Avian scale development: XI. Immunoelectron microscopic localization of alpha and beta keratins in the scutate scale

Epithelial-mesenchymal interactions play important roles in morphogenesis, histogenesis, and keratinization of the vertebrate integument. In the anterior metatarsal region of the chicken, morphogenesis results in the formation of distinct overlapping scutate scales. Recent studies have shown that the dermis of scutate scales is involved in the expression of the beta keratin gene products, which characterize terminal differentiation of the epidermis on the outer scale surface (Sawyer et al.: Dev. Biol. 101:8-18, '84; Shames and Sawyer: Dev. Biol. 116:15-22, '86; Shames and Sawyer: In A.A. Moscona and A. Monroy (eds), R.H. Sawyer (Vol. ed): Current Topics in Developmental Biology. Vol. 22: The Molecular and Developmental Biology of Keratins. New York: Academic Press, pp. 235-253, '87). Since alpha and beta keratins are both found in the scutate scale and are members of two different multigene families, it is important to know the precise location of these distinct keratins within the epidermis. In the present study, we have used protein A-gold immunoelectron microscopy with antisera made against avian alpha and beta keratins to specifically localize these keratins during development of the scutate scale to better understand the relationship between dermal cues and terminal differentiation. We find that the bundles of 3-nm filaments, characteristic of tissues known to produce beta keratins, react specifically with antiserum which recognizes beta keratin polypeptides and are found in the embryonic subperiderm that covers the entire scutate scale and in the stratum intermedium and stratum corneum making up the platelike beta stratum of the outer scale surface. Secondly, we find that 8-10-nm tonofilaments react specifically with antiserum that recognizes alpha keratin polypeptides and are located in the germinative basal cells and the lowermost cells of the stratum intermedium of the outer scale surface, as well as in the embryonic alpha stratum, which is lost from the outer surface of the scale at hatching. The alpha keratins are found throughout the epidermis of the inner surface of the scale and the hinge region. Thus, the present study further supports the hypothesis that the tissue interactions responsible for the formation of the beta stratum of scutate scales do not directly activate the synthesis of beta keratins in the germinative cells but influence these cells so that they or their progeny will activate specific beta keratin genes at the appropriate time and place.     

Avian scale development. X. Dermal induction of tissue-specific keratins in extraembryonic ectoderm

Epidermal-dermal tissue interactions regulate morphogenesis and tissue-specific keratinization of avian skin appendages. The morphogenesis of scutate scales differs from that of reticulate scales, and the keratin polypeptides of their epidermal surfaces are also different. Do the inductive cues which initiate morphogenesis of these scales also establish the tissue-specific keratin patterns of the epidermis, or does the control of tissue-specific keratinization occur at later stages of development? Unlike feathers, scutate and reticulate scales can be easily separated into their epidermal and dermal components late in development when the major events of morphogenesis have been completed and keratinization will begin. Using a common responding tissue (chorionic epithelium) in combination with scutate and reticulate scale dermises, we find that these embryonic dermises, which have completed morphogenesis, can direct tissue-specific statification and keratinization. In other words, once a scale dermis has acquired its form, through normal morphogenesis, it is no longer able to initiate morphogenesis of that scale, but it can direct tissue-specific stratification and keratinization of a foreign ectodermal epithelium, which itself has not undergone scale morphogenesis.     

Differences in the histogenesis and keratin expression of avian extraembryonic ectoderm and endoderm recombined with dermis

The responses of the chorionic ectoderm and allantoic endoderm (from 8-day chick embryos) to dermal induction were compared through tissue recombinants grafted onto the chorioallantoic membrane. The chorionic epithelium formed the appropriate epidermis with a fully developed stratum corneum in response to both spur and scutate scale dermises. Analysis of these recombinant epidermal tissues by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that tissue-specific expression of the alpha (alpha) and beta (beta) keratin polypeptides occurred. In addition, indirect immunofluorescence studies with antisera to alpha or beta keratins showed that the beta stratum, which characterizes the epidermis of spurs and scutate scales, was formed, and the alpha keratins were distributed as in the normal epidermal tissues. In contrast, although the allantoic endoderm became stratified in association with either spur or scutate scale dermis, a stratum corneum with a beta stratum did not develop. SDS-PAGE analysis demonstrated that while the characteristic beta keratins of scutate scales and spur were not detected, most of the alpha keratins normally elaborated by these structures were present, suggesting that even without histogenesis of a stratum corneum the expression of alpha keratins of endoderm could be regulated in a tissue-specific manner by dermis. This study also demonstrated that there are differences in the abilities of the chorionic and allantoic epithelia to respond to the same dermal cues, which may reflect earlier restrictions in their developmental potentials.     

Avian scale development. XIII. Epidermal germinative cells are committed to appendage-specific differentiation and respond to patterned cues in the dermis.

The ability of the germinative cell population of scutate scale epidermis to continue to generate cells that undergo their appendage-specific differentiation (beta stratum formation), when associated with foreign dermis, was examined. Tissue recombination experiments were carried out which placed anterior metatarsal epidermis (scutate scale forming region) from normal 15-day chick embryos with either the anterior metatarsal dermis from 15-day scaleless (sc/sc) embryos or the dermis from the metatarsal footpad (reticulate scale forming region) of 15-day normal embryos. Neither of these dermal tissues are able to induce beta stratum formation in the simple ectodermal epithelium of the chorion, however, the footpad dermis develops an appendage-specific pattern during morphogenesis of the reticulate scales, while the sc/sc dermis does not. Morphological and immunohistological criteria were used to assess appendage-specific epidermal differentiation in these recombinants. The results show that the germinative cell population of the 15-day scutate scale epidermis is committed to generating suprabasal cells that follow their appendage-specific pathways of histogenesis and terminal differentiation. Of significance is the observation that the expression of this determined state occurred only when the epidermis differentiated in association with the footpad dermis, not when it was associated with the sc/sc dermis. The consistent positioning of the newly generated beta strata to the apical regions of individual reticulate-like appendages demonstrates that the dermal cues necessary for terminal epidermal differentiation are present in a reticulate scale pattern. The observation that beta stratum formation is completely missing in the determined scutate scale epidermis when associated with the sc/sc dermis adds to our understanding of the sc/sc defect. The present data support the conclusion of earlier studies that the anterior metatarsal dermis from 15-day sc/sc embryos lacks the ability to induce beta stratum formation in a foreign epithelium. In addition, these observations evoke the hypothesis that the sc/sc dermis either lacks the cues (generated during scutate and reticulate scale morphogenesis) necessary for terminal differentiation of the determined scutate scale epidermis or inhibits the generation of a beta stratum.     

Region-specific expression of scutate scale type beta keratins in the developing chick beak.

This study shows that different patterns of scutate scale type beta keratins are accumulated in the three adjacent structures of the embryonic chick beak: periderm, egg tooth, and cornified beak. The cornified beak accumulates all of the beta keratins of scutate scale except pp2,3. The periderm, which is the outermost, multilayered covering of the whole embryonic beak, accumulates only beta keratins 2,3, and p2,3 of the scutate scale pattern. The egg tooth, which is the rounded elevation on the dorsal surface of the upper beak, and the embryonic claw accumulate greatly reduced levels of 2,3 and p2,3 compared to scutate scale. Like cornified beak, the claw does not accumulate pp2,3, but both tissues express a potentially new beta keratin, beta keratin 8. Neither the histidine rich "fast" proteins (HRPs), which are expressed in embryonic scutate scales and feathers, nor the avian cytokeratin associated proteins (cap-1 and cap-2), which are expressed in scutate and reticulate scales, are expressed in any of the embryonic beak structures or in the claw. The implications of these findings with regard to regulation of terminal differentiation of avian skin are discussed.     

Biochemical identification and immunological localization of two non-keratin polypeptides associated with the terminal differentiation of avian scale epidermis

Summary The expression of two previously uncharacterized polypeptides produced in epidermal cells of chick reticulate and scutate scales during late embryonic scale histogenesis and in hatchling birds has been studied biochemically and immunologically. These polypeptides have been identified by two-dimensional pH gradient gel electrophoresis as basic in charge, with apparent molecular weights of 20 and 23 kD, and they have been characterized immunologically and by amino acid analysis as non-keratin in nature. Monoclonal antibodies which react with both polypeptides have been used for immunohistochemical and immunogold electron-microscopic localization. Immunoreactivity was observed in suprabasal cells of reticulate scale epidermis, where it codistributed with bundles of -type cytokeratins in the -keratin-rich layers of epidermis known as the alpha stratum and in suprabasal cells of the outer epidermal surface of scutate scales, where it codistributed with -and -type keratin filament bundles in the -keratin-rich layers of epidermis known as the beta stratum.     

Avian scale development. IX. Scale formation by scaleless (sc/sc) epidermis under the influence of normal scale dermis

Unlike normal scutate scales whose outer and inner epidermal surfaces elaborate β (β-keratins) and α (α-keratins) strata, respectively, the scaleless mutant's anterior metatarsal epidermis remains flat and elaborates only an α stratum. Reciprocal epidermal-dermal recombinations of presumptive scale tissues from normal and mutant embryos have demonstrated that the scaleless defect is expressed only by the epidermis. In fact, the scaleless anterior metatarsal epidermis is unable to undergo placode formation. More recently, it has been determined that the absence of epidermal placode morphogenesis into a definitive scale ridge actually results in the establishment of a scale dermis which is incapable of inducing the outer and inner epidermal surfaces of scutate scales. Can the initial genetic defect in the scaleless anterior metatarsal epidermis be overcome by replacing the defective dermis with a normal scutate scale dermis, i.e., a dermis with scale ridges already present? Or, are the genes involved in the production of a β stratum regulated by events directly associated with morphogenesis of the epidermal placode? In the present study, we combined scaleless anterior metatarsal epidermis (stages 36 to 42) with normal scutate scale dermis (stage 40, 41, or 42) old enough to have acquired its scutate scale-inducing ability. After 7 days of growth as chorioallantoic membrane grafts, we observed grossly and histologically, typical scutate scales in these recombinant grafts. Electron microscopic and electrophoretic analyses have verified that these recombinant scales are true scutate scales. The scaleless mutation, known to be expressed initially by the anterior metatarsal epidermis, can be overcome by exposing this epidermis to appropriate inductive cues, i.e., cues that direct the differentiation of the outer and inner epidermal surfaces of the scutate scales and the production of specific structural proteins. We have determined that the time between stages 38 and 39 is the critical period during which the normal scutate scale dermis acquires these inductive abilities.     

Keratinization of the outer surface of the avian scutate scale: Interrelationship of alpha and beta keratin filaments in a cornifying tissue

Summary The outer surface of adult Gallus domesticus scutate scale was studied as a model for epidermal cornification involving accumulation of both alpha and beta keratins. Electron-microscopic analysis demonstrated that the basal cells of the adult epidermis contained abundant lipid droplets and that filament bundles and desmosomes were distributed throughout the cell layers. Indirect immunofluorescence microscopy and double-labeling immunogold-electron microscopy confirmed that the stratum germinativum contained alpha keratin but not beta keratin. Beta keratins were first detected in the stratum intermedium and were always found intermingled with filament bundles of alpha keratin. As the differentiating cells moved into the outer regions of the stratum intermedium and the stratum corneum, the large mixed keratin filament bundles labeled increasingly more with beta keratin antiserum and relatively less so with alpha keratin antiserum. Sodium dodecyl sulfate-polyacrylamide gel analysis of vertical layers of the outer surface of the scutate scale confirmed that cells having reached the outermost layers of stratum corneum had preferentially lost alpha keratin. The mixed bundles of alpha and beta keratin filaments were closely associated with desmosomes in the lower stratum intermedium and with electron-dense aggregates in the cytoplasm of cells in the outer stratum intermedium. Using anti-desmosomal serum it was shown that these cytoplasmic plaques were desmosomes.     

Avian skin development and the evolutionary origin of feathers

The discovery of several dinosaurs with filamentous integumentary appendages of different morphologies has stimulated models for the evolutionary origin of feathers. In order to understand these models, knowledge of the development of the avian integument must be put into an evolutionary context. Thus, we present a review of avian scale and feather development, which summarizes the morphogenetic events involved, as well as the expression of the beta (beta) keratin multigene family that characterizes the epidermal appendages of reptiles and birds. First we review information on the evolution of the ectodermal epidermis and its beta (beta) keratins. Then we examine the morphogenesis of scutate scales and feathers including studies in which the extraembryonic ectoderm of the chorion is used to examine dermal induction. We also present studies on the scaleless (sc) mutant, and, because of the recent discovery of "four-winged" dinosaurs, we review earlier studies of a chicken strain, Silkie, that expresses ptilopody (pti), "feathered feet." We conclude that the ability of the ectodermal epidermis to generate discrete cell populations capable of forming functional structural elements consisting of specific members of the beta keratin multigene family was a plesiomorphic feature of the archosaurian ancestor of crocodilians and birds. Evidence suggests that the discrete epidermal lineages that make up the embryonic feather filament of extant birds are homologous with similar embryonic lineages of the developing scutate scales of birds and the scales of alligators. We believe that the early expression of conserved signaling modules in the embryonic skin of the avian ancestor led to the early morphogenesis of the embryonic feather filament, with its periderm, sheath, and barb ridge lineages forming the first protofeather. Invagination of the epidermis of the protofeather led to formation of the follicle providing for feather renewal and diversification. The observations that scale formation in birds involves an inhibition of feather formation coupled with observations on the feathered feet of the scaleless (High-line) and Silkie strains support the view that the ancestor of modern birds may have had feathered hind limbs similar to those recently discovered in nonavian dromaeosaurids. And finally, our recent observation on the bristles of the wild turkey beard raises the possibility that similar integumentary appendages may have adorned nonavian dinosaurs, and thus all filamentous integumentary appendages may not be homologous to modern feathers.     

Expression of the cell adhesion molecules, L-CAM and N-CAM during avian scale development

To examine the involvement of cell adhesion molecules in the inductive epithelial-mesenchymal interactions during avian scale development, a study of the spatiotemporal distribution of L-CAM and N-CAM was undertaken. During scutate scale development, L-CAM and N-CAM are expressed together in cells of the transient embryonic layers destined to be lost at hatching. The ongoing linkage of the cells of these layers by both CAMs sets them apart, early in development, as unique cell populations. L-CAM and N-CAM were also expressed simultaneously at the basal surface of the early germinative cells where signal transduction is presumed to occur. In spite of the differences in cell shape, adhesion, density and proliferative state between populations of epidermal placode and interplacode cells, the expression of L-CAM and N-CAM appeared to be uniform and nondiscriminating for these discrete cell lineages. The same pattern of L-CAM and N-CAM expression was observed during morphogenesis of reticulate scales that develop without placode formation. While L-CAM and N-CAM are present during the early stages of scale development and most likely function in cell adhesion, the data do not support a role for these adhesion molecules in the formation of the morphogenetically critical placode and interplacode cell populations. In both scale types, L-CAM became predominantly epithelial, and N-CAM became predominantly dermal as histogenesis occurred. Initially, N-CAM was concentrated near the basal lamina where it may be involved in the reciprocal epidermal-dermal interactions required for morphogenesis. However, as development of the scales progressed, N-CAM disappeared from the tissues. L-CAM expression continued in the epidermis and was intense on all suprabasal cells undergoing differentiation into either an alpha-stratum or beta-stratum. However, L-CAM was more prevalent on the basal cells of alpha-keratinizing regions than on the basal cells of beta-keratinizing regions.     

Development and keratinization of the epidermis in the common lizard, Anolis carolinensis

Epithelial-mesenchymal interactions play important roles in the development of the vertebrate integument with its diverse appendages. As a result of these interactions, specific morphogenetic events occur which result in the formation of distinct epidermal appendages. Following the early morphogenetic events involving cell proliferation and movement, other developmental events such as stratification, histotypic differentiation, and terminal cytodifferentiation occur in the epidermis. Using the common lizard Anolis carolinensis, we are seeking to obtain a better understanding of the relationship between the various developmental events and the expression of alpha and beta keratins, with the aim of eventually understanding the mechanisms by which tissue-specific keratinization patterns are established in the integument. As a first step, we have used immunoblot analyses and indirect immunofluorescence procedures with antisera specific for either alpha or beta keratins to determine the temporal and spatial appearance of these keratins at specific developmental stages. We have found that: 1) There are relatively low molecular weight alpha keratin polypeptides present in the epidermis early in development as morphogenesis is taking place. 2) After morphogenesis occurs and histogenesis is well under way, the alpha keratins which characterize the adult epidermis appear. 3) Only alpha keratins are found in the basal cells of all regions of the epidermis. 4) beta keratins are found only in the suprabasal layers of well-developed scales and show region-specific distribution in overlapping scales.     

Avian feather development: relationships between morphogenesis and keratinization

Morphogenesis and expression of the alpha and beta keratin polypeptides are controlled by epidermal-dermal interactions during development of avian skin derivatives. We have examined the relationship between morphogenesis of the embryonic feather and expression of the feather alpha and beta keratins by routine histology, indirect-immunofluorescence, and SDS-PAGE. Initially beta keratins are expressed only in the feather sheath. Following barb ridge morphogenesis beta keratins can be detected in the barb ridge, coincident with the differentiation of barb ridge cells into eight distinct morphological types. Beta keratinization occurs in gradients; from feather apex to base, and from periphery of the barb ridge to the interior. The onset of beta keratinization in the barb ridges is paralleled by an increase in the major feather beta keratin polypeptides, as detected by SDS-PAGE. The alpha keratins are present in both the periderm and feather sheath at early stages of feather development, but become greatly reduced after hatching, when the down feather emerges from the sheath.     

Differential expression of keratin genes during mouse development

Suprabasal layers of the newborn mouse epidermis contain two mRNAs of 2.0 and 2.4 kb which are translated into keratins of 59 and 67 kDa, respectively. To study their expression during development, cDNA sequences corresponding to the 2.0- and the 2.4-kb mRNAs were cloned, characterized by hybridization selection assay, and used as probes to detect keratin sequences in polyadenylated RNA from Day 11, 13, 15, and 17 embryos. In RNA from Day 11 of gestation, two RNAs of 2.8 and 1.8 kb were identified. They were found to have homologies with both epidermal RNAs, suggesting that they are coding for proteins of the keratin family. These two sequences were not detected in sample of later stages. RNAs comigrating with the two epidermal keratin RNAs were identified only in Day 15 and 17 embryos indicating that their expression was induced between Day 13 and 15. Finally, the localization of the 59-kDa keratin mRNA was examined by in situ hybridization. The spinous and granulous cell layers were found to be heavily covered with grains while other regions of the tissue sections were unlabeled. All these results support the hypothesis of a sequential expression of keratins during differentiation of epidermal cells and suggest that proteins related to the keratins expressed specifically in keratinizing cells are expressed earlier during development.     

The initial expression and patterned appearance of tenascin in scutate scales is absent from the dermis of the scaleless (sc/sc) chicken.

Morphogenesis of the anterior metatarsal skin (scutate scale region), from 9.5 to 12 days of development, results in the formation of orderly patterned scale ridges. It is after the initial formation of the Definitive Scale Ridge that the characteristic outer and inner epidermal surfaces differentiate. The hard, plate-like beta stratum, with its unique beta keratins, characterizes the epidermis of the outer surface, while the epidermis of the inner surface elaborates an alpha stratum. The anterior metatarsal region of the scaleless mutant does not undergo scale morphogenesis. Therefore, scale ridges do not form nor do the outer and inner epidermal surfaces with their characteristic beta and alpha strata. We have found that the extracellular matrix molecule, tenascin, first appears in the scutate scale dermis at 12 days of development when the scale ridge is established. Tenascin is found in the dermis only under the scale ridge and is not associated with the dermal-epidermal junction. Tenascin is not found in scaleless anterior metatarsal dermis at this time. As outgrowth of the Definitive Scale Ridge takes place, tenascin distribution correlates closely with the formation of the outer epidermal surface of each scale ridge. By 16 days of development tenascin is also found in close association with the dermal-epidermal junction. Tenascin does not appear in scaleless anterior metatarsal dermis until 16 days of development and then it is randomly and sparsely distributed at the dermal-epidermal junction. Tenascin's initial appearance and pattern of distribution in the scutate scale dermis and its abnormal expression in the scaleless dermis suggest that morphogenesis plays a significant role in regulation of its expression.     

Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.     

Avian scale development. VII. Normal keratinization follows abnormal morphogenesis of reticulate scales from the “scaleless” mutant

The scutate scales are entirely missing in chick embryos homozygous for the gene, “scaleless.” Reticulate scales of this mutant are present; however, they have undergone abnormal morphogenesis into irregular mounds and crevices. The pattern of keratinization seen along the anterior metatarsus of normal embryos differs dramatically from that seen along the anterior metatarsus of scaleless embryos. In contrast, we find that the unique pattern of keratinization seen in the epidermal cells of normal reticulate scales is retained in mutant reticulate scales, even though these scales are morphologically abnormal. We believe that differences in the initial tissue interactions (which establish the inductive ability of the dermis) of these two types of scales are responsible for the differences seen in their responses to the scaleless gene. The pleiotropic nature of the scaleless gene is discussed.     

Immunocytochemical localization and biochemical analysis of alpha and beta keratins in the avian lingual epithelium

The alpha and beta keratins are found as 10-nm and 3-nm cytoplasmic filaments, respectively. While the alpha keratins are produced in essentially all vertebrate epithelia (Franke et al.: Exp. Cell Res., 116:429-445, 1978; Sun et al.: Proc. Natl. Acad. Sci. USA, 76:2813-2817, 1979), the beta keratins have been demonstrated only in specific epithelial tissues of birds and reptiles (Sawyer et al.: In: Biology of the Integument: Vertebrates. J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards, eds. Springer-Verlag, Berlin, Vol. 2, pp. 194-238, 1986; Landmann: In: Biology of the Integument: Vertebrates. J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards, eds. Springer-Verlag, Berlin, Vol. 2, pp. 150-187, 1986). Recently, Homberger and Brush (Zoomorphology, 106:103-114, 1986) have demonstrated that within the lingual epithelium of parrots, beta keratins are expressed exclusively in the anterior ventral region. While it is well established that epidermal-dermal interactions are important for the regional expression of the beta keratin genes in the avian scutate scales and feathers, little is known about the expression of beta keratins in other epithelial structures such as the tongue. We have used biochemical and immunocytochemical techniques to analyze the alpha and beta keratins of the lingual epithelium of the chick as an initial step in the characterization of this model system for developmental studies. We have found that alpha keratins are present throughout the lingual epithelium. The anterior ventral epithelium contains alpha keratin polypeptides characteristic of skin-type differentiation, while the epithelium of the dorsal and posterior ventral regions contains alpha keratin polypeptides characteristic of esophageal-type differentiation (O'Guin et al.: In: Current Topics in Developmental Biology: The Molecular and Developmental Biology of Keratins. A.A. Moscona and A. Monroy, eds. R.H. Sawyer, vol. ed. Academic Press, New York, Vol. 22, pp. 282-306, 1987). Beta keratins are produced only in the differentiated epithelial cells of the anterior ventral region of the tongue. Immunoelectron microscopy demonstrates that the alpha and beta keratins of the stratum intermedium and corneum of the anterior ventral region are found together in the large filament bundles characteristic of this region. The preexistence of the alpha keratins in the cells destined to produce beta keratins as well as the colocalization of these keratins in the filament bundles of these cells suggests that a functional relationship may exist between the alpha and beta keratins.