========= ANNALS
or ANATOMY =========
Development of the chick tongue. A scanning electron microscopical investigation Silvia Gheri Bryk, Sandra Zecchi Orlandini, Gherardo Gheri and Eleonora Sgambati Dipartimento di Anatomia umana ed Istologia, Universita di Firenze, Viale Morgagni 85, 1-50134 Firenze, Italia
Summary. 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.
Key words: Tongue - Chick - Surface epithelium - SEM
The aim of the present study was to investigate, by means of SEM, the three-dimensional aspects of the lingual epithelium of the chick between the 8th day of incubation and hatching and during the early postincubation period.
Material and methods The anlagen of the tongue of White Leghorn chick embryos from the 8th to the 21st day of incubation (stages 34-46 of Hamburger and Hamilton, 1951) were removed by dividing them immediately anterior to the aditus ad laringem, five on each day of incubation. The specimens were carefully washed in physiological saline solution (0.85%) and fixed in glutaraldehyde as described by Litke and Low (1977) at 4°C for 2 - 3 hours. Some specimens were removed 3 days after hatching and underwent the same procedure. After postfixation in 1 % OS04 in phosphate buffer for 2 hours, the fragments were subjected to the routine procedure for SEM. As controls, 2 anlagen of the tongue were obtained on each day examined, washed briefly in physiological solution (0.85%), fixed in Carnoy and processed for L.M.
Introduction
Results
Little information is available on the development of the tongue epithelium during embryonic life (Dourov et al. 1981; Tedde Piras and Mazzarella 1985). As far as the bird tongue is concerned, many investigations on the general morphology, lingual glands and taste buds in adult birds have been reported (pigeon, Moore and Elliott 1944; fowl, Tucker 1964 a, b, c: chick, Susi 1969; Ganchrow and Ganchrow 1985, 1987; various birds, Navalade and Varute 1977), whereas no data are available on the three-dimensional features of the lingual mucous membrane and its evolution during the development of the chick.
On the 8th day of incubation, the dorsal surface of the tongue was relatively smooth and regular. Thereafter it became much more irregular, owing to the presence of almost straight longitudinal folds and increasingly deep furrows. On the 8th-9th day of incubation the central part of the dorsal surface of the tongue (lingual body) appeared to be covered by undifferentiated epithelial cells, with domeshaped apices separated from one another by deep grooves (Fig. 1) which day by day became increasingly shallower. The cell surface was regularly covered by short microvilli. Later the epithelial cells became more flattened, and on
Ann. Anat. (1992) 174: 531-534 Gustav Fischer Verlag Jena
Fig. 1. Chick embryo tongue on the 8th day of incubation. Dorsum. Dome-shaped apices with microvilli on the superficial epithelial layer. X 2 295 Fig. 2. Chick embryo tongue on the 14th day of incubation. Dorsum. The cell apices are flat and the nuclear shadow stands out clearly. X 1125 Fig. 3. Chick embryo tongue on the 15th day of incubation. Lingual apex. The microplicae are arranged parallel to each other. X 2250. Fig. 4. Chick embryo tongue on the 21st day of incubation. Root. Openings of the posterior lingual glands. X 270. about the 10th day the microvilli appeared to be less numerous in the central part of the cell apex. From the 13th day onwards the cell apices were completely flat and the cell boundaries characterized by slight ridges, owing to the overlapping of the plasmalemma of adjacent cells (Fig. 2). On the cell surface, the nuclear shadow could be clearly recognized. The apical specializations were now mainly represented by microplicae, showing a labyrinthine arrangement, although short microvilli were visible in those zones where the cell apices appeared to be still slightly dome-shaped. From the 18th-19th day of incubation, the epithelium of the superficial layer of the dorsal part of the tongue displayed desquamative phenomena. A similar pattern was detectable at the lingual dorsum in the 3 day-old chick, as well as at the level of the borders and apex of the tongue. On the 15th day of incubation, the apical microplicae of the cells in the region of the apex showed a particular arrangement which appeared mostly to take up a parallel orientation (Fig. 3).
On the lingual body, borders and apex, we never found structures or cavities resembling the openings of glandular excretory ducts. On the 8th day of incubation the posterior part of the tongue was separated from the anterior part by a low smooth-surfaced ridge. The epithelial cells lining the crest showed flattened apices with short and scattered microvilli. On the 12th day of incubation slight elevations on the lingual surface, lined with cells with dome-shaped apices and well defined borders were detectable in some regions. These elevations became more prominent during the following days and, by the 15th-16th day, had developed a lumen, giving rise to the outlets of the posterior lingual glands (Fig. 4). The epithelial regions between the glandular openings underwent some degree of desquamation even during the last few days of incubation. The crest which separated the anterior and posterior parts of the tongue began to show indentations by the 12th day of incubation. During the following days the crest became
532
characterized by the presence of finger-like, posteriorly directed spines. On the 20th-21st day the spines were separated from the anterior part of the tongue by an M -shaped furrow. The superficial epithelial layer lining the spines was characterized by flattened cells which were irregular in shape and size. The mean diameter of the cell apices was extremely variable, ranging from 20 !Lm to 60 !Lm. Furthermore, the apices were covered by a labyrinthine network of microplicae. The lingual spines were also characterized by desquamation, even if less that observed on the dorsal surface of the tongue. Such desquamative phenomena gave a tile-like appearance to the superficial layer of the epithelium.
Discussion The bird tongue differs from the mammalian organ in certain functional aspects. Modem avian species lack teeth and do not masticate. The front part of the tongue acts mainly as a prehensile organ for conveying solid food from the anterior region of the beak to the posterior palate and tongue regions. The food is conveyed into the pharynx by the combined movements of the tongue and the basi branchial muscles (Botte and Palagelli 1982), and at the same time the food is moistened by saliva. Therefore the presence of taste buds, which in adult birds (Bath 1906; Saito 1966; Ganchrow and Ganchrow 1985) are associated with the excreting ducts of the salivary glands, is relatively rare. The three-dimensional characteristics of the lingual epithelium undergo morphological changes during development, chiefly represented by a flattening of superficial epithelial cells with the presence, at first , of an apical labyrinth of microplicae and later by desquamative phenomena . Minor regional differences can be observed, such as the typical, almost parallel arrangement of the microplicae at the lingual apex. We have never observed either the taste bud-like structures, reported by Ganchrow and Ganchrow (1985), or glandular outlets on the dorsal surface of the tongue. Under the light microscope, a large number of glands have been observed in the substance of the lingual body, opening by means of ducts onto the ventral surface of the tongue (Gheri Bryk et al. 1992). At the level of the lingual root, several different morphogenetic events take place. The crest, which separates the body from the root, will give rise to the lingual spines, the functional significance of which is not yet sufficiently clear. According to Anastasi et al. (1977) the spines might be considered to be "sensorial fringes" whereas, according to Hodges (1974), they could, in the adult bird, be regarded as lingual papillae. Our observations with the light microscope have never confirmed the presence of taste buds on the lingual spines. Moreover, their architecture is very rudimentary, and without any epithelial and/or mesenchymal specialization . Posterior to the spines, the lingual root undergoes significant changes during maturation, mostly represented by the differentiation of the posterior salivary glands which characterizes the developmental pattern of this area during most of
the embryonic period. At the level of the anlage of the salivary gland openings, secretory activity is detectable. Observations under light microscope immediately before hatching show that this secretion is chiefly mucous in type. It is noteworthy that desquamative phenomena at the superficial layer of the epithelium, which are particularly abundant on the lingual body in the last 3 days of incubation and immediately after hatching, are barely detectable at the root of the tongue.
References Anastasi G, Lorenzini C, Spinella S (1977) Sull'organogenesi della lingua nel polIo: studio al M.S.E. Atti Accademia Peloritana dei Pericolanti. Classe Scienze Medico-Biologiche 64: 47-61 Bath W (1906) Die Geschmacksorgane der Vogel und Krokodile. Arch Biotonll: 5-47 Botte V, Pelagalli GV (1982) Anatomia funzionale degli uccelli domestici. Ed Edi Ermes, Milano Dourov N, Milaire J, Arys A (1981) Etude en microscopie electronique abalayage de la surface de la muqueuse linguale de I' embryon et du foetus humain. Bull Grop int Rech sc Stomat et Odont 24: 219-223 Ganchrow D, Ganchrow JR (1985) Number and distribution of taste buds in the oral cavity of hatching chicks. Physiol Behav 34: 889-894 Ganchrow D, Ganchrow JR (1987) Taste bud development in chickens (Gallus gallus domesticus) . Anat Rec 218: 88-93 Gheri Bryk S, Gheri G, Sgambati E, Orlandini GE (1992) Histochemical detection of sugar residues in chick embryo developing lingual glands with horseradish-peroxidase conjugated lectins . Acta histochem 92: 127-137 Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo . J Morphol 88 : 49-92 . Hodges RD (1974) The histology of the fowl. Ed Academic Press, London pp 35-112 Litke LL, Low FN (1977) Fixative tonicity for scanning electron miscroscopy of delicate chick embryos . Am J Anat 148: 121-127 Moore CA, Elliot R (1944) Numerical and regional distribution of taste buds on the tongue of the pigeon. Anat Rec 88: 449 Navalade MN , Varute AT (1977) Histochemical studies on the mucins of the vertebrate tongues . XI: Histochemical analysis of mucosubstances in the lingual glands and taste buds of some birds . Acta histochem 60: 18-31 Saito I (1966) Comparative anatomical studies of the oral organs of the poultry . V. Structures and distribution of taste buds of the fowl. Bull Fac Agric 13: 95-102 (quoted by Ganchrow) Susi FR (1969) Keratinization in the mucosa of the ventral surface of the chicken tongue. J Anat 105: 477-486 Tedde Piras A, Mazzarella V (1985) Caratteristiche della superficie dela lingua fetale umana osservata al microscopio a scansione. Arch Ital Anat Embriol90: 17-30 Tucker R (1964 a) Contributions to the development and histogenesis of the salivary gland in birds . II. Development of the dorsal lingual gland in the tongue of the fowl. Z mikros anat Forsch 71 : 158-171 Tucker R (1964b): Contributions to the development and histogenesis of the salivary gland in birds . 1II. The marginal lingual gland and epithelium of the margin of the tongue in the fowl. Z mikros anat Forsch 71: 305 - 309
533
Tucker R (1964 c) Contributions to the development and histogenesis of the salivary gland in birds. IV. The intermandibular gland and its relationship to the papillary system in the tongue of the fowl. Z mikros anat Forsch 71: 310- 330
Tucker R (1966) Differentiation of epithelial and connective tissue components in the tongue of Gallus domesticus. Res vet Sci 7: 1-16 Accepted July 7, 1989
534
or ANATOMY =========
Development of the chick tongue. A scanning electron microscopical investigation Silvia Gheri Bryk, Sandra Zecchi Orlandini, Gherardo Gheri and Eleonora Sgambati Dipartimento di Anatomia umana ed Istologia, Universita di Firenze, Viale Morgagni 85, 1-50134 Firenze, Italia
Summary. 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.
Key words: Tongue - Chick - Surface epithelium - SEM
The aim of the present study was to investigate, by means of SEM, the three-dimensional aspects of the lingual epithelium of the chick between the 8th day of incubation and hatching and during the early postincubation period.
Material and methods The anlagen of the tongue of White Leghorn chick embryos from the 8th to the 21st day of incubation (stages 34-46 of Hamburger and Hamilton, 1951) were removed by dividing them immediately anterior to the aditus ad laringem, five on each day of incubation. The specimens were carefully washed in physiological saline solution (0.85%) and fixed in glutaraldehyde as described by Litke and Low (1977) at 4°C for 2 - 3 hours. Some specimens were removed 3 days after hatching and underwent the same procedure. After postfixation in 1 % OS04 in phosphate buffer for 2 hours, the fragments were subjected to the routine procedure for SEM. As controls, 2 anlagen of the tongue were obtained on each day examined, washed briefly in physiological solution (0.85%), fixed in Carnoy and processed for L.M.
Introduction
Results
Little information is available on the development of the tongue epithelium during embryonic life (Dourov et al. 1981; Tedde Piras and Mazzarella 1985). As far as the bird tongue is concerned, many investigations on the general morphology, lingual glands and taste buds in adult birds have been reported (pigeon, Moore and Elliott 1944; fowl, Tucker 1964 a, b, c: chick, Susi 1969; Ganchrow and Ganchrow 1985, 1987; various birds, Navalade and Varute 1977), whereas no data are available on the three-dimensional features of the lingual mucous membrane and its evolution during the development of the chick.
On the 8th day of incubation, the dorsal surface of the tongue was relatively smooth and regular. Thereafter it became much more irregular, owing to the presence of almost straight longitudinal folds and increasingly deep furrows. On the 8th-9th day of incubation the central part of the dorsal surface of the tongue (lingual body) appeared to be covered by undifferentiated epithelial cells, with domeshaped apices separated from one another by deep grooves (Fig. 1) which day by day became increasingly shallower. The cell surface was regularly covered by short microvilli. Later the epithelial cells became more flattened, and on
Ann. Anat. (1992) 174: 531-534 Gustav Fischer Verlag Jena
Fig. 1. Chick embryo tongue on the 8th day of incubation. Dorsum. Dome-shaped apices with microvilli on the superficial epithelial layer. X 2 295 Fig. 2. Chick embryo tongue on the 14th day of incubation. Dorsum. The cell apices are flat and the nuclear shadow stands out clearly. X 1125 Fig. 3. Chick embryo tongue on the 15th day of incubation. Lingual apex. The microplicae are arranged parallel to each other. X 2250. Fig. 4. Chick embryo tongue on the 21st day of incubation. Root. Openings of the posterior lingual glands. X 270. about the 10th day the microvilli appeared to be less numerous in the central part of the cell apex. From the 13th day onwards the cell apices were completely flat and the cell boundaries characterized by slight ridges, owing to the overlapping of the plasmalemma of adjacent cells (Fig. 2). On the cell surface, the nuclear shadow could be clearly recognized. The apical specializations were now mainly represented by microplicae, showing a labyrinthine arrangement, although short microvilli were visible in those zones where the cell apices appeared to be still slightly dome-shaped. From the 18th-19th day of incubation, the epithelium of the superficial layer of the dorsal part of the tongue displayed desquamative phenomena. A similar pattern was detectable at the lingual dorsum in the 3 day-old chick, as well as at the level of the borders and apex of the tongue. On the 15th day of incubation, the apical microplicae of the cells in the region of the apex showed a particular arrangement which appeared mostly to take up a parallel orientation (Fig. 3).
On the lingual body, borders and apex, we never found structures or cavities resembling the openings of glandular excretory ducts. On the 8th day of incubation the posterior part of the tongue was separated from the anterior part by a low smooth-surfaced ridge. The epithelial cells lining the crest showed flattened apices with short and scattered microvilli. On the 12th day of incubation slight elevations on the lingual surface, lined with cells with dome-shaped apices and well defined borders were detectable in some regions. These elevations became more prominent during the following days and, by the 15th-16th day, had developed a lumen, giving rise to the outlets of the posterior lingual glands (Fig. 4). The epithelial regions between the glandular openings underwent some degree of desquamation even during the last few days of incubation. The crest which separated the anterior and posterior parts of the tongue began to show indentations by the 12th day of incubation. During the following days the crest became
532
characterized by the presence of finger-like, posteriorly directed spines. On the 20th-21st day the spines were separated from the anterior part of the tongue by an M -shaped furrow. The superficial epithelial layer lining the spines was characterized by flattened cells which were irregular in shape and size. The mean diameter of the cell apices was extremely variable, ranging from 20 !Lm to 60 !Lm. Furthermore, the apices were covered by a labyrinthine network of microplicae. The lingual spines were also characterized by desquamation, even if less that observed on the dorsal surface of the tongue. Such desquamative phenomena gave a tile-like appearance to the superficial layer of the epithelium.
Discussion The bird tongue differs from the mammalian organ in certain functional aspects. Modem avian species lack teeth and do not masticate. The front part of the tongue acts mainly as a prehensile organ for conveying solid food from the anterior region of the beak to the posterior palate and tongue regions. The food is conveyed into the pharynx by the combined movements of the tongue and the basi branchial muscles (Botte and Palagelli 1982), and at the same time the food is moistened by saliva. Therefore the presence of taste buds, which in adult birds (Bath 1906; Saito 1966; Ganchrow and Ganchrow 1985) are associated with the excreting ducts of the salivary glands, is relatively rare. The three-dimensional characteristics of the lingual epithelium undergo morphological changes during development, chiefly represented by a flattening of superficial epithelial cells with the presence, at first , of an apical labyrinth of microplicae and later by desquamative phenomena . Minor regional differences can be observed, such as the typical, almost parallel arrangement of the microplicae at the lingual apex. We have never observed either the taste bud-like structures, reported by Ganchrow and Ganchrow (1985), or glandular outlets on the dorsal surface of the tongue. Under the light microscope, a large number of glands have been observed in the substance of the lingual body, opening by means of ducts onto the ventral surface of the tongue (Gheri Bryk et al. 1992). At the level of the lingual root, several different morphogenetic events take place. The crest, which separates the body from the root, will give rise to the lingual spines, the functional significance of which is not yet sufficiently clear. According to Anastasi et al. (1977) the spines might be considered to be "sensorial fringes" whereas, according to Hodges (1974), they could, in the adult bird, be regarded as lingual papillae. Our observations with the light microscope have never confirmed the presence of taste buds on the lingual spines. Moreover, their architecture is very rudimentary, and without any epithelial and/or mesenchymal specialization . Posterior to the spines, the lingual root undergoes significant changes during maturation, mostly represented by the differentiation of the posterior salivary glands which characterizes the developmental pattern of this area during most of
the embryonic period. At the level of the anlage of the salivary gland openings, secretory activity is detectable. Observations under light microscope immediately before hatching show that this secretion is chiefly mucous in type. It is noteworthy that desquamative phenomena at the superficial layer of the epithelium, which are particularly abundant on the lingual body in the last 3 days of incubation and immediately after hatching, are barely detectable at the root of the tongue.
References Anastasi G, Lorenzini C, Spinella S (1977) Sull'organogenesi della lingua nel polIo: studio al M.S.E. Atti Accademia Peloritana dei Pericolanti. Classe Scienze Medico-Biologiche 64: 47-61 Bath W (1906) Die Geschmacksorgane der Vogel und Krokodile. Arch Biotonll: 5-47 Botte V, Pelagalli GV (1982) Anatomia funzionale degli uccelli domestici. Ed Edi Ermes, Milano Dourov N, Milaire J, Arys A (1981) Etude en microscopie electronique abalayage de la surface de la muqueuse linguale de I' embryon et du foetus humain. Bull Grop int Rech sc Stomat et Odont 24: 219-223 Ganchrow D, Ganchrow JR (1985) Number and distribution of taste buds in the oral cavity of hatching chicks. Physiol Behav 34: 889-894 Ganchrow D, Ganchrow JR (1987) Taste bud development in chickens (Gallus gallus domesticus) . Anat Rec 218: 88-93 Gheri Bryk S, Gheri G, Sgambati E, Orlandini GE (1992) Histochemical detection of sugar residues in chick embryo developing lingual glands with horseradish-peroxidase conjugated lectins . Acta histochem 92: 127-137 Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo . J Morphol 88 : 49-92 . Hodges RD (1974) The histology of the fowl. Ed Academic Press, London pp 35-112 Litke LL, Low FN (1977) Fixative tonicity for scanning electron miscroscopy of delicate chick embryos . Am J Anat 148: 121-127 Moore CA, Elliot R (1944) Numerical and regional distribution of taste buds on the tongue of the pigeon. Anat Rec 88: 449 Navalade MN , Varute AT (1977) Histochemical studies on the mucins of the vertebrate tongues . XI: Histochemical analysis of mucosubstances in the lingual glands and taste buds of some birds . Acta histochem 60: 18-31 Saito I (1966) Comparative anatomical studies of the oral organs of the poultry . V. Structures and distribution of taste buds of the fowl. Bull Fac Agric 13: 95-102 (quoted by Ganchrow) Susi FR (1969) Keratinization in the mucosa of the ventral surface of the chicken tongue. J Anat 105: 477-486 Tedde Piras A, Mazzarella V (1985) Caratteristiche della superficie dela lingua fetale umana osservata al microscopio a scansione. Arch Ital Anat Embriol90: 17-30 Tucker R (1964 a) Contributions to the development and histogenesis of the salivary gland in birds . II. Development of the dorsal lingual gland in the tongue of the fowl. Z mikros anat Forsch 71 : 158-171 Tucker R (1964b): Contributions to the development and histogenesis of the salivary gland in birds . 1II. The marginal lingual gland and epithelium of the margin of the tongue in the fowl. Z mikros anat Forsch 71: 305 - 309
533
Tucker R (1964 c) Contributions to the development and histogenesis of the salivary gland in birds. IV. The intermandibular gland and its relationship to the papillary system in the tongue of the fowl. Z mikros anat Forsch 71: 310- 330
Tucker R (1966) Differentiation of epithelial and connective tissue components in the tongue of Gallus domesticus. Res vet Sci 7: 1-16 Accepted July 7, 1989
534
