 16 weeks. Any delay in fusion may result in sub-epithelial OO defects, such as the altered migration of the mesenchymal cells. Sub-epithelial (non-visible) defects of the orbicularis oris muscle represent the mildest form of cleft lip, and such defects are part of the phenotypic spectrum of CL/P. This defect usually is visualized as a ridge of tissue resembling a scar on the upper lip along the philtrum. ^[98]

Histological studies have demonstrated that such defects extend to the muscle fibres of the superior OO muscle. A method using high-resolution ultrasonography (USG) was developed to visualize the OO muscle non-invasively. ^[99] Significant differences in the defects of the OO are found in the first-degree relatives of CL/P individuals and controls. The OO muscle defect detected by ultrasound is consistent with the histological examination of cadavers. ^[99] Interestingly, the Bmp4 knockout mouse model shows bilateral cleft lip at E14.5, although this condition occurs at a rate of 22% after birth, ^[100] suggesting the initial cleft lip is rescued or healed in utero, leaving only the subepithelial OO defect. Potential mutations in BMP4 were found in two individuals with OO defects and none in the controls. ^[101] The strong evidence that OO discontinuities are indeed part of the phenotypic spectrum of CL/P provides an important clue for the clinical recurrence risk estimation for families with members affected with CL/P.

The mildest form of CP is termed a "submucosal cleft palate," described as a bifid uvula, palatal muscle diastasis and a notch in the posterior surface of the hard palate. ^[102] Defects in the nasopharyngeal anatomy and/or physiology may lead to velopharyngeal incompetence (VPI). Although most VPI is caused by CP, the population prevalence of VPI due to other causes is estimated to be approximately 2.5%. ^[103] In such cases, VPI may be caused by submucosal muscular defects of the levator veli palatini or musculus uvulae. Most of the soft palate muscles are derived from myotome cells, which first invade pharyngeal arch 4 and then migrate to the palate, carrying their innervations from the vagus nerve. One muscle (tensor veli palatini) is derived from myotome cells that first invade arch 1 and are innervated by the trigeminal nerve. ^[104] In the mouse, the tensor veli palatine, levator veli palatini, medial pterygoid, and lateral pterygoid muscles are identified as myogenic fields as early as gestational day 15. The palatoglossus, palatopharyngeus, and musculus uvulae, however, are not clearly visible. ^[105] In principle, the presence of these anatomical features in unaffected individuals may signify an elevated risk for producing clefts in offspring. ^[106]

Sumo Modification of Signalling Pathways in Palatogenesis

The molecular understanding of NS CL/P is further complicated when one considers that large differences in penetrance often occur when the same mutations are placed on different mouse strains, indicating a potential role for both genetic and/or environmental modifiers in the pathogenesis of CL/P. Several lines of evidence point to the involvement of the small ubiquitin-like modifier (SUMO) posttranslational modification machinery. ^[107] A surprisingly specific role in oro-facial development has been revealed for protein modification by the SUMO, which might hint at a possible interaction with environmental factors. Small ubiquitin-related modifiers belong to the ubiquitin-related protein family, and SUMO proteins are ubiquitously expressed throughout the eukaryotic kingdom. ^[108] SUMO1 shows strong expression in the MEE of the secondary palate. ^[109] A translocation breakpoint interrupting SUMO1 was found in a patient with CLP. ^[109] The causative nature of the translocation defect has been confirmed in SUMO1-deficient mice having a distinct CP phenotype. ^[109] Furthermore, it was recently shown that mutations in TBX22 have a profound effect on its ability to be "sumoylated," which is at least partially responsible for its loss of function. ^[110] Other SUMO targets include Smad4, Msx1, p63, Pax9, Eya1 and FGF signalling. ^[107] It seems likely that some of these factors may manifest through the disturbance of the SUMO pathway. De-stabilizing the normal balance of expression and activity for genes such as TBX22, MSX1, SATB2, and P63 during early pregnancy is likely to provide a high-risk environment for the occurrence of CL/P. Elucidating the relationship among environmental factors, the SUMO pathway, and the networks of craniofacial genes influenced by this post-transcriptional modification may be crucial to our understanding of the idiopathic forms of oro-facial clefts.

A-P Gradient of Molecular Signalling in Palatal Development

Multiple genes are critical for the development of the anterior region of the palate. Msx1, Bmp4, Bmp2, Fgf10, and Shox2 have restricted expression patterns in the anterior region of the palate. ^[45] In addition to the differential gene expression patterns along the A-P axis of the developing palate, there is also mesenchymal heterogeneity between the medial and lateral regions of the palatal shelf. The odd, skipped related genes Osr1 and Osr2 are expressed in a medial-lateral gradient in the palatal shelf. The mutation of the Osr2 gene results in the compromised development of the medial aspect of the palatal shelf and retards palatal shelf elevation. ^[60],[111] The expression of Fgfr2 is focused on the medial aspect of the developing palatal shelf, suggesting a possible functional significance in regulating its development and elevation.

An important discovery has been the confirmation of genetic heterogeneity along the anterior-posterior and medial-lateral axes of the developing palate. ^[48] This heterogeneity may provide a differential regulatory mechanism for the fusion of the anterior vs. posterior region of the palate. MEE cells undergo apoptosis at different times during palatal fusion. It has been shown that the apoptosis of MEE cells is triggered by palatal shelf contact in the anterior region, whereas it is initiated before any contact between the opposing shelves in the posterior region. ^[38] This difference may be the result of dissimilar molecular signals in the palatal mesenchyme along the anteroposterior axis that instruct different fates to the palatal epithelium. ^[112] Recent studies have demonstrated that constant and reciprocal interactions between palatal epithelium and CNC-derived mesenchyme are responsible for setting up this genetic heterogeneity along the AP axis and are crucial for normal palatal development and fusion. ^{[25],[45],[113]} The specific gene expression patterns in the posterior region of the palatal mesenchyme are less understood. Fgfr2 is expressed in the epithelium, and the CNC-derived mesenchyme is found in the middle and posterior palate. FGF8 signalling selectively induces the expression of Pax9 in the posterior region of the palatal mesenchyme. The loss of Pax9 results in a palatal shelf development defect and a cleft palate ^[48],[58]

Conclusion

It is clear from this and other review of literature that CL/P is caused by many factors, including both genes and environment. Gene targeting technology and basic conventional techniques using animal models led to the identification of genes associated with known and unknown aetiologic factors. In some cases, the human gene deficiency was identified first and replicated in an animal model, but in other cases, animal models led the way to understand gene/environment interactions. It is also clear from this extensive list of possible contributing genes that the molecular and cellular interactions associated with CL/P are not all understood. Fortunately, some subclinical changes in facial features may lead to a greater understanding of the gene/environment interactions in cranio-facial development.^[186]

Acknowledgments

We thank Dr. Lynne Opperman for discussions about palate ossification and for permitting the use of a figure from one of her publications on palatal suture development. We thank Jeanne Santa Cruz for editorial assistance. We also thank the publishers for permission to reprint figures from previous papers. The gene tables are an ongoing project.

References

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