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Differentiation of foetal rat tongue homografts in the anterior chamber of the eye
h&s
oral Biol. Vol. 16,pp. 51-57, 1971. Pergamon Press.Printed in GreatBritain.
DIFFERENTIATION OF FOETAL RAT TONGUE HOMOGRAFTS IN THE ANTERIOR CHAMBER OF THE EYE A. I. FARBMAN Department of Biostructure, Northwestern University, Chicago, Illinois 60611, U.S.A. Summary-Slices of foetal rat tongue were transplanted into the anterior chamber of the eye of adult rats and harvested 5-19 days later. Within a few days, the homografts became vascularized and encysted. Differentiation of filiform papillae, the larnina propria and the 3-dimensional arrangement of muscle fibers simulated the in400 pattern. Fungiform papillae and taste buds did not differentiate. The homografts were invaded by bundles of nerve fibres with and without myelin.
INTRODUCTION THE
anterior chamber of the eye is a favorable site for the growth and development of homografts, particularly when the donor tissue is foetal or embryonic. In the present experiments, fragments of rat foetal tongue were grafted into the anterior chamber of the eye in order to see whether the tissues in the graft, particularly tongue epithelium, developed in a manner similar to that in situ. The ultimate aim of these experiments was to determine whether the anterior chamber would be a suitable site for experiments on the differentiation of the various cell lines found in lingual epithelium, e.g., those involved in the two keratinization patterns of the filiform papilla (FARBMAN, 1970) and those that differentiate into taste bud cells under the influence of a gustatory nerve (FARBMAN, 1965). MATERIALS
AND
METHODS
(a) Preparation of the transplant
Pregnant Wistar rats were anaesthetized with ether in the 14th or 15th day of gestation. The day when sperm was found in the vagina was counted as day zero. All of the following procedures were carried out under aseptic conditions. The foetuses were removed, immediately decapitated and the heads were placed in a sterile Petri dish containing Hanks’ balanced salt solution with penicillin (50 units/ml) and streptomycin (50 mcg/ml). One or two foetal tongues from each litter were removed intact and processed for light and/or electron microscope examination as described below. Each of the remaining tongues was sliced in a horizontal plane so that the dorsal surface and underlying mesenchyme were separated from the ventral part. An 18 gauge spinal needle, which had been modified by shortening the bevel, was prepared by drawing into it a small amount of melted 1 per cent agar and allowing the agar to 51
52
A. I. FARBMAN
solidify. The slice of dorsal tongue was then drawn into the needle and injected into the anterior chamber of the eye of a host animal. (b) Preparation of the host animal The host animals were chosen from the same strain as the donors, but no attempt was made to use a carefully inbred homogeneous strain of animals because grafts to the anterior chamber generally do not elicit an immune reaction (MEDAWAR,1948). Although some grafts in this experiment showed the signs of rejection (heavy round cell infiltration) about 60 per cent of a total of 45 grafts survived and grew well for up to 19 days with no histological signs of infection or host rejection. Only the healthy grafts are described in the next section. Some of the host animals were prepared by removing the left superior cervical sympathetic ganglion about 7 days before the grafting operation. This was done to prevent growth of sympathetic nerves into the graft together with blood vessels. For the grafting operation, the host animals were anaesthetized with ether and their corneas incised near the cornea-sclera junction by insertion of the point of a No. 11 BardParker blade. The cornea1 incision was made just wide enough to accommodate the insertion of the spinal needle containing the tongue slice. After the fluid had drained, the tongue slice was injected into the anterior chamber of the host animal’s eye and the animal was allowed to recover. Usually grafts were placed in both eyes. Host animals were examined daily after placement of the grafts and those that had become infected were discarded. Five to nineteen days after operation, the host animals were anaesthetized with ether, the grafts removed from the anterior chamber, fixed in 10 per cent neutral buffered formalin, dehydrated, cleared and embedded in parat%. Seven-micron sections were made and stained with haematoxylin and eosin. In addition, some grafts were prepared for examination with the electron microscope by fixation for 2 hr in cold 1 per cent 0~0~ buffered at pH 7.4 with 0.1 m phosphate. These specimens were processed and embedded in Epon 812 (LUFT, 1961). Thin sections were made with a diamond knife on a Sorvall M-l microtome, stained with uranyl acetate (WATSON, 1958) and lead citrate (REYNOLDS,1963) and examined in a Siemens Elmiskop 1. RESULTS At the time of grafting, i.e., in the fourteenth or fifteenth foetal day, the rat tongue had a smooth epithelium consisting essentially of 2 layers, a cuboidal to low columnar basal layer and a superficial flattened layer (Figs. 1 and 2). The epithelial cells had a relatively large nucleus with prominent nucleoli and cytoplasm in which free ribosomes were prominent. Small rod-shaped mitochondria, scattered profiles of endoplasmic reticulum and Golgi apparatus were present. Occasionally, superflcial cells that contained a moderately dense ground cytoplasm were seen. A few small desmosomes were seen on adjoining epithelial cell membranes. The epithelium rested on a flimsy basement lamina and hemidesmosomes were usually seen on the cell membranes facing the basement lamina. The lamina propria was characterized by very few fibrils and primitive connective
TONGUE
HOMOGRAFTS
IN ANTERIOR
CHAMBER
53
tissue cells widely separated from each other. None of the cells in deeper layers contained myofilaments although at low magnification in the light microscope there was some suggestion of alignment of the cells in the presumptive muscle area. Within 24-28 hr after placement, the grafts had become well vascularized and most of them looked healthy. Those that had obvious infections were discarded. In histological section, the epithelium of the graft was seen to be disposed around a cystic cavity containing keratinized cell ghosts (Fig. 3). The cyst varied in size according to how long the graft was allowed to remain, i.e., the older grafts had larger cysts. The epithelium lining the cysts was thrown up into filiform papillae, the morphology of which was virtually identical to that usually seen on rat tongue after birth (FARBMAN,1970). The papillae were curved, conical structures with two patterns of epithelial keratinization clearly distinguishable with the electron microscope (Fig. 4). On the convex side of the curve, a stratum granulosum, containing keratohyalin granules, was seen beneath the keratinized layer and the pattern of keratinization was generally similar to that usually seen in oral epithelium. However, the epithelium on the concave side of the papilla contained no stratum granulosum; the cells deep to the stratum corneum contained densely packed masses of tonofilaments and prominent ribosomes in a rather electron-translucent cytoplasm. Their structure was similar to that in tissues forming hard keratin, such as hair cortex. Some of the papillae on the cystic surface were broader than the typical filiform papillae (Fig. 5); these resembled in some respects the fungiform papilla type seen on rat tongue. However, taste buds, usually seen on rat fungiform papillae were absent in the grafts. Moreover, when serial sections of these broader papillae were made, in virtually every case the epithelium was thrown up into a pointed tip with the dual pattern of keratinization as described above for the filiform papillae. The lamina propria of the graft contained an assortment of typical connective tissue cells and fibres (Fig. 6). Well-developed striated muscle was present in the grafts and arranged in 3 planes as is usual in tongue. In addition, fat cells were frequently seen in the deeper muscle layers. Occasionally, small bundles of nerve containing both myelinated and unmyelinated fibres were seen in the stalk through which host blood vessels entered the graft. In addition, they were seen in the muscular portion of the graft (Fig. 7) and sometimes approached but did not enter the epithelium. The nerves were seen in grafts harvested from sympathectomized as well as control rats. DISCUSSION Homotransplants of foetal rat tongue into the anterior chamber of the eye grew and differentiated reasonably well. In many respects, differentiation proceeded as it would have in situ. The epithelium at the time of grafting was of a primitive type and showed no morphological signs of its ultimate papillary form. After 5 days or more in the anterior chamber, the epithelium differentiated into filiform papillae with the dual pattern of keratinization usually seen on rat tongue epithelium (FARBMAN,1970). The broad papillae seen in the homografts resembled fungiform papillae in size, but the epithelium on most of them had a filiform-type point and a corresponding dual
54
A. I. FARBMAN
pattern of keratinization. The typical fungiform papilla type of epithelium, namely a keratohyalin-containing, soft keratin forming epithelium, with a taste bud on the top was not seen. The absence of the taste bud itself was not surprising because there were no gustatory nerves growing into the graft to induce its formation. Those nerves that were seen in the stalks of some grafts probably grew in with the vessels and may have been sprouts from sensory or possibly parasympathetic nerves in the iris or sclera; they were seen in both the sympathectomized and non-sympathectomized rats. In an earlier discussion of taste bud development (FARBMAN,1965), it was suggested that the development of the fungiform papilla may be initiated without the influence of nerve, and that the latter may subsequently enter the forming papilla to induce taste bud formation. The present results neither confirm nor contradict this possibility. The broad papillae seen in the homografts may represent structures that began their development as fungiform papillae and continued to grow in size in the usual way, but, in the absence of nerve, did not assume their characteristic pattern of keratinization nor did they acquire gustatory cells. On the other hand, it is also possible that they were atypical filiform papillae formed perhaps by distortion. The latter is the more likely possibility because in several instances many of them were encountered adjacent to each other; in situ, it is rare to find fungiform papillae immediately adjacent to each other without several intervening filiform papillae (FISH et al., 1944; FARBMAN,1965). Striated muscle differentiated fairly well in the grafts and assumed a three-dimensional orientation as is usually seen in tongue. However, even after more than 2 weeks in the anterior chamber, some of the striated muscle cell nuclei in the graft were centrally located. These findings provide an interesting contrast to those of EASTLICK and ANDERSON(1944) who placed chick limb homografts into anterior chambers and found that striated muscle failed to differentiate at all. They concluded that the anterior chamber was not a favorable site for differentiation of muscle. COMMENT AND CONCLUSION The primary reason for initiating this study, as stated above, was to determine whether the anterior chamber of the eye would provide a favorable site for experiments on differentiation of the lingual epithelium. It seems quite clear the filiform papilla epithelium differentiates and keratinizes in a manner similar to that in vim and that further experimentation on differentiation of filiform papillae can be done using the experimental system described. However, the design of future experiments on taste bud differentiation requires a site completely free of host nerves. Unfortunately, this requirement was not met under the present experimental conditions. Alternatively, other experiments were attempted in which the tongue transplants were placed in specially constructed Millipore filter diffusion chambers modified slightly from the type described by ALGIRE et al. (1954). The chamber was placed in the peritoneal cavity of a host animal. The rationale for this was that the tissue could be nourished by diffusion of host fluids and, therefore, would grow and differentiate within the chamber but would be completely free from any contact with host cells because of the small pore-size (0.45 pm) of the filter material. Unfortunately, within 4-7 days the
TONGUE
HOMOGRAFIX
IN ANTERIOR
CHAMBER
55
transplant lost its integrity as an organ as the cells spread along the inner surfaces of the chamber. Because of this, the diffusion chamber method was unsuitable for any of the studies on epithelial differentiation. At present, experiments are in progress to grow lingual epithelium and mesenchyme in an organ culture system. Preliminary findings have revealed that this is feasible and both filiform papillae and taste buds have been successfully grown in organ culture from foetal tongue fragments. These results will be reported separately. Acknowledgements-The author would like to thank Mr. MARTIN ZIEGNJSR and Mrs. KATHERINE WALSH for their excellent assistance with these experiments. This work was supported by Grant No. NB 06181 from the National Institutes of Health and by a Grant from the Dysautonomia Society, New York. Receipt of a Research Career Development Award No. l-K3-DE-17, 654 from the NationalInstitutes of Health is acknowledged.
R&&---Des tranches de langue de rat foetal ont et& transplant&s dans la chambre anterieure de l’oeil de rats adultes et cueillies 5-19 jours plus tard. Apr&s quelques jours les homogreffes devinrent vasularisees et enkystees. La differentiation des papilles, la lamina propria et l’arrangement tri-dimensionnel simulaient les aspects in-vivo. Les papilles fungiformes et les pores gustatifs ne se sont pas dilferentib. Les homogreffes ont CtC invadees par des faisceaux de fibres nerveuses avec et sans myeline.
Zusammenfassung-Streifen von Rattenfotuszungen wurden in den Augenvorhof von ausgereiften Ratten tiberpflanzt und 5-19 Tage spater geemtet. Innerhalb weniger Tage erwiesen sich die &srpflanzungen als mit Geweben versehen und eingekapselt. Differenzierung von filiform Pap&n, der Lamina propria und der 3dimensionalen Anordmmg von Muskelfasem simulierte das in-vivo Bild. Schwammartige Papillen und die Geschmackskiirperchen entwickelten sich nicht verschieden. In die Verpflanzungen waren Btindel von Nervenfasem mit oder ohne Myelin eingedrungen.
REFERENCES AMIRE, G. H., WEAVER,J. M. and PREHN, R. T. 1954. Growth of cells in oivo in diffusion chambers. I. Survival of homoarafts in immunized mice. J. nut. Cancer 1st. 15.493-507. EASTLICK,H. L. and ANDERSON,R. H. 1944. Studies on transplanted embryonic limbs of the chick. II. The development of limb primordia within the anterior chamber of the eye. J. Morphol. 75, l-9. FARBMAN,A. I. 1965. Electron microscope study of the developing taste bud in rat fungiform papilla. Devel. Biol. 11, 110-135. F-MAN, A. I. 1970. The dual-pattern of keratinization in filiform papillae on rat tongue. J. An&. 106, 233-242. Frsrr, H. S., MAUINE, P. D. and RICHTER, C. P. 1944. The anatomy of the tongue of the domestic Norway rat. I. The skin of the tongue; the various papillae; their number and distribution. Anat. Rec. 89,429440. LUFP, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. biochem. Cytol. 24,297-301.
56
A. I. FARBNAN
MEDAWAR, P. B. l!W. Immunity to homologous grafted skin. III. The fate of skin grafts transplanted to the brain, to subcutaneous tissue and to the anterior chamber of the eye. Br. J. exp. Pathol. 29, 58-69. REYNOLDS,E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17,208-212. WATSON, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. biochem. OtoI. 4,475-478.
TONGUE HOMOGRAFTS IN ANTERIOR CHAMBER
FIG. 1. Photomicrograph
rat foetus. A two-layered
of a section of the dorsal surface of the tongue from a 14-day x 400 epithelium overlies a highly cellular mesenchyme.
FIG. 2. Electron micrograph
of an area of 16day foetal rat tongue epithelium from the same specimen as that illustrated in Fig. 1. The cells contain relatively large nuclei and the most prominent cytoplasmic constituents are free ribosomes and some mitochondria. x 7500
PLATE 1
A.O.B. f.p. 56
A.I.FARBMAN
PLATE 2
TONGUE HOMOGRAETS IN ANTERIOR CHAMBER
PLATE2 FIG. 3. Photomicrograph of a graft grown in the anterior chamber for 14 days. The epithelium is thrown up into keratinizing filiform papillae (f) which project into a flattened cyst cavity (c), filled with intensely staining keratinized cells. Muscle fibres (m) pass horizontally and vertically and some are cut in cross-section (mx), thus demonstrating the 3-dimensional arrangement of their fibres. x 100 FIG. 4. Electron micrograph of a cross-section through a tiliform papilla from a graft grown 7 days in the anterior chamber. In the upper part, cells containing keratohyahn granules (g) are seen. In the lower part, the hard keratin-producing cells containing dense tonofilaments (t), prominent free ribosomes, and no keratohyalin granules, are seen. x8000
57
TONGUE
HOMOGRAFTS
IN ANTERIOR
CHAMBER
FIG. 5. Photomicrograph of a broad papilla with a pointed tip. These papillae resemble fungiform papillae in size but have the dual pattern of keratinization typical of filiform papillae. x 400 FIG. 6. Electron micrograph of the lamina propria of a graft grown 14 days in the anterior chamber. The edge of the epithelium is seen across the top and along the right side of the field. The lamina propria contains fibroblast-type cells, containing much granular endoplasmic reticulum. Scattered connective tissue fibres are seen between cells. x 8500
PLATE
3
A.O.B. f.p. 58
A.I.
FARBMAN
FIG. 7. Electron micrograph of a graft grown 14 days in the anterior chamber. Muscle x 8000 cells (m), myelinated nerves (n) and unmyelinated nerves (u) are clearly seen.
oral Biol. Vol. 16,pp. 51-57, 1971. Pergamon Press.Printed in GreatBritain.
DIFFERENTIATION OF FOETAL RAT TONGUE HOMOGRAFTS IN THE ANTERIOR CHAMBER OF THE EYE A. I. FARBMAN Department of Biostructure, Northwestern University, Chicago, Illinois 60611, U.S.A. Summary-Slices of foetal rat tongue were transplanted into the anterior chamber of the eye of adult rats and harvested 5-19 days later. Within a few days, the homografts became vascularized and encysted. Differentiation of filiform papillae, the larnina propria and the 3-dimensional arrangement of muscle fibers simulated the in400 pattern. Fungiform papillae and taste buds did not differentiate. The homografts were invaded by bundles of nerve fibres with and without myelin.
INTRODUCTION THE
anterior chamber of the eye is a favorable site for the growth and development of homografts, particularly when the donor tissue is foetal or embryonic. In the present experiments, fragments of rat foetal tongue were grafted into the anterior chamber of the eye in order to see whether the tissues in the graft, particularly tongue epithelium, developed in a manner similar to that in situ. The ultimate aim of these experiments was to determine whether the anterior chamber would be a suitable site for experiments on the differentiation of the various cell lines found in lingual epithelium, e.g., those involved in the two keratinization patterns of the filiform papilla (FARBMAN, 1970) and those that differentiate into taste bud cells under the influence of a gustatory nerve (FARBMAN, 1965). MATERIALS
AND
METHODS
(a) Preparation of the transplant
Pregnant Wistar rats were anaesthetized with ether in the 14th or 15th day of gestation. The day when sperm was found in the vagina was counted as day zero. All of the following procedures were carried out under aseptic conditions. The foetuses were removed, immediately decapitated and the heads were placed in a sterile Petri dish containing Hanks’ balanced salt solution with penicillin (50 units/ml) and streptomycin (50 mcg/ml). One or two foetal tongues from each litter were removed intact and processed for light and/or electron microscope examination as described below. Each of the remaining tongues was sliced in a horizontal plane so that the dorsal surface and underlying mesenchyme were separated from the ventral part. An 18 gauge spinal needle, which had been modified by shortening the bevel, was prepared by drawing into it a small amount of melted 1 per cent agar and allowing the agar to 51
52
A. I. FARBMAN
solidify. The slice of dorsal tongue was then drawn into the needle and injected into the anterior chamber of the eye of a host animal. (b) Preparation of the host animal The host animals were chosen from the same strain as the donors, but no attempt was made to use a carefully inbred homogeneous strain of animals because grafts to the anterior chamber generally do not elicit an immune reaction (MEDAWAR,1948). Although some grafts in this experiment showed the signs of rejection (heavy round cell infiltration) about 60 per cent of a total of 45 grafts survived and grew well for up to 19 days with no histological signs of infection or host rejection. Only the healthy grafts are described in the next section. Some of the host animals were prepared by removing the left superior cervical sympathetic ganglion about 7 days before the grafting operation. This was done to prevent growth of sympathetic nerves into the graft together with blood vessels. For the grafting operation, the host animals were anaesthetized with ether and their corneas incised near the cornea-sclera junction by insertion of the point of a No. 11 BardParker blade. The cornea1 incision was made just wide enough to accommodate the insertion of the spinal needle containing the tongue slice. After the fluid had drained, the tongue slice was injected into the anterior chamber of the host animal’s eye and the animal was allowed to recover. Usually grafts were placed in both eyes. Host animals were examined daily after placement of the grafts and those that had become infected were discarded. Five to nineteen days after operation, the host animals were anaesthetized with ether, the grafts removed from the anterior chamber, fixed in 10 per cent neutral buffered formalin, dehydrated, cleared and embedded in parat%. Seven-micron sections were made and stained with haematoxylin and eosin. In addition, some grafts were prepared for examination with the electron microscope by fixation for 2 hr in cold 1 per cent 0~0~ buffered at pH 7.4 with 0.1 m phosphate. These specimens were processed and embedded in Epon 812 (LUFT, 1961). Thin sections were made with a diamond knife on a Sorvall M-l microtome, stained with uranyl acetate (WATSON, 1958) and lead citrate (REYNOLDS,1963) and examined in a Siemens Elmiskop 1. RESULTS At the time of grafting, i.e., in the fourteenth or fifteenth foetal day, the rat tongue had a smooth epithelium consisting essentially of 2 layers, a cuboidal to low columnar basal layer and a superficial flattened layer (Figs. 1 and 2). The epithelial cells had a relatively large nucleus with prominent nucleoli and cytoplasm in which free ribosomes were prominent. Small rod-shaped mitochondria, scattered profiles of endoplasmic reticulum and Golgi apparatus were present. Occasionally, superflcial cells that contained a moderately dense ground cytoplasm were seen. A few small desmosomes were seen on adjoining epithelial cell membranes. The epithelium rested on a flimsy basement lamina and hemidesmosomes were usually seen on the cell membranes facing the basement lamina. The lamina propria was characterized by very few fibrils and primitive connective
TONGUE
HOMOGRAFTS
IN ANTERIOR
CHAMBER
53
tissue cells widely separated from each other. None of the cells in deeper layers contained myofilaments although at low magnification in the light microscope there was some suggestion of alignment of the cells in the presumptive muscle area. Within 24-28 hr after placement, the grafts had become well vascularized and most of them looked healthy. Those that had obvious infections were discarded. In histological section, the epithelium of the graft was seen to be disposed around a cystic cavity containing keratinized cell ghosts (Fig. 3). The cyst varied in size according to how long the graft was allowed to remain, i.e., the older grafts had larger cysts. The epithelium lining the cysts was thrown up into filiform papillae, the morphology of which was virtually identical to that usually seen on rat tongue after birth (FARBMAN,1970). The papillae were curved, conical structures with two patterns of epithelial keratinization clearly distinguishable with the electron microscope (Fig. 4). On the convex side of the curve, a stratum granulosum, containing keratohyalin granules, was seen beneath the keratinized layer and the pattern of keratinization was generally similar to that usually seen in oral epithelium. However, the epithelium on the concave side of the papilla contained no stratum granulosum; the cells deep to the stratum corneum contained densely packed masses of tonofilaments and prominent ribosomes in a rather electron-translucent cytoplasm. Their structure was similar to that in tissues forming hard keratin, such as hair cortex. Some of the papillae on the cystic surface were broader than the typical filiform papillae (Fig. 5); these resembled in some respects the fungiform papilla type seen on rat tongue. However, taste buds, usually seen on rat fungiform papillae were absent in the grafts. Moreover, when serial sections of these broader papillae were made, in virtually every case the epithelium was thrown up into a pointed tip with the dual pattern of keratinization as described above for the filiform papillae. The lamina propria of the graft contained an assortment of typical connective tissue cells and fibres (Fig. 6). Well-developed striated muscle was present in the grafts and arranged in 3 planes as is usual in tongue. In addition, fat cells were frequently seen in the deeper muscle layers. Occasionally, small bundles of nerve containing both myelinated and unmyelinated fibres were seen in the stalk through which host blood vessels entered the graft. In addition, they were seen in the muscular portion of the graft (Fig. 7) and sometimes approached but did not enter the epithelium. The nerves were seen in grafts harvested from sympathectomized as well as control rats. DISCUSSION Homotransplants of foetal rat tongue into the anterior chamber of the eye grew and differentiated reasonably well. In many respects, differentiation proceeded as it would have in situ. The epithelium at the time of grafting was of a primitive type and showed no morphological signs of its ultimate papillary form. After 5 days or more in the anterior chamber, the epithelium differentiated into filiform papillae with the dual pattern of keratinization usually seen on rat tongue epithelium (FARBMAN,1970). The broad papillae seen in the homografts resembled fungiform papillae in size, but the epithelium on most of them had a filiform-type point and a corresponding dual
54
A. I. FARBMAN
pattern of keratinization. The typical fungiform papilla type of epithelium, namely a keratohyalin-containing, soft keratin forming epithelium, with a taste bud on the top was not seen. The absence of the taste bud itself was not surprising because there were no gustatory nerves growing into the graft to induce its formation. Those nerves that were seen in the stalks of some grafts probably grew in with the vessels and may have been sprouts from sensory or possibly parasympathetic nerves in the iris or sclera; they were seen in both the sympathectomized and non-sympathectomized rats. In an earlier discussion of taste bud development (FARBMAN,1965), it was suggested that the development of the fungiform papilla may be initiated without the influence of nerve, and that the latter may subsequently enter the forming papilla to induce taste bud formation. The present results neither confirm nor contradict this possibility. The broad papillae seen in the homografts may represent structures that began their development as fungiform papillae and continued to grow in size in the usual way, but, in the absence of nerve, did not assume their characteristic pattern of keratinization nor did they acquire gustatory cells. On the other hand, it is also possible that they were atypical filiform papillae formed perhaps by distortion. The latter is the more likely possibility because in several instances many of them were encountered adjacent to each other; in situ, it is rare to find fungiform papillae immediately adjacent to each other without several intervening filiform papillae (FISH et al., 1944; FARBMAN,1965). Striated muscle differentiated fairly well in the grafts and assumed a three-dimensional orientation as is usually seen in tongue. However, even after more than 2 weeks in the anterior chamber, some of the striated muscle cell nuclei in the graft were centrally located. These findings provide an interesting contrast to those of EASTLICK and ANDERSON(1944) who placed chick limb homografts into anterior chambers and found that striated muscle failed to differentiate at all. They concluded that the anterior chamber was not a favorable site for differentiation of muscle. COMMENT AND CONCLUSION The primary reason for initiating this study, as stated above, was to determine whether the anterior chamber of the eye would provide a favorable site for experiments on differentiation of the lingual epithelium. It seems quite clear the filiform papilla epithelium differentiates and keratinizes in a manner similar to that in vim and that further experimentation on differentiation of filiform papillae can be done using the experimental system described. However, the design of future experiments on taste bud differentiation requires a site completely free of host nerves. Unfortunately, this requirement was not met under the present experimental conditions. Alternatively, other experiments were attempted in which the tongue transplants were placed in specially constructed Millipore filter diffusion chambers modified slightly from the type described by ALGIRE et al. (1954). The chamber was placed in the peritoneal cavity of a host animal. The rationale for this was that the tissue could be nourished by diffusion of host fluids and, therefore, would grow and differentiate within the chamber but would be completely free from any contact with host cells because of the small pore-size (0.45 pm) of the filter material. Unfortunately, within 4-7 days the
TONGUE
HOMOGRAFIX
IN ANTERIOR
CHAMBER
55
transplant lost its integrity as an organ as the cells spread along the inner surfaces of the chamber. Because of this, the diffusion chamber method was unsuitable for any of the studies on epithelial differentiation. At present, experiments are in progress to grow lingual epithelium and mesenchyme in an organ culture system. Preliminary findings have revealed that this is feasible and both filiform papillae and taste buds have been successfully grown in organ culture from foetal tongue fragments. These results will be reported separately. Acknowledgements-The author would like to thank Mr. MARTIN ZIEGNJSR and Mrs. KATHERINE WALSH for their excellent assistance with these experiments. This work was supported by Grant No. NB 06181 from the National Institutes of Health and by a Grant from the Dysautonomia Society, New York. Receipt of a Research Career Development Award No. l-K3-DE-17, 654 from the NationalInstitutes of Health is acknowledged.
R&&---Des tranches de langue de rat foetal ont et& transplant&s dans la chambre anterieure de l’oeil de rats adultes et cueillies 5-19 jours plus tard. Apr&s quelques jours les homogreffes devinrent vasularisees et enkystees. La differentiation des papilles, la lamina propria et l’arrangement tri-dimensionnel simulaient les aspects in-vivo. Les papilles fungiformes et les pores gustatifs ne se sont pas dilferentib. Les homogreffes ont CtC invadees par des faisceaux de fibres nerveuses avec et sans myeline.
Zusammenfassung-Streifen von Rattenfotuszungen wurden in den Augenvorhof von ausgereiften Ratten tiberpflanzt und 5-19 Tage spater geemtet. Innerhalb weniger Tage erwiesen sich die &srpflanzungen als mit Geweben versehen und eingekapselt. Differenzierung von filiform Pap&n, der Lamina propria und der 3dimensionalen Anordmmg von Muskelfasem simulierte das in-vivo Bild. Schwammartige Papillen und die Geschmackskiirperchen entwickelten sich nicht verschieden. In die Verpflanzungen waren Btindel von Nervenfasem mit oder ohne Myelin eingedrungen.
REFERENCES AMIRE, G. H., WEAVER,J. M. and PREHN, R. T. 1954. Growth of cells in oivo in diffusion chambers. I. Survival of homoarafts in immunized mice. J. nut. Cancer 1st. 15.493-507. EASTLICK,H. L. and ANDERSON,R. H. 1944. Studies on transplanted embryonic limbs of the chick. II. The development of limb primordia within the anterior chamber of the eye. J. Morphol. 75, l-9. FARBMAN,A. I. 1965. Electron microscope study of the developing taste bud in rat fungiform papilla. Devel. Biol. 11, 110-135. F-MAN, A. I. 1970. The dual-pattern of keratinization in filiform papillae on rat tongue. J. An&. 106, 233-242. Frsrr, H. S., MAUINE, P. D. and RICHTER, C. P. 1944. The anatomy of the tongue of the domestic Norway rat. I. The skin of the tongue; the various papillae; their number and distribution. Anat. Rec. 89,429440. LUFP, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. biochem. Cytol. 24,297-301.
56
A. I. FARBNAN
MEDAWAR, P. B. l!W. Immunity to homologous grafted skin. III. The fate of skin grafts transplanted to the brain, to subcutaneous tissue and to the anterior chamber of the eye. Br. J. exp. Pathol. 29, 58-69. REYNOLDS,E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17,208-212. WATSON, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. biochem. OtoI. 4,475-478.
TONGUE HOMOGRAFTS IN ANTERIOR CHAMBER
FIG. 1. Photomicrograph
rat foetus. A two-layered
of a section of the dorsal surface of the tongue from a 14-day x 400 epithelium overlies a highly cellular mesenchyme.
FIG. 2. Electron micrograph
of an area of 16day foetal rat tongue epithelium from the same specimen as that illustrated in Fig. 1. The cells contain relatively large nuclei and the most prominent cytoplasmic constituents are free ribosomes and some mitochondria. x 7500
PLATE 1
A.O.B. f.p. 56
A.I.FARBMAN
PLATE 2
TONGUE HOMOGRAETS IN ANTERIOR CHAMBER
PLATE2 FIG. 3. Photomicrograph of a graft grown in the anterior chamber for 14 days. The epithelium is thrown up into keratinizing filiform papillae (f) which project into a flattened cyst cavity (c), filled with intensely staining keratinized cells. Muscle fibres (m) pass horizontally and vertically and some are cut in cross-section (mx), thus demonstrating the 3-dimensional arrangement of their fibres. x 100 FIG. 4. Electron micrograph of a cross-section through a tiliform papilla from a graft grown 7 days in the anterior chamber. In the upper part, cells containing keratohyahn granules (g) are seen. In the lower part, the hard keratin-producing cells containing dense tonofilaments (t), prominent free ribosomes, and no keratohyalin granules, are seen. x8000
57
TONGUE
HOMOGRAFTS
IN ANTERIOR
CHAMBER
FIG. 5. Photomicrograph of a broad papilla with a pointed tip. These papillae resemble fungiform papillae in size but have the dual pattern of keratinization typical of filiform papillae. x 400 FIG. 6. Electron micrograph of the lamina propria of a graft grown 14 days in the anterior chamber. The edge of the epithelium is seen across the top and along the right side of the field. The lamina propria contains fibroblast-type cells, containing much granular endoplasmic reticulum. Scattered connective tissue fibres are seen between cells. x 8500
PLATE
3
A.O.B. f.p. 58
A.I.
FARBMAN
FIG. 7. Electron micrograph of a graft grown 14 days in the anterior chamber. Muscle x 8000 cells (m), myelinated nerves (n) and unmyelinated nerves (u) are clearly seen.