We report the identity of a major component of Tritoninsoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.

Intermediate filament (IF) proteins are components of the cytoskeleton of virtually all vertebrate cell types. Sequence analysis has allowed at least five different classes of IF proteins to be identified (Steinert and Roop, 1988). A wealth of information is now available describing detailed expression patterns of the individual IF proteins from each class in most tissue types. In contrast, very little is known about the expression of IF proteins in embryos, particularly during the early cleavage stages. In this paper we extend our studies on the IF cytoskeleton of early Xenopus embryos.

We have previously made an antibody, Z9, against the non-helical carboxy terminus of Xenopus vimentin expressed as a fusion protein (Torpey et al. 1990). In tissue sections of larvae, this antibody stained cell types known to express vimentin in other species. The antibody did not cross-react with any epithelia, which contain cytokeratins, or with muscle or nerve cells, which contain desmin and neurofilaments respectively. Z9 also showed staining of tissue sections of Xenopus oocytes and early blastulae. Staining was particularly concentrated in an area of the cytoplasm known as the germ plasm (Smith and Williams, 1975). We observed less intense staining in other areas of the cytoplasm.

These observations led us to believe that vimentin was present in Xenopus oocytes and early embryos, in agreement with previous studies (Godsave et al. 1984a; Tang et al. 1988). This was confirmed by immunoblotting experiments (Torpey et al. 1990). However, Z9 also cross-reacted with two Triton-insoluble proteins in oocytes and early embryos that were not vimentin. This was surprising, since the antibody seemed only to react with vimentin in larvae, even though many different IF proteins are expressed by this stage (Jamrich et al. 1987; LaFlamme et al. 1988; Sharpe, 1988; Henman et al. 1989a, b; Fouquet et al. 1988). We were therefore uncertain whether the staining of oocyte and early embryo tissue sections represented the distribution of vimentin at these stages, or a combination of vimentin and the other, presumably vimentin-related, proteins.

In this paper we show, by direct micro-sequencing, that one of these vimentin-related proteins is a basic (type II) cytokeratin. Our sequence is virtually identical to parts of the predicted amino acid sequence of the cDNA XCK1(8) (Franz and Franke, 1986). This cDNA encodes the equivalent of the mammalian type II cytokeratin 8, found in simple epithelia, and is expressed in oocytes. The protein product of this mRNA has not been directly demonstrated in oocytes or early embryos, although various antibodies have been found that cross-react with cytokeratin-like networks at these stages (Franz et al. 1983; Godsave et al. 1984b, 1986; Klymkowsky et al. 1987). We describe a new antibody raised against the non-helical carboxy terminus of XCK1(8). This antibody, called CK7, recognises both of the proteins that we have previously identified, indicating that they are both type II cytokeratins, but does not cross-react with vimentin. CK7 stains a cytoskeletal network in oocytes and early embryos that is distinct from that stained by Z9. We conclude that Xenopus oocytes and early embryos contain two complementary IF networks.

Protein microsequencing

Triton extracts of early blastulae (stage 5; Nieuwkoop and Faber, 1956) were separated by 2-dimensional (2D) electrophoresis as previously described (Tang et al. 1988; Torpey et al. 1990). About 100 embryos were loaded onto each of 36 IEF tube gels. Each tube gel was then cut into 3 equal pieces, the middle one containing the protein of interest (protein ‘z’; Torpey et al. 1990). Three such segments were loaded on top of 6%-12% SDS-containing gels, and protein ‘2’ was localised by staining with Coomassie blue. The appropriate dots were excised and washed with water until the pH exceeded 6. All 36 dots were then combined and processed exactly according to Rasmussen et al. 1991). Sequencing was carried out using an Applied Biosystems model 470A sequenator.

Antibody production

A Ddel fragment from positions 1286–1522 of the clone XCK1(8) (Franz and Franke, 1986; kindly provided by Dr W. Franke) was purified from a 6% acrylamide-TBE gel, and the ends filled in using the Klenow fragment of Escherichia coli DNA polymerase I following standard procedures (Sambrook et al. 1989). This fragment was ligated into the Smai site of the vector pGEX 1 (Pharmacia). The identity and orientation of the clone were confirmed by double-stranded sequencing using Sequenase (United States Biochemical Corp.). Fusion proteins consisting of an amino-terminal Mr 26,000 portion of the enzyme glutathione-S-transferase and a carboxy-terminal XCK1(8) fragment were produced in E. coli DH5α and purified by following exactly the protocols of Smith and Johnson (1988). Yields of Img fusion protein per 100ml bacterial culture were routinely obtained. Antibodies were raised in two Dutch rabbits, each initially injected subcutaneously with 200μg fusion protein emulsified in complete Freund’s adjuvant. Subsequent immunisations were given at 4-week intervals with 100μg protein in incomplete adjuvant. Following two such boosts, serum from both rabbits strongly stained epithelial tissues in sections of Xenopus larvae (see Results). Antibodies were affinity purified as previously described (Torpey et al. 1990).

Immunoblot analysis of embryos

Triton extracts, electrophoresis and immunoblots were all performed according to Torpey et al. (1990).

Preparation of mRNA and embryo injection

Synthetic vimentin mRNA was prepared and injected into oocytes as described by Torpey et al. (1990). In the present study, 2ng of vimentin mRNA was injected into manually defolliculated oocytes, which were subsequently cultured for 20 hours. The oocytes were then treated with 2mg/ml collagenase H (Boehringer) to remove follicle cells prior to biochemical analysis (Tang et al. 1988).

RNase protection assays

Oocyte and embryo RNA was extracted as described by Gurdon et al. (1985). A suitable probe was prepared by ligating an Alul fragment (positions 739–852) of the clone XCK1(8) (Franz and Franke, 1986) into pSP73 (Promega). This construct (CK22) was linearised with BglR, and RNA was synthesised using bacteriophage T7 polymerase, as described by Krieg and Melton (1987). The 5S probe was synthesised by transcribing the plasmid pSP 5S (Sharpe et al. 1987) with SP6 polymerase, after cutting with PvuII. Protection assays were performed using 2 oocyte or embryo equivalents of RNA, according to Krieg and Melton (1987).

Immunocytochemistry

Larval stages, fixed in 2% TCA for 24 hours, and oocytes fixed for 24 hours in 100% ethanol were embedded in PEDS (polyethyleneglycol 400 distearate + 1% cetyl alcohol) wax, and 7–10μm sections cut (Godsave et al. 1984a). Sections were dewaxed in an acetone series, rinsed in PBS and flooded with blocking buffer (10% horse serum, 4% BSA, in PBS). After 10 minutes the blocking buffer was replaced by the appropriate antibody solution, and the slides incubated overnight at 4°C, or for 2·4 hours at room temperature. After 3×5 min washes in washing buffer (PBS containing 1% horse serum), the slides were incubated with fluorescein conjugated antirabbit antibody (FITC GAR, Nordic, diluted 1:50) for 1–2 hours. The slides were then washed 3×5 min in washing buffer, with 0.01% eriochrome black included in the final wash to eliminate yolk autofluorescence, and mounted in 90% glycerol/PBS containing 100mg/ml l,4-diazabicyclo(2,2,2)oc-tane (DABCO). Slides were photographed using a Zeiss Axiophot microscope onto Ilford XP1 film.

Microsequencing of Mr 57,s000 protein

We have previously identified two vimentin-related proteins in Triton extracts of Xenopus oocytes and early embryos by their cross-reaction with the antibody Z9 (Torpey et al. 1990). We chose to characterise the more abundant of these (Mr 57,000) by direct microsequencing. Initial attempts using protein blotted onto PVDF membranes from 2D gels proved unsuccessful, presumably because the protein was blocked at the N terminus. We therefore collected this protein from 36 2D gels stained with Coomassie blue, as described above. The gel pieces were then processed according to Rasmussen et al. (1991), resulting in a number of HPLC-purified tryptic fragments. The most abundant of these was selected for microsequencing.

The HPLC fraction selected contained three fragments. It was possible to resolve the N-terminal sequence of each of these on the basis of their relative abundance in the sample. The three sequences obtained (Pl, P2 and P3) are shown in Fig. 1. Comparison of these sequences with the predicted amino acid sequence of a Xenopus cytokeratin clone (XCK1(8); Franz and Franke, 1986) known to be expressed in oocytes, revealed considerable similarities (Fig. 1). Pl differs at 2 positions out of 12, and P2 is interrupted by an isoleucine residue in XCK1(8). The identity of one residue of P3 was unclear from our data, but in XCK1(8) is occupied by a serine.

Fig. 1.

Sequences of three tryptic fragments obtained from the Mr 57,000 protein, arbitarily named Pl, P2 and P3. A comparison of these with XCK1(8) is shown. Amino acid positions within the XCK1(8) clone are given above each comparison, mismatches are in bold type.

Fig. 1.

Sequences of three tryptic fragments obtained from the Mr 57,000 protein, arbitarily named Pl, P2 and P3. A comparison of these with XCK1(8) is shown. Amino acid positions within the XCK1(8) clone are given above each comparison, mismatches are in bold type.

This result strongly suggests that the Mr 57,000 protein is closely related to the XCK1(8) protein, or, allowing for error in the sequence, is this protein. We presume that the Mr 56,000 protein is closely related, and that the two proteins are the products of two nonallelic XCK1(8) genes.

Production of an anti-XCKl(8) antibody

The XCK1(8) protein has not been previously identified in Xenopus oocytes and embryos, although its mRNA is known to be present throughout development (Franz and Franke, 1986; Fouquet et al. 1988). To investigate further the expression of this protein we produced a polyclonal antiserum by immunising rabbits with part of the non-helical C terminus of XCK1(8) expressed as a fusion protein (see Materials and methods). This part of the molecule was chosen because of its low homology with the closely related Xenopus cytokeratin CK55/56, expressed during embryogenesis but not in oocytes (Fouquet et al. 1988) and also with Xenopus vimentin (Herrman et al. 1989a). The antibody is called CK7, and has been used affinity-purified in all the experiments in this study.

The specificity of the antibody was determined using two-dimensional (2D) immunoblotting of Triton extracts of oocytes and embryos. The antibody blots two pairs of proteins of Mr 56,000 and 57,000 throughout development (Fig. 2). The positions of these proteins correspond exactly to those we had previously identified as ‘vimentin-related’ and, in the case of the Mr 57,000 protein, the one we have microsequenced. This result supports our conclusion that these two proteins are the products of two non-allelic XCK1(8) genes, although it is formally possible that there is a second, as yet unidentified, type II keratin expressed in oocytes. 2D immunoblots of later stages show a very strong cross-reaction with the same two proteins (Fig. 2C). In particular, no other proteins in the Afr 55,000 to 57,000 range are detected, even though other cytokeratins of this Mr are expressed at these stages (Fouquet et al. 1988). Three smaller more acidic proteins do crossreact, although their position suggests that they may be fragments of the XCK1(8) keratin. The antibody does not cross-react with vimentin. The position and relative abundance of vimentin is shown on Comassie blue-stained gels corresponding to the immunoblot of stage 38 (Fig. 2D).

Fig. 2.

2-D immunoblots of Triton-insoluble extracts from stage 5 (A), stage 17 (B), and stage 38 (C)Xenopus embryos, using affinity-purified CK7 antibody (2μg/ml). Mr 56,000 and 57,000 proteins can be seen in each case (indicated in A). Arrows in C mark three smaller, more acidic cross-reacting proteins. D shows a Coomassie blue stain of stage 38 Tritoninsoluble proteins. CK7 immunoreactive proteins are arrowed, and the position of vimentin shown (v).’ Loadings: A-C, 5 embryos; D, 20 embryos.

Fig. 2.

2-D immunoblots of Triton-insoluble extracts from stage 5 (A), stage 17 (B), and stage 38 (C)Xenopus embryos, using affinity-purified CK7 antibody (2μg/ml). Mr 56,000 and 57,000 proteins can be seen in each case (indicated in A). Arrows in C mark three smaller, more acidic cross-reacting proteins. D shows a Coomassie blue stain of stage 38 Tritoninsoluble proteins. CK7 immunoreactive proteins are arrowed, and the position of vimentin shown (v).’ Loadings: A-C, 5 embryos; D, 20 embryos.

To demonstrate further that CK7 does not cross-react with vimentin, we compared immunoblots stained with both this antibody and Z9, our anti-vimentin antibody. Z9 blots three proteins of MT 55,000, 56,000 and 57,000 in Triton extracts of oocytes (Fig. 3B). The Mr 55,000 protein is vimentin (Torpey et al. 1990), and a blot of oocytes that have been injected with synthetic vimentin mRNA shows considerable enhancement of this band (Fig. 3B). When these same samples are blotted with CK7, only the MT 56,000 and 57,000 proteins are detected, even though vimentin is vastly over expressed in the sample of oocytes injected with synthetic Viml mRNA (Fig. 3A).

Fig. 3.

(A and B) show immunoblots of Triton-insoluble proteins from 10 defollicuiated oocytes, either uninjected (–) or injected with 5μg of synthetic vimentin mRNA 20 hours prior to preparation of the extract (+). A is blotted with affinity-purified CK7 antibody (2μg/ml), and B with affinity-purified Z9 (5μg/ml). Arrows show Mr 55, 56 and 57,000 proteins. Overexpressed vimentin is detected by Z9 (B; lane 1), but not by CK7 (A; lane 1). C shows Triton extracts of 5 stage 5, 10, 17, 23 embryos, and 2 stage 38 embryos blotted with CK7 (2,μg/ml).

Fig. 3.

(A and B) show immunoblots of Triton-insoluble proteins from 10 defollicuiated oocytes, either uninjected (–) or injected with 5μg of synthetic vimentin mRNA 20 hours prior to preparation of the extract (+). A is blotted with affinity-purified CK7 antibody (2μg/ml), and B with affinity-purified Z9 (5μg/ml). Arrows show Mr 55, 56 and 57,000 proteins. Overexpressed vimentin is detected by Z9 (B; lane 1), but not by CK7 (A; lane 1). C shows Triton extracts of 5 stage 5, 10, 17, 23 embryos, and 2 stage 38 embryos blotted with CK7 (2,μg/ml).

Expression of CK1 (8) protein during development

We have shown by 2D immunoblotting that the CK7 antibody is specific for the two XCK1(8) proteins. These two proteins can also be seen as two bands on a ID immunoblot. We can therefore use CK7 in ID blots to analyse the expression of the MT 56,000 and 57,000 XCK1(8) proteins throughout development, confident that the antibody is not cross-reacting with other cytokeratins that may be of a similar Mr, for example CK55/56 (Fouquet et al. 1988). Triton extracts from different stages of development blotted with CK7 are shown in Fig. 3C. Both XCK1(8) proteins can be seen at all the stages shown. In the early blastula, as in the oocyte (Fig. 3A,C) the Mr 57,000 form is the more abundant. Both forms decrease in abundance during cleavage, so that there is less XCK1(8) protein at the onset of gastrulation than in the blastula, but begin to increase by stage 17. Thereafter the levels of both proteins rise rapidly, although it is the Mr 56,000 form that is the more abundant at these later stages (Fig. 3C). Note that in this experiment the antibody does not significantly blot any proteins smaller than XCK1(8), presumably because there was little degradation of the protein while preparing the extract.

These levels of protein correlate well with the levels of XCK1(8) mRNA. The amount of mRNA falls during cleavage from a peak in the oocyte to reach a minimum in the blastula, as shown by RNase protection (Fig. 4). Embryonic transcription of XCK1(8) mRNA seems to begin at the onset of gastrulation, and high levels have accumulated by stage 19. It is not possible to detect two different transcripts for the MT 56,000 and 57,000 variants using either this assay, or Northern analysis (not shown).

Fig. 4.

RNase protection assay using the CK22 probe (see Materials and methods) demonstrating the levels of XCK1(8) during development (CK). RNA recovery is controlled for using a 5 S RNA probe (5 S). Size markers are 220 and 150 nucleotide fragments of 32P-labelled HinfL- digested pBR322.

Fig. 4.

RNase protection assay using the CK22 probe (see Materials and methods) demonstrating the levels of XCK1(8) during development (CK). RNA recovery is controlled for using a 5 S RNA probe (5 S). Size markers are 220 and 150 nucleotide fragments of 32P-labelled HinfL- digested pBR322.

Distribution of XCK1 (8) protein in oocytes and embryos

We have used immunohistochemistry to analyse the distribution of the XCK1(8) proteins in larval stages, and in the oocyte and early embryo. In swimming tadpoles both CK7 and Z9 show staining within the CNS (central nervous system), although each stains a different population of cells. Vimentin is found within the radial glia cells, and in cells surrounding the nerve cord (Fig. 5B; Torpey et al. 1990). Cells of the floor plate are also strongly positive for vimentin. In contrast, CK7 stains only a few cells within the floor plate and also in the roof plate (Fig. 5A). It is not clear whether vimentin and cytokeratin are expressed in different cells within the floor plate, or whether the two proteins are distributed asymetrically in the same cells, even if the sections are observed at a higher magnification (not shown). CK7 stains the notocord very brightly (Fig. 5A. Staining is seen round the circumference of the notocord, and also within the septa running through it. There is also strong staining in the gut epithelia and the epithelia lining the mesentry (Figs 5D and 6B). These tissues are negative when stained with Z9, which instead stains connective tissue within the gut wall (Fig. 6A), and a group of cells in the wall of the aorta, around the somite, and in the developing kidney (Fig. 5E). In short, the two antibodies show a complementary pattern of vimentin and XCK1(8) expression. One important consequence of these results is that Z9 does not seem to cross-react with XCK1(8) protein under these conditions, even though there is a cross-reaction in immunoblot experiments (Torpey et al. 1990).

Fig. 5.

Comparison of CK7 (A, D) and Z9 (B, E) staining of larval tissues. (A, B and C) show the neural tube (nt), notocord (no), and somites (s) stained with 40μg/ml CK7 (A), 80μg/ml Z9 (B). (C) is the same section as in B viewed under phase contrast. (D, E and F) show the dorsal body wall and aorta (a), somites (s), and mesentry (m) stained with CK7 (D), Z9 (E). (F) is the same section as in E viewed under phase contrast showing the aorta (a), somite (s), kidney (k) and mesentry (m). Vimentin staining is seen only around the somite, in cells within the developing kidney, and the wall of the aorta. All these tissues are negative with CK7 (D).×l60.

Fig. 5.

Comparison of CK7 (A, D) and Z9 (B, E) staining of larval tissues. (A, B and C) show the neural tube (nt), notocord (no), and somites (s) stained with 40μg/ml CK7 (A), 80μg/ml Z9 (B). (C) is the same section as in B viewed under phase contrast. (D, E and F) show the dorsal body wall and aorta (a), somites (s), and mesentry (m) stained with CK7 (D), Z9 (E). (F) is the same section as in E viewed under phase contrast showing the aorta (a), somite (s), kidney (k) and mesentry (m). Vimentin staining is seen only around the somite, in cells within the developing kidney, and the wall of the aorta. All these tissues are negative with CK7 (D).×l60.

Fig. 6.

Further analysis of CK7 and Z9 in larval tissues. High power view of three different sections of a loop of gut: (A) under phase contrast, (B) stained with 40μg/ml CK7, and (C) stained with 80μg/ml Z9. Lu, lumen of gut. ×600.

Fig. 6.

Further analysis of CK7 and Z9 in larval tissues. High power view of three different sections of a loop of gut: (A) under phase contrast, (B) stained with 40μg/ml CK7, and (C) stained with 80μg/ml Z9. Lu, lumen of gut. ×600.

CK7 cross-reacts with fine filaments within the oocye cortex (Fig. 7B,C), and with a sparse network of fibres within the yolky cytoplasm (Fig. 7C). Vimentin is absent from the cortex in the animal pole of the oocyte, but is found in thick strands extending from the subcortical cytoplasm to the nuclear envelope (Fig. 7A; Torpey et al. 1990). The vegetal pole of the oocyte also contains cortical cytokeratin filaments, although staining within the deeper cytoplasm is virtually absent (Fig. 7E). The cortical CK1(8) staining is distinct from Z9, which shows strong cross-reaction with the germ plasm in the cortex (Fig. 7D). In addition Z9 stains a pattern of small spherical masses in the vegetal cytoplasm, which are not stained by CK7. Both antibodies crossreact with cells in the follicle layers surrounding the oocyte.

Fig. 7.

Immunostaining of sections of ethanol-fixed ovary with Z9 at 80μg/ml (A and D) and CK7 at 40μg/ml (B, C and E). (A and B) show Z9 (A) and CK7 (B) staining of the animal pole of stage 6 oocytes (gv, germinal vesicle). (C) shows CK7 staining of two oocyte corticies (f, folíele cell layers). (D and E) Views of the vegetal pole stained with Z9 (D; gp, germ plasm) and CK7 (E). Cytokeratin staining is arrowed. Note that both antibodies stain cells within the folíele cell layers (f). ×550.

Fig. 7.

Immunostaining of sections of ethanol-fixed ovary with Z9 at 80μg/ml (A and D) and CK7 at 40μg/ml (B, C and E). (A and B) show Z9 (A) and CK7 (B) staining of the animal pole of stage 6 oocytes (gv, germinal vesicle). (C) shows CK7 staining of two oocyte corticies (f, folíele cell layers). (D and E) Views of the vegetal pole stained with Z9 (D; gp, germ plasm) and CK7 (E). Cytokeratin staining is arrowed. Note that both antibodies stain cells within the folíele cell layers (f). ×550.

The complementary distributions of vimentin and cytokeratin can be better seen in grazing sections through the animal pole of the oocyte. Once again the follicle cells stain strongly. CK7 staining is very bright, and concentrated in the cortex, with a few fine filaments in the deeper cytoplasm (Fig. 8A). In contrast, vimentin is seen only in the deeper cytoplasm, with no staining whatsoever in the animal cortex (Fig. 8B). CK7 staining remains localised to the cortex throughout the early cleavage stages and up to gastrulation, and is best observed using whole-mount immunocytochemistry (Klymkowsky et al. 1987; Dent and Klymkowsky, 1989). Fig. 8D shows one vegetal blastomere of a stage 7 embryo. A network of fine filaments extends over the whole cortical suface of the cell, with particular concentrations at areas of cell contact. This pattern is identical to that obtained with the antibody 1H5 by Klymkowsky et al. (1987), who report the pattern of surface cytokeratin staining during early development. We presume that 1H5 cross-reacts with XCK1(8). Immunoblots with 1H5 of insoluble proteins from Xenopus A6 cells show a cross-reaction with a broad band of Mr about 56,000 (Klymkowsky et al. 1987).

Fig. 8.

(A, B and C) Grazing sections through the animal pole of a fully grown oocyte stained with CK7 (A; f, follicle cell layers) and with Z9 (B). (C) is the same section as in A viewed under phase contrast. (D) shows CK7 staining in the surface of one vegetal blastomere of a stage 7 embryo stained as a whole mount. ×200.

Fig. 8.

(A, B and C) Grazing sections through the animal pole of a fully grown oocyte stained with CK7 (A; f, follicle cell layers) and with Z9 (B). (C) is the same section as in A viewed under phase contrast. (D) shows CK7 staining in the surface of one vegetal blastomere of a stage 7 embryo stained as a whole mount. ×200.

We have microsequenced an Mr 57,000 protein, previously identified as one of a pair of vimentin-related proteins, and found it to be a type II cytokeratin. This result was a little surprising, since these proteins were originally identified by their crossreaction with the anti-vimentin antibody Z9 (Torpey et al. 1990). This antibody was made against the COOH terminus of vimentin, a region that has little homology’ to Xenopus cytokeratins expressed early in development, such as XCK1(8), and XenCK 55/56 (Franz and Franke, 1986; Fouquet et al, 1988). It is possible that this cross-reaction results from the relative abundance of cytokeratins compared to vimentin in oocytes. At later stages, when both proteins are more abundant, Z9 blots vimentin, giving only a weak reaction with cytokeratin (Torpey et al. 1990).

To analyse the expression of these cytokeratins, we have made an antibody against the COOH terminus of one, using the cDNA clone XCK1(8). The predicted amino acid sequence of this clone is very similar to the sequence of the Mr 57,000 protein shown in this paper. We presume that one of the Mr 56,000 and 57,000 proteins is the XCK1(8) protein, and the other a nonallelic variant, reflecting the duplicated nature of the Xenopus genome (Kobel and DuPasquier, 1986). Overexpression of XCK1(8) mRNA in oocytes results in the synthesis only of a MT 56,000 protein, as judged by autoradiography of 35S-labelled Triton extracts (data not shown), making it likely that the Mr 57,000 protein is the product of a different gene, probably a non-allelic form. The antibody, CK7, reacts with both proteins, and is specific for these cytokeratins. It does not crossreact with vimentin even when vimentin is specifically overexpressed.

Our analysis of XCK1(8) during development shows that both protein and mRNA decline throughout cleavage from being relatively abundant oocyte components, but subsequently reach high levels. We also find that the level of XCK1(8) mRNA falls by stage 42, in agreement with Fouquet et al. (1988). This presumably reflects the restriction in expression of this cytokeratin from many tissues to liver and intestine in later stages. Our results show very clearly levels of XCK1(8) mRNA at different stages, and in particular early stages, that have not been clear from previous reports (Franz and Franke, 1986; Fouquet et al. 1988).

We have used the CK7 antibody to study the distribution of the Mr 56,000 and 57,000 XCK1(8) keratins in larvae, oocytes and early embryos, and to make a comparison with the staining pattern of Z9, our anti-vimentin antibody. Two things are clear from the staining patterns of these antibodies in larvae. Firstly, CK7 does not cross-react with vimentin, or Z9 with cytokeratin. The two patterns are distinct even in tissues that contain both cytokeratin and vimentin; for example, within the floor plate of the neural tube or the gut. This shows that, using these histological methods, each antibody is specific for its target protein. The second observation is that CK7 stains only a subset of tissues that might be expected to contain cytokeratin. The apical surface of the gut epithelia, the mesentry and notocord are the most brightly staining structures, together with a few cells within the neural tube. There was no staining in the epidermis, which is known to express a set of keratin genes activated at gastrulation (Jamrich et al. 1987). These keratins presumably replace the XCK1(8) keratins in the surface epithelial cells of the developing embryo, whilst zygotic XCK1(8) expression is restricted to developing simple epithelial structures within the embryo.

Spatially distinct staining patterns of vimentin and cytokeratin are also evident in oocytes. Vimentin is largely restricted to the oocyte cytoplasm deep to the cortex, the only exception being staining localised to the germ plasm in the vegetal cortex (Torpey et al. 1990). Cytokeratin is almost completely localised to the oocyte cortex, with only a few fine filaments in the deeper cytoplasm. Early blastulae inherit an IF cytoskeleton in this pattern from the oocyte. Most blastomeres contain at least some vimentin (Torpey et al. 1990), but only the superficial cells will inherit a keratin cytoskeleton, as seen by whole-mount immunocytochemistry. This represents a functional distinction between the outermost cells of the blastula, which have the properties of a simple epithelia, and the deeper cells, which do not. The concentration of cytokeratin in the cortex may reflect a role in the oocyte itself, as well as localisation of the cytokeratin for later function in the embryo.

The distribution of cytokeratin in oocytes and early embryos that we see with CK7 is similar to that observed in a number of other studies. Franz et al. (1983) have used an unspecified antibody raised against a bovine keratin to show an IF network in Xenopus oocytes, although they failed to find vimentin with similar anti-vimentin antibodies. Heterologous antibodies have also been used by Godsave et al. (1984b, 1986) to demonstrate cytokeratins in adult Xenopus tissues and in oocytes. Once again cortical staining was observed, although there were varying degrees of crossreaction with epidermis, glandular tissue and the deeper oocyte cytoplasm, suggesting that the antibodies used cross-reacted with a number of different keratins. Finally, Klymkowsky et al. (1987) have used the monoclonal antibody 1H5, raised against insoluble proteins from the Xenopus kidney line XLKE A6. This antibody reacts with a broad band of Mt 56,000 in immunoblots of extracts of these cells, and stains a cortical pattern identical to CK7. We presume that 1H5 is specific for the XCK1(8) keratins at these early stages, and that careful immunoblots of oocyte extracts would show a cross-reaction with two discrete proteins of Afr 56,000 and 57,000. Note that A6 cells also express XenCK 55/56 (Fouquet et al. 1988), so 1H5 may also cross-react with this protein.

The staining patterns of cytokeratin and vimentin in oocytes differ in form as well as distribution. Cytokeratins are arranged in an almost geometric array of fine filaments (this study; and Klymkowsky et al. 1987) as would be expected from studies on mammalian cell types (see Steinert and Roop, 1988). Vimentin staining can be broadly divided into three types, none of which is usual. Staining in the animal pole is in the form of thick strands extending from the sub-cortical cytoplasm to the germinal vesicle. In the vegetal pole, we see staining in the germ plasm, and in bright dots in the deeper cytolplasm. We do not know the form of the IF polymers present in any of these three structures, although staining in the form of the spherical masses is seen in cultured cells that have been transfected with cDNAs encoding mutant IF proteins incapable of forming filaments (Raats et al. 1990; Chin et al. 1991). These dots could therefore represent some form of store of vimentin in the oocyte.

Our results provide a direct link between the XCK1(8) mRNA and the patterns of cytokeratin staining reported in Xenopus oocytes. Our CK7 antibody is specific for the protein product of XCK1(8) mRNA and a highly related keratin at all stages of development. We have demonstrated a cortical network of cytokeratin filaments identical to that reported by Klymkowsky et al. (1987). We show that, in addition to this network, oocytes contain a deeper vimentin IF system. We were unable to be certain of this in our previous work (Torpey et al. 1990), since we were not certain that Z9 was cross-reacting only with vimentin in oocyte sections. We can now say that Z9 is specific for vimentin when used to stain tissue sections, despite its cross-reaction with cytokeratin in immunoblots. We are therefore certain that oocyte germ plasm contains vimentin. The challenge now is to establish what role these two IF networks have in development.

We are extremely grateful to M. Puype and Dr J. Vandekerckhove for providing both the facilities and expertise for protein sequencing. We thank Dr Werner Franke for providing the XCK1(8) clone, Dr Colin Sharpe for the 5 S RNA probe, and Kim Goldstone for technical assistance. We are very grateful to the Wellcome Trust for financial support.

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