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CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE |
Epithelial-Mesenchymal Transformation during Craniofacial Development
P. Kang1 and
K.K.H. Svoboda2,*
1 Graduate Endodontics Department and
2 Biomedical Sciences Department, Texas A&M University System, Baylor College of Dentistry, 3302 Gaston Ave., Dallas, TX 75266, USA;
Correspondence: * corresponding author, ksvoboda{at}bcd.tamhsc.edu
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ABSTRACT
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Epithelial to mesenchymal phenotype transition is a common phenomenon during embryonic development, wound healing, and tumor metastasis. This transition involves cellular changes in cytoskeleton architecture and protein expression. Specifically, this highly regulated biological event plays several important roles during craniofacial development. This review focuses on the regulation of epithelial-mesenchymal transformation (EMT) during neural crest cell migration, and fusion of the secondary palate and the upper lip. Abbreviations used in this paper: BMP, bone morphogenic protein; CCFSE, 5 (and 6) carboxy 2,7' dichlorofluorescein diacetate succinimidyl ester; CNC, cranial neural crest; DiI, 1,1-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate; EMT, epithelial-mesenchymal transformation; FGF, fibroblast growth factor; ILK, integrin-linked kinase; LEF1, Lymphoid enhancer factor-1; MEE, medial edge epithelia; MFS, mean fusion score; MMP, matrix metalloproteinase; PDK, 3-phosphoinostide-dependent protein kinase; Pax, paired box-1 to -9; PI-3 kinase, phosphatidylinositol-3 kinase; Ptc, patched; PTEN, phosphatase and tensin homolog deleted on chromosome ten; Shh, Sonic hedgehog; Tbx, T-box family; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase.
Key Words: palate • epithelial-mesenchymal transformation (EMT) • TGFβ • PI-3 kinase • Akt • Smad
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INTRODUCTION
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Cell phenotype transformations, from epithelial to mesenchymal (epithelial-mesenchymal transformation, EMT), have been well-documented in embryonic development, wound healing, and tumor metastasis. Epithelia serve as the boundary between the external environment and the internal structures, while mesenchymal cells are found in the connective tissue compartment. The epithelial barrier function is partly supported by firm cell-cell junctions, such as tight junctions and desmosomes. In addition, epithelial cells normally have apical-basal polarity and attach to basal lamina by hemidesmosomes. In contrast, mesenchymal cells are more mobile and surrounded by extracellular matrix. They have anterior-posterior polarity, and they form only transient contacts with their neighboring cells. A phenotype transformation from epithelial to mesenchymal requires a regulated gene expression sequence.
EMT and the opposite, mesenchymal-epithelial transformation occur during normal developmental processes. One example is EMT during one of the earliest developmental events—gastrulation—that involves the invagination of epiblast-derived cells to form mesoderm (Sanders and Prasad, 1989). Also, during neurulation, as the cranial neural folds elevate, cranial neural crest (CNC) cells migrate away from an embryonic neuroepithelial layer by changing their cell-cell adhesion and shape. The cells then migrate to specific destinations after basal lamina degradation (Weston, 1982; Duband et al., 1995). In addition, several other developmental processes—such as sclerotome formation (Solursh et al., 1979), and cardiac cushion mesenchyme development (Runyan and Markwald, 1983; Potts and Runyan, 1989; Boyer et al., 1999b)—require EMT. In contrast, mesenchymal-epithelial transformations occur in the formation of somites, kidneys, and caudal or secondary neural tubes (Griffith et al., 1992).
The EMT phenotype change occurs through a regulated sequence of events that can be divided into stages. EMT can occur from simple, stratified, or fused epithelia. The cranial neural crest (CNC) transforms from a single layer of neuroepithelium (Fig. 1 ), whereas palate and lip fusion occurs when 2 epithelial sheets fuse and then transform through EMT (Fig. 1). Both tissues have a loss of cell-cell attachment, breakdown of basal lamina, and increased mobility. However, the palate is complicated by the process of 2 epithelial surfaces first bonding to each other (Fig. 1) after the surface cells have sloughed and before proceeding through EMT. During the mid-1990s, several reviews were published on EMT in development and pathogenesis (Duband et al., 1995; Hay, 1995; Hay and Zuk, 1995; Shuler, 1995; Guarino et al., 1999; Boyer et al., 2000).
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Figure 1. Comparison of cranial neural crest and palate EMT. Cranial neural crest (CNC) is derived from a single layer of epithelial cells located at the transition zone between neuroepithelium and surface ectoderm (Weston et al., 2004). The cells have increased Slug, and decreased Sox2 expression. The cells lose cell-cell adhesion molecules [(N-cam or E-Cadherin (E-Cad)] and increase actin (RhoB) and extracellular matrix remodeling (Mmp2) proteins and growth factor receptors (PdgfR ). After the crest cells have moved away from the neuroepithelium, they increase expression of the HNK-1 epitope (Del Barrio and Nieto, 2004). Palate EMT requires several more steps than CNC, since the periderm cells are sloughed through apoptosis, two epithelial sheets fuse at the apical membranes, and some cells may move to the oral or nasal epithelium. The cells increase Snail, Tgfβ3 signal transduction through Smad2, PI-3 kinase, and MMP, while decreasing E-cadherin. In both tissues, cell adhesion proteins are repressed, and matrix degradation and mesenchymal or tissue-specific proteins are increased. A confocal image of a single optical plane through palatal MEE cells that were labeled with CCFSE and then cultured for 72 hrs demonstrates the extensive remodeling at the seam as cells undergo EMT.
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Describing CNC and palatal EMT will be the focus of this review. In addition, the current information on gene expression changes and signal transduction pathways involved in these events will be discussed. First, we will introduce the various experimental models that have been used to study EMT in craniofacial and other tissues. This will be followed by a review of the literature on CNC, and palate- and lip-fusion EMT. The signaling molecules and signal transduction pathways will be discussed, focusing mainly on the Tgfβ family of proteins. Finally, the clinical applications and implications of future research will be explored.
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EXPERIMENTAL MODELS
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Several organ culture models and three-dimensional culture methods have been used for the study of EMT, including cardiac cushion, palate, and lip organ culture models. The gelling collagen tissue culture model has also been a useful tool for studying the gain and loss of function experiments with transient gene expression in vitro. Finally, the contributions of transgenic animals to our understanding of EMT mechanisms and human disorders will be discussed.
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ORGAN CULTURE MODELS
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The embryonic cardiac cushion has been one of the models used for the study of EMT mechanisms. In embryonic hearts, endothelial cells transform and migrate into mesenchyme to form the future valves and septa. When this tissue is placed in a three-dimensional culture system, the endocardial cells migrate into the collagen gel (Runyan and Markwald, 1983). Antibodies or/and antisense oligonucleotides to transforming growth factor β2 (Tgfβ2), β3 (Tgfβ3), Tgfβ receptor types II (TβrII) and III (TβrIII), differentially inhibited EMT during avian or mouse embryonic cardiac cushion formation (Runyan et al., 1992; Boyer et al., 1999a; Boyer and Runyan, 2001; Camenisch et al., 2002). Antibodies specific for Tgfβ2 blocked cell-cell separation, while antibodies to Tgfβ3 blocked mesenchymal invasion but not separation. These different experimental outcomes support the hypothesis that EMT is a multistep process. More recently, this model was used to show that Mox-1 played a necessary role to sustain EMT (personal communication, R. Runyan). Numerous growth factors and signaling pathways have been reported to control proliferation and EMT, and many parallel the pathways reported for the CNC and palate.
Several craniofacial research groups studied the regulation of EMT using organ cultures of embryonic rodent or chicken palates and lips. In most of the experiments, the palates were harvested at palate elevation stages [mice, E13-14 (Figs. 2A, 2B); rats, E16; and chickens, E8] (Shuler and Schwartz, 1986; Griffith and Hay, 1992; Shuler et al., 1992; Sun et al., 1998b, 2000; Kang and Svoboda, 2002, 2003). The palatal shelves were removed from the maxilla and placed on a substrate, either in pairs or as single palatal shelves, and then maintained in static organ culture at the air-medium interface in media for 2–72 hrs (Fig. 2C) (Kang and Svoboda, 2002, 2003). In vitro, palates fused and EMT progressed, with complete mesenchymal confluence by 72 hrs. Chicken palates required additional Tgfβ3 (Sun et al., 1998b), whereas rodent palates and chicken lips (Sun et al., 2000) did not need additional growth factors. The transformation and migration of epithelia were investigated by a variety of techniques. For example, tissues were incubated with lipophilic markers such as 5 (and 6) carboxy 2,7' dichlorofluorescein diacetate succinimidyl ester (CCFSE; Figs. 1 , 3) (Griffith and Hay, 1992; Kang and Svoboda, 2002, 2003), or with 1,1-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) (Shuler et al., 1992) to label the epithelia prior to culture. Labeled cells were then visualized by fluorescent or TEM microscopy after different culture periods. The design of this type of experiment was not complicated; however, major disadvantages included lack of label incorporation into the basal medial edge epithelium (MEE) cells and loss of labeling through time. Another type of cell lineage study used epithelia infected with virus in vitro or in vivo, and the labeling was then detected after static organ culture (Martinez-Alvarez et al., 2000a). Unfortunately, the palatal cells have been difficult to infect with viruses consistently.
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Figure 2. Experimental models for the study of EMT. (A) Embryonic mouse palatal shelves were dissected from the 12.5-gestational-day mouse embryos. (B) The palatal shelves are in the horizontal position, but are not touching or fused at this stage. (C) The dissected palatal shelves were placed in organ culture on a filter at the air-media interface for 20 to 72 hrs in the static organ culture model (Kang and Svoboda, 2002). (D) The palatal region can also be cultured in suspension cultures. The palatal region was dissected by a horizontal incision made through the oral opening (A - solid line), and the upper part of the head was resected by a second incision made parallel to the first at the level of the eyes (dashed lines in A and dashed line in B). The tongue and brain tissue were removed from the explants. The palatal explants were placed in 50-mL penicillin bottles (n = 3 to 6 explants/bottle) on a roller bottle culture system (Shiota et al., 1990; Chou et al., 2004). In this system, the palatal shelves fuse in 24 hrs.
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Using a different approach that cultured mouse maxilla with some associated tissues in suspension (Figs. 2A, 2D), Shiota’s group established that the anterior, middle, and posterior palates may close by different mechanisms (Chou et al., 2004). This group used carbon particles to mark palatal locations prior to cultivation for up to 48 hrs. They found that the midpalate closes by rotation and medial elongation, whereas the anterior and posterior regions appeared to have more remodeling (Chou et al., 2004).
In new experiments comparing the suspension culture (Fig. 2D) with the traditional static cultures (Fig. 2C) described above, Takigawa and Shiota found that MEE cells can disappear throughout the medial edge, even without contact and adhesion to the opposing MEE in suspension culture (Takigawa and Shiota, 2004). Apoptosis, EMT, and epithelial cell migration all occurred at various stages of MEE cell disappearance, including the transient formation and disappearance of epithelial triangles and islets in the suspension cultures. In contrast, MEE cells showed poor differentiation in static culture in a CO2 incubator. Furthermore, mouse and human amniotic fluids were found to prevent MEE cell differentiation in the cultured single palatal shelf, although paired palatal shelves fused successfully even in the presence of amniotic fluid. These experiments demonstrate that the palatal shelves behave differently when separated from the maxilla, and that maintaining them in their three-dimensional space may be critical to our understanding of the mechanisms of palatogenesis. Clearly, more experiments examining individual cell fates need to be conducted before we can fully understand palatal fusion.
More recently, a transgenic mouse has been developed that expresses β-galactosidase (β-gal) in the CNC cells (Chai et al., 2000) (see complete description in the ‘transgenic mouse’ section). Palates from these embryos were incubated in DiI and then cultured according to the static organ culture method. In the resulting tissues, all CNC were labeled blue, and surface epithelium was labeled red (DiI). Non-labeled cells were neither CNC nor epithelial in origin. These mice will be very valuable in future studies using other markers, and as controls for selective gene knockouts or other alterations (Ito et al., 2003).
Several studies using in vitro models have provided information on the necessary molecules that facilitate EMT. One in vitro model used the transformation of adult and embryonic epithelial cells (notochord, lens, cornea, limb ectoderm, and thyroid) into fibroblast or mesenchymal cells, when they were cultured in three-dimensional (3D) gelling type I collagen (Greenburg and Hay, 1982, 1986, 1988). The lens epithelial cells changed phenotype by decreasing cell-cell adhesion, increasing cell-matrix adhesion, and switching from epithelial to fibroblast intermediate filaments. The lens epithelial apical cell surface extended filopodia into the surrounding collagenous matrix. These cells began producing type I collagen and stopped producing crystallins, type IV collagen, and laminin. The apical surface started expressing β1 integrin, and the mesenchymal cells up-regulated 5β1 integrin and started producing fibronectin (Zuk and Hay, 1994). It was found that blocking the cell-matrix interaction with antibodies to β1 integrin partially inhibited transformation of lens epithelial cells to fibroblasts in collagen gels (Zuk and Hay, 1994). In contrast, thyroid follicle cells extended filopodia from the basal cell surface and decreased thyroglobulin and cytokeratin (cell-type-specific proteins) in 3D type I collagen gel cultures. The transformed cells also increased expression of vimentin, an intermediate filament family found in fibroblastic cell types (Greenburg and Hay, 1988), demonstrating that the cells lost epithelial-specific proteins. The ECM composition also played an important role, since culturing the thyroid follicle cells in a 3D gelling basal lamina extract (Matrigel) prevented the EMT phenotype change (Greenburg and Hay, 1988).
During EMT, epithelial cells lose polarity and differentiated cell-cell contacts, to become mobile mesenchymal cells. In contrast, when cells transform from mesenchyme to epithelial phenotype, they need to increase differentiated cell-cell contacts. The loss of cell-cell junctions was observed in normal MDCK cells incubated in neutralizing antibodies directed toward E-cadherin (Behrens et al., 1989). The gene for E-cadherin has been shown to cause fibroblastic cell lines to become epithelioid in culture. To determine if the E-cadherin gene could cause a definitive embryonic mesenchyme to transdifferentiate into an epithelial phenotype, investigators co-transfected primary corneal fibroblasts by impact-loading with plasmids containing E-cadherin (Vanderburg and Hay, 1996). The fibroblasts expressing E-cadherin aggregated, localized E-cadherin to lateral surfaces, and formed stratified epithelia that developed zonulae occludentes and adherents. These epithelial cells expressed connexin 43, cytokeratin, and desmoplakin, and developed desmosomes. However, the cells continued to contain vimentin intermediate filaments, and the basement membranes did not develop, even though the cells synthesized laminin and type IV collagen. These types of cell culture experiments established that cell phenotype (epithelial vs. mesenchymal) was determined by both the extracellular environment and the gene expression program of the cells. Furthermore, the expression of cell-cell junction proteins such as E-cadherin was instrumental in determining the cell type (Hay, 1995; Hay and Zuk, 1995).
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TRANSGENIC ANIMALS
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Palatal clefting has been a popular model for the study of EMT during craniofacial development. Twenty or more genes have been related to facial clefting defects (Slavkin, 1995; Thyagarajan et al., 2003). These include genes for growth factors, extracellular matrix, signaling molecules, and homeobox genes. A recent review has a detailed list of the genes responsible for craniofacial defects, including palatal clefts (Thyagarajan et al., 2003). Two recent studies have identified genes associated with cleft palate in humans: The transcription factor, interferon regulatory factor 6 (IRF6), is responsible for the autosomal-dominant disorder, Van der Woude syndrome (VWS) (Murray and Schutte, 2004), and nonsense mutations and deletions in the fibroblast growth factor receptor 1 (FGFR1) gene have been identified with Kallmann syndrome (Dode et al., 2003).
Several transgenic mice have been developed that have been instructive for the understanding of the genetic component of palatal clefts. Several signaling molecules are necessary for initiating palate growth, including Fgf10, expressed in the mesenchyme, and its receptor FgfR2b, in the epithelium. The activation of FgfR2b mediates the expression of sonic hedgehog (Shh) in the epithelium (Rice et al., 2004). Msx1 was identified as having a critical role in mediating epithelial-mesenchymal interactions during craniofacial bone and tooth development. The animals that do not express this protein (Msx1–/–) have extensive craniofacial defects, including cleft secondary palate and deficiency of alveolar mandible and maxilla, with failure of tooth development and abnormalities of the nasal, frontal, and parietal bones, and malleus in the middle ear (Satokata and Maas, 1994).
In Tgfβ3–/– mice, the secondary palate cleft was their only craniofacial defect. Interestingly, in organ culture, the palatal shelves from these mutated mice were able to fuse in the presence of exogenous Tgfβ3 (Taya et al., 1999). The authors suggested that the one role of Tgfβ3 was to regulate the formation of epithelial filopodia prior to fusion. In a separate study, the medial edge epithelial surfaces of embryonic days 12, 13, and 14 mouse palatal shelves from homozygous null (Tgfβ3–/–), heterozygous (Tgfβ3+/–), or homozygous normal (Tgfβ3+/+) mice were observed with Environmental Scanning Electron Microscopy (ESEM) (Martinez-Alvarez et al., 2000a). These investigators found that, in mice that were heterozygous or homozygous-normal, the MEE had surface bulging. However, the surface bulges were not seen in the Tgfβ3–/– embryos (Martinez-Alvarez et al., 2000a). In addition, the MEE was thinner and had fewer apoptotic cells (Martinez-Alvarez et al., 2000a).
The Shuler laboratory examined the phenotype of highly back-crossed (12 generations) Tgfβ3–/– animals compared with Tgfβ3–/+ and wild-type mice and found that 100% of Tgfβ3–/– newborns had cleft secondary palate, 91.4% complete cleft, and 8.6% partial cleft. In the partial-cleft-palate newborn mice, fusion occurred only between the 2nd and 5th rugae. No epithelium remained in the midline fusion region. In addition, some of the heterozygous animals (8.8% of Tgfβ3+/–) and homozygous normal (2.5% of TGF-β3+/+) newborns had a failure of fusion between the primary and secondary palates ( 2 test, 0.1 > p > 0.05). Failure of fusion of the primary palate with the secondary palate was also identified in Tgf-β3+/– adult mice. Third, complete cleft palate was not detected in either Tgfβ3+/– or Tgfβ3+/+ newborns (Cui et al., 2004).
Investigators used another transgenic mouse in a two-component genetic system (Cre-Flox or Cre-Lox) for marking the progeny of the CNC during tooth and mandible development, using the Wnt1 promoter. Wnt1 transgene expression was limited to the migrating neural crest cells that were derived from the developing dorsal central nervous system. The second mouse line carried a deletion of the Tgfbr2 receptor, so that progeny had decreased Tgfβ signaling in only the CNC. The resulting fetuses had a complete cleft of the secondary palate, calvaria agenesis, and other skull defects, with complete phenotype penetrance (Ito et al., 2003). Disruption of the Tgfβ signaling pathway did not negatively affect CNC migration. It was determined that the cleft palate in these mutant mice resulted from decreased cell proliferation due to decreased cyclin D1 expression within the CNC-derived palatal mesenchyme. The MEE of the mutant palatal shelf remained functionally competent for EMT when the palatal shelves were placed in contact in vitro (Ito et al., 2003).
This particular mouse is very valuable, since it has added a marker (β-gal) to the CNC with a conditional reporter allele (Ito et al., 2003). Other transgenic mice have been engineered with either this reporter or green fluorescent protein (GFP) derivatives for cell lineage and ablation studies. The advantage of the Cre/LoxP system was that the genes could be selectively over- or underexpressed in a single tissue type, rather than the whole animal (Chai et al., 2000).
Several laboratories collaborated on a study using 3 mutant animals in a cross-breeding strategy to explore the role of Fgf10–/–, FGF receptor (Fgfr2B–/–), and Sonic hedgehog (Shh) mutant mice, all of which exhibited cleft palate, to show that Shh is a downstream target of Fgf10/Fgfr2b signaling (Rice et al., 2004). They demonstrated that mesenchymally expressed Fgf10 activated its receptor, Fgfr2b, in the epithelium. Furthermore, they determined that the receptor, Fgfr2b, mediates the epithelial expression of Shh that signals back to the mesenchyme to target molecules including bone morphogenetic protein (Bmp2) and patched (Ptc). They confirmed this by demonstrating that cell proliferation was decreased in the palatal epithelium and mesenchyme of Fgfr2b–/– mice. These results reveal a new role for Fgf signaling in mammalian palate development and demonstrated a coordinated epithelial-mesenchymal interaction essential during the initial stages of palate development (Rice et al., 2004).
During the 2004 General Session of the International Association for Dental Research, several laboratories reported their latest research on either transgenic mice (Tgfβ3–/–, Odd-skipped transcription factor 1,2 Osr2), naturally occurring mutants (Dancer-Tbx10), or down-regulation of a specific mRNA with antisense oligonucleotides (snail) (Bush et al., 2004; Cui et al., 2004). The Osr2–/– mice exhibit failure of midface development, including a missing cranial base, open eyelids, and palatal shelves that do not grow vertically or elevate as well as those of wild-type control animals (Lan et al., 2004). An investigation into the proliferation level of the mesenchymal cells in the palatal shelves demonstrated that the Osr2–/– mice had 25% fewer dividing cells. The investigators also reported that Pax9 was down-regulated in the Osr2–/– palatal shelves, while Tgfβ3, Msx1, Bmp4, and Tbx2 were unchanged compared with those in wild-type control mice (Lan et al., 2004).
The Dancer (Dc) mouse is a spontaneous mutation that exhibits cleft lip and palate, since the palatal shelves do not elevate, while heterozygote animals are predisposed to teratogen-induced clefting. By the use of PCR, RT-PCR, in situ hybridization, and Southern hybridization, the gene responsible for this mutation was identified as the Tbx10 gene. It was demonstrated that the Dc mutation was caused by the insertional translocation of a chromosome 10 fragment into the Tbx10 first intron. This insertion resulted in the ectopic expression of a mutant-specific Tbx10 transcript. Transgenic recapitulation of the Dc phenotype further confirmed that it is caused by Tbx10 mis-expression (Bush et al., 2004). In summary, several transgenic mice have been developed that will be useful tools for improving our understanding of important palate and EMT genes. However, each animal must be evaluated to determine if the cleft phenotype is a primary effect of the transgenic manipulation, or a secondary effect caused by other physical factors.
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CRANIAL NEURAL CREST EMT
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Before the closure of the neural folds in the mammalian head, the induced neural crest cells break away from an embryonic epithelial layer of the dorsal neural tube by changing their shape and properties, from those of typical neuroepithelial cells to those of mesenchymal cells (Fig. 1). In a recent meeting celebrating the contributions of James A. Weston to the understanding of the neural crest, Dr. Weston proposed that the CNC were derived from an early population of non-neuronal ectodermal cells (Erickson, 2004; Weston et al., 2004). The controversy appears to be centered on confusion over what portion of the neural fold gives rise to the neural crest. Weston’s group provides evidence that the cells at the edge of the neural fold express E-cadherin and PdgfR; therefore, the ectomesenchyme may be from the non-neuronal epithelium in the neural fold (Weston et al., 2004). This theory is certain to engage many investigators in the next few years. This review will concentrate on the transition from epithelial cell types, since both neuroepithelia and surface ectoderm must complete EMT. (A recent special issue of Developmental Dynamics was dedicated to this topic, "Special focus on the neural crest and the contributions of James A. Weston".)
A major difference between cells of the CNC and those of the trunk is that the CNC cells are patterned with level-specific instructions in the head, whereas those of the trunk do not appear to be pre-programmed. In the cranial region, the CNC migrate in streams throughout the cranial mesenchyme, with a level-specific instruction, to reach their final destinations. Extensive transplantation experiments demonstrated that the maintenance of this segmental characteristic is very important in the patterning of head development (Noden, 1983; Couly et al., 1998; Ferguson et al., 2000). Recently, in vitro studies have suggested that all neural crest cells have the potential to form skeletal elements (McGonnell and Graham, 2002); however, the CNC may play a key role in organizing the innervation of the hindbrain by the cranial sensory ganglia (Graham et al., 2004).
Many research groups have been interested in investigating neural crest cell differentiation. The CNC cells are multipotent stem-like cells, which respond to temporospatially expressed signals and become ‘committed’. Candidate regulators include growth factors—members of the Tgfβ family (Delannet and Duband, 1992), Fgfs (Kinoshita et al., 1993; Baird, 1994), Pdgf (Morrison-Graham et al., 1992), and Wnt (homologous to Wingless in Drosophilae) gene products (Nusse and Varmus, 1992; Augustine et al., 1993). The role of the Tgfβ family in CNC developmental processes has recently been reviewed (Chai et al., 2003). The involvement of several signal transduction molecules and transcription factors has also been reported (Duband et al., 1995).
The paired maxillary processes and mandibular prominences of the first branchial arch give rise mainly to the structures of the upper and lower jaws. The neural crest component of the maxilla derives from the forebrain and midbrain, while that of the mandible arises from the midbrain and hindbrain (rhombomeres 1 and 2). These CNC cells contribute mainly to the following structures in the first branchial arch: palate and maxilla, dermis and fat of skin, dental papilla, Schwann cells of peripheral nerves, melanocytes, and some connective tissue. A recent study demonstrated that the conditional ablation of the TβrII gene in the cranial neural crest lineage resulted in clefting of the secondary palate and calvarial defects. The pathogenesis of cleft palate in these mice appears to be related to impairment of cell proliferation (Ito et al., 2003).
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PALATAL FUSION
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As the precursor of the secondary palate, the lateral palatine processes appear during the 6th week in human embryos. Initially, the 2 palatal shelves grow downward, lateral to the tongue. At this point, the tongue is narrow and tall, almost completely filling the oral-nasal cavity, and reaches the nasal septum. During the 7th week, the 2 palatal shelves dramatically change their positions and elevate to a horizontal position above the dorsum of the tongue (Greene and Pratt, 1976; Shaw et al., 1991; Johnston and Bronsky, 1995; Long, 1998; Vieira and Orioli, 2001; Murray and Schutte, 2004).
Palatal shelves are composed of predominantly CNC-derived mesenchymal and ectomesenchymal cells covered by craniopharyngeal ectoderm. Initially, 2 epithelial layers cover the surfaces of the developing palatal shelves as a primary boundary (periderm and basal cells). The basal epithelia on the edges of the 2 opposing shelves are defined as medial edge epithelia (MEE) (Figs. 1, 2). During palatal morphogenesis, the MEE of the approximating palatal shelves fuse at the apical borders of the basal cells to form a midline epithelial seam. The epithelial cell seam rapidly disappears and establishes mesenchyme continuity across the intact horizontal palate. At almost the same time as the midline epithelial cells disappear, the epithelia on the nasal aspect of the palate (NE) differentiate into pseudostratified ciliated columnar cells, while those on the oral aspect (OE) become stratified squamous cells. Another structure involved in the formation of the palate is the nasal septum. It reaches the level of the palatal shelves when the latter fuse to form the definitive secondary palate (Ferguson, 1978, 1988; Fitchett and Hay, 1989; Griffith and Hay, 1992; Takigawa and Shiota, 2004).
Several theories have been proposed to explain how these 2 epithelium-covered palatal shelves fuse into one continuous structure. Some consistent features have been observed in various morphological studies. The sequence of events appears to be: loss of the superficial periderm cells, adhesion of the basal cells, basement membrane breakdown, and disappearance of the epithelial cells. One theory proposed that all of the epithelial cells died (Farbman, 1968; Hayward, 1969; Smiley, 1970; Hudson and Shapiro, 1973; Shah and Chaudhry, 1974; Pratt and Martin, 1975; Shah et al., 1991), another proposed that the epithelial cells migrated to the oral or nasal surface (Carette and Ferguson, 1992), and, more recently, it was proposed that the epithelial cells transformed into mesenchymal cells (Fitchett and Hay, 1989; Shuler et al., 1991, 1992) and continued to populate the palate, but as a different cell type.
The cell death theory has been resurrected in 2 publications (Martinez-Alvarez et al., 2000b; Cuervo and Covarrubias, 2004). Apoptotic cells (TUNEL-positive) were observed in the MEE cell population prior to and during seam formation from albino Swiss mice killed during various stages of palatogenesis in vivo. In addition, the authors identified macrophages, in the MEE seam, apparently phagocytosing the dying cell population. However, these same images showed that over 80% of the MEE cells were healthy and not TUNEL-positive. In a more recent study, mouse palatal explants were cultured in suspension (Fig. 2D) in the presence of apoptosis enzyme inhibitors, and the palates fused normally (Takahara et al., 2004). The authors suggested that the static culture system (Fig. 2C) used by other groups may contribute to increased cell death. In other experiments, using the static culture system, the MEE were infected with a retrovirus expressing LacZ. Although not all of the MEE cells were infected, the virus experiments clearly showed that some MEE cells died, while other cells transformed into mesenchyme. Some of the retrovirally labeled MEE cells migrated into mesenchyme and not only were found some distance from the midline, but also were TUNEL-negative (Martinez-Alvarez et al., 2000b).
Using the lipophilic cellular markers DiI or CCFSE, several groups have successfully traced the fate of MEE (Griffith and Hay, 1992; Shuler et al., 1992). A more recent cell lineage study that labeled the CNC with β-gal (Wnt1-Cre/R26R mouse) and epithelium with DiI definitely demonstrated that the MEE were maintained as mesenchymal cells (Ito et al., 2003).
The transformation of MEE occurs through many biological events, which have been distinguished into the following sequence: First, the superficial layer of periderm cells sloughed off through an apoptosis mechanism (Martinez-Alvarez et al., 2000b). After the periderm cells died, the underlying basal cells produced desmosomes and adhered, forming a midline seam (Fig. 1) (Ferguson, 1988; Fitchett and Hay, 1989). The midline epithelial seam then lost cell-cell junctions and became mesenchymal. Included in these changes was the loss of the epithelium-specific adhesion molecules E-cadherin and syndecan-1 that were localized to the basolateral surfaces of the MEE at embryonic day 14 in the mouse. Twelve hours later, when a midline seam had formed, syndecan-1 and E-cadherin were still present on the basal and lateral epithelial surfaces, and remained after the seam broke up into epithelial islands. Expression of both molecules was lost simultaneously and abruptly when EMT occurred (Sun et al., 1998a). The cells also switched expression of intermediate filaments from keratin to vimentin (Fitchett and Hay, 1989; Shuler et al., 1991). In addition, the cells began producing enzymes responsible for matrix degradation, including matrix metalloproteinases (Mmp) and tissue inhibitors of metalloproteinases (Timps). These proteins have been identified by gelatin zymography and reverse zymography of MEE. Specifically, Mmp-2 showed a significant elevation during palatal fusion (Morris-Wiman et al., 1999; Mansell et al., 2000). The increase in these degradative enzymes led to the basement membrane breakdown, allowing the transformed cells to become confluent with the palatal shelf mesenchyme (Griffith and Hay, 1992).
As palate morphogenesis proceeded, the MEE cells decreased mitosis (Cui et al., 2003), changed shape as they lost cell-cell junctions, and produced numerous filopodia, extending from the basal surface, that extended between basement membrane breaks (Boyer et al., 2000). The cell movement was driven by re-arrangement of the cytoskeleton and formation of new cell-substratum contacts (Boyer et al., 2000). In a cell lineage study in which the epithelial cells were labeled with CCFSE, the investigators observed CCFSE bodies in the mesenchymal cells, indicating that these cells were basal epithelial in origin (Fig. 1) (Griffith and Hay, 1992).
Several mechanisms have been developed to scale or grade the amount of EMT in palatal specimens, including counting the number of MEE cells (Nawshad and Hay, 2003; Cuervo and Covarrubias, 2004), or developing a scale system (Kang and Svoboda, 2002; Takahara et al., 2004). Our scale system was based upon the characteristics of palatal fusion divided into 5 stages (1–5) according to histo-morphological observations, including confocal analysis of CCFSE, hematoxylin and eosin (H&E)-stained paraffin sections, and laminin immunohistochemical staining (Fig. 3, Table 1) (Kang and Svoboda, 2002). Single optical confocal images obtained from the middle of the tissues were analyzed for fusion. The location, continuity, and intensity of CCFSE-labeled MEE were compared anterior-posteriorly in the horizontal plane (Figs. 3A–3E). Non-fused or partially fused samples had more intense, continuous, or even 2 epithelial layers of CCFSE-labeled cells (Figs. 3A–3C), while the fused palates had very discrete labeling in the fusion zone (Figs. 3D, 3E), indicating that EMT had occurred in these palates. Palatal samples were also processed as individual cross-sections for H&E staining, and MEE were analyzed in the coronal plane (Figs. 3F–3J). The amount of persistent MEE and degree of mesenchymal confluence were compared between treatment groups. To establish that the basement membrane had completed degradation, the investigators analyzed cross-sections by immunohistochemical localization of laminin, a marker for basal lamina (Figs. 3K–3O). The amount and continuity of laminin were greater in non- or partially fused samples.
Each cultured palate was scored from 1–5, and the mean fusion score (MFS) was determined for the palates cultured for 72 hrs. The investigators calculated the MFS by multiplying the palate stage (1–5) times the number of embryos for each stage, and dividing the sum by the total number of embryos in that group. Groups with higher fusion scores had MFS closer to 5. Based on this scale, it was determined that different signal transduction inhibitors could have an impact on palatal fusion (Fig. 3P). We have also used the scoring method to determine that nicotine decreased palatal fusion in the mouse palate in vitro (Kang and Svoboda, 2003).
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LIP FUSION
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Chicken upper lip forms by growth and fusion of the bi-maxillary processes of the first branchial arch. Unlike the chicken secondary palate, the upper lip naturally fuses in the midline. An in vivo study with CCFSE and TEM demonstrated that the chicken upper lip fused by EMT mechanisms (Sun et al., 2000). It was shown that, as in the palate, the periderm of the two-layered embryonic epithelium sloughed off before fusion of the basal cells. The authors labeled the cells undergoing programmed cell death by staining the fragmented DNA (TUNEL), followed by ultrastructural analysis, and confirmed that the periderm cells died. In addition, they determined that the basal cells (lip MEE) transformed into mesenchyme morphologically similar to that of the palate (Sun et al., 2000). Interestingly, the chicken lip does not express Tgfβ 1–3 and, unlike the mouse or chicken palate, did not require Tgfβ for EMT and complete fusion in vitro. The authors suggested that factors other than Tgfβ, such as Shh, could be responsible for EMT and fusion of the upper lip. Shh was observed in the lip epithelium at the time of fusion (Helms et al., 1997). The addition of agents inhibiting Shh, including retinoic acid and Shh antibodies, blocked the normal fusion of maxillary and nasofrontal processes and resulted in bilateral cleft lip in chickens (Helms et al., 1997; Hu and Helms, 1999), indicating that different genes may regulate EMT in specific craniofacial structures.
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REGULATION OF EMT
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The regulation of EMT is critical during dynamic developmental processes and post-natal homeostasis. In 1989, Hay postulated that the master gene(s) are turned on in epithelia by changes in the environment to initiate EMT (Master Gene theory). These changes in the environment include growth factors, cell adhesion molecules, extracellular matrix, the surface receptors and downstream signal transduction events, and transcription factors (Table 2). Evidence is mounting, in both developmental systems and tumorigenesis studies, that Twist (Kang and Massagué, 2004; Yang et al., 2004), Snail (Cano et al., 2000; Ip and Gridley, 2002; Ohkubo and Ozawa, 2004), and Slug (Bastidas et al., 2004; Del Barrio and Nieto, 2004) may be the transcription factors that control EMT through regulating cell adhesion proteins, although the surface growth factor receptors and subsequent signal transduction pathways may be upstream of the transcription factors (Kang and Massagué, 2004).
The Tgfβ super-family includes many small proteins that are multifunctional (controlling growth, migration, and differentiation) during both embryonic development and post-natal tissue homeostasis (Massagué, 1998; Whitman, 1998; Massagué et al., 2000). The cellular responses to Tgfβ during craniofacial development and CNC regulation have been extensively studied and recently reviewed (Chai et al., 2003).
During mammalian palate development, Tgfβ isoforms 1, 2, 3, TβrII, and TβrIII were detected in the MEE by in situ hybridization (Fitzpatrick et al., 1990; Pelton et al., 1990a,b) or immunohistochemistry (Cui et al., 1998; Cui and Shuler, 2000). Of particular interest was the highly localized expression of Tgfβ3 RNA and, to a lesser extent, that of Tgfβ1 and Tgfβ2 in the MEE and the nasal septum epithelium, which were destined to undergo EMT (Pelton et al., 1990b). Antisense oligodeoxynucleotides or neutralizing antibodies to Tgfβ3, but not Tgfβ1 or Tgfβ2, resulted in the failure of palatal fusion in vitro (Brunet et al., 1995). In addition, Tgfβ3 transgenic and knockout mice have cleft palate as their only craniofacial birth defect (Kaartinen et al., 1995; Proetzel et al., 1995). When palatal shelves from Tgfβ3 knockout mice were cultured in static organ cultures (Fig. 2C), the midline epithelia failed to go through EMT (Kaartinen et al., 1997; Taya et al., 1999). In addition, although the chicken has a naturally open palate, the cultured chicken palatal shelves fused when Tgfβ3 was added to the medium (Sun et al., 1998b). Therefore, it has been concluded that Tgfβ3 is an essential growth factor inducing EMT during palatal fusion, indicating that it may be the master gene that stimulates transcription repression of cell-cell proteins. Investigators have proposed that possible mechanisms of Tgfβ3-induced palatal fusion include the regulation of fusion by inducing cell membrane filopodia on MEE prior to shelf contact (Taya et al., 1999). Second, the regulation of extracellular matrix degradation by modulation of the production of tissue inhibitor of metalloproteinase-2 (Timp-2), Mmp13, and Mmp2 has been proposed (Blavier et al., 2001). More recently, it was found that Tgfβ3 is necessary for inhibiting MEE proliferation during EMT (Cui et al., 2003). However, a new study cultured single (unpaired) mouse palatal shelves by suspension and static culture methods and found that MEE cells could disappear throughout the medial edge, even without contact and adhesion to the opposing MEE in suspension culture. MEE cell behavior in the suspension culture demonstrated that apoptosis, EMT, and epithelial cell migration all occurred at various stages of MEE cell disappearance, including the transient formation and disappearance of epithelial triangles and islets (Takigawa and Shiota, 2004).
Several research groups have started to investigate the TGFβ3-stimulated intracellular signaling molecules that are responsible for EMT during palatal fusion. Smad2 expression was detected during palatal fusion (Cui et al., 2000). In a later investigation, the authors suggested that phosphorylation of Smad2 may be necessary for Tgfβ3 down-regulation of MEE proliferation (Cui et al., 2003). Interestingly, overexpression of Smad2 in the Tgfβ3–/– mouse did not completely rescue secondary palate clefts (Cui et al., 2005). There is also evidence, from the studies of mammary epithelial cell culture, that down-regulation of Smad signaling decreased Smad-dependent growth and transcriptional responses; however, the down-regulation did not affect Tgfβ-mediated stress fiber formation and EMT (Bhowmick et al., 2001). In another recent study, Hay’s group used dominant-negative Smad4 and dominant-negative Lymphoid enhancer factor-1 (LEF1) to demonstrate that Tgfβ3 used Smads to up-regulate synthesis of Lef1 and to activate Lef1 transcription during the induction of palatal EMT. When phospho-Smad2 and -Smad4 were in the nucleus, Lef1 was activated without β-catenin (Nawshad and Hay, 2003; Nawshad et al., 2004a). This work indicates that palatal EMT does not depend on β-catenin (Nawshad and Hay, 2003; Nawshad et al., 2004b) and that Smads can activate Lef1 without β-catenin (Labbe et al., 2000).
As an alternative downstream Tgfβ signaling effector, PI-3 kinase has been identified in actin re-organization and Tgfβ-mediated EMT (Metzner et al., 1996; Sugiura and Berditchevski, 1999; Bakin et al., 2000). Activated PI-3 kinase phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), which recruits its downstream effectors to the plasma membrane. Along with the small GTPases Rac and Rho, PIP3 activates several serine/threonine kinases, such as 3-phosphoinositide-dependent protein kinases (Pdks) (Alessi et al., 1998; Le Good et al., 1998). Pdk1 activates Pkc (Pullen et al., 1998) and targets protein kinase B (Pkb, also known as Akt) (Burgering and Coffer, 1995), while integrin-linked kinase (ILK), a newly found Pdk, also activates Akt (Hannigan et al., 1997). Upon stimulation, Akt migrates to and anchors the membrane (Andjelkovic et al., 1997). Subsequently, activated Akt detaches from the plasma membrane and translocates into the cytoplasm and nucleus, regulating cell survival, protein synthesis, and cell cycle (Kandel and Hay, 1999). It also appears that PI-3 kinase possesses both lipid kinase and protein kinase activity (Carpenter et al., 1993) and may directly control the activities of individual components of the Ras/Raf/Erk-mitogenic pathway by forming a complex with signal proteins.
The consequences of PI-3 kinase activation are numerous, including effects on cell cycle progression, suspension-mediated apoptosis, cell migration, and alterations in cell-cell adhesion (Roymans and Slegers, 2001). As a downstream effecter of Tgfβ signaling, PI-3 kinase is involved in actin re-organization, Mmp production, and cell mobility (Metzner et al., 1996; Sugiura and Berditchevski, 1999). A specific inhibitor of PI-3 kinase, LY294002, completely blocked Tgfβ-mediated phosphorylation of Smad2, cell migration, and partially blocked EMT in mammary epithelial cell cultures (Bakin et al., 2000). In addition, other experiments demonstrated that Mmp2 production after integrin 3β1 stimulation was PI-3-kinase-dependent (Sugiura and Berditchevski, 1999). Since Mmps are necessary for breaking down basement membrane, it was speculated that blocking PI-3 kinase activity inhibited cell migration and Mmp production, two essential steps of EMT. In addition, the overexpression of a PI-3 kinase downstream effecter, ILK, induced anchorage-independent epithelial cell growth, loss of E-cadherin expression, and EMT (Radeva et al., 1997; Wu et al., 1998). ILK was also implicated in TGFβ-induced fibroblastic conversion of highly metastatic cells (Janji et al., 1999).
Using static palate organ cultures, we found that, during palatal fusion in vitro, EMT was dependent on PI-3 kinase activity within the 72-hour culture time studied (Figs. 1C, 3P) (Kang and Svoboda, 2002). However, it was possible that inhibiting PI-3 kinase delayed but did not completely block EMT, since some of the palates were partially fused (Kang and Svoboda, 2002).
In addition to PI-3 kinase, other signaling pathways can also be activated by Tgfβ. An investigation of Tgfβ1-mediated disassembly of epithelial cell-cell junctions demonstrated a link between the Tgfβ/Smad pathway and alterations of β-catenin/E-cadherin phosphorylation (Tian and Phillips, 2002). A later study by the same group transiently transfected epithelia with Smad2/4 or Smad3/4 expression vectors, but did not alter cell phenotype (Tian et al., 2003). These results suggested that the Wnt pathway may be a further potential signaling pathway mediating downstream events following Tgfβ receptor binding. As part of the epithelial cytoskeleton, β-catenin binds to E-cadherin. The activity of β-catenin is controlled by a large number of binding partners that affect its stability and localization, which can be modulated by many signaling agents, such as Wnt, Ras, and PI-3 kinase (Espada et al., 1999; Willert et al., 1999). Upon release from the complex, β-catenin relocates into the nucleus and interacts with transcription factors such as T-cell factor/lymphoid enhancer factor-1 (TCF/LEF-1). Stabilized nuclear β-catenin has been shown to induce EMT in tumor cells in vitro (Kim et al., 2002). However, it was recently demonstrated that β-catenin does not translocate to the nucleus in palatal MEE (Nawshad and Hay, 2003).
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TRANSCRIPTION FACTORS
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Throughout this review, transcription factors have been discussed in context with the experimental model, transgenic animals, or signal transduction pathways. It is beyond the scope of this review to provide an exhaustive discussion of transcription factors; therefore, we will concentrate on specific proteins that have been identified specifically in EMT (Table 2).
There are a great many transcriptional partners that affect Tgf-β’s choice of target genes (DNA binding co-factors) and their effect on their transcription (co-activators, co-repressors) (Shi and Massagué, 2003). Cbfa1, Cbfa3, Stat3, and Jun are examples of DNA-binding co-factors, Cbp and P300 are examples of the co-activators, and Tgif, c-Ski, SnoN, and Evi-1 are some of the known co-repressors of the Tgf-β signaling system (Massagué et al., 2000; Massagué, 2003).
There is mounting evidence that Lef1 may be one of the controlling transcription factors that determines EMT cell fate (Nawshad and Hay, 2003). It was recently shown that Lef1 transcription is up-regulated via a Smad2/4 mechanism during palatal EMT. Furthermore, using laser capture microscopy, this group demonstrated that mRNA was also quantitatively increased in the MEE cells (Nawshad et al., 2004b).
Another transcription factor, Fsp1, was also shown to be involved in EMT (Okada et al., 1997). Although a murine fibroblast-specific protein, Fsp1 expression was induced in renal proximal tubular epithelial cells when treated with growth factors (Egf/Tgfβ) and/or extracellular matrix protein (Collagen I). The epithelia having de novo expression of Fsp1 displayed mesenchymal phenotypic morphology and protein expression. In addition, this EMT was abolished when cells were treated with Fsp1 antisense oligomers.
As stated previously, Twist, Snail, and Slug appear to down-regulate cell-cell adhesion proteins in developmental and tumorigenesis models (Kang and Massagué, 2004). Snail represses transcription of E-cadherin and activates mesenchymal differentiation, such as vimentin and fibronectin (Cano et al., 2000; Schlessinger and Hall, 2004; Zhou et al., 2004). Recent experiments show that Snail regulation is balanced between EMT-promoting growth factors and blocking factors, begging the question, Can Snail serve as the master gene, since it regulates cell adhesion molecules?
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ECM BREAKDOWN
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In addition to changes in growth factors, the modulation of the extracellular environment also includes the maintenance and degradation of ECM, which is mediated, in part, by Mmps and Timps. Temporospatial expression of Mmps 2, 3, 7, 9, and 13, and Timps 1 and 2, was observed during murine palatal fusion (Morris-Wiman et al., 1999, 2000). In the palatal fusion zone of Tgfβ3-deficient mice, Timp-2 was completely absent; Mmp-2 and Mmp-13 had reduced levels (Blavier et al., 2001). Upon exposure to Mmp inhibitor (BB 3103), the murine palatal shelves failed to fuse in culture (Brown et al., 2002).
Other mechanisms of Tgfβ3 included the regulation of extracellular matrix degradation (Blavier et al., 2001). The investigators compared the expression of several Mmps, including a cell-membrane-associated Mmp (Mt1-Mmp) and Timp-2, in normal and Tgfβ3–/– mice. They found that Mmps and Timp-2 were highly expressed by wild-type MEE. Mmp-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (Mmp-2) was expressed by mesenchymal cells neighboring the MEE. In contrast, Tgfβ3–/– mice had complete absence of Timp-2 in the midline and expressed significantly lower levels of Mmp-13 and slightly reduced levels of Mmp-2. In support of a role for Tgfβ3 in regulating matrix breakdown, Mmp-13 expression was strongly induced by Tgfβ3 in palatal fibroblasts. It was also shown that blocking Mmps or exposure of palates to higher concentrations of Timp-2 caused palatal fusion failure. The MEE cells did not transform into mesenchyme, indicating that ECM degradation by Mmps was a necessary step for palatal fusion (Blavier et al., 2001).
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CLINICAL/TERATOLOGY
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The etiology of cleft palate is generally considered multifactorial; however, a recent study has identified IRF6, a transcription factor, as responsible for the autosomal-dominant disorder, Van der Woude syndrome (Murray and Schutte, 2004; Zucchero et al., 2004), a model for isolated cleft lip and palate. Other candidate genes related to cleft palate include: TGF, TGFβ2 and 3, MSX1, B-cell lymphoma 3 (Bcl3), FGFR1, and the retinoic acid receptor alpha (RARA) (Vieira and Orioli, 2001; Murray and Schutte, 2004).
Disturbances at any stage during palate development—i.e., defective palatal shelf growth, failed or delayed shelf elevation, and failure of shelf fusion—can result in cleft palate (Ferguson, 1988; Murray and Schutte, 2004). As one of the most common congenital craniofacial defects, cleft palate occurs in approximately 1 per 750 live births in the United States (Fogh-Andersen, 1971; Hay, 1971; Murray and Schutte, 2004). Cleft palate may not be life-threatening, but many functions can be disturbed because of the structures involved, such as feeding, digestion, speech, middle-ear ventilation, hearing, respiration, and facial and dental development. These problems can also cause emotional, psychosocial, and educational problems. In addition, cleft palate is an economic burden, since it costs an average of $100,000 per patient for the entire treatment, amounting to $697 million per year in the United States (Morbidity and Mortality Weekly Report, 1995).
Children with oral clefting need to see a variety of specialists who will work together as a team, including surgeons, dentists, audiologists, neurologists, pediatricians, psychologists, speech therapists, ophthalmologists, and otolaryngologists. Treatment usually begins in infancy and often continues through early adulthood, and although different experts focus on the patient’s different needs at different stages, the entire team of experts follows through multiple surgeries, growth, and development. There have been many medical advances in the treatment of cleft palate. A possibility of in utero surgical correction has been explored. A recent study has documented that TGFβ3 aids in the ‘scarless’ repair of cleft palate (Weinzweig et al., 1999, 2002).
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CONCLUSION AND FUTURE DIRECTIONS
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In summary, we have discussed the experimental models and the current knowledge of EMT mechanisms, especially during craniofacial development. It is the orchestration of cytokines, extracellular matrix, cell-surface proteins, signaling molecules, and transcription factors that makes EMT a masterpiece.
We are just beginning to uncover the myth of this cellular phenotype transition that plays important roles during development and homeostasis. There have been advancements in research tools since the discovery of the EMT phenomenon. Laser-capture microscopy has aided researchers in the investigation of the temporospatial molecular changes of specific cell groups (Nawshad et al., 2004b). Newer transfection techniques have enabled more accurate cell-lineage studies to be conducted. In addition, exciting findings in signaling pathways are being investigated and published daily (Murray and Schutte, 2004). It will be just a matter of time until the pathway leading to EMT will be mapped and understood.
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ACKNOWLEDGMENTS
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We thank Jesus Acevedo, Tamara Field, Jan Lalor, Steve Lin, and Petra Moessner for their technical assistance. We are also grateful to the Baylor Oral Health Foundation, the Tobacco Endowment Fund (Texas A & M University System; Grant Number 304-202850-4013), and NIH EY08886 (KKHS) for supporting the study.
Received for publication June 15, 2004.
Accepted for publication March 18, 2005.
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Journal of Dental Research, Vol. 84, No. 8,
678-690 (2005)
DOI: 10.1177/154405910508400801
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ABSTRACT
INTRODUCTION
M LY294002-treated tissues. However, the 1- and 10-µM LY294002-treated palates were significantly different from controls (p 
0.01).