Early development and innervation of taste bud-bearing papillae on the rat tongue.

Early development of fungiform papillae on the fetal rat tongue was examined: (1) to determine whether morphogenesis of the taste bud-bearing fungiform papillae is induced by nerve and (2) to study the growth pattern of the two sensory nerves that innervate the papilla. The papillae first appear on the 15th day of gestation (E15; E1 is the day when the dam is sperm positive) in rows parallel to the midline sulcus. There appears to be a medial-lateral and an anterior-posterior gradient in the sequence of papilla differentiation. The epithelium of the early papilla resembles a multilayered placode topped by a flattened surface periderm. Close examination of the peridermal cells at the apex of the papillae reveals that the cells have fewer surface microvilli and their cytoplasm is more electron opaque than that of similar cells in interpapillary regions. The basal cells in the placode-like epithelium differ from those in interpapillary regions in that they are postmitotic and have more mitochondria. At later stages, the papilla acquires a mesenchymal core and nerves grow into the core. Results from organ culture experiments of tongue fragments taken from E14 fetuses indicate that morphogenesis of fungiform papillae is initiated in the absence of sensory nerve influence, but the nerve exerts a trophic effect on their maintenance. The two sensory nerves of the tongue, the chorda tympani and the lingual branch of the trigeminal nerve, enter the tongue mesenchyme at E14 and grow toward the epithelium. By E15 the chorda tympani branches have reached the developing fungiform papillae, by E16 many have entered the papilla, and by E17 they have penetrated the epithelium at the papilla apex. Their fibers are associated exclusively with the cells at the papilla apex, where the taste bud will develop. The trigeminal nerve ramifies beneath the surface of the entire epithelium by E15. Later, it, too, sends branches into fungiform papillae; these ascend along the trunk of the chorda tympani and at E17 terminate in the connective tissue core around the chorda tympani field. The results are compatible with the notion that the tongue epithelium exerts a general tropic effect on growing axons of both sensory nerves, and the epithelial cells of the fungiform papilla apex exert a similar effect to which only the chorda tympani axons are responsive.

Differentiation and maturation of the sensory hair bundles in the fetal and postnatal vestibular receptors of the mouse: a scanning electron microscopy study.

By means of scanning electron microscopy, the differentiation and maturation of sensory hair bundles have been studied in the ampullar cristae of the mouse during development from gestational day 13 (GD13) to postnatal day 10 (PD10). Two gradients of ciliary differentiation were demonstrated, one from the apex to the base and the other from the center to the periphery. The different hair bundles that appeared on the crista during the fetal period originated in an initial ciliary stage found in regions undergoing differentiation, differentiation, which was visible at the apex of the crista starting on GD14. This stage of ciliary development is gradually followed by a juvenile one at GD15. Starting on GD18, the maturation of hair bundles in the central apical area differed from that in the peripheral areas and prefigured the regional specialization of the crista found in the adult stage. In the discussion, we suggest that when the vestibular epithelial cells begin to exhibit ciliary differentiation, they are simultaneously contacted by nerve fibers.

Organ cultures of embryonic rat tongue support tongue and gustatory papilla morphogenesis in vitro without intact sensory ganglia.

Taste buds on the mammalian tongue are confined to the epithelium of three types of gustatory papillae: the fungiform, circumvallate, and foliate. The gustatory papillae are composed of an epithelium that covers a broad connective tissue core, with extensive innervation to taste bud and nongustatory epithelial locations. Although the temporal sequence of gustatory papilla development is known for several species, factors that regulate initiation, growth, and maintenance of the papillae are not understood. We tested the hypothesis that sensory innervation is required for the initial formation and early morphogenesis of fungiform papillae in a patterned array. An organ culture of the embryonic rat tongue was developed to provide an in vitro system for studying mechanisms involved in fungiform papilla morphogenesis in patterns on the anterior tongue. Tongues were dissected from embryos at 13 days of gestation (E13), a time when the tongue has not yet fully formed and gustatory papillae have not yet appeared, and at 14 days of gestation (E14), when the tongue is well formed and papillae make their initial morphological appearance. Dissected tongues were maintained at the gas/liquid interface in standard organ culture dishes, fed with DMEM/F12 plus 2% B-27 supplement and 1% fetal bovine serum. After 1, 2, 3, or 6 days in culture, tongues were processed for scanning electron or light microscopy, or immunocytochemistry. Tongues cultured from E13 or E14 underwent extensive morphogenesis and growth in vitro. Furthermore, fungiform papillae developed on these tongues on a culture day equivalent to E15 in vivo; that is, after 2 days for cultures begun at E13 and 1 day for those begun at E14. Because E15 is the characteristic time for gustatory papilla formation in the intact embryo, results demonstrate that the cultured tongues retain important temporal information related to papilla development. In addition, fungiform papillae formed in the tongue cultures in the stereotypic pattern of rows. The papillae were large structures with epithelial and mesenchymal cell integrity, and an intact epithelial basement membrane was indicated with laminin immunoreactivity. The cultures demonstrate that gustatory papilla morphogenesis can progress in the absence of an intact sensory innervation. To exclude a potential developmental role for autonomic ganglion cells that are located in the posterior rat tongue, cultures consisting of only the anterior half of E14 tongues were established. Fungiform papilla development progressed in half tongues in a manner directly comparable to whole tongue cultures. Therefore, robust, reproducible development of fungiform papillae in patterns is supported in rat tongue cultures from E13 or E14, without inclusion of intact sensory or major, posterior tongue autonomic ganglia. This is direct evidence that papillae will form and develop further in vitro without sensory ganglion support. The data also provide the first detailed account of in vitro development of the entire embryonic tongue.

Evidence for stimulus access to taste cells and nerves during development: an electron microscopic study.

We have examined developing taste buds in fungiform papillae of rats from the 18th day of gestation (E18) to the 15th postnatal day (P15). Nerve processes were seen in the epithelium of E18 rats before taste buds were obvious. At E20, early taste buds were visible, but were embedded within the epithelium, i.e., their cells were shielded from the oral cavity by overlying squamous epithelium. At this stage, the epithelium on the lateral aspects of the fungiform papillae was keratinized, but that overlying the taste bud was not. Some taste bud cells at E20 contained synapse-like structures near their contacts with nerve processes. In postnatal animals, keratinized epithelial cells were seen overlying taste buds, but taste pores were not observed until P10. How, then, do stimuli reach the taste cells and elicit physiological and behavioral responses as reported by others? The keratinized epithelium overlying the buds was unlike that on the lateral aspect of the papilla in at least one significant way. Few lamellated bodies were present in intercellular spaces beneath the stratum corneum, whereas these were abundant in the corresponding location within epithelium on the slope of the papilla. Although some were present within the squamous epithelium overlying the bud, they apparently were not released into the intercellular space. These lipid-rich lamellated bodies are thought to represent the water barrier of the epithelium, i.e., the barrier which prevents aqueous solutions from passing through the epithelium. We determined that the keratinized epithelium overlying the taste bud was permeable to a tracer, lanthanum nitrate, whereas that on the lateral surface was not. Lanthanum was visualized around taste cells and around nerve profiles within and near the taste bud. We propose that the absence of an aqueous permeability barrier in the epithelium overlying taste buds likely explains the ability of tastants to reach the taste bud cells and nerves in the developmental period before pore formation.

Early innervation and differentiation of hair cells in the vestibular epithelia of mouse embryos: SEM and TEM study.

Early afferent innervation and differentiation of sensory vestibular cells were studied in mouse embryos from gestation day (GD) 13 to 16. Afferent neurites were found as early as GD 13 in the epithelium when there were no clearly differentiated sensory cells. By GD 14 the earliest sensory cells which exhibited short hair bundles at their luminal pole were then contacted by afferent endings at their basal part. On GD 15 nerve endings establishing specialized synaptic contacts, characterized by asymmetrical membrane densities and synaptic bodies, were observed. At this stage, microtubules contacting the presynaptic membranes, as well as coated vesicles were found. On GD 16 the hair cells were multi-afferented and numerous synaptic bodies were found. These results showing a concomitance between the hair cell differentiation and the establishment of nerve contacts are discussed with particular respect to nerve-hair cell interactions during sensory differentiation. This study does not point to a primary induction of vestibular hair cell differentiation by nerve endings, but it is consistent with the possibility that the ingrowth of nerve fibers is one of many factors that influence the differentiation of receptor cells. With respect to synapse formation, it is assumed that the location of synaptic bodies at presynaptic densities is determined by the arrival of afferent nerve endings.

The pattern of ciliary development in fetal mouse vestibular receptors. A qualitative and quantitative SEM study.

The development of vestibular receptors in the mouse was studied by scanning electron microscopy between the 13th gestation day to birth. On the 13th gestation day, the utricle was entirely covered with microvilli, which were often grouped around small kinocilia at the center of the macula. The vertical cristae were not clearly differentiated at this stage. On the 15th gestation day, the opposite orientation of ciliary tufts in the utricle indicated the beginnings of the striola. During the whole period studied, gradients in differentiation of ciliary tufts were observed between the center and the periphery of the utricle, and the top and base of the cristae. The auxiliary structures (otolithic membrane and cupula) began to appear at the same time as the first ciliary tufts differentiated. Otoliths, still immature, were only observed as from the 16th gestation day. Differentiation of ciliary tufts on the utricle appeared to be progressive during the fetal period. However, between the 16th and 17th gestation days, a pause in the differentiation of ciliary tufts was registered. A day later, there was a pause in the increase of the utricular sensory surface, which coincided with a temporary stabilization of the decrease in the thickness of the sensory epithelium.

Freeze-fracture study of the vestibular hair cell surface during development.

The ciliary arrangement and external surface of the vestibular receptor cell were studied in their immature stages. In the first stages, immature stereocilia are similar to the microvilli of the adjacent supporting cells. Later, when the cilia grow, a geometric arrangement of the stereocilia occurs, while the peripheral microvilli disappear. The size of the hair cell apex increases. The biggest and more bulbous cells correspond to type I hair cells, but in the newborn cat it is difficult to distinguish accurately type II flat hair cells from immature hair cells.

Postnatal development of vestibular receptor surfaces in the rat.

The development of vestibular receptor surfaces was studied during the postnatal period in the rat on the 1st, 7th, 13th, 32nd and 75th days after birth. Cristae and utricles increase and change their shapes, up to day 13 for the cristae and day 32 for the utricles. Cristae hair bundles are less developed than those of the utricles on the 1st day after birth, with evidence of ciliogenesis being present in the cristae. There is an increase in hair bundle length in both organs that appears complete by the 32nd day after birth. These results are discussed in relation to the ultrastructural and electrophysiological studies concerning the postnatal maturation of the vestibular receptors.

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.

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.

Immunocytological characterization of the expression of cell adhesion molecule L1 during early innervation of mouse otocysts.

Doubts exist as to whether afferent nerve fibers exert a neurotrophic effect on the differentiation of sensory cells in the developing vestibular neuroepithelium. To determine whether innervation of hair cells precedes their differentiation, we have used the L1 adhesion molecule as a marker for axons. The detection of L1 on afferent axons in the otic vesicle of mouse embryos on gestation day 11 shows that nerve fibers penetrate the neuroepithelium before the sensory cells differentiate. L1-immunoreactivity of nerve endings also reveals the considerable fiber ramification on gestation days 14 and 15, i.e., corresponding to the first stages of sensory cell differentiation. The expression of L1 at successive stages of nerve fiber growth in the neuroepithelium, such as fasciculation and ramification, is not consistent with the previous role proposed for L1 as a fascicule-promoting factor and raises the possibility that other mechanisms are involved in L1 mediated adhesion.