Immunocytochemical localization of the L1 and N-CAM cell adhesion molecules and their shared carbohydrate epitope L2/HNK-1 in the developing and differentiated gustatory papillae of the mouse tongue

Summary The localization of the cell adhesion molecules L1 and N-CAM, and their shared carbohydrate epitope L2/HNK-1, was investigated at the light and electron microscopic levels in developing and adult fungiform and circumvallate gustatory papillae of the mouse tongue.At embryonic day 13, the earliest stage investigated, the tongue epithelium was still undifferentiated and was not yet innervated by sensory fibres. At this stage none of the three molecules was detectable within the tongue epithelium. At embryonic day 15 the primordia of the gustatory papilla became unequivocally discernible when the papillary epithelium was already innervated by few sensory axons. At this stage N-CAM was the first molecule expressed on epithelial cells and was confined to those parts of the papillary epithelium destined to become the chemosensory cells of the taste buds. The sensory axons were N-CAM-, L1- and L2/HNK-1-positive when fasciculating or contacting their accompanying Schwann cells or the cells of the papillary epithelium. Contacts between Schwann cells were also prominently labelled by antibodies to the three antigens. The mesenchymal tissue underlying the prospective sensory epithelium expressed N-CAM at all embryonic stages, but ceased to be N-CAM positive within the first six postnatal days. From embryonic day 16 onward a weak L1 immunoreactivity was detectable within the basal and intermediate layers of the lingual epithelium and remained present in adulthood.Cytodifferentiation of epithelial cells into spindle-shaped sensory cells and organization into taste buds began at postnatal day two. Simultaneously, L1 and L2/HNK-1 immunoreactivity increased on taste bud cells and N-CAM disappeared from the non-sensory extragemmal parts of the papillary epithelium. At approximately postnatal day six, taste bud formation was complete and the pattern of cell adhesion molecule expression was comparable to that found in the adult in that L1 was strongly expressed on the apposing surfaces of all cells, whereas N-CAM was confined to cell contacts between a subpopulation of intragemmal cells. The L2/HNK-1 epitope was visible on the surfaces of taste bud cells, on intragemmal axons, and in a small portion of extracellular matrix directly underlying the taste buds, but was no longer expressed on those parts of the sensory fibres embedded in the subepithelial mesenchyme. The L2/HNK-1 epitope may thus be regarded as a cell surface marker for the cellular elements of mature taste buds. The highly sialylated form of N-CAM was not detectable at any stage investigated.The observations suggest that the expression of the three molecules within the papillary epithelium follows rather than precedes the innervation by sensory axons and does not, therefore, reflect the gustatory epithelium's susceptibility to innervation as found for N-CAM in the neuromuscular system. The spatio-temporal expression of N-CAM, however, is suggestive of its influence on the differentiation of taste bud cells. Apart from axon-axon and axon-Schwann cell interactions L1 might be involved in interactions between gustatory cells and sensory nerve terminals and, surprisingly, also between non-sensory epithelial cells, whereas the L2/HNK-1 epitope may be implicated in the maintenance of the characteristic cytoarchitecture of the differentiated taste buds.     

扬子鳄胚胎舌表面的扫描电镜观察

在扫描电镜下观察了孵化第３６ｄ至第６２ｄ孵出的扬子鳄（Ａｌｌｉｇａｔｏｒｓｉｎｅｎｓｉｓ）胚胎舌表面的形态变化过程。舌表面于孵化第３８ｄ形成少数大的隆起与凹陷，第４刚开始出现舌乳头。舌上皮细胞的表面在第３６～４２ｄ为圆形，表面光滑，中央凹陷，以后逐渐变成扁平细胞，第５２ｄ细胞呈规则的多边形，表面微绒毛清晰，细胞中央有一向外隆起的圆形或卵圆形核区，第５６ｄ后老化的舌上皮出现脱落现象。孵化第４２ｄ舌后部及中部的上皮内陷形成较大的舌腺腺孔。第４８～５６ｄ中，较小的舌腺孔显著增多，而大舌腺孔数目无明显增加。第６２ｄ多数大舌腺孔内可见有粘液样分泌物。舌表面味蕾的形成很迟，第５６ｄ才出现Ⅰ、Ⅱ、Ⅲ型发育中的味蕾，第６２ｄ形成部分成熟的味蕾。本文对扬子鳄舌腺及味蕾的形态发生特点作了讨论。     

Two generations of the tongue and gustatory organs in the development of Hynobius dunni Tago

In the development of Hynobius dunni there are two consecutive generations of the tongue and two generations of gustatory organs (taste buds and taste disks). The anlage of the developing secondary tongue appears just in front of the free ending of the primary tongue beginning at the larval developmental stage 62. From stage 67, a gradual reduction in the anterior part of the gill skeleton that supports the primary tongue occurs as the developing secondary tongue replaces the primary one. The lining of the entire oropharyngeal cavity of larvae contains only gustatory organs of the taste bud (TB) type. In younger larvae, the sensory area of a TB has a diameter of between 10 and 13 microm, while in older larvae, TBs reach 16-18 microm in diameter. After metamorphosis, some gustatory organs in the secondary tongue with a sensory area of 26-36 microm in diameter appear. In older animals they may reach as much as 56-71 microm. In other regions of the oropharyngeal epithelium than the tongue, these organs have an ellipsoid shape with a major axis of about 50 microm. On the basis of the cytomorphological criteria established previously, these organs were designated as taste disks. Thus, the presence of two generations of gustatory organs is characteristic of some urodeles, as well as frogs.     

Development of the chick tongue. A scanning electron microscopical investigation.

With the aim of clarifying certain morphological aspects of lingual development, chick tongues between the 8th day of incubation and hatching and also during the early postincubation period were investigated by means of the Scanning Electron Microscope. During this time, the tongue anlage underwent some remarkable morphogenetic changes, mostly involving the superficial epithelium but also including the appearance of the lingual glands at the level of the lingual root. With regards to the epithelium, it was possible to observe that in the first days of the incubation period examined, the superficial cells appeared dome-shaped, with microvilli on the apical surface; later they tended to become more flattened, and the microvilli were replaced by a thick net of microplicae. During the final days of incubation, and after hatching desquamative phenomena became evident. At no site of the tongue rudiment were taste buds ever observed, possibly because of the different functional role played by the avian tongue in comparison with that of the mammals.     

Morphological differentiation of taste organs in the ontogeny of Salamandra salamandra

Morphological studies presented here provide additional cytological evidence that in the postnatal development of Salamandra salamandra there are two successive generations of taste organs: premetamorphic taste buds (TBs) in larval forms and taste disks (TDs) in postmetamorphic animals. The TBs have been found in the epithelium of the whole oropharyngeal cavity of larval forms, while in adults TDs appear only at the end of metamorphosis. The TDs can be papillary (or fungiform) on the soft (secondary) tongue and non-papillary outside the tongue. Two main cyto-morphological criteria distinguishing TDs from TBs have been established: (1) high differentiation of "nonsensory" components of a taste organ into several kinds of cells (often named "associate cells")--at least mucous cells and, separating them, wing cells; (2) a considerably larger area of the sensory epithelium than that in TBs, as the consequence of the large size of the mucous cells. In contrast to TDs each TB consists of longitudinally elongated supporting cells and taste cells, as well as of horizontally oriented basal cells, adjacent to the basement membrane. The sensory area in TBs measures 10-12 microm in diameter, while that in TDs has diameter of 45-90 microm. The anlage of the secondary tongue appears as a small folding of the floor epithelium just in front to the tip of the primary tongue in larvae 3 cm long, and is definitely formed in an animal with body length of about 6 cm.     

Morphological differentiation of taste organs in the ontogeny of Salamandra salamandra

Morphological studies presented here provide additional cytological evidence that in the postnatal development of Salamandra salamandra there are two successive generations of taste organs: premetamorphic taste buds (TBs) in larval forms and taste disks (TDs) in postmetamorphic animals. The TBs have been found in the epithelium of the whole oropharyngeal cavity of larval forms, while in adults TDs appear only at the end of metamorphosis. The TDs can be papillary (or fungiform) on the soft (secondary) tongue and non-papillary outside the tongue. Two main cyto-morphological criteria distinguishing TDs from TBs have been established: (1) high differentiation of ”nonsensory” components of a taste organ into several kinds of cells (often named ”associate cells”) – at least mucous cells and, separating them, wing cells; (2) a considerably larger area of the sensory epithelium than that in TBs, as the consequence of the large size of the mucous cells. In contrast to TDs each TB consists of longitudinally elongated supporting cells and taste cells, as well as of horizontally oriented basal cells, adjacent to the basement membrane. The sensory area in TBs measures 10–12 µm in diameter, while that in TDs has diameter of 45–90 µm. The anlage of the secondary tongue appears as a small folding of the floor epithelium just in front to the tip of the primary tongue in larvae 3 cm long, and is definitely formed in an animal with body length of about 6 cm.     

The epithelium of the tongue of Ambystoma mexicanum. Ultrastructural and histochemical aspects.

The distribution pattern of taste buds and goblet cells and histochemical and ultrastructural aspects of the tongue epithelium of Ambystoma mexicanum are here described. This study is also concerned with the developmental stages and origins of the epithelial cells. Pavement cells and goblet cells of the stratum superficiale are replaced by basal cells of the stratum germinativum in larvae and neotenous adults. The pavement cells of the larvae are characterized by a marginal layer of mucin grana. Decompaction of the mucins occurs immediately before extrusion in the adult. The larval goblet cell type (type I), which is also present in the adult, contains unfused grana of irregular shape. At the tip of the tongue, a further type (type II) of goblet cells is found. In the type II cells the intracellular secretory grana fuse to a single homogeneous mass. Leydig cells of the tongue epithelium are discerned by light microscopy first in the semi-adult, apparently correlated with partial metamorphosis. In the course of ontogenesis and induced metamorphosis the secretion changes to neutral glycoconjugates. The mucins of the pavement cells change first followed by those of the goblet cells. The glands of the secondary tongue show a dorso-ventral pattern of varying secretory qualities. Taste buds are found at the anterior margin of the tongue and along the base of the gill clasps in the early larva. They are already distributed all over the tongue at the end of the early larval phase.     

The epithelium of the tongue of Ambystoma mexicanum: Ultrastructural and histochemical aspects

The distribution pattern of taste buds and goblet cells and histochemical and ultrastructural aspects of the tongue epithelium of Ambystoma mexicanum are here described. This study is also concerned with the developmental stages and origins of the epithelial cells. Pavement cells and goblet cells of the stratum superficiale are replaced by basal cells of the stratum germinativum in larvae and neotenous adults. The pavement cells of the larvae are characterized by a marginal layer of mucin grana. Decompaction of the mucins occurs immediately before extrusion in the adult. The larval goblet cell type (type I), which is also present in the adult, contains unfused grana of irregular shape. At the tip of the tongue, a further type (type II) of goblet cells is found. In the type II cells the intracellular secretory grana fuse to a single homogeneous mass. Leydig cells of the tongue epithelium are discerned by light microscopy first in the semi-adult, apparently correlated with partial metamorphosis. In the course of ontogenesis and induced metamorphosis the secretion changes to neutral glycoconjugates. The mucins of the pavement cells change first followed by those of the goblet cells. The glands of the secondary tongue show a dorso-ventral pattern of varying secretory qualities. Taste buds are found at the anterior margin of the tongue and along the base of the gill clasps in the early larva. They are already distributed all over the tongue at the end of the early larval phase.     

Organogenesis of the orbital glands in the lizard Podarcis s. sicula: a histological, histochemical and ultrastructural study

The orbital glands of the lizard Podarcis s. sicula are represented by the anterior and posterior lacrimal glands and the Harderian gland. The anlage of the Harderian gland appears on about the 22nd day of development in the form of a short tubule projecting from the conjunctival epithelium. This event is coincident with the appearance of the nictitating membrane. At this stage the mesenchymal cells surrounding the glandular blastema proliferate at a high rate and form a definite sac, later occupied by both the Harderian gland and the anterior lacrimal glands. At the 26th day of development, the glandular blastema forms acini at its distal end. The prospective glandular cells are not yet differentiated histologically. At the 36th day of development, differentiated serous glandular cells become visible. At the 41st day of development, the acini fill up the preformed mesenchymal sac. Only at this stage does the most medial part of the gland differentiate into mucous-secreting anterior lacrimal gland. At the same time, a small primordium of the posterior lacrimal gland can be seen in the posterior commissure of the eye. The appearance of junctional complexes between epithelial cells and mesenchymal cells in the early developmental stages supports the role of the mesenchyme in the differentiation of the glandular cells. Since the glandular anlage differentiates laterally into Harderian gland and medially into anterior lacrimal gland, spatial and temporal differences seem to exist in the inductive process. Furthermore, a concentration gradient of the inductive substance(s) may be envisaged, since an intermediate zone is present between the Harderian gland and the anterior lacrimal gland, consisting of mixed glandular cells containing both mucous and serous secretory granules.     

神经纤维丝蛋白、神经烯醇化酶和突触素在人胚胎舌组织中的表达

目的 探讨神经纤维丝蛋白（NF）、神经烯醇化酶(NSE)和突触素(SYN)在人胚胎舌组织不同发育阶段的分布特征。 方法 应用免疫组织化学法,检测第2～4个月胎龄段共16份人胚胎舌组织内NF、NSE和SYN蛋白的表达,分析其变化规律。 结果 第2～4个月龄段,NF、NSE和SYN蛋白在人胚舌组织内均有阳性表达。随着胎龄的增大,NF、NSE和SYN在舌组织内阳性表达数量增多,表达强度逐渐增强。第2个月龄时,NF、NSE和SYN蛋白呈少量弱阳性表达,阳性表达强度值分别是135.83±24.62、136.57±15.23和139.84±21.40。第3个月龄时,NF、NSE和SYN阳性表达强度值分别是96.04±23.37、94.89±22.52和90.65±21.08。第4个月胎龄时,NF、NSE和SYN阳性表达强度值分别是79.02±20.90、76.78±21.27和83.43±25.90。应用 One-Way ANOVA和 LSD-t统计学方法,分析第2～4个月龄段人胚胎舌组织内NF、NSE和SYN蛋白的各自阳性表达强度值,P＜0.01。 结论 第2～4个月龄段,人胚胎舌组织内NF、NSE和SYN的表达强度值随胎龄增大而降低,它们均参与调控人胚胎舌内神经系统和舌肌的分化发育。     

Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium

We investigated the relationship between mouse taste bud development and innervation of the soft palate. We employed scanning electron microscopy and immunohistochemistry using antibodies against protein gene product 9.5 and peripherin to detect sensory nerves, and cytokeratin 8 and α-gustducin to stain palatal taste buds. At E14, nerve fibers were observed along the medial border of the palatal shelves that tracked toward the epithelium. At E15.5, primordial stages of taste buds in the basal lamina of the soft palate first appeared. At E16, the taste buds became large spherical masses of columnar cells scattered in the soft palate basal lamina. At E17, the morphology and also the location of taste buds changed. At E18–19, some taste buds acquired a more elongated shape with a short neck, extending a variable distance from the soft palate basal lamina toward the surface epithelium. At E18, mature taste buds with taste pores and perigemmal nerve fibers were observed on the surface epithelium of the soft palate. The expression of α-gustducin was demonstrated at postnatal day 1 and the number of pored taste buds increased with age and they became pear-shaped at 8 weeks. The percent of pored fungiform-like papillae at birth was 58.3% of the whole palate; this increased to 83.8% at postnatal day 8 and reached a maximum of 95.7% at 12 weeks. The innervation of the soft palate was classified into three types of plexuses in relation to taste buds: basal nerve plexus, intragemmal and perigemmal nerve fibers. This study reveals that the nerve fibers preceded the development of taste buds in the palate of mice, and therefore the nerve fibers have roles in the initial induction of taste buds in the soft palate.     

The fine morphology of the basal cell in the frog's taste organ

We investigated the fine morphology of basal cell in the frog's taste organ by means of transmission electron microscopy. Results show that basal cells are placed at the base of the disc and are highly polarized; the cell body is peripherally located and a long cell process reaches the central region of the taste disc without branching. The cell body contains the nucleus, the Golgi apparatus and large lysosomes; junctions between more peripherally located 'marginal' cells prevent passage of macromolecules from oral ambient to basal cells as shown by horse-radish peroxidase experiments. The cell process, running just over the basement membrane in the taste disc epithelium, is rich in microtubules, filaments and clustered secretory granules arranged near the plasmalemma. Nerves interrupting the basement membrane make synaptic-like junctions with basal cells. The cell process ends in the central region of the taste disc; here, the basal cell is expanded to contain filaments, secretory granules and mitochondria in characteristic arrangement and contacts intraepithelial nerve endings as well as basal processes of sensory and supporting cells. Marginated granules are found where basal cell contacts nerve ending and also where nerve and sensory cells contact basal cell. Our findings are consistent with the hypothesis that basal cells are under nerve control and that they may have a diffuse (paracrine) influence on neighbouring cells in the frog's taste organ.     

Structural diversification of the gustatory organs during metamorphosis in the alpine newt Triturus alpestris

Gustatory organs of the taste bud type occur in the epithelial lining of the oropharyngeal cavity of alpine newt larvae. They resemble the taste buds of bony fish, both in appearance (as revealed by scanning electron microscopy) and in detailed internal structure (seen on transmission electron micropscopy). During metamorphosis, at stage 55 of development, the secondary tongue (i.e. the soft tongue) is well formed and the anlages of taste discs are clearly apparent. Somewhat later, taste discs also appear in the epithelial lining outside the tongue, paralleling the disappearance of the taste buds. Well-developed taste discs of the newt differ from taste buds mainly by their structurally diversified set of 'associate cells' (mucous, wing and glial cells), which have no synaptic contact with nerve fibres. These cells accompany the neurosensory cellular components of the taste disc, i.e. the taste receptor cells and basal cells. This indicates that gustatory organs in metamorphosed newts, regardless of their small dimensions, fulfil the criteria established for taste discs previously defined in other Caudata and Anura species. Therefore, in the development of the newt there are two subsequent types of gustatory organs and two generations of the tongue: primary, in the larvae, and secondary, in metamorphosed animals.     

Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage

Thymic epithelial cells, which create a three-dimensionally organized meshwork structure peculiar to the thymus, develop from simple epithelia of the third pharyngeal pouch and cleft during organogenesis. We comparatively investigated the thymus anlages of normal and nude mice by immunohistochemical analysis with regard to epithelial organization and distribution of hematopoietic progenitor cells at early stages of organogenesis. Our results show that development of the mouse thymus anlage at early stages can be subdivided into at least two stages by the differences in epithelial organization, i.e. stratified epithelial stage on embryonic day (Ed) 11 and clustered epithelial stage on Ed12. At the former stage, hematopoietic progenitor cells are accumulated in the mesenchymal layer of the thymus anlage, and at the latter stage progenitor cells enter the epithelial cluster and proliferate. In nude mice, hematopoietic progenitor cells are found in the mesenchymal layer on Ed11.5, but they are not observed among epithelial cells on Ed12, even though epithelial cells form a cluster structure. The present results suggest that aberrant development of the nude mouse thymus anlage occurs at the clustered epithelial stage and that epithelial cells of the nude anlage lack the ability to induce the entrance of hematopoietic progenitor cells into the epithelial cluster.     

The early development of the median thyroid gland of the mouse

Summary The mesobranchial area and the median thyroid anlage of embryonic albino mice were investigated from the somite stage 4 to 40 (81/2–10 days of gestation). In stage I (5–25 somites), there is an unequal growth and differentiation of the epithelium in the floor of the pharynx, whereby a mesobranchial area with a stratified or pseudostratified epithelium is formed. This area is distinct from the remaining pharyngeal epithelium, among other things by an apical microfilament system in the superficial epithelial cells. It is found just basal to a row of plump cytoplasmic protrusions, which extend into the lumen of the pharynx. In stage II (26–40 somites), the cranial part (median thyroid anlage) of the mesobranchial area thickens in relation to the caudal part and grows down into the underlying mesenchyme. The filament system is concentrated in the superficial cell layer of the median thyroid anlage at the beginning of stage II and disappears during downgrowth.In both stages, but most pronounced in stage II, there is a population of 0.1–5 intracellular bodies, which occasionally contain the remains of organelles. The larger bodies, which often contain the remains of nuclei, are usually found peripherally while the smaller ones are more evenly distributed. Acid phosphatase can often be demonstrated histochemically in small bodies, while larger bodies are usually without reaction. Cells with pycnotic nuclei and/or degenerated cytoplasmic components are regularly found. Acid phosphatase can also be demonstrated in Golgi complexes and surrounding vesicles. Basal to the epithelium, bodies are occasionally found which may possibly have been extruded from that tissue.This work was supported by a grant (A 1/65) from Danish Medical Research Coucil.     

Development of the larval lung of Salamandra salamandra L.

Summary The development of the lung in larvae of Salamandra salamandra L. was studied at different developmental stages: from the youngest forms to those entering the metamorphosis. The entire developmental process was divided into three stages. At stage I the lung is filled with fluid and lined by undifferentiated cells. Its wall contains the unramified pulmonary artery and vein. Numerous lamellar and electron dense bodies at different stages of transformation occur inside the lung. At stage II the lung is filled with air and the lining cells start to differentiate: those located along the pulmonary vein change thier shape into a columnar form and start to transform into the ciliated cells. The pulmonary wall contains numerous capillaries separated from the lumen of the lung by a thick epithelial layer. A monolayer film appears at the luminal surface of the alveolar lining layer. At stage III ciliogenesis begins in cells located along the pulmonary artery. The internal surface of the lung remains smooth, with the exception of the pulmonary artery and vein that protrude toward the lumen of the lung, together with the accompanying epithelium. Goblet cells appear in the ciliated epithelium and in the deeper layers of the latter occasional endocrine-like cells can be found. The remaining lung surface is lined by a respiratory epithelium composed of a single type of pneumocytes. The capillaries protrude toward the lumen of the lung and the meshes of capillary network are occupied by cell bodies of pneumocytes. A typical air-blood barrier is in the process of being formed.This study was supported by a grant no 476/II from the Polish Academy of Sciences     

Morphogenesis of rat lingual filiform papillae

The morphogenesis of filiform papillae on rat tongue was investigated with the electron microscope. Tongue rudiments were first seen on the 12th day of gestation. At 15–17 days, dermal papillae had formed and were arranged in hexagonal array on the dorsal lingual surface. Capping each dermal papilla was a two-layered epithelium that protruded slightly above the lingual surface, thus forming the early filiform papilla. In the next stage of development, at 18–19 days of gestation, the epithelium lining the papilla had differentiated into two cell populations, one producing hard keratin, the other producing soft keratin. Some of the keratinized epithelial cells assumed a position at an acute angle to the tongue surface and extended deep into the epithelium. In the next stage, 20–21 days, a cleft appeared within these angularly oriented cells. This resulted in the division of the epithelium into keratin-lined individual filiform papillae. Finally, the individual papillae increased in size to the adult form.     

Relationship between genital ridge formation and settlement site of primordial germ cells in chick embryos

Chick embryos from stage 15 to stage 18, which is the most frequent extravasation period, were investigated by means of serial sections and light microscopy in order to learn the detailed relationship between the settlement sites of the primordial germ cells (PGCs) and the forming genital ridge. PGCs circulating in the vascular system came out of the small vessels in the splanchnopleure posterior to the vitelline artery. This PGC extravasation was limited to an area about 1.2 mm caudal to the vitelline artery. After the extravasation, the PGCs entered the neighboring thickened coelomic epithelium of the splanchnopleure. This thickened epithelium, which had incorporated the PGCs, changed the location toward the future gonadal site with the advance of development. At stage 16, the thickened portion of the epithelium was located in the splanchnopleure; then it moved toward the somatopleure via the coelomic angle; finally, at stage 18, this epithelium occupied the region between coelomic angle and the mesonephros which corresponded to the future genital ridge. This means that the thickened epithelium of the splanchnopleure which initially incorporated the PGCs becomes the superficial epithelium of the genital ridge in more advanced stages. The thickened portion of the epithelium of the splanchnopleure at stage 16 is thought to be the definitive gonadal anlage.     

Protein gene-product 9.5 in developing mouse circumvallate papilla: comparison with neuron-specific enolase and calcitonin gene-related peptide

The present study was made to investigate the ontogeny of protein gene-product 9.5 (PGP 9.5)-like immunoreactivity (-LI) in the developing mouse circumvallate papilla (CVP), and its distribution was compared to that of neuron-specific enolase (NSE) and calcitonin gene-related peptide (CGRP). In adult CVP, PGP 9.5-LI was observed in the subgemmal nerve plexus; some thin PGP 9.5-like immunoreactive (-IR) nerve fibers penetrated taste buds and apical epithelium. PGP 9.5-LI was also observed in the spindle-shaped cells in taste buds, and a small number of round- or oval-shaped ganglionic cells in the lamina propria. The distribution of NSE-LI was comparable to that of PGP 9.5-LI. CGRP-LI was observed in the nerve fibers only; distribution of CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers, although the number of CGRP-IR nerve fibers was smaller than that of PGP 9.5-IR nerve fibers. At least six developmental stages were defined with regard to the developmental changes in the distribution of PGP 9.5-LI from embryonic day (E) 12 to adulthood: Stage I (E12–13) — a dense nerve plexus of PGP 9.5-IR nerve fibers was detected in the lamina propria beneath the core of newly-formed papilla. Stage II (E14–16) — thin PGP 9.5-IR nerve fibers penetrated the apical epithelium, and a few round-shaped cells in the apical epithelium also displayed PGP 9.5-LI. Stage III (E17–18) — thin PGP 9.5-IR nerve fibers penetrated the inner lateral epithelium of the trench. Stage IV [Postnatal day (P) 0–3] many PGP 9.5-IR nerve fibers penetrated the outer lateral epithelium of the trench; later in this stage, taste buds appeared. Stage V (P5–10) — a small number of PGP 9.5 IR cells in the taste buds appeared, and their number increased gradually. Stage VI (PI4-adult) — the number of PGP 9.5-IR taste cells increased and reached the adult level, while the number of PGP 9.5-IR nerve fibers decreased. The development of NSE-LI was similar to that of PGP 9.5-LI. CGRP-IR nerve fibers were detected at E12 in the lamina propria, and the development of the intraepithelial CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers. The present results indicate that invasion by nerve fibers of the epithelium of lingual papillae occurs in a complex manner, and that these nerve fibers may participate in the formation of the taste buds.