Development of fungiform papillae, taste buds, and their innervation in the hamster.

Fungiform taste buds in mature hamsters are less subject to neurotrophic influences than those of other species. This study evaluates taste-bud neurotrophism during development in hamsters by examining the relation between growing nerves and differentiating fungiform papillae. Chorda tympani (CT) or lingual (trigeminal) nerve (LN) fibers were labelled with Lucifer Yellow as they grew into (CT fibers) or around (LN fibers) developing taste buds. Developing fungiform papillae and taste pores were counted with the aid of a topical tongue stain. The tongue forms on embryonic days (E) 10.5-11 and contains deeply placed CT and LN fibers but no papillae. By E12, the tongue epithelium develops scattered elevations. These "eminences" selectively become innervated by LN fibers that grow to the epithelium earlier and in larger numbers than CT fibers. Definitive fungiform papillae form rapidly during E13-14 and become heavily innervated by LN fibers. Intraepithelial CT fibers, rare at E13, invariably innervate fungiform papillae containing nascent taste buds at E14. During E14-15 (birth = E15-16), most papillae contain taste buds with pores, extensive perigemmal LN innervation, and extensive intragemmal CT innervation. At birth, numbers of fungiform papillae and taste pores are adultlike. The results show that fungiform eminences begin forming in the absence of innervation. The subsequent differentiation of definitive fungiform papillae and their innervation by LN fibers occur synchronously, prior to the differentiation of taste buds and their CT innervation. The hamster is precocious (e.g., compared to rat) in terms of LN development and the structural maturity of the anterior tongue at birth.

The innervation of the primate fungiform papilla--development, distribution and changes following selective ablation.

The development of the terminal parts of the chorda tympani nerve, lingual nerve and cranial sympathetics in the macaque fungiform papillae were studied by light- and electron microscopy. Their respective distributions in the intra- and extragemmal compartments of papillae from adult macaques were examined following selective ablation of each nerve. Prior to midgestation, a single bundle of unmyelinated axons which contained numerous axoaxonic synapses passed through the subepithelial connective tissue and ramified in the single nascent chemosensory corpuscle and surrounding non-gustatory epithelium. Following midgestation, additional chemosensory corpuscles appeared, possibly by division of existing corpuscles, myelination of axons was begun, axoaxonic synapses were eliminated, and nerve terminals appeared in the subepithelial connective tissue as free nerve endings and coiled simple nerve endings. In the perinatal period, coiled simple endings, corpuscular receptors and Meissner corpuscles were present in the papilla core. Large numbers of intra-epithelial nerve endings were present in the extragemmal epithelium throughout development. Tonofilament collars ensheathed intra-epithelial axons and 80-100 nm dense core granules, occupying adjacent epithelial cells, appeared to be sequestered near such axons. Experimental selective ablation indicated that the terminal parts of chorda tympani fibers were present only within chemosensory corpuscles. In contrast, lingual nerve endings were present both in the extragemmal epithelium and chemosensory corpuscles and also were the sole supply of corpuscular receptors. Sympathetics appeared to be sparsely distributed in the papilla core. Intra-epithelial axons degenerated within 24 h following transection, while axons with Schwann or lamellar cell sheaths or myelin persisted for at least 3 days.

An unnamed branch of the lingual nerve: gingival branch.

Our main aim was to study the mylohyoid nerve, but during cadaveric dissections an unnamed branch of the lingual nerve was encountered incidentally. Dissections of sublingual and pterygomandibular spaces on 13 cadavers preserved in formalin showed an unnamed branch present bilaterally in 11 specimens, which had not been identified before in any of the anatomical textbooks. The branch extended horizontally from the medial mandibular cortex at the level of the retromolar pad to mesial of the lower first molars-second premolars. It was supplying the lingual periosteum, gingiva, and mucosa that were overlying the medial alveolar process. The mean (SD) diameter of the left and right branches was 0.66 (0.1) mm at the branching side. The mean (SD) length of the right and left sides was 28.7 (4.4) mm. The mean (SD) distance from the alveolar crest was 5.8 (0. 9) mm. The lingual nerve supplies the lingual soft tissues; however, none of the anatomical textbooks mention such a subdivision or a branch supplying that part of the oral cavity. We describe the site and the morphological characteristics of this unnamed branch, and recommend that it be named "the gingival branch of the lingual nerve".

Expression of the basal cell markers of taste buds in the anterior tongue and soft palate of the mouse embryo.

Although embryonic expression of Shh in the fungiform papilla placodes has a critical role in fungiform papilla patterning, it remains unclear whether its appearance indicates the differentiation of the basal cells of taste buds. To examine the embryonic development of the basal cells, the expression of Shh, Prox1, and Mash1 was determined in the anterior tongue and soft palate in mouse embryos by in situ hybridization. In the anterior tongue, Prox1 was coexpressed with Shh from the beginning of Shh expression in the fungiform papilla placodes at E12.5. Shh was expressed in the soft palate in a band-like pattern in the anteriormost region and in a punctate pattern in the posterior region at E14.5. The number (21.4 +/- 4.3, at E14.5) of locations where Shh was observed (i.e., spots) rapidly increased and reached a peak level (54.8 +/- 4.0 at E15.5). Also in the soft palate, Prox1 was coexpressed with Shh from the beginning of Shh expression. These results suggest that basal cell differentiation occurs synchronously with the patterning of Shh spots both in the anterior tongue and in the soft palate. In contrast, Mash1 expression lagged behind the expression of Shh and Prox1 and began after the number of Shh spots had reached its peak level in the soft palate. Furthermore, immunohistochemistry of PGP9.5 and Shh revealed that epithelial innervation slightly preceded Mash1 expression both in the tongue and in the soft palate. This is the first report describing the time courses of the embryonic expression of basal cell markers of taste buds.

Development of the sensory nerves to the dorsum of the tongue in staged human embryos.

To determine in selected, staged human embryos the time period and manner of formation of the sensory nerves to the mucosa on the dorsum of the tongue, cranial nerves V(3) and IX and their lingual branches were traced microscopically. These two cranial nerves along with cranial nerve VII are present in the dorsal part of their respective pharyngeal arch 1, 2, and 3 at Stage 13. All three nerves grow ventrally at Stage 16, and the lingual branches of V(3) and IX become closer to the developing tongue region. The lingual fibers begin to enter the tongue substance at Stage 17 when they appear as large short branches that terminate 100-400 microm from the dorsal mucosal basement membrane; of the fibers that are in the tissue deep to the surface mucosa, those in the posterior tongue segment terminate the closest and those in the anterior segment the farthest. Chorda tympani nerve fibers are joined with V(3) at Stage 20. At this stage nerve fibers reach the basement membrane in the posterior tongue segment and by Stage 23 they terminate at the basement membrane throughout the tongue, being sparsest in the anterior segment. Primordial papillae appear as small mucosal projections throughout the tongue at Stage 20 and are innervated by Stage 23. The study found that innervation to the dorsum of the tongue develops during the second month of prenatal life. During the 4-week period, Stages 13-23, an innervation pattern was observed based on the relative time fibers terminate at the basement membrane. The pattern begins caudally in the root, then moves rostrally lateral to the middle tongue segment, proceeding then to the tip of the tongue. The last area to become innervated is the medial part of the middle segment or central portion of the tongue.

Initial innervation of embryonic rat tongue and developing taste papillae: nerves follow distinctive and spatially restricted pathways.

The rat tongue has an extensive, complex innervation from four cranial nerves. However, the precise developmental time course and spatial routes of these nerves into the embryonic tongue are not known, although this knowledge is crucial for studying mechanisms that regulate development and innervation of the lingual taste organs, gustatory papillae and resident taste buds. We determined the initial spatial course of nerves in the developing tongue and papillae, and tested the hypothesis that sensory nerves first innervate the tongue homogeneously and then retract to more densely innervate papillae and taste buds. Antibodies to GAP-43 and neurofilaments were used to label nerve fibers in rat embryo heads from gestational day 11 through 16 (E11-E16). Serial sagittal sections were traced and reconstructed to follow paths of each nerve. In E11 rat, geniculate, trigeminal and petrosal ganglia were labeled and fibers left the ganglia and extended toward respective branchial arches. At E13 when the developing tongue is still a set of tissue swellings, the combined chorda/lingual, hypoglossal and petrosal nerves approached the lingual swellings from separate positions. Only the chorda/lingual entered the tongue base at this stage. At E14 and E15, the well-developed tongue was innervated by all four cranial nerves. However, the nerves maintained distinctive entry points and relatively restricted mesenchymal territories within the tongue, and did not follow one another in common early pathways. Furthermore, the chorda/lingual and glossopharyngeal nerves did not set up an obvious prepattern for gustatory papilla development, but rather seemed attracted to developing papillae which became very densely innervated compared to surrounding epithelium at E15. To effect this dense papilla innervation, sensory nerves did not first innervate the tongue in a homogeneous manner with subsequent retraction and/or extensive redirection of fibers into the taste organs. Results contribute to a set of working principles for development of tongue innervation. Points of entry and initial neural pathways are restricted from time of tongue formation through morphogenesis, suggesting distinctive lingual territories for each nerve. Thus, sensory and motor nerves distribute independently of each other, and sensory innervation to anterior and posterior tongue remains discrete. For taste organ innervation, gustatory papillae are not induced by a prepatterned nerve distribution. In fact, papillae might attract dense sensory innervation because neither chorda/lingual nor glossopharyngeal nerve grows homogeneously to the lingual epithelium and then redistributes to individual papillae.

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.

Distinctive spatiotemporal expression patterns for neurotrophins develop in gustatory papillae and lingual tissues in embryonic tongue organ cultures.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs are expressed in the developing rat tongue and taste organs in specific spatiotemporal patterns. BDNF mRNA is present in the early lingual gustatory papilla epithelium, from which taste buds eventually arise, prior to the arrival of gustatory nerve fibers at the epithelium, whereas NT-3 initially distributes in the mesenchyme. However, a direct test for neural dependence of neurotrophin expression on the presence of innervation in tongue has not been made, nor is it known whether the patterns of neurotrophin expression can be replicated in an in vitro system. Therefore, we used a tongue organ culture model that supports taste papilla formation while eliminating the influence from sensory nerve fibers, to study neurotrophin mRNAs in lingual tissues. Rat tongue cultures were begun at embryonic day 13 or 14 (E13, E14), and BDNF, NT-3, nerve growth factor (NGF) and neurotrophin-4 (NT-4) mRNAs were studied at 0, 2, 3 and 6 days in culture. BDNF transcripts were localized in the gustatory epithelium of both developing fungiform and circumvallate papillae after 2 or 3 days in culture, and NT-3 transcripts were in the subepithelial mesenchyme. The neurotrophin distributions were comparable to those in vivo at E13-E16. In 6-day tongue cultures, however, BDNF transcripts in anterior tongue were not restricted to fungiform papillae but were more widespread in the lingual epithelium, while the circumvallate trench epithelium exhibited restricted BDNF labeling. The NT-3 expression pattern shifted in 6-day organ cultures in a manner comparable to that in the embryo in vivo, and was expressed in the lingual epithelium as well as mesenchyme. NGF mRNA expression was subepithelial throughout 6 days in cultures. NT-4 mRNA was not detected. The neurotrophin mRNA distributions demonstrate that temporospatial localization of neurotrophins observed during development in vivo is retained in the embryonic tongue organ culture system. Furthermore, initial neurotrophin expression in the developing lingual epithelium, mesenchyme, and/or taste papillae is not dependent on intact sensory innervation. We suggest that patterns of lingual neurotrophin mRNA expression are controlled by the influence of local tissue interactions within the tongue at early developmental stages. However, the eventual loss of restricted BDNF mRNA localization from fungiform papillae in anterior tongue suggests that sensory innervation may be important for restricting the localized expression of neurotrophins at later developmental stages, and for maintaining the unique phenotypes of gustatory papillae.

Taste placodes are primary targets of geniculate but not trigeminal sensory axons in mouse developing tongue.

Tongue embryonic taste buds begin to differentiate before the onset of gustatory papilla formation in murine. In light of this previous finding, we sought to reexamine the developing sensory innervation as it extends toward the lingual epithelium between E 11.5 and 14.5. Nerve tracings with fluorescent lipophilic dyes followed by confocal microscope examination were used to study the terminal branching of chorda tympani and lingual nerves. At E11.5, we confirmed that the chorda tympani nerve provided for most of the nerve branching in the tongue swellings. At E12.5, we show that the lingual nerve contribution to the overall innervation of the lingual swellings increased to the extent that its ramifications matched those of the chorda tympani nerve. At E13.0, the chorda tympani nerve terminal arborizations appeared more complex than those of the lingual nerve. While the chorda tympani nerve terminal branching appeared close to the lingual epithelium that of the trigeminal nerve remained rather confined to the subepithelial mesenchymal tissue. At E13.5, chorda tympani nerve terminals projected specifically to an ordered set of loci on the tongue dorsum corresponding to the epithelial placodes. In contrast, the lingual nerve terminals remained subepithelial with no branches directed towards the placodes. At E14.5, chorda tympani nerve filopodia first entered the apical epithelium of the developing fungiform papilla. The results suggest that there may be no significant delay between the differentiation of embryonic taste buds and their initial innervation.