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Extracellular matrix |
This review concentrates on mechanisms of palatogenesis. This includes theories of shelf elevation, the role of matrix and identification of molecules and growth factors, which have key roles. The areas where failure to develop could potentially lead to clefting are highlighted. A key part of shelf fusion is the breakdown of the medial edge epithelium, a process that is probably dependent on enzymes involved in matrix turnover. There is good evidence that the matrix metalloproteinases may provide a common link to the multiple genetic and environmental factors that are known to cause clefting.
Keywords: Development of the mammalian palate, cleft lip and palate
J.R.Coll.Surg.Edinb., 45, December 2000, 351-358
The anomaly of cleft lip and palate occurs in approximately 1 in 700 live births and is associated with a significant degree of morbidity.1, 2 The incidence varies depending on race, geography, gender, and cleft type; it is more common among Indian and Oriental populations (2.3 per thousand total clefts) and least common among Afro-Caribbean groups (0.6 per thousand total clefts).2 There are about 1000 new cases each year in Great Britain and these patients are committed to lengthy treatment from a wide variety of healthcare workers. The aetiology of clefts is not known, but genetic and environmental factors are involved.3 Variation of cleft expressions in ethnic groups provides evidence for genetic differences in susceptibility. This view is reinforced by a Danish study which showed that cleft recurrence decreased with a change in partner rather than exposure to pollution or toxins.4 Cleft palate has been associated with numerous loci in the mammalian species and include Msx1, transforming growth factor a (TGF-a) and TGF-b3, suggesting no single gene is responsible.5,6,7
NORMAL PALATE DEVELOPMENT
The organisation of the face requires tissues to proliferate, fuse and differentiate. The polarising signal candidates expressed in craniofacial primordia include sonic hedgehog (shh), its putative receptor patched, fibroblast growth factor 8 (FGF-8) and bone morphogenetic protein 2 (BMP-2).8 Evidence exists that the teratogen, retinoic acid exerts some of its effects on craniofacial development through the disruption of the shh signalling pathway. Mutations in human shh9,10 and deletions in the shh locus of mice,11 result in loss of midline structures or holoprosencephaly. Non-mutant mice provide a further means to examine craniofacial development with a wide range of other craniofacial anomalies frequently expressed. Transforming growth factor-b 3 (TGFb-3) has a broad spectrum of biological activities. Homozygous TGF-b3 -/- mice die within a day of birth from delayed pulmonary development.12 Strikingly, and unlike other null mutants, they have a failure of palatal shelf fusion and no other craniofacial abnormality.
In humans, palate development begins towards the end of the fifth week of intra-uterine life and is complete at about twelve weeks. The critical period is from the end of the sixth week to the beginning of the ninth week. In normal palate development, mesenchymal cells from the neural crest migrate to the primitive oral cavity forming the maxillary processes in association with the craniopharyngeal ectoderm.
The primary palate arises from the fusion of two medial nasal prominences that form the intermaxillary segment, which develops towards the end of the fifth week of intra-uterine life in humans. It consists of two portions: a labial component that forms the philtrum of the upper lip and a triangular palatal component of bone that includes the four maxillary incisor teeth. The primary palate extends posteriorly to the incisive foramen. In humans, the secondary palate comprises at least 90% of the hard and soft palates. Development of the intact secondary palate is a dynamic process which, for the purpose of this article, has been arbitrarily split into three stages (Figure 1). Stage I of secondary palate development is characterised by formation of the palatal shelves from the maxillary processes. These shelves are orientated vertically, either side of the developing tongue (Figure 1). It is not known why the shelves attain this vertical orientation. Ferguson (1981) proposed that the direction of shelf growth be related to the amount of space available in the oronasal cavity during the period of palatogenesis.13 He suggested that the horizontal growth of palatal shelves in alligators from the outset of their development is related to the small size of the alligator tongue, the absence of cheeks, and the movement of the lower jaw beneath the premaxillary bulge. He postulated that the evolution of the large muscular mammalian tongue constrains the shelves to grow vertically until sufficient space can be created in the oronasal cavity.
At a precise developmental stage, (Stage II, Figure 1 and 2), these vertical palatal shelves elevate to a horizontal position above the dorsum of the tongue.14 This event occurs rapidly, possibly in a matter of hours.
Figure 1: Schematic diagram of coronal section through a developing face showing three stages of secondary palate development. At stage I the palatal shelves are vertical, they elevate in stage II and fuse in stage III.
Figure 2: Haematoxylin and eosin stained section of a developing foetus where the palatal shelves have elevated with the tongue lying inferiorly. Although there has been elevation the shelves have still to fuse.
Stage III of secondary palate development involves fusion of the medial edge epithelium (MEE) of the approximating palatal shelves with each other via numerous desmosome contacts to form a midline palatal seam. This then separates the oral and nasal cavities (Figure 1). Keratin fibrils and desmosomes are upregulated in the medial edge epithelium seam at this point, presumably to strengthen the bond between the newly adherent medial edge epithelium cells. This seam rapidly degenerates, a process characterised by loss of complex cytokeratins and basement membrane components such as laminin and desmosomes, and by an increase of vimentin-rich connective tissue, tenascin, proteoglycan and collagen expression.14 The increase in vimentin is particularly interesting as it is generally found as a mesenchymal intermediate filament.15 Medial edge epithelium degeneration allows mesenchymal cells to flow across the now intact horizontal palate. There have been several theories on the mechanism(s) of medial edge epithelium degeneration. Shapiro and Sweney (1969) suggested that it occurred as a result of programmed cell death.16 Gartner et al. (1978) disputed this on the grounds that there was no evidence of any cellular debris or phagocytic activity at any time during this process. Furthermore, some evidence of metabolic activity occurring within so called apoptotic cells was described. A radical postulate by Fitchett and Hay (1989) offered the possibility that the medial edge epithelium cells migrate into the body of the mesenchyme and transform into mesenchymal cells.18 This process is known as epithelial-mesenchyme transformation (EMT). An alternative view is that medial edge epithelium cells migrate nasally and orally out of the medial edge epithelium seam and become incorporated into the oral and nasal epithelia on the palatal surface.19 In vitro organ culture studies have suggested that TGF-b1 and TGF-b2 accelerate the palatal shelf fusion process.20,21 However it has been shown that neutralizing antibodies to TGF-b1 and TGF-b2 appear to have no effect on this fusion process. On the other hand, antibodies to TGF-b3 block palatal shelf fusion.22
Transforming growth factor-b3 is known to be abundantly expressed by rodent medial edge epithelial cells; TGF-b3-null mice exhibit an incompletely penetrant failure of the palatal shelves to fuse, leading to cleft palate.7, 23 Rescue experiments have demonstrated the importance of TGF-b3 in palate development, possibly through the induction of filopodia on the surface of medial edge epithelial cells.24
On completion of stage III, the epithelia on the nasal aspect of the palate are pseudostratified ciliated columnar cells whilst those on the oral aspect of the palate are stratified squamous, non-keratinizing cells.
Cleft palate may result from disturbances at any stage of palate development: defective palatal shelf growth, delayed or failed shelf elevation, defective shelf fusion, failure of medial edge epithelium cell death, post-fusion rupture and failure of mesenchymal consolidation and differentiation, have all been postulated.25 Sun et al (1998a) suggested that lack of intimate palatal shelf contact after elevation is one possible cause, and recent research into palate development has concentrated on fusion of the shelves rather than elevation.26 Ferguson (1981), on the other hand, took the view that failure of palatal shelf elevation may be responsible for 90% of palatal clefting.13
MECHANISM OF MAMMALIAN PALATAL SHELF ELEVATION
Several theories have been proposed to account for palatal shelf elevation in humans. Palatal shelves may play an entirely passive role and elevate as a result of some extrinsic force. Alternatively, there may be an active role for the shelves during the process of palatogenesis. It is possible that a combination of extrinsic and intrinsic factors are required with some components playing a more prominent role than others.
In principle, an intrinsic force generated within the palatal shelves reaches a threshold level which exceeds the force of resistance culminating in shelf elevation. Elevation of the palatal shelves is rapid with a swinging ‘flip-up’ mechanism in the anterior one third of the palate and an oozing remodelling ‘flow’ mechanism in the posterior two-thirds of the palate.27 The intrinsic shelf elevating force is complex, but Pratt et al (1973) proposed that in rat palatogenesis, the chief component appears to be a regionally specific accumulation of glycosaminoglycans (GAGs), predominantly hyaluronan, as development proceeds.28 Hyaluronan consists of highly electrostatically charged, open coil molecules, capable of binding up to ten times their own weight in water and small changes in its concentration result in large changes in osmotic pressure. Therefore, a regional accumulation of hyaluronan may result in an increase in water content of the extracellular matrix (ECM), leading to an increase in turgidity of the vertical palatal shelves and the production of an elevating force.29 This postulate is supported by the observation that in the developing mouse palate there is more hyaluronan in the anterior portion of the palate than in the posterior region. There is also more sulphated GAGs (primarily chondroitin sulphate) in the future oral aspect than the future nasal aspect.30 Singh et al (1994) investigated the GAG composition in rat palatal shelves at various stages of palatogenesis.31 They identified hyaluronan, heparan sulphate, and chondroitin-4-sulphate, but found no evidence of dermatan sulphate or chondroitin-6-sulphate. They also found a significant increase in the percentage of hyaluronan at the time of palatal shelf reorientation and fusion, followed by an immediate decrease in the anterior and posterior palatal regions.31 Also, they found no significant differences in GAG composition in the presumptive hard and soft palates at the time of shelf elevation. Unfortunately in the rat, it is only the anterior region of the palate which elevates, the posterior region assumes horizontal orientation at the outset of palato-genesis.13,32 The work by Singh et al (1994), therefore, neither proves nor disproves the notion that changes in GAG composition generates a shelf-elevating force.31 It is possible that the production of higher amounts of hyaluronan before shelf elevation could be the major influence on morpho-genesis with the decrease after elevation reflecting the fact that this task has been completed. It is also conceivable that the apparent lack of change in the total amount of GAGs during palatogenesis may mask other events that are responsible for palate elevation. For example, similar GAG changes may be required pre- and post-elevation in order to maintain the elevated palatal position prior to fusion of the two processes. It has also been demonstrated that epidermal growth factor (EGF),33 and some members of the TGF-b family stimulate palatal mesenchymal cells to produce hyaluronan.34
The separation of cells as a result of an increase in extracellular matrix hydration, secondary to increased hyaluronan content, may also be important in preventing cell-cell and cell-matrix interactions at this stage of development. Thus, if adjacent cells adhere to one another too strongly, this may inhibit palatal shelf elevation. In the later stages of palate development, such interactions are critical and an increase in cell-cell interactions corresponds with a decrease in hyaluronan content and an increase in cell density.
It has also been proposed that an increase in vascularity of the developing palate may be responsible for production of an ‘erectile’ force.35 The contraction of elastic fibres36 and/or skeletal muscle fibres37 are other potential mechanisms for this process.
The epithelium surrounding the shelves may also play an important role in reorientation of the palatal shelves. Bulleit and Zimmerman (1985) discovered that after removal of the oral epithelium in vitro, a significant inhibition of palatal shelf reorientation occurred.38 By contrast, disruption of the epithelium on the presumptive nasal palatal surface had no effect on the degree of movement. Hence, the epithelium present on the oral aspect of the hard palate may be a major factor in generating the intrinsic force but how this is achieved is unclear. Luke (1984) proposed that there may be an unequal cell division in the palatal epithelium, the oral epithelium dividing more rapidly than the nasal epithelium, with a resultant differential force augmenting an intrinsic palatal shelf elevating force.39 The ‘erectile’ shelf elevating force may be partly directed by stout bundles of type I collagen which run down the centre of the vertical shelf from its base to its tip. Bulleit and Zimmerman (1985) suggested that the epithelial covering and associated basement membrane of the oral aspect of the palatal shelves play a major role in the reorientation of the secondary palate, although the mechanisms by which this is achieved are unknown.38 The alignment of mesenchymal cells within the core of the palatal shelf may further serve to direct the elevating force.40 Palatal mesenchymal cells are contractile and can also secrete several neurotransmitters including serotonin and acetylcholine.40,41 These neurotransmitters can affect both mesenchymal cell contractility and GAG degradation, it is possible, therefore, that these neuro-transmitters play a modulatory role in palate morphogenesis.
During the period of palatal shelf elevation, there is a decrease in the width of the oral cavity and the maxilla, but no change in the vertical dimension. However, as the palatal shelves come in contact and fuse, the oro-nasal height increases markedly whilst there is little change in its width.42 The tongue muscles also become functional at around the time of palatal shelf elevation.43
EXTRACELLULAR MATRIX
Remodelling of the extracellular matrix is an essential element in many areas of development.44 Surprisingly, the composition of extracellular matrix during the different stages of palatogenesis has received little attention.
Palate development will inevitably include the synthesis and degradation of palatal shelf extracellular matrix. A key enzyme involved in matrix metabolism is matrix-metalloproteinase-2 (MMP-2) which, was originally known to degrade gelatin, but has since been found to hydrolyse type IV collagen (present in basement membranes), elastin, entactin, fibronectin, and galectin-3. Recently, Ashworth et al (1999) have demonstrated that MM-2 can also degrade fibrillin molecules, the principal and probably most fundamental component of extracellular matrix elastic microfibrils.45 Of particular significance to the palate, MMP-2 is capable of cleaving native type I collagen into N-terminal " and C-terminal ¼ fragments, a function typical of the collagenases.46 Collagen types I, III, and V are the major collagens produced by murine palates in vitro.47 The catabolism of these macromolecules is an essential feature in the overall maintenance of all connective tissues and this probably includes the developing palate.
Matrix-metalloproteinase-2 can also regulate extracellular matrix metabolism through its interaction with cell surface molecules including the vitronectin receptor integrin avb3.48 The integrins are a large family of cell adhesive glycoprotein receptors comprising over 20 members that are capable of forming complexes with a variety of extracellular matrix components. Integrins offer a means of communication between the matrix and its resident cells via their cytoskeletal apparatus; it is thought that such communication is likely to play a significant role in extracellular matrix regulation, cell adhesion and migration.49
The wide distribution of MMP-2, its interactions with integrins, the broad substrate specificity, the peculiar mechanisms of expression and activation, as well as its ability to adhere to cellular and matrix macromolecules (via the fibronectin-like type II domain), suggest that the processing and activation of latent MMP-2 depends upon the cooperative interactions between a number of different molecules in the pericellular environment. It may be that these interactions allow MMP-2 to play an important role in the regulation of cell-cell and cell-matrix interactions, which probably occur during the positional changes of the palatal shelves during palatogenesis.
In addition to their essential role as regulators of the extracellular matrix, the MMPs have a wide variety of other functions. Matrix-metalloproteinase-3, for example, appears to have a role in maintenance of epithelial phenotype.50 Where there is excessive MMP-3 activity the process of epithelial-mesenchymal transformation (EMT) is seen51 and this may be relevant to medial edge epithelium degeneration during palatogenesis.18
The role of MMPs in palate development, as yet, are unknown. Some global data on MMPs and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) in craniofacial development exist with a good indication that inhibitors of MMP activity affect mandibular morphogenesis, glossogenesis and cartilage formation.44 Expression of the MMP's and TIMP's in craniofacial development is tissue specific with strong temporal and spatial changes.52 This latter group provided good evidence of changes in MMP-2 levels in a variety of craniofacial structures. It is known that the candidate molecules involved in palatogenesis (EGF and TGF-b) also regulate MMP and TIMP synthesis.53, 54 The tissue specificty of MMPs and TIMPs may also have some relation to changes in medial edge epithelium, be it epithelial migration or transformation. The contiguous cells at the medial edge epithelium seam are joined by cell-cell adhesion molecules of which the major molecules are E-cadherin, an integral membrane glycoprotein,55 and syndecan-1, a heparan sulphate proteoglycan.56 Recent work suggests that there is simultaneous loss of expression of syndecan-1 and E-cadherin during epithelial-mesenchymal transformation at the seam of the palate.57 The MMP, stromelysin-1 (SL-1), may be pivotal in this process since induction of its expression results in cleavage of the cadherin, loss of epithelial phenotype and a stable epithelial mesenchymal transformation.50 Transforming growth factor-a. is also expressed at the medial edge epithelium and it is known that cleft palate in humans is associated with the TGF-a locus.52,58,59
Transforming growth factor-a is a homologue of EGF and the receptor (EGFR) is upregulated as the medial edge epithelium seam degenerates. Seminal work by Miettenen et al.(1999) created an EGFR knockout model. In the homozygote (EGFR-/-) there was decreased MMP expression in palate explants compared with wild-type explants.6 Misdulation of EGFR controls MMP activity and suggests MMP's may be involved in seam disruption. Potentially, the disruption of MMPs may be a pivotal mechanism for the large number of environmental and growth factors which can cause clefting.
EPITHELIAL - MESENCHYMAL TRANSFORMATION
Electron microscopy and cell lineage studies performed during the latter stages of palatogenesis support phenotypic transformation coincident with medial edge epithelium loss and mesenchymal continuity.18,60 Epithelial-mesenchymal transformation enables epithelial cells to invade the extracellular matrix, a property which they do not normally possess.60 This has been demonstrated in vitro by placing individual epithelial cells on top of extracellular matrix where they remain cuboidal in shape with flattened basal surfaces, forming a non-invasive cell laminate maintained through cell-to-cell adhesion molecules.61 In contrast, mesenchymal cells in contact with extracellular matrix exhibit front-end/backend polarity, elongation and extracellular matrix invasion. The process of epithelial-mesenchymal transformation is a key feature in several developmental processes; in the amniotic embryo it is the process by which the embryonic mesoderm and endoderm are formed from the flat epithelial disc known as the epiblast. It is the process which also enables presumptive muscle cells to detach from their somites and migrate to definitive locations in the face and appendages of higher vertebrates.62,63 The process also occurs in the m¸llerian duct of the kidney, during disappearance of the ectodermal branchial clefts and in the elimination of the ducts of the thyroid and other endocrine glands.18 It is a feature of involution of the mammary gland; a normal, physiological process that is well recognised as involving extensive extracellular matrix degradation.64 The transformation of epithelia can be determined in fully differentiated epithelium, namely tumourigenesis, where epithelial cells are produced locally made and systematically disseminate.65
Fitchett and Hay (1989) studied medial edge epithelium degeneration in secondary palate formation and demonstrated a change in phenotype, of the cells present in the seam, from epithelial to mesenchymal. In addition, they found an increase in the expression of vimentin, an intermediate filament protein commonly found in mesenchymal tissue but rarely in epithelial cells, suggesting that transformation had occurred. The major cell-cell adhesion molecules, E-cadherin and syndecan-1, rapidly and abruptly disappear once epithelial-mesenchymal transformation has commenced.57 Desmosomes and keratin are probably also downregulated in the medial edge epithelium, at the same time as E-cadherin and syndecan-1. They also showed that the rodent palatal shelves adhere and transform to mesenchyme under the influence of TGF-b3, which has been implicated in cleft palate.7,26 During the process of transformation, epithelial cells are somehow stimulated to express a specific sequence of genes, the so-called mesenchymal gene programme,60 that is responsible for production of the mesenchymal phenotype and their resultant motile activity.66 The most likely candidate for the master controlling gene of this programme is FSP1, however, shh has also been implicated as have several oncogenes including v-src and c-fos.67,68 Activation of this mesenchymal gene programme produces cell front-end/backend polarity, as well as stimulating vimentin, collagen, and actin cortex/integrin/extracellular matrix interactions,66 all features synonymous with mesenchymal tissue. The role of the transformation process, therefore, is to remove unwanted epithelia at the medial edges of the fusing palatal shelves and produce cells with the capacity to invade the extracellular matrix. It is possible that MMP-3, is an essential developmental cue enabling this process to occur during the latter stages of palate development.
Osteogenic blastemata for the palatal processes of the maxillary and palatine bones differentiate in the mesenchyme of the hard palate whilst several patterned myogenic blastemata develop in the soft palate. Palatal mineralisation occurs through a process of intramembranous ossification involving the gradual deposition of hydroxyapatite upon a template comprised largely of type I collagen (Figure 3).
Figure 3: A fused palate where the medial edge epithelium has disappeared and the palate mesenchyme is organising to form the definitive palate
Ossification is seen where mesenchymal cells condense, the surrounding tissue vascularizes and cells differentiate into osteoblasts. There are a number of signalling molecules involved in this process and these include the BMPs, core binding factor (cbf) FGFs and the hedgehog proteins which seem to be responsible for regulating patterning of undifferentiated mesenchyme. Bone morphogenetic protein-6 and the transcription factor gli1 are also expressed during intramembranous bone formation.69
The intact palate is essential for normal oral function and speech. As our understanding of the molecular and biological mechanisms of normal palate development increases, potential prevention strategies for clefting may be applicable.70
Copyright date: 19 October 2000
Correspondence: Professor J.R. Sandy, Division of Child Dental Health, University of Bristol Dental School, Lower Maudlin Street, Bristol U.K.
E-mail: Jonathan.Sandy@bris.ac.uk
©2000 The Royal College of Surgeons of Edinburgh, J.R.Coll.Surg.Edinb. 45, 6: 351-358