Chem. Senses 24: 7-17,
1999
© Oxford University Press
The Effects of ß-Bungarotoxin on the Morphogenesis of Taste Papillae and Taste Buds in the Mouse
Department of Orthodontics, University of Florida Dental College, Gainesville, FL, USA
Correspondence to be sent to: Joyce Morris-Wiman, Department of Orthodontics, Box 100444, JHMHC, University of Florida Dental College, Gainesville, FL 32610-0444, USA. e-mail: morris-wiman{at}dental.ufl.edu
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Abstract |
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Although it has been long accepted that innervation by a taste nerve is essential for maintenance of taste buds, it is not clear what role, if any, innervation plays in the morphogenesis of taste papillae and taste bud development. The following study was undertaken to determine what effects lack of sensory innervation have on the development of taste papillae and the formation of taste buds in the mouse. Timed-pregnant female mice (n = 3) at gestational day 12 (gd12) were anesthetized and a 1 µl solution (1 µg/µl) of ß-bungarotoxin (ß-BTX), a neurotoxin that disrupts sensory and motor neuron development, was injected into the amniotic cavity of two embryos per dam. Two shams were injected with PBS. Fetuses were harvested at gd18, 1 day before birth, and four ß-BTX-injected embryos, two shams and two controls were fixed in buffered paraformaldehyde. Serial sections were examined for the presence and morphology of taste papillae and taste buds. No nerve profiles were observed in ß-BTX-injected tongues. Although circumvallate papillae were present on ß-BTX tongues, only five fungiform papillae could be identified. Taste buds were present on a large percentage of fungiform papillae profiles (24% and on circumvallate papillae in sham and control fetuses; in contrast, no taste buds were associated with taste papillae in ß-BTX fetuses. These results implicate a significant role for innervation in taste papillae and taste bud morphogenesis.
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Introduction |
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Taste buds on the dorsal surface of the tongue are only found in association with taste papillae. Thus taste papillae are unique in the tongue in their ability to support the formation and to maintain taste buds. Evidence exists that taste papillae play a major role in determining the number, location and size of taste buds, as well as their maintenance Mistretta et al., 1988
; Mistretta, 1991; Robinson and
Winkles, 1991; Whitehead et al., 1998). A recent
study has determined the origin of taste bud cells to be the local epithelium of the gustatory
papillae (Stone et al., 1995), further linking taste papillae and
taste bud morphogenesis. However, what factors might control morphogenesis of taste papillae
or taste buds have yet to be totally elucidated. It is generally accepted that the presence of taste
nerves is required for the maintenance of taste buds and taste papillae in the adult. Disruption of
normal innervation to the taste papillae results in total loss of taste buds and the formation of
keratinized spines on papillae, reminiscent of those on filiform papillae (Oakleyet al., 1990). However, it is unclear what role innervation plays in taste bud
induction and maturation, or in the formation and differentiation of taste papillae.
Evidence from several studies provides support for a nerve-independent initiation of taste
papillae formation. The initial stages of taste papillae development occur before nerve fibers
come into contact with the dorsal tongue surface (Farbman and Mbiene, 1991; Whitehead and Kachele, 1994). Embryonic tongue
fragments explanted to organ culture before nerve ingrowth have been reported to form
fungiform papillae (Farbman and Mbiene, 1991; Mbieneet al., 1997). However, in another study neural presence was required for the
differentiation of circumvallate papillae (J. Morris-Wiman et al., submitted for
publication ). Evidence for a nerve-dependent initiation of taste bud formation also comes from
several sources. Although taste papillae will form in culture in the absence of innervation, taste
buds do not (Farbman and Mbiene, 1991; Mbiene et al., 1997). Taste bud formation in vivo occurs only after the appearance of
sensory nerves. Hosley and co-workers (Hosley and Oakley, 1987; Hosleyet al., 1987) established a critical period for taste
initiation by demonstrating that the early disruption of taste nerves in postnatal rat pups was
associated with decreased numbers of taste buds in the adult. More recently molecular studies
examining the distribution and effects of nerve growth factors have established that
temporospatial patterns of distribution of the neurotrophic factors, BDNF and NT3 correlate with
taste papillae and bud morphogenesis (Nosrat and Olson, 1995;Nosrat et al., 1996,1997;Zhang et al., 1997). Papillae and taste buds in mice lacking these factors are
malformed and decreased in number. In contrast to the remise that taste buds require innervation
for their maturation and maintenance is work done on amphibian taste bud development (Barlow et al., 1996).
In order to investigate the role of innervation in mouse taste bud and taste papillae
morphogenesis, we have utilized an experimental paradigm first developed by Harris (1981) in which fetuses were injected with ß-bungarotoxin
(ß-BTX) to produce aneural musculature. ß-BTX consists of two subunits, a
phospholipase subunit and a K+ channel binding subunit. The combined
effects of the two subunits are the destruction of sensory and motor neurons (Kwong et al., 1995). To produce aneural tongues, we injected fetuses on
gestational day 12 (gd12) with ß-BTX. At this time taste papilla primordia or taste placodes
have not yet formed on the dorsal surface of the tongue and sensory nerve fibers have not yet
reached the dorsal tongue. It had been previously shown that injection of rat embryos at a
comparable age resulted in the complete destruction of sensory and motor innervation (McCaig et al., 1987). ß-BTX-injected fetuses were
harvested at gd 18 (1 day before birth) and examined for the presence of neural elements, taste
papillae and taste buds. Our results show an almost total lack of fungiform papillae and taste
buds in ß-BTX-injected fetuses, suggesting that innervation plays a major role in the
morphogenesis of these structures. In contrast, circumvallate papillae did form, indicating that
the morphogenesis of these structures may be nerve-independent.
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Materials and methods |
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Bungarotoxin administration
Using the procedure of Wilson and Harris (1993), individual mouse
embryos were injected with ß-bungarotoxin (ß-BTX) on gd12. This age was chosen
because in the rat, injection of this toxin on or later than gd14, an age comparable to gd12.5 in
mice, permanently destroys the entire peripheral nervous system. Timed pregnant mice (n = 3) were anesthetized by MetaFane inhalation. After sterile preparation of the
surgical site on the abdomen, a horizontal incision was made through the skin and the abdominal
layers, exposing the uterine horns. Embryos were counted, numbering from the attachment of the
ovary, and 1 µl of ß-BTX (1 mg/ml in sterile PBS) was injected into the amniotic
cavities of two embryos adjacent to the right ovary (ß-BTX) and 1 µl of sterile PBS
was injected into the amniotic cavities of two embryos adjacent to the left ovary (shams). The
embryos were impaled by hand with pulled micropipettes that had been fractured to produce
diameters in a range of 0.11–0.14 mm and the ß-BTX or saline expelled using a
Narishige microinjection system. Following injections, the abdominal wall of the dam was
closed
with nylon suture. On gd18—1 day prior to birth—the embryos were recovered;
injected embryos were identified from their position within the uterine horn. (The embryos were
recovered before birth because dams eliminate pups that do not suckle.) The embryos were
measured for crown–rump length and examined for evidence of an aneural state. This was
determined using a tail/foot pinch to produce spontaneous activity, including limb and jaw
movements. The effectiveness of ß-BTX was also evaluated by examining the embryos for
wrist drop, cervical kyphosis and a smaller crown rump size. These characteristics have been
previously shown to be associated with the creation of aneural embryos by ß-BTX (Condon et al., 1990). The tongues of sham and injected embryos
were examined for the presence of fungiform and circumvallate papillae.
Histological procedures and analysis
Embryos (four ß-BTX, two shams and two controls) from three different dams were fixed in 4% paraformaldehyde in .01 M phosphate buffer overnight at 4°. Their heads were dehydrated in graded alcohols and embedded in glycol methacrylate (JB-4 plus, Polysciences, Warrington, PA). Two-micron frontal sections were collected at 60 µm intervals and mounted on two sets of subbed slides. One set was stained with Toluidine blue; the other set was silver-stained using a protocol modified from Bielschowsky. Sections were examined for the presence of neural elements, taste papillae and taste buds. Fungiform papillae were identified in sections from sham, control and ß-BTX fetuses based on size and shape. Taste buds were identified as a `ball-like' collection of lightly staining cells within the apical epithelium of fungiform or circumvallate papillae or within the palatal epithelium. For all sections containing taste papillae and taste buds, images were acquired with a CCD low-light digitizing camera (Optronics) and analyzed using Image Pro Plus (Media Cybernetics) analysis software. Specifically, fungiform number and area and taste bud number were measured in ß-BTX, sham and control fetuses. Statistically significant differences among ß-BTX, sham and control fetuses were assessed using an ANOVA for repeated measures and, when significant, multiple post-hoc comparisons were made to determine which groups were different.
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Results |
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Fetuses that had been injected with ß-BTX on gd12 were characterized by a distinctive cervical kyphosis, wrist drop, an elongated snout and a decreased crown–rump length (Figure 1
A, B). They lacked any motor activity in response to tail or foot
pinch and were slightly edematous. They also had palatal clefts, probably as the result of tongue
immobility. Histological examination of silver-stained and Toluidine blue-stained frontal
sections of the heads from ß-BTX-injected fetuses confirmed the absence of both motor and
sensory nerves within the tongue (Figure 2B). In contrast, nerve bundles
corresponding to lingual and hypoglossal nerves were prominent in the body of the tongue of
control and sham fetuses (Figure 2A). Cranial nerve ganglia were
present,
although significantly reduced in size and lacking associated nerve bundles. In addition, the jaw
musculature in effected embryos was undeveloped, accounting for the narrow snouts of these
embryos. ß-BTX exposure had no effect on the formation of salivary glands, the
morphology
of the oral mucosa or tooth formation. There were also no adverse effects seen in cranial bone or
cartilage development, or in the development of whisker or other epidermal-associated structures.
Shams (saline injected) were indistinguishable from uninjected controls in size and showed no
developmental abnormalities either on macroscopic or microscopic examination.
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Effects of ß-BTX exposure on tongue morphogenesis
Tongues from control and sham fetuses at gd18 were indistinguishable in size and morphology.
In contrast, the tongues from ß-BTX-injected fetuses were smaller and appeared flaccid. On
histological examination the dorsal epithelium of ß-BTX-injected fetuses appeared to be
fairly normal, with well-developed filiform papillae (Figure 3C),
although the dorsal tongue surface was thrown into folds. This presence of furrows within the
dorsal mucosa appeared to be the result of the paucity of muscle within the tongue of affected
fetuses. The muscle content of the anterior tongue was similar in ß-BTX-injected fetuses to
control and sham. In all three, muscle fibers in the anterior tongue were interspersed with loose
connective tissue (Figure 3A, B, C). However, in control and sham
fetuses, muscle formed the bulk of the posterior tongue with very little connective tissue
packaging. Muscle formation was severely affected in the posterior tongue of ß-BTX fetuses
as evidenced by the increased number of primary myotubes and the decreased muscle fiber area
as compared with controls (Figure 3D, E). This lack of muscle within the
posterior tongue resulted in n increased flaccidity of the affected tongue and the abnormal tongue
shape characteristic of ß-BTX fetuses.
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Fungiform papillae morphogenesis in ß-BTX fetuses
No differences were observed in either number or morphology of fungiform papillae between
control and sham fetuses at gd18. In both, fungiform papillae could be identified as
mushroom-shaped structures that projected above adjacent smaller filiform papillae (Figure 4A, B). No differences were found in the number of fungiform papillae
that contained taste buds (Figure 6) or in the degree of differentiation
between taste buds from control and sham fetuses (Figure 4A, B). The
tongues of ß-BTX-injected fetuses contained only a few structures that might be called
fungiform papillae at gd18. These were located primarily in the anterior tongue and were
distinguished from adjacent filiform papillae by their slightly enlarged bases. In only one of these
structures did the apical epithelium show any organization reminiscent of the first stages of taste
bud formation (Figure 4C). In this papilla, some specialization of the
apical epithelium, similar to that described for primordial taste buds, was apparent. The mean
area of sham papillae did not differ significantly from the mean of control papillae (Table 1). In contrast, although the sample size was small (n =
5),
the mean area of ß-BTX papillae was ignificantly different from control and sham papillae (P < 0.01).
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Circumvallate papillae morphogenesis in ß-BTX fetuses
The morphology of circumvallate papillae at gd18 was identical in control and sham fetuses
(Figure 5A, B). In both, the core of the papillae was highly vascularized
and well innervated, and numerous taste buds could be identified in the epithelium covering the
papilla apex. Circumvallate papillae also formed in ß-BTX fetuses (Figure 5C). However, these papillae had an elongated apex covered by a thinned epithelium.
More importantly, these papillae lacked taste buds in the apical epithelium at gd18. Although the
papilla core was well vascularized, nerve fibers were absent.
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Palatal taste buds in ß-BTX fetuses
Numerous taste buds were observed in the palatal epithelium of control and sham fetuses at
gd18 (Figure 5
A, B). No differences were found in the morphology or
number of palatal taste buds from control and sham fetuses (Figure 6). In
both, palatal taste buds closely approximated adult taste buds in form. In contrast, only a few
`taste bud-like' cells were identified in the palatal epithelium of
ß-BTX-injected fetuses at gd18 (Figure 5D).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In utero exposure to the neurotoxin ß-BTX resulted in the disruption of normal innervation to the tongue. Affected tongues lacked fungiform papillae and taste buds. In contrast, circumvallate papillae did form, although they also lacked taste buds. These results indicate that the normal morphogenesis of fungiform papillae and taste buds requires the presence of a nerve; in contrast, formation of circumvallate papillae may not.
Effects of ß-bungarotoxin
ß-BTX is one of several neurotoxic proteins contained in the venom of the snake Bungarus multicinctus. It is a heterodimeric protein consisting of a phospholipase subunit
linked by a disulfide bond to a K+ channel binding sub-unit that is a member
of the Kunitz protease superfamily (Kwong et al., 1995). The
mechanism of action of ß-BTX is unclear, but is believed to be the result of the lipolytic
action of the phospholipase targeted to the presynaptic membrane by the Kunitz module.
ß-BTX exposure does not necessarily kill neurons; the destruction of neurons is secondary
to
the destruction of axons and is dependent on the time of injection (McCaig et al., 1987). Evidence exists showing that developing neuronal processes are
particularly sensitive to the toxin (Abe et al., 1976) and that
exposure to ß-BTX during embryonic development can effectively destroy sensory, motor
and sympathetic neurons (Hirokawa, 1978). When rat embryos were
exposed to a single injection of ß-BTX on gd14, all motor and sensory nerves were
destroyed within the limb and trunk musculature (Ross et al.,1987;McCaig et al., 1987;Harris et al., 1989; Ashbyet al. 1993; Wilson
and Harris, 1993). When examined shortly before
birth, the injected fetuses were completely paralyzed and had no histologically identifiable nerve
trunks. In rat embryos injected in utero with ß-BTX, increased motoneuron cell
death corresponded to a time of injection that matched the target-dependent stage of motoneuron
development, indicating a sensitive period for the actions of ß-BTX on nerve fibers (McCaig et al., 1987).
Effects of ß-BTX exposure on fungiform papillae and taste bud morphogenesis
In this study, fetuses exposed to ß-BTX on gd12, when examined 1 day prior to birth at gd18, had aneural tongues. Examination of cranial ganglia showed a decrease in neuronal cell number and the complete absence of projecting nerve bundles. These observations indicate that exposure of gd12 embryos to ß-BTX effected the death of neuronal cell bodies within the cranial sensory ganglia and destruction of nerve fibers, both sensory and motor, innervating the tongue. In addition, innervation to the entire craniofacial region was affected. Embryos into which sterile PBS had been injected on gd12 showed no deficits or abnormalities in the innervation of the tongue or in the appearance of taste structures. Thus, the effects of ß-BTX injection were due to the actions of the neurotoxin and not to the in utero manipulations.
The aneural tongues in exposed fetuses lacked fungiform papillae (Figure 4C) and taste buds in all regions of the tongue and palate (Figures 4C and 5C, D). It is generally accepted that the initiation of
taste papillae morphogenesis is nerve independent. The first fungiform primordia, placodes or
eminences, form on the dorsal tongue late on d12 in the mouse, before nerve fibers have
extended into the dorsal tongue (unpublished observation). As taste placodes acquire a
mesenchymal core, nerve fibers infiltrate and extend towards the papilla apex. Nerve fibers do
not penetrate the apical epithelium of the papillae until gd15. Exposure of embryos to
ß-BTX on gd12 would have resulted in the destruction of sensory fibers before their entry
into the dorsal tongue. Thus, this exposure would prevent any or all inductive interactions
between nerve fibers and the dorsal tongue epithelium that might be required for the initiation of
papillae formation, as well as for the further maturation of papillae and taste bud initiation.
However, because exposed fetuses were only examined at gd18, it cannot be determined from the
results of this study whether lack of innervation to the dorsal tongue blocked the initiation of
papillae formation or prevented papillae maintenance and further maturation. Therefore, the
results of this study neither support nor rule out a role for innervation in papillae initiation.
Whereas it is generally assumed that the initial stage of papillae morphogenesis—the
formation of taste placodes or eminences—occurs in the absence of innervation, evidence
from several sources indicates that the maintenance and further maturation of papillae to form
taste buds is nerve dependent. If sensory innervation to taste papillae is interrupted in the adult
rat, taste buds are lost and fungiform papillae acquire apical spines reminiscent of filiform
papillae (Oakley et al., 1990;Nagato et al.,
1995), suggesting that the maintenance of normal morphology of taste papilla is
dependent on innervation. The conversion of fungiform papillae to a filiform phenotype would
require the papillary epithelium to reprogram keratin production from characteristic
`soft' cytokeratins (Oakley et al., 1990;Sawaf et al., 1990;Knapp et al., 1995;Zhang et al., 1995) to the `hard'
keratins present in the apical spine (Heid et al., 1988; Sawaf et al., 1990). Oakley and colleagues (1990) have postulated that one role innervation may play in the development of taste
papillae and their maintenance may be to specify keratin type or rather to prevent the formation
of inappropriate types that may disrupt or prevent taste bud formation. Few fungiform papillae
were identified in ß-BTX-exposed fetuses. However, the removal of normal innervation to
developing fungiform papillae could disrupt events in the programming of these papillae to
produce specific keratins, resulting in their phenotypic conversion to default filiform structures.
This conversion could explain the dearth of recognizable fungiform papillae in
ß-BTX-exposed embryos.
Support for nerve-dependent maintenance of taste papillae also comes from studies of BDNF
knockout mice. Atypical forms of fungiform papillae were observed in mice lacking the
neurotrophin BDNF that were identical to those seen in nerve transection studies by Oakley and
others (Oakley et al., 1990, 1993). These
papillae were characterized as lacking taste buds and having a filiform appearance (Mistretta et al., 1997;Nosrat et al., 1997; Zhang et al., 1997). BDNF knockout mice have a significantly
decreased innervation of taste papillae and taste buds. Fungiform papillae numbers in these mice
are reduced, and have an altered morphology. The proportion of atrophic papillae increases
postnatally, indicating that fungiform papillae are not maintained. In rat embryos, innervation of
the taste epithelium by taste nerves occurs concomitantly with the expression of mRNA for the
neurotrophic factor BDNF, suggesting that taste epithelial cells produce this factor to support the
ingrowing taste nerve fibers (Nosrat and Olson, 1995; Nosrat et al. 1996). In adult mice, BDNF is expressed in areas where mature taste
cells are located, indicating that BDNF may be required for the maintenance of taste cell
innervation. Thus it has been postulated that when taste buds do not produce the proper taste
neurotrophic factor, BDNF, the trophic function of the nerve disappears and taste buds and
papillae are not maintained (Mistretta et al., 1997;Nosrat et al., 1997;Zhang et al., 1997). The
results of the BDNF knockout studies and the nerve transection studies described above support a
role for innervation in papillae maintenance and provide mechanisms through which innervation
might mediate taste papillae and bud survival. In this model, the malformed and decreased
numbers of fungiform papillae observed in ß-BTX-exposed fetuses would be viewed as
resulting from the absence of any trophic support by sensory innervation to papillary epithelium
and the subsequent conversion of fungiform papillae to the default filiform phenotype.
Taste buds were not present either on taste papillae or in the palatal epithelium in
ß-BTX-exposed embryos (Figures 4C and 5C, D). A few `primordial-like' taste bud cells were infrequently observed,
suggesting the possibility that perhaps taste bud initiation had occurred in the absence of
innervation in these few cases. However, the numbers of these cells are too few to warrant any
conclusion. It should be noted that the results of in vitro manipulation of taste bud
morphogenesis in an amphibian model do provide support for a nerve-independent initiation of
taste bud formation (Barlow and Northcutt, 1995, 1997;Barlow et al., 1996). However, although the
amphibian model provides a unique opportunity for manipulation of taste bud development,
results from this model may not be directly applicable to mammalian taste bud development.
Tissues in these animals process a degree of morphogenetic plasticity not observed in mammals.
For example, amphibians can regenerate limbs even in the absence of innervation (Filoni et al., 1995); mammals merely wound heal. Therefore, although taste
buds may form without innervation in an amphibian, the evidence from studies examining taste
bud morphogenesis in mammals indicates that innervation may be mandatory in this system.
Effects of ß-BTX exposure on circumvallate papillae morphogenesis
In contrast to the complete lack of fungiform papillae in ß-BTX fetuses, circumvallate
papillae were observed in all the affected embryos. These papillae had an altered morphology;
they lacked taste buds and had a clearly different shape from shams or controls. This observed
difference in the sensitivity of fungiform and circumvallate papillae morphogenesis to the effects
of ß-BTX indicates that events in the formation and maturation of these taste papillae may
be distinct. Although in vitro fungiform papillae formation in embryonic tongue
fragments from which nerve had been excluded was highly reproducible, the presence of
circumvallate papillae in these explants was highly variable (Mbiene et al.,
1997). In contrast, when portions of the branchial arches that form the tongue were
explanted to roller tube culture, the formation of circumvallate papillae was highly reproducible
but the formation of fungiform papillae was variable (J. Morris-Wiman et al., submitted
for publication). In addition, the maturation of circumvallate placodes to raised papillae in this
culture system was found to be dependent on the presence of intact ganglia. This contrast in the
ability of these different in vitro models to support fungiform versus circumvallate
papillae morphogenesis may be due to differences in both the culture methodologies used and the
embryonic origins of fungiform and circumvallate papillae. Both fungiform and circumvallate
papillae support taste bud formation and maintenance, but their origin differs significantly and
processes inherent to their development and maintenance may also be distinct. Differences in the
embryonic origin of fungiform and circumvallate papillae and their innervation might also
explain the contrasting results observed with ß-BTX. However, an alternative explanation
for the differences observed in fungiform and circumvallate susceptibility to ß-BTX
exposure could lie in possible non-specific effects that ß-BTX might have on the taste
periphery. Indeed, it cannot be totally ruled out that this toxin has a direct or local effect on the
tongue epithelium separate from its effects on its innervation.
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Acknowledgments |
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This study was supported in part by a USPHS grant DC01657 form the National Institute of Deafness and Communication Disorders
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Accepted September 30, 1998
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