Functional redundancy and gustatory development in bdnf null mutant mice

In the mouse nasopalate papilla and in the trenches of the foliate and vallate papillae, taste buds accumulated primarily during the first 2 weeks after birth. Null mutation for brain-derived neurotrophic factor caused extensive death of embryonic taste neurons, with the secondary outcome that most taste buds failed to form. However not all taste neurons died; functional redundancy rescued a variable number. The primary research objective was to identify the likely site of the taste neuron rescue factor that substituted for BDNF. In this quest taste bud abundance served as a useful gauge of taste neuron abundance. The proportion of taste buds that developed was variable and uncorrelated among the nasopalate, vallate, and foliate gustatory papillae within each bdnf null mutant mouse. Thus, in spite of shared IXth nerve innervation, the vallate and foliate papillae independently varied in residual gustatory innervation. This variation rules against the rescue of gustatory neurons by system-wide factors or by factors acting on the IXth ganglion or nerve trunk. Therefore it is likely that surviving BDNF-deprived taste neurons were stochastically rescued by a redundant neurotrophic factor at the level of the local gustatory epithelium. These findings broaden the classic expectation that target tissue supplies only a single neurotrophic factor that can sustain sensory (taste) neurons.     

Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs.

Sensory ganglia that innervate taste buds and gustatory papillae (geniculate and petrosal) are reduced in volume by about 40% in mice with a targeted deletion of the gene for brain-derived neurotrophic factor (BDNF). In contrast, the trigeminal ganglion, which innervates papillae but not taste buds on the anterior tongue, is reduced by only about 18%. These specific alterations in ganglia that innervate taste organs make possible a test for roles of lingual innervation in the development of appropriate number, morphology, and spatial pattern of fungiform and circumvallate papillae and associated taste buds. We studied tongues of BDNF null mutant and wild-type littermates and made quantitative analyses of all fungiform papillae on the anterior tongue, the single circumvallate papilla on the posterior tongue, and all taste buds in both papilla types. Fungiform papillae and taste buds were reduced in number by about 60% and were substantially smaller in diameter in mutant mice 15-25 days postnatal. Remaining fungiform papillae were selectively concentrated in the tongue tip region. The circumvallate papilla was reduced in diameter and length by about 40%, and papilla morphology was disrupted. Taste bud number in the circumvallate was reduced by about 70% in mutant tongues, and the remaining taste buds were smaller than those on wild-type tongues. Our results demonstrate a selective dependence of taste organs on a full complement of appropriate innervation for normal growth and morphogenesis. Effects on papillae are not random but are more pronounced in specific lingual regions. Although the geniculate and petrosal ganglia sustain at least half of their normal complement of cell number in BDNF -/- mice, remaining ganglion cells do not substitute for lost neurons to rescue taste organs at control numbers. Whereas gustatory ganglia and the taste papillae initially form independently, our results suggest interdependence in later development because ganglia derive BDNF support from target organs and papillae require sensory innervation for morphogenesis.     

Gustatory innervation and bax-dependent caspase-2: participants in the life and death pathways of mouse taste receptor cells

In the adult mouse tongue, an average of 11% of the gustatory receptor cells are replaced each day. In investigating homeostatic cell death mechanisms in gustatory renewing epithelium, we observed that taste receptor cells were selectively immunopositive for the bcl-2 family death factor, Bax, and for the protease Caspase-2 (Nedd2/Ich1). We determined that 8-10% of the taste receptor cells of the vallate papilla were Bax positive and that 11% were Caspase-2 positive. Some of these immunopositive taste cells had apoptotic morphological defects. Within the subset of vallate taste cells immunopositive for either Caspase-2 or Bax, up to 79% coexpressed both death factors. Bax and Caspase-2 first appeared in occasional vallate taste receptor cells on the same postnatal day-the day after birth. bax null mutation markedly reduced gustatory Caspase-2 immunoexpression. These observations suggest that taste cell death pathways utilize p53, Bax, and Caspase-2 to dispose of aged receptor cells. Apart from reducing Caspase-2 expression, Bax deficiency also altered taste organ development. bax(-/-) mice had a more profusely innervated vallate papilla, which grew to be 25% longer and taller, with the mean taste bud containing more than twice the normal number of taste cells. This augmentation of taste organ development with increased innervation is complementary to the well-documented reduction in taste organ development with sparse innervation. We propose that additional taste neurons survived programmed cell death in Bax-deficient mice, thereby providing an inductive boost to vallate gustatory development.     

A sensitive period for the neural induction of taste buds

Taste buds mature postnatally in the vallate papilla of the rat and reach a mean number of 610 by day 90. Although taste buds are neurotrophically dependent, the presence of widespread bilateral innervation permits more than 80% of the 610 vallate taste buds to survive after one IXth nerve is removed in adults. However, after a IXth nerve is removed at 0-3 d postpartum, about two-thirds of the vallate taste buds fail to develop. In the present investigation, the timing of the neural induction of taste buds was examined by unilaterally removing the IXth nerve at 12 different postnatal ages, from 0 to 75 d. Unilateral denervation revealed the existence of a sensitive period that is maximal from 0 to 10 d, when unilateral or bilateral interruption of the IXth nerve profoundly impairs the formation of taste buds. The number of taste buds that form is nonlinearly dependent upon the number of axons; at low levels of innervation, a doubling of the number of myelinated axons quintuples the number of taste buds. Thus, taste axons interact synergistically. In studying regeneration, we found that axons of both neonatal and adult IXth nerves elongate approximately 1.8 mm/d. Taste buds were re-formed more rapidly and a higher proportion were bilaterally innervated when regenerating axons and the sites of former taste buds were numerous. The proportion of bilaterally innervated taste buds could be approximated from the likelihood of random overlap of axons from the right and left IXth nerves. The greater ease with which taste buds are re-formed than developed suggests that taste bud regeneration does not recapitulate taste bud development.     

Regeneration of taste buds after reinnervation of a denervated tongue papilla by a normally nongustatory nerve

Taste buds degenerate and disappear after transection of their sensory nerve supply, and they differentiate anew from epithelial cells (e.g., lingual) following regeneration of sensory but not motor or autonomic axons. A controversy exists as to whether only gustatory sensory nerves can cause buds to reform or whether any sensory nerve can perform this function. This issue arose because the results of cross-innervation studies revealed a specificity whereas grafting data demonstrated a nonspecificity. A retest of specificity in the cross-reinnervation situation was performed by reinnervating the denervated vallate papilla of adult rat tongue with a sensory branch of the vagus nerve that is not normally gustatory. It was found that taste buds disappeared and remained lost from acutely and chronically denervated papilla. However, some buds were found 90–100 days after reinnervation by the normally nongustatory vagus nerve branch. Transection of the regenerated vagus nerve resulted in the loss of innervation and the degeneration of taste buds from reinnervated papilla indicating that this nerve had supported buds. These results show that a normally nongustatory nerve can induce the formation of taste buds after its axons grow into appropriate tissue. It appears that the ability to support taste buds is a nonspecific, rather than a specific, property of sensory nerve.     

Distribution of keratin 8-containing cell clusters in mouse embryonic tongue: evidence for a prepattern for taste bud development

The initiation of the morphogenesis of gustatory papillae is independent of innervation. To address the question of whether taste bud formation is associated with gustatory papilla morphogenesis, we examined developing tongues in mouse embryos from embryonic day 11 to birth. Despite the smooth morphological appearance of the lingual dorsal surface at 13 days of gestation, we observed embryonic taste bud primordia as discrete collections of cytokeratin 8-positive and elongated cells in epithelial placodes in the anterior tongue. In subsequent stages until birth, cytokeratin 8 continues to be expressed in embryonic taste buds distributed in punctuate patterns at regular intervals along rows that are symmetrically located on both sides of the median sulcus in the dorsal anterior developing tongue. Embryonic taste buds were observed in the developing circumvallate papillae from 15.5 days of gestation until birth. The dorsal epithelium of the anterior tongue is not innervated when embryonic taste buds first occur. The increased numbers of embryonic taste buds in developing fungiform papillae until birth are not correlated with the neural invasion of the epithelium. Thus, taste buds occur prenatally more likely independently of the innervation.     

Cellular expression of alpha-gustducin and the A blood group antigen in rat fungiform taste buds cross-reinnervated by the IXth nerve.

Although taste buds are trophically dependent on their innervation, cross-reinnervation experiments have shown that their gustatory sensitivities are determined by the local epithelium. Both the gustatory G-protein, alpha-gustducin, and the cell-surface carbohydrate, the A blood group antigen, are expressed by significantly fewer fungiform than vallate taste cells in the rat. In these experiments, one side of the anterior portion of the tongue was cross-reinnervated by the IXth nerve in order to determine whether the molecular expression of taste bud cells is determined by the epithelium from which they arise or by the nerve on which they are trophically dependent. The proximal portion of the IXth nerve was anastomosed to the distal portion of the chorda tympani (CT) nerve using fibrin glue (IX-CT rats). Control animals had the CT cut and reanastomosed using the same technique (CT-CT rats), or had the CT avulsed from the bulla and resected to prevent regeneration (CTX rats). The animals survived for 12 weeks postoperatively, and the tongues were removed, stained with methylene blue, and the fungiform taste pores counted on both sides. Tissue from the anterior 5 mm of the tongue was cut into 50-microm sections, which were incubated with antibodies against alpha-gustducin and the human blood group A antigen. In both CT-CT and IX-CT rats, there was regeneration of fungiform taste buds, although in both groups there were significantly fewer taste buds on the operated side of the tongue. The normal vallate papilla had a mean of 8.37 alpha-gustducin-expressing cells and 5.22 A-expressing cells per taste bud, whereas the fungiform papillae contained 3.06 and 0.23 cells per taste bud, respectively. In both CT-CT and IX-CT rats there was a normal number of cells expressing alpha-gustducin or the A antigen in regenerated taste buds; in the CTX animals there was a significant decrease in the expression of these markers. These results demonstrate that the molecular phenotype of taste bud cells is determined by the local epithelium from which they arise and not by properties of the innervating nerve.     

NCAM expression by subsets of taste cells is dependent upon innervation

The expression of the neural cell adhesion molecule (NCAM) and distinct carbohydrate groups by cells of the taste buds of the rat vallate papilla was investigated by immunohistochemical and biochemical techniques. We employed antibodies against (1) the extracellular (mAb 3F4) and cytoplasmic (mAb 5B8) portions of the NCAM polypeptide, (2) the highly sialylated form of NCAM (mAb 5A5), (3) carbohydrate epitopes associated with glycosylated NCAM forms in the rat (mAb 2B8) or frog (mAb 9-OE) olfactory system, and also (4) the Lewis^b blood group carbohydrate epitope (mAb CO431). NCAM mRNA was demonstrated by polymerase chain reaction (PCR) in samples of the vallate papilla, suggesting the presence of NCAM in cells of the taste buds. Antibodies against NCAM (mAbs 3F4 and 5B8) recognized a subset (about 20%) of cells within the vallate taste buds; fibers of the glossopharyngeal nerve, including those innervating the gustatory epithelium, were NCAM immunoreactive. Taste bud cells did not express polysialic acid (mAb 5A5), but mAb 5A5 immunoreactivity was observed on fibers of the IXth nerve, including a few that entered the taste buds. All or nearly all of the cells within the vallate taste buds were immunoreactive to mAb 2B8, whereas mAbs 9-OE and CO431 reacted with subsets of cells. The carbohydrates recognized by mAbs 2B8 and 9-OE were also abundantly expressed in the ducts and acini of the lingual salivary glands. Bilateral crush of the IXth nerve resulted in the loss of expression of all of these molecules from the gustatory epithelium. If cells of the taste bud express NCAM during their final stage(s) of differentiation, then NCAM could play a role(s) in the growth of gustatory axons toward their target epithelial cells and in the recognition between the nerve fibers and mature taste receptor cells, or among the taste bud cells themselves. © 1993 Wiley-Liss, Inc.     

Expression of the neural cell adhesion molecule (NCAM) and polysialic acid during taste bud degeneration and regeneration

Taste receptor cells are replaced throughout life, accompanied by continuing synaptogenesis between newly formed taste cells and first-order gustatory fibers. The neural cell adhesion molecule (NCAM) is expressed by a subset of taste cells in adult rodents and appears on gustatory nerve fibers during development prior to differentiation of the taste buds. We employed antibodies against the extracellular domain of the NCAM polypeptide (mAb 3F4) and against polysialic acid (PSA) residues found on embryonic forms of NCAM (mAb 5A5) to investigate the relationship between the expression of these molecules and the innervation of taste buds in adult rats. In unoperated rats, anti-NCAM recognized a subset of cells within the vallate taste buds and also the fibers of the glossopharyngeal (IXth) nerve, including those innervating the gustatory epithelium. Taste bud cells did not express PSA but mAb 5A5 immunoreactivity was observed on some fibers of the IXth nerve, including a few that entered the taste buds. Bilateral crush of the IXth nerve resulted in the loss of NCAM expression from the gustatory epithelium within 8 days. As IXth nerve fibers reinnervated the epithelium, NCAM expression was seen first in the nerve, followed by increased expression in the epithelium as the taste cells differentiated from their precursors. PSA expression by fibers of the IXth nerve did not return to normal until well after the regeneration of the vallate taste buds. The present results demonstrate that taste cell expression of NCAM is dependent upon innervation by the IXth nerve and that NCAM expression appears in the nerve prior to its expression in the differentiating epithelium during regeneration. The occurrence of a similar temporal sequence in the developing taste system suggests that NCAM could play a role in cell-cell interactions that are important for the differentiation of the taste epithelium. Ongoing taste cell turnover and synaptogenesis between IXth nerve fibers and newly differentiating taste cells also requires recognition and adhesion, in which NCAM could play a role. © 1994 Wiley-Liss, Inc.     

Support of trigeminal sensory neurons by nonneuronal p75 neurotrophin receptors

The p75 neurotrophin receptor (p75NTR) binds all four mammalian neurotrophins, including neurotrophin-3 (NT-3) required for the development of select sensory neurons. This study demonstrated that many gustatory and somatosensory neurons of the tongue depend upon p75NTR. Each of thousands of filiform papillae at the front of the tongue as well as each somatosensory prominence at the back of the tongue has a small cluster of p75NTR-positive epithelial cells that is targeted by somatosensory innervation. This expression of p75NTR by epithelial target cells required NT-3 but not adult innervation. NT-3-secreting cells were adjacent to the p75NTR-positive target cells of each somatosensory organ, as demonstrated in NT-3(lacZneo) transgenic mice. In NT-3 null mutant mice, there were few lingual somatosensory neurons. In p75NTR null mutant mice, the lingual somatosensory axons were likewise absent or had deficient terminal arborizations. Cell culture indicated that substrate p75NTR can influence neuronal outgrowth. Specifically, dissociated trigeminal sensory neurons more than doubled their neurite lengths when grown on a lawn of p75NTR-overexpressing fibroblasts. This enhancement of neurite outgrowth by fibroblast p75NTR raises the possibility that epithelial target cell p75NTR may help to promote axonal arborization in vivo. The co-occurrence in p75NTR null mice of a 35% reduction in geniculate ganglion taste neurons and a shortfall of taste buds is consistent with the established role of gustatory innervation in prompting mammalian taste receptor cell differentiation.     

Regeneration of taste buds in tongue grafts after reinnervation by neurons in transplanted lumbar sensory ganglia

Taste buds disappear after denervation and reappear after nerve regeneration. Sensory neurons are responsible since reinnervation by motor or autonomic fibers of peripheral nerve fail to induce bud regeneration. However, we do not know whether some neurons in all sensory ganglia can support buds or whether gustatory (i.e., taste bud inducing) neurons are localized to specific cranial ganglia. The present study was therefore pefrormed to determine whether neurons in transplanted spinal ganglia could support taste buds similarly to those in transplanted cranial ganglia. Grafts of lumbar or vagal nodose ganglia were combined with grafts of tongue's vallate papillae in the anterior chamber of rats' eyes and the papillae examined for taste buds 35 days later. Neurons were present in all transplanted ganglia, and all papillae reinnervated by them contained regenerated taste buds. Nerve fibers could be traced from the transplanted ganglia to the epithelium of the tongue grafts which bore the regenerated taste buds. Papillae transplanted without ganglia lacked buds. These findings indicate that some neurons in all sensory ganglia can induce taste bud formation. The present results could occur if gustatory neurons are intrinsically present in all sensory ganglia, but an alternative interpretation is that the tongue grafts transformed some neurons into gustatory neurons and, hence, that neuronal plasticity is involved.     

The neural induction of taste buds in the salivary ducts of the lingual gland of von Ebner

Taste buds appear in the vallate papilla when tongue grafts are combined with sensory ganglia grafts in the anterior chamber of rats' eyes. After 50 days, many of the tongue grafts (but not the ganglion grafts) developed cysts, the papilla atrophied, and fewer taste buds were found in them than at earlier postoperative times. A study was therefore undertaken to determine whether exteriorizing the tongue graft (i.e., placing it in the confines of a corneal biopsy opening with the epithelial surface of the papilla facing outward) would better preserve the morphology of long-term grafts. In isogenic adult Lewis rats, tongue grafts were exteriorized alone or with nodose ganglia and examined after 90 to 100 days. The tongue grafts were better preserved irrespective of whether they were transplanted with or without a nodose ganglion. In addition, taste buds were now found in the epithelium of the ducts of von Ebner's lingual salivary gland. These glands lie just below the vallate papilla and because their ducts empty into the base of the papilla, portions of the ducts are unavoidably contained in tongue grafts. It appeared that intrinsic nerve fibers of the eye, just like nerve fibers from ganglia, induced the formation of ductal buds since ductal buds appeared in ten of 12 tongue grafts transplanted without added ganglia. Of significance was the observation that intense cholinesterase activity was seen in intragemmal nerve fibers of ganglionic neurons whereas no such activity was seen in intragemmal fibers when they were derived from intrinsic nerve fibers of the eye. These results indicate that exteriorization of tongue grafts better preserves their structure, that epithelium in the ducts of von Ebner's gland can be transformed into taste bud cells, that intrinsic nerve fibers of the eye can induce taste bud formation, and that there are two types of sensory neurons (based on intragemmal cholinesterase activity of their nerve fibers) which can cause taste bud development.     

Taste bud-derived BDNF maintains innervation of a subset of TrkB-expressing gustatory nerve fibers

Taste receptor cells transduce different types of taste stimuli and transmit this information to gustatory neurons that carry it to the brain. Taste receptor cells turn over continuously in adulthood, requiring constant new innervation from nerve fibers. Therefore, the maintenance of innervation to taste buds is an active process mediated by many factors, including brain-derived neurotrophic factor (BDNF). Specifically, 40% of taste bud innervation is lost when Bdnf is removed during adulthood. Here we speculated that not all gustatory nerve fibers express the BDNF receptor, TrkB, resulting in subsets of neurons that vary in their response to BDNF. However, it is also possible that the partial loss of innervation occurred because the Bdnf gene was not effectively removed. To test these possibilities, we first determined that not all gustatory nerve fibers express the TrkB receptor in adult mice. We then verified the efficiency of Bdnf removal specifically in taste buds of K14-CreER:Bdnf mice and found that Bdnf expression was reduced to 1%, indicating efficient Bdnf gene recombination. BDNF removal resulted in a 55% loss of TrkB-expressing nerve fibers, which was greater than the loss of P2X3-positive fibers (39%), likely because taste buds were innervated by P2X3 +/TrkB − fibers that were unaffected by BDNF removal. We conclude that gustatory innervation consists of both TrkB-positive and TrkB-negative taste fibers and that BDNF is specifically important for maintaining TrkB-positive innervation to taste buds. In addition, although taste bud size was not affected by inducible Bdnf removal, the expression of the γ subunit of the ENaC channel was reduced. So, BDNF may regulate expression of some molecular components of taste transduction pathways.     

Role of brain-derived neurotrophic factor in target invasion in the gustatory system.

Brain-derived neurotrophic factor (BDNF) is a survival factor for different classes of neurons, including gustatory neurons. We have studied innervation and development of the gustatory system in transgenic mice overexpressing BDNF under the control of regulatory sequences from the nestin gene, an intermediate filament gene expressed in precursor cells of the developing nervous system and muscle. In transgenic mice, the number and size of gustatory papillae were decreased, circumvallate papillae had a deranged morphology, and there was also a severe loss of lingual taste buds. Paradoxically, similar deficits have been found in BDNF knock-out mice, which lack gustatory neurons. However, the number of neurons in gustatory ganglia was increased in BDNF-overproducing mice. Although gustatory fibers reached the tongue in normal numbers, the amount and density of nerve fibers in gustatory papillae were reduced in transgenic mice compared with wild-type littermates. Gustatory fibers appeared stalled at the base of the tongue, a site of ectopic BDNF expression, where they formed abnormal branches and sprouts. Interestingly, palatal taste buds, which are innervated by gustatory neurons whose afferents do not traverse sites of ectopic BDNF expression, appeared unaffected. We suggest that lingual gustatory deficits in BDNF overexpressing mice are a consequence of the failure of their BDNF-dependent afferents to reach their targets because of the effects of ectopically expressed BDNF on fiber growth. Our findings suggest that mammalian taste buds and gustatory papillae require proper BDNF-dependent gustatory innervation for development and that the correct spatial expression of BDNF in the tongue epithelium is crucial for appropriate target invasion and innervation.     

Neural induction of taste buds

Bilateral innervation allows more than 80% of the 610 vallate taste buds to survive removal of one IXth nerve in adult rats. Removal of both IXth nerves in neonatal or adult rats results in the absence of taste buds. In studying development, we found that removing or crushing one IXth nerve in three-day-old neonates profoundly decreased the number of vallate taste buds that subsequently developed. Specifically, after removal of one IXth nerve at 3 days, only 228 taste buds formed, compared with 496 taste buds that one nerve would maintain in adults. Thus, during normal development, the right and left IXth nerves interact synergistically, as at least 150 more taste buds develop than predicted by the sum of the independent action of each IXth nerve. This suggests that vallate taste buds are induced by the IXth nerve. A second example of synergism, representing evidence for the neural induction of taste buds, came from experiments in which we crushed the left IXth nerve 3 days after birth and found that these regenerated IXth nerve axons induced 4 times as many taste buds in the presence of the normal right IXth nerve (118 taste buds) as in its early absence (30 taste buds). We conclude that taste buds are neurally induced and that axons of the IXth nerve interact synergistically in inducing them, rather than competing for targets. We propose that in development innervated progenitor cells form stem cells which lead to taste bud cells.     

Dystonin deficiency reduces taste buds and fungiform papillae in the anterior part of the tongue

The anterior part of the tongue was examined in wild type and dystonia musculorum mice to assess the effect of dystonin loss on fungiform papillae. In the mutant mouse, the density of fungiform papillae and their taste buds was severely decreased when compared to wild type littermates (papilla, 67% reduction; taste bud, 77% reduction). The mutation also reduced the size of these papillae (17% reduction) and taste buds (29% reduction). In addition, immunohistochemical analysis demonstrated that the dystonin mutation reduced the number of PGP 9.5 and calbindin D28k-containing nerve fibers in fungiform papillae. These data together suggest that dystonin is required for the innervation and development of fungiform papillae and taste buds.     

Temporal and spatial patterns of tenascin and laminin immunoreactivity suggest roles for extracellular matrix in development of gustatory papillae and taste buds

Gustatory papillae are complex organs that are composed of 1) an epithelium, 2) specialized sensory cells within the epithelium (the taste buds), 3) a broad connective core, and 4) sensory innervation. During papilla development, cells in the various tissue compartments must divide, aggregate, detach, migrate, and reaggregate in relation to each other, but factors that regulate such steps are poorly understood and have not been extensively studied. All of these processes potentially require participation of the extracellular matrix. Therefore, we have studied temporal and spatial patterns of immunoreactivity for two extracellular matrix molecules, tenascin and laminin, in the developing fungiform and circumvallate papillae of fetal, perinatal, and adult sheep tongue. To determine relations of tenascin and laminin to sensory innervation, we used an antibody to growth-associated protein (GAP-43) to label growing nerves. Immunocytochemical distributions of tenascin and laminin alter during development in a manner that reflects morphogenesis rather than histologic boundaries of the taste papillae. In early fungiform papillae, tenascin immunoreactivity is very weak within the mesenchyme of the papilla core. However, there is a subsequent shift to an intense, restricted localization in the apical papilla core only—directly under taste bud-bearing regions of the papilla epithelium. In early circumvallate papillae, tenascin immunoreactivity is patchy within the papilla core and within the flanking, nongustatory papillae. Later, immunoreactivity is restricted to the perimeter of the central papilla core, under epithelium that contains developing taste buds. In fungiform and circumvallate papillae, the shift in tenascin immunolocalization is associated with periods of taste bud formation and multiplication within the papilla epithelium and with extensive branching of the sensory innervation in the papilla apex. Laminin immunoreactivity, although it is continuous throughout the basement membrane of general lingual epithelium, is interrupted in the epithelial basement membrane of early fungiform and circumvallate papillae in regions where taste buds are forming. The breaks are large in young fetuses, when taste buds first develop, and are evidenced later as punctate disruptions. Heparan sulfate proteoglycan immunoreactivity confirms that these are basement membrane discontinuities. GAP-43 label coincides with innervation of the papilla core and is most extensive in regions where tenascin immunoreactivity is weak or absent. GAP-43 immunoreactivity is also found in early taste buds: Later, it is extensive within more mature multiple taste buds, presumably in relation to synaptogenesis. We propose that tenascin has a role in promoting deadhesion of cells in the papilla epithelium during periods of taste bud formation and multiplication. Discontinuities in the epithelial basement membrane under developing taste buds, indicated with laminin and heparan sulfate proteoglycan immunoreactivity, may interact to facilitate taste bud morphogenesis and multiplication, to permit access of papilla innervation to the forming taste buds, and/or to allow epithelial/mesenchymal interactions during papilla and taste bud development. © 1996 Wiley-Liss, Inc.     

Neurocalcin-immunoreactive neurons in the petrosal ganglion innervate the taste bud

The distribution and origin of neurocalcin-immunoreactive (NC-ir) nerve fibers in the taste bud and carotid body were examined by an immunofluorescence method. In the circumvallate papilla of the tongue, NC-ir nerve fibers made subepithelial nerve plexuses and occasionally penetrated the taste bud. However, the carotid body was devoid of ir nerve fibers. In the petrosal ganglion, 32% of neurons were immunoreactive for NC. Such neurons were mostly medium-sized to large, and scattered throughout the ganglion. In the superior cervical and intralingual ganglia, numerous ir varicose fibers surrounded postsynaptic neurons. However, NC-ir could not be detected in cell bodies of these neurons. The retrograde tracing method indicated that NC-ir petrosal neurons innervated taste buds in the circumvallate papilla. NC-ir neurons may have a gustatory function in the petrosal ganglion.     

An in vitro model for the study of taste papillae morphogenesis using branchial arch explants

It is generally accepted that innervation is required for the maintenance of taste papillae and taste buds, but it is not entirely clear what role, if any, innervation plays in papillae and taste bud formation. Events in taste papillae formation and differentiation take place almost entirely in utero and, therefore, the study of the role of innervation in these events requires a suitable in vitro model. In the past, investigators have made use of various culture techniques to study mammalian taste papillae development in vitro and the role of innervation in this process with varying success. All of these models examined papillae development in isolated tongue or tongue fragments and have lacked the ability to manipulate the innervation of developing taste papillae in these explants. We have established a protocol for an in vitro model of taste papillae morphogenesis using branchial arch explants and roller tube culture methodology. Our results demonstrate that this model supports the morphogenesis of the circumvallate papilla with an integrated nerve. In addition, the use of branchial arch explants allows the inclusion or exclusion of geniculate and petrosal ganglia to examine directly the effects of the presence or absence of innervation on papillae formation and maintenance.