Table of contents

DPP homologs and morphogenesis: Facial patterning

The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. Expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2 (Drosophila homologs: Muscle segment homeobox); ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. This activation of gene expression is correlated with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel's cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of Sonic Hedgehog and bifurcation of the palatine bone. Application of BMP-2 can increase cell proliferation of the mandibular primordia. This work suggests that BMP-2 and BMP-4 are part of a signaling cascade that controls outgrowth and patterning of the facial primordia (Barlow, 1997).

Growth factor-mediated signaling has been implicated in the regulation of epithelial-mesenchymal interactions during organogenesis. Bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta superfamily, is expressed in the presumptive dental epithelium at the initiation of tooth development. Subsequently, epithelial signaling leads to mesenchymal induction of BMP-4 expression. To address the role of this factor, BMP-4-releasing agarose beads were added to dental mesenchyme in culture. These beads induce a translucent mesenchymal zone similar to that induced by dental epithelium. Three transcription factors (Msx-1, Msx-2, and Egr-1) whose expression is governed by epithelial signaling are induced in response to BMP-4. BMP-4 also induces its own mesenchymal expression. These findings support the hypothesis that BMP-4 mediates epithelial-mesenchymal interactions during early tooth development (Vainio, 1993).

Pax9 is a marker for prospective tooth mesenchyme prior to the first morphological manifestation of odontogenesis. The sites of Pax9 expression in the mandibular arch are positioned by the combined activity of two signals, one (FGF8) that induces Pax9 expression and the other (BMP2 and BMP4) that prevents this induction. Thus it appears that the position of the teeth is determined by a combination of two different types of signaling molecules produced in wide but overlapping domains rather than by a single localized inducer. It is suggested that a similar mechanism may be used for specifying the sites of development of other organs. For example, BMP2 and BMP4 can antagonize FGF function in the developing mouse limb bud (Neubuser, 1997).

Growth factor-mediated signaling has been implicated in the regulation of epithelial-mesenchymal interactions during organogenesis. Bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta superfamily, is expressed in the presumptive dental epithelium at the initiation of tooth development. Subsequently, epithelial signaling leads to mesenchymal induction of BMP-4 expression. To address the role of this factor, BMP-4-releasing agarose beads were added to dental mesenchyme in culture. These beads induce a translucent mesenchymal zone similar to that induced by dental epithelium. Three transcription factors (Msx-1, Msx-2, and Egr-1) whose expression is governed by epithelial signaling are induced in response to BMP-4. BMP-4 also induces its own mesenchymal expression. These findings support the hypothesis that BMP-4 mediates epithelial-mesenchymal interactions during early tooth development (Vainio, 1993).

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).

The enamel knot, a transient epithelial structure, appears at the onset of mammalian tooth shape development. Until now, the morphological, cellular and molecular events leading to the formation and disappearance of the enamel knot have not been described. The cessation of cell proliferation in the enamel knot in mouse molar teeth is linked with the expression of the cyclin-dependent kinase inhibitor p21. p21 expression is induced by bone morphogenetic protein 4 (BMP-4) in isolated dental epithelia. Since BMP-4 is expressed only in the underlying dental mesenchyme at the onset of the enamel knot formation, these results support the role of the cyclin-dependent kinase inhibitors as inducible cell differentiation factors in epithelial-mesenchymal interactions. The expression of p21 in the enamel knot is followed by BMP-4 expression, and subsequently by apoptosis of the differentiated enamel knot cells. Three-dimensional reconstructions of serial sections after in situ hybridization and Tunel-staining indicate an exact codistribution of BMP-4 transcripts and apoptotic cells. Apoptosis is stimulated by BMP-4 in isolated dental epithelia, but only in one third of the explants. It is concluded that BMP-4 may be involved both in the induction of the epithelial enamel knot, as a mesenchymal inducer of epithelial cyclin-dependent kinase inhibitors, and later in the termination of the enamel knot signaling functions by participating in the regulation of programmed cell death. These results show that the life history of the enamel knot is intimately linked to the initiation of tooth shape development and support the role of the enamel knot as an embryonic signaling center (Jernvall, 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).

The development of many organs depends on sequential epithelial-mesenchymal interactions, and the developing tooth germ provides a powerful model for elucidating the nature of these inductive tissue interactions. In Msx1-deficient mice, tooth development arrests at the bud stage when Msx1 is required for the expression of Bmp4 and Fgf3 in the dental mesenchyme. To define the tissue requirements for Msx1 function, tissue recombinations were performed between wild-type and Msx1 mutant dental epithelium and mesenchyme. Through the E14.5 cap stage of tooth development, Msx1 is required in the dental mesenchyme for tooth formation. After the cap stage, however, tooth development becomes Msx1 independent, although there is a further late function of Msx1 in odontoblast and dental pulp survival. These results suggest that prior to the cap stage, the dental epithelium receives an Msx1-dependent signal from the dental mesenchyme that is necessary for tooth formation. To further test this hypothesis, Msx1 mutant tooth germs were first cultured with either BMP4 or with various FGFs for two days in vitro and then grown under the kidney capsule of syngeneic mice to permit completion of organogenesis and terminal differentiation. Using an in vitro culture system, it has been shown that BMP4 stimulates the growth of Msx1 mutant dental epithelium. Using the more powerful kidney capsule grafting procedure, it has now been shown that when added to explanted Msx1-deficient tooth germs prior to grafting, BMP4 rescues Msx1 mutant tooth germs all the way to definitive stages of enamel and dentin formation. Collectively, these results establish a transient functional requirement for Msx1 in the dental mesenchyme that is almost fully supplied by BMP4 alone, and not by FGFs. In addition, they formally prove the postulated downstream relationship of BMP4 with respect to Msx1, establish the non-cell-autonomous nature of Msx1 during odontogenesis, and disclose an additional late survival function for Msx1 in odontoblasts and dental pulp (Bei, 2000).

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signaling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal. A target gene for Bmp4 signaling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. The temporal and spatial expression of these two genes was compared to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. Changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localized up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, it was confirmed that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. The mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, a reciprocal series of interactions act to restrict expression of both genes to future sites of tooth formation, creating a positive feedback loop that maintains expression of both genes in tooth mesenchymal cells (Tucker, 1998a).

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).

Mammalian dentitions are highly patterned, with different types of teeth positioned in different regions of the jaws. BMP4 is an early oral epithelial protein signal that directs odontogenic gene expression in mesenchyme cells of the developing mandibular arch. BMP4 was shown to inhibit expression of the homeobox gene Barx-1 and to restrict expression to the proximal, presumptive molar mesenchyme of mouse embryos at embryonic day 10. The inhibition of BMP signaling early in mandible development by the action of exogenous Noggin protein results in ectopic Barx-1 expression in the distal, presumptive incisor mesenchyme and a transformation of tooth identity from incisor to molar (Tucker, 1998b).

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling proteins are expressed in the developing tooth germ and interact with one another to mediate the inductive tissue interactions. Among these proteins are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). The interactions among these signaling proteins during early tooth development have been investigated. Bmp4 is expressed in dental epithelium at the initiation stage (E11.5) and then shifts to the dental mesenchyme shortly afterwards at the early bud stage (E12.5). This shift of Bmp4 expression pattern coincides with the shift in tooth developmental potential between tissue layers, indicating that BMP4 may constitute one component of the odontogenic potential. Indeed, BMP4 can induce in the dental mesenchyme the morphological changes and expression of a number of genes, including the transcription factors Msx1, Msx2, Lef1 and Bmp4 itself, mimicking the effect of the early dental epithelium. The mesenchymally expressed Bmp4 is believed to exert its function upon the dental epithelium as a feedback signal for further tooth development. The expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ and Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin results in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted in the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein are able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrate that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4- soaked beads repress Shh and Bmp2 expression in the wild-type dental epithelium. Ectopic expression of human Bmp4 in the dental mesenchyme driven by the mouse Msx1 promoter restores Shh expression in the Msx1 mutant dental epithelium but represses Shh in the wild-type tooth germ in vivo. This regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression is unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, these data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis (Zhang, 2000).

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).

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).

Expression studies of bone morphogenetic proteins (BMPs) and Noggin (a BMP antagonist) in the embryonic chicken face have suggested that BMP signals are important for closure of the upper lip or primary palate. Noggin expression is restricted to the frontonasal mass epithelium but is reduced at the corners of the frontonasal mass (globular processes) just prior to fusion with the adjacent maxillary prominences. Gain- and loss-of-function experiments were performed to determine the role of BMPs in lip formation. Noggin treatment leads to reduced proliferation and outgrowth of the frontonasal mass and maxillary prominences and ultimately to the deletion of the maxillary and palatine bones. The temporary block in BMP signaling in the mesenchyme also promotes epithelial survival. Noggin treatment also upregulates expression of endogenous BMPs, therefore whether increasing BMP levels would lead to the same phenotype was investigated. A BMP2 bead was implanted into the globular process and a similar phenotype to that produced by Noggin resulted. However, instead of a decrease in proliferation, defects were caused by increased programmed cell death, first in the epithelium and then in the mesenchyme. Programmed cell death was induced primarily in the lateral frontonasal mass with very little cell death medial to the bead. The asymmetric cell death pattern was correlated with a rapid induction of Noggin in the same embryos, with transcripts complementary to the regions with increased cell death. A requirement has been demonstrated for endogenous BMP in the proliferation of facial mesenchyme. Furthermore, mesenchymal signals are seen to promote either survival or thinning of the epithelium. In vivo, it has been demonstrated that BMP homeostasis is regulated by increasing expression of ligand or antagonist and that certain mechanisms may help to protect the embryo from changes in growth factor levels during development or after exposure to teratogens (Ashique, 2002).

The vertebrate skull vault forms almost entirely by the direct mineralization of mesenchyme, without the formation of a cartilaginous template -- a mechanism called membranous ossification. Dlx5 gene mutation leads to cranial dismorphogenesis. In avians, little is known about the genetic regulation of cranial vault development. In this study, Dlx5 expression and regulation were analyzed during skull formation in the chick embryo. Dlx5 expression pattern is compared with that of several genes involved in mouse cranial suture regulation. This provides an initial description of the expression in the developing skull of the genes encoding the secreted molecules BMP 2, BMP 4, BMP 7, the transmembrane FGF receptors FGFR 1, FGFR 2, FGFR 4, the transcription factors Msx1, Msx2, and Twist, as well as Goosecoid and the early membranous bone differentiation marker osteopontin. Dlx5 is activated in proliferating osteoblast precursors, before osteoblast differentiation. High levels of Dlx5 transcripts are observed at the osteogenic fronts (OFs) and at the edges of the suture mesenchyme, but not in the suture itself. Dlx5 expression is initiated in areas where Bmp4 and Bmp7 genes become coexpressed. In a calvarial explant culture system, Dlx5 transcription is upregulated by BMPs and inhibited by the BMP-antagonist Noggin. In addition, FGF4 activates Bmp4 but not Bmp7 gene transcription and is not sufficient to induce ectopic Dlx5 expression in the immature calvarial mesenchyme (Holleville, 2003).

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Overexpression of follistatin, a proposed BMP antagonist, in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin is continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicate that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation (Wang, 2004).

BMP signals and the specification of olfactory and lens placodes

Spatial gradients of extracellular signals are implicated in the patterning of many different tissues. Much less is known, however, about how differences in time of exposure of progenitor cells to patterning signals can influence different cell fates. Bone morphogenetic protein (BMP) signals are known to pattern embryonic ectoderm. The olfactory and lens placodes are ectodermal structures of the vertebrate head. By using an explant assay of placodal cell differentiation, evidence is provided that BMP signals are required and sufficient to induce olfactory and lens placodal cells from progenitor cells located at the anterior neural plate border. Evidence is also provided that time of exposure of these progenitor cells to BMP signals plays a key role in the differential specification of olfactory and lens placodal cells (Sjödal, 2007).

Previous results in Xenopus, zebrafish, and chick have suggested that the generation of border region cells, which later can generate different placodal cell types and neural crest cells, requires suppression or intermediate levels of BMP activity. It has also been suggested that the generation of placodal progenitor cells depends on an interaction between epidermal and neural cells. However, the current results indicate that BMP signals can directly induce Six1+ and Dlx5+ placodal progenitors in prospective neural cells. Moreover, the results provide evidence that at stage 4, BMP activity is required and sufficient to induce both olfactory and lens placodal cells, but that levels of BMP signals do not mediate the differential induction, implying that other molecular events may discriminate between lens and olfactory placodal progenitors at this stage. By the neural fold stage, stage 8, the generation of olfactory placodal cells has become independent of further exposure to BMP signals, whereas the generation of lens cells requires continued exposure to BMP signals. Moreover, at this stage, cells can switch between an olfactory and lens placodal fate in response to changes in BMP activity, providing evidence that at stage 8 BMP signals promote the generation of lens cells at the expense of olfactory placodal cells. These results indicate that time of exposure of cranial placodal progenitors to BMP signals plays a key role in the differential specification of olfactory and lens placodal cells. In agreement with these results, at stage 4 pSmad-1/5/8 is detected in a domain where both prospective olfactory and lens placodal cells are positioned, while at stage 8/9 pSmad-1/5/8 is preferentially detected in the prospective lens ectoderm compared to the prospective olfactory placodal region. Consistently in intact embryos, ectopic BMP activity blocks the generation of olfactory placodal cells. Conversely, blockade of BMP signaling inhibits the generation of lens cells, but under these conditions no ectopic olfactory placodal cells were generated, which may indicate that when BMP signaling is suppressed, prospective lens ectoderm respond to other signals that inhibit the generation of olfactory placodal cells (Sjödal, 2007).

DPP homologs and morphogenesis: Eye development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance. The expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, it has been demonstrated that the addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, the expression of several early lens placode-specific markers were examined in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway that is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. It is concluded that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation (Wawersik 1999).

The bone morphogenetic protein (BMP) expression in vertebrates suggests a reiterative function for these molecules during eye development. However, genetic analysis in mice has provided only partial information. Using the chick embryo as a model system, possible additional functions of BMP4 during optic cup formation have been examined. The expression pattern of Bmp4 and Bmp7 are described, and, in contrast to the mouse, the prospective lens placode ectoderm expresses high levels of Bmp4 but no Bmp7. After optic vesicle invagination, Bmp4 is expressed in the prospective dorsal neural retina, where BmprIA, BmprII, and Smad1, components of the BMP4 signal transduction pathway, are also expressed. TUNEL analysis shows that the dorsal optic cup is the site of a spatiotemporally restricted apoptosis, which parallels the expression not only of Bmp4 but also of Msx1 and Msx2, genes implicated in BMP4-mediated apoptosis. The use of optic vesicle cultures as well as in ovo local addition of BMP4 and its antagonist Noggin proves that the local activity of BMP4 is responsible for programmed cell death in the dorsal optic cup. In addition, Noggin is able to reduce the rate of cell proliferation in the dorsal part of the optic cup whereas BMP4 increases the number of BrdU-positive cells in retina cultures. These results provide evidence that BMP4 contributes to eye development by promoting cell proliferation and programmed cell death (Trousse, 2001).

Fibroblast growth factor receptor (Fgfr) signaling plays a role in lens induction. Three distinct experimental strategies were used: (1) using small-molecule inhibitors of Fgfr kinase activity, it has been shown that both the transcription level and protein expression of Pax6, a transcription factor critical for lens development, is diminished in the presumptive lens ectoderm; (2) transgenic mice (designated Tfr7) that express a dominant-negative Fgf receptor exclusively in the presumptive lens ectoderm show defects in formation of the lens placode at E9.5 but in addition, showe reduced levels of expression for Pax6, Sox2 and Foxe3, all markers of lens induction; (3) by performing crosses between Tfr7 transgenic and Bmp7-null mice, it has been shown that there is a genetic interaction between Fgfr and Bmp7 signaling at the induction phases of lens development. This is manifested as exacerbated lens development defects and lower levels of Pax6 and Foxe3 expression in Tfr7/Tfr7, Bmp7+/- mice, when compared with Tfr7/Tfr7 mice alone. Since Bmp7 is an established lens induction signal, this provides further evidence that Fgfr activity is important for lens induction. This analysis establishes a role for Fgfr signaling in lens induction and defines a genetic pathway in which Fgfr and Bmp7 signaling converge on Pax6 expression in the lens placode with the Foxe3 and Sox2 genes lying downstream (Faber, 2001).

The role of Bmp signaling in development of the mouse lens has been investigated using several experimental strategies. First, it has been shown that the Bmp ligand inhibitor noggin can suppress the differentiation of primary lens fiber cells in explant culture. Second, a dominant-negative form of the type 1 Bmp family receptor Alk6 (Bmpr1b -- Mouse Genome Informatics) has been expressed in the lens in transgenic mice and it has been shown that an inhibition of primary fiber cell differentiation can be detected at E13.5. Interestingly, the observed inhibition of primary fiber cell development is asymmetrical and appears only on the nasal side of the lens in the ventral half. Expression of the inhibitory form of Alk6 is driven either by the alphaA-cystallin promoter or the ectoderm enhancer from the Pax6 gene in two different transgenes. These expression units drive transgene expression in distinct patterns that overlap in the equatorial cells of the lens vesicle at E12.5. Despite the distinctions between the transgenes, they cause primary fiber cell differentiation defects that are essentially identical, which implies that the equatorial lens vesicle cells are responding to Bmp signals in permitting primary fiber cells to develop. Importantly, E12.5 equatorial lens vesicle cells show cell-surface immunoreactivity for bone-morphogenetic protein receptor type 2 and nuclear immunoreactivity for the active, phosphorylated form of the Bmp responsive Smads. This indicates that these cells have the machinery for Bmp signaling and were responding to Bmp signals. It is concluded that Bmp signaling is required for primary lens fiber cell differentiation and, given the asymmetry of the differentiation inhibition, that distinct differentiation stimuli may be active in different quadrants of the eye (Faber, 2002).

The retina produces factors that promote the differentiation of lens fiber cells, and members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families have been identified as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. Phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delays lens fiber cell elongation and increases cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects are inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 are able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants is stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor (Belecky-Adams, 2002).

The ciliary body in the vertebrate eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During eye development, the optic vesicle grows out from the diencephalon and invaginates to form the optic cup. The optic cup gives rise to the neural retina, the retinal pigment epithelium (RPE), and the epithelia of the iris and the ciliary body. The ciliary epithelium differentiates from the two layers of neuroepithelial cells at the rim of the optic cup. The unpigmented inner layer is continuous with the neural retina and the iris while the pigmented outer layer lies between the RPE and the outer iris. During eye development, the ciliary epithelium folds to form the ciliary processes, while the mesenchymal cells of neural crest origin differentiate into the connective tissue of the ciliary body and part of the ciliary muscle. The non-pigmented ciliary epithelium secretes fibrillins that are the primary component of the ciliary zonules, the suspensory ligaments of the lens. Primary functions of the ciliary body include: (1) secretion of aqueous humor and glycoproteins of the vitreous body, (2) maintenance of the intraocular pressure, and (3) controlling the shape of the lens through the ciliary muscle and the ciliary zonules. The molecular signals that control morphogenesis of the ciliary body are largely unknown. Lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye (Zhao, 2002).

In vertebrates, the neuroepithelium of the optic vesicle is initially multipotential, co-expressing a number of transcription factors that are involved in retinal pigment epithelium (RPE) and neural retina (NR) development. Subsequently, extrinsic signals emanating from the surrounding tissues induce the separation of the optic vesicle into three domains: the optic stalk/nerve, the NR and the RPE. Bone morphogenetic proteins (BMPs) are sufficient and essential for RPE development in vivo. Bmp4 and Bmp7 are expressed in the surface ectoderm overlying the optic vesicle, the surrounding mesenchyme and/or presumptive RPE during the initial stages of eye development. During the initial stages of chick eye development the microphthalmia-associated transcription factor (Mitf), important for RPE development, is expressed in the optic primordium that is covered by the BMP-expressing surface ectoderm. Following BMP application, the optic neuroepithelium, including the presumptive optic stalk/nerve and NR domain, develop into RPE as assessed by the expression of Otx2, Mitf, Wnt2b and the pigmented cell marker MMP115. By contrast, interfering with BMP signalling prevents RPE development in the outer layer of the optic cup and induces NR-specific gene expression (e.g., Chx10). These results show that BMPs are sufficient and essential for RPE development during optic vesicle stages. A model is proposed in which the BMP-expressing surface ectoderm initiates RPE specification by inducing Mitf expression in the underlying neuroepithelium of the optic vesicle (Muller, 2007).

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal-distal and dorsal-ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, the Smoothened (Smo) conditional knockout (CKO) mouse line was used. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. Expression was examined of various genes, such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. These data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology (Zhao, 2010).

RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling

The retinal pigment epithelium (RPE) is indispensable for vertebrate eye development and vision. In the classical model of optic vesicle patterning, the surface ectoderm produces fibroblast growth factors (FGFs) that specify the neural retina (NR) distally, whereas TGFbeta family members released from the proximal mesenchyme are involved in RPE specification. However, it was previously proposed that bone morphogenetic proteins (BMPs) released from the surface ectoderm are essential for RPE specification in chick. This study now shows that the BMP- and Wnt-expressing surface ectoderm is required for RPE specification. Wnt signalling from the overlying surface ectoderm is involved in restricting BMP-mediated RPE specification to the dorsal optic vesicle. Wnt2b is expressed in the dorsal surface ectoderm and subsequently in dorsal optic vesicle cells. Activation of Wnt signalling by implanting Wnt3a-soaked beads or inhibiting GSK3beta at optic vesicle stages inhibits NR development and converts the entire optic vesicle into RPE. Surface ectoderm removal at early optic vesicle stages or inhibition of Wnt, but not Wnt/beta-catenin, signalling prevents pigmentation and downregulates the RPE regulatory gene Mitf. Activation of BMP or Wnt signalling can replace the surface ectoderm to rescue MITF expression and optic cup formation. Evidence is provided that BMPs and Wnts cooperate via a GSK3beta-dependent but beta-catenin-independent pathway at the level of pSmad to ensure RPE specification in dorsal optic vesicle cells. A new dorsoventral model of optic vesicle patterning is proposed, whereby initially surface ectoderm-derived Wnt signalling directs dorsal optic vesicle cells to develop into RPE through a stabilising effect of BMP signalling (Steinfeld, 2013).

DPP homologs and morphogenesis: Ear development

A mature inner ear is a complex labyrinth containing multiple sensory organs and nonsensory structures in a fixed configuration. Any perturbation in the structure of the labyrinth will undoubtedly lead to functional deficits. Therefore, it is important to understand molecularly how and when the position of each inner ear component is determined during development. To address this issue, each axis of the chick otocyst at embryonic day 2.5 (E2.5), stage 16-17, was changed systematically at an age when axial information of the inner ear is predicted to be fixed based on gene expression patterns. Transplanted inner ears were analyzed at E4.5 for gene expression of BMP4 (bone morphogenetic protein), SOHo-1 (sensory organ homeobox-1), Otx1 (cognate of Drosophila orthodenticle gene), p75NGFR (nerve growth factor receptor) and Msx1 (muscle segment homeobox), or at E9 for their gross anatomy and sensory organ formation. The results show that axial specification in the chick inner ear occurs later than expected and patterning of sensory organs in the inner ear is first specified along the anterior/posterior (A/P) axis, followed by the dorsal/ventral (D/V) axis. Whereas the A/P axis of the sensory organs is fixed at the time of transplantation, the A/P axis for most non-sensory structures is not specified and is still able to be re-specified according to the new axial information from the host. The D/V axis for the inner ear was not fixed at the time of transplantation. The asynchronous specification of the A/P and D/V axes of the chick inner ear suggests that sensory organ formation is a multi-step phenomenon, rather than a single inductive event. The expression patterns of BMP4 in rotated ears is consistent with a role for BMP4 in the specification of sensory organs. Similarly, Otx1 and SOHo-1 gene expressions are always found associated with the formation of the cochlear and semicircular canals, respectively, suggesting that their gene products may play a role in the specification of these inner ear structures (Wu, 1998).

A mature inner ear is a complex labyrinth containing multiple sensory organs and nonsensory structures in a fixed configuration. Any perturbation in the structure of the labyrinth will undoubtedly lead to functional deficits. Therefore, it is important to understand molecularly how and when the position of each inner ear component is determined during development. To address this issue, each axis of the chick otocyst at embryonic day 2.5 (E2.5), stage 16-17, was changed systematically at an age when axial information of the inner ear is predicted to be fixed based on gene expression patterns. Transplanted inner ears were analyzed at E4.5 for gene expression of BMP4 (bone morphogenetic protein), SOHo-1 (sensory organ homeobox-1), Otx1 (cognate of Drosophila orthodenticle gene), p75NGFR (nerve growth factor receptor) and Msx1 (muscle segment homeobox), or at E9 for their gross anatomy and sensory organ formation. The results show that axial specification in the chick inner ear occurs later than expected and patterning of sensory organs in the inner ear is first specified along the anterior/posterior (A/P) axis, followed by the dorsal/ventral (D/V) axis. Whereas the A/P axis of the sensory organs is fixed at the time of transplantation, the A/P axis for most non-sensory structures is not specified and is still able to be re-specified according to the new axial information from the host. The D/V axis for the inner ear was not fixed at the time of transplantation. The asynchronous specification of the A/P and D/V axes of the chick inner ear suggests that sensory organ formation is a multi-step phenomenon, rather than a single inductive event. The expression patterns of BMP4 in rotated ears is consistent with a role for BMP4 in the specification of sensory organs. Similarly, Otx1 and SOHo-1 gene expressions are always found associated with the formation of the cochlear and semicircular canals, respectively, suggesting that their gene products may play a role in the specification of these inner ear structures (Wu, 1998).

Bone morphogenetic protein 4 (Bmp4) is expressed during multiple stages of development of the chicken inner ear. At the otocyst stage, Bmp4 is expressed in each presumptive sensory organ. Bmp4 is expressed in the three presumptive cristae in mice (cristae are sensory epithelia of the semicircular canals serving as the end organ for the sense of balance) and in all eight presumptive sensory organs (the basilar papilla, macula utriculi, macula sacculi, three cristae, lagena, and macula neglecta), as well as in the mesenchymal cells surrounding the region of the otocyst that is destined to form the semicircular canals. After the formation of the gross anatomy of the inner ear, Bmp4 expression persists in some sensory organs and restricted domains of the semicircular canals. To address the role of this gene in inner ear development, BMP4 function(s) were blocked by delivering one of its antagonists, Noggin, to the developing inner ear in ovo. Exogenous Noggin was delivered to the developing otocyst by using a replication-competent avian retrovirus encoding the Noggin cDNA (RCAS-N) or implanting beads coated with Noggin protein. Noggin treatment results in a variety of phenotypes involving both sensory and nonsensory components of the inner ear. Among the nonsensory structures, the semicircular canals are the most sensitive and the endolymphatic duct and sac most resistant to exogenous Noggin. Noggin affects the proliferation of the primordial canal outpouch, as well as the continual outgrowth of the canal after its formation. In addition, Noggin affects the structural patterning of the cristae, possibly via a decrease of Msx1 and p75NGFR expression. These results suggest that BMP4 and possibly other BMPs are required for multiple phases of inner ear development (Chang, 1999).

Semicircular canal formation in the chicken inner ear can be divided into four stages: (1) outgrowth; (2) patterning; (3) resorption, and (4) continual growth. The anterior and posterior canals are both derived from the vertical outpouch, and the lateral canal is derived from the horizontal outpouch. The vertical outpouch starts to form around E3.5. At E5.5, the presumptive posterior canal, via differential growth, is positioned at an approximately right angle to the presumptive anterior canal. Starting at E6, the opposing epithelia from each outpouch come together and form a fusion plate, which later 'resorbs', leaving behind a canal. The process of resorption is not totally understood and involves programmed cell death and possibly retraction of epithelial cells. By E7, the three semicircular canals have acquired the mature pattern but the size of the canals continues to increase until E16 (Chang, 1999).

Based on these normal developmental processes, the malformed canals of RCAS-N-infected inner ears observed at E7 could be due to a defect in the primary outgrowth of the canal outpouch and/or resorption. To address this question, infected inner ears were harvested at intermediate stages. A defect in the canal plate is evident as early as E5, indicating that Noggin affects the primary outgrowth of the canal pouch. At E6 to E6.5, a reduction in the size of the canal pouch is more extensive, expanding into the presumptive common crus area. The normal resorption process that occurs in the center of the canal plate at this stage seems to progress normally in the infected inner ears. Noggin beads mimic RCAS-N-induced phenotypes. Implantation of Noggin beads to either the anterior or the posterior side of the otocyst results in inner ear defects in closest proximity to the beads. Noggin reduces proliferation of the canal outpouch. Since the location and delivery of exogenous Noggin can be better controlled using Noggin beads instead of RCAS-N, the bead implantation approach was to address mechanisms underlying the canal outpouch defect. Following bead implantation into the mesenchyme by the anterior region of otocysts, inner ears were analyzed for proliferation using BrdU labeling and programmed cell death using TUNEL staining. At all time points tested, there was a significant decrease in BrdU labeling in treated ears, compared to controls. Noggin induces apoptosis in the canal outpouch as a late effect. At 24 h postimplantation, a reduction in the thickness of the otic epithelium in the implanted side is observed. No apoptosis, however, is evident in the epithelium at this stage. By 36 h postimplantation (E5 to E5.5), apoptotic cells in control inner ears are sparse, whereas in the Noggin-treated inner ears, apoptotic cells are observed frequently. The size of the canal outpouch is also reduced by the site of bead implantation. Taken together, the BrdU labeling and TUNEL staining results show that reduction in cell proliferation is the primary cause for the defects observed in the canal outpouch at 24 h, followed by an increase in apoptosis at 36 h postimplantation (Chang, 1999).

Noggin also affects the continual outgrowth of formed semicircular canals. Implantation of two or three Noggin-soaked beads to E7 or E7.5 inner ears results in the absence or occlusion of the canal 2 days postimplantation. Normal canals exhibit PCNA staining as two foci, whereas affected canals show a diffused staining pattern by 1 day postimplantation and a disorganized histology by 2 days postimplantation. In a normal canal at E9, Bmp4 and Msx1 are expressed in distinct regions within the canal epithelium. In canals exposed to Noggin, Bmp4 expression in the canal epithelium is normal or slightly increased. In the mesenchyme, however, the increase in Bmp4 expression surrounding the bead is dramatic. In contrast, Msx1 expression in the canals is greatly decreased (Chang, 1999).

Sensory structures are more resistant to Noggin treatment than nonsensory structures, based on the fact that multiple beads are required in order to elicit a phenotype in sensory structures. This could be partly due to a relatively higher level of Bmp4 expression in the sensory versus nonsensory structures at E4. Nevertheless, within 24 h of Noggin treatment, expression of p75NGFR and Msx1, two presumptive crista markers, is reduced. In contrast, expression of more general sensory organ markers such as Fng and Bmp4 is not affected. These results are consistent with the observation that the cristae do form in Noggin-treated ears, but their structural patterning is affected, possibly via the decrease of Msx1 and p75NGFR expression. However, no apparent defects of the cristae were reported in Msx1 knockout mice. Due to the lack of molecular markers specific for the developing maculae of the utricle and saccule (sensory hair cells involved in the sense of equilibrium), it is difficult to evaluate whether changes in these sensory organs are also a consequence of changes in gene expression. Furthermore, Bmp4 expression in the two maculae is transient and Bmp4 transcripts are no longer detectable by E9, whereas Bmp4 expression persists until E12 in the cristae. In the basilar papilla (the sensory organs of the cochlea), Bmp4 expression becomes restricted to sensory hair cells. The significance of this expression for cochlear hair cell formation is not clear, and this pattern of expression is not conserved in mice. Sensory hair cells seemed to form normally in Noggin-treated ears and early expression patterns of Fng and Bmp4. However, some aberrant hair cell patterns have been observed in the basilar papilla, which warrants further investigation (Chang, 1999).

Dorsal and ventral aspects of the mammalian eye are distinct from the early stages of development. The developing eye cup grows dorsally, and the choroidal fissure is formed on its ventral side. Retinal axons from the dorsal and ventral retina project to the ventral and dorsal tectum, respectively. Expression of the Tbx5 gene, an optomotor blind homolog, in the chick eye is first detected at stage 11 throughout the retina, with the strongest signal in the dorsal retina. Expression is later confined to the dorsal eye cup. Tbx5 is expressed in both retinal pigment epithelium (RPE) and neural retina (NR) at these early stages. Although expression in RPE starts to fade out at embryonic day 10, robust expression is maintained in the dorsal NR (all layers except the nerve fiber layer at the inner surface of the retina). Misexpression of Tbx5 induces dorsalization of the ventral side of the eye and altered projections of retinal ganglion cell axons. Thus, Tbx5 is involved in eye morphogenesis and is a topographic determinant of the visual projections between retina and tectum (Koshiba-Takeuchi, 2000).

When mouse BMP4 is misexpressed in the ventral half, round eyes are formed with expansion of Tbx5 expression in the ventral half. In such eyes, expression of Vax and Pax2 was repressed. Misexpression of BMP4 induces profound effects on eye morphology. These observations indicate that BMP4 acts upstream of Tbx5. The Pax2, Vax, EphrinB1, and EphrinB2 genes act downstream of the Tbx5 gene. This genetic hierarchy seems in good accordance with the timing of expression of BMP4, Tbx5, Pax2, EphrinB1, and EphrinB2 (stage 10+, 11, 11, 13 to 15, and 13 to 15, respectively). Misexpression of Tbx5 in the ventral side of the eye induces marked changes of the retinotectum projection without obvious morphological alteration. This rules out the possibility that the effect of Tbx5 misexpression on the projection is secondary to the dorsalization, because the exit of retinal axons into the optic nerve and the optic nerves themselves are formed normally in virus-infected eyes. Hence, it is concluded that the signaling cascade mediated by Tbx5 plays a key role in both eye morphogenesis and the visual projection (Koshiba-Takeuchi, 2000).

In the mouse embryo, Dlx5 is expressed in the otic placode and vesicle, and later in the semicircular canals of the inner ear. In mice homozygous for a null Dlx5/LacZ allele, a severe dysmorphogenesis of the vestibular region is observed, characterized by the absence of semicircular canals and the shortening of the endolymphatic duct. Minor defects are observed in the cochlea, although Dlx5 is not expressed in this region. Cristae formation is severely impaired; however, sensory epithelial cells, recognized by calretinin immunostaining, are present in the vestibular epithelium of Dlx5-/- mice. The maculae of utricle and saccule are present but cells appear sparse and misplaced. The abnormal morphogenesis of the semicircular canals is accompanied by an altered distribution of proliferating and apoptotic cells. In the Dlx5-/- embryos, no changes in expression of Nkx5.1(Hmx3), Pax2, and Lfng have been seen, while expression of bone morphogenetic protein-4 (Bmp4) is drastically reduced. Notably, BMP4 has been shown to play a fundamental role in vestibular morphogenesis of the chick embryo. It is proposed that development of the semicircular canals and the vestibular inner ear requires the independent control of several homeobox genes, which appear to exert their function via tight regulation of BMP4 expression and the regional organization of cell differentiation, proliferation, and apoptosis (Merlo, 2002).

The vertebrate inner ear consists of a complex labyrinth of epithelial cells, surrounded by a bony capsule. The molecular mechanisms coordinating the development of the membranous and bony labyrinths are largely unknown. Using avian retrovirus encoding Noggin (RCAS-Noggin) or beads soaked with Noggin protein, it has been shown that bone morphogenetic proteins (BMPs) are important for the development of the otic epithelium in the chicken inner ear. Using two recombinant avian retroviruses, dominant negative and constitutively active forms of BMP receptors IB (BMPRIB), it has been shown that BMPs, possibly acting through BMPRIB, are important for otic capsule formation. Bmp2 is strongly expressed in the prospective semicircular canals starting from the canal outpouch stage, suggesting that BMP2 plays an important role in canal formation. In addition, by correlating expression patterns of Bmps, their receptors, and localization of phosphorylated R-Smad (phospho R-Smad) immunoreactivity, an indicator of BMP activation, it has been shown that BMPs emanating from the otic epithelium influence chondrogenesis of the otic capsule including the cartilage surrounding the semicircular canals (Chang, 2002).

Inner ear sensory organs and VIIIth cranial ganglion neurons of the auditory/vestibular pathway derive from an ectodermal placode that invaginates to form an otocyst. In the mouse otocyst epithelium, Tbx1 suppresses neurogenin 1-mediated neural fate determination and is required for induction (Otx1) or proper patterning of gene expression (Bmp4) related to sensory organ morphogenesis. Tbx1 loss-of-function causes dysregulation of neural competence in otocyst regions linked to the formation of either mechanosensory or structural sensory organ epithelia. Subsequently, VIIIth ganglion rudiment form is duplicated posteriorly, while the inner ear is hypoplastic and shows neither a vestibular apparatus nor a coiled cochlear duct. It is proposed that Tbx1 acts in the manner of a selector gene to control neural and sensory organ fate specification in the otocyst (Raft, 2004).

DPP homologs and morphogenesis: Pituitary and hypothalamus development

Pituitary gland development serves as an excellent model system in which to study the emergence of distinct cell types from a common primordium in mammalian organogenesis. The role of the morphogen Sonic hedgehog (SHH) in outgrowth and differentiation of the pituitary gland has been investigated using loss- and gain-of-function studies in transgenic mice. Shh is expressed throughout the ventral diencephalon and the oral ectoderm, but its expression is subsequently absent from the nascent Rathke's pouch as soon as it becomes morphologically visible, creating a Shh boundary within the oral epithelium. Oral ectoderm/Rathke's pouch-specific 5' regulatory sequences (Pitx1HS) from the bicoid related pituitary homeobox gene (Pitx1) were used to target overexpression of the Hedgehog inhibitor Hip (Huntingtin interacting protein) to block Hedgehog signaling. It was found that SHH is required for proliferation of the pituitary gland. In addition, evidence is provided that Hedgehog signaling, acting at the Shh boundary within the oral ectoderm, may exert a role in differentiation of ventral cell types (gonadotropes and thyrotropes) by inducing Bmp2 expression in Rathke's pouch, which subsequently regulates expression of ventral transcription factors, particularly Gata2. Furthermore, the data suggest that Hedgehog signaling, together with FGF8/10 signaling that arises from the dorsally located infundibulum, synergizes to regulate expression of the LIM homeobox gene Lhx3, which has been proved to be essential for initial pituitary gland formation. Thus, SHH appears to exert effects on both proliferation and cell-type determination in pituitary gland development (Treier, 2001).

In the developing chick hypothalamus, Shh and BMPs are expressed in a spatially overlapping, but temporally consecutive, manner. This study demonstrates how the temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells. These studies reveal a requirement for a dual action of BMPs: first, the inhibition of GliA function through Gli3 upregulation; and second, activation of a Smad5-dependent BMP pathway. Previous studies have shown a requirement for spatial antagonism of Shh and BMPs in early CNS patterning; this study proposes that neural pattern elaboration can be achieved through a versatile temporal antagonism between Shh and BMPs (Ohyama, 2008).

This analysis makes a number of key points. First, it provides a novel insight into how cellular diversity can be achieved within the embryo in response to a limited repertoire of signalling molecules. In addition to the well-accepted view that BMP signal opposes Shh activity in a spatial manner, ventrally derived Bmp7 signalling can oppose Shh signalling in a temporal manner to specify ventral progenitors within the hypothalamus. The deployment of the two signals in this versatile temporal manner in turn leads to novel modules of transcription factor expression, in order to achieve elaborate cellular diversity. This work adds to the growing body of data suggesting that cell fate in the neural tube is governed through the temporal integration of, and adaptation to, signalling ligands (Ohyama, 2008)

DPP homologs and development of sensory domains in the developing tongue

The regenerative capacity of many placode-derived epithelial structures makes them of interest for understanding the molecular control of epithelial stem cells and their niches. This study investigated the interaction between the developing epithelium and its surrounding mesenchyme in one such system, the taste papillae and sensory taste buds of the mouse tongue. Follistatin (FST) was identified as a mesenchymal factor that controls size, patterning and gustatory cell differentiation in developing taste papillae. FST limits expansion and differentiation of Sox2-expressing taste progenitor cells and negatively regulates the development of taste papillae in the lingual epithelium: in Fst-/- tongue, there is both ectopic development of Sox2-expressing taste progenitors and accelerated differentiation of gustatory cells. Loss of Fst leads to elevated activity and increased expression of epithelial Bmp7; the latter effect is consistent with BMP7 positive autoregulation, a phenomenon this study demonstrated directly. FST and BMP7 influence the activity and expression of other signaling systems that play important roles in the development of taste papillae and taste buds. In addition, using computational modeling, it was shown how aberrations in taste papillae patterning in Fst-/- mice could result from disruption of an FST-BMP7 regulatory circuit that normally suppresses noise in a process based on diffusion-driven instability. Because inactivation of Bmp7 rescues many of the defects observed in Fst-/- tongue, it is concluded that interactions between mesenchyme-derived FST and epithelial BMP7 play a central role in the morphogenesis, innervation and maintenance of taste buds and their stem/progenitor cells (Beites, 2009).

Table of contents

decapentaplegic: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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