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.
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.
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.
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