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- EXPERIMENTAL PROCEDURES
Fungiform papillae are epithelial taste organs that form on the tongue, requiring differentiation of papillae and inter-papilla epithelium. We tested roles of epidermal growth factor (EGF) and the receptor EGFR in papilla development. Developmentally, EGF was localized within and between papillae whereas EGFR was progressively restricted to inter-papilla epithelium. In tongue cultures, EGF decreased papillae and increased cell proliferation in inter-papilla epithelium in a concentration-dependent manner, whereas EGFR inhibitor increased and fused papillae. EGF preincubation could over-ride disruption of Shh signaling that ordinarily would effect a doubling of fungiform papillae. With EGF-induced activation of EGFR, we demonstrated phosphorylation in PI3K/Akt, MEK/ERK, and p38 MAPK pathways; with pathway inhibitors (LY294002, U0126, SB203580) the EGF-mediated decrease in papillae was reversed, and synergistic actions were shown. Thus, EGF/EGFR signaling by means of PI3K/Akt, MEK/ERK, and p38 MAPK contributes to epithelial cell proliferation between papillae; this biases against papilla differentiation and reduces numbers of papillae. Developmental Dynamics 237:2378–2393, 2008. © 2008 Wiley-Liss, Inc.
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- EXPERIMENTAL PROCEDURES
Taste papilla development and patterning require interactive programs both for induction of the specific organ and differentiation of inter-papilla epithelium (Mistretta and Liu,2006). Whereas the development of fungiform papillae in their distinctive pattern has long been noted (Mistretta,1972,1998; Mbiene et al.,1997), there is not a clear understanding of molecular events in papilla patterning. EGF is a potent secreted factor that has reported roles in spacing other epithelial specializations including hair (Mak and Chan,2003), feather (Atit et al.,2003), and denticle (Urban et al.,2004), but potential regulatory roles for EGF in fungiform papilla patterning have not been studied. Therefore, distinctions or developmental generalizations between EGF actions in skin vs. lingual specialized organs are not known. Here, we demonstrate roles of EGF and EGFR in defining the inter-papilla space in embryonic rat tongue; report EGF effects in lingual epithelial cell proliferation; and, identify intracellular signaling pathways that mediate EGF effects.
The mammalian tongue hosts three types of taste papillae: fungiform, circumvallate, and foliate, each with a unique location, morphology, and innervation to resident taste buds. Fungiform papillae develop in diagonal rows on the anterior two-thirds of the rodent tongue, from a homogeneous epithelium that covers the three lingual swellings at embryonic day (E) 13 in rat (Mistretta,1972,1991; Mbiene et al.,1997) or E11.5–E12 in mouse (Kaufman,1992). Approximately 1 day later, E14, when lingual swellings have merged into a spatulate tongue, papilla placodes are first identified as focal cell clusters. By E15, the tongue has a distinctive topography and fungiform papillae are in rows on anterior tongue (Mistretta,1972; Mbiene et al.,1997). The nontaste, heavily keratinized filiform papillae that cover inter-papilla epithelium in the postnatal tongue are not visible until approximately E20. Furthermore, histologically defined, early taste buds are not seen in rodent papillae until just before birth; taste bud development is essentially postnatal (Mistretta,1991; Hill,2001).
Functional roles are known for Sonic hedgehog (SHH; Hall et al.,2003; Mistretta et al.,2003; Liu et al.,2004); bone morphogenetic protein (BMP) 2, 4, and 7 and NOGGIN (Zhou et al.,2006); SOX2 (Okubo et al.,2006); and WNT10b (Iwatsuki et al.,2007; Liu et al.,2007) in regulating the number and distribution of fungiform papillae. These factors have stage-specific effects and can induce or inhibit papilla development. However, in these studies there has not been attention to the inter-papilla epithelium and in fact, little is known about regulation of inter-papilla epithelial differentiation in patterning. There are specific innervation patterns to taste papillae compared with inter-papilla, nontaste epithelium (Mistretta,1998; Hill,2001). Therefore, to understand development of sensory functions, it is important to know how differentiation programs arise for gustatory organs vs. filiform papilla domains. EGF has prominent roles in cell survival, proliferation, and differentiation (Woodburn,1999; Harris et al.,2003; Shilo,2005), and, therefore, could have dual functions in papilla and inter-papilla epithelial development.
Aberrant morphology in surviving, EGFR null mutant mice previously suggested a role for EGF in fungiform papilla development (Miettinen et al.,1995; Threadgill et al.,1995; Sun and Oakley,2002). However, the mice had compromised face and tongue integrity that limited conclusions about EGF effects on papillae. In organ culture, there is a unique opportunity for direct study of tongue and taste papilla development in a quantitative manner, without confounding effects from oral-facial deformities. The entire tongue progresses from three lingual swellings (at E13) to a spatulate (E14) and larger (E15–E16) tongue, and taste papillae form with retention of spatial, temporal, and molecular information that is similar to in vivo development (Mbiene et al.,1997; Nosrat et al.,2001; Liu et al.,2004). This culture system now is widely used to understand papilla development (Hall et al.,2003; Mistretta et al.,2003; Okubo et al.,2006; Zhou et al.,2006; Iwatsuki et al.,2007).
In the present study, we first identify specific EGF and EGFR locations during tongue and papilla development. Then, we investigate EGF effects in tongue cultures begun at two early embryonic stages, when tongue epithelium is homogenous and not differentiated to papilla or inter-papilla fates (E13) and just after prepapilla placodes have begun to emerge (E14). We show that exogenous EGF regulates patterning by reducing papilla number, and that EGF action on fungiform papillae is mediated by means of EGFR. Furthermore, we demonstrate that EGF/EGFR action increases inter-papilla cell proliferation and can over-ride SHH signaling disruption that doubles the number of fungiform papillae. Mediating the epithelial effects, EGFR-induced intracellular signaling cascades including phosphatidylinositol 3-kinase (PI3K)/Akt, MEK/ERK, and p38 MAPK cascades are shown to have specific roles. Together, results show new roles for EGF signaling by means of EGFR, in regulating fungiform papillae and tongue epithelium development. For the first time, specific intracellular cascades are identified in mediating papilla development.
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- EXPERIMENTAL PROCEDURES
The fungiform papilla is a taste organ that develops early in the embryo to provide a specialized tissue home for eventual taste bud differentiation on the anterior tongue; therefore, at some point in papilla development, taste cell progenitor epithelium resides within the papillae (Mistretta and Liu,2006). Covering the remaining anterior tongue dorsum is the developing inter-papilla epithelium that will differentiate to form nongustatory, filiform papillae. To regulate taste papilla development and pattern, then, factors effective in emergence of the taste organ itself, and the lingual tissue between organs, must be active.
Here, we demonstrate that EGF signaling through EGFR is a key regulator of the inter-papilla epithelium and number of fungiform papillae. EGF is in early, embryonic tongue epithelium and remains distributed throughout lingual and differentiating papilla epithelium. In contrast, EGFR is progressively restricted to inter-papilla epithelium and essentially is absent from developing and advanced papillae. This restricts principal EGF action to the inter-papilla epithelium. Exogenous EGF in E13 or E14 tongue cultures regulates papilla pattern by reducing numbers of papillae, whereas inhibition of endogenous EGFR increases fungiform papilla numbers and fuses adjacent papillae, effectively eliminating an inter-papilla space. In the embryo, epithelial cell proliferation is substantially reduced in emerging papilla placodes and developing papillae, compared with the highly proliferative, inter-papilla tongue epithelium where EGFR is localized. Indeed added EGF stimulates further proliferation of inter-papilla epithelial cells in tongue cultures. EGF can block the doubling of differentiated fungiform papillae that results from disruption of SHH signaling, further indicating a bias to maintain inter-papilla epithelium. We propose that alteration of epithelial cell differentiation programs is a primary mechanism underlying EGF effects, which holds inter-papilla cells in a proliferative cycle and thereby inhibits cell differentiation programs for fungiform papilla formation. The specific effects of EGF/EGFR-mediated papilla patterning act through intracellular cascades, including PI3K/Akt, MEK/ERK, and p38 MAPK. Furthermore, interactive roles of MEK/ERK with PI3K/Akt and with p38 MAPK are apparent.
EGF Signaling Through EGFR and Papilla Effects
EGF is abundant in saliva, approximately 1 μg/ml, which continually bathes the tongue and promotes health of oral tissues (Noguchi et al.,1991). Whereas EGF in saliva has important roles in maintaining fungiform papilla integrity in adult (Morris-Wiman et al.,2000), we found that endogenous EGF is present throughout the embryonic epithelium. In embryonic rodent, the submandibular salivary gland is functionally differentiated before birth (Hoffman et al.,2002), so exogenous EGF also is potentially available to developing oral tissues.
Although not quantified, reduced or aberrant papillae were observed in stunted tongues with thin epithelium in EGFR null mutant, postnatal surviving mice (Miettinen et al.,1995; Threadgill et al.,1995). Building on these prior studies, Sun and Oakley (2002) made a detailed study of taste bud loss in fungiform papillae in EGFR null mutants and in contrast to prior reports did not observe a reduction in papillae, but did report an unspecified number of fungiform papillae with keratinized spines. This is similar to aberrant fungiform papillae in mice with salivary gland removal (Morris-Wiman et al.,2000). Different results across studies are not unexpected because the EGFR loss-of-function phenotype is reportedly highly variable and dependent on the genetic background (Kasper et al.,2006). In sum, postnatal null mutants show that signaling through EGFR is important in maintenance of taste and nontaste papilla and tongue epithelium but provide no clear picture of EGF signaling effects in papilla formation and lingual epithelial differentiation.
EGFR belongs to a family of ErbB receptor tyrosine kinases (Harris et al.,2003; Jorissen et al.,2003): ErbB1 (the EGFR itself, HER-1), ErbB2 (Neu, or HER-2), ErbB3 (HER-3), and ErbB4 (HER-4). In rats, ErbB1–3 have been detected in adult taste bud cells in all three types of taste papillae, and also in E16–20 papillae (McLaughlin,2000). ErbB2 individually cannot bind any known ligand and ErbB3 can only signal in a complex (Harris et al.,2003; Jorissen et al.,2003; Shilo,2005). In the present study, we focused on EGFR, which is the receptor for EGF binding and has a stage-specific localization in inter-papilla epithelium.
We identified a progressive, embryonic restriction of EGFR to inter-papilla tongue epithelium where it is intensely expressed, in contrast to distribution of EGF throughout tongue epithelium. We further demonstrated that EGF action is through EGFR. The specific distribution of EGFR in inter-papilla epithelium indicates that EGF is a spacing factor for fungiform papillae, because EGF acts to increase proliferation in epithelium that is between the papillae. In addition, developmental effects of the EGFR inhibitor, Compound 56, are to increase papilla number and fusion, in support of the conclusion that EGF/EGFR plays a physiological role in papilla patterning. In the present study we focused on EGFR, which is the receptor for EGF binding and has a specific localization in inter-papilla epithelium. Although EGFR generally undergoes homodimerization (Shilo,2005), we cannot exclude that other ErbB receptors expressed in tongue epithelium that do not act as homeodimers, form heterodimers with EGFR, for example, EGFR/ErbB2, as in skin and hair follicle development (Mak and Chan,2003).
Epithelial Cell Phenotypes of Fungiform Papillae and EGF/EGFR Function
The early fungiform papilla forms as a placode and develops through epithelial–mesenchymal remodeling (Mistretta,1998). Signaling in the epithelium reportedly determines position of newly formed papillae (Kim et al.,2003) and in this study our focus has been on epithelial events in particular. At papilla initiation (E14–E15, rat), epithelial cells clustered in the placode apex already are different in shape and organelle density from surrounding cells (Farbman and Mbiene,1991). Moreover, epithelial cells in placodes and early papillae are mitotically quiescent (Farbman and Mbiene,1991; Mbiene and Roberts,2003; Zhou et al.,2006). In contrast, we show that the surrounding lingual epithelium is in a proliferative state (Fig. 5). The data suggest that placode and early papilla epithelial cells are no longer in the cell cycle, reflecting differentiation.
EGFR-activated signaling stimulates cell cycle progression, regulates cell shape and motility, and inhibits apoptosis (Woodburn,1999; Olayioye et al.,2000; Yarden and Sliwkowski,2001). The specific distribution of EGFR in inter-papilla tongue epithelium, where cells are proliferating, and absence of EGFR in embryonic fungiform papillae, where epithelial cells are not proliferating, suggest roles for EGFR in determining epithelial cell fate and, thus, in spacing fungiform papillae. There is a dramatic increase in cell proliferation in the inter-papilla region with addition of EGF in culture. Furthermore, EGF can block the effect of SHH signal disruption, to double the number of fungiform papillae. Together, our data support the hypothesis that EGF/EGFR activation leads to increased cell cycle progression while inhibiting differentiation to a papilla pathway; this would prevent formation of fungiform papillae and thus reduce papilla number.
From our prior studies, we know that the inter-papilla epithelium is competent to form fungiform papillae (Mistretta et al.,2003). Therefore, we had proposed that regulatory factors must act directly or by means of other signaling factors to suppress fungiform papilla formation and enable patterned spacing of papillae. Our current data provide strong evidence for EGF/EGFR signaling in suppressing papilla formation in part by sustaining cell proliferation between papillae.
EGF in Development of Epithelial Specializations: Feather, Hair, and Denticle
EGF and EGFR are in chick embryo skin before feather placodes form, and then are reduced in placodes but maintained in the inter-bud epidermis (Atit et al.,2003). In culture, EGF stimulates epidermal proliferation and expands inter-bud EGFR gene expression, with a concurrent loss of feather bud gene expression. Conversely, EGFR inhibitors result in loss of inter-bud fate and lead to feather bud fusion. In hair follicles, EGFR is absent from epidermal cells over dermal condensates that mark the first stage of follicle development (Green and Couchman,1984). EGF inhibits formation of hair buds in embryonic mouse skin culture (Kashiwagi et al.,1997). In transgenic mice that constitutively express EGF in skin, hair follicle development is retarded in postnatal animals and the epidermis is thickened (Mak and Chan,2003). Overall, reports suggest that EGFR directs epidermal cells to an inter-feather or inter-follicle fate, whereas inhibition of EGFR leads to feather or hair follicle differentiation. In Drosophila epidermis, belts of hair-like denticles alternate with smooth cuticle. Reduced EGFR signaling increases inter-denticle apoptosis and leads to fusion of adjacent denticle belts (Urban et al.,2004), indicating a conserved effect of EGF in epidermal organ formation.
Distributions and effects of EGF/EGFR signaling in the tongue epithelium during papilla development are similar to those in skin and outer cuticle, during feather, hair follicle, and denticle formation. EGFR expression is in inter-papilla epithelium, and activation with EGF results in increased cell proliferation between papillae; this leads to expansion of inter-papilla space and loss of papillae. EGFR inhibition induces increased number and fusion of papillae. Our data add the taste papilla as an epithelial specialization that relies on EGF/EGFR signaling for patterning, and demonstrates common EGF/EGFR effects in developing tongue epithelium, an oral mucosa, compared with skin.
Intracellular Pathways and Synergistic Roles in EGF/EGFR Signaling
EGF/EGFR signaling results in simultaneous activation of several intracellular pathways, which can be functionally linked (Jorissen et al.,2003). We studied PI3K/Akt, MEK/ERK, and p38 MAPK in papilla development, pathways widely associated with cell survival, proliferation, differentiation, migration, and death that are preferentially activated in response to growth factors or cell stress (Jorissen et al.,2003; Downward,2004; Roux and Blenis,2004).
Signaling in tongue cultures.
We detected phosphorylated Akt, ERK1/2, and p38 MAPK in lingual epithelium of nontreated E14+2 day cultures with immunohistochemistry and Western blots, suggesting active endogenous signaling in embryonic tongue. With EGF in tongue culture medium, immunoproducts of phosphorylated Akt, ERK1/2, or p38 MAPK were more intense in the epithelium compared with controls, implicating all three signaling cascades in the EGF effect on fungiform papilla development. Increased kinase intensity was especially pronounced in inter-papilla epithelium, consistent with expression of EGFR in this location.
In support of data from immunoreactions, in Western blot assays exogenous EGF effected a dramatic increase in levels of phosphorylated Akt and ERK1/2 in the epithelium of E14+2 day cultures. Furthermore, when a specific inhibitor for each kinase was used (LY294002 or U0126), Akt and ERK1/2 phosphorylation was completely blocked without change in total kinase level.
However, no significant change in phosphorylated p38 MAPK was observed in Western blots, in contrast to increased lingual immunoproducts of phosphorylated p38 MAPK. In addition, when SB203580 was used to block signaling through p38 MAPK, the phosphorylation of p38 MAPK was not inhibited in Western blot analysis. This is similar to reports demonstrating that SB203580 inhibits activity of p38 MAPK by blocking activation of downstream factors, but not the activation/phosphorylation of p38 MAPK itself (Godl et al.,2003; Morel et al.,2005). SB203580 inhibits p38α and β splice variants of p38 MAPK (Kumar et al.,2003); p38α reportedly is the most physiologically important variant, but p38β has suggested roles in protecting against apoptosis (Yang et al.,2004). Clearly, p38 MAPK pathways are complex and further experiments are required to understand the SB203580 inhibition of p38 MAPK activity in our tongue culture system.
Functional effects and synergistic actions on papilla number.
With inhibitors to PI3K, MEK/ERK, or p38 MAPK signaling, we found that any inhibitor alone did not alter papilla number and pattern in culture without exogenous EGF. However, with combined inhibitors, there was a dramatic increase in papilla number indicating synergistic signaling actions in endogenous papilla patterning. The MEK/ERK cascade may be a primary component in these synergies because alteration of papilla number occurred only when MEK/ERK inhibition was in conjunction with PI3K/Akt or p38 MAPK inhibition; combined use of inhibitors of the latter two kinases did not have an additive effect.
In a concentration-dependent manner, any one of the inhibitors, LY294002 for PI3K/Akt, U0126 for MEK/ERK, or SB203580 for p38 MAPK, blocked the effect of exogenous EGF in reducing fungiform papilla number. Moreover, at 3 μM concentration, which is not effective alone, combined U0126 with LY294002 or SB203580 blocked the EGF-induced decrease in papilla number. Use of LY294002 with SB203580 did not block EGF effects. This further demonstrates a synergistic role of MEK/ERK with PI3K/Akt and p38 MAPK in regulating the EGF-mediated effect on papilla pattern. Additive effects among these cascades are noted in other systems (Larsen et al.,2003; She et al.,2005). Furthermore, sensitivity to tryosine kinase inhibition is dependent on cell context and can alter with and without growth factor stimulation (Irmer et al.,2007). Therefore, differences in concentration and synergistic parameters when inhibitors are used without or with EGF stimulation are not unexpected.
While other secreted proteins might affect papilla development through the PI3K/Akt and MEK/ERK and p38 MAPK signaling cascades that we have localized in developing tongue epithelium and papillae, these other potential effects have not yet been studied. We have clearly shown that exogenous EGF will not only lead to phosphorylation of these kinases, but also that when these pathways are blocked specifically, EGF no longer alters papilla number.
EGF Signaling and Interactions With Other Pathways in Fungiform Papilla Development
Cell cycle progression assessed by proliferation in embryonic tongue and tongue cultures is pronounced between papilla placodes or papillae, and is virtually absent within placodes or papillae. We propose that primary effects of EGF/EGFR activation on papilla spacing and pattern are by means of signaling in the inter-papilla epithelium, through PI3K/Akt, MEK/ERK, and p38 MAPK cascades involved in cell survival, proliferation, differentiation, migration, and/or apoptosis (Fig. 9A). If PI3K/Akt, MEK/ERK, or p38 MAPK signaling is inhibited, more fungiform papillae form in EGF stimulated cultures. Our data are congruent with the idea that EGFR-mediated EGF regulation of papilla number and pattern acts through signaling in the epithelium between papillae. An inter-papilla epithelial fate is promoted, rather than a papilla differentiation pathway.
Figure 9. Epidermal growth factor (EGF) signaling effects in inter-papilla epithelium, and model to compare EGF and bone morphogenetic protein (BMP) in fungiform papilla reduction. A: EGF signaling is progressively constrained to inter-papilla epithelium by restricted location of EGF receptor (EGFR) to inter-papilla tissue. Signaling through PI3K/Akt, MEK/ERK, and p38 MAPK cascades can interact and potentially regulate cells in the inter-papilla epithelium (for survival, proliferation, differentiation, or apoptosis), effectively biasing away from a fungiform papilla fate. B: The model indicates that both exogenous EGF (current study) and BMPs (BMP2, 4, or 7; Zhou et al.,2006b) reduce numbers of fungiform papillae in embryonic tongue. EGF signaling expands the inter-papilla space through a proliferative effect on between-papilla epithelium that biases cells away from papilla differentiation. In contrast, excess BMP effects a thinner, nonproliferative inter-papilla epithelium. Thus, whereas both EGF and BMP prevent papilla formation, different mechanisms are used. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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In addition to EGF signaling in the inter-papilla epithelium, we previously have demonstrated that BMP2, 4, or 7 reduces formation of fungiform papillae (Zhou et al.,2006). Comparison of EGF and BMP effects in reducing papilla number is informative. In cultures with implanted beads, BMPs lead to thinning and much reduced proliferation in the tongue epithelium (Zhou et al.,2006). The BMP antagonist noggin, on the other hand, elicits formation of multiple papillae and a thicker, highly proliferative epithelium. BMP signaling effects, then, are very different from those of EGF, although both lead to reduced papillae. Whereas EGF promotes cell proliferation in inter-papilla epithelium and biases away from fungiform papilla differentiation, BMP reduces cell survival and proliferation and inhibits papilla formation (Fig. 9B). Clearly, these are factors that must be balanced in developing tongue epithelium for patterned formation of taste organs.
Furthermore, counter to and/or interacting with EGF signaling can be stage- and concentration-specific effects of SHH (Liu et al.,2004), NOGGIN (Zhou et al.,2006), or WNT molecules (Iwatsuki et al.,2007) in papilla formation. We have shown that EGF can block SHH signaling effects on papilla formation. In extending our results, it will be important to determine whether, when, and how EGF, BMP, NOGGIN, SHH, and WNT signaling interact in papilla and inter-papilla epithelial formation, and how these interactions might be distinctive in accessing various intracellular tyrosine kinase cascades.