Joshua M. Hall
Cell & Developmental
Biology
Comprehensive Examination Proposal
Contents:
Mammalian lingual taste buds develop solely within specialized gustatory
papillae. These gustatory papillae develop in mice between the 12th and 16th
embryonic days in a specific spatial and temporal pattern: parasagittal rows of
fungiform papillae arise on anterior portion of the tongue, while a single
circumvallate papilla forms more posteriorly on the midline. Initial
investigation of the expression patterns of the developmental signaling
molecules Sonic hedgehog (Shh), its receptor Patched (Ptc), and the
Shh-activated transcription factor Gli1 shows the genes for these molecules to
be specifically expressed within developing fungiform and circumvallate
papillae. This suggests a role for Shh in papillary patterning and/or
development. The focus of the proposed study is to examine the role of Shh in
papillary development. Specifically, this work will address the hypothesis that
Shh directs the growth and morphogenesis of taste papillae.
Aim 1: Characterize the expression patterns of Shh signaling pathway members and possible interacting signaling molecules during lingual and papillary development in vivo and in vitro.
Taste papillae develop on cultured tongue primordia explanted from embryonic
rats and mice with normal spatial and morphological characteristics. The spatial
and temporal patterns of Shh,
Ptc and Gli1 expression in intact embryos and explant tongue
cultures have been examined by in situ hybridization. In addition, the
patterns of BMP4 and BDNF expression will be determined by the
use of transgenic mice expressing LacZ markers for these two genes. Expression
patterns should correlate with each other and with papillary development.
Aim 2: Determine the effects of exogenous activation of Shh signaling during lingual development.
Shh signaling will be ectopically activated in vitro by placement of
Shh-saturated beads onto cultured tongue explants. Beads will be placed at
various locations on the tongue and at various stages of lingual development.
Papillary morphogenesis will be monitored both grossly and by examination of
expression of Ptc, BMP4 and BDNF. Ectopic activation of
Shh signaling in vivo will be achieved through injection of
Shh-expressing retrovirus into the mouth of developing embryos. These
experiments will test whether Shh plays an inductive role in papillary and/or
taste bud development.
Aim 3: Determine the effects of inactivation of Shh signaling during lingual development.
Shh signaling will be blocked in vitro by general or focal
application of cyclopamine or anti-Shh antibodies to explant tongue cultures.
Cyclopamine is a steroidal alkaloid compound that blocks Shh signaling
downstream of receptor binding. To complement this, Shh signaling will also be
blocked in vivo by administration of cyclopamine to pregnant mice at
appropriate times in gestation. Combined with the experiments from Aim 2, these
experiments should determine the definitive action of Shh in papillary and
gustatory development.
Upon successful completion of the above experiments, we should have a clearer understanding of the early steps in the development of the mammalian taste system. In addition to expanding our knowledge of the development of one of our basic sensory systems, this work can serve as a model for sensory organogenesis. Many vertebrate organs develop in a fashion similar to taste papillae. Taste papillae, teeth, feathers and whiskers all form through epithelial-mesenchymal interactions and are innervated subsequent to induction of morphogenesis. The mechanisms at work in one of these systems may also play a part in the development of any of the others.
Many specialized organs are formed from epithelial sheets. In vertebrates,
these include feathers, teeth, and lingual papillae. Development of each of
these epithelial specializations involves a complex series of cellular
interactions. First, a set of cells within a relatively undifferentiated
epithelium must be specified to become a particular organ. Next, these cells
must begin to form that organ. Additionally, the differentiating cells must
interact constantly with underlying mesenchymal cells to ensure the proper
growth and development of the entire organ.
Taste Papillae
Four types of lingual papillae are present on the tongues of mammals: fungiform, circumvallate, foliate, and filiform. Each type of papilla has distinct morphological characteristics and distribution pattern along the tongue. Of these types, fungiform, circumvallate and foliate papillae contain taste buds responsible for taste transduction and are referred to as taste papillae. Filiform papillae are thought to serve tactile functions in the tongue (Nosrat et al. 1996) and fill in the epithelium between the other papillae.
Development of taste papillae occurs prenatally, from 6-9 weeks gestation in humans and from E12-E16 in mice (Mistretta 1991). Development of these structures occurs in a very specific spatial and temporal pattern, leading to the suggestion that an organ patterning process is at work during tongue development. Fungiform papillae form in parasagittal rows on the anterior portion of the tongue. The medial rows of fungiform papillae form prior to more lateral ones and within these rows the more anterior papillae develop first (Farbman and Mbiene 1991). The single circumvallate papilla forms on the midline at the oral-pharyngeal border of the tongue. The foliate papillae form on the lateral edge of the tongue just anterior to the oral-pharyngeal border.
Although each type of papilla is morphologically distinct, the initial
events in their development are histologically similar (Mistretta 1991;
Fujimoto et al. 1993). The papillae begin as
placodal thickenings from local increased proliferation of epithelial cells. The
papillary epithelium then begins to grow down into the underlying mesenchyme and
evaginates into a raised structure. At the same time, nerve bundles of the
trigeminal and facial nerves begin to grow into the tongue and reach the lingual
epithelium. Taste buds form from local papillary epithelium and appear at about
the time of parturition (Mistretta 1991;
Barlow and Northcutt 1995;
Stone et al. 1995). The normal development of
taste buds appears to require innervation in rodents (Fritzsch et al. 1997;
Oakley et al. 1998). However, studies of
cultured explants of embryonic rat tongue have shown that papillary
morphogenesis is not dependent on innervation (Farbman
and Mbiene 1991; Mbiene et al. 1997).
Papillary Patterning
The patterned epithelial morphogenesis of taste papillae resembles development of other specialized epithelial structures, particularly teeth and feather buds. In development of both of these structures, there are many intercellular signaling interactions which specify the organ pattern and induce morphogenesis (Chuong 1993; Kratochwil et al. 1996). One signaling molecule known to operate in both feather and tooth development is Sonic hedgehog (Shh) (Bitgood and McMahon 1995; Nohno et al. 1995; Vaahtokari et al. 1996). Shh signaling occurs through the membrane-bound Patched (Ptc) receptor and has been shown to be involved in many aspects of vertebrate development (reviewed in Hammerschmidt et al. 1997 and discussed below). Recent studies in mice have reported expression of Shh in the tongue during the period of papillary morphogenesis (Bitgood and McMahon 1995). In preliminary studies described below, we have confirmed Shh expression in the tongue, and have found Shh to be exclusively localized to developing taste papillae. We have also seen expression of Ptc in the same regions. These findings suggest that Shh signaling may play a role in papillary morphogenesis.
Other signaling molecules also are expressed in developing taste papillae.
Brain-derived neurotrophic factor (BDNF) is expressed in the epithelium of
developing taste papillae in rats and is thought to support incoming gustatory
innervation (Nosrat and Olson 1995). As this
signaling molecule is also expressed in fully-formed taste buds (Nosrat, Ebendal et al. 1996), expression of BDNF
is thought to be an early marker of gustatory epithelial differentiation. A
member of the bone morphogenic protein family of signaling molecules,
BMP4, is also expressed in developing taste papillae (Bitgood and McMahon 1995). Like Shh, both of
these molecules may be involved in papillary patterning, induction and/or
morphogenesis. It is not clear which, if any, of these molecules is first
expressed in presumptive papillary regions and is therefore a candidate for
inducing papillary formation.
Sonic hedgehog as a developmental signal
Shh was first identified in mice on the basis of its homology with the Drosophila signaling molecule hedgehog (hh) (Echelard et al. 1993) and is well conserved across vertebrate species, including human, rat, chicken, Xenopus, and zebrafish (Hammerschmidt et al. 1997). Initial examination of Shh found it to be highly expressed by the notochord and posterior aspect of the limb bud, embryonic tissues thought to act as organizers for body patterning (Echelard et al. 1993).
Shh from the notochord is responsible for dorsal-ventral patterning of the developing central nervous system (CNS) and somites. Ectopic expression of chick Shh in mice and rat Shh in Xenopus causes ectopic differentiation of neural tube cells into floor plate, normally a ventral structure (Echelard et al. 1993; Roelink et al. 1994). Additionally, purified Shh induces floor plate at high concentrations and motor neurons, another ventral CNS structure, at low concentrations in cultured chick neural plate explants (Roelink et al. 1995). In somites, ventral cell fates are induced by, and dorsal fates antagonized by, diffusible Shh signal from the notochord as evidenced by ectopic Shh application and millipore barrier experiments with mouse presomitic mesoderm explants (Fan and Tessier-Lavigne 1994).
Another system in which the organizer function of Shh has been well studied is vertebrate limb development. In the limb bud, Shh is expressed in the posterior region that corresponds to the classically identified zone of polarizing activity (ZPA). Grafting of Shh-expressing quail cells to chick limb induces digit duplications identical to ZPA grafting, indicating Shh is the posteriorizing signal in the developing limb (Chang et al. 1994). Shh plays an important organizational role involving reciprocal interactions with other signaling centers in the developing limb. Shh is involved in a feedback loop with FGF-4 produced by the apical ectodermal ridge thought to be responsible for proximo-distal limb pattern specification (Laufer et al. 1994). Thus, in limb, CNS and somite development, Shh plays an important signaling role by specifying body and organ axes.
In developing teeth, hair, and feathers, Shh is thought to play a slightly
different signaling role, but it is still involved in reciprocal interactions
between tissues. Like taste papilae, these organs develop from epithelial
placodes and then evaginate or invaginate to form a distinct structure with a
mesenchymal core (Fristrom 1988;
Lumsden 1988; Chuong
1993). In each of these, Shh is expressed early on in the placodal
epithelium of the developing organ (Bitgood and
McMahon 1995;
Nohno et al. 1995;
Iseki et al. 1996). Shh is believed to be
involved in the epithelial-mesenchymal signaling that directs the formation of
these structures and involves other signaling molecule families such as
fibroblast growth factors (FGFs) and bone morphogenic proteins (BMPs) (Koyama et al. 1996;
Vaahtokari et al. 1996;
Jung et al. 1998).
Mechanisms of Shh signaling
Much of what is known about the mechanisms of Shh signaling was found initially in studies of Drosophila hedgehog and has been confirmed in vertebrate systems. Upon expression, both Hh and Shh undergo signal sequence cleavage and autoproteolysis into a 25-27 kD C-terminal domain and a 19-20 kD N-terminal domain (Lee et al. 1994; Bumcrot et al. 1995). All known long- and short-range Shh signaling activity is dependent on the N-terminal 19 kD Shh domain, even though both the N- and C-terminal domains are secreted by Shh-expressing cells (Bumcrot et al. 1995; Lopez-Martinez et al. 1995; Martí et al. 1995). During autoproteolysis, a lipophilic moiety is added to the N-terminal domain, which has been shown to be a cholesterol molecule in Drosophila (Porter et al. 1996). Resemblance of embryonic lethal Shh knockout mice to holoprosencephalic mice born to mothers treated with cholesterol synthesis inhibitors argues for a similar cholesterol requirement in vertebrate Shh function (Chiang et al. 1996; Porter et al. 1996).
Hh signaling is mediated, at least in part, by the Patched (Ptc) protein (reviewed in Tabin and McMahon 1997). Ptc is an integral membrane protein that binds to the Shh N-terminal protein as the Shh receptor (Hahn et al. 1996; Marigo et al. 1996). Upon binding Shh, Ptc protein inhibits the function of another membrane protein, Smoothened (Smo) (Stone et al. 1996). Inhibition of Smo triggers downstream events that activate transcription of Shh target genes, one of which is Ptc itself (Marigo et al. 1996). Thus, Ptc expression is one immediate effect of Shh signaling.
Transcription of Hh target genes is dependent on the Cubitus interruptus transcription factor which has three vertebrate homologs, designated Gli1, Gli2 and Gli3. Initial studies of Gli1 expression indicated that it is expressed in Shh target tissues (Hui et al. 1994). More recently, Gli1 expression patterns have been shown to be upregulated by Shh like Ptc (Marigo et al. 1996). Also, ectopic expression of an active form of Gli1 has shown that Gli1 can activate Ptc expression in chick embryos and HNF-ß expression in rat neural stem cells, both targets of Shh signaling (Marigo et al. 1996; Sasaki et al. 1997). These results strengthen the argument that Gli1 is responsible for Shh response in vertebrates.
Another factor involved in Shh signaling is the cAMP-dependent protein
kinase A (PKA), which appears to be a negative regulator of Shh that acts
independently of Ptc (reviewed in Kalderon 1995).
Pharmacologic activators of PKA antagonize the effects of Shh on presomitic
mesoderm explants (Fan et al. 1995).
Conversely, expression of dominant-negative PKA regulatory subunits in mouse and
zebrafish activates transcriptional targets of Shh signalling (Concordet et al. 1996;
Epstein et al. 1996). At this point it is
unclear what the developmental signals are that regulate PKA, but it clearly
plays a critical role in Shh signaling.
Possible roles of Shh in taste papilla development
As discussed above, the most well-characterized functions of Shh are induction of ventral cell fates in CNS and somitic mesoderm and specifying the anterior-posterior axis of the developing limb. With Shh expressed in developing taste papillae, what could its role be in the tongue? Specification of overall tongue pattern or polarity is unlikely since Shh is seen in individual papilla progenitors. A more likely possibility is that Shh is somehow helping to establish or specify papillary cell fates. Like Shh from the notochord, Shh may be inducing papillary-type epithelium. Alternatively, papillary morphogenesis may be induced by another signal and each papilla may be using Shh to repress papillary development in the immediate vicinity. Since Shh is known to function in short-range (ZPA, floor plate) and long-range (motor neurons, sclerotome) signaling, both of the roles proposed above for Shh are possible and may coexist. A third possiblity is that Shh may be involved in the epithelial-mesenchymal interactions of papillary development. Finally, Shh may be responsible for specifying differentiation of gustatory epithelium and taste buds, independent of papillary morphogenesis.
The experiments proposed here are intended to determine which, if any, of
these functions Shh has in papillary development and test the hypothesis that
Shh directs the growth and morphogenesis of taste papillae. Delineation of Shh
function in this system will allow deeper understanding of the development of
the mammalian taste system. Because mammalian taste buds develop exclusively in
taste papillae, papillary morphogenesis is an important first step in formation
of this major sensory system. Additionally, investigation of papillary
development may reveal mechanisms of organogenesis applicable to other
epithelial structures. Shh expression has been detected in developing
teeth, hair and whiskers (Bitgood and McMahon 1995),
all of which form from epithelial invaginations or evaginations similar to taste
papillae (Fristrom 1988). Thus, the proposed
work can provide insight into specific and general mechanisms of organogenesis.
As an initial means of determining the role of Shh in lingual and papillary development, a detailed study of the expression patterns of Shh, Ptc, and Gli1 has been done on embryonic tongues, E11-E18. Both whole and sectioned tongues were analyzed by non-radioactive in situ hybridization. Results from this study are presented in Hall et al. (in press), also available online at http://www.uchsc.edu/rmtsc/finger/josh/jcnpaper.
Expression of Shh, Ptc, and Gli1 begins broadly in the tongue primordium early in the 12th embryonic day and becomes progressively localized to regions in and around taste papillae. Shh is detectable only in epithelial cells throughout lingual development. Expression of Shh changes from its initial broad pattern and is predominantly expressed in the center of fungiform and circumvallate papillae by E13.5. Shh expression within the central cells of the papillary epithelium continues through at least E18. Ptc and Gli1 expression is similar to that of Shh. Ptc expression, however, has a slightly different distribution than Shh as it is expressed in larger regions surrounding developing fungiform papillae than Shh. Ptc is expressed in the cells underlying Shh-expressing areas throughout papillary morphogenesis. At later stages of papillary development, Ptc expression is lower in, or absent from, the Shh-expressing centers of fungiform papillae.
Both Ptc and Gli1 are transcriptionally activated in response to Shh in tissues adjacent to Shh signaling centers (Goodrich et al. 1996; Marigo et al. 1996; Marigo et al. 1996; Sasaki et al. 1997). Thus, expression of Ptc indicates those cells or tissues that are actively responding to the Shh signal. Because Ptc is expressed both within the epithelium and in the underlying mesenchyme of developing papillae, Shh may play a role in papillary morphogenesis by signaling to one or both of these tissues. Also, the location of Shh expression at later stages in tongue development corresponds to the eventual locations of lingual taste buds. These 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.
Although papillae develop in a patterned manner in denervated tongue
explants (Farbman and Mbiene 1991;
Mbiene et al. 1997), to confirm intrinsic
patterning of lingual epithelium the relationship of innervation to
Shh expression was investigated using antibodies to the pan-neuronal
marker, PGP 9.5 (ubiquitin terminal hydrolase). Nerve fibers grow towards
papillary placodes during lingual development, but do not contact the basement
membrane until E14, after the presumptive papillary pattern of Shh
expression has been established. Papillae are densely innervated by E16. This
result supports the conclusion that neuronal induction is not responsible for
patterning papillary development.
Aim 1: Characterize the expression patterns of Shh signaling pathway members and possible interacting signaling molecules during lingual and papillary development in vivo and in vitro.
In order to determine the role of Shh signaling in papillary development, we
must first provide a detailed description of the expression patterns of the
components of the Shh pathway and other markers of papillary development.
Particular attention will be given to the time course of changes in expression
patterns as papillae form. In addition, it will be important to determine which
cell types, and which subgroups of these, are expressing Shh pathway members as
well as the interrelated signaling molecule BMP4 and the neurotrophin BDNF.
Preliminary studies described above have shown the lingual expression patterns
of Shh, Ptc and Gli1 in E11-E18 embryos. A closer
characterization of the expression patterns of BMP4 and BDNF in
comparison to Shh expression and the characterization of expresion of
all of these genes in vitro is still needed.
a.) BMP4 and BDNF
The lingual expression patterns for each of these genes will be studied
using transgenic animals expressing the ß-galactosidase gene, lacZ, in
place of one of the alleles for the respective gene. BDNF-lacZ mice have been
given to us by the Jones lab, University of Colorado and BMP4-lacZ mice were a
gift of the Hogan lab, Vanderbilt University. Visualization of ß-galactosidase
using X-gal is a common means of assessing gene expression in such transgenic
animals, both on a gross and histologic level (Gossler
and Zachgo 1993). In situ hybridization (ISH) of digoxigenin-labeled
antisense RNA probes for BMP4 and BDNF will be used to confirm
the pattern of gene expression in embryonic tongues. Using transgenic lacZ
animals has an advantage over ISH in that it is much faster and easier to
visualize expression and leaves tissue morphology more intact. Additionally, the
X-gal staining can be combined with ISH, allowing comparison of expression of
BMP4 or BDNF with Shh or other genes (Tajbakhsh and Houzelstein 1995). Both
BMP4 and BDNF are known to be expressed in developing fungiform
and circumvallate papillae (Bitgood and McMahon
1995; Nosrat and Olson 1995), and B.
Hogan, unpublished data). However, it is not known when either gene begins to be
expressed in developing papillae. If either gene is expressed in a punctate
fashion prior to Shh, ~E12.5, it may serve as a potential inductive
signal for Shh or papillary patterning and morphogenesis.
b.) Establishment and characterization of an in vitro model of mouse papillary development
In order to test hypotheses concerning the role of Shh signaling in
papillary morphogenesis, it is important to have a model developmental system in
which Shh signaling can be manipulated. Growth and development of rat tongue
primordia can be supported for up to one week in organ culture. These tongues
develop fungiform and circumvallate papillae with normal morphology and
distribution (Farbman and Mbiene 1991;
Mbiene et al. 1997). The rat tongue culture
system will be adapted to support murine E12-E14 tongue primordia and ISH for
Shh and Ptc will be used to confirm its similarity to in
vivo development. Normal expression of BMP4 and BDNF will be
confirmed by culture of transgenic, lacZ expressing enbryonic tongues.
Alternatively, if development of embryonic mouse tongues cannot be supported in
culture or does not appear normal, the ISH can be performed on the cultured rat
tissue (Roelink et al. 1994;
Nosrat and Olson 1995). In either case, it is
expected that the expression patterns of each of the genes investigated will
match those seen in vivo.
Aim 2: Determine the effects of exogenous activation of Shh signaling during lingual development.
Classically, studies of developmental signaling look to determine the
phenotypic effects of exogenous activation or blockade of that signaling
mechanism. These experiments will provide a test of the inductive capabilities
of Shh in papillary and taste bud morphogenesis.
a.) In vitro
Developing limb bud and tooth culture systems have used agarose beads soaked
in Shh protein as exogenous activators of Shh signaling (Lopez-Martinez et al. 1995;
Hardcastle et al. 1998). N-terminal Shh
protein will be produced by bacterial cells and purified according to (Lopez-Martinez et al. 1995). Affi-gel beads
(Bio-Rad) will be saturated with the bacterially-produced protein and placed on
tongue cultures within the developing lingual epithelium. Control experiments
will include: no bead application; non-protein saturated bead application; and
application of beads impregnated with albumin. Variables for bead placement will
include the location of the bead, placement of the bead on or within the lingual
epithelium, as well as the embryonic age at which bead cultures are started.
Bead-cultured tongues will be assayed by ISH for Ptc expression
(normally induced by Shh) to test whether the exogenous Shh is acting in its
normal signaling capacity and also whether the surrounding epithelium is
responsive to Shh signal. Bead placement on cultured BDNF-lacZ or BMP4-lacZ
tongues will allow expression of these genes to be assayed as a marker for taste
papillary differentiation. If Shh is an inductive signal for papillary
morphogenesis, papilla-like structures, with approprate patterns of BMP4
and BDNF expression, should form in close proximity to the bead or one
large papilla should develop at the exact location of the bead. Also, Shh should
induce BDNF expression if it is the inducer of papillary development. If
Shh is involved in epithelial-mesenchymal interactions, mesenchymal growth or
condensation may occur below the bead without attendant epithelial changes.
Alternatively, if Shh is involved in specifying papillary patterning, the high
concentration of Shh near the bead may disrupt the normal spacing of papillae
such that there is an abnormally wide area devoid of papillae around the bead.
This result would suggest that Shh is involved in sending some sort of lateral
inhibitory signal. Finally, Shh-induced expression of BDNF without
attendant morphological changes may indicate a role for Shh in specifying
gustatory epithelial differentiation. Combinations of these results may occur as
well, suggesting a dual role for Shh signaling in the tongue. Absence of
response to Shh beads at later stages may indicate that there is a "window"
of Shh responsiveness in the tongue similar to that proposed for feather
development (Jung et al. 1998). Absence of
any effect of exogenous Shh will argue against a role for Shh in papillary
morphogenesis.
b.) In vivo
One method that has been used to induce ectopic gene expression in mouse
embryos is infection of with retroviral vectors (Soriano
et al. 1986;
Price 1993). A particularly useful retroviral
vector has been engineered which cause infected cells to express Shh as
well as green fluorescent protein (GFP) as a lineage marker (Fan et al. 1997). These replication-deficient
vectors infect murine epithelial cells with high efficiency (P. Khavari,
personal communication) and will be used to induce ectopic
Shh expression in the embryonic tongue. To test whether the virus
infects mouse tongue, virus will be applied to cultured tongue explants and
these will be assayed for Shh and GFP expresion. Virus application to
cultures will also serve as an alternative means of ectopic activation of Shh
signaling in vitro and should yield results similar to Shh bead
placement. After confirming lingual infection, appropriate amounts of virus will
be injected intra-amniotically into the developing mouth at E11-12 according to
(Price 1993), producing a limited number of
ectopic Shh-expressing loci on the embryonic tongue. Embryonic tongues
will then be collected at E13, E14, and E16 and analyzed for papillary
morphology as well as Shh and Ptc expression. Virally-induced
Shh expression can be delineated from endogenous Shh by the GFP
lineage tracer. This will provide an in vivo correlate to the ectopic
Shh application described above and should achieve similar results. Ectopic
expression of Shh in utero can also be used to assess Shh involvement in taste
bud development. Infected embryos can be allowed to develop to P1-2, when taste
buds normally appear in the tongue. If Shh is responsible for differentiation of
gustatory epithelium and taste buds, then extra-papillary taste buds may develop
in these mice.
Aim 3: Determine the effects of inactivation of Shh signaling during lingual development.
Complementary to activation of Shh signaling, blockade of Shh will further
test the role of Shh in lingual and papillary development. These experiments
will show if Shh is necessary for papillary or taste bud development.
a.) In vitro
Anti-Shh antibodies have been used in neural tube explant culture to block
Shh signaling (Martí et al. 1995).
Such antibodies will be applied to cultured tonges both generally and focally.
Approptiate amounts of antibody will be added to the culture media of some
cultured tongues to block Shh signaling througout the tongue, while agarose
beads saturated with anti-Shh antibody will be placed within the epithelium of
other tongues to block Shh more locally. A second means of inhibiting Shh signal
is the use of the steroidal alkaloid cyclopamine, which blocks Shh signaling
downstream of Ptc receptor binding (Incardona et
al. 1998). Cyclopamine will be applied to tongue cultures in the same manner
as anti-Shh antibodies -- both generally in the culture media and focally using
agarose beads. Activation of protein kinase A (PKA) is yet another method of
blocking Shh signaling in vitro (Fan et al. 1995).
Forskolin will be added to culture medium to increase intracellular cAMP levels
and thereby activate PKA. This will cause a global blockade of Shh signaling
downstream of Ptc binding. Control experiments will include application of other
antibodies to the tongue, application of non-Shh blocking steroidal alkaloids,
such as veratramine, and addition of the inert compound 1,9-dideoxyforskolin to
culture media. Tongues will again be assayed for Ptc expression to
verify the absence of Shh signaling. Because Ptc expression is activated
by Shh, absence of Ptc expression will confirm Shh signal blockade. BDNF
and BMP4 expression will also be monitored using cultured transgenic
tongues to indicate taste papillary specfication and/or differentiation. Both
methods of blocking Shh signal should yield similar results. Lack of papillary
development will suggest that Shh does indeed play an inductive role in this
system. If BDNF or BMP4 is expressed in presumptive papillary
regions, but no papillary growth occurs, Shh will be implicated in papillary
growth and differentiation only and not establishing papillary patterning. If
Shh is involved with papillary border specification or lateral inhibition of
papillary growth, then these experiments should show a greater number of closely
spaced papillae. Absence of effects will again argue against Shh involvement in
papillary morphogenesis.
b.) In vivo
Cyclopamine is known to have teratogenic effects on sheep, hamsters, and chicks which resemble the effects of Shh mutation or deletion in humans and mice (Binns et al. 1963; Keeler 1978; Chiang et al. 1996; Incardona et al. 1998). Administration of cyclopamine to gastrulation-stage embryos blocks Shh signaling and prevents normal neural tube patterning (Incardona et al. 1998). Cyclopamine will be delivered to pregnant mice begining just slightly before, and at the time of, tongue morphogenesis, E11-16, in order to block lingual Shh signaling. Embryonic tongues will be collected and assessed for papillary morphogenesis as well as expression of Ptc, BDNF, and BMP4. Results should be similar to those seen in vitro and will confirm the role for Shh determined using the explant culture system. Some cyclopamine-administered mice will be allowed to develop to P1-2 and will be assayed for the presence of taste buds both morphologically and by immunohistochemical markers. If taste buds are present on the tongue, with or without papillae, then Shh is not required for differentiation of gustatory epithelium. If taste buds are absent, but papillae present, then Shh is more likely involved in later stages of gustatory development and required for taste bud differentiation. Lack of both taste buds and papillae will suggest a role for Shh in papillary morphogenesis, but will not clarify the role of Shh in taste bud formation as it is not clear whether lingual taste bud formation is dependent on papillary formation.
An alternative strategy for blocking Shh signaling is administration of
cholesterol synthesis inhibitors to pregnant mice as cyclopamine blocks Shh
signaling independent of cholesterol synthesis (Incardona
et al. 1998). Using this method of blocking Shh makes timing the actual
point of Shh inhibition more difficult as embryos will have to use up their
cholesterol stores before Shh signaling is affected. Also, inhibition of
cholesterol synthesis may have other metabolic effects on lingual development
which mask the effects of Shh blockade. However, if the timing of adminstration
of these drugs is worked out, similar results to those of cyclopamine
administration would be expected.
| Aim 1. Expression studies | ||
| a.) BMP4 and BDNF | 3 months | |
| b.) Establishment and characterization of culture system | 5 months | |
| Aim 2. Activation of Shh | ||
| a.) In vitro | 6 months | |
| b.) In vivo | 6 months | |
| Aim 3. Inhibition of Shh | ||
| a.) In vitro | 6 months | |
| b.) In vivo | 4 months | |
| TOTAL PROJECTED TIME: | 30 months | |
Barlow, L. A. and R. G. Northcutt (1995). "Embryonic origin of amphibian taste buds." Developmental Biology 169: 273-285.
Binns, W., et al. (1963). "A congenital cyclopian-type malformation in lambs induced by maternal ingestion of a range plant, Veratrum californicum." Am. J. Vet. Res. 24: 1164-1174.
Bitgood, M. J. and A. P. McMahon (1995). "Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo." Developmental Biology 172: 126-138.
Bumcrot, D. A., et al. (1995). "Proteolytic processing yields two secreted forms of sonic hedgehog." Molecular & Cellular Biology 15: 2294-2303.
Chang, D. T., et al. (1994). "Products, genetic linkage and limb patterning activity of a murine hedgehog gene." Development 120: 3339-3353.
Chiang, C., et al. (1996). "Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function." Nature 383: 407-413.
Chuong, C. (1993). "The making of a feather: homeoproteins, retinoids and adhesion molecules." Bioessays 15: 513-521.
Concordet, J. P., et al. (1996). "Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning." Development 122: 2835-2846.
Echelard, Y., et al. (1993). "Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity." Cell 75: 1417-1430.
Epstein, D. J., et al. (1996). "Antagonizing cAMP-dependent protein kinase A in the dorsal CNS activates a conserved Sonic hedgehog signaling pathway." Development 122: 2885-2894.
Fan, C. M., et al. (1995). "Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway." Cell 81: 457-465.
Fan, C. M. and M. Tessier-Lavigne (1994). "Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog." Cell 79: 1175-1186.
Fan, H., et al. (1997). "Induction of basal cell carcinoma features in transgenic human skin expressing Sonic Hedgehog." Nature Medicine 3: 788-792.
Farbman, A. I. and J. P. Mbiene (1991). "Early development and innervation of taste bud-bearing papillae on the rat tongue." Journal of Comparative Neurology 304: 172-186.
Fristrom, D. (1988). "The cellular basis of epithelial morphogenesis. A review." Tissue & Cell 20: 645-690.
Fritzsch, B., et al. (1997). "Mice with a targeted disruption of the neurotrophin receptor trkB lose their gustatory ganglion cells early but do develop taste buds." International Journal of Developmental Neuroscience 15: 563-576.
Fujimoto, S., et al. (1993). "Pre- and postnatal development of rabbit foliate papillae with special reference to foliate gutter formation and taste bud and serous gland differentiation." Microscopy Research & Technique 26: 120-132.
Goodrich, L. V., et al. (1996). "Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog." Genes & Development 10: 301-312.
Gossler, A. and J. Zachgo (1993). Gene and enhancer trap screens in ES cell chimeras. In Gene Targeting: A practical approach. A. L. Joyner, Ed. Oxford, IRL Press: 181-228.
Hahn, H., et al. (1996). "A mammalian patched homolog is expressed in target tissues of sonic hedgehog and maps to a region associated with developmental abnormalities." Journal of Biological Chemistry 271: 12125-12128.
Hammerschmidt, M., et al. (1997). "The world according to hedgehog." Trends in Genetics 13: 14-21.
Hardcastle, Z., et al. (1998). "The Shh signalling pathway in tooth development: defects in Gli2 and Gli3 mutants." Development 125: 2803-2811.
Hui, C. C., et 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 postimplantation development." Developmental Biology 162: 402-413.
Incardona, J. P., et al. (1998). "The teratogenic veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction." Development 125: 3553-3562.
Iseki, S., et al. (1996). "Sonic hedgehog is expressed in epithelial cells during development of whisker, hair, and tooth." Biochemical & Biophysical Research Communications 218: 688-693.
Jung, H.-S., et al. (1998). "Local inhibitory action of BMPs and their relationships with activators in feather formation: Implications for periodic patterning." Developmental Biology 196: 11-23.
Kalderon, D. (1995). "Morphogenetic signalling. Responses to hedgehog." Current Biology 5: 580-582.
Keeler, R. (1978). "Cyclopamine and related steroidal alkaloid teratogens: their occurrence, structural relationship, and biologic effects." Lipids 13: 708-715.
Koyama, E., et al. (1996). "Polarizing activity, Sonic hedgehog, and tooth development in embryonic and postnatal mouse." Developmental Dynamics 206: 59-72.
Kratochwil, K., et al. (1996). "Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development." Genes & Development 10: 1382-1394.
Laufer, E., et al. (1994). "Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud." Cell 79: 993-1003.
Lee, J. J., et al. (1994). "Autoproteolysis in hedgehog protein biogenesis." Science 266: 1528-1537.
Lopez-Martinez, A., et al. (1995). "Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage." Current Biology 5: 791-796.
Lumsden, A. G. (1988). "Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ." Development. Supplement 103: 155-169.
Marigo, V., et al. (1996). "Biochemical evidence that patched is the Hedgehog receptor." Nature 384: 176-179.
Marigo, V., et al. (1996). "Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development." Developmental Biology 180: 273-283.
Marigo, V., et al. (1996). "Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb." Development 122: 1225-1233.
Martí, E., et al. (1995). "Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants." Nature 375: 322-325.
Mbiene, J. P., et al. (1997). "Organ cultures of embryonic rat tongue support tongue and gustatory papilla morphogenesis in vitro without intact sensory ganglia." Journal of Comparative Neurology 377: 324-340.
Mistretta, C. M. (1991). Developmental neurobiology of the taste system. In Taste and Smell in Health and Disease. T. V. Getchell, Ed. New York, Raven Press: 35-64.
Nohno, T., et al. (1995). "Involvement of the Sonic hedgehog gene in chick feather formation." Biochemical & Biophysical Research Communications 206: 33-39.
Nosrat, C. A., et al. (1996). "Differential expression of brain-derived neurotrophic factor and neurotrophin 3 mRNA in lingual papillae and taste buds indicates roles in gustatory and somatosensory innervation." Journal of Comparative Neurology 376: 587-602.
Nosrat, C. A. and L. Olson (1995). "Brain-derived neurotrophic factor mRNA is expressed in the developing taste bud-bearing tongue papillae of rat." Journal of Comparative Neurology 360: 698-704.
Oakley, B., et al. (1998). "The morphogenesis of mouse vallate gustatory epithelium and taste buds requires BDNF-dependent taste neurons." Brain Res. Dev. Brain Res. 105: 85-96.
Porter, J. A., et al. (1996). "Cholesterol modification of hedgehog signaling proteins in animal development." Science 274: 255-259.
Price, J. (1993). Introduction of genes using retroviral vectors. In Essential Developmental Biology: A Practical Approach. C. D. Stern and P. W. H. Holland, Eds. Oxford, IRL Press: 179-190.
Roelink, H., et al. (1994). "Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord." Cell 76: 761-775.
Roelink, H., et al. (1995). "Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis." Cell 81: 445-455.
Sasaki, H., et al. (1997). "A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro." Development 124: 1313-1322.
Soriano, P., et al. (1986). "Tissue-specific and ectopic expression of genes introduced into transgenic mice by retroviruses." Science 234: 1409-1413.
Stone, D. M., et al. (1996). "The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog." Nature 384: 129-134.
Stone, L. M., et al. (1995). "Taste receptor cells arise from local epithelium, not neurogenic ectoderm." Proceedings of the National Academy of Sciences of the United States of America 92: 1916-1920.
Tabin, C. J. and A. P. McMahon (1997). "Recent advances in Hedgehog signalling." Trends in Cell Biology 7: 442-446.
Tajbakhsh, S. and D. Houzelstein (1995). "In situ hybridization and beta-galactosidase: a powerful combination for analysing transgenic mice." Trends Genet 11: 42.
Vaahtokari, A., et al. (1996). "The enamel knot as a signaling center in the developing mouse tooth." Mechanisms of Development 54: 39-43.