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Jul 20, 2005 - of non-syndromic cleft palate and the mechanics of normal palatogenesis. Key words cleft palate; ... Present addresses. *Department of Oral Biology, School of Dentistry, University of Oslo, ..... In wild-type mice, an auto- regulatory loop ..... Dudas M, Nagy A, Laping NJ, Moustakas A, Kaartinen V (2004).

J. Anat. (2005) 207, pp655–667

REVIEW Blackwell Publishing, Ltd.

Regional regulation of palatal growth and patterning along the anterior–posterior axis in mice Sylvia A. Hilliard,1 Ling Yu,1 Shuping Gu,1* Zunyi Zhang1† and Yi Ping Chen1,2 1 Division of Developmental Biology, Department of Cell and Molecular Biology, and Center for Bioenvironmental Research, Tulane University, New Orleans, USA 2 College of Bioengineering, Fujian Normal University, China

Abstract Cleft palate is a congenital disorder arising from a failure in the multistep process of palate development. In its mildest form the cleft affects only the posterior soft palate. In more severe cases the cleft includes the soft (posterior) and hard (anterior) palate. In mice a number of genes show differential expression along the anterior–posterior axis of the palate. Mesenchymal heterogeneity is established early, as evident from Bmp4-mediated induction of Msx1 and cell proliferation exclusively in the anterior and Fgf8-specific induction of Pax9 in the posterior palate alone. In addition, the anterior palatal epithelium has the unique ability to induce Shox2 expression in the anterior mesenchyme in vivo and the posterior mesenchyme in vitro. Therefore, the induction and competence potentials of the epithelium and mesenchyme in the anterior are clearly distinct from those in the posterior. Defective growth in the anterior palate of Msx1–/– and Fgf10–/– mice leads to a complete cleft palate and supports the anteriorto-posterior direction of palatal closure. By contrast, the Shox2–/– mice exhibit incomplete clefts in the anterior presumptive hard palate with an intact posterior palate. This phenotype cannot be explained by the prevailing model of palatal closure. The ability of the posterior palate to fuse independent of the anterior palate in Shox2–/– mice underscores the intrinsic differences along the anterior–posterior axis of the palate. We must hitherto consider the heterogeneity of gene expression and function in the palate to understand better the aetiology and pathogenesis of non-syndromic cleft palate and the mechanics of normal palatogenesis. Key words cleft palate; epithelial–mesenchymal interaction; growth factor; palatogenesis; transcription factor.

Introduction The secondary palate develops late during organogenesis of higher vertebrates. The process begins on day 45 in humans, day 17 in alligators, day 6 in chick and embryonic day (E) 11.5 in mice (Ferguson, 1988). Arising as bilateral outgrowths of the maxilla, the palatal shelves extend from the anterior to the posterior along

Correspondence Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA. T: +1 504 865 5587; F: +1 504 865 6785; E: [email protected] Present addresses *Department of Oral Biology, School of Dentistry, University of Oslo, Oslo, Norway; †Shanghai Research Center for Biomodel Organism, China. Accepted for publication 20 July 2005

the lateral walls of the oropharynx (Fig. 1A,B). These palatal shelves are formed of mesenchyme mainly of neural crest as well as mesodermal origin in association with craniopharyngeal ectoderm (Ito et al. 2003). In mammals, growth of the palatal shelves causes their vertical extension on either side of the tongue (Fig. 1C–F). Guided by what appears to be both mechanical and morphogenetic forces, the palatal shelves elevate at a definite time and become horizontally positioned over the tongue. Continued directed growth causes the approximation of the horizontally occurring palatal shelves (Fig. 1G,H). Palatogenesis culminates when the shelves make contact, adhere and fuse along the midline, forming an epithelial seam that is later replaced by mesenchyme to form the definitive palate (Fig. 1I–L). The definitive palate fuses with the primary palate and the nasal septum in the anterior to middle regions

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Fig. 1 Developmental stages of the secondary palate in mouse. (A,C,E,G,I,K) Oral view of the palate from E11.5 to E16.5 as viewed under a stereo microscope. (B,D,F,H,J,L) Haematoxylin–eosin-stained sections of the palate in frontal view of the corresponding stages. In A, C, E, G and I, the anterior end points to the top, whereas in K the anterior is to the left. Abbreviations: ns, nasal septum; pp, primary palate; ps, palatal shelf; rg, rugae; SP, secondary palate; T, tongue; tg, tooth germ. The white dotted lines in A, C, E and G depict the margins of the palatal shelves.

and thus separates the oral and the nasal regions of the oropharynx. Variations on this theme have been reported in amphibians, most reptiles and birds (Ferguson, 1988). In birds, the shelves develop horizontally from the start. Another notable deviation seen in birds and in some reptiles is the persistence of the epithelial seam on the shelves, generating a naturally cleft palate. Therefore, from a phylogenetic perspective the cleft palate is an ancestral trait with respect to an intact palate. The formation of the mammalian secondary palate is a highly regulated and complex process. Disruption of palate development could occur at any one of the multiple morphogenetic steps involved. Retarded growth of the palatal shelves, impaired shelf elevation, failure of the bilateral shelves to contact, adhere or fuse medially, post-fusion rupture and failure of the mesenchyme to differentiate appropriately are the commonly recognized causes of a pathological cleft (Ferguson, 1988; Sperber, 2001; Cohen, 2002; Francis-West et al. 2003). Orofacial clefts include syndromic and non-syndromic forms of cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO), respectively. A highly prevalent

congenital anomaly, orofacial clefts, can result from genetic and environmental perturbations. Also of relevance in the prevalence of orofacial clefts are factors such as geographical origin, racial and ethnic backgrounds, and socio-economic status. Syndromic forms of cleft palate constitute 50–55% of cases (Jones, 1988; Koillinen et al. 2005). Non-syndromic forms of cleft palate occur once in every 1000 live human births while cleft lip with or without cleft palate occurs at a frequency of 1 : 700 in the Caucasian population (Gorlin et al. 2001; Rice et al. 2004; Koillinen et al. 2005). In the early part of the 20th century, Veau devised a classification system for orofacial clefts (Shprintzen, 2002). This classification recognizes four major subtypes of orofacial clefts: clefts of the soft palate (type I); complete clefts of the palate extending posteriorly from the incisive foramen (type II); complete unilateral cleft lip and cleft palate (type III); and complete bilateral cleft lip and cleft palate (type IV). However, this classification system appears to be an oversimplification and has its limitations. Attempts to establish a universally applicable system of classifying orofacial clefts has since generated several models (Kernahan, 1971;

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Kriens, 1989; Schwartz et al. 1993; Smith et al. 1998). However, such classification schemes are inadequate for therapeutic and research purposes without the consideration of aetiological heterogeneity of orofacial clefts as well as any and all associated anomalies or syndromes (Shprintzen, 2002). An understanding of the genetic and molecular components of palate development is aimed at devising advanced preventative and therapeutic strategies while awareness of the environmental aetiologies will provide risk counselling and management alternatives. Genetic approaches used to identify the susceptible gene loci include genetic linkage and segregation studies in humans and gene knockout approaches in animal models. Although a number of genes have been linked to non-syndromic cleft palate in humans, very little work has been conducted on the pathogenesis of cleft palate in humans (Johnston & Bronsky, 1995).

Mouse as a model system for studying the genetic control of palatogenesis and cleft palate formation Humans and rodents share great similarity in palatogenesis (Schüpbach, 1983). Recent loss-of-function analyses and transgenic studies in mice have linked a growing number of genes to cleft palate formation. These mouse models would be useful to unveil the molecular and cellular mechanisms for palatogenesis in mammals. The advent of molecular biology has spawned the study of hierarchies involving growth factors and their receptors, transcription factors, and extracellular molecules within intricate signalling pathways that guide specific morphogenetic events. Among them, members of the TGF-β superfamily and TGF-α/ EGF family and retinoic acid (RA) have been studied quite extensively in palatogenesis. Tgf-α is expressed in the medial edge epithelia (MEE) of fusing palatal shelves, whereas the gene encoding the EGF receptor (Egfr) is expressed in the degenerating MEE (Iamaroon et al. 1996). In palate cultures, EGF and TGF-α promote extracellular matrix synthesis and mesenchymal cell migration (Dixon & Ferguson, 1992). The role of the TGF-α/EGF signalling pathway in palatogenesis is manifested by Egfr-knockout mice in which cleft palate appears due to a failed or delayed fusion of the palatal shelves (Miettinen et al. 1999). RA is known to play a key role in craniofacial development (Morriss-Kay, 1993; Brickell & Thorogood, 1997). RA induces cleft

palate when administered to pregnant mice during early stages of palatogenesis and is a known human teratogen (Johnston & Bronsky, 1995). Disruption of the RA signalling pathway either by the deletion of genes encoding RA receptors or by the expression of dominant negative RA receptor mutants also results in cleft palate in mice (Damm et al. 1993; Lohnes et al. 1993, 1994). RA is crucial for the proper fusion of facial primordia. The vitamin A deficiency syndrome (VAD) is characterized by facial defects that include cleft lip and palate and microophthalmia (Mark et al. 1995; Morriss-Kay & Sokolova, 1996). Ectopically high levels of RA interfere with neural crest survival and/or migration, causing severe hypoplasia of the branchial arches and facial clefting (Lammer et al. 1985; Sulik et al. 1988; Abbott & Pratt, 1991; Morriss-Kay, 1993). The incidence and severity of the craniofacial malformations encountered are affected by genetic susceptibility, dosage and timing of exposure to the teratogen. Elevated levels of RA inhibit the expression of Shh and its receptor patched leading to truncated growth of the frontonasal and maxillary processes and resultant bilateral clefting of the lip and primary palate in the chick embryo (Helms et al. 1997). Exposure to RA at the palatal shelf outgrowth stage severely compromises palatal mesenchyme organization and cell turnover rates, preventing growth and elevation of the palatal shelves (Degitz et al. 1998; Suwa et al. 2001). It has been reported recently that exogenous RA arrests palatal mesenchyme cells at the G1/S checkpoint while promoting apoptosis in a dose-dependent manner (Yu et al. 2005). Endogenous RA has an essential role in the fusion of palatal shelves by regulating the levels of apoptosis in the MEE (Cuervo et al. 2002). In summary, at physiological concentrations RA has an obligatory role in the early and late phases of craniofacial morphogenesis. The first indication for a role of TGF-β in mammalian palatogenesis was suggested by the localization of TGF-β mRNAs and proteins in the developing mouse palate (Heine et al. 1987; Lehnert & Akhurst, 1988; Sharpe & Ferguson, 1988; Fitzpatrick et al. 1990; Pelton et al. 1990). During mouse palatogenesis, both Tgf-β1 and Tgf-β3 are expressed in the MEE of the growing and fusing palatal shelves, whereas Tgf-β2 expression is restricted to the mesenchyme beneath the MEE (Fitzpatrick et al. 1990; Pelton et al. 1990). Once the epithelial seam is disrupted and the cells lose their epithelial phenotype by transformation into mesenchymal cells, the expression of Tgf-β1 and Tgf-β3 is lost. The

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temporal and spatial expression pattern of Tgf-βs suggests a role for TGF-β family members in the growth and fusion of palatal shelves. Indeed, functional analysis by gene targeting has demonstrated a crucial requirement for TGF-β in palate development. Although Tgfβ1–/– mice do not develop cleft palate (Shull et al. 1992; Kulkarni et al. 1993), both Tgf-β2 and Tgf-β3 mutant mice do. Cleft palate was observed in the Tgf-β2–/– mice with a relatively low incidence (Sanford et al. 1997). By contrast, Tgf-β3–/– mice exhibit a cleft secondary palate phenotype with 100% penetration (Kaartinen et al. 1995; Proetzel et al. 1995). The generation of cleft palate in the Tgf-β3–/– mice is apparently due to a failed fusion of the palatal shelves, which could be rescued by exogenous addition of TGF-β3 in an in vitro culture system (Brunet et al. 1995; Taya et al. 1999). The role of TGF-β3 in palatal fusion is further supported by studies showing that exogenous TGF-β3 can induce abnormal fusion of cultured chick palatal shelves that normally do not fuse by promoting transformation of palatal MEE to mesenchyme (Sun et al. 1998). TGF-β3 is thus specifically required for adhesion and fusion of the palatal shelves, probably by enhancing the transformation of the MEE cells and inducing apoptosis in the MEE (Kaartinen et al. 1997; Sun et al. 1998; Martínez-Álvarez et al. 2000; Blavier et al. 2001; Cuervo et al. 2002; Gato et al. 2002; Tudela et al. 2002). Mutations in TGF-β3 have been associated with several cases of isolated cleft palate in humans (Lidral et al. 1998). Examination of the downstream components of the TGF-β3 pathway was undertaken to understand better their functional contribution to palatogenesis and the aetiopathogenesis of secondary cleft palate in Tgf-β3–/– mice. Expression studies show that TGF-β type I receptor (TβR-I) is expressed ubiquitously in the palatal epithelium while TGF-β type II receptor (TβR-II) is expressed throughout the epithelium and cranial neural crest-derived mesenchyme of the palate. By contrast, TGF-β type III receptor (TβR-III) was restricted specifically to the MEE (Cui et al. 1998; Cui & Shuler, 2000; Ito et al. 2003). Among the TβR-I receptors, Alk-1, Alk-2 and Alk-5 but not Alk-7 are detected in the palate (Dudas et al. 2004). Cui et al. (2003) found that TGF-β3-mediated phosphorylation of Smad2 inhibits MEE cell proliferation, a prerequisite for palatal fusion. Significantly, over-expression of Smad2 in vivo and constitutive activation of Alk-5/Smad2 in vitro can rescue the cleft palate phenotype of Tgf-β3 homozygous null mice (Dudas et al. 2004; Cui et al. 2005). TGF-β signals can be transduced by Smad-dependent

(canonical) and Smad-independent (non-canonical) pathways. However, a comparison of Smad-dependent pathways with Smad-independent pathways demonstrates an absolute requirement for the former in the process of palate fusion (Dudas et al. 2004). The Smadindependent pathways are required but not sufficient for TGF-β-mediated palate fusion (Bakin et al. 2000; Yu et al. 2002). Moreover, there is evidence of crosstalk between the Wnt and canonical TGF-β signalling pathways during epithelial–mesenchymal transition of the palatal epithelial seam (Nawshad & Hay, 2003). Apart from its role in palate fusion, TGF-β signalling via TβR-II is required at an early stage to support the proliferation of cranial neural crest-derived mesenchymal cells of the palate (Ito et al. 2003). Recently, ten families were identified with heterozygous missense mutations in genes encoding either the type I or the type II receptor for TGF-β (Loeys et al. 2005). Individuals in these families present with an autosomal, dominantly inherited syndrome marked by pleiotropic defects, including hypertelorism, cleft palate and bifid uvula. This study implicates TGF-β signalling in the pathogenesis of multiple human disorders primarily involving cardiovascular, craniofacial, neurocognitive and skeletal development. Activins/Inhibins represent a subdivision of the TGF-β superfamily. Activin-βA is expressed in the mesenchyme of the developing mouse face (Feijen et al. 1994). Knockout of the Activin-βA gene in mice results in cleft palate probably due to a defect in the growth of the facial primordia (Matzuk et al. 1995a). Similarly, disruption of the Activin signalling pathway by knocking out the Activin type II receptor, ActRcII, also causes craniofacial abnormalities, including cleft palate (Matzuk et al. 1995b). A null mutation in the gene encoding Follistatin, an Activin binding protein, results in palate defects similar to those seen in the Activin-βA mutants (Matzuk et al. 1995c). These results thus implicate a role for Activin-mediated signalling in the development of the facial primordia, including the palatal shelf. Expression of a number of Bmp genes has been detected in the mouse craniofacial region (Lyons et al. 1990, 1995; Jones et al. 1991). Bmp2 and Bmp4 transcripts are localized to the epithelium and mesenchyme of the palate at E12.5. At E13.5, Bmp4 expression is only found in the palate mesenchyme subjacent to the MEE whereas the Bmp2 expression pattern remains unaltered (Zhang et al. 2002). However, Bmp2 or Bmp4 mutant mice die well before the formation of the palate (Winnier et al. 1995; Zhang & Bradley, 1996), providing

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no information on the role of these factors in palatogenesis. Mice deficient for Bmp7 do not exhibit obvious defects in the craniofacial region (Dudley et al. 1995; Luo et al. 1995). The lack of craniofacial abnormalities in the Bmp-7-deficient mice indicates that Bmp-7 is not crucial for craniofacial development. Mice deficient for Bmpr-IA die before the formation of the palate (Mishina et al. 1995). However, mice deficient for Bmpr-IB do not exhibit a defect in the palate (Baur et al. 2000; Yi et al. 2000). Genetic perturbations in either MSX1 or p63, known targets of Bmp signalling, show CL/P and CPO within the same pedigree (van den Boogaard et al. 2000; Barrow et al. 2002). Recent studies have uncovered distinct roles for Bmp signalling in lip and secondary palate morphogenesis, providing a molecular basis for understanding mixed orofacial clefts in human patients (Liu et al. 2005). Conditional inactivation of Bmp4 causes isolated cleft lip. However, conditional inactivation of Bmpr1A (Alk3) causes CL/P owing to growth and patterning defects in the palate and premature apoptosis in the edge epithelium of the nasal processes (Liu et al. 2005). It was previously demonstrated that a signalling pathway involving Bmp4, Msx1, Shh and Bmp2 (detailed later in this review) is necessary for normal growth and fusion of palatal shelves in the mouse (Zhang et al. 2002). Several members of the fibroblast growth factor (FGF) family, including Fgf-1, -2, -4, -5, -8, -9 and -12, are expressed in the craniofacial region (for review, see Francis-West et al. 1998; Colvin et al. 1999). Fgfs may participate in the regulation of facial growth, as Fgfs can partially substitute for epithelial signals in their ability to support outgrowth of the facial primordia and can increase proliferation in micromass cultures of the frontonasal mass primordia (Richman & Crosby, 1990; Richman et al. 1997). Lee et al. (2001) reported the expression of Fgfr1 and Fgfr2 through all stages of palate development, from initiation through to complete fusion of the palatal shelves, in the mouse embryo. Fgf receptor expression also persists during maturation and differentiation of the palate. In humans, during shelf elevation FGF4, FGFR1, FGFR2 and the downstream effector STAT1 are present throughout the palatal epithelia (Britto et al. 2002). Like FGF4, FGF2 is also expressed in the epithelium although it shows strict localization to the oral palatal epithelium. Subsequent to the apposition of the palatal shelves, FGF2 and FGF4 are co-expressed with their receptors FGFR1–3 and TGFβ3 in the epithelial islands of the seam. During

membranous ossification of the anterior palate there is up-regulation of FGF-2, -4, FGFR-1, FGFR-2 and TGFβ in the osteogenic blastemata. Although FGF7 expression has not been detected in the human palate, in mice its expression is found subjacent to nasal palatal epithelium. There is some overlap with Fgf10 expression in the dorsalmost aspect of the palate near the bend between the palatal shelf and the cranial base (Rice et al. 2004). Direct evidence that supports a role for FGF signalling in mammalian palatogenesis derives from studies on disruption of the Fgf10/Fgfr2-mediated signalling pathway (Celli et al. 1998; Martínez-Álvarez, 2000; Rice et al. 2004; Alappat et al. 2005). A block of the Fgfr2mediated signalling pathway in mice, by either overexpression of soluble dominant-negative Fgfr2 or knockout of the IIIb isoform of Fgfr2, results in severe craniofacial defects, including cleft palate. Disruption of Fgf10/ Fgfr2b signalling causes diminished proliferation and survival in the palatal epithelium and/or mesenchyme, leading to an elevation defect (Rice et al. 2004; Alappat et al. 2005). Regulation of epithelial Shh, Jagged-2 and Tgfβ3 by Fgf10 signalling ensures proper development of the murine palate (Alappat et al. 2005). Shh–/– mice develop very severe midline patterning defects such as holoprosencephaly and cyclopia, precluding any assessment of palate development (Chiang et al. 1996). In the developing mouse palate, Shh expression is detected in the precursor cells of the rugae epithelia from E11.5 to E16.5 (Bitgood & McMahon, 1995). Conditional inactivation of Shh specifically in the epithelium results in a wide-open cleft palate phenotype, indicating the critical involvement of Shh in palate development (Rice et al. 2004). A similar approach targeting Smo function in the epithelium does not disrupt palatogenesis. This confirms that Shh in the palatal epithelium signals to the underlying mesenchyme tissue, consistent with the detection of Smo expression in the mesenchyme at E13.0. Previous studies show that Shh functions as a critical mitogenic factor for the palate mesenchyme through its coordination of Fgf10 and Msx1–Bmp4 signalling pathways (Zhang et al. 2002; Rice et al. 2004). Furthermore, mutations in downstream components of the Shh signalling pathway, Gli2 or Gli3, cause facial abnormalities, including cleft palate (Mo et al. 1997). Transcription factors, particularly homeodomain proteins, have been implicated in palatogenesis. For example, mice deficient for Msx1, Lhx8, Pax9, Pitx1 or

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Pitx2 exhibit cleft palate (Satokata & Maas, 1994; Peters et al. 1998; Lu et al. 1999; Szeto et al. 1999; Zhao et al. 1999). In humans, mutations in the MSX1 gene cause orofacial clefting and tooth agenesis, consistent with the phenotype observed in the Msx1 mutant mice (Vastardis et al. 1996; van den Boogaard et al. 2000). Palatal shelves in the Msx1 mutant mice become elevated, but do not make contact and fuse (Satokata & Maas, 1994). Similarly, the Lhx8 mutants have shelves that become elevated and yet fail to make contact and fuse in the midline (Zhao et al. 1999). Cleft palate resulting from failure of palatal shelves to elevate is encountered in Pax9 and Pitx1 mutants (Peters et al. 1998; Szeto et al. 1999). Targeted deletion of many other transcription factor genes have resulted in a cleft palate, including Sim2, Foxf2 (LUN), Rae28, Hic1, Alx3/ 4, Titf2 and Tbx1 (Takihara et al. 1997; De Felice et al. 1998; Carter et al. 2000; Beverdam et al. 2001; Jerome & Papaioannou, 2001; Shamblott et al. 2002; Wang et al. 2003). The Sim2–/– mice develop two kinds of palatal abnormalities (Shamblott et al. 2002). Seventyfive per cent of the offspring develop full clefts of the secondary palate where the palatal shelves are elevated and yet do not make contact owing to outgrowth defects, whereas the remaining animals manifest a partial cleft specifically in the posterior with abnormally thin secondary palate. It is now believed that variations on the extent of clefting in humans and mice could be the result of underlying molecular differences along the different axes of the developing palate. This idea is gaining momentum in the wake of growing evidence showing the regional diversification of signalling pathways along the anterior and posterior palate.

Differential gene expression along the anterior–posterior and medial–lateral axes of the developing palate Elevation, maturation and fusion of the palatal shelves follow an anterior to posterior sequence (Taya et al. 1999; Dudas et al. 2004). Most clinical cases of clefts involving the anterior hard palate almost always include the posterior soft palate (Sperber, 2001). In its mildest form, for instance bifid uvula, the cleft affects only the posterior-most structure associated with the soft palate. In more severe cases, the clefts extend from the posterior further into the anterior. By contrast, one rarely encounters clefts of the hard palate alone with the soft palate intact (Schüpbach, 1983). These very

rare malformations are characterized by oval-shaped openings in the hard palate and may occur in association with occult submucous clefts in the soft palate (Fara, 1971; Mitts et al. 1981; Schüpbach, 1983). Incomplete hard palate clefts were initially believed to be the result of post-fusion rupture (Fara, 1971; Mitts et al. 1981). However, one of seven reported human cases of incomplete hard palate clefts appeared to have a welldeveloped soft palate, disproving the post-fusion rupture hypothesis (Schüpbach, 1983). Using rat as an animal model to induce incomplete hard palate clefts experimentally, Schüpbach (1983) showed that the soft palate could fuse independent of the hard palate. Recently, Shox2–/– mice were characterized with this rare type of cleft in the presumptive hard palate coinciding with the domain of Shox2 expression in the wild-type anterior palate (Yu et al. 2005). Notably, an identical expression pattern exhibited by human SHOX2 in the anterior palate makes it a candidate gene for this rare type of cleft. These data imply that fusion in the anterior and posterior parts of the palate probably proceeds by discrete molecular mechanisms. In humans and rodents, closure of the palate begins medially at the earliest sites of contact, proceeding both anterior and posterior from it until fusion is complete along its entire length (Francis-West et al. 2003). This sequence of palatal closure could be the result of regionally distinct signalling pathways being activated along the anterior–posterior (A–P) axis. The existence of heterogeneity in the developing palate became evident from regional differences in the morphology of the palate epithelium. The palate develops pseudostratified, ciliated, columnar epithelia on its nasal aspect and stratified non-keratinizing, squamous epithelia on its oral side (Ferguson, 1988). Cuervo et al. (2002) found that apoptosis of MEE cells in the anterior palate is triggered by contact, whereas in the posterior apoptosis occurs prior to any contact between the opposing shelves. Early tissue recombination studies demonstrated that signals originating in the mesenchyme confer different fates to the palatal epithelium (Ferguson & Honig, 1984). We now realize that these gross morphological differences could be the result of molecular heterogeneity established early along the different axes (A–P and medial–lateral) of the developing palate. This notion is fast gaining acceptance as an increasing number of studies show regionally restricted expression of signalling pathway components all with relevance to growth and patterning events in

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Fig. 2 Spatially restricted gene expression in the developing palate in mice. (A) Shox2 shows strong localization to the anterior palate at E13.5. (B) Msx1 is expressed in the anterior palate mesenchyme at E12.5. (C) No Msx1 expression is detected in the posterior palate mesenchyme of the same stage. (D) Mesenchymal expression of Fgf10 in the anterior palate at E12.5. (E) Fgf10 is absent in the posterior palate at E12.5. Abbreviations: md, mandible; ns, nasal septum; ps, palatal shelf; T, tongue; tg, tooth germ.

Fig. 3 The anterior and posterior palatal mesenchyme responds differently to diffusible growth factors in vitro. (A) Bmp4-soaked beads can induce Msx1 expression in the anterior palatal mesenchyme from E12.5. (B) Bmp4soaked beads fails to induce Msx1 expression in the posterior palatal mesenchyme from E12.5. (C) Fgf8soaked beads cannot induce Pax9 in the anterior palatal mesenchyme from E12.5. (D) Fgf8-soaked beads induce Pax9 expression in the posterior palatal mesenchyme. (E,F) Exogenous Bmp-4 induces cell proliferation in the anterior palatal explants from E12.5 (E) but not the posterior palate (F). Arrows in A and D indicate gene expression and arrows in E and F show positive and weak BrdU labelling, respectively.

the palatal shelves (Zhang et al. 2002; Herr et al. 2003; Alappat et al. 2005; Cui et al. 2005). The existence of intrinsic molecular differences between the anterior and posterior palate is best exemplified by gene expression and regulation studies in mice (Figs 2 and 3). During early outgrowth stages (E12.5–E13.5) of the palate, the expression of Msx1, Bmp4 and Bmp2 is restricted to the anterior palate (Zhang et al. 2002).

Notably, all of them are absent from the posterior palate. Although delimited to the anterior palate, they all show characteristic spatial–temporal localization. At E12.5 Bmp4 and Bmp2 are expressed in the epithelium and mesenchyme of the palate; by E13.5 whereas expression of Bmp2 remains unaltered, that of Bmp4 is lost from the epithelium and is found only in the anterior mesenchyme subjacent to the MEE. Msx1 expression

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Fig. 4 Expression of Fgfr2 in E13.5 palatal shelf. (A) Fgfr2 is expressed in the epithelium of the anterior palate, including the MEE (arrow), but is absent from the palatal mesenchyme. (B,C) Fgfr2 is expressed in the epithelium including the MEE (black arrows) and the mesenchyme of the middle (B) and posterior (C) portions of the palate. Note the restricted expression in the medial (nasal) half of the palatal mesenchyme (red arrows).

is strictly mesenchymal at E12.5 (Fig. 2B,C) and E13.5. Unlike Bmp4, Bmp2 and Msx1, the expression of Shh is seen in the anterior and posterior palate, but in unique regions. Surprisingly, the localization of Shh to the MEE is unique to the anterior palate at E12.5 and E13.5. In the posterior, Shh transcripts mark the presumptive rugae epithelia (oral epithelia) at E13.5. Joining the repertoire of anterior-specific factors are Fgf10 and Shox2 (Alappat et al. 2005; Yu et al. 2005; Fig. 2A,D,E). Fgf10 shows robust expression in the anterior mesenchyme from E11.5 to E13.5 of palate development with little to no expression in the posterior palate (Alappat et al. 2005; see Fig. 2D,E). Similarly, the posterior-most limit of Shox2 expression coincides with the level of the first molar tooth germ in mice between E11.5 and E14.5 (Yu et al. 2005; Fig. 2A). Again, in the anterior region, Fgfr2 transcripts are restricted to the entire palatal epithelium, with no expression detected in the palatal mesenchyme (Fig. 4A). By contrast, in the middle and posterior regions, Fgfr2 is expressed in the palatal epithelium, including the MEE and the mesenchyme (Fig. 4B,C). It is interesting to note that the mesenchymal Fgfr2 expression is restricted to the medial half of the palatal shelf, suggesting the existence of mesenchymal heterogeneity between the medial (nasal) and lateral (oral) halves of the palate as well. Unfortunately, compared with the anterior there is precious little known of posterior-specific gene markers of the palate. Tbx22, the mouse orthologue of the human X-linked cleft palate gene, shows restricted expression in the posterior palatal shelf before fusion (Braybrook et al. 2001; Herr et al. 2003). This regionally distinct localization of Tbx22 transcripts could be a testament of innate genetic differences in the anterior and posterior palate. However, an analysis of the expression of mouse Tbx22 by another group makes no mention of its restriction to the posterior palate (Bush et al. 2002).

Regional regulation and function of genes along the A–P axis of the developing palate As is common to other organs, the development of the palate involves reciprocal signalling between the epithelium and the mesenchyme (Ferguson & Honig, 1984; Slavkin et al. 1984). Regionalized regulation of growth and patterning by such interactions is gaining acceptance as a developmental strategy used during palatogenesis in mice. It has been shown that the anterior and posterior palatal mesenchyme explants respond very differently in terms of gene expression and cellular behaviour to diffusible growth factors. Application of Bmp4 induces Msx1 and Bmp4 in the E12.5 anterior palatal mesenchyme but it fails to do so in the posterior palatal mesenchyme (Zhang et al. 2002; Fig. 3A,B). Similarly, the anterior palatal mesenchyme but not the posterior mesenchyme responds to exogenous Shh by the expression of Bmp2. This induction of Bmp2 is independent of Bmp4 and Msx1 expression. BrdU analyses revealed that Shh, Bmp2 and Bmp4 exert mitogenic influences strictly on the anterior palate mesenchyme explants (Zhang et al. 2002; this paper, Fig. 3E,F). The mitogenic activity of epithelial Shh is mediated by mesenchymally expressed Bmp2 in the palate. Notably, Bmp4 and Shh do not exert the same effect on the posterior palatal mesenchyme. Explants of the posterior palatal mesenchyme instead respond to Fgf8 induction by expressing Pax9 as in the dental mesenchyme (Fig. 3C,D). The endogenous inducer of Pax9 expression in the palate is yet to be identified. A recent study demonstrates that the palatal epithelium from the anterior domain can induce ectopic Shox2 expression in the posterior palatal mesenchyme (Yu et al. 2005). Among the signalling factors present in the anterior palatal epithelium, Bmp activity is necessary but not sufficient for the induction of Shox2 expression in the palatal mesenchyme. Thus, there are clear differences in inductive and

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competent potentials between the anterior and posterior palate. The heterogeneity along the A–P axis of the palatal shelves is established apparently early in palatogenesis and exists in both the palatal epithelium and the mesenchyme. Examination of complex epithelial–mesenchymal interactions in the developing secondary palate continues to unravel the aetiology of clefts. Notably, the complete cleft seen in the Msx1 null mutant mice stems from a proliferation defect in the anterior of the palate (Zhang et al. 2002). Msx1 is localized to the anterior palatal mesenchyme. In wild-type mice, an autoregulatory loop involving Msx1 and Bmp4 induces the expression of Shh in the MEE of that region. Epithelial Shh induces Bmp2 in the underlying anterior mesenchyme and together they regulate cellular proliferation of the anterior palate. Targeted disruption of Msx1 results in a loss of Bmp4, Bmp2 and Shh, leading to a significantly reduced level of cell proliferation in the anterior palate. Cell proliferation in the posterior palate is unaffected in these mutants. So although the palatal shelves in the Msx1–/– mice elevate normally they fail to make contact in the anterior, leading to a complete cleft phenotype. Furthermore, the ectopic expression of Bmp4, the downstream target of Msx1, in a pattern reminiscent of its endogenous expression not only restores proliferation in the anterior palatal mesenchyme but also allows fusion of the palatal shelves to occur normally. Similarly, Fgf10 expression in the anterior mesenchyme acts in parallel to the Bmp4–Msx1 pathway, inducing Shh expression and cell proliferation, and supporting cell survival in the anterior palatal epithelium and/or mesenchyme (Rice et al. 2004; Alappat et al. 2005). There is some speculation that the delimitation of Shh expression to the anterior palatal epithelium may be one component that establishes early A–P polarity in the palate (Rice et al. 2004). A combination of diminished proliferation of the maxillary mesenchyme and defective patterning along the A–P axis results in a complete cleft palate in the Bmpr1A conditional null mutant mice (Liu et al. 2005). It was demonstrated that Bmp signalling via Bmpr1A is essential for normal palatal growth and highly regulated spatial–temporal expression of Barx1 and Pax9 required for the fusion of the palatal shelves. These studies demonstrate that defective growth in the anterior palate causes complete cleft palate, and support the model that palatal closure follows an anterior-toposterior direction. However, in a recent study, it was

shown that mice lacking Shox2 have an incomplete cleft within the anterior palate, while the posterior palate including the soft palate closes and fuses normally (Yu et al. 2005). BrdU labelling experiments have revealed substantially lower levels of cell proliferation in the anterior palate where Shox2 is normally expressed. Cell proliferation remains unaltered in the posterior palate. Although the defective cell proliferation pattern in the developing palate of the Shox2 mutant is similar to that in the Msx1 mutant, the cleft palate phenotype observed is different. The phenotype seen in Shox2 mutant mice provides the first genetic evidence in support of the idea that the posterior palate can fuse independent of fusion of the anterior palate. Thus, a revision of the prevailing model on the palatal closure sequence might be necessary. In humans and mice, Tgfβ3 function is essential to achieve successful fusion of the palatal shelves (Kaartinen et al. 1995, 1997; Proetzel et al. 1995; Lidral et al. 1998; Taya et al. 1999; Martínez-Álvarez et al. 2000). Of the Tgf-β type I receptors, Alk-1, Alk-2 and Alk-5 are expressed in the palatal epithelium and mesenchyme (Dudas et al. 2004). Curiously, in the posterior palate, the expression of Alk-5, the main transducer of Tgfβ signals, is absent from the medial edge epithelium (before fusion) and the medial epithelial seam (during fusion). Constitutive activation of Alk-5 in the epithelium of Tgfβ3–/– palate explants achieves rescue of the fusion defect by a Smad2-dependent pathway. In a corollary approach, inactivation of Alk-5 in wild-type palate explants, using a specific inhibitor or a dominant negative approach, impedes fusion of the shelves only in the anterior wild-type palate (Dudas et al. 2004). As would be expected from the endogenous expression pattern of Alk-5 in the palate, the Alk-5 inhibitor does not prevent fusion in the posterior region. Taking these data together, Tgf-β3 signals transduced by Alk-5/Smad2 are essential for fusion of the anterior palate. In addition, Smad2 activation in the anterior and posterior palate appears to involve different mechanisms. Activation of Alk-5 at E13.5 rescues the fusion defect in the anterior and posterior regions but by E14.0 rescue is more pronounced in the posterior palate of the Tgfβ3–/– mice (Dudas et al. 2004). Such difference in the degree of rescue achieved expounds the normally occurring direction of palate maturation and fusion. Smad2 over-expression achieves a near complete rescue of the cleft palate phenotype in Tgfβ3 null mutant mice (Cui et al. 2005). The minor persistence of clefts in the anterior- and

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posterior-most regions of the secondary palatal shelves in these mutants is attributed to differences that are intrinsic to the anterior and posterior regions of the palate. Thus, the functionality of Tgf-β3 signalling is believed to extend beyond the activation of Smad2 in these regions. In addition, there may be other signalling pathways that complement Tgf-β3 function in the regions in question. By contrast, there are genes that function in medial– lateral patterning of the palate such as odd skippedrelated genes, Osr2 and Osr1 (Lan et al. 2004). Osr2 is expressed throughout the A–P extent of the palate mesenchyme with a lateral to medial gradient of expression. Unlike Msx1 null mutants, growth retardation in the palate of Osr2–/– mice occurs along the entire A–P axis. Growth impairment in the absence of Osr2 gene function is more pronounced in the medial aspect of the palate and is accompanied by the down-regulation of Pax9, misexpression of Osr1 and loss of Tgfb3 gene expression. Osr2 has been implicated in medial–lateral patterning events and elevation of the palatal shelves. All such reports suggest that regionally controlled growth and patterning events ensure the success of palatogenesis concomitant with extensive growth and morphogenetic events of the craniofacial complex as a whole.

Conclusion A thorough understanding of cleft palate aetiology warrants careful consideration of the dynamic heterogeneity of gene expression and function along the different axes of the developing palate. The heterogeneity in the developing palate may well be the underpinning that guides the mechanics of palatal shelf growth and fusion.

Acknowledgements The work described in this review from the authors’ laboratory was supported by grants from the NIH (R01DE12329, R01DE14044, R01DE15123, P60DE13076) and the Wall Fund of Tulane University.

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