THE JOURNAL OF COMPARATIVE NEUROLOGY 406:143155 (1999)

Expression of Sonic Hedgehog, Patched, and Gli1 in Developing Taste Papillae of the Mouse
JOSHUA M. HALL,* JOAN E. HOOPER, AND THOMAS E. FINGER Department of Cellular and Structural Biology and Medical Scientist Training Program, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT Lingual taste buds form within taste papillae, which are specialized structures that develop in a characteristic spatial and temporal pattern. To investigate the signaling events responsible for patterning and morphogenesis of taste papillae, the authors examined the time course and distribution of expression of several related developmental signaling genes as well as the time course of innervation of taste papillae in mouse embryos from embryonic day 12 (E12) to E18. Lingual expression of the signaling molecule Sonic hedgehog (Shh), its receptor Patched (Ptc), and the Shh-activated transcription factor Gli1 were assayed by using in situ hybridization. Shh is expressed broadly in the lingual epithelium at E12 but becomes progressively restricted to developing circumvallate and fungiform papillary epithelia. Shh is expressed specically within the central cells of the papillary epithelium starting at E13.5 and persisting through E18. Ptc and Gli1 expression follow a pattern similar to that of Shh. Compared with Shh, Ptc is expressed in larger regions surrounding the central papillary cells and also in the mesenchyme underlying Shh-expressing epithelium. Innervation of taste papillae was examined by using the panneuronal antibody to ubiquitin carboxyl terminal hydrolase (protein gene product 9.5). Nerves reach the basal lamina of developing taste papillae at E14 to densely innervate the papillary epithelium by E16. Thus, the pattern of Shh expression within developing taste papillae is established prior to innervation, ruling out neuronal induction of papillae. The results suggest that the Shh signaling pathway may be involved in: 1) establishing papillary boundaries in taste papilla morphogenesis, 2) papillary epithelial-mesenchymal interactions, and/or 3) specifying the location or development of taste buds within taste papillae. J. Comp. Neurol. 406:143155, 1999. 1999 Wiley-Liss, Inc.
Indexing terms: developmental signaling; epithelial patterning; in situ hybridization; protein gene product 9.5; taste bud

The gustatory system is unique in that its receptor cells develop from local epithelium rather than from neurogenic ectoderm, as in other sensory systems (Barlow and Northcutt, 1995; Stone et al., 1995). In mammals, taste receptor cells are organized into taste buds that, on the tongue, are located within specialized papillae (Mistretta, 1991). In rodents, taste papillae develop prenatally, whereas taste buds appear subsequently within these papillae at about the time of parturition (Paulson et al., 1985; Mistretta, 1991). Thus, the formation of papillae is an important rst step in the development of the gustatory system in mammals. Four types of lingual papillae are present on the tongues of mice (and other rodents): fungiform, circumvallate, foliate, and liformthe rst three of these contain taste buds and are referred to as taste papillae. Morphogenesis of fungiform and circumvallate papillae occurs in a stereo1999 WILEY-LISS, INC.

typed pattern on the tongue during prenatal development, from embryonic day 12 (E12) to E16 in mice (Paulson et al., 1985; Mistretta, 1991). Fungiform papillae form in longitudinal rows on the anterior portion of the tongue, with the medial rows forming prior to more lateral rows and with the more anterior papillae developing rst within each row (Paulson et al., 1985; Farbman and Mbiene, 1991). A single circumvallate papilla forms on the midline at the oralpharyngeal border of the tongue. The foliate papillae form

Grant sponsor: NIH; Grant numbers: DC00244 and GM08497. *Correspondence to: Joshua M. Hall, Department of Cellular and Structural Biology, 4200 East 9th Avenue, Box B111, Denver, CO 80262. E-mail: halljosh@york.uchsc.edu Received 7 July 1998; Revised 18 November 1998; Accepted 10 December 1998

144 on the lateral edge of the tongue at the level of the intermolar eminence. Although each type of papilla is morphologically distinct, the initial events in their development are histologically similar. Taste papillae begin as placodal thickenings in the lingual epithelium (Paulson et al., 1985; Farbman and Mbiene, 1991; Mistretta, 1991; Fujimoto et al., 1993). The placodal epithelium then begins to grow into the underlying mesenchyme and evaginates into a raised structure. At the same time, nerve bundles begin to grow into the tongue and ultimately reach the lingual epithelium (Farbman and Mbiene, 1991; Whitehead and Kachele, 1994). It is noteworthy that studies of cultured explants of embryonic rat tongue have shown that the fungiform papillae initially develop normally despite the absence of innervation (Farbman and Mbiene, 1991; Mbiene et al., 1997), suggesting that papillary morphogenesis is independent of innervation. However, innervation does seem to be necessary in mammals for completion of normal taste bud development (Oakley et al., 1998). The early stages in the formation of taste papillae resemble those of other specialized epithelial structures, particularly teeth and feather buds. In both of those developing systems, a cascade of intercellular signaling interactions species organ position and induces morphogenesis (Chuong, 1993; Kratochwil et al., 1996). One signaling molecule that is known to operate in vertebrate patterning and tissue induction is sonic hedgehog (Shh), a homolog of the Drosophila hedgehog (Hh) signaling molecule. Shh signaling uses the transmembrane patched (Ptc) protein as a receptor and the Gli1 transcription factor as part of its signal transduction mechanism (Chen and Struhl, 1996; Hahn et al., 1996; Marigo et al., 1996a). Shh is well known for inducing ventral neural and somitic tissues and for specifying limb anterior-posterior polarity (Hammerschmidt et al., 1997). Shh also appears to be involved in many other developmental processes, including whisker, tooth, and even lung formation (Bitgood and McMahon, 1995; Bellusci et al., 1997; Hammerschmidt et al., 1997). Recent studies in mice have reported expression of Shh in the tongue during the period of taste papillary morphogenesis prior to the formation of the taste buds (Bitgood and McMahon, 1995). This raises the possibility that Shh is one of the signals active in patterning the lingual epithelium. Because Shh is involved in several developmental processes (Hammerschmidt et al., 1997), it could play multiple roles with regard to papillary development. For example, Shh could act as a polarizing signal as it does in the limb bud (Johnson et al., 1994), establishing tongue polarity for subsequent papillary development. It also could act as an inductive signal as it does in the neural tube (Echelard et al., 1993; Roelink et al., 1995), inducing development of lingual epithelium along papillary cell fates. Shh could be active in epithelial-mesenchymal interactions or in the establishment of papillary spacing or borders as well. Finally, Shh could be involved in later processes of lingual gustatory development, potentially specifying the location of and/or inducing taste receptor cell differentiation. To clarify the role that Shh plays in the development of taste papillae, we conducted a detailed study of the timing and distribution of lingual expression of members of the Shh signaling pathway from E11 to E16.5 in mice. This corresponds to the period of lingual organogenesis and

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TABLE 1. In Situ Hybridization Probes Used in the Study Probe
Sonic hedgehog Patched Gli1 Gli3

Length
640 bp 841 bp 800 bp 1.6 kb

From
L. Goodrich, Stanford University L. Goodrich, Stanford University A. Joyner, New York University A. Joyner, New York University

morphogenesis of fungiform and circumvallate papillae. We also used a panneuronal antibody to ubiquitin carboxyl terminal hydrolase (protein gene product 9.5; PGP 9.5), to study papillary innervation in relation to timing of expression of Shh pathway genes. We report here that Shh, Ptc, and Gli1 expression occurs throughout the early lingual epithelium and becomes progressively restricted to the regions in and around the developing fungiform and circumvallate papillae prior to innervation of the papillary epithelium.

MATERIALS AND METHODS Embryo collection and staging

Timed pregnant CD-1 mice were obtained from Charles River (Wilmington, MA). Use of these animals was approved by the University of Colorado Health Sciences Center Animal Care and Use Committee and conformed to National Institutes of Health guidelines. The mice were overdosed with halothane, decapitated on appropriate days from 10 days to 18 days postcoitus, and embryos were collected into 4% phosphate-buffered paraformaldehyde (PFA; 0.1 M), pH 7.2. Embryos were xed overnight at 4C and staged by crown-rump (CR) length according to Rugh (1968) and by other morphologic features (Kaufman, 1992). The tissue was dehydrated by sequential washes in 50%, 100%, 100% methanol and stored at 20C. When they were used, embryos were washed sequentially with 50% methanol and phosphate-buffered saline (PBS; 0.01 M sodium phosphate and 0.13 M NaCl), pH 7.4, plus 0.1% Tween-20 (PBST; three times). Heads, jaws, or tongues were then dissected out for hybridization. For sections, some dissected tissue was equilibrated with 20% sucrose in 4% PFA then cut into 1520 m sagittal sections and placed on Superfrost slides (Fisher Scientic, Pittsburgh, PA). Following wholemount expression analysis, the lingual developmental stage was assessed by morphology and tongue length (circumvallate to tip). Comparison of these data with CR length showed that CR length was an accurate predictor of relative lingual development.

Wholemount and section in situ hybridization

Whole or sectioned tissue was washed in PBST and treated for 1020 minutes with proteinase K (10 g/ml in PBST). It was washed twice in PBST and rexed for 20 minutes in 4% PFA. Embryos were then prehybridized for 2 hours at 42C in hybridization buffer containing 50% formamide; 5 hybridization buffer (20 buffer 3M NaCl, 100 mM ethylenediamine tetraacetic acid, 100 mM 1,4-Piperazinediethane sulfonic acid, pH 6.8); 1 Denhardts; 250 g/ml sheared salmon sperm DNA; 250 g/ml poly-A; and 0.1% Tween-20. Digoxigenin-labeled sense and antisense riboprobes were produced from pBluescript II vectors as described elsewhere (Hui, 1994; Goodrich et al., 1996). Details about the probes are listed in Table 1. Hybridization was performed overnight at 60C in hybrid-

Shh, Ptc, AND Gli1 IN DEVELOPING TASTE PAPILLAE ization buffer containing 5.5% dextran sulphate and 0.2 0.5 g/ml riboprobe. Excess probe was removed by sequential washes in 2 standard saline citrate (SSC; three times at 60C), 0.2 SSC (three times at 60C), and 1:1 0.2 SSC:0.1 M phosphate buffer (PB) and PB (twice). Nonspecic binding in the tissue was blocked for 12 hours in 10% sheep serum, 4% dry milk, 2 mg/ml bovine serum albumin, and 0.3% Triton X-100 in 0.1 M PB. After this treatment, the tissue was incubated overnight with antidigoxigenin antibody conjugated to alkaline phosphatase diluted 1:1,000 in blocking solution. Excess antibody was removed by washes in 0.1 M PB, and the tissue was equilibrated with color buffer containing 100 mM Tris, pH 9.5; 50 mM MgCl2; 100 mM NaCl; and 0.1% Tween 20. Antibody was visualized by using the 4-Nitro blue tetrazolinium chloride/5-Bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) blue color reaction. Prior to photography, the tissue was rexed in 4% PFA. Following hybridization, some wholemount tongues were cryoprotected by incubation in 25% sucrose in 4% PFA overnight and then cut on a cryostat into 20 M sections.

145 oor is not signicantly above background (not shown). Early on E12, a tongue primordium is visible, and Shh is expressed diffusely throughout this early tongue. A dark staining region of higher Shh expression is present just anterior to the foramen caecum, corresponding to the future location of the circumvallate papilla. Broad lingual expression persists as the tongue grows until around E12.5. At this time, Shh expression is comparatively diminished along the midline of the tongue, and areas of higher expression can be seen in rows parallel to the long axis of the tongue. This is the same pattern and location in which the rst fungiform papillae develop (Paulson et al., 1985; Farbman and Mbiene, 1991). Sections of hybridized tongues at this age show that Shh expression is localized to lingual epithelial cells and is absent from the underlying mesenchyme (Fig. 3A). The region of dark staining at the location of the developing circumvallate papilla persists. As the late E12 tongue grows, Shh expression continues to be focused into the developing papillae. During E13 and E14, the tongue takes on the curvature and shape of a fully developed mouse tongue. At E13, the rows of higher Shh expression at the presumed sites of fungiform papillae, which are seen rst at E12.5, have condensed into more discrete regions of expression (not shown). Beginning at E13.5 and continuing throughout gestation, Shh expression is tightly localized to the regions of developing fungiform and circumvallate papillae (Fig. 2A). The broad Shh expression seen previously has diminished and is not signicantly above background outside of taste papilla regions. Sections of hybridized tongues at this stage show that Shh expression is localized to small groups of columnar epithelial cells that are ve to seven cells across (Fig. 3C) and that apparently correspond to the placodal thickenings that eventually give rise to fungiform papillae (Mistretta, 1991). The restricted Shh expression in taste papillae persists through the end of gestation. From E16 onward, Shh is expressed in the central core of the developing papillary epithelium (Figs. 2E, 4A). Older tongues are not amenable to wholemount in situ hybridization, but hybridization to sectioned tissue shows that Shh expression remains localized to the central set of cells on the surface of the fungiform papillae through E18 (Fig. 4E). Ptc. The expression of Ptc in the developing tongue correlates with that of Shh, as observed in other systems (Goodrich et al., 1996). The results of wholemount in situ hybridization with Ptc antisense RNA probes are shown in Figures 1 and 2. Ptc expression is not detectable above background during fusion of the mandibular arches at E11.5 (not shown). Broad Ptc expression occurs throughout the tongue primordium early on E12. By E12.5, expression is obviously higher in the regions that will form taste papillae (Fig. 1D). Thus, in the earliest stages of tongue development (E12E13), the pattern of Ptc expression mirrors that of Shh. In sections of tongues at these stages, Ptc expression is present in both the lingual epithelium and its underlying mesenchyme (Fig. 3B). This is the rst time at which Shh and Ptc are expressed in different cell populations within the tongue. Ptc expression, similar to Shh expression, continues over time to coalesce into regions around taste papillae (Fig. 2B). By E13.5, signicant Ptc expression is present only in regions of developing taste papillae. However, the expression pattern around these papillae is distinct from that of Shh. The regions of Ptc expression near the

PGP 9.5 immunohistochemistry

After rehydration and sectioning, some tongues were analyzed for presence of the neuronal marker PGP 9.5 (ubiquitin carboxyl terminal hydrolase). Sections were rinsed in 0.1 M PB and blocked for 1 hour in 10% donkey serum in 0.1 M PB plus 0.3% Triton X-100. Incubation with primary rabbit anti-PGP 9.5 (1:1,000; Biogenesis, Poole, United Kingdom) occurred overnight at 4C in a humidied chamber. After rinsing with 0.1 M PB, sections were incubated with lissamine rhodamine sulfonyl chloride (LRSC)-conjugated donkey anti-rabbit immunoglobulin G (1:100; Jackson, West Grove, PA) for 12 hours at room temperature. Slides were rinsed with 0.1 M PB and coverslipped with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL). Immunouorescence and Nomarski images were collected with an Olympus Fluoview confocal microscope (Tokyo, Japan). All gures were composed using Adobe Photoshop software (version 4.0.1; Adobe Systems, Mountain View CA) with only overall adjustment of levels and color balance for each image. Immunouorescence/Nomarski overlay images were created within Photoshop by using layering effects.

RESULTS Lingual morphogenesis in the mouse

The basic progression of mouse tongue development can be seen in Figures 1 and 2. Our studies in mice conrm that, as described by Paulson et al. (1985), murine tongue development is morphologically similar to that described in rat (Slavkin et al., 1989; Farbman and Mbiene, 1991; Mbiene et al., 1997). A tongue bud is rst visible early on E12 and quickly becomes a distinct tongue by E12.5. It lengthens and takes on both lateral and dorsal-ventral curvature during E13E14, achieving the adult spatulate shape by E16.

Lingual expression patterns of Shh signaling pathway members

Shh. Figure 1A,B shows the results of wholemount in situ hybridization for Shh in developing tongues of embryos ages E12 and E12.5. At E11, there is no distinguishable tongue, and the hybridization signal from the oral

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Fig. 1. Expression of sonic hedgehog (Shh), patched (Ptc), and Gli1 in early stages of tongue development in mice. Dorsal views of embryonic day 12 (E12) [crown-rump (C-R) length 78 mm; A,C,E], and E12.5 (C-R length 89 mm; B,D,F) tongues assayed for expression of these genes by wholemount in situ hybridization. Anterior is to the right. A: E12 jaw showing broad lingual Shh expression as well as Shh expression in the presumptive circumvallate papilla (arrow) and

developing incisors (arrowhead). B: E12.5 lower jaw with Shh expression clearing along the midline of the tongue and parasagittal rows of expression on either side. C: E12 tongue with broad lingual Ptc expression. D: E12.5 Ptc expression. Note the lower expression along the midline mirroring Shh expression. E: E12 Gli1 expression. F: E12.5 Gli1 expression. Scale bars 500 m.

Fig. 2. Expression of Shh, Ptc, and Gli1 in intermediate and late-stage developing tongues of mice. AC: Dorsal views of E14 (C-R length 10.511.2 mm) and E14.5 (C-R length 1112 mm) tongues assayed for Shh, Ptc, and Gli1 expression by wholemount in situ hybridization. A: E14.5 tongue showing Shh expression predominantly in developing circumvallate (arrow) and fungiform (arrowheads) papillae. B: E14.5 tongue Ptc expression. C: E14 tongue Gli1 expression. Both Ptc and Gli1 expression mirror Shh expression. DG: Dorsal views of E16.5 (C-R length 15.016.5 mm) tongues assayed for

Shh and Ptc expression by wholemount in situ hybridization. D: E16.5 tongue showing Shh expression predominantly in circumvallate (arrow) and fungiform papillae. E: Detail of fungiform papilla showing Shh expression. F: E16.5 tongue showing Ptc expression. G: Detail of fungiform papilla showing Ptc expression. Note the larger area of Ptc expression compared with Shh expression and the lower Ptc expression in the central core of the papilla. Scale bars 500 m in AD,F, 50 m in E,G.

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Fig. 3. Shh and Ptc are expressed in different tissue types in the tongues of early and intermediate stage mice. Sagittal sections of E12.5 (A,B) and E14 (CF) tongues assayed for Shh expression (A,C,E) or Ptc expression (B,D,F) by in situ hybridization. A: Shh is expressed throughout the lingual epithelium at E12.5. B: Ptc is expressed in both the lingual epithelium and the mesenchyme. C: E14 tongue showing

Shh expression in a developing fungiform papilla. D: At E14, Ptc is expressed in the fungiform epithelium and in the underlying mesenchyme. E: Shh expression in E14 circumvallate papillary epithelium. F: Ptc is expressed in E14 circumvallate epithelium and mesenchyme. Scale bars 50 m.

fungiform papillae are more diffuse and larger than the regions expressing Shh centered in the developing papillae. Also, Ptc expression near the circumvallate papilla

forms a ring surrounding the center of the papilla rather than being centered in the papilla like Shh. In sections, expression of Ptc is less discrete than that of Shh. Ptc

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Fig. 4. Shh and Ptc are expressed in taste papillae throughout gestation. Sagittal sections of tongues from E15E18 mice assayed for Shh and Ptc expression by in situ hybridization. A: E16 tongue showing Shh expression in the central core of a fungiform papilla. B: E16.5 tongue showing a broader region of Ptc expression surround-

ing a fungiform papilla. C: E15 tongue showing Shh expression in the circumvallate epithelium. D: E16.5 tongue showing both epithelial and mesenchymal Ptc expression around the circumvallate papilla. E: Shh expression at the surface of an E18 fungiform papilla. F: Ptc is also expressed in E18 fungiform papillae. Scale bars 50 m.

expression extends beyond the columnar epithelial cells of the developing fungiform papillae laterally 1015 m in the lingual epithelium and vertically 510 m into the mesenchymal cells of the tongue (Fig. 3D).

At later stages of lingual development, Ptc expression changes slightly, although it remains localized to taste papillae. By E16.5, expression near the circumvallate papilla is within the entire region of the papilla (Fig. 2F).

150 In the fungiform papillae, Ptc expression at E16.5 is clearly in an annular pattern around each papilla: lower in the center of the papillae and darker around the edge (Fig. 2G). This is the reverse of Shh expression at this age, which is focused discretely in the center of the developing papilla (Fig. 2E). Sectioned tongues show that Ptc expression continues to be localized to a larger region within fungiform papillae, extending both laterally and vertically beyond where Shh is expressed. Lower Ptc expression is seen in the epithelium in the center of the developing papillae, consistent with the annular pattern seen in whole tongues (Fig. 4B). Ptc expression in the circumvallate papillae is in both the epithelium and the underlying mesenchymal cells at this stage (Fig. 4D). Again like Shh, Ptc is expressed in fungiform (Fig. 4F) and circumvallate (not shown) papillae through E18. Gli1 and Gli3. Gli1 is the transcription factor responsible, at least in part, for cellular responses to Shh (Mart et al., 1995; Hammerschmidt et al., 1997). In the tongue, Gli1 expression mirrors that of Ptc shown in Figures 1 and 2. Gli1 is expressed broadly in the early tongue primordium at E12 and is localized progressively to taste papillae in a pattern resembling that of Ptc. Gli1 expression does not decrease in nonpapillary regions as quickly as Shh or Ptc; some broad Gli expression persists through E13 (Fig. 2C). At later stages of lingual development, Gli1 expression exists in an annular pattern surrounding each fungiform papilla, again similar to Ptc (not shown). Gli3 is another member of the Gli transcription factor family. It is not thought to be involved in Shh response and can act antagonistically to Shh and Gli (Marigo et al., 1996b). We do not see Gli3 expression above background levels in the tongue.

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DISCUSSION
Development of lingual taste receptors can be divided into two stages. First, taste papillae form on the tongue in a characteristic spatial and temporal pattern. This process occurs from E12.5 to E16 in mice and requires several steps: an inductive event, specifying the locations of taste papillae; proliferation and evagination of the developing papillary epithelium; and growth of a supportive mesenchymal core (Farbman and Mbiene, 1991; Mistretta, 1991). Second, taste buds develop within the papillary epithelium following papillary morphogenesis. This process may require an additional inductive event to specify taste receptor cell differentiation and must be coordinated with innervation of the lingual epithelium (Farbman and Mbiene, 1991; Oakley, 1991). The lingual expression patterns of Shh signaling pathway members relative to morphologic stages are summarized in Figure 7. These results indicate that Shh may function in both stages of gustatory development.

Shh signaling in papillary morphogenesis

Expression of Shh, Ptc, and Gli1 begins broadly in the tongue primordium early on E12 and becomes progressively localized to regions in and around taste papillae. Shh is detectable only in epithelial cells throughout lingual development, and its expression is located exclusively in the center of fungiform and circumvallate papillae by E13.5. Ptc expression has a slightly different distribution than Shh: it is expressed in larger regions surrounding developing fungiform papillae than Shh. Ptc expression is lower in or absent from the Shh-expressing centers of fungiform papillae at later stages of papillary development. Ptc also is expressed in mesenchyme underlying Shh-expressing areas throughout papillary morphogenesis. Both Ptc and Gli1 are activated transcriptionally in response to Shh in tissues adjacent to Shh signaling centers (Goodrich et al., 1996; Marigo et al., 1996a,c; Sasaki et al., 1997). Thus, expression of Ptc or Gli1 indicates those cells or tissues that are actively responding to the Shh signal. Because Ptc is expressed both within the lingual epithelium and in the underlying mesenchyme, Shh may play a role in papillary morphogenesis by signaling to one or both of these tissues. In the epithelium, Shh may be involved in establishing papillary boundaries. This situation is exemplied in Drosophila, in which Hh is responsible for specifying embryonic parasegment borders (Kalderon, 1995). Reciprocal signals between Hh and wingless establish two adjacent rows of cells that follow separate developmental fates. In the same way, reciprocal signals between Shh and another signaling molecule, produced by lingual epithelium around the perimeter of the developing papillae, may establish a boundary for papillary development. The tight localization of Shh to an easily identied set of papillary epithelial cells suggests that a boundary formation or restriction process is active in papillary development. Alternatively, Shh could specify papillary boundaries within the lingual epithelium in a concentration-dependent manner as in the developing neural tube. In neural tube development, high levels of Shh from the notochord promote oor plate differentiation, whereas lower concentrations induce motor neuron cell fates (Roelink et al., 1995). Shh may act in a similar fashion by specifying papillary epithelial growth and differentiation at a certain

Innervation of developing taste papillae

The time course of innervation of papillary epithelium from E13 to E16 was examined by immunohistochemistry for the neuronal marker PGP 9.5 (ubiquitin carboxyl terminal hydrolase). At E13, nerve bers can be seen growing toward the surface of the anterior tongue and just below the circumvallate placode (Fig. 5AD). Nerves have not yet reached the epithelium at this time. At E14, immunouorescence extends to the basement membrane just below developing fungiform and circumvallate papillary epithelium (Fig. 5EH). The sites of nerve growth to the epithelium are restricted to the developing circumvallate and fungiform papillae; no bers appear to be innervating nonpapillary epithelium. Nerves begin to penetrate into the papillary epithelium by E15 (Fig. 6AD). By E16, the entire circumvallate and central core of the fungiform papillary epithelia are densely innervated by nerve bers (Fig. 6EH). Taken together with the in situ hybridization data presented above, it appears that Shh expression is localized to taste papillae prior to nerve ber contact with the lingual epithelium. Shh expression is localized specically to the developing circumvallate papillae as early as E12 and to the developing fungiform papillae by E13.5. Nerve bers are not present at the papillary epithelium until E14 and do not innervate the entire papillary epithelium until E16.

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Fig. 5. Lingual innervation at E13 and E14. Protein gene product 9.5 (PGP 9.5; ubiquitin carboxyl terminal hydrolase) immunouorescence (left column) and Nomarski image overlays (right column) showing the extent of papillary innervation. AD: E13 tongues showing developing fungiform (A,B) and circumvallate (C,D) papillae.

The lingual epithelium is not innervated at this time. EH: E14 tongues showing developing fungiform (E,F) and circumvallate (G,H) papillae. Nerve bers have reached the basement membrane below the forming papillae. Scale bars 50 m.

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Fig. 6. Papillary innervation at E15 and E16. PGP 9.5 immunouorescence (left column) and Nomarski image overlays (right column) showing the extent of papillary innervation. AD: E15 tongues showing fungiform (A,B) and circumvallate (C,D) papillae. Nerve

bers have begun to penetrate the papillary epithelium. EH: E16 tongues showing fungiform (E,F) and circumvallate (G,H) papillae. The papillary epithelium is densely innervated by this time. Scale bars 50 m.

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Fig. 7. Time line of lingual development. Comparison of expression patterns of Shh, Ptc, and Gli1 with lingual innervation and morphology. At E12, a tongue bud is visible with broad epithelial Shh expression and broad epithelial and mesenchymal Ptc expression. The tongue is a distinct structure by E12.5. At E12.5, expression of all three genes has cleared from the midline and exists in parasagittal rows along the tongue as well as in the location of the future circumvallate papilla. Prior to and at E13, nerves have not reached the lingual epithelium. From E13 to E16, the tongue elongates and takes on adult shape. At E13.5, Shh is found only in developing papillary

epithelium. Ptc is expressed in papillary precursor epithelium and mesenchyme. Expression of Shh and Ptc remains restricted to taste papillae from this time onward. At E14, nerve bers have reached the basement membrane below developing papillae. At E15, papillae are visible on the tongue, and nerve bers begin to penetrate the papillary epithelium. Papillae are densely innervated by E16. At E16 and beyond, Shh is expressed only in the central epithelial core of fungiform papillae, whereas Ptc is found in a wider region surrounding the center of each papilla.

threshold concentration. In this model, cells at the boundaries of the developing papillae would follow nonpapillary fates as the Shh concentration decreases with increasing distance from Shh-expressing centers. Shh might also play a role in the epithelial-mesenchymal interactions of papillary morphogenesis. Papillary development requires coordination between epithelium and mesenchyme, which grows and differentiates to form the supportive core of the papilla (Farbman and Mbiene, 1991; Mistretta, 1991; Fujimoto et al., 1993). Expression of Ptc in the mesenchyme underlying developing taste papillae indicates that this tissue is responding to the epithelial Shh signal. The epithelial Shh signal could be responsible for induction or support of growth of the mesenchymal core of taste papillae. Shh-mediated epithelial-mesenchymal interactions are well known in other systems. During tooth formation, Shh is expressed in the epithelial enamel knot, which acts as a signaling center for organization of dental epithelialmesenchymal interactions (Vaahtokari et al., 1996). Epithelial Shh expression with corresponding mesenchymal Ptc expression also has been observed in developing whisker barrels (Bitgood and McMahon, 1995; Iseki et al., 1996; Platt et al., 1997) and feather buds (Nohno et al., 1995; Jung et al., 1998). These structures develop in a manner similar that of taste papillae, in that they begin as placodal epithelial thickenings and gain a mesenchymal core as they grow (Fristrom, 1988). The involvement of Shh in epithelial-mesenchymal interactions in such similar structures supports the idea that it is involved also in epithelialmesenchymal signaling in taste papillae.

epithelium of the circumvallate papilla. This distribution coincides with the eventual locations of taste buds. Taste buds form postnatally, initially within the dorsal epithelium of the circumvallate papilla and in the centers of fungiform papillae (Mistretta, 1991). These are the same sites at which Shh expression concentrates on E16.5 in fully formed lingual papillae. Thus, Shh may be involved in the morphogenesis of taste buds. Support for this proposed role of Shh comes from studies of neurotrophic factors within the developing tongue. The distribution of lingual Shh expression after E15.5 matches that of brain-derived neurotrophic factor (BDNF) at the same stage (Nosrat and Olson, 1995). BDNF is believed to support gustatory nerves that grow into developing taste buds (Nosrat et al., 1996; Zhang et al., 1997). If it is assumed that BDNF is an early marker of differentiation of the gustatory epithelium, then Shh is in the right place at the right time to be responsible for specication or support of gustatory development. However, Shh probably is not solely responsible for taste bud histogenesis, because that process requires innervation to proceed normally in mammals (Oakley, 1991; Fritzsch et al., 1997; Mbiene et al., 1997; Oakley et al., 1998). Shh could be inducing competence to form taste receptor cells within the central papillary epithelial cells, which then differentiate further due to interaction with gustatory nerves.

Taste papilla patterning

Gustation is the only sensory system in which the receptor cells are not derived from neurogenic ectoderm. Instead, the receptors of the gustatory system arise from local epithelium (Barlow and Northcutt, 1995; Stone et al., 1995). Whereas taste buds develop in the absence of papillae in the epiglottis and palate, lingual taste buds develop solely within taste papillae (Mistretta, 1991). Thus, the positioning of, and possibly the development of,

Shh in taste bud determination

From E15 through E16.5, Shh expression remains in the lingual epithelium, restricted to a small set of cells in the center of each fungiform papilla and throughout the

154 taste receptor cells in the tongue may be dependent on the formation of taste papillae. Taste papillary morphogenesis is a process intrinsic to the tongue. In rats, taste papillae develop in the normal spatial and temporal sequence in the absence of innervation (Farbman and Mbiene, 1991; Mbiene et al., 1997), so neural induction is not responsible for taste papilla patterning. This conclusion is supported by comparing the time course of papillary innervation to Shh expression (Figs. 2, 5). Discrete localization of Shh to developing papillae occurs by E12.5 in the presumptive circumvallate papilla and by E13.5 in fungiform papillary precursors. However, nerve bers have not yet reached the lingual epithelium by E13 and extend only to the level of the basement membrane at E14. So, Shh expression in the papillary precursor regions is established prior to their innervation. Thus, papillae are specied independent of neuronal inuence. If papillary development is initiated independently within the tongue, then how is the pattern established? If Shh were patterning papillae by self-restriction, then one would expect to see an evenly-spaced, random distribution of papillae. The actual distribution of taste papillae is too regular and stereotyped for this. The process of papilla patterning, as monitored by Shh expression, occurs in at least two steps. The rst step is restriction of precursor regions to parasagittal rows along either side of the tongue. Following this, individual papillae are generated within these rows. This is reminiscent of the patterning of feathers in birds, another type of epithelial specialization. Feather buds also arise as Shh-expressing placodal thickenings in a patterning process intrinsic to the epidermal epithelium (Chuong, 1993; Nohno et al., 1995). Prior to expression of Shh within feather placodes, Shh and Fibroblast Growth Factor 4 (FGF-4) are expressed in a stripe running along the dorsal midline. This stripe then changes to become discrete Shh-expressing regions that form feather buds (Jung et al., 1998). This process is thought to occur through epithelial-mesenchymal interactions that are mediated by Shh and other signaling molecules (Jung et al., 1998). A similar process involving Shh may be responsible for taste papilla patterning. Taste papilla, feather, and tooth development are all alike in that these structures begin as placodal thickenings and then evaginate or invaginate to form a raised structure with a mesenchymal core (Fristrom, 1988; Lumsden, 1988; Mistretta, 1991; Chuong, 1993; Fujimoto et al., 1993). In addition, Shh is expressed in tooth and feather primordia as well as in taste papilla precursors (Bitgood and McMahon, 1995; Nohno et al., 1995; Fig 1A). Feather position is thought to be specied by interaction between FGF-4, Bone Morphogenic Proteins (BMP-2 and BMP-4), and Shh (Jung et al., 1998). Recent studies also indicate that tooth position is specied by overlap of mesenchymal FGF-8 and BMP signals (Neubuser et al., 1997). A patterning process involving overlap of several intercellular signals may be active in the developing tongue. Investigation of the role of these other signaling families in the tongue may help to further explain papillary morphogenesis and lingual development.

J.M. HALL ET AL. plasmids, to A. Joyner at New York University for the Gli1 and Gli3 clones, and to L. Barlow for critical reading and comments on this paper. We also thank A. Ribera for use of her laboratory facilities and photographic equipment. This work was supported by NIDCD grant DC00244 to T.E.F. and training grant GM08497 to J.M.H.

LITERATURE CITED
Barlow LA, Northcutt RG. 1995. Embryonic origin of amphibian taste buds. Dev Biol 169:273285. Bellusci S, Furuta Y, Rush MG, Henderson R, Winnier G, Hogan BL. 1997. Involvement of sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 124:5363. Bitgood MJ, McMahon AP. 1995. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 172:126138. Chen Y, Struhl G. 1996. Dual roles for patched in sequestering and transducing hedgehog. Cell 18:553563. Chuong C. 1993. The making of a feather: homeoproteins, retinoids and adhesion molecules. Bioessays 15:513521. Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J, McMahon, JA McMahon AP. 1993. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75:14171430. Farbman AI, Mbiene JP. 1991. Early development and innervation of taste bud-bearing papillae on the rat tongue. J Comp Neurol 304:172186. Fristrom D. 1988. The cellular basis of epithelial morphogenesis. A review. Tissue Cell 20:645690. Fritzsch B, Sarai PA, Barbacid M, Silos-Santiago I. 1997. Mice with a targeted disruption of the neurotrophin receptor trkB lose their gustatory ganglion cells early but do develop taste buds. Int J Dev Neurosci 15:563576. Fujimoto S, Yamamoto K, Yoshizuka M, Yokoyama M. 1993. Pre- and postnatal development of rabbit foliate papillae with special reference to foliate gutter formation and taste bud and serous gland differentiation. Microsc Res Tech 26:120132. Goodrich LV, Johnson RL, Milenkovic L, McMahon JA, Scott MP. 1996. Conservation of the hedgehog/patched signaling pathway from ies to mice: induction of a mouse patched gene by hedgehog. Genes Dev 10:301312. Hahn H, Christiansen J, Wicking C, Zaphiropoulos PG, Chidambaram A, Gerrard B, Vorechovsky I, Bale AE, Toftgard R, Dean M, Wainwright B. 1996. A mammalian patched homolog is expressed in target tissues of sonic hedgehog and maps to a region associated with developmental abnormalities. J Biol Chem 271:1212512128. Hammerschmidt M, Brook A, McMahon AP. 1997. The world according to hedgehog. Trends Genet 13:1421. Hui CC, Slusarski D, Platt KA, Holmgren R, Joyner AL. 1994. Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during post-implantation development. Dev Biol 162:402413. Iseki S, Araga A, Ohuchi H, Nohno T, Yoshioka H, Hayashi F, Noji S. 1996. Sonic hedgehog is expressed in epithelial cells during development of whisker, hair, and tooth. Biochem Biophys Res Commun 218:688693. Johnson RL, Riddle RD, Laufer E, Tabin C. 1994. Sonic hedgehog: a key mediator of anterior-posterior patterning of the limb and dorso-ventral patterning of axial embryonic structures. Biochem Soc Trans 22:569574. Jung H-S, Francis-West PH, Widelitz RB, Jiang T-X, Ting-Berreth S, Tickle C, Wolpert L, Chuong C-M. 1998. Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning. Dev Biol 196:1123. Kalderon D. 1995. Morphogenetic signalling. Responses to hedgehog. Curr Biol 5:580582. Kaufman MH. 1992. The atlas of mouse development. London: Academic Press. Kratochwil K, Dull M, Farinas I, Galceran J, Grosschedl R. 1996. Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development. Genes Dev 10:13821394. Lumsden AG. 1988. Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ. Development 103(Suppl):155169.

ACKNOWLEDGMENTS
The authors thank K. Anderson for expert technical assistance and sectioned in situ data. We are grateful to L. Goodrich at Stanford University for the Shh and Ptc cDNA

Shh, Ptc, AND Gli1 IN DEVELOPING TASTE PAPILLAE

Marigo V, Davey RA, Zuo Y, Cunningham JM, Tabin CJ. 1996a. Biochemical evidence that patched is the hedgehog receptor. Nature 384:176179. Marigo V, Johnson RL, Vortkamp A, Tabin CJ. 1996b. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev Biol 180:273283. Marigo V, Scott MP, Johnson RL, Goodrich LV, Tabin CJ. 1996c. Conservation in hedgehog signaling: induction of a chicken patched homolog by sonic hedgehog in the developing limb. Development 122:12251233. Mart E, Takada R, Bumcrot DA, Sasaki H, McMahon AP. 1995. Distribu tion of Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121:25372547. Mbiene JP, Maccallum DK, Mistretta CM. 1997. Organ cultures of embryonic rat tongue support tongue and gustatory papilla morphogenesis in vitro without intact sensory ganglia. J Comp Neurol 377:324340. Mistretta CM. 1991. Developmental neurobiology of the taste system. In: Getchell TV, editor. Taste and smell in health and disease. New York: Raven Press. p 3564. Neubuser A, Peters H, Balling R, Martin GR. 1997. Antagonistic interac tions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90:247255. Nohno T, Kawakami Y, Ohuchi H, Fujiwara A, Yoshioka H, Noji S. 1995. Involvement of the sonic hedgehog gene in chick feather formation. Biochem Biophys Res Commun 206:3339. Nosrat CA, Olson L. 1995. Brain-derived neurotrophic factor mRNA is expressed in the developing taste bud-bearing tongue papillae of rat. J Comp Neurol 360:698704. Nosrat CA, Ebendal T, Olson L. 1996. Differential expression of brainderived neurotrophic factor and neurotrophin 3 mRNA in lingual papillae and taste buds indicates roles in gustatory and somatosensory innervation. J Comp Neurol 376:587602. Oakley B. 1991. Neuronal-epithelial interactions in mammalian gustatory epithelium. Ciba Found Symp 160:277287. Oakley B, Brandemihl A, Cooper D, Lau D, Lawton A, Zhang C. 1998. The morphogenesis of mouse vallate gustatory epithelium and taste buds

155
requires BDNF-dependent taste neurons. Brain Res Dev Brain Res 105:8596. Paulson RB, Hayes TG, Sucheston ME. 1985. Scanning electron microscope study of tongue development in the CD-1 mouse fetus. J Craniofac Genet Dev Biol 5:5973. Platt KA, Michaud J, Joyner AL. 1997. Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between ies and mice. Mech Dev 62:121 135. Roelink H, Porter JA, Chiang C, Tanabe Y, Chang DT, Beachy PA, Jessell TM. 1995. Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81:445455. Rugh R. 1968. The mouse: its reproduction and development. Minneapolis: Burgess. Sasaki H, Hui C, Nakafuku M, Kondoh H. 1997. A binding site for Gli proteins is essential for HNF-3beta oor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 124:1313 1322. Slavkin HC, Bringas P Jr., Sasano Y, Mayo M. 1989. Early embryonic mouse mandibular morphogenesis and cytodifferentiation in serumless, chemically dened medium: a model for studies of autocrine and/or paracrine regulatory factors. J Craniofac Genet Dev Biol 9:185205. Stone LM, Finger TE, Tam PP, Tan SS. 1995. Taste receptor cells arise from local epithelium, not neurogenic ectoderm. Proc Natl Acad Sci USA 92:19161920. Vaahtokari A, Aberg T, Jernvall J, Keranen S, Thesleff I. 1996. The enamel knot as a signaling center in the developing mouse tooth. Mech Dev 54:3943. Whitehead MC, Kachele DL. 1994. Development of fungiform papillae, taste buds, and their innervation in the hamster. J Comp Neurol 340:515530. Zhang C, Brandemihl A, Lau D, Lawton A, Oakley B. 1997. BDNF is required for the normal development of taste neurons in vivo. Neuroreport 8:10131017.