Changes in neurotrophin-3 messenger RNA expression patterns in the prenatal rat tongue suggest guidance of developing somatosensory nerves to their final targets

While brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) has been localized in the developing gustatory epithelium, little information is available about neurotrophin-3 (NT-3) mRNA expression pattern in the prenatal developing gustatory and lingual epithelium. In the present study, using in situ hybridization histochemistry, we report on NT-3 mRNA expression in the tongue of rats. At embryonic day (E) 13–17, NT-3 mRNA was expressed subepithelially in the periphery of the developing tongue, as well as among developing muscle. At E19, there was a shift in the expression of NT-3 mRNA. It was then expressed in the surface epithelium of the developing tongue in the developing filiform papillae and, in higher concentrations, in top-surface and fringe epithelium of the developing circumvallate papillae, and top- and lateral-surface epithelium of the developing fungiform papillae. NT-3 mRNA expression in areas rich in somatosensory innervation of the tongue, as well as its specific expression in defined regions compared with BDNF, and the decreased labeling noted from prenatal and early postnatal animals to adults indicate a specific role for NT-3 in the development of lingual somatosensory innervation, as well as for maintenance of this innervation.     

Lingual deficits in neurotrophin double knockout mice

Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophin family and are expressed in the developing and adult tongue papillae. BDNF null-mutated mice exhibit specific impairments related to innervation and development of the gustatory system while NT-3 null mice have deficits in their lingual somatosensory innervation. To further evaluate the functional specificity of these neurotrophins in the peripheral gustatory system, we generated double BDNF/NT-3 knockout mice and compared the phenotype to BDNF^?/? and wild-type mice. Taste papillae morphology was severely distorted in BDNF^?/?xNT-3^?/? mice compared to single BDNF^?/? and wild-type mice. The deficits were found throughout the tongue and all gustatory papillae. There was a significant loss of fungiform papillae and the papillae were smaller in size compared to BDNF^?/? and wild-type mice. Circumvallate papillae in the double knockouts were smaller and did not contain any intraepithelial nerve fibers. BDNF^?/?xNT-3^?/? mice exhibited additive losses in both somatosensory and gustatory innervation indicating that BDNF and NT-3 exert specific roles in the innervation of the tongue. However, the additional loss of fungiform papillae and taste buds in BDNF^?/?xNT-3^?/? mice compared to single BDNF knockout mice indicate a synergistic functional role for both BDNF-dependent gustatory and NT-3-dependent somatosensory innervations in taste bud and taste papillae innervation and development.     

Epithelial overexpression of BDNF or NT4 disrupts targeting of taste neurons that innervate the anterior tongue

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are essential for the survival of geniculate ganglion neurons, which provide the sensory afferents for taste buds of the anterior tongue and palate. To determine how these target-derived growth factors regulate gustatory development, the taste system was examined in transgenic mice that overexpress BDNF (BDNF-OE) or NT4 (NT4-OE) in basal epithelial cells of the tongue. Overexpression of BDNF or NT4 caused a 93 and 140% increase, respectively, in the number of geniculate ganglion neurons. Surprisingly, both transgenic lines had severe reduction in fungiform papillae and taste bud number, primarily in the dorsal midregion and ventral tip of the tongue. No alterations were observed in taste buds of circumvallate or incisal papillae. Fungiform papillae were initially present on tongues of newborn BDNF-OE animals, but many were small, poorly innervated, and lost postnatally. To explain the loss of nerve innervation to fungiform papillae, the facial nerve of developing animals was labeled with the lipophilic tracer DiI. In contrast to control mice, in which taste neurons innervated only fungiform papillae, taste neurons in BDNF-OE and NT4-OE mice innervated few fungiform papillae. Instead, some fibers approached but did not penetrate the epithelium and aberrant innervation to filiform papillae was observed. In addition, some papillae that formed in transgenic mice had two taste buds (instead of one) and were frequently arranged in clusters of two or three papillae. These results indicate that target-derived BDNF and NT4 are not only survival factors for geniculate ganglion neurons, but also have important roles in regulating the development and spatial patterning of fungiform papilla and targeting of taste neurons to these sensory structures.     

Neurotrophins and their receptors in rat peripheral trigeminal system during maxillary nerve growth

We examined the expression of the neurotrophins (NTFs) and their receptor mRNAs in the rat trigeminal ganglion and the first branchial arch before and at the time of maxillary nerve growth. The maxillary nerve appears first at embryonic day (E)10 and reaches the epithelium of the first branchial arch at E12, as revealed by anti-L1 immunohistochemistry. In situ hybridization demonstrates, that at E10- E11, neurotrophin-3 (NT-3) mRNA is expressed mainly in the mesenchyme, but neurotrophin-4 (NT-4) mRNA in the epithelium of the first branchial arch. NGF and brain-derived neurotrophic factor (BDNF) mRNAs start to be expressed in the distal part of the first brachial arch shortly before its innervation by the maxillary nerve. Trigeminal ganglia strongly express the mRNA of trkA at E10 and thereafter. The expression of mRNAs for low-affinity neurotrophin receptor (LANR), trkB, and trkC in trigeminal ganglia is weak at E10, but increases by E11-E12. NT-3, NT-4, and more prominently BDNF, induce neurite outgrowth from explant cultures of the E10 trigeminal ganglia but no neurites are induced by NGF, despite the expression of trkA. By E12, the neuritogenic potency of NGF also appears. The expression of NT-3 and NT-4 and their receptors in the trigeminal system prior to target field innervation suggests that these NTFs have also other functions than being the target-derived trophic factors.     

Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning: fungiform papillae double in number and form in novel locations in dorsal lingual epithelium

From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.     

Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers

During development, axons of the chorda tympani nerve navigate to fungiform papillae where they penetrate the lingual epithelium, forming a neural bud. It is not known whether or not all chorda tympani axons initially innervate fungiform papillae correctly or if mistakes are made. Using a novel approach, we quantified the accuracy with which gustatory fibers successfully innervate fungiform papillae. Immediately following initial targeting (E14.5), innervation was found to be incredibly accurate: specifically, 94% of the fungiform papillae on the tongue are innervated. A mean of five papillae per tongue were uninnervated at E14.5, and the lingual tongue surface was innervated in 17 places that lack fungiform papillae. To determine if these initial errors in papillae innervation were later refined, innervation accuracy was quantified at E16.5 and E18.5. By E16.5 only two papillae per tongue remained uninnervated. Innervation to inappropriate regions was also removed, but not until later, between E16.5 and E18.5 of development. Therefore, even though gustatory fibers initially innervate fungiform papillae accurately, some errors in targeting do occur that are then refined during later embryonic periods. It is likely that trophic interactions between gustatory neurons and developing taste epithelium allow appropriate connections to be maintained and inappropriate ones to be eliminated.     

Blood vascular architecture of the rat lingual papillae with special reference to their relations to the connective tissue papillae and surface structures: a light and scanning electron microscope study

Subepithelial blood vessels of the rat lingual papillae and their spatial relations to the connective tissue papillae and surface structures were demonstrated by light and scanning electron microscopy. In the rat, four types of papillae were distinguished on the dorsal surface of the tongue, i.e. the filiform, fungiform, foliate and circumvallate papillae. Vascular beds of various appearance were found in all four types of lingual papillae: a simple or twisted capillary loop in the filiform papilla; a basket- or petal-like network in the fungiform papilla; a ring-like network in the foliate papilla, and a conglomerated network surrounded by double heart-shaped capillary networks in the circumvallate papilla. These characteristic vascular beds corresponded to the shape of the connective tissue papillae and surface structures. The vascular bed beneath the gustatory epithelium in the fungiform, foliate and circumvallate papilla consisted of fine capillary networks next to the taste buds.     

Epithelial overexpression of BDNF and NT4 produces distinct gustatory axon morphologies that disrupt initial targeting

Most fungiform taste buds fail to become innervated when BDNF or NT4 is overexpressed in the basal layer of tongue epithelium. Here, we examined when and how overexpression of BDNF and NT4 disrupt innervation to fungiform papillae. Overexpression of either factor disrupted chorda tympani innervation patterns either before or during the initial innervation of fungiform papillae. NT4 and BDNF overexpression each disrupted initial innervation by producing different gustatory axon morphologies that emerge at distinct times (E12.5 and E14.5, respectively). Chorda tympani nerve branching was reduced in NT4 overexpressing mice, and neuronal fibers in these mice were fasciculated and remained below the epithelial surface, as if repelled by NT4 overexpression. In contrast, many chorda tympani nerve branches were observed near the epithelial surface in mice overexpressing BDNF, and most were attracted to and invaded non-taste filiform papillae instead of gustatory papillae. These results suggest that BDNF, but not NT4, normally functions as a chemoattractant that allows chorda tympani fibers to distinguish their fungiform papillae targets from non-gustatory epithelium. Since BDNF and NT4 both signal through the p75 and TrkB receptors, trophin-specific activation of different internal signaling pathways must regulate the development of the distinct gustatory axon morphologies in neurotrophin-overexpressing mice.     

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.     

Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development

Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor.     

Spacing patterns on tongue surface-gustatory papilla

The dorsal surface of the mammalian tongue is covered with four kinds of papillae, fungiform, circumvallate, foliate and filiform papillae. With the exception of the filiform papillae, these types of papillae contain taste buds and are known as the gustatory papillae. The gustatory papillae are distributed over the tongue surface in a distinct spatial pattern. The circumvallate and foliate papillae are positioned in the central and lateral regions respectively and the fungiform papillae are distributed on the anterior part of the tongue in a stereotyped array. The patterned distribution and developmental processes of the fungiform papillae indicate some similarity between the fungiform papillae and the other epithelial appendages, including the teeth, feathers and hair. This is because 1) prior to the morphological changes, the signaling molecules are expressed in the fungiform papillae forming area with a stereotyped pattern; 2) the morphogenesis of the fungiform papillae showed specific structures in early development, such as epithelial thickening and mesenchymal condensation and 3) the fungiform papillae develop through reciprocal interactions between the epithelium and mesenchymal tissue. These results led us to examine whether or not the early organogenesis of the fungiform papillae is a good model system for understanding both the spacing pattern and the epithelial-mesenchymal interaction during embryogenesis.     

Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4

Taste buds and the peripheral nerves innervating them are two important components of the peripheral gustatory system. They require appropriate connections for the taste system to function. Neurotrophic factors play crucial roles in the innervation of peripheral sensory organs and tissues. Both brain-derived neurotrophic factor (BDNF) null-mutated and neurotrophin-4 (NT-4) null-mutated mice exhibit peripheral gustatory deficits. BDNF and NT-4 bind to a common high affinity tyrosine kinase receptor, TrkB (NTRK-2), and a common p75 neurotrophin receptor (NGFR). We are currently using a transgenic mouse model to study peripheral taste system development and innervation in the absence of both TrkB ligands. We show that taste cell progenitors express taste cell markers during early stages of taste bud development in both BDNF^−/−xNT-4^−/− and wild-type mice. At early embryonic stages, taste bud progenitors express Troma-1, Shh, and Sox2 in all mice. At later stages, lack of innervation becomes a prominent feature in BDNF^−/−xNT-4^−/− mice leading to a decreasing number of fungiform papillae and morphologically degenerating taste cells. A total loss of vallate taste cells also occurs in postnatal transgenic mice. Our data indicate an initial independence but a later permissive and essential role for innervation in taste bud development and maintenance. This work was supported by NIH-NIDCD R01-RDC007628.     

Regional expression patterns of taste receptors and gustducin in the mouse tongue

In order to understand differences in taste sensitivities of taste bud cells between the anterior and posterior part of tongue, it is important to analyze the regional expression patterns of genes related to taste signal transduction on the tongue. Here we examined the expression pattern of a taste receptor family, the T1r family, and gustducin in circumvallate and fungiform papillae of the mouse tongue using double-labeled in situ hybridization. Each member of the T1r family was expressed in both circumvallate and fungiform papillae with some differences in their expression patterns. The most striking difference between fungiform and circumvallate papillae was observed in their co-expression patterns of T1r2, T1r3, and gustducin. T1r2-positive cells in fungiform papillae co-expressed T1r3 and gustducin, whereas T1r2 and T1r3 double-positive cells in circumvallate papillae merely expressed gustducin. These results suggested that in fungiform papillae, gustducin might play a role in the sweet taste signal transduction cascade mediated by a sweet receptor based on the T1r2 and T1r3 combination, in fungiform papillae.     

哺乳动物舌面味蕾的发育

根据近年来有关大鼠、小鼠味觉发育方面的大量研究,对哺乳动物味蕾(taste buds)发育的情况进行了综述和讨论.哺乳动物舌面上的味蕾分布在菌状乳头(fungiform papillae,FF)、叶状乳头(foliate papillae,FL)、轮廓状乳头(circumvallate papillae,CV)之中,味蕾细胞(taste bud cells)不断地进行着周期性的更新,味蕾的形态、数量和功能随动物随年龄而变化.有关味孔头的研究表明,味乳头(gustatory papillae)在味蕾形成和维持味蕾存在及正常发育方面有着独特的功能.味乳头和味蕾的发育过程与细胞信号分子(signaling molecules)、味觉神经(gustatory nerve fibers)等许多因素有着密切的关系,其中有些作用机理至今尚无定论.     

NT4/5 mutant mice have deficiency in gustatory papillae and taste bud formation.

Neurotrophins are key determinants for controlling the survival of peripheral neurons during development. Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT4/5) exert their action through a common trkB receptor but independently support gustatory sensory neurons. To assess the role of NT4/5 during development, we examined the postnatal development and maintenance of fungiform taste buds in mice carrying a deletion of NT4/5. The absence of NT4/5 results in embryonic deficits in gustatory innervation and a reduced number of fungiform papillae at birth. No degenerative deficits of fungiform papillae were observed for the first 3 weeks of postnatal development. However, these remaining fungiform papillae were smaller in appearance and many did not contain taste pores. By postnatal day 60, there was 63% decrease in the number of fungiform papillae, and remaining papillae were smaller in size or modified into filiform-like spines. These papillae had either no taste bud or a taste bud with a reduced number of taste cells compared to controls. These findings demonstrate that the NT4/5 gene functions in the maintenance of fungiform gustatory papillae and raises the possibility for an earlier role in development.     

Expression patterns of neurotrophic factor mRNAs in developing human teeth

Neurotrophic factors regulate survival, differentiation, growth and plasticity in the nervous system. In addition, based on their specific and shifting temporospatial expression patterns, neurotrophic factors have been implicated in morphogenetic events during tooth development in rodents. To determine whether these findings in rodents could be related to humans, we have now studied nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-line derived neurotrophic factor (GDNF), and neurturin (NTN) mRNA expression patterns in developing human teeth during gestational weeks 6.5-11. Using in situ hybridization histochemistry, we found distinct and specific patterns of neurotrophin and GDNF mRNA expression in the developing human teeth. NGF mRNA labeling was weak and confined predominantly to the dental papilla. BDNF mRNA labeling was stronger than NGF mRNA and was seen in the mesenchyme located lateral to the dental organ, as well as in epithelial structures (inner dental epithelium and enamel knot). NT-3 mRNA was observed in the dental papilla and in the area of the cervical loop. NT-4 mRNA was expressed in both oral and dental epithelia in all stages studied. GDNF mRNA was found in the dental follicle and at different sites in the inner dental epithelium. Weak NTN mRNA labeling was also found in the developing teeth. Based on these findings, we suggest that neurotrophins, GDNF and NTN might be involved in morphogenetic events during early stages of tooth development in humans. Protein gene product (PGP) 9.5-immunoreactive nerve fibers were observed in the dental follicle by 11 weeks coinciding with the labeling for neurotrophic factor mRNAs in this structure. This suggests that these neurotrophic factors might be involved in the innervation of dental structures. The rich expression of neurotrophic factors in developing dental tissues suggests that developing, or possibly adult, dental tissue might be used as an allograft source of trophic support for diseases of the nervous system.