Five kinds of monoclonal antibodies that are specific for the epidermis of Xenopus embryos were produced. Epidermis-specific antibodies were used to investigate the spatial and temporal expressions of epidermal antigens during embryonic and larval development. The cells that were recognized by the antibodies at the larval stage are as follows: all of the outer epidermal cells and cement gland cells were recognized by the antibody termed XEPI-1, all of the outer and inner epidermal cells, except the cement gland cells, were recognized by XEPI-2 antibody, the large mucus granules and the apical side of the outer epidermal cells, except for the ciliated epidermal cells, were recognized by XEPI-3 antibody, the large mucus granules and basement membrane were recognized by XEPI-4 antibody, and the small mucus granules contained in the outer epidermal cells as well as extracellular matrices were recognized by the antibody termed XEPI-5. All of the epidermal antigens, except XEPI-4, were first detected in the epidermal region of the late gastrula or early neurula. The XEPI-4 antigen was first detected in stage-26 tail-bud embryos. None of these antigens were expressed by the neural tissues at any time during embryonic development. Only the XEPI-2 antigen continued to be expressed after metamorphosis, while the expression of the other antigens disappeared during or before metamorphosis. The specificity of the antibodies allowed us to classify the epidermal cells into four types in early epidermal development. The four types of epidermal cells are (1) the outer epidermal cells that contain small mucus granules, (2) the ciliated epidermal cells, (3) the outer epidermal cells that contain large mucus granules and (4) the inner sensorial cells.
In the early development of Xenopus, the epidermal tissues are derived primarily from the animal-ventral blastomeres and partially from the animal-dorsal and the vegetal-ventral blastomeres of an 8-cell-stage embryo (Masho & Kubota, 1986). After several cleavages, the prospective epidermal area occupies more than half of the animal hemisphere and spreads toward the ventral side of the early gastrula (Keller, 1975). During gastrulation, the surface of the embryo is gradually covered by the presumptive ectoderm due to morphogenetic movements. The dorsal ectoderm, in contact with inductive chordamesoderm, thickens to form the neural plate which is subsequently folded into the neural tube. After the completion of neurulation the epidermis covers the entire surface of the embryo.
Previously, a detailed SEM study of gastrulation showed that during epiboly the multiple layers of the presumptive epidermal region interdigitate radially into two distinct layers (Keller, 1980), which are termed the outer epithelial layer and the inner sensorial layer (Nieuwkoop & Faber, 1956). The epidermis consists of two layers until metamorphosis, a period when the number of epidermal layers increases to about five layers. Later during metamorphosis, the outer layers of the larval skin are shed. After metamorphosis, at stage 66, the adult epidermis comprises more than five layers.
In the course of epidermal development as described above, several kinds of epidermal cells are differentiated (Fox, 1984), therefore, it is likely that many molecules must participate in the process. To investigate epidermal differentiation, molecular markers specific for the various kinds of epidermal cells are needed.
Several epidermal markers in amphibian embryos have been reported thus far: epimucin, a major glycoprotein in Ambystoma that is an epidermal receptor for peanut agglutinin (Slack, 1985) and a group of epidermis-specific antigens identified by monoclonal antibodies (Jones, 1985; Jones & Woodland, 1986; Akers et al. 1986). Furthermore, the transcription of cytokeratin genes has been used to investigate the differentiation of epidermal cells (Jamrich et al. 1987).
In the present investigation, we have obtained five kinds of monoclonal antibodies that recognize different antigens expressed by epidermal cells. Immunohistochemical observations on the spatial and temporal expression of epidermal antigens showed that four types of epidermal cells can be identified in Xenopus embryos.
Materials and methods
Collection of eggs
Eggs of Xenopus laevis were obtained through artificial mating after the injection of human chorionic gonadotropin into the dorsal lymph sac at a dose of 300 i.u. for females and of 200 i.u. for males. Embryos were staged according to Nieuwkoop & Faber (1956).
Preparation of antigens
We used two kinds of antigens for immunization. Embryos at stages 35 –38 were homogenized in modified Steinberg’s solution buffered to pH 7 ·0 by 3 mm-Hepes-NaOH in place of Tris-HCl. The centrifugation of the homogenate at 1600g for 10 min resulted in the separation of the homogenate into five layers. The five layers, from the top of the centrifuge tube to the bottom of the tube are as follows: the layer that contained lipid droplets, the clear layer of cytoplasm, the grey coloured layer of cytoplasm, the layer containing pigment granules and the layer that contained yolk platelets. The layer of clear cytoplasm was centrifuged again at 8000g for 10 min to pellet any residual yolk platelets and to discard the top layer of the lipid droplets. After the lipid and the pellet were discarded, the remaining clear cytoplasm was used as immunogen.
For the second source of antigens, neural and epidermal tissues were dissected from stage-33 to -38 larvae using forceps. Neural and epidermal tissues collected from 200 embryos were placed in modified Steinberg’s solution and forced through a 25-gauge syringe. The suspension of disrupted neural and epidermal tissues was used as the second kind of immunogen.
Production of hybridomas
BALB/c mice were injected intraperitoneally three times with 0 ·4 ml of the clear cytoplasm or the suspension of disrupted neural and epidermal tissues every two to four weeks. Three days after the last immunization, murine spleen cells were fused with myeloma cells (P3 ×63Ag8U-l) in the presence of 50% (w/v) polyethylene glycol 4000 (Nakarai Chem. Co. Ltd, Kyoto). Cells were suspended in DMEM (Dulbecco’s modified Eagle medium) after cell fusion and plated into 96-well plastic dishes. After approximately 12h, DMEM that contains HAT (hypoxanthine-aminopterin-thymidine) was added to the wells to raise hybridomas according to the method of Littlefield (1964). Approximately one week later, antibodies were screened by staining histological sections of stage-35/-36 embryos using indirect immunofluorescent microscopy. Hybridomas that secreted epidermis-specific antibodies were cloned on the feeder cells (thymus or spleen cells) by picking up a single cell under an inverted microscope with a fine-tipped Pasteur pipette.
Fixation and embedding
Whole embryos at various stages or excised epidermal tissues were fixed in 100% methanol for 40 min at −20 °C. Next, the fixative was replaced with 100% ethanol at −20 °C. Soon after the replacement, the samples were left at room temperature for 15 min to warm gradually the samples. The ethanol was replaced with fresh absolute ethanol at room temperature. After 15 min the samples were incubated in 50% polyester wax (Steedman, 1957; BDH Chem. Ltd) in absolute ethanol for 30 min followed by impregnation with 100 % polyester wax for 1 h at 40 °C. Samples were embedded in the polyester wax and stored at 4 °C.
Sectioning and staining
Embedded samples were sectioned at 18 °C using a microtome. 10 μm sections were adhered to cover slips (4 ·5 ×24mm) using 0 ·1 % amylopectin. After the sections dried overnight at room temperature, they were soaked in 100 % ethanol for 15 min and washed with phosphate-buffered saline (PBS). The sections were incubated with 100 /A of monoclonal supernatant for 1 h, washed with PBS and incubated with 10 μl of FITC-conjugated rabbit antimouse 1gG (Miles-Yeda Ltd) for 30 min. After the sections were washed with PBS for 15 min, they were mounted with 80% glycerol in PBS, and examined under an epifluorescence microscope.
Immunoelectron microscopy was performed as described by Asada-Kubota (1988).
The proteins that were contained in the clear cytoplasm, obtained by centrifugation of homogenized stage-35/-36 embryos, and contained in homogenized epidermal tissues (stage 54) were separated on SDS-polyacrylamide gels and blotted onto nitrocellulose sheets. The nitrocellulose sheets were soaked in 0·5 % skimmed milk and cut into strips. Strips were incubated with the hybridoma culture supernates, washed three times with PBS containing 0·1% Tween 20 and washed three times with PBS without Tween 20. Strips were incubated with 35S-labelled anti-mouse 1gG (Amersham), washed with PBS, dried and exposed for 5 days to Fuji RX X-ray film at −80°C.
Peanut lectin blocking
Peanut lectin blocking was carried out by incubating sections with peanut lectin (Hohnen Oil Co. Ltd, Tokyo) at 100 μg ml−1 for 30 min and washing them with PBS prior to staining the sections with the monoclonal antibodies and FITC-conjugated anti-mouse 1gG.
Sections were incubated overnight at 37°C with 4mi.u. neuraminidase (Seikagaku Kogyo Co. Ltd, Tokyo) in 0·4 ml 50mm-sodium acetate (pH 6·0), 80mm-NaCl, and 5mm-CaCl2, before the sections were incubated with the monoclonal antibodies and FITC-conjugated anti-mouse 1gG.
Five kinds of monoclonal antibodies (XEPI-1, XEPI-2, XEPI-3, XEPI-4 and XEP1-5) that recognized Xenopus epidermal antigens were obtained. The staining pattern of each antibody was examined at various stages from the fertilized egg (stage 1) to the frog at postmetamorphosis (stage 66). Each of the monoclonal antibodies showed a characteristic staining pattern that made it possible to locate specific antigens in epidermal cells. The spatial and temporal expression of the antigens recognized by the five antibodies are shown in Figs 1–11 and Table 1.
Expression and localization of epidermal antigens during embryonic development
Transverse or sagittal sections of Xenopus embryos from stage 1 to stage 40 were stained with the antibodies. None of the antibodies reacted with neural tissues at any stages of development that were examined.
XEPI-1 antibody did not bind to sections until after stage 12. A weak but distinct binding to the epidermal region of the embryo was first observed at stage . The staining became stronger as development proceeded (Fig. 1). XEPI-1 was never bound to any tissue other than the epidermis. The antigen recognized by XEPI-1 was localized exclusively in the outer epithelial layer. Furthermore, the apical side of the outer epithelial cells was the most heavily stained part of the cell. All of the cells that constitute the outer epithelial layer were stained at each developmental stage up to the time of metamorphosis. Asada-Kubota (1988) observed by immunoelectron microscopic study the binding of XEPI-1 on the moderately electron-dense bodies and the cortical dense materials in the outer epithelial cells. Cement gland cells were also recognized by the XEPI-1 antibody (Fig. 1D). Among five antibodies that were tested, only the XEPI-1 stained the cement gland cells.
The antigen recognized by XEPI-2 first appeared in the epidermal region at stage 13. In the neurula, the antigen recognized by XEPI-2 was found mainly in the apical side of the outer layer and very weakly in the inner layer (Fig. 2A,B). In the tail-bud embryos, the antigen could be clearly detected in both layers (Fig. 2C). The apical side of the outer layer was stained more strongly at this stage. In the larvae (stage 36), both layers were intensely stained with XEPI-2 (Fig. 2D,E).
Weak binding of the XEPI-2 antibody to the intersegmental region of somites from stage 26 to stage 34 was also observed (Fig. 2C).
The antigen that was recognized by XEPI-3 first appeared on the apical side of the outer epidermal layer at stage 13 and was strongly expressed up to the larval stage (Fig. 3). It was characteristic that the staining pattern was interrupted by the ciliated epidermal cells that are scattered throughout the outer layer (Fig. 3C,D,E). Beginning at stage 36 and continuing up to the time of metamorphosis, there was also detected binding of XEPI-3 to the large mucus granules contained in some of the epidermal cells (Fig. 3D.E).
The epidermal antigen that was recognized by XEPI-4 could not be detected until stage 26. At stage 28, there was binding of XEPI-4 to intermediate-sized granules contained in some of the outer epithelial cells (Fig. 4A). In stage-36 larvae, these granules increased in size and were stained more heavily with XEPI-4 (Fig. 4B,C). The granules seemed to leak out from the cell (Fig. 4C).
The XEPI-4 antibody also bound to the notochordal sheath from stage 18 and to the basement membrane from stage 28 (Fig. 4).
The epidermal antigen was first detected in the epidermal region at stage 13 (Fig. 5B). The apical side of the outer epithelial cells was heavily stained (Fig. 5B-E) whereas the ciliated epidermal cells were not stained (Fig. 5D,E).
The XEPI-5 antibody recognized not only epidermal tissues but also extracellular matrices (ECM). XEPI-5 was bound to the ECM in the blastocoel of blastulae (Fig. 5A). Also, this antibody was bound to the ECM that surrounds the notochord, the neural tube, and the somites at later stages (Fig. 5B-D).
Immunoelectron microscopic observation showed that XEPI-5 was bound to small mucus granules contained in the apical side of the outer epithelial cells (Fig. 5F) and the ECM.
Comparison of distribution of antigens at stage 36
The localization of the five different epidermal antigens described above were compared in the stage-36 larva (Fig. 6).
The distribution of the XEPI-1 antigen was confined to the outer epithelial cells (Fig. 6A). The XEPI-2 antigen was detected in both the outer epithelial cells and the inner sensorial cells (Fig. 6B). The XEPI-3 antibody was bound to the apical side of the outer epidermal cells, except for the ciliated cells. Also, XEPI-3 was bound to the large granules contained in some of the outer epithelial cells (Fig. 6C). Fig. 6D shows the staining pattern after sections were stained with the XEPI-4 antibody. Fig. 6C and D are photomicrographs of the adjacent sections of serial sections. These photomicrographs show that the XEPI-4 antibody was bound to the same large granules as those that were stained using the XEPI-3 antibody. Also, the XEPI-4 antibody was bound to the basement membrane (Fig. 6D). The XEPI-5 antigen was localized in the small mucus granules on the apical side of the outer epithelial cells. Ciliated epidermal cells were not stained with XEPI-5 antibody (Fig. 6E).
Thus, we could identify at least four kinds of epidermal cells in Xenopus larvae at stage 36 by combining the staining patterns of these monoclonal antibodies. The four kinds of epidermal cells were (1) the outer epidermal cells that contained small mucus granules, (2) the ciliated epidermal cells, (3) the outer epidermal cells that contained large mucus granules and (4) the inner sensorial cells.
Expression and localization of epidermal antigens during and after metamorphosis
Transverse sections of the larval body from stage 46 to stage 49 and those of the dorsal skin from stage 52 to stage 66 were stained with the antibodies described above. At stage 46, the epidermis still comprises two epidermal layers. The number of epidermal layers increases during metamorphosis. At stage 66, the epidermis comprises more than five layers.
The XEPI-1 antigen was detected in the outermost layer of the epidermis until stage 61 (Fig. 7A-D). In all larvae between stage 52 and 56, the expression of the XEPI-1 antigen was suppressed in some of the cells in the outermost layer (Fig. 7B). At stage 62, the outermost epidermal layer that expressed the XEPI-1 antigen, was being shed (Fig. 7E). At stage 64, there was no binding of the XEPI-1 antibody to the epidermis (Fig. 7F).
From stages 46 to 49, both the outer epithelial and the inner sensorial layer were stained with the XEPI-2 antibody. The latter was stained more heavily than the former (Fig. 8B). After stage 49, the number of epidermal layers increased. All the layers continued to express XEPI-2 up to stage 60 (Fig. 8C). At stage 58, the primordia of the multicellular granular and mucus glands were seen beneath the epidermis (Fig. 8C). The cells that constituted these glands were also stained. Thereafter the staining became weaker, while it persisted in the cells that faced the epidermal layers up to stage 64 (Fig. 8C-E). From stage 62, the outer layers of epidermis gradually lost the XEPI-2 antigen (Fig. 8D-F). At stage 66, only the innermost layer of epidermis could be stained (Fig. 8F).
Among the five monoclonal antibodies tested in the present investigation, only the XEPI-2 antibody stained the adult epidermis.
The antigen that was recognized by the XEPI-3 antibody situated on the apical side of the outer epithelial layer continued to be expressed but the staining was very weak (Fig. 9A-D). After the sloughing of the outer epidermis, the XEPI-3 antigen could no longer be detected (Fig. 9E).
The large mucus granules contained in the outer epithelial layer were also stained with the XEPI-3 antibody in stage-36, -40, -46 and -48 larvae (Fig. 9A). At stage 50, vacuolation began in the cells that were located between the outermost layer and the basal layer of the epidermis. Within the vacuolated cells, large mucus granules were stained with the XEPI-3 antibody (Fig. 9B) and the number of stained granules increased up to stage 60 (Fig. 9C,D). The stained vacuolated cells were located beneath the outermost cell layer. At the time that the larval skin was shed the stained vacuolated cells disappeared (Fig. 9D,E).
Also, the XEPI-4 antibody was bound to the basement membrane during and after metamorphosis (Fig. 10).
The antigen that was identified by the XEPI-5 antibody on the apical side of the outer epithelial layer disappeared after stage 40 (Fig. 11A-C). However, the XEPI-5 antibody was bound transiently to some of the outermost epidermal cells (Fig. 11D). After the sloughing of the outer epidermal layers, there was no binding of the XEPI-5 antibody to the epidermis (Fig. 11E,F).
Molecular aspects of epidermal antigens
The result of Western blotting analysis of the soluble proteins that were obtained from stage-35/-36 larvae showed that the XEPI-1 antibody recognized one major protein band having an estimated relative molecular mass of 250 × 103 (Fig. 12B). The soluble proteins extracted from stage-54 larval skin were used for Western blotting analysis using the XEPI-2 antibody. A total of six protein bands having estimated Mrs between 45 and 67×103 were resolved by the XEPI-2 antibody (Fig. 12D). As for the other three antibodies, we could not detect any bands by the Western blotting technique. The reason for this may be ( 1 ) these antigens are not proteins but glycolipids or other molecules. (2) the antigens could not be detected using Western blots because their antigenicity was altered during electrophoresis or by blotting technique or (3) the antigens could not be solubilized using the sample buffer we employed.
In order to determine whether the five monoclonal antibodies recognized peanut lectin receptors (see Slack, 1985), sections of stage-36 embryos were preincubated with peanut lectin prior to being stained with monoclonal antibodies. Peanut lectin blocked the binding of only the XEPI-4 antibody to the large mucus granules, while the binding of the XEPI-4 antibody to the basement membrane was not blocked (data not shown). This suggests that the XEPI-4 antigen in the large mucus granules is the same as one of the peanut lectin receptors reported by Slack 1985.
Neuraminidase treatment was carried out prior to staining sections with monoclonal antibodies in order to determine whether there were any antigens masked with sialic acid that could be recognized by the five antibodies (see Slack, 1985). The results show that none of the antigens were masked with sialic acid that could be recognized by these antibodies.
Epidermal cell types in the embryonic epidermis
We have obtained five monoclonal antibodies against epidermal antigens. The spatial and temporal expressions of these antigens in normal embryos show that at least four types of epidermal cells exist in the larval epidermis. The four types of epidermal cells are (1) the outer epidermal cells that contain small mucus granules, (2) the ciliated epidermal cells, (3) the outer epidermal cells that contain large mucus granules and (4) the inner sensorial cells. Among these cell types, the cells that contained small mucus granules were identified by the XEPI-5 antibody (Fig. 5). The appearance of the XEPI-5 antigen (stage 13) was slightly earlier than that of the small mucus granules that were observed at the ultrastructural level (stage 14) (Billett & Gould, 1971). The ciliated epidermal cells were detected by the XEPI-1 antibody and by the interruption in the binding of the XEPI-3 and the XEPI-5 antibodies that stain the outer epithelial layer (Fig. 6). The cells that contained large mucus granules were recognized by the XEPI-3 antibody and the XEPI-4 antibody (Fig. 6). Since their temporal patterns of staining were slightly different and the binding of only the XEPI-4 antibody to the large mucus granules could be blocked by peanut lectin, it may be concluded that the XEPI-3 and XEPI-4 antibodies recognize different antigens contained in the same granules. These cells that contained large mucus granules appeared in the outer epithelial layer at the tail-bud stage and these granules became larger as development proceeded (Fig. 4). The staining pattern of the granules recognized by the XEPI-3 and XEPI-4 antibodies at later stages suggests that these granules may be secreted (Figs 3, 4). The inner epidermal cells were recognized by the XEPI-2 antibody, but not by any of the other antibodies at any stages of development (Fig. 6). Among the five antibodies tested, the XEPI-2 antibody is the most useful tool for investigating the differentiation of the epidermis in early Xenopus development because this antibody recognizes all four types of epidermal cells. The monoclonal antibodies described in the present investigation were used to study the differentiation of the four types of epidermal cells in isolated and explanted outer and inner ectoderm (Itoh & Kubota, in preparation).
Differentiation of epidermis
The mechanism that is responsible for the differentiation of the epidermis can be discussed in terms of several models such as localization of cytoplasmic determinants, cell-cell interaction, and induction. Ectodermal explants produced from around the animal pole region of the early gastrulae were shown to differentiate into epidermal cells and express epidermal markers (Slack, 1984, 1985). Jones & Woodland (1986) showed that animal pole explants of Xenopus cultured from the 8-cell stage onwards express a specific epidermal antigen and that embryos that were either disaggregated or incubated in cytochalasin B after the midblastula stage do not require cell interactions, Ca2+ and cell divisions for epidermal differentiation to occur. Furthermore, Jones & Woodland (1987) showed that animal cap cells at stage 10 or later develop exclusively into ectodermal tissues even if they are transplanted into ectopic positions such as the blastocoel or the vegetal pole of the host embryos. These investigations suggest that the animal pole region of the embryo at cleavage stage differentiates autonomously into epidermis when cultured in isolation, although cellular interactions and cell divisions up to the midblastula stage are necessary. Further, the cells constituting the animal pole region of the embryo at stage 10 or later are determined to develop into the ectodermal tissues.
Cytokeratin gene transcripts have been detected in animal pole cells of stage-9 blastulae and in the entire ectoderm, including prospective neural area of the early gastrula (Jamrich et al. 1987). The transcription in the neural area was suppressed when in contact with the involuting chordamesoderm during gastrulation. Recently, neural differentiation has been investigated using neural-specific markers, N-CAM gene transcripts (Kintner & Melton, 1987) or using a polyclonal antibody that was produced against purified N-CAM (Jacobson & Rutishauser, 1986; Balak et al. 1987). These studies demonstrated that the level of N-CAM RNA increases during gastrulation when the mesoderm comes in contact with the ectoderm and that N-CAM was first detected at the neural plate stage (stage 14/15). Together, these investigations show that the cells that constitute the animal pole region become specified at the late blastula stage to express cytokeratin genes in the presumptive epidermal and neural cells. Furthermore, if the ectoderm is induced by the underlying mesoderm, transcription of the cytokeratin genes is suppressed, whereas the N-CAM genes are activated.
In the present study, four of the epidermal cell markers (XEPI-1, 2, 3, and 5) were detected in the epidermal region of the late gastrula (stage 121) or early neurula (stage 13), but never in the neural region. This result is consistent with those previously obtained using other kinds of epidermal markers (Slack, 1985; Jones & Woodland, 1986; Akers et al. 1986) . Therefore, it is suggested that the epidermis of the late gastrula embryo expresses molecular markers that are characteristic of differentiated cells before it expresses morphological features that are typical to the epidermal cells (Billett & Gould, 1971).
Expression of epidermal antigens during metamorphosis
From the feeding tadpole stage and onwards, the number of epidermal layers increases from two to approximately five. In all cases, the XEPI-1 antigen was expressed in the outermost layer and then disappeared with the sloughing of the outer epidermal layers before the adult stage (Fig. 7). In contrast, the XEPI-2 antigen was expressed in both layers of the epidermis during early development and in all of the epidermal layers up to stage 60. From stage 60 and later, the outer layers did not express the XEPI-2 antigen. In the adult epidermis, only the innermost layer expressed the XEPI-2 antigen (Fig. 8). These results suggest that the increase in the number of epidermal layers was due to proliferation of the inner epidermal cells at earlier stages. The cells that constituted the granular and mucus glands that are located underneath the innermost layer also expressed the XEPI-2 antigen (Fig. 8). This result is consistent with the description that the gland cells originate from the inner epidermal cells (Nieuwkoop & Faber, 1956).
The vacuolated cells (termed Leydig cells) were located between the outermost layer and the inner layers before metamorphosis and these cells contained large mucus granules that were recognized by the XEPI-3 and XEPI-4 antibodies (Figs 9, 10). At earlier larval stages, both the XEPI-3 and XEPI-4 antibodies recognized the large mucus granules contained in some of the cells that constitute the outer epithelial layer (Figs 3, 4). However, it remains to be investigated whether these granules are the same as those present in the Leydig cells.
Molecular nature of the epidermal antigens
In the present investigation, Western blotting analysis showed that the XEPI-1 antibody detected one major protein band having an estimated Mr of 250×103 (Fig. 12B). Jones (1985) showed, using the method of immunoprecipitation, that an epidermis-specific monoclonal antibody termed 2F7-C7 recognizes one major protein band that has an Mr greater than 220× 103. The staining patterns of XEPI-1 and 2F7-C7 are similar because (1) both antibodies detected epidermal antigens starting at the late gastrula stage, (2) the antigens recognized by both these antibodies were restricted to the outer epithelial layer of epidermis and (3) the cement gland cells were recognized by both XEPI-1 and 2F7-C7 antibodies. In order to determine whether the XEPI-1 antibody recognizes the same antigen as the 2F7-C7 antibody, we compared them directly in the same conditions by staining polyester wax sections, by Western blotting and by immunoelectron microscopy. The results of these experiments indicate that both antibodies showed the same staining patterns at light microscopic and ultra-microscopic levels and both antibodies recognized the same band in Western blotting (data not shown). Therefore, it can be concluded that the antigen recognized by the XEPI-1 antibody is the same as that recognized by the 2F7-C7 antibody. The XEPI-1 antibody is different from a monoclonal antibody (Epi-1) that was produced by Akers et al. (1986). By Western blot analysis, Epi-1 recognized an epidermal glycoprotein that has an Mr of approximately 300×103. Furthermore, Epi-1 did not bind to cement gland cells. Although the function of the XEPI-1 antigen is not known, it is a component of MEB (moderately electron-dense bodies) and is secreted to the surface of the embryo (Asada-Kubota, 1988).
Using the Western blotting technique, the XEPI-2 antibody recognized a total of six protein bands having estimated Mrs between 45 and 67×103 from proteins obtained from stage-54 larval epidermis (Fig. 12D). This pattern is similar to that of keratins (Ellison et al. 1985). The distributions of cytokeratins in early Xenopus development were investigated using polyclonal antibodies that recognized most of the type I embryonic cytokeratins (Jamrich et al. 1987) . The temporal distribution of the type 1 cytokeratins is different from that of the XEPI-2 antigens, but both antibodies are alike in that they recognized the inner and outer epidermal two layers. These results suggest that the XEPI-2 antigen may be composed of cytokeratins.
The XEPI-3 and XEPI-4 antigens could not be detected using the method of Western blotting. However, peanut lectin blocking studies showed that the XEPI-4 antigen in the large mucus granules is one of the peanut lectin receptors. Slack (1985, Fig. 4b) showed that these granules as well as the outer surface of epidermis were recognized by FITC-PNA (peanut agglutinin).
Slack (1985) has shown that PNA receptors exist in the ECM as well as in the epidermis. In the axolotl, the epidermal receptor is a glycoprotein, termed epimucin, and has an estimated Mr of 170×103. Furthermore, the ECM receptor was shown to contain fibronectin and other components. In Xenopus, the epidermal receptor is known to be a highly polydisperse glycoprotein. The present investigation showed that the XEPI-5 antibody was bound not only to the epidermis but also to the ECM (Fig. 5). However, the binding of the XEPI-5 antibody was not blocked by preincubation of peanut lectin and neuraminidase treatment had no effect on the staining pattern of the XEPI-5 antibody. Furthermore, the cement gland cells and the vitelline membrane were recognized by FITC-PNA but not by the XEPI-5 antibody. These results show that the XEPI-5 antigen is most likely different from the peanut lectin receptor.
The five monoclonal antibodies that we have described in this report are being used to investigate the specification and differentiation of the epidermis.
We wish to thank Dr N. Satoh for his encouragement during the course of this work. We thank Professor M. Yoneda for his critical reading of this manuscript. We thank Drs H. Fujisawa, S. Takagi, T. Nishikata and I. Mita-Miyazawa for their technical advice for raising monoclonal antibodies. Thanks are also due to Dr M. Asada-Kubota for her kind gift of immunoelectron micrographs. We thank Dr E. A. Jones for her kind gift of the 2F7-C7 antibody. We thank Dr W. R. Bates for his helpful comments on the manuscript.