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First published online April 10, 2009
doi: 10.1242/10.1242/dev.029090
1 Department of Cell and Developmental Biology and the Rocky Mountain Taste and
Smell Center, University of Colorado School of Medicine, Aurora, CO 80045,
USA.
2 Department of Anatomical Sciences and Neurobiology, University of Louisville
School of Medicine, Louisville, KY 40292, USA.
* Author for correspondence (e-mail: linda.barlow{at}ucdenver.edu)
Accepted 2 March 2009
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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Key words: Taste bud development, CreER, Shh, Wnt1Cre, Bdnf, Genetic inducible, Fate mapping, Tamoxifen, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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):
fungiform papillae are arrayed on the anterior tongue, whereas foliate
papillae and a single circumvallate papilla in rodents reside posteriorly.
Taste receptor cells, like neurons, possess voltage-gated channels, generate
action potentials, and release neurotransmitter onto post-synaptic nerves
(Finger et al., 2005;
Roper, 1992). However, unlike
neurons or other receptor cell types, taste cells continually regenerate in
adults (Beidler, 1953;
Beidler and Smallman, 1965;
Farbman, 1980;
Delay et al., 1986).
Furthermore, taste buds differ from other sensory receptors developmentally,
arising from local epithelia rather than from neurogenic ectoderm
(Barlow and Northcutt, 1995;
Stone et al., 1995). Taste
buds are thus unique among sensory receptor cells with characteristics of both
epithelia and neurons.
Each taste bud comprises a heterogeneous population of 
100 cells
belonging to three differentiated cell types (I, II and III) that can be
identified via expression of marker proteins. Type I cells, assumed to be
support cells, express a specific ectoATPase, NTPDase2
(Bartel et al., 2006). Types II
and III are taste receptor cells proper; type II cells express the
transmembrane receptors and signaling machinery to transduce sweet, bitter and
umami tastes (Clapp et al.,
2001; Miyoshi et al.,
2001), whereas type III cells are sour detectors
(Huang et al., 2006;
Huang et al., 2008) and
function as relay cells (Yang et al.,
2000a; Roper,
2007). These three taste cell types are maintained by continuous
renewal from the progenitor population, the characteristics of which remain
unclear. However, birthdating analyses suggest that taste cells arise from
either adjacent basal epithelial cells, intragemmal basal cells within taste
buds, or edge cells located laterally outside of taste buds proper
(Beidler and Smallman, 1965;
Delay et al., 1986;
Miura et al., 2006;
Nakayama et al., 2008;
Okubo et al., 2008).
Taste buds differentiate late in embryogenesis, well after the tongue is
innervated. This sequence of innervation followed by differentiation has
suggested that taste buds are induced by nerves. In axolotls, an aquatic
salamander, the taste periphery is organized simply, with taste buds embedded
directly in the epithelium (Takeuchi et
al., 1997). In a test of the neural induction model using axolotl
embryos, taste buds arise without contact by either nerves
(Barlow et al., 1996;
Stone, 1940) or cranial
mesenchyme (Barlow and Northcutt,
1997); rather, specification and patterning of amphibian taste
buds is intrinsic to oral epithelium, and occurs early in development via
cell-cell signaling (Parker et al.,
2004; Barlow,
2001). Analysis of X-inactivation transgenic female mice also
supports an epithelial origin of taste buds in mammals
(Stone et al., 1995).
Restriction of mammalian lingual taste buds to papillae adds a level of
complexity to the development of these composite taste organs. In mice, taste
organ formation begins at mid-gestation, when focal thickenings called taste
placodes arise in the lingual epithelium. Placodes evaginate and develop a
mesenchymal core, transforming morphologically into taste papillae
(Farbman and Mbiene, 1991).
Taste buds differentiate within papillae and begin to express taste cell
type-specific markers around birth (Krimm
and Barlow, 2008). Because placodes transform into papillae,
followed by taste bud differentiation, it has been inferred that taste
placodes develop into taste papillae, which in turn produce taste buds from a
subset of papillary epithelial cells
(Mistretta and Liu, 2006).
However, the precise relationship between placodes, papillae and buds has not
been tackled experimentally.
Discerning how these structures are related to one another is particularly
important in the context of the role of innervation in mammalian taste
development. Taste placodes, and, at least initially, taste papillae develop
independently of innervation (Farbman and
Mbiene, 1991; Hall et al.,
1999; Hall et al.,
2003), whereas taste bud differentiation appears to be nerve
dependent (Oakley and Witt,
2004). These findings have been obtained primarily from short-term
explant culture, where taste placodes and papillae differentiate
morphologically and molecularly, despite a lack of innervation
(Farbman, 1972;
Hall et al., 2003;
Mbiene et al., 1997;
Mistretta et al., 2003;
Nosrat et al., 2001). In
particular, Sonic Hedgehog (Shh) is expressed in early taste placodes, and,
consistent with neural independence, Shh is expressed in taste papillae that
form in cultured lingual explants. However, the fate of the Shh-expressing
cells is unknown, and, thus, Shh has been interpreted to be a marker of taste
papillae, rather than of taste buds (Hall
et al., 1999; Hall et al.,
2003; Liu et al.,
2004).
To define the lineage relationship of taste placodes to taste papillae and
to taste buds, we crossed mice carrying a drug-sensitive Cre recombinase
fusion protein under the Shh promoter
(Harfe et al., 2004) with
R26RLacZ reporter mice (Soriano,
1999), and tracked the fate of taste placode cells in both
embryonic and adult taste organs. We show here that Shh-expressing placodes
are taste bud progenitors, which give rise exclusively to cells within taste
buds but not to taste papillae. Furthermore, Shh-expressing progenitors give
rise to at least two differentiated taste cell types as well as to intragemmal
basal and adjacent edge cells in adult mice. However, this contribution is
transient; within a few months of birth, placodally derived cells are lost
from mature taste buds. We demonstrate that the neural crest does not
compensate for this loss, and does not contribute to taste buds at any stage.
Finally, we show that the specification, patterning and probable early
differentiation of taste bud progenitors are not affected by reduced gustatory
innervation caused by a null Bdnf mutation.
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DISCUSSION
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) were a
gift from Clifford Tabin, Harvard Medical School. R26RLacZ
(Soriano, 1999) and
Wnt1Cre (Danielian et al.,
1998) mice were obtained from Trevor Williams (University of
Colorado School of Dentistry, USA). Bdnf+/- mice
(Jones et al., 1994) were
obtained from Thomas Finger (University of Colorado School of Medicine, USA).
ShhcreERT2 mice are maintained on the C57BL/6 background; the
background of all other lines is mixed. Mice and embryos were genotyped as
described previously (Soriano,
1999; Harfe et al.,
2004; Danielian et al.,
1998; Jones et al.,
1994), and maintained and sacrificed in accordance with protocols
approved by the Institutional Animal Care and Use Committee at the University
of Colorado Denver School of Medicine.
Fate mapping of Shh-expressing taste placodes and lingual mesenchyme
ShhcreERT2 males were crossed with homozygous R26RLacZ
females, and double transgenic progeny were assayed for β-galactosidase.
Embryos and pups with the R26RLacZ allele served as controls. Midday on the
day of an observed plug was considered embryonic day (E) 0.5. Pregnant dams
were dosed intra-peritoneally once between E12.5-E14.5 with 4-5 mg tamoxifen
(T-5648, Sigma), which was prepared and administered as previously described
(Nakamura et al., 2006).
Embryos were recovered on desired days of gestation, and staged according to
Kaufman (Kaufman, 1999). For
fate mapping the lingual mesenchyme, Wnt1cre males were crossed with
homozygous R26RlacZ females, and embryos (E13.5) and adults (6 weeks, 4
months) with both alleles were assayed for β-galactosidase.
In situ hybridization, immunofluorescence and β-galactosidase histochemistry
For Shh mRNA expression, wild-type tongues were fixed overnight in
4% PFA, and processed for in situ hybridization as described
(Hall et al., 1999) with
hybridization of the Shh probe (640 bp, L. Goodrich, Stanford
University) and stringency washes at 62°C. For Shh immunodetection, live
tongues were cultured in mouse anti-Shh (30 µg/ml, 5E1, DSHB) and processed
as described previously (Hall et al.,
2003).
To analyze Cre-mediated recombination, embryos were harvested at various
times after tamoxifen dose and assayed for β-galactosidase (β-gal)
by X-gal staining or processed immunofluorescently with guinea pig
anti-β-gal (1:1000) (Yee et al.,
2003). For whole-mount X-gal, dissected embryonic tongues were
fixed briefly in 0.25% glutaraldehyde and stained in X-gal solution
(Harfe et al., 2004).
Alternatively, embryonic tongues were processed first as whole mounts for
anti-Shh immunofluorescence, followed by sectioning and β-gal
immunofluorescence.
Tongues from P0 pups were fixed in 0.2% PFA overnight and stained for X-gal after sectioning. Some P0 sections were double labeled with anti-β-gal and rat anti-cytokeratin 8 (CK8, Troma-1; 1:25, DSHB). Light fixation obviated antigen retrieval required when tongues were fixed more strongly, but required less dilute antibody (see below).
To detect innervation in Bdnf-/- and +/+ embryos, E14.5 tongues immunostained with anti-Shh were fixed in 4% paraformaldehyde overnight, sectioned and immunostained with the neurite marker rabbit anti-Gap43 (1:200; Chemicon; MAB347), followed by goat anti-rabbit Cy3 antibody (1:500; Jackson Immunoresearch; 111-165-006). For detecting CK8 immunoreactivity at E18.5 in wild-type versus Bdnf-/- tongues, embryos were perfused with 4% paraformaldehyde, their tongues embedded in paraffin, sectioned at 8 µm, mounted on slides, dewaxed, rehydrated and treated with proteinase K (antigen retrieval). Sections were incubated with anti-CK8 at 1:100 for 1 hour at 37°C, followed by biotinylated goat anti-rat antiserum and streptoavidin-Cy3.
Lineage tracing in adult mice
Postnatal animals at 2 and 6 weeks, and 4 and 7 months with both Cre and
R26RLacZ were transcardially perfused with 4% PFA, post-fixed in 4% PFA
overnight at 4°C and cryosectioned (12 µm). Sections were double
immunostained with guinea pig anti-β-gal and taste cell type-specific
rabbit antisera: (1) anti-NTPDase2 (1:1000, a gift from L. G. Lavoie and J.
Se'vigny); (2) anti-PLCβ2 (1:1000, Santa Cruz; sc-206); (3) anti-N-CAM
(1:1000, Chemicon; AB5032); or (4) anti-PGP 9.5 (1:1000, Abd Serotec,
7863-0504). Wnt1cre;R26RLacZ adult tongue sections were immunostained with a
cocktail of NTPDase2, PLCβ2, and NCAM antisera to label simultaneously
all three taste cell types, and guinea pig anti-β-gal antiserum, followed
by the appropriate fluorescently conjugated secondaries (1:500, Molecular
Probes, Invitrogen).
Image acquisition
Bright-field or multichannel fluorescent images were acquired with an
Axiocam CCD camera and Axioplan fluorescence microscope with Axiovision
software (Zeiss, Germany). Z-stack confocal images were acquired at
0.75 µm through 12 µm cryosections using a laser-scanning Olympus
Fluoview confocal microscope with Fluoview Software. Images were saved as
TIFFs, contrast adjusted and cropped, and figures compiled using Adobe
Photoshop CS2.
Data analysis and quantitation
Shh-descendent cells in taste buds or epithelial clones at P0 were counted
in pups treated with tamoxifen at E12.5 (n=3) or E14.5
(n=3). To avoid double counting of 30 µm diameter taste buds at
P0, labeled cells in every third section were counted (3x12 µm=36
µm intervals). Taste buds with β-gal-immunoreactive (IR) cells at 2
weeks (n=3), 6 weeks (n=4) or 4-7 months (n=3)
postnatal were tallied from counts made at every 5th section (5x12
µm=60 µm interval) to avoid overcounting of adult taste buds (50 µm
diameter). The average number of β-gal-IR cells per labeled taste bud at
each postnatal time point was tallied from 20 randomly selected taste buds per
stage across animals. The average number of double-labeled cells for each
taste cell marker (anti-NTPDase, anti-PLCß2, anti-NCAM or anti-PGP9.5,
and anti-β-gal) was tallied at 6 weeks postnatal in 20 taste buds from
each of four animals. Means and standard errors were calculated using
Microsoft Excel.
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To assess the extent of taste placode innervation at E14.5, serial sections immunostained for both Shh and Gap43 were examined and each Shh-labeled papilla was categorized as innervated - anti-Gap43-IR labeled fibers penetrating Shh-labeled epithelial cells or not innervated - Gap43-IR fibers completely absent, or only reaching the papilla core mesenchyme.
The number of differentiating taste buds at E18.5, identified via CK8 immunoreactivity, was quantified in serial sections. Each taste bud was carefully followed through all sections it occupied, such that each taste bud was counted only once. Comparisons between wild-type and Bdnf-/- mice were made using a t-test.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
To discern the fate of these Shh-expressing taste placode cells, Cre
recombination was activated via tamoxifen at E12.5. When treated embryos were
examined at E14.5, β-gal activity was evident in the epithelium of taste
placodes (Fig. 1A,D,E, red
arrows). Extra-placodal epithelial cells were also labeled
(Fig. 1A,D, red arrowheads),
and were presumed to be general epithelial cells, which expressed Shh at E12.5
when tamoxifen was injected, but had turned off Shh by E14.5. Consistent with
this idea (and see below), in embryos given tamoxifen at E13.5 and examined at
E15.5, most labeled cells were limited to taste papillae
(Fig. 1B,F, red arrows), with
many fewer extra-papillary labeled cells
(Fig. 1B,G, red arrowheads). In
addition, sectioned material showed that Shh-descendant cells, even at these
early stages, contribute only a central population of cells in papillae
(Fig. 1F), and that
Shh-descendant cells are densely innervated
(Fig. 1H); both features
suggested that placodes contribute to taste buds, and not papillae. Similar
results were obtained when the palatal epithelium of embryos injected 48 hours
prior was examined (see Fig. S1 in the supplementary material). Importantly,
X-gal reaction product was never observed in embryos lacking Cre
(Fig. 1C). Moreover,
β-galactosidase activity was routinely observed in positive control
tissues where Shh is known to be expressed at E12.5, such as vibrissae and
hair follicles (data not shown)
(St-Jacques et al., 1998).
Shh-descendent cells within taste placodes continue to express Shh, whereas extra-placodal daughter cells cease Shh expression
Although Shh-descendent cells progressively restrict to placodes, many
β-gal-labeled cells were quite close to taste placodes, as well as in
extra-placodal epithelium when Cre was induced at E12.5. Labeling of
extra-placodal cells suggested that these cells were either: (1) being
recruited by and to Shh-expressing placodes, thus contributing to taste organ
genesis; or (2) turning off Shh expression and pursuing a lingual epithelial
fate. Thus, we examined Shh protein expression in both types of Shh-descendent
cells. In placodes, most Shh-descendent cells persisted in expressing Shh
(Fig. 2A-C), although some
cells at the edge of Shh-descendent clusters (β-gal-IR) ceased to express
Shh (Fig. 2A-C, yellow
arrowhead). By contrast, all extra-placodal cells indelibly marked with
β-galactosidase lacked Shh (Fig.
2D,E). We could not determine, however, whether Shh-descendent
cells immediately adjacent to placodes are recruited, or cease Shh expression
and become epithelial.
Shh expressing taste placodes contribute to taste buds and not taste papillae at birth
To identify the postnatal fate of Shh-expressing taste placodes, tongues of
embryos treated with tamoxifen at E12.5 (n=3) or E14.5 (n=3)
were collected at birth (P0). Irrespective of the timing of tamoxifen
treatment, β-gal-expressing cells were confined predominantly to taste
papilla apices. Furthermore, labeled cells resembled taste buds; they formed
onion-shaped clusters, and individual cells had a characteristic fusiform
shape (Fig. 3A-C, white
asterisks). Anti-cytokeratin 8 (CK8), a marker for taste cells
(Zhang et al., 1995;
Asano-Miyoshi et al., 2008;
Okubo et al., 2008), confirmed
that these labeled cells were taste cells. In most taste buds, several
β-gal-IR cells were also CK8-IR (Fig.
3D-F), indicating that Shh-expressing taste placodes give rise to
taste cells at birth. However, double labeling was not complete; some
β-gal-IR cells were not CK8-IR, and vice versa. Cells solely
β-gal-IR probably represent taste cells that had not yet differentiated,
yet were nonetheless descendant from taste placodes. Conversely, cells
immunoreactive only for CK8 could result from mosaic labeling of taste
placodes at E12.5, or could correspond to type III taste cells, which we
failed to label in this study (see below).
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Importantly, although most taste buds in papillae were labeled at P0, and epithelial cell clones were also encountered (Fig. 3G), papillary epithelial cells adjacent to labeled taste buds rarely expressed β-galactosidase. Labeling of non-taste bud regions of fungiform papillae was restricted to the acellular keratinized layer above labeled taste buds (Fig. 3B,C, red arrows), probably owing to epithelial differentiation and ultimate loss of these Shh-descendent cells.
Shh-expressing taste placode cells give rise to at least two differentiated taste cell types, as well as to intragemmal basal and perigemmal edge cells
Mature taste buds comprise three differentiated cell types (I, II and III)
with discrete functions, and are maintained by continuous renewal from a
population of intragemmal basal and/or perigemmal edge cells. Thus, we next
asked whether Shh-expressing taste placodes give rise to all cell types, or
are restricted in the cell type(s) they produce. Mice were examined at 2 weeks
(n=3), 6 weeks (n=4) and 4-7 months (n=3)
postnatally, after tamoxifen treatment at E12.5. The number of labeled taste
buds per tongue at 2 and 6 weeks was strikingly lower than that encountered at
P0, and the number of labeled cells per bud was also reduced as mice aged
(Table 1). Compared with 85%
labeling of taste buds at P0, only 49% and 41% of taste buds were labeled at 2
and 6 weeks, respectively. The number of labeled cells per taste bud also
declined during this period, from six labeled cells per taste bud profile at
P0, to only two cells per bud at 6 weeks. By 4 months, Shh-descendent cells
were virtually absent from taste buds
(Table 1).
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50% of taste cells per bud
(Bartel et al., 2006), and type
II cells represent 
20% of taste cells
(Ma et al., 2007), this double
labeling frequency would seem to infer that taste placodes give rise
predominantly to type II cells. However, we probably undercounted NTPDase-IR
cells descendent from taste placodes: because distinguishing type I cell
bodies from their extensive processes is difficult, as the protein localizes
to the membrane, we only counted double labeled cells when β-gal-IR
filled an NTPDase-IR surround. Thus, we conclude here only that embryonic
Shh-expressing taste placodes give rise to both type I and II taste cells.
Despite the reasonable frequency with which NCAM-IR type III cells and
β-gal-IR cells were detected in taste buds
(Fig. 4J-L), double-labeled
type III cells were never encountered. Using another marker, PGP9.5, which is
expressed by a subset of type II and III cells
(Yee et al., 2001;
Ma et al., 2007), we also did
not find double-labeled cells (Fig.
4G-I). This outcome suggests that either: (1) taste placodes are
lineage restricted, and do not give rise to type III and type II/III PGP9.5-IR
cells; or (2) type III and PGP9.5-IR cells arise from Shh-expressing
progenitors, but the reduction in β-gal-IR cells at 6 weeks
(Table 1), combined with the
relatively sparse distribution of these cell types (<10%)
(Ma et al., 2007), results in
a low probability of detecting double-labeled cells.
In addition to β-gal-IR fusiform taste cells, we encountered β-gal labeled intragemmal basal cells (Fig. 4A-F, arrowheads) and perigemmal edge cells (Fig. 4D-F, double arrows). These cell types were identified via their shape and location, and their lack of taste cell immunomarker expression. For example, β-gal-IR intragemmal basal cells are polygonal and abut the basal lamina (Fig. 4A-F), whereas the β-gal-IR perigemmal edge cells are located along the lateral margin of the taste bud, and are small and crescent shaped (Fig. 4D-F).
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), this study did not extend to postnatal
stages. To test this idea, we indelibly labeled neural crest cells, including
those contributing to tongue mesenchyme
(Chai et al., 2000) in
Wnt1Cre;R26RLacZ mice, and then searched for labeled neural crest cells in
taste buds and papillae at embryonic and adult stages. At E13.5,
X-gal-positive neural crest mesenchyme is clearly demarcated from both the
X-gal-negative lingual epithelium (Fig.
5A,B) and lingual musculature derived from somitic mesoderm
(Fig. 5A, arrows)
(Chai et al., 2000). Staining
of Wnt1Cre;R26RLacZ adult tongues for anti-β-gal and taste cell
immunomarkers at 6 weeks (Fig.
5C,D) and 4 months (Fig.
5E,F) showed that neural crest-derived mesenchyme extends into the
taste papilla core but does not contribute to taste buds or to papillary
epithelium (Fig. 5C-F). At 4
months, in contrast to the loss of Shh-descendent cells in taste buds
(Table 1), there is no loss of
β-gal in neural crest-derived cells of the tongue.
Development of taste bud progenitors in embryos which lose gustatory innervation is indistinguishable from that of controls
Several groups have shown that development of taste placodes is initially
nerve independent; tongue explants develop taste placodes that evaginate as
taste papillae and express Shh
(Hall et al., 2003;
Liu et al., 2004). However,
these cultures do not survive sufficiently long to monitor taste bud
differentiation. Thus, we examined Shh-expressing taste progenitors within
emerging papillae at E14.5 in embryos homozygous null for the gene encoding
the neurotrophin BDNF (Fig. 6),
which lose gustatory innervation early on
(Nosrat et al., 1997;
Krimm, 2007). The
distribution, total number, and size of Shh-expressing taste placodes did not
differ between Bdnf+/+ and Bdnf-/-
embryos at E14.5 (Fig. 6A-F),
yet the number of innervated placodes was dramatically reduced in the
Bdnf-/- embryos (Fig.
6D,G).
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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).
Immature taste buds only become evident in papillary epithelium late in
gestation, with fully differentiated taste buds present within 1 week of birth
(Mistretta, 1991). This
developmental sequence of placodes transforming into papillae, followed by
differentiation of taste buds, has suggested that placodes are papilla
precursors, which subsequently organize taste bud progenitors within the
apical epithelium (Mistretta and Liu,
2006). We show here, using inducible Cre-lox lineage tracing, that taste placodes do not give rise to papillae, but rather contribute almost exclusively to taste buds. At postnatal stages, embryonic Shh-expressing taste placodes are progenitors for the majority of taste cells, giving rise to both type I and II cells, as well as to intragemmal basal and perigemmal edge cells, although we have yet to identify type III cells as being descended from taste placodes. Unexpectedly, Shh-expressing progenitor cells and their descendents, although evident at birth, begin to disappear prior to weaning, and are completely absent in adults. This loss of placodal cells is not compensated for by a neural crest contribution, either embryonically or postnatally. Finally, we show that taste bud progenitors develop in vivo independently of nerve contact, in that specification, patterning and likely initial differentiation of Shh-expressing progenitors cells remains constant, even when BDNF-dependent taste innervation is lost genetically.
Taste bud progenitors are specified early, and may organize subsequent papilla morphogenesis
Taste placodes express several signaling factors, including Shh, Bmp and
Wnt/β-catenin, and their expression continues as taste papillae undergo
morphogenesis (Hall et al.,
1999; Iwatsuki et al.,
2007; Jung et al.,
1999; Liu et al.,
2007). Alteration of each of these pathways during placode
formation and papillary morphogenesis in vitro affects the patterning of taste
placodes and resultant papillae. For example, both Shh and Bmp inhibit
(Hall et al., 2003;
Mistretta et al., 2003;
Zhou et al., 2006), whereas
Wnt/β-catenin promotes placode development, including expression of Shh,
in cultured tongue explants (Iwatsuki et
al., 2007; Liu et al.,
2007). However, as lingual explants do not survive until taste
buds differentiate, this in vitro approach has lead only to the conclusion
that these pathways regulate papilla development
(Mbiene and Roberts,
2003).
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). Thus, the
increased number of Shh-expressing progenitors in embryonic tongues correlates
well with increased taste buds in greatly expanded fungiform papillae observed
at birth. Given our new interpretation of early events in taste bud
patterning, we speculate that Shh-expressing taste bud progenitors may
function as transient signaling centers within the epithelium, which in turn
regulate subsequent development of taste buds and papillae. Intriguingly,
these taste progenitor cells share a number of similarities with known
signaling centers, such as the tooth enamel knot
(Obara and Lesot, 2007), and
the apical ectodermal ridge of the developing limb
(Antalikova et al., 1989).
Specifically, taste placodes express a number of secreted signaling factors
(see above), are mitotically quiescent during embryogenesis
(Farbman and Mbiene, 1991;
Mbiene and Roberts, 2003;
Zhou et al., 2006) and
contribute only transiently to postnatal taste buds (this study).
We must also formally consider, however, that postnatal loss of embryonic
taste progenitors may be an artifact of our fate-mapping technique. The
R26RLacZ mouse is widely used in developmental studies, owing to ubiquitous
expression from the R26 promoter throughout life
(Friedrich and Soriano, 1991;
Zambrowicz et al., 1997). In
studies of thymic development using the Wnt1Cre;R26LacZ mice, neural
crest-derived cells were detected in embryonic but not postnatal thymus
(Jiang et al., 2000;
Yamazaki et al., 2005). In a
recent report, however, Wnt1Cre;R26YFP reporter mice were employed, and neural
crest-derived mesenchyme persisted in adult thymus
(Foster et al., 2008). These
authors suggested that the lacZ gene itself was silenced, owing to
heavy methylation of the lacZ-coding sequence
(Chevalier-Mariette et al.,
2003). To date, a loss of lacZ transcription has not been
reported in epithelial lineage studies (e.g.
Berton et al., 2000). Our
results, moreover, tend to reject an artifactual loss of embryonic taste
progenitors postnatally. First, we see no loss of β-gal in neural
crest-derived cells of the tongue mesenchyme up to 4 months of age, suggesting
that, in lingual tissue immediately adjacent to taste buds, the lacZ
reporter is not silenced. Second, the loss of labeled taste cells in adults
follows a comparable time course across animals, already evident at P14 and
complete by 4 months, implying a regulated, rather than a stochastic,
process.
Are Shh-expressing taste bud progenitors lineage restricted?
Taste buds are a heterogeneous population of three differentiated cells
types (I, II and III), which can be identified via expression of distinct
proteins: type I glial-like cells; type II sweet, bitter and umami detectors;
and type III sour detectors and putative relay cells
(Clapp et al., 2001;
Clapp et al., 2004;
Yang et al., 2000b;
Huang et al., 2006;
Roper, 2006). The entire taste
bud cell population has been estimated to arise embryonically from 7-13
progenitor cells, and then is continually renewed throughout adult life from a
proliferative progenitor pool within the papillary epithelium
(Stone et al., 2002;
Okubo et al., 2008). However,
the cell lineage for taste cells generated at any stage is completely unknown.
One view is that taste cell types represent separate lineages, which remain
distinct throughout the life of each cell
(Farbman, 1965).
Alternatively, taste cells may have a common lineage, with different cell
types representing different stages in the lifespan of a single cell
(Delay et al., 1986). More
recently, type I cells have been proposed to arise from a dedicated lineage,
whereas types II and III have an intermingled relationship, with a subset of
type III cells giving rise to type II cells
(Miura et al., 2006).
Our fate-mapping studies indicate that Shh-expressing embryonic taste bud
progenitors generate both type I and II cells, suggesting that they share a
common embryonic lineage. This leaves the issue of type III cell lineage
unresolved. We cannot rule out the possibility that type III cells may descend
from this same progenitor pool, as we may be looking for a very rare event.
Embryonically labeled progenitor cells are steadily lost postnatally, and
combined with the low frequency of type III cells within taste buds
(Ma et al., 2007), the chance
that double-labeled type III taste cells would be detected is very low (<1
double labeled type III cell per animal). We must therefore look at many more
experimental animals in order to encounter an example of this lineage
relationship. Alternatively, type III cells may have a distinct embryonic
origin.
To address this possibility, we asked whether taste buds receive a cellular
contribution from the neural crest, either embryonically or postnatally as the
taste placode-descendant cells are lost; perhaps type III cells arise uniquely
from neural crest? Although an embryonic neural crest contribution has been
excluded in mouse circumvallate papilla epithelium in early embryos
(Jitpukdeebodintra et al.,
2002), as well as in early development of amphibian taste buds
(Barlow and Northcutt, 1995),
our studies extend these results. Using the Wnt1Cre line, we confirm extensive
neural crest in the lingual mesenchyme
(Chai et al., 2000), including
that of taste papillae; however, in no case and at no time did we observe
neural crest-derived cells within taste buds, let alone in lingual
epithelium.
Although not derived from neural crest, type III cells may develop via
processes that differs from that of types I and II, perhaps because of their
distinct morphology and life history within taste buds. Type III cells are the
only taste cell type to form conventional synapses with sensory neurons
conveying taste information to the CNS
(Roper, 2006;
Yang et al., 2000a), and may
also be the most long-lived cell within buds. Although the average lifespan of
rodent taste cells is 10 days, 4-week-old cells with a type III morphology
have been documented in adults (Hamamichi
et al., 2006). One final explanation for our failure to label type
III cells as placodal descendents is that they may arise from a non-Shh
expressing population, which is induced by signals from the taste placodes.
This recruitment of type III cells or their progenitors could occur
embryonically, or early on in postnatal life. However, they must be recruited
before postnatal day 4, when we first observe differentiated type III cells
(T. Glover, H. Nguyen and L.A.B., unpublished). In any case, the identity of
Type III taste cell progenitors remains unknown.
Neural dependence of taste bud development
In amphibians, development of taste buds can occur without nerves
(Barlow et al., 1996).
Similarly, cultured lingual explants from rodents form taste placodes and
papillae, which express crucial signaling molecules, despite the lack of
innervation (Hall et al.,
2003; Farbman and Mbiene,
1991; Nosrat et al.,
2001; Mistretta et al.,
2003). A number of groups have also used an in vivo genetic
approach to address this issue. Brain-derived neurotrophic factor (BDNF) is
expressed in developing taste papillae
(Nosrat and Olson, 1995;
Nosrat et al., 1996), and loss
of BDNF results in embryonic loss of taste sensory neurons
(Jones et al., 1994;
Liebl et al., 1997). In
tongues of these mutant mice examined 1-2 weeks postnatally, lingual
innervation to taste buds is indeed lost, and taste buds and papillae are
dramatically reduced (Nosrat et al.,
1997). However, we show here that this reported postnatal effect
is probably due to a postnatal role of either Bdnf or neural
maintenance of taste progenitor cells, rather than to an absolute requirement
for either BDNF or nerve contact for taste progenitor induction and
initial taste bud differentiation. Specifically, specification and patterning
of taste bud progenitors does not differ between wild type and
Bdnf-/- mice, despite our finding that most of these
progenitors are not successfully innervated during development. Furthermore, 4
days later at birth, taste cells, as defined by CK8 expression, do not differ
significantly between control and mutant embryos. However, because of the high
variability in CK8-IR taste bud numbers at this later stage in both mutants
and controls, it is possible that gustatory innervation may impact taste bud
progenitor differentiation at this time. Nonetheless, specification and
patterning of mammalian taste bud progenitors, and probably the initial
differentiation of taste buds, are independent of both BDNF and gustatory
innervation.
The development of mammalian taste buds and papillae is typically
considered to be similar to that of other epithelial appendages, such as
teeth, hair follicles and feathers, which require extensive interactions
between epithelium and mesenchyme (Chuong
et al., 2000; Pispa and
Thesleff, 2003). The observation that mammalian taste placodes
give rise to taste buds only, and not to papillae, is reminiscent of taste
organ formation in axolotls, where taste buds lack papillae and are embedded
directly in the lingual epithelium
(Takeuchi et al., 1997).
Development of axolotl taste organs occurs independently of mesenchyme
(Barlow and Northcutt, 1997),
and, moreover, has been shown to be an early epithelium-intrinsic process
(Parker et al., 2004). Thus,
we speculate that mammalian taste progenitors are also specified via an
epithelial event, which is probably independent of mesenchyme.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/9/1519/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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