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EVOLUTIONARY HOMOLOGS

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FGF and head and skull development

LIM-homeobox containing (Lhx) genes encode transcriptional regulators that play critical roles in a variety of developmental processes. Two identifed genes belong to a novel subfamily of mammalian Lhx genes, designated Lhx6 and Lhx7. The overall similarity between Lhx6 and Lhx7 is 75%, with the homeodomain and the LIM domains being the most conserved regions (95% and 74% identity, respectively. Sequence comparison of the Lhx6 and Lhx7 homeodomains to those of other LIM homeodomain proteins shows that they are more similar to each other than to other members of this family, indicating that they constitute a novel subfamily of the Lhx protein family. Whole-mount in situ hybridization shows that Lhx6 and Lhx7 are expressed during mouse embryogenesis in overlapping domains of the first branchial arch and the basal forebrain. More specifically, expression of Lhx6 and Lhx7 is detected prior to initiation of tooth formation in the presumptive oral and odontogenic mesenchyme of the maxillary and mandibular processes. During tooth formation, expression is restricted to the mesenchyme of individual teeth. Using explant cultures, expression of Lhx6 and Lhx7 in mandibular mesenchyme is under the control of signals derived from the overlying epithelium; such signals are absent from the epithelium of the non-odontogenic second branchial arch. Expression studies and bead implantation experiments in vitro provide strong evidence that Fgf8 is primarily responsible for the restricted expression of Lhx6 and Lhx7 in the oral aspect of the maxillary and mandibular processes. In the telencephalon, expression of both genes is predominantly localized in the developing medial ganglionic eminences, flanking a Fgf8-positive midline region. It is suggested that Fgf8, Lhx6 and Lhx7 are all key components of signaling cascades that determine morphogenesis and differentiation in the first branchial arch and the basal forebrain (Grigoriou, 1998).

The FGFR2 gene is of particular interest in the context of craniofacial development. Dominantly acting missense mutations located mainly in the IgIIIa/IIIc domain or in the IgII/IgIII linker region are associated with a variety of craniosynostosis syndromes. Common to all phenotypes is the consistency with which the coronal suture, as compared to other sutures, shows premature fusion. Some of these syndromes, as well as non-syndromic coronal craniosynostosis, have also been found to be associated with equivalent mutations of FGFR1 or FGFR3. Abnormal FGFR signaling has also been implicated in the coronal craniosynostosis seen in Saethre-Chotzen syndrome, which is due to a mutation of TWIST: the TWIST gene product is a transcription factor that appears to be a prerequisite for FGFR signaling during mesoderm formation, required for the expression of FGFRs. Many of these FGFR-related craniosynostosis syndromes additionally show abnormalities of the digits of the hands and feet, but achondroplasia and related syndromes affecting the growth plates of long-bones have to date only been associated with mutation of FGFR3 (Iseki, 1997 and references).

In the fetal mouse skull vault, Fgfr2 transcripts are most abundant at the periphery of the membrane bones; they are mutually exclusive with those of osteopontin (an early marker of osteogenic differentiation) but coincide with sites of rapid cell proliferation. Fibroblast growth factor type 2 (FGF2) protein, which has a high affinity for the FGFR2 splice variant associated with craniosynostosis, is locally abundant; but it is present at low levels in Fgfr2 expression domains and at high levels in adjacent differentiated areas. Implantation of FGF2-soaked beads onto the fetal coronal suture by ex utero surgery results in ectopic osteopontin expression, encircled by Fgfr2 expression, after 48 hours. It is suggested that increased FGF/FGFR signaling in the developing skull, whether due to FGFR2 mutation or to ectopic FGF2, shifts the cell proliferation/differentiation balance towards differentiation by enhancing the normal paracrine down-regulation of Fgfr2. In this model, a minor shift in the timing of the proliferation to diffentiation switch (due to loss of FGFR function or heightened levels of FGF) would favor differentiation and would result in the premature loss of the stem cell population and the induction of abnormal coronal suture (Iseki, 1997).

The mesenchymal cells that contribute to oral and facial hard tissues are derived from cranial neural crest cells, whereas limb mesenchyme cells are derived from axial mesoderm. The outgrowth of facial processes has been compared with limb bud outgrowth; the tooth bud enamel knot has been identified as a signaling center with similarities to both the limb ZPA and AER signaling centers. In addition, several homeobox-containing genes have been implicated in both branchial arch and limb development, such as members of the Msx, Dlx and Lim-homeobox families. The expression of the Lim-domain gene Lhx-6, and its closely associated family member Lhx-7 are largely restricted to anterior mesenchymal cells of the mandibular and maxillary arches. Clim-2 (NLI, Lbd1) is one of two related mouse proteins that interact with Lim-domain homeoproteins. In the mouse, embryonic expression of Clim-2 is particularly pronounced in facial ectomesenchyme and limb bud mesenchyme in association with Lim genes Lhx-6 and Lmx-1, respectively. In common with both these Lim genes, Clim-2 expression is regulated by signals from overlying epithelium. In both the developing face and the limb buds Fgf-8 is identified as the likely candidate signaling molecule that regulates Clim-2 expression. In the mandibular arch, as in the limb, Fgf-8 functions in combination with CD44, a cell surface binding protein that has been considered to be a hyaluronan receptor. Blocking CD44 binding results in inhibition of Fgf8-induced expression of Clim-2 and Lhx-6. CD44 has been shown to be required for presentation of Fgf-8 to its receptor, rather than as a hyaluronan receptor. Regulation of gene expression by Fgf8 in association with CD44 is thus conserved between limb and mandibular arch development (Tucker, 1999b).

The roles of the BMP antagonists Chordin and Noggin in development of the mandible, which is derived from the first branchial arch (BA1) were examined. Both genes are expressed in the pharynx during early mandibular outgrowth and later in the mandibular process. Mice mutant for either Nog or Chd have only mild mandibular defects; however, double mutant pups exhibit a range of mandibular truncation phenotypes, from normal to agnathia. A few embryos homozygous null for both genes survive to late gestation; many are agnathic, though a few have significant mandibular outgrowth. In mandibular explants, ectopic BMP4 rapidly induces expression of both Chd and Nog, consistent with results obtained in vivo with mutant embryos. FGF8 is a survival factor for cells populating the mandibular bud. Excess BMP4 represses Fgf8 transcription in mandibular explants. Embryos lacking these BMP antagonists often show a strong reduction in Fgf8 expression in the pharyngeal ectoderm, and increased cell death in the mandibular bud. It is suggested that the variable mandibular hypoplasia in double mutants involves increased BMP activity downregulating Fgf8 expression in the pharynx, decreasing cell survival during mandibular outgrowth (Stottmann, 2001).

Fgf8 is required for normal development of the nasal region. Here, a candidate approach has been used to identify genes that are induced in chick nasal mesenchyme in response to FGF signaling. Using an explant culture system, expression of the transcription factors Tbx2, Erm, Pea3, and Pax3, but not Pax7, in nasal mesenchyme has been shown to be regulated by ectodermal signals in a stage-dependent manner. Using beads soaked in recombinant FGF protein and an FGF receptor antagonist, it has been demonstrated that FGF signaling is necessary and sufficient for expression of Tbx2, Erm, Pea3, and Pax3, but that it has no effect on Pax7 expression. Within the nasal mesenchyme, competence to respond to FGF signaling is initially widespread and uniform but becomes restricted to regions normally exposed to FGF at later stages of development, coincident with changes in FGF receptor expression. Finally, evidence is provided that FGF8 also regulates Erm and Pea3 expression in the nasal placodes. Together, these results identify Tbx2, Erm, Pea3, and Pax3 as downstream targets of FGF signaling in the facial area and suggest that these genes may mediate some of the effects of FGF8 during development of the nasal region (Firnberg, 2002).

Normal growth and morphogenesis of the cranial vault reflect a balance between cell proliferation in the sutures and osteogenesis at the margins of the cranial bones. In the clinical condition craniosynostosis, the sutures fuse prematurely as a result of precocious osteogenic differentiation and craniofacial malformation results. Mutations in several fibroblast growth factor receptor (FGFR) genes have now been identified as being responsible for the major craniosynostotic syndromes. A grafting technique was used to manipulate the levels of endogenous FGF-2 ligand in embryonic chick cranial vaults and thereby perturb morphogenesis. Implantation of beads loaded with FGF-2 does not affect normal cranial development at physiological concentrations, although they elicit a morphogenetic response in the limb. Implantation of beads loaded with a neutralizing antibody to FGF-2 generates a concentration-dependent response. When a single bead was implanted, the grafts grew to a massive size as a result of increased cell division in the tissue. With greater inactivation of FGF-2 protein (two to three beads implanted), all further bone differentiation and cell proliferation is blocked. These data further support the emerging idea that the intensity of FGF-mediated signaling determines the developmental fate of the skeletogenic cells in the cranial vault. High and low levels correlate with differentiation and proliferation, respectively. A balance between the two ensures normal cranial vault morphogenesis. This is consistent with the observation that several FGFR mutations causing craniosynostosis result in constitutive activation of the receptor (Moore, 2002).

FGFs and tooth morphogenesis

In mammals, rostral ectomesenchyme cells of the mandibular arch (first brachial arch) give rise to odontogenic cells, while more caudal cells form the distal skeletal elements of the lower jaw. Neural crest-derived mesenchyme from sources outside the mandibular arch is capable of forming teeth but only when recombined with early rostral (oral) epithelium from the first branchial arch. Signals from the epithelium are required for the development of odontogenic and skeletogenic mesenchyme cells. Rostral-caudal polarity is first established in mandibular branchial arch ectomesenchymal cells by a signal, Fgf-8, from the rostral epithelium. All neural crest-derived ectomesenchymal cells are equicompetent to respond to Fgf-8. The restriction into rostral (Lhx-7-expressing) and caudal (Gsc-expressing) domains is achieved by cells responding differently according to their proximity to the source of the signal. Once established, spatial expression domains and cell fates are fixed and maintained by Fgf-8 in conjunction with another epithelial signal, endothelin-1, and by positional changes in ectomesenchymal cell competence to respond to the signal. Fgf-8 is not the only signaling molecule present in oral epithelium. Bmp-4 and Shh are both expressed early in oral epithelium in regions that overlap those of Fgf-8. Bmp-4 is involved in upreglating several different ectomesenchymal genes including Msx-1. Bmp-4 has been suggested to act antagonistically with Fgf-8 to produce localized sites of ectomesenchyme that express Pax-9 and specify where teeth will develop. Shh is expressed in oral epithelium and subsequently localized to very small sites that correspond to presumptive dental epithelium. Given that different signals are present in oral epithelium it is not surprising that a single signal, Fgf-8, is unable to convert non-odontogenic tissue, such as caudal (aboral) mesenchme to an odontogenic fate (Tucker, 1999a).

There has been rapid progress recently in the identification of signaling pathways regulating tooth development. It has become apparent that signaling networks involved in Drosophila development and the development of structures such as limbs are also used in tooth development. Teeth are epithelial appendages formed in the oral region of vertebrates; their early developmental anatomy resembles that of other strucures, such as hairs and glands. The neural crest origin of tooth mesenchyme has been confirmed and recent evidence suggests that specific combinations of homeobox genes expressed in the neural crest cells may regulate the types of teeth and their patterning. Signaling molecules in the Shh, FGF, BMP and Wnt families appear to regulate the early steps of tooth morphogenesis. Certain transcription factors associated with these pathways have been shown to be necessary for tooth development. BMP-2 and BMP-4 as well as BMP-7 are expressed in the early dental epithelium: interestingly, the expression of BMP-4 shifts to the mesenchyme at the time when the instructive capacity shifts from the epithelium. BMP2 and BMP-4 stimulate expression of the homeobox-containing transcription factors MSX1 and MSX-2. FGF-3 expression is confined to dental papilla mesenchyme and is downregulated as morphogenesis advances. FGF-4, FGF-8 and FGF-9 are expressed exclusively in dental epithelial cells. Their respective receptors are present in both epithelial and mesenchymal tissues in the tooth. The FGF's also use cell surface heparan sulfate proteoglycans as receptors. Msx-1 also appears to participate in the FGF signaling pathway. Several FGFs upregulate Msx-1 expression in the dental mesenchyme when applied in vitro. FGFs are potent stimulators of cell proliferation: they stimulate cell division both in dental mesenchyme and epithelium at several stages in tooth morphogenesis. Several of the conserved signals are also transiently expressed in the enamel knots in the dental epithelium. The enamel knots are associated with the characteristic epithelial folding morphogenesis, which is responsible for the development of tooth shape. It is currently believed that the enamel knots function as signaling centers, regulating the development of tooth shape. Enamel knots constitute a specific ectodermal cell lineage; it has been proposed that enamel knots determine the site of the first cusp of teeth and that they regulate the formation of other cusps in molar teeth (Thesleff, 1997).

To elucidate the roles of fibroblast growth factors (FGF) in the regulation of tooth morphogenesis the expression patterns of Fgf-4, -8, and -9 have been analyzed by insitu hybridization in the developing mouse molar and incisor tooth germs from initiation to completion of morphogenesis. The expression of these Fgfs is confined to dental epithelial cells at stages when epithelial-mesenchymal signaling regulates critical steps of tooth morphogenesis. Fgf-8 and Fgf-9 mRNAs are present in the oral epithelium of the first branchial arch at E10. One day later, expression becomes more restricted to the area of presumptive dental epithelium and persists there until the start of epithelial budding. Fgf-8 mRNAs are not detected later in the developing tooth. Fgf-4 and Fgf-9 expression is upregulated in the primary enamel knot, a putative signaling center regulating tooth shape. Subsequently, Fgf-4 and Fgf-9 are expressed in the secondary enamel knots at the sites of tooth cusps. Fgf-9 expression spreads from the primary enamel knot within the inner enamel epithelium where it remains until E18. In the continuously growing incisors Fgf-9 expression persists in the epithelium of the cervical loops. The effects of FGFs were analyzed on the expression of the homeobox-containing transcription factors Msx-1 and Msx-2, which are associated with tissue interactions and regulated by the dental epithelium. Locally applied FGF-4, -8, and -9 stimulates intensely the expression of Msx-1 but not Msx-2 in the isolated dental mesenchyme. It is suggested that the three FGFs act as epithelial signals mediating inductive interactions between dental epithelium and mesenchyme during several successive stages of tooth formation. This data suggest roles for FGF-8 and FGF-9 during initiation of tooth development, and for FGF-4 and FGF-9 during regulation of tooth shape. FGF-9 may also be involved in differentiation of odontoblasts. The coexpression of Fgfs with other signaling molecules including Shh and several Bmps and their partly similar effects suggest that the FGFs participate in the signaling networks during odontogenesis (Kettunen, 1998).

During early tooth development, multiple signaling molecules are expressed in the dental lamina epithelium; these molecular signals induce the dental mesenchyme. One signal, BMP4, has been shown to induce morphologic changes in dental mesenchyme and mesenchymal gene expression via Msx1, but BMP4 cannot substitute for all the inductive functions of the dental epithelium. To investigate the role of FGFs during early tooth development, the expression of epithelial and mesenchymal Fgfs was examined in wild-type and Msx1 mutant tooth germs and the ability of FGFs to induce Fgf3 and Bmp4 expression was tested in wild-type and Msx1 mutant dental mesenchymal explants. Fgf8 expression is preserved in Msx1 mutant epithelium while that of Fgf3 is not detected in Msx1 mutant dental mesenchyme. Moreover, dental epithelium as well as beads soaked in FGF1, FGF2 or FGF8 induce Fgf3 expression in dental mesenchyme in an Msx1-dependent manner. These results indicate that, like BMP4, FGF8 constitutes an epithelial inductive signal capable of inducing the expression of downstream signaling molecules in dental mesenchyme via Msx1. However, the BMP4 and FGF8 signaling pathways are distinct. BMP4 cannot induce Fgf3 nor can FGFs induce Bmp4 expression in dental mesenchyme, even though both signaling molecules can induce Msx1 and Msx1 is necessary for Fgf3 and Bmp4 expression in dental mesenchyme. In addition, the effects of FGFs and BMP4 were investigated on the distal-less homeobox genes Dlx1 and Dlx2 and the relationship between Msx and Dlx gene function was clarified in the developing tooth. Dlx1,Dlx2 double mutants exhibit a lamina stage arrest in maxillary molar tooth development. Although the maintenance of molar mesenchymal Dlx2 expression at the bud stage is Msx1-dependent, both the maintenance of Dlx1 expression and the initial activation of mesenchymal Dlx1 and Dlx2 expression during the lamina stage are not. Moreover, in contrast to the tooth bud stage arrest observed in Msx1 mutants, Msx1,Msx2 double mutants exhibit an earlier phenotype closely resembling the lamina stage arrest observed in Dlx1,Dlx2 double mutants. These results are consistent with functional redundancy between Msx1 and Msx2 in dental mesenchyme and support a model whereby Msx and Dlx genes function in parallel within the dental mesenchyme during tooth initiation. Indeed, as predicted by such a model, BMP4 and FGF8, epithelial signals that induce differential Msx1 and Msx2 expression in dental mesenchyme, also differentially induce Dlx1 and Dlx2 expression, and do so in an Msx1-independent manner. These results integrate Dlx1, Dlx2 and Fgf3 and Fgf8 into the odontogenic regulatory hierarchy along with Msx1, Msx2 and Bmp4, and provide a basis for interpreting tooth induction in terms of transcription factors which, individually, are necessary but not sufficient for the expression of downstream signals and therefore must act in specific combinations (Bei, 1998).

Members of the Pitx/RIEG family of homeodomain-containing transcription factors have been implicated in vertebrate organogenesis. In this study, the expression and regulation of Pitx1 and Pitx2 during mouse tooth development was examined. Pitx1 expression is detected in early development in a widespread pattern, in both epithelium and mesenchyme, covering the tooth-forming region in the mandible, and is then maintained in the dental epithelium from the bud stage to the late bell stage. Pitx2 expression, on the other hand, is restricted to the dental epithelium throughout odontogenesis. Interestingly, from E9.5 to E10.5, the expression domains of Pitx1 and Pitx2, in the developing mandible, overlap with that of Fgf8 but are exclusive to the zone of Bmp4 expression. Bead implantation experiments demonstrate that ectopic expression of Fgf8 can induce/maintain the expression of both Pitx1 and Pitx2 at E9.5. In contrast, Bmp4-expressing tissues and BMP4-soaked beads are able to repress Pitx1 expression in mandibular mesenchyme and Pitx2 expression in the presumptive dental epithelium, respectively. However, the effects of FGF8 and BMP4 are transient. It thus appears that the early expression patterns of Pitx1 and Pitx2 in the developing mandible are regulated by the antagonistic effects of FGF8 and BMP4 such that the Pitx1 and Pitx2 expression patterns are defined. These results indicate that the epithelial-derived signaling molecules are responsible not only for restricting specific gene expression in the dental mesenchyme, but also for defining gene expression in the dental epithelium (St.Amand, 2000).

Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in the cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult mouse incisor stem cells. To illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, incisor development was analyzed in Fgf10-deficient mice, and the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures was analyzed. The incisor germs of FGF10-null mice proceeds to cap stage normally. However, at a later stage, the cervical loop is not formed. The absence of the cervical loop is due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, the growth of dental epithelium from incisor explants of FGF10-null mice was estimated by organ culture. The dental epithelium of FGF10-null mice shows limited growth, although the epithelium of wild-type mice appears to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induces apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescues the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs (Harada, 2002).

Teeth develop as epithelial appendages, and their morphogenesis is regulated by epithelial-mesenchymal interactions and conserved signaling pathways common to many developmental processes. A key event during tooth morphogenesis is the transition from bud to cap stage, when the epithelial bud is divided into specific compartments distinguished by morphology as well as gene expression patterns. The enamel knot, a signaling center, forms and regulates the shape and size of the tooth. Mesenchymal signals are necessary for epithelial patterning and for the formation and maintenance of the epithelial compartments. The expression of Notch pathway molecules was studied during the bud-to-cap stage transition of the developing mouse tooth. Lunatic fringe expression is restricted to the epithelium, where it forms a boundary flanking the enamel knot. The Lunatic fringe expression domains overlapped only partly with the expression of Notch1 and Notch2, which are coexpressed with Hes1. The regulation of Lunatic fringe and Hes1 was examined in cultured explants of dental epithelium. The expression of Lunatic fringe and Hes1 depend on mesenchymal signals and both are positively regulated by FGF-10. BMP-4 antagonizes the stimulatory effect of FGF-10 on Lunatic fringe expression but has a synergistic effect with FGF-10 on Hes1 expression. Recombinant Lunatic fringe protein induced Hes1 expression in the dental epithelium, suggesting that Lunatic fringe can act also extracellularly. Lunatic fringe mutant mice do not reveal tooth abnormalities, and no changes were observed in the expression patterns of other Fringe genes. It is concluded that Lunatic fringe may play a role in boundary formation of the enamel knot and that Notch-signaling in the dental epithelium is regulated by mesenchymal FGFs and BMP (Mustonen, 2002).

Fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition were investigated with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. The tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocks dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remains intact as judged by normal pitx2 expression, not only is the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 is as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. These results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme (Jackman, 2004).

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