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

Abstract

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.

Wnt signaling interacts with Shh to regulate taste papilla development

“…To more closely examine the interactions of Wnt and Shh signaling molecules during taste papilla development, we turned to organ culture of embryonic tongues. Rodent tongues in culture have been shown to closely mimic in vivo spatial and temporal stages of morphogenesis and molecular expression, and to support the development of taste papillae (22,23). Because the expression of Shh and Wnt signaling components in mice is seen only after E12.5, we maintained stage E12.5 (or E11.5; data not shown) tongues in culture for up to 2 days under varying treatment conditions.…”

Sonic hedgehog signaling pathway in vertebrate epithelial appendage morphogenesis: perspectives in development and evolution

“…Similarly, Ptc, Gli1, Bmp-2 and Bmp-4 are expressed in the dorsal epithelium and share similar punctate patterns at regular intervals which are often symmetrically located in rows at both sides of the median sulcus. At E 14, small eminencies on the anterior part of the tongue are distinguished as the first morphological indications of fungiform papillae through a dissecting microscope [45]. Shh transcripts continue to be expressed in the developing fungiform papillae in discrete spot patterns at E 14-15.…”

Roles of innervation in developing and regenerating orofacial tissues

“…In vitro studies have also shown that taste buds cannot develop without innervation [20]. Although the different types of papillae can develop in the tongue of the rat when cultured in vitro in the absence of innervation, the development of functional taste buds is compromised [20,28]. Taken together these in vivo and in vitro data suggest that innervation is essential for the development of taste buds.…”