expression ofsonic hedgehog,patched, andgli1 in developing taste papillae of the mouse

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

ABSTRACTLingual taste buds form within taste papillae, which are specialized structures that

develop in a characteristic spatial and temporal pattern. To investigate the signaling eventsresponsible for patterning and morphogenesis of taste papillae, the authors examined thetime course and distribution of expression of several related developmental signaling genes aswell as the time course of innervation of taste papillae in mouse embryos from embryonic day12 (E12) to E18. Lingual expression of the signaling molecule Sonic hedgehog (Shh), itsreceptor Patched (Ptc), and the Shh-activated transcription factor Gli1 were assayed by usingin situ hybridization. Shh is expressed broadly in the lingual epithelium at E12 but becomesprogressively restricted to developing circumvallate and fungiform papillary epithelia. Shh isexpressed specifically within the central cells of the papillary epithelium starting at E13.5 andpersisting 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 cellsand also in the mesenchyme underlying Shh-expressing epithelium. Innervation of tastepapillae was examined by using the panneuronal antibody to ubiquitin carboxyl terminalhydrolase (protein gene product 9.5). Nerves reach the basal lamina of developing tastepapillae at E14 to densely innervate the papillary epithelium by E16. Thus, the pattern of Shhexpression within developing taste papillae is established prior to innervation, ruling outneuronal induction of papillae. The results suggest that the Shh signaling pathway may beinvolved in: 1) establishing papillary boundaries in taste papilla morphogenesis, 2) papillaryepithelial-mesenchymal interactions, and/or 3) specifying the location or development of tastebuds within taste papillae. J. Comp. Neurol. 406:143–155, 1999. r 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 cellsdevelop from local epithelium rather than from neurogenicectoderm, as in other sensory systems (Barlow and North-cutt, 1995; Stone et al., 1995). In mammals, taste receptorcells are organized into taste buds that, on the tongue, arelocated within specialized papillae (Mistretta, 1991). Inrodents, taste papillae develop prenatally, whereas tastebuds appear subsequently within these papillae at aboutthe time of parturition (Paulson et al., 1985; Mistretta,1991). Thus, the formation of papillae is an important firststep in the development of the gustatory system in mam-mals.

Four types of lingual papillae are present on the tonguesof mice (and other rodents): fungiform, circumvallate,foliate, and filiform—the first three of these contain tastebuds and are referred to as taste papillae. Morphogenesisof fungiform and circumvallate papillae occurs in a stereo-

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 longitu-dinal rows on the anterior portion of the tongue, with themedial rows forming prior to more lateral rows and withthe more anterior papillae developing first within each row(Paulson et al., 1985; Farbman and Mbiene, 1991). A singlecircumvallate papilla forms on the midline at the oral-pharyngeal 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 Struc-

tural Biology, 4200 East 9th Avenue, Box B111, Denver, CO 80262.E-mail: [email protected]

Received 7 July 1998; Revised 18 November 1998; Accepted 10 December1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 406:143–155 (1999)

r 1999 WILEY-LISS, INC.

on the lateral edge of the tongue at the level of theintermolar eminence.

Although each type of papilla is morphologically dis-tinct, the initial events in their development are histologi-cally similar. Taste papillae begin as placodal thickeningsin the lingual epithelium (Paulson et al., 1985; Farbmanand Mbiene, 1991; Mistretta, 1991; Fujimoto et al., 1993).The placodal epithelium then begins to grow into theunderlying mesenchyme and evaginates into a raisedstructure. At the same time, nerve bundles begin to growinto the tongue and ultimately reach the lingual epithe-lium (Farbman and Mbiene, 1991; Whitehead and Kachele,1994). It is noteworthy that studies of cultured explants ofembryonic rat tongue have shown that the fungiformpapillae initially develop normally despite the absence ofinnervation (Farbman and Mbiene, 1991; Mbiene et al.,1997), suggesting that papillary morphogenesis is indepen-dent of innervation. However, innervation does seem to benecessary in mammals for completion of normal taste buddevelopment (Oakley et al., 1998).

The early stages in the formation of taste papillaeresemble those of other specialized epithelial structures,particularly teeth and feather buds. In both of thosedeveloping systems, a cascade of intercellular signalinginteractions specifies organ position and induces morpho-genesis (Chuong, 1993; Kratochwil et al., 1996). Onesignaling molecule that is known to operate in vertebratepatterning and tissue induction is sonic hedgehog (Shh), ahomolog of the Drosophila hedgehog (Hh) signaling mol-ecule. Shh signaling uses the transmembrane patched(Ptc) protein as a receptor and the Gli1 transcription factoras part of its signal transduction mechanism (Chen andStruhl, 1996; Hahn et al., 1996; Marigo et al., 1996a). Shhis well known for inducing ventral neural and somitictissues and for specifying limb anterior-posterior polarity(Hammerschmidt et al., 1997). Shh also appears to beinvolved in many other developmental processes, includ-ing whisker, tooth, and even lung formation (Bitgood andMcMahon, 1995; Bellusci et al., 1997; Hammerschmidt etal., 1997).

Recent studies in mice have reported expression of Shhin the tongue during the period of taste papillary morpho-genesis prior to the formation of the taste buds (Bitgoodand McMahon, 1995). This raises the possibility that Shhis one of the signals active in patterning the lingualepithelium. Because Shh is involved in several developmen-tal processes (Hammerschmidt et al., 1997), it could playmultiple roles with regard to papillary development. Forexample, Shh could act as a polarizing signal as it does inthe limb bud (Johnson et al., 1994), establishing tonguepolarity for subsequent papillary development. It alsocould act as an inductive signal as it does in the neuraltube (Echelard et al., 1993; Roelink et al., 1995), inducingdevelopment of lingual epithelium along papillary cellfates. Shh could be active in epithelial-mesenchymal inter-actions or in the establishment of papillary spacing orborders as well. Finally, Shh could be involved in laterprocesses of lingual gustatory development, potentiallyspecifying the location of and/or inducing taste receptorcell differentiation.

To clarify the role that Shh plays in the development oftaste papillae, we conducted a detailed study of the timingand distribution of lingual expression of members of theShh signaling pathway from E11 to E16.5 in mice. Thiscorresponds to the period of lingual organogenesis and

morphogenesis of fungiform and circumvallate papillae.We also used a panneuronal antibody to ubiquitin carboxylterminal hydrolase (protein gene product 9.5; PGP 9.5), tostudy papillary innervation in relation to timing of expres-sion of Shh pathway genes. We report here that Shh, Ptc,and Gli1 expression occurs throughout the early lingualepithelium and becomes progressively restricted to theregions in and around the developing fungiform andcircumvallate papillae prior to innervation of the papillaryepithelium.

MATERIALS AND METHODS

Embryo collection and staging

Timed pregnant CD-1 mice were obtained from CharlesRiver (Wilmington, MA). Use of these animals was ap-proved by the University of Colorado Health SciencesCenter Animal Care and Use Committee and conformed toNational Institutes of Health guidelines. The mice wereoverdosed with halothane, decapitated on appropriatedays from 10 days to 18 days postcoitus, and embryos werecollected into 4% phosphate-buffered paraformaldehyde(PFA; 0.1 M), pH 7.2. Embryos were fixed overnight at 4°Cand 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 220°C. When theywere used, embryos were washed sequentially with 50%methanol and phosphate-buffered saline (PBS; 0.01 Msodium phosphate and 0.13 M NaCl), pH 7.4, plus 0.1%Tween-20 (PBST; three times). Heads, jaws, or tongueswere then dissected out for hybridization. For sections,some dissected tissue was equilibrated with 20% sucrosein 4% PFA then cut into 15–20 µm sagittal sections andplaced on Superfrost slides (Fisher Scientific, Pittsburgh,PA).

Following wholemount expression analysis, the lingualdevelopmental stage was assessed by morphology andtongue length (circumvallate to tip). Comparison of thesedata with CR length showed that CR length was anaccurate predictor of relative lingual development.

Wholemount and section in situ hybridization

Whole or sectioned tissue was washed in PBST andtreated for 10–20 minutes with proteinase K (10 µg/ml inPBST). It was washed twice in PBST and refixed for 20minutes in 4% PFA. Embryos were then prehybridized for2 hours at 42°C in hybridization buffer containing 50%formamide; 5 3 hybridization buffer (20 3 buffer 5 3 MNaCl, 100 mM ethylenediamine tetraacetic acid, 100 mM1,4-Piperazinediethane sulfonic acid, pH 6.8); 1 3 Den-hardt’s; 250 µg/ml sheared salmon sperm DNA; 250 µg/mlpoly-A; and 0.1% Tween-20. Digoxigenin-labeled sense andantisense riboprobes were produced from pBluescript IIvectors as described elsewhere (Hui, 1994; Goodrich et al.,1996). Details about the probes are listed in Table 1.Hybridization was performed overnight at 60°C in hybrid-

TABLE 1. In Situ Hybridization Probes Used in the Study

Probe Length From

Sonic hedgehog 640 bp L. Goodrich, Stanford UniversityPatched 841 bp L. Goodrich, Stanford UniversityGli1 800 bp A. Joyner, New York UniversityGli3 1.6 kb A. Joyner, New York University

144 J.M. HALL ET AL.

ization buffer containing 5.5% dextran sulphate and 0.2–0.5 µg/ml riboprobe. Excess probe was removed by sequen-tial washes in 2 3 standard saline citrate (SSC; threetimes at 60°C), 0.2 3 SSC (three times at 60°C), and1:1 0.2 3 SSC:0.1 M phosphate buffer (PB) and PB (twice).Nonspecific binding in the tissue was blocked for 1–2 hoursin 10% sheep serum, 4% dry milk, 2 mg/ml bovine serumalbumin, and 0.3% Triton X-100 in 0.1 M PB. After thistreatment, the tissue was incubated overnight with antidi-goxigenin antibody conjugated to alkaline phosphatasediluted 1:1,000 in blocking solution. Excess antibody wasremoved by washes in 0.1 M PB, and the tissue wasequilibrated with color buffer containing 100 mM Tris, pH9.5; 50 mM MgCl2; 100 mM NaCl; and 0.1% Tween 20.Antibody was visualized by using the 4-Nitro blue tetrazo-linium chloride/5-Bromo-4-chloro-3-indolyl-phosphate(NBT/BCIP) blue color reaction. Prior to photography, thetissue was refixed in 4% PFA. Following hybridization,some wholemount tongues were cryoprotected by incuba-tion in 25% sucrose in 4% PFA overnight and then cut on acryostat into 20 µM sections.

PGP 9.5 immunohistochemistry

After rehydration and sectioning, some tongues wereanalyzed for presence of the neuronal marker PGP 9.5(ubiquitin carboxyl terminal hydrolase). Sections wererinsed in 0.1 M PB and blocked for 1 hour in 10% donkeyserum in 0.1 M PB plus 0.3% Triton X-100. Incubation withprimary rabbit anti-PGP 9.5 (1:1,000; Biogenesis, Poole,United Kingdom) occurred overnight at 4°C in a humidi-fied chamber. After rinsing with 0.1 M PB, sections wereincubated with lissamine rhodamine sulfonyl chloride(LRSC)-conjugated donkey anti-rabbit immunoglobulin G(1:100; Jackson, West Grove, PA) for 1–2 hours at roomtemperature. Slides were rinsed with 0.1 M PB andcoverslipped with Fluoromount G (Southern Biotechnol-ogy Associates, Birmingham, AL). Immunofluorescenceand Nomarski images were collected with an OlympusFluoview confocal microscope (Tokyo, Japan).

All figures were composed using Adobe Photoshop soft-ware (version 4.0.1; Adobe Systems, Mountain View CA)with only overall adjustment of levels and color balance foreach image. Immunofluorescence/Nomarski overlay im-ages were created within Photoshop by using layering effects.

RESULTS

Lingual morphogenesis in the mouse

The basic progression of mouse tongue development canbe seen in Figures 1 and 2. Our studies in mice confirmthat, as described by Paulson et al. (1985), murine tonguedevelopment is morphologically similar to that describedin rat (Slavkin et al., 1989; Farbman and Mbiene, 1991;Mbiene et al., 1997). A tongue bud is first visible early on E12and quickly becomes a distinct tongue by E12.5. It lengthensand takes on both lateral and dorsal-ventral curvature duringE13–E14, achieving the adult spatulate shape by E16.

Lingual expression patterns of Shh signalingpathway members

Shh. Figure 1A,B shows the results of wholemount insitu hybridization for Shh in developing tongues of em-bryos ages E12 and E12.5. At E11, there is no distinguish-able tongue, and the hybridization signal from the oral

floor is not significantly above background (not shown).Early on E12, a tongue primordium is visible, and Shh isexpressed diffusely throughout this early tongue. A darkstaining region of higher Shh expression is present justanterior to the foramen caecum, corresponding to thefuture location of the circumvallate papilla. Broad lingualexpression persists as the tongue grows until aroundE12.5. At this time, Shh expression is comparativelydiminished along the midline of the tongue, and areas ofhigher expression can be seen in rows parallel to the longaxis of the tongue. This is the same pattern and location inwhich the first fungiform papillae develop (Paulson et al.,1985; Farbman and Mbiene, 1991). Sections of hybridizedtongues at this age show that Shh expression is localizedto lingual epithelial cells and is absent from the underly-ing mesenchyme (Fig. 3A). The region of dark staining atthe location of the developing circumvallate papilla per-sists. As the late E12 tongue grows, Shh expressioncontinues to be focused into the developing papillae.

During E13 and E14, the tongue takes on the curvatureand shape of a fully developed mouse tongue. At E13, therows of higher Shh expression at the presumed sites offungiform papillae, which are seen first at E12.5, havecondensed into more discrete regions of expression (notshown). Beginning at E13.5 and continuing throughoutgestation, Shh expression is tightly localized to the regionsof developing fungiform and circumvallate papillae (Fig.2A). The broad Shh expression seen previously has dimin-ished and is not significantly above background outside oftaste papilla regions. Sections of hybridized tongues at thisstage show that Shh expression is localized to small groupsof columnar epithelial cells that are five to seven cellsacross (Fig. 3C) and that apparently correspond to theplacodal thickenings that eventually give rise to fungiformpapillae (Mistretta, 1991).

The restricted Shh expression in taste papillae persiststhrough the end of gestation. From E16 onward, Shh isexpressed in the central core of the developing papillaryepithelium (Figs. 2E, 4A). Older tongues are not amenableto wholemount in situ hybridization, but hybridization tosectioned tissue shows that Shh expression remains local-ized to the central set of cells on the surface of thefungiform papillae through E18 (Fig. 4E).

Ptc. The expression of Ptc in the developing tonguecorrelates with that of Shh, as observed in other systems(Goodrich et al., 1996). The results of wholemount in situhybridization with Ptc antisense RNA probes are shown inFigures 1 and 2. Ptc expression is not detectable abovebackground during fusion of the mandibular arches atE11.5 (not shown). Broad Ptc expression occurs through-out the tongue primordium early on E12. By E12.5,expression is obviously higher in the regions that will formtaste papillae (Fig. 1D). Thus, in the earliest stages oftongue development (E12–E13), the pattern of Ptc expres-sion mirrors that of Shh. In sections of tongues at thesestages, Ptc expression is present in both the lingualepithelium and its underlying mesenchyme (Fig. 3B). Thisis the first time at which Shh and Ptc are expressed indifferent cell populations within the tongue.

Ptc expression, similar to Shh expression, continuesover time to coalesce into regions around taste papillae(Fig. 2B). By E13.5, significant Ptc expression is presentonly in regions of developing taste papillae. However, theexpression pattern around these papillae is distinct fromthat of Shh. The regions of Ptc expression near the

Shh, Ptc, AND Gli1 IN DEVELOPING TASTE PAPILLAE 145

Fig. 1. Expression of sonic hedgehog (Shh), patched (Ptc), and Gli1in early stages of tongue development in mice. Dorsal views ofembryonic day 12 (E12) [crown-rump (C-R) length 7–8 mm; A,C,E],and E12.5 (C-R length 8–9 mm; B,D,F) tongues assayed for expressionof these genes by wholemount in situ hybridization. Anterior is to theright. A: E12 jaw showing broad lingual Shh expression as well as Shhexpression in the presumptive circumvallate papilla (arrow) and

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


Fig. 2. Expression of Shh, Ptc, and Gli1 in intermediate andlate-stage developing tongues of mice. A–C: Dorsal views of E14 (C-Rlength 10.5–11.2 mm) and E14.5 (C-R length 11–12 mm) tonguesassayed for Shh, Ptc, and Gli1 expression by wholemount in situhybridization. A: E14.5 tongue showing Shh expression predomi-nantly in developing circumvallate (arrow) and fungiform (arrow-heads) papillae. B: E14.5 tongue Ptc expression. C: E14 tongue Gli1expression. Both Ptc and Gli1 expression mirror Shh expression. D–G:Dorsal views of E16.5 (C-R length 15.0–16.5 mm) tongues assayed for

Shh and Ptc expression by wholemount in situ hybridization. D: E16.5tongue showing Shh expression predominantly in circumvallate (ar-row) and fungiform papillae. E: Detail of fungiform papilla showingShh expression. F: E16.5 tongue showing Ptc expression. G: Detail offungiform papilla showing Ptc expression. Note the larger area of Ptcexpression compared with Shh expression and the lower Ptc expres-sion in the central core of the papilla. Scale bars 5 500 µm in A–D,F, 50µm in E,G.

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

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

Fig. 3. Shh and Ptc are expressed in different tissue types in thetongues of early and intermediate stage mice. Sagittal sections ofE12.5 (A,B) and E14 (C–F) tongues assayed for Shh expression (A,C,E)or Ptc expression (B,D,F) by in situ hybridization. A: Shh is expressedthroughout the lingual epithelium at E12.5. B: Ptc is expressed in boththe lingual epithelium and the mesenchyme. C: E14 tongue showing

Shh expression in a developing fungiform papilla. D: At E14, Ptc isexpressed in the fungiform epithelium and in the underlying mesen-chyme. E: Shh expression in E14 circumvallate papillary epithelium.F: Ptc is expressed in E14 circumvallate epithelium and mesenchyme.Scale bars 5 50 µm.


expression extends beyond the columnar epithelial cells ofthe developing fungiform papillae laterally 10–15 µm inthe lingual epithelium and vertically 5–10 µm into themesenchymal cells of the tongue (Fig. 3D).

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

Fig. 4. Shh and Ptc are expressed in taste papillae throughoutgestation. Sagittal sections of tongues from E15–E18 mice assayed forShh and Ptc expression by in situ hybridization. A: E16 tongueshowing 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 thecircumvallate epithelium. D: E16.5 tongue showing both epithelialand mesenchymal Ptc expression around the circumvallate papilla.E: Shh expression at the surface of an E18 fungiform papilla. F: Ptc isalso expressed in E18 fungiform papillae. Scale bars 5 50 µm.


In the fungiform papillae, Ptc expression at E16.5 isclearly in an annular pattern around each papilla: lower inthe 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 developingpapilla (Fig. 2E). Sectioned tongues show that Ptc expres-sion continues to be localized to a larger region withinfungiform papillae, extending both laterally and verticallybeyond where Shh is expressed. Lower Ptc expression isseen in the epithelium in the center of the developingpapillae, consistent with the annular pattern seen inwhole tongues (Fig. 4B). Ptc expression in the circumval-late papillae is in both the epithelium and the underlyingmesenchymal 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 respon-sible, at least in part, for cellular responses to Shh (Martıet al., 1995; Hammerschmidt et al., 1997). In the tongue,Gli1 expression mirrors that of Ptc shown in Figures 1 and2. Gli1 is expressed broadly in the early tongue primor-dium at E12 and is localized progressively to taste papillaein a pattern resembling that of Ptc. Gli1 expression doesnot decrease in nonpapillary regions as quickly as Shh orPtc; some broad Gli expression persists through E13 (Fig.2C). At later stages of lingual development, Gli1 expres-sion exists in an annular pattern surrounding each fungi-form papilla, again similar to Ptc (not shown).

Gli3 is another member of the Gli transcription factorfamily. It is not thought to be involved in Shh response andcan act antagonistically to Shh and Gli (Marigo et al.,1996b). We do not see Gli3 expression above backgroundlevels in the tongue.

Innervation of developing taste papillae

The time course of innervation of papillary epitheliumfrom E13 to E16 was examined by immunohistochemistryfor the neuronal marker PGP 9.5 (ubiquitin carboxylterminal hydrolase). At E13, nerve fibers can be seengrowing toward the surface of the anterior tongue and justbelow the circumvallate placode (Fig. 5A–D). Nerves havenot yet reached the epithelium at this time. At E14,immunofluorescence extends to the basement membranejust below developing fungiform and circumvallate papil-lary epithelium (Fig. 5E–H). The sites of nerve growth tothe epithelium are restricted to the developing circumval-late and fungiform papillae; no fibers appear to be innervat-ing nonpapillary epithelium. Nerves begin to penetrateinto the papillary epithelium by E15 (Fig. 6A–D). By E16,the entire circumvallate and central core of the fungiformpapillary epithelia are densely innervated by nerve fibers(Fig. 6E–H).

Taken together with the in situ hybridization datapresented above, it appears that Shh expression is local-ized to taste papillae prior to nerve fiber contact with thelingual epithelium. Shh expression is localized specificallyto the developing circumvallate papillae as early as E12and to the developing fungiform papillae by E13.5. Nervefibers are not present at the papillary epithelium until E14and do not innervate the entire papillary epithelium untilE16.

DISCUSSION

Development of lingual taste receptors can be dividedinto two stages. First, taste papillae form on the tongue ina characteristic spatial and temporal pattern. This processoccurs from E12.5 to E16 in mice and requires severalsteps: an inductive event, specifying the locations of tastepapillae; proliferation and evagination of the developingpapillary epithelium; and growth of a supportive mesenchy-mal core (Farbman and Mbiene, 1991; Mistretta, 1991).Second, taste buds develop within the papillary epitheliumfollowing papillary morphogenesis. This process may re-quire an additional inductive event to specify taste recep-tor cell differentiation and must be coordinated withinnervation of the lingual epithelium (Farbman and Mbi-ene, 1991; Oakley, 1991). The lingual expression patternsof Shh signaling pathway members relative to morphologicstages are summarized in Figure 7. These results indicatethat Shh may function in both stages of gustatory develop-ment.

Shh signaling in papillary morphogenesis

Expression of Shh, Ptc, and Gli1 begins broadly in thetongue primordium early on E12 and becomes progres-sively localized to regions in and around taste papillae.Shh is detectable only in epithelial cells throughout lin-gual development, and its expression is located exclusivelyin the center of fungiform and circumvallate papillae byE13.5. Ptc expression has a slightly different distributionthan Shh: it is expressed in larger regions surroundingdeveloping fungiform papillae than Shh. Ptc expression islower in or absent from the Shh-expressing centers offungiform papillae at later stages of papillary develop-ment. Ptc also is expressed in mesenchyme underlyingShh-expressing areas throughout papillary morphogen-esis. Both Ptc and Gli1 are activated transcriptionally inresponse to Shh in tissues adjacent to Shh signalingcenters (Goodrich et al., 1996; Marigo et al., 1996a,c;Sasaki et al., 1997). Thus, expression of Ptc or Gli1indicates those cells or tissues that are actively respondingto the Shh signal. Because Ptc is expressed both within thelingual epithelium and in the underlying mesenchyme,Shh may play a role in papillary morphogenesis by signal-ing to one or both of these tissues.

In the epithelium, Shh may be involved in establishingpapillary boundaries. This situation is exemplified inDrosophila, in which Hh is responsible for specifyingembryonic parasegment borders (Kalderon, 1995). Recipro-cal signals between Hh and wingless establish two adja-cent rows of cells that follow separate developmental fates.In the same way, reciprocal signals between Shh andanother signaling molecule, produced by lingual epithe-lium around the perimeter of the developing papillae, mayestablish a boundary for papillary development. The tightlocalization of Shh to an easily identified set of papillaryepithelial cells suggests that a boundary formation orrestriction process is active in papillary development.

Alternatively, Shh could specify papillary boundarieswithin the lingual epithelium in a concentration-depen-dent manner as in the developing neural tube. In neuraltube development, high levels of Shh from the notochordpromote floor plate differentiation, whereas lower concen-trations induce motor neuron cell fates (Roelink et al.,1995). Shh may act in a similar fashion by specifyingpapillary epithelial growth and differentiation at a certain


Fig. 5. Lingual innervation at E13 and E14. Protein gene product9.5 (PGP 9.5; ubiquitin carboxyl terminal hydrolase) immunofluores-cence (left column) and Nomarski image overlays (right column)showing the extent of papillary innervation. A–D: E13 tonguesshowing developing fungiform (A,B) and circumvallate (C,D) papillae.

The lingual epithelium is not innervated at this time. E–H: E14tongues showing developing fungiform (E,F) and circumvallate (G,H)papillae. Nerve fibers have reached the basement membrane belowthe forming papillae. Scale bars 5 50 µm.


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

fibers have begun to penetrate the papillary epithelium. E–H: E16tongues showing fungiform (E,F) and circumvallate (G,H) papillae.The papillary epithelium is densely innervated by this time. Scalebars 5 50 µm.


threshold concentration. In this model, cells at the bound-aries of the developing papillae would follow nonpapillaryfates as the Shh concentration decreases with increasingdistance from Shh-expressing centers.

Shh might also play a role in the epithelial-mesenchy-mal interactions of papillary morphogenesis. Papillarydevelopment requires coordination between epitheliumand mesenchyme, which grows and differentiates to formthe supportive core of the papilla (Farbman and Mbiene,1991; Mistretta, 1991; Fujimoto et al., 1993). Expression ofPtc in the mesenchyme underlying developing taste papil-lae indicates that this tissue is responding to the epithelialShh signal. The epithelial Shh signal could be responsiblefor induction or support of growth of the mesenchymal coreof taste papillae.

Shh-mediated epithelial-mesenchymal interactions arewell known in other systems. During tooth formation, Shhis expressed in the epithelial enamel knot, which acts as asignaling center for organization of dental epithelial-mesenchymal interactions (Vaahtokari et al., 1996). Epithe-lial Shh expression with corresponding mesenchymal Ptcexpression also has been observed in developing whiskerbarrels (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 mannersimilar that of taste papillae, in that they begin as placodalepithelial thickenings and gain a mesenchymal core asthey grow (Fristrom, 1988). The involvement of Shh inepithelial-mesenchymal interactions in such similar struc-tures supports the idea that it is involved also in epithelial-mesenchymal signaling in taste papillae.

Shh in taste bud determination

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

epithelium of the circumvallate papilla. This distributioncoincides with the eventual locations of taste buds. Tastebuds form postnatally, initially within the dorsal epithe-lium of the circumvallate papilla and in the centers offungiform papillae (Mistretta, 1991). These are the samesites at which Shh expression concentrates on E16.5 infully formed lingual papillae. Thus, Shh may be involvedin the morphogenesis of taste buds.

Support for this proposed role of Shh comes from studiesof neurotrophic factors within the developing tongue. Thedistribution of lingual Shh expression after E15.5 matchesthat of brain-derived neurotrophic factor (BDNF) at thesame stage (Nosrat and Olson, 1995). BDNF is believed tosupport gustatory nerves that grow into developing tastebuds (Nosrat et al., 1996; Zhang et al., 1997). If it isassumed that BDNF is an early marker of differentiationof the gustatory epithelium, then Shh is in the right placeat the right time to be responsible for specification orsupport of gustatory development. However, Shh probablyis not solely responsible for taste bud histogenesis, becausethat process requires innervation to proceed normally inmammals (Oakley, 1991; Fritzsch et al., 1997; Mbiene etal., 1997; Oakley et al., 1998). Shh could be inducingcompetence to form taste receptor cells within the centralpapillary epithelial cells, which then differentiate furtherdue to interaction with gustatory nerves.

Taste papilla patterning

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

Fig. 7. Time line of lingual development. Comparison of expressionpatterns of Shh, Ptc, and Gli1 with lingual innervation and morphol-ogy. At E12, a tongue bud is visible with broad epithelial Shhexpression and broad epithelial and mesenchymal Ptc expression. Thetongue is a distinct structure by E12.5. At E12.5, expression of allthree genes has cleared from the midline and exists in parasagittalrows along the tongue as well as in the location of the futurecircumvallate papilla. Prior to and at E13, nerves have not reached thelingual epithelium. From E13 to E16, the tongue elongates and takeson adult shape. At E13.5, Shh is found only in developing papillary

epithelium. Ptc is expressed in papillary precursor epithelium andmesenchyme. Expression of Shh and Ptc remains restricted to tastepapillae from this time onward. At E14, nerve fibers have reached thebasement membrane below developing papillae. At E15, papillae arevisible on the tongue, and nerve fibers begin to penetrate the papillaryepithelium. Papillae are densely innervated by E16. At E16 andbeyond, Shh is expressed only in the central epithelial core offungiform papillae, whereas Ptc is found in a wider region surroundingthe center of each papilla.


taste receptor cells in the tongue may be dependent on theformation of taste papillae.

Taste papillary morphogenesis is a process intrinsic tothe tongue. In rats, taste papillae develop in the normalspatial and temporal sequence in the absence of innerva-tion (Farbman and Mbiene, 1991; Mbiene et al., 1997), soneural induction is not responsible for taste papilla pattern-ing. This conclusion is supported by comparing the timecourse of papillary innervation to Shh expression (Figs. 2,5). Discrete localization of Shh to developing papillaeoccurs by E12.5 in the presumptive circumvallate papillaand by E13.5 in fungiform papillary precursors. However,nerve fibers have not yet reached the lingual epithelium byE13 and extend only to the level of the basement mem-brane at E14. So, Shh expression in the papillary precur-sor regions is established prior to their innervation. Thus,papillae are specified independent of neuronal influence.

If papillary development is initiated independentlywithin the tongue, then how is the pattern established? IfShh were patterning papillae by self-restriction, then onewould expect to see an evenly-spaced, random distributionof papillae. The actual distribution of taste papillae is tooregular and stereotyped for this. The process of papillapatterning, as monitored by Shh expression, occurs in atleast two steps. The first step is restriction of precursorregions to parasagittal rows along either side of thetongue. Following this, individual papillae are generatedwithin these rows. This is reminiscent of the patterning offeathers in birds, another type of epithelial specialization.Feather buds also arise as Shh-expressing placodal thick-enings in a patterning process intrinsic to the epidermalepithelium (Chuong, 1993; Nohno et al., 1995). Prior toexpression of Shh within feather placodes, Shh and Fibro-blast Growth Factor 4 (FGF-4) are expressed in a striperunning along the dorsal midline. This stripe then changesto become discrete Shh-expressing regions that formfeather buds (Jung et al., 1998). This process is thought tooccur through epithelial-mesenchymal interactions thatare mediated by Shh and other signaling molecules (Junget al., 1998).

A similar process involving Shh may be responsible fortaste papilla patterning. Taste papilla, feather, and toothdevelopment are all alike in that these structures begin asplacodal thickenings and then evaginate or invaginate toform a raised structure with a mesenchymal core (Fris-trom, 1988; Lumsden, 1988; Mistretta, 1991; Chuong,1993; Fujimoto et al., 1993). In addition, Shh is expressedin tooth and feather primordia as well as in taste papillaprecursors (Bitgood and McMahon, 1995; Nohno et al.,1995; Fig 1A). Feather position is thought to be specifiedby interaction between FGF-4, Bone Morphogenic Pro-teins (BMP-2 and BMP-4), and Shh (Jung et al., 1998).Recent studies also indicate that tooth position is specifiedby overlap of mesenchymal FGF-8 and BMP signals (Neu-buser et al., 1997). A patterning process involving overlapof several intercellular signals may be active in thedeveloping tongue. Investigation of the role of these othersignaling families in the tongue may help to furtherexplain papillary morphogenesis and lingual development.

ACKNOWLEDGMENTS

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

plasmids, to A. Joyner at New York University for the Gli1and Gli3 clones, and to L. Barlow for critical reading andcomments on this paper. We also thank A. Ribera for use ofher laboratory facilities and photographic equipment. Thiswork was supported by NIDCD grant DC00244 to T.E.F.and training grant GM08497 to J.M.H.

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