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H5 variant in various tissues of adult Xenopus laevis
Cell Differentiation, 16 (1985) 109-117
109
Elsevier ScientificPublishersIreland,Ltd. CDF 00252
Immunohistochemical distribution of the histone HI°/H5 variant in various tissues of adult Xenopus laevis A.F.M. M o o r m a n and P.A.J. de Boer Department of Anatomy and Embryology, University of Amsterdam, Meibergdreef15, 1105 AZ Amsterdam, The Netherlands
(Accepted9 October1984)
The cellular distribution of the histone H I ° / H 5 variant has been examined immunohistochemically in various tissUes of adult Xenopus laevis, using monoclonal antibodies against this variant that was isolated from erythrocyte nuclei. The H I ° / H 5 variant appears not to be erythrocyte-specific and appears to be present in all cell types of liver, stomach, and skin. In contrast, in oocyte nuclei the H I ° / H 5 variant cannot be detected, whereas they do contain HI; the nuclei of spermatogenic cells contain the Hl°/I-I5 variant, but probably less than the somatic cells. In Xenopus no H1 ° variant distinct from H5 seems to occur and the H I ° / H 5 variant apparently may perform a functional role related to mammalian H1 °. histone H I ° / H 5 ; tissue specificity; X. laevls;, monoclonal antibodies Introduction In 1951 Stedman and Stedman proposed that histones might be involved in the process of cellular differentiation. Evidence has accumulated since in support of this hypothesis, although a causality has not yet been established. In particular, variants of the lysine-ric.h histone protein family have been described that are developmentally regulated (Kinkade, 1969; Arceci et al., 1976; Gjerset et al., 1982). The picture of lysine-rich histone variants that has emerged from these and other data has several intriguing aspects. First, a lysine-rich histone variant, histone H1 °, has been described to be present in increased amounts in mammalian tissues with low mitotic activity (Panyim and Chalkley, 1969; Pehrson and Cole, 1980; Smith and Johns, 1980; Gjerset et al., 1982). Based on these data a role has been suggested for H1 ° in the regulation of cell proliferation and/or maintenance of the differentiated condition.
Second, an extreme example of tissue specificity of a histone is histone H5 that is present in the nucleated erythrocytes of several avian species (Neelin and Butler, 1961; Huang et al., 1977). The precise function of H5 is unknown, but it is somehow linked to the progressive condensation of chromatin and to the repression of genome activity during erythropoiesis (Sung, 1977). An analogous histone has been reported in the nucleated erythrocytes of fish, amphibia and reptiles (Tsai and Hnilica, 1975; Mild and Neelin, 1977; Destr6e et al., 1979). However, substantial qualitative and quantitative species-specific differences between the proteins in question have been observed, whereas the exclusive occurrence of histone H5 in the erythrocytes is questionable (Risley and Eckhardt, 1981), or has not been established. Finally, the highly conserved, functionally important, central globular regions of mammalian H1 ° and avian H5 share an extensive homology, with respect to amino acid sequence, to protein conformation, and to immunoreactivity that is dis-
0045-6039/85/$03.30 © 1985 ElsevierScientificPublishersIreland, Ltd.
110 tinct from that of H1 (Smith et al., 1980; Cary et al., 1981; Mura and Stollar, 1981a; Pehrson and Cole, 1981; Allan et al., 1982). These data suggest that H1 ° and H5 constitute a histone subclass, structurally and functionally distinct from the H1 subclass (Pehrson and Cole, 1981). Since H5 seems to be restricted to the nucleated erythrocytes of the non-mammalian vertebrates one might expect the presence of a H1 ° variant in these taxonomic classes as well to perform a function similar to that of the mammalian H1 °. A presumptive H1 °, distinct from H5, has been described in trout and chicken, but these data are confusing and controversial (cf. Seyedin and Cole, 1981, and Srebreva and Zlatanova, 1983; cf. Smith et al., 1981, and Srebreva et al., 1983). Clearly amino acid sequence data are essential. In Xenopus laevis a H1 ° variant distinct from the presumptive H5 has not been described. Risley and Eckhardt (1981) have described a protein, HIE, that is present in liver, intestine and testis and is indistinguishable from the presumptive H5. Recently we reported an immunological homology between Xenopus H5 and chicken H5 and reported the preparation of monoclonal antibodies to this protein that we denote in this paper as the H I ° / H 5 variant (Moorman et al., 1984). Now we communicate a detailed immunohistochemical study and demonstrate the presence of this H I ° / H 5 variant in a variety of adult cell types except oocytes. These data could point to a related functional condition both in X. laevis and in mammals with respect to the H1 ° variant. Materials and Methods
A n tisera As a probe for the H I ° / H 5 variant a monoclonal antibody against Xenopus histone 'H5' (clone 106) was used (Moorman et al., 1984). A monoclonal antibody to rat-liver arginase (clone 92.13) of the same immunoglobulin class (IgG2a) as the 'H5' antibody was used as a negative control (Charles et al., 1983), whereas as a positive control a monoclonal (clone 16.10) or a polyclonal anti-Xenopus histone H1A serum was used (Moorman et al., 1984). Peroxidase anti-peroxidase was
prepared according to Sternberger (1974) and kindly provided by Drs. C. Pool and W. van Raamsdonk (University of Amsterdam).
Immunohistochemical procedures Adult animals (Xenopus laevis laevis (Daud!n)) were anaesthetized by immersion in a solution of 0.1% MS222 (Sandoz). Small cubes of tissue were rapidly frozen in Freon 22 and freeze-substituted in methacarn, a mixture of chloroform, methanol and acetic acid ( 3 : 6 : 1 ) (Puchtler et al., 1970) under continuous gentle rotation at - 4 0 ° C for at least 20 days. Freeze-substituted tissue was transferred to chloroform and embedded in paraffin (Merck). Sections of 7 ~m were made, applied to object and stored at 4°C. Prior to staining sections were passed through xylol, ethanol, 3% hydrogen peroxide, to remove endogenous peroxidase, graded alcohols and water. Sections were preincubated for 30 min in 10 mM Tris-HC1, 5 mM EDTA, 150 mM NaC1, 0.25% gelatin, 0.05% Tween-20 (pH 7.4) to reduce background staining. The immunological reactions were performed in 10 mM sodium phosphate, 150 mM NaCI (pH 7.4) at room temperature as described previously (Moorman et al., 1984), using the peroxidase anti-peroxidase method (Sternberger, 1974). Briefly, incubations were performed with: (1) monoclonal antibody (1:10 diluted); (ii) rabbit anti-mouse immunoglobulin serum (1:500 diluted); (iii) goat anti-rabbit immunoglobulin serum (1 : 150 diluted); (iv) peroxidase anti-peroxidase (1:1000 diluted); (v) diaminobenzidine and hydrogen peroxide. Sections were then dehydrated in graded alcohol series, counterstained with light green and mounted in malinol. Photography was performed with a Zeiss photomicroscope, using a green filter and Ilford Pan-F film.
Results
To find an appropriate fixative, a large number of fixation conditions were tried out on small cubes of Xenopus liver tissue. Briefly, immunogenicity was preserved reasonably in cryostat sections when methanol, ethanol, para-formaldehyde or
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methacarn were used as fixative. However, morphology was insufficiently preserved, probably due to the high fat content of amphibian livers. On the other hand, morphology was much better preserved by fixation followed by preparation of paraffin sections, but immunogenicity was relatively poor; the best results were obtained with ethanol or methacarn as fixative. Freeze-substitution in methacarn followed by embedding in paraffin was found to be superior, both with respect to morphology and to antigenicity. Actually, freeze-substitution can be considered as a combination of both methods mentioned above. Using the freeze-substitution fixation technique a large number of tissues were investigated. The results obtained with liver are demonstrated in Fig. 1. Liver morphology has been quite well preserved. Like a typical adult amphibian liver (Elias and Sherrick, 1969) the Xenopus liver
consists of interconnected, reticular liver plates, usually two cells thick and surrounded by liver sinusoids, filled completely with erythrocytes. The bile capillaries form strongly branching intercellular clefts between the plates of hepatocytes. It is clear from the immunohistochemical reactions that all nuclei from the hepatocytes, erythrocytes and endothelial cells react with both anti-H1A and anti-' H5'. The specificity of the sera used has been shown in detail previously (Moorman et al., 1984). The immunohistochemical reactions are considered to be specific: (i) only the nuclei react; (ii) supernatant from the non-immunoglobulin secreting myeloma strain SP2/0 used for fusion does not react (Fig. 1A); (iii) monoclonal anti-rat liver arginase of the same immunoglobulin class as the anti-histone sera used does not react (see Fig. 2C); (iv) unrelated tissue, like chicken erythrocytes, does not react with anti-Xenopus H1A or 'H5' (Moor-
Fig. 1. X laevis liver sections stained with culture medium from: (A) myeloma strain SP2/0 that does not produce immunoglobulin; (B) clone 16.10 (anti-H1A); (C) clone 106 (anti-'H5'); and (D) clone 106 (anti-'H5') smaller magnification, hn = hepatocyte nucleus; en = erythrocyte nucleus; ecn = endothelial cell nucleus; bc = bile capillary; ls = liver sinusoid.
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i,i
Fig. 2. Transverse sections through the stomach wall of adult X. laevis incubated with (A, B) clone 106 (anti-'H5') and (C) clone 92.13 (anti-rat-liver arginase). (A) Cross section through the entire wall. (B, C) Detail of a gastric pit. (1) Epithelium with mucous cells; (2) lamina propria with tubular gastric glands; (3) muscularis mucosae; (4) submucosa; (5) circular muscular layer; (6) longitudinal muscular layer; (7) serosa; (8) pancreas.
man et al., 1984); (v) rat-liver sections do not react with anti-Xenopus histone H1A or 'H5', but do react with a monoclonal anti-rat H1 ° (Moorman and Lamers, unpublished observations).
Essentially the same results were obtained with sections of stomach (Fig. 2) and skin (Fig. 3), despite a great variety of cell types in these tissues. In stomach the nuclei of the epithelial ceils, glands,
Fig. 3. Skin section of the abdominal wall incubated with (A) polyclonal anti-H1A serum, and (B) clone 106 (anti-'H5'). sc = stratum corneum; sg = stratum germinativum; bm = basement membrane; mg = mucous gland; sg = serous gland.
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muscular cells and connective tissue react with the 'H5' antibody (Fig. 2A, B), whereas anti-rat-liver arginase of the same immunoglobulin class does not (Fig. 2C). At all dilutions of the primary 'H5' antibody applied, the nuclei of all cell types of stomach are equally densely stained. Heterogeneity in staining behaviour could not be observed. In skin tissue all nuclei including those of the epidermis, dermis and glands react both with antiH1A (Fig. 3A) and with anti-'H5' (Fig. 3B). Compared with somatic cells the nuclei of the germ cells revealed quite different staining behaviour with the 'H5'-antibody. First, as shown in Fig. 4A, reaction of the 'H5' antibody with the oocyte nuclei could not be detected, whereas the nuclei of the surrounding connective tissue and the erythrocytes do react with this antibody. On the other hand, oocyte nuclei react with the H1A-antibody though the staining seems restricted to the amplified nucleoli (Fig. 4B). The surrounding tissue reacts, as expected, with the anti-H1A serum as well. Second, spermatogenic cells, at all stages of development except mature sperm, react with both the anti-histone 'H5' serum (Fig. 5C) and the anti-histone H1A serum (Fig. 5B), whereas a monoclonal anti-rat-liver arginase of the same immunoglobulin class as the anti-histone 'H5' does not (Fig. 5A). The cross sections through the testis from an adult frog (Fig. 5) nicely show the seminiferous tubules with the so-called 'cysts' of
spermatogenic cells with synchronous development, the spermatids with small nuclei, and the spermatocytes with larger nuclei. As mentioned above the staining with the anti'H5' serum, as shown in Fig. 5C, seems homogeneous, i.e., all nuclei seem to react equally well. This pattern of staining was only seen under optimized conditions, i.e., 1 : 10 diluted primary monoclonal antibody, 1:500 diluted rabbit anti-mouse immunoglobulin serum (secondary reaction), 1:150 diluted goat anti-rabbit immunoglobulin and 1:1000 diluted peroxidase anti-peroxidase (Moorman et al., 1984). However, the interesting observation was made that the use of lower concentrations of the primary or secondary antibody resulted in a different staining behaviour. A sharp decrease in staining intensity of the nuclei of the spermatogenic cells can be observed, compared with the nuclei of the surrounding connective tissue, suggesting that the histone 'H5' content of the spermatogenic nuclei is lower than that of the somatic nuclei (Fig. 5D). The results, as presented in Fig. 5D, were obtained using a dilution of the primary monoclonal 'H5' antibody of 1:10 and a 1:8000 dilution of the rabbit anti-mouse immunoglobulin serum. Such a phenomenon was observed with all anti-'H5' clones in spermatogenic cells only and not in liver, stomach or skin tissue. It was never observed using several dilutions of the anti-H1A serum. In this context we stress the importance of the
Fig. 4. Section t h r o u g h X. laevis o v a r y i n c u b a t e d with (A) clone 106 ( a n t i - ' H 5 ' ) a n d (B) p o l y c l o n a l a n t i - H I A serum, o = oocyte: gv = g e r m i n a l vesicle (nucleus); n = nucleoli.
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Fig. 5. Transversesection through the seminiferous tubules of the testis from an adult X. laevis, incubated with: (A) anti-rat-liver arginase clone (92.13); (B) polyclonal anti-H1A serum; (C) monoclonal anti-'H5' (clone 106), secondary rabbit anti-mouse immunoglobulin serum 1:500 diluted; and (D) monoclonalanti-'H5' (clone 106), secondaryantiserum 1:8000 diluted.
use of a range of antibody dilutions in immunohistochemical studies. We have found, for instance, that antibodies that are able to detect a heterogeneity of carbamoyl-phosphate synthetase according to the vascular architecture in adult rat liver (Gaasbeek Janzen et al., 1984) fail to detect heterogeneity in neonatal livers at similar antibody concentrations, but do detect heterogeneity at higher antibody dilutions.
Discussion In X. laevis erythrocytes a lysine-rich histone occurs (denoted as H I ° / H 5 variant in this paper) that is immunologically related to chicken histone H5 (Moorman et al., 1984; Smith et al., 1984). Careful biochemical extractions and detailed elec-
trophoretic analyses have made it seem very likely that a protein indistinguishable from this variant, denoted as H1E, is present in liver, intestine and testis also (Risley and Eckhardt, 1981). The immunohistochemical procedure, as described in this paper, has permitted us to establish unambiguously the presence of this histone H I ° / H 5 variant in all somatic tissues that were examined and has excluded the possibility that the presence of this variant is due to erythrocyte contamination. The latter possibility had to be considered very seriously, as clearly illustrated in Fig. 1: most of the surface of the section is occupied by the large hepatocytes, but most of the nuclei are erythrocyte nuclei. It is noteworthy that erythrocyte nuclei in tissue sections react much better than smears of red blood cells (Moorman et al., 1984), apparently because the histone antigens are much better
115 accessible to immunoglobulin in sections than smears. In agreement with our previous observations on smears of red blood cells (Moorman et al., 1984), no heterogeneity of anti-H5 staining could be detected. Thus in Xenopus we have no indications for a heterogeneity in chromatin conformation as suggested for chicken erythrocyte nuclei (Mura and Stollar, 1981b). We consider the observed staining with the monoclonal 'H5' antibody as evidence for the presence of the H I ° / H 5 variant; first, because of the rigorously tested antibody specificity (Moorman et al., 1984) and second, because of the method specificity as summarized in the Results section. Essentially the same results were obtained with the fluorescence technique (data not shown). One of the advantages of an immunohistochemical approach is that it gives information on the cellular distribution of a given protein within a tissue. It is clear from the results presented that the nuclei of all cell types of the somatic tissues examined contain the histone H I ° / H 5 variant. Thus, a histone H5, defined as 'restricted to erythrocyte nuclei' seems not to occur in Xenopus. This raises the question of whether the Xenopus H I ° / H 5 variant has to be considered as functionally similar to mammalian H1 °. The immunological relationship between these histones (Smith et al., 1984) points to a common, conserved structure with a similar function. On the other hand, it is not immediately apparent from the observed cellular distribution in Xenopus that the Xenopus H I ° / H 5 variant is characteristic of tissues with low mitotic activity, as is the case for mammalian H1 °. This point deserves some consideration. First, in oocyte nuclei the H I ° / H 5 variant cannot be detected, whereas the surrounding tissues react with the anti-H5 serum, suggesting that this histone is not present in the amplified nucleoli at least, although oocytes are not very active in DNA synthesis or cell division. A trivial explanation is that the immunohistochemical procedure used did not allow the detection of histone 'H5' in oocyte nuclei. Another explanation might be that the H I ° / H 5 variant is absent in oocytes, because this cell is optimally prepared for the very rapid divisions just after fertilization. Also, the rapidly di-
viding, early stages of development do not contain the H I ° / H 5 variant, whereas H1A was present in all nuclei (Moorman et al., in preparation). Thus, if the H I ° / H 5 variant is involved in the cessation of DNA synthesis or mitotic activity, oocytes have solved this problem in another way. Previously, we showed that Xenopus oocytes have a large store of histone H1A (Van Dongen et al., 1983). Nevertheless, histone H1A could be detected only in the nucleoli located characteristically just beneath the nuclear membrane. Apparently, the way of storage prevents immunohistochemical detection with the procedures we have applied. The presence of histone HIA in the nucleoli at all stages of oocyte development, some of which are highly active in ribosomal RNA synthesis, is interesting in view of the discussions on the composition of active chromatin (see, e.g., Weisbrod, 1982) and dearly demonstrates that the absence of a lysine-rich histone is not a prerequisite for active chromatin. Second, the cells of the various stages of spermatogenesis undoubtedly react with the antiH5 serum. This reaction seems less intense compared with that of the surrounding connective tissue. This became apparent if higher antibody dilutions were used, suggesting that the H I ° / H 5 variant is present in lower amounts. Biochemical extractions have not revealed the presence of this variant in spermatogenic cells (Risley and Eckhardt, 1981), but if this variant represented approx. 1% of total histone content, its presence would easily have remained undetected. Thus, in testis the H I ° / H 5 variant seems more abundant in the somatic cells with low mitotic activity, analogous to mammalian H1 °. But both the nondividing spermatids and the dividing spermatogenic cells also contain the H I ° / H 5 variant. Third, whereas one can expect a difference in mitotic activity between stomach epithelium and the other stomach cells or skin germinative layer and the other skin cells, no difference in staining between the 'H5' and the H1A antiserum could be detected, suggesting no heterogeneity with respect to the distribution of the H I ° / H 5 variant. In conclusion, in somatic tissues from adult X. laeois the H I ° / H 5 variant seems a specialized subfraction of the lysine-rich histones that is not
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restricted to amitotic cells or cells with low mitotic activity and the specific function of which remains unknown. This condition is different from that in birds where H5 seems restricted to the nucleated erythrocytes and may be more like that in mammals. The presence of mammalian H1 ° has been suggested to be indirectly linked to cell proliferation and a role for H1 ° has been proposed in the termination of differentiation and/or maintenance of the differentiated state (Gjerset al., 1982). Our data indicate that the absence of the Xenopus HI°/H5 variant is not a prerequisite for mitotic activity, but whether this variant plays a similar role as proposed for mammalian H1 ° has to be established. The absence of this variant in early embryonic stages (Moorman and de Boer, in preparation) could point in this direction. A precise description of the appearance of the HI°/H5 variant during embryonic development is currently being carried out and may indicate a role for the HI°/H5 variant.
Acknowledgements We thank Mrs. M.Th. Linders and C.C. Verhoek-Pocock for expert technical assistance, Dr. W.H. Lamers and Prof. Dr. R. Charles for their continuous interest and many fruitful discussions, Dr. W. van Raamsdonk for much histochemical advice, Mrs. F. de Jong and C. Hersbach for help on microscopy and photography and Mrs. J. Husslage for secretarial help.
References Allan, J., B.J. Smith, B. Dunn and M. Bustin: Antibodies against the folding domain of histone H5 cross-react with H1 ° but not with H1. J. Biol. Chem. 257, 10533-10535 (1982). Arceci, R.J., D.R. Senger and P.R. Gross: The programmed switch in lysine-rich histone synthesis at gastrulation. Cell 9, 171-178 (1976). Cary, P.D., M.L. Hines, E.M. Bradbury, B.J. Smith and E.W. Johns: Conformational studies of histone H1 ° in comparison with histones H1 and H5. Eur. J. Biochem. 120, 371-377 (1981). Charles, R., A. de Graaf, P.A.J. de Boer, W.H. Lamers and A.F.M. Moorman: Use of monoclonal antibodies for the
identification of different rat-liver arginases. Proc. 24th Dutch Fed. Meet., 76 (1983). Destrre, O.H.J., H.J. Hoenders, A.F.M. Moorman and R. Charles: Histones of Xenopus laevis erythrocytes. Biochim. Biophys. Acta 577, 61-70 (1979). Elias, H. and J.C. Sherrick: Morphology of the Liver (Academic Press, New York) (1969). Gaasbeek Janzen, J.W., W.H. Lamers, A.F.M. Moorman, A. de Graaf, J.A. Los and R. Charles: Immunohistochemical localization of carbamoyl-phosphate synthetase (ammonia) in adult rat fiver. J. Histochem. Cytochem. 32, 557-564 (1984). Gjerset, R., C. Gorka, S. Hasthorpe, J.J. Lawrence and H. Eisen: Developmental and hormonal regulation of protein H1 ° in rodents. Proc. Natl. Acad. Sci. USA 79, 2333-2337 (1982). Huang, P.C., L.P. Branes, C. Mura, V. Quagliarello and P. Kropkowski-Bohdan: Histone H5 in developing chick erythroid cells. In: The Molecular Biology of the Mammalian Genetic Apparatus, ed. P. Ts'o (Elsevier/NorthHolland, Amsterdam) (1977). Kinkade, J.M.: Qualitative species differences and quantitative tissue differences in the distribution of lysine-rich histones. J. Biol. Chem. 244, 3375-3386 (1969). Miki, B.L.A. and J.M. Neelin: Comparison of the histones from fish erythrocytes. Can. J. Biochem. 55, 1220-1227 (1977). Moorman, A.F.M., P.A.J. de Boer, M.Th. Linders and R. Charles: The histone H5 variant in Xenopus laevis. Cell Differ. 14, 113-123 (1984). Mura, C.V. and B.D. Stollar: Serological detection of homologies of H1 ° with H5 and H1 histones. J. Biol. Chem. 256, 9767-9769 (1981a). Mura, C.V. and B.D. Stollar: Fluorescence-activated sorting of isolated nuclei. Exp. Cell Res. 135, 31-37 (1981b). Neelin, J.M. and G.C. Butler: A comparison of histones from chicken tissues by zone electrophoresis in starch gel. Can. J. Biochem. Physiol. 39, 485-491 (1961). Panyim, S. and R. Chalkley: A new histone found only in mammalian tissues with little cell division. Biocfiem. Biophys. Res. Commun. 37, 1042-1049 (1969). Pehrson, J. and R.D. Cole: Histone H1 ° accumulates in growth-inhibited cultured cells. Nature (London) 285, 43-44 (1980). Pehrson, J.R. and R.D. Cole: Bovine H1 ° histone subfractions contain an invariant sequence which matches histone H5 rather than HI. Biochemistry 20, 2298-2301 (1981). Puchtler, H., F.S. Waldrop, S.N. Meloan, M.S. Terry and H.M. Conner: M e t h a c a r n ( m e t h a n o l - c a r n o y ) fixation. Histochemie 21, 97-116 (1970). Risley, M.S. and R.A. Eckhardt: H1 histone variants in Xenopus laevis. Dev. Biol. 84, 79-87 (1981). Seyedin, S.M. and R.D. Cole: H1 histones of trout. J. Biol. Chem. 256, 442-444 (1981). Smith, B.J. and E.W. Johns: Isolation and characterization of subfractions of nuclear protein H1 °. FEBS Lett. 110, 25-29 (1980). Smith, B.J., Y. Cook, E.W. Johns and R.A. Weiss: Absence of
117 H1 ° from quiescent chicken cells. FEBS Lett. 135, 77-80 (1981). Smith, B.J., M.R. Harris, C.M. Sigournay, E.L.V. Mayes and M. Bustin: A survey of H1 °- and H5-1ike protein structure and distribution in higher and lower eukaryotes. Eur. J. Biochem. 138, 309-317 (1984). Smith, B.J., J.M. Walker and E.W. Johns: Structural homology between a mammalian H1 ° subfraction and avian erythrocyte-specific histone H5. FEBS Lett. 112, 42-44 (1980). Srebreva, L. and J. Zlatanova: Occurrence of histone Hl°-re lated protein fraction in trout liver. Biochim. Biophys. Acta 740, 163-168 (1983). Srebreva, L.N., N.B. Andreeva, K.G. Gasaryan, R.G. Tsanev and J. Zlatanova: Presence of histone Hl°-related fraction in chicken liver. Differentiation 25, 113-120 (1983).
Stedman, E. and E. Stedman: The basic proteins of cell nuclei. Philos. Trans. R. Soc. London Ser. B. 235, 565-595 (1951). Sternberger, L.A.: Immunocytochemistry (Prentice-Hall, New Jersey) (1974). Sung, M.T.: Phosphorylation and dephosphorylation of histone V (H5): controlled condensation of avian erythrocyte chromatin. Biochemistry 16, 286-291 (1977). Tsal, Y.H. and L.S. Hnilica: Tissue-specific histones in the erythrocytes of chicken and turtle. Exp. Cell Res. 91, 107-112 (1975). Van Dongen, W.M.A.M., A.F.M. Moorman and O.H.J. Destr6e: The accumulation of the maternal pool of histone H1A during oogenesis in Xenopus laevis. Cell Differ. 12, 257-264 (1983). Weisbrod, S.: Active chromatin. Nature (London) 297,289-295 (1982).
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Elsevier ScientificPublishersIreland,Ltd. CDF 00252
Immunohistochemical distribution of the histone HI°/H5 variant in various tissues of adult Xenopus laevis A.F.M. M o o r m a n and P.A.J. de Boer Department of Anatomy and Embryology, University of Amsterdam, Meibergdreef15, 1105 AZ Amsterdam, The Netherlands
(Accepted9 October1984)
The cellular distribution of the histone H I ° / H 5 variant has been examined immunohistochemically in various tissUes of adult Xenopus laevis, using monoclonal antibodies against this variant that was isolated from erythrocyte nuclei. The H I ° / H 5 variant appears not to be erythrocyte-specific and appears to be present in all cell types of liver, stomach, and skin. In contrast, in oocyte nuclei the H I ° / H 5 variant cannot be detected, whereas they do contain HI; the nuclei of spermatogenic cells contain the Hl°/I-I5 variant, but probably less than the somatic cells. In Xenopus no H1 ° variant distinct from H5 seems to occur and the H I ° / H 5 variant apparently may perform a functional role related to mammalian H1 °. histone H I ° / H 5 ; tissue specificity; X. laevls;, monoclonal antibodies Introduction In 1951 Stedman and Stedman proposed that histones might be involved in the process of cellular differentiation. Evidence has accumulated since in support of this hypothesis, although a causality has not yet been established. In particular, variants of the lysine-ric.h histone protein family have been described that are developmentally regulated (Kinkade, 1969; Arceci et al., 1976; Gjerset et al., 1982). The picture of lysine-rich histone variants that has emerged from these and other data has several intriguing aspects. First, a lysine-rich histone variant, histone H1 °, has been described to be present in increased amounts in mammalian tissues with low mitotic activity (Panyim and Chalkley, 1969; Pehrson and Cole, 1980; Smith and Johns, 1980; Gjerset et al., 1982). Based on these data a role has been suggested for H1 ° in the regulation of cell proliferation and/or maintenance of the differentiated condition.
Second, an extreme example of tissue specificity of a histone is histone H5 that is present in the nucleated erythrocytes of several avian species (Neelin and Butler, 1961; Huang et al., 1977). The precise function of H5 is unknown, but it is somehow linked to the progressive condensation of chromatin and to the repression of genome activity during erythropoiesis (Sung, 1977). An analogous histone has been reported in the nucleated erythrocytes of fish, amphibia and reptiles (Tsai and Hnilica, 1975; Mild and Neelin, 1977; Destr6e et al., 1979). However, substantial qualitative and quantitative species-specific differences between the proteins in question have been observed, whereas the exclusive occurrence of histone H5 in the erythrocytes is questionable (Risley and Eckhardt, 1981), or has not been established. Finally, the highly conserved, functionally important, central globular regions of mammalian H1 ° and avian H5 share an extensive homology, with respect to amino acid sequence, to protein conformation, and to immunoreactivity that is dis-
0045-6039/85/$03.30 © 1985 ElsevierScientificPublishersIreland, Ltd.
110 tinct from that of H1 (Smith et al., 1980; Cary et al., 1981; Mura and Stollar, 1981a; Pehrson and Cole, 1981; Allan et al., 1982). These data suggest that H1 ° and H5 constitute a histone subclass, structurally and functionally distinct from the H1 subclass (Pehrson and Cole, 1981). Since H5 seems to be restricted to the nucleated erythrocytes of the non-mammalian vertebrates one might expect the presence of a H1 ° variant in these taxonomic classes as well to perform a function similar to that of the mammalian H1 °. A presumptive H1 °, distinct from H5, has been described in trout and chicken, but these data are confusing and controversial (cf. Seyedin and Cole, 1981, and Srebreva and Zlatanova, 1983; cf. Smith et al., 1981, and Srebreva et al., 1983). Clearly amino acid sequence data are essential. In Xenopus laevis a H1 ° variant distinct from the presumptive H5 has not been described. Risley and Eckhardt (1981) have described a protein, HIE, that is present in liver, intestine and testis and is indistinguishable from the presumptive H5. Recently we reported an immunological homology between Xenopus H5 and chicken H5 and reported the preparation of monoclonal antibodies to this protein that we denote in this paper as the H I ° / H 5 variant (Moorman et al., 1984). Now we communicate a detailed immunohistochemical study and demonstrate the presence of this H I ° / H 5 variant in a variety of adult cell types except oocytes. These data could point to a related functional condition both in X. laevis and in mammals with respect to the H1 ° variant. Materials and Methods
A n tisera As a probe for the H I ° / H 5 variant a monoclonal antibody against Xenopus histone 'H5' (clone 106) was used (Moorman et al., 1984). A monoclonal antibody to rat-liver arginase (clone 92.13) of the same immunoglobulin class (IgG2a) as the 'H5' antibody was used as a negative control (Charles et al., 1983), whereas as a positive control a monoclonal (clone 16.10) or a polyclonal anti-Xenopus histone H1A serum was used (Moorman et al., 1984). Peroxidase anti-peroxidase was
prepared according to Sternberger (1974) and kindly provided by Drs. C. Pool and W. van Raamsdonk (University of Amsterdam).
Immunohistochemical procedures Adult animals (Xenopus laevis laevis (Daud!n)) were anaesthetized by immersion in a solution of 0.1% MS222 (Sandoz). Small cubes of tissue were rapidly frozen in Freon 22 and freeze-substituted in methacarn, a mixture of chloroform, methanol and acetic acid ( 3 : 6 : 1 ) (Puchtler et al., 1970) under continuous gentle rotation at - 4 0 ° C for at least 20 days. Freeze-substituted tissue was transferred to chloroform and embedded in paraffin (Merck). Sections of 7 ~m were made, applied to object and stored at 4°C. Prior to staining sections were passed through xylol, ethanol, 3% hydrogen peroxide, to remove endogenous peroxidase, graded alcohols and water. Sections were preincubated for 30 min in 10 mM Tris-HC1, 5 mM EDTA, 150 mM NaC1, 0.25% gelatin, 0.05% Tween-20 (pH 7.4) to reduce background staining. The immunological reactions were performed in 10 mM sodium phosphate, 150 mM NaCI (pH 7.4) at room temperature as described previously (Moorman et al., 1984), using the peroxidase anti-peroxidase method (Sternberger, 1974). Briefly, incubations were performed with: (1) monoclonal antibody (1:10 diluted); (ii) rabbit anti-mouse immunoglobulin serum (1:500 diluted); (iii) goat anti-rabbit immunoglobulin serum (1 : 150 diluted); (iv) peroxidase anti-peroxidase (1:1000 diluted); (v) diaminobenzidine and hydrogen peroxide. Sections were then dehydrated in graded alcohol series, counterstained with light green and mounted in malinol. Photography was performed with a Zeiss photomicroscope, using a green filter and Ilford Pan-F film.
Results
To find an appropriate fixative, a large number of fixation conditions were tried out on small cubes of Xenopus liver tissue. Briefly, immunogenicity was preserved reasonably in cryostat sections when methanol, ethanol, para-formaldehyde or
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methacarn were used as fixative. However, morphology was insufficiently preserved, probably due to the high fat content of amphibian livers. On the other hand, morphology was much better preserved by fixation followed by preparation of paraffin sections, but immunogenicity was relatively poor; the best results were obtained with ethanol or methacarn as fixative. Freeze-substitution in methacarn followed by embedding in paraffin was found to be superior, both with respect to morphology and to antigenicity. Actually, freeze-substitution can be considered as a combination of both methods mentioned above. Using the freeze-substitution fixation technique a large number of tissues were investigated. The results obtained with liver are demonstrated in Fig. 1. Liver morphology has been quite well preserved. Like a typical adult amphibian liver (Elias and Sherrick, 1969) the Xenopus liver
consists of interconnected, reticular liver plates, usually two cells thick and surrounded by liver sinusoids, filled completely with erythrocytes. The bile capillaries form strongly branching intercellular clefts between the plates of hepatocytes. It is clear from the immunohistochemical reactions that all nuclei from the hepatocytes, erythrocytes and endothelial cells react with both anti-H1A and anti-' H5'. The specificity of the sera used has been shown in detail previously (Moorman et al., 1984). The immunohistochemical reactions are considered to be specific: (i) only the nuclei react; (ii) supernatant from the non-immunoglobulin secreting myeloma strain SP2/0 used for fusion does not react (Fig. 1A); (iii) monoclonal anti-rat liver arginase of the same immunoglobulin class as the anti-histone sera used does not react (see Fig. 2C); (iv) unrelated tissue, like chicken erythrocytes, does not react with anti-Xenopus H1A or 'H5' (Moor-
Fig. 1. X laevis liver sections stained with culture medium from: (A) myeloma strain SP2/0 that does not produce immunoglobulin; (B) clone 16.10 (anti-H1A); (C) clone 106 (anti-'H5'); and (D) clone 106 (anti-'H5') smaller magnification, hn = hepatocyte nucleus; en = erythrocyte nucleus; ecn = endothelial cell nucleus; bc = bile capillary; ls = liver sinusoid.
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i,i
Fig. 2. Transverse sections through the stomach wall of adult X. laevis incubated with (A, B) clone 106 (anti-'H5') and (C) clone 92.13 (anti-rat-liver arginase). (A) Cross section through the entire wall. (B, C) Detail of a gastric pit. (1) Epithelium with mucous cells; (2) lamina propria with tubular gastric glands; (3) muscularis mucosae; (4) submucosa; (5) circular muscular layer; (6) longitudinal muscular layer; (7) serosa; (8) pancreas.
man et al., 1984); (v) rat-liver sections do not react with anti-Xenopus histone H1A or 'H5', but do react with a monoclonal anti-rat H1 ° (Moorman and Lamers, unpublished observations).
Essentially the same results were obtained with sections of stomach (Fig. 2) and skin (Fig. 3), despite a great variety of cell types in these tissues. In stomach the nuclei of the epithelial ceils, glands,
Fig. 3. Skin section of the abdominal wall incubated with (A) polyclonal anti-H1A serum, and (B) clone 106 (anti-'H5'). sc = stratum corneum; sg = stratum germinativum; bm = basement membrane; mg = mucous gland; sg = serous gland.
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muscular cells and connective tissue react with the 'H5' antibody (Fig. 2A, B), whereas anti-rat-liver arginase of the same immunoglobulin class does not (Fig. 2C). At all dilutions of the primary 'H5' antibody applied, the nuclei of all cell types of stomach are equally densely stained. Heterogeneity in staining behaviour could not be observed. In skin tissue all nuclei including those of the epidermis, dermis and glands react both with antiH1A (Fig. 3A) and with anti-'H5' (Fig. 3B). Compared with somatic cells the nuclei of the germ cells revealed quite different staining behaviour with the 'H5'-antibody. First, as shown in Fig. 4A, reaction of the 'H5' antibody with the oocyte nuclei could not be detected, whereas the nuclei of the surrounding connective tissue and the erythrocytes do react with this antibody. On the other hand, oocyte nuclei react with the H1A-antibody though the staining seems restricted to the amplified nucleoli (Fig. 4B). The surrounding tissue reacts, as expected, with the anti-H1A serum as well. Second, spermatogenic cells, at all stages of development except mature sperm, react with both the anti-histone 'H5' serum (Fig. 5C) and the anti-histone H1A serum (Fig. 5B), whereas a monoclonal anti-rat-liver arginase of the same immunoglobulin class as the anti-histone 'H5' does not (Fig. 5A). The cross sections through the testis from an adult frog (Fig. 5) nicely show the seminiferous tubules with the so-called 'cysts' of
spermatogenic cells with synchronous development, the spermatids with small nuclei, and the spermatocytes with larger nuclei. As mentioned above the staining with the anti'H5' serum, as shown in Fig. 5C, seems homogeneous, i.e., all nuclei seem to react equally well. This pattern of staining was only seen under optimized conditions, i.e., 1 : 10 diluted primary monoclonal antibody, 1:500 diluted rabbit anti-mouse immunoglobulin serum (secondary reaction), 1:150 diluted goat anti-rabbit immunoglobulin and 1:1000 diluted peroxidase anti-peroxidase (Moorman et al., 1984). However, the interesting observation was made that the use of lower concentrations of the primary or secondary antibody resulted in a different staining behaviour. A sharp decrease in staining intensity of the nuclei of the spermatogenic cells can be observed, compared with the nuclei of the surrounding connective tissue, suggesting that the histone 'H5' content of the spermatogenic nuclei is lower than that of the somatic nuclei (Fig. 5D). The results, as presented in Fig. 5D, were obtained using a dilution of the primary monoclonal 'H5' antibody of 1:10 and a 1:8000 dilution of the rabbit anti-mouse immunoglobulin serum. Such a phenomenon was observed with all anti-'H5' clones in spermatogenic cells only and not in liver, stomach or skin tissue. It was never observed using several dilutions of the anti-H1A serum. In this context we stress the importance of the
Fig. 4. Section t h r o u g h X. laevis o v a r y i n c u b a t e d with (A) clone 106 ( a n t i - ' H 5 ' ) a n d (B) p o l y c l o n a l a n t i - H I A serum, o = oocyte: gv = g e r m i n a l vesicle (nucleus); n = nucleoli.
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Fig. 5. Transversesection through the seminiferous tubules of the testis from an adult X. laevis, incubated with: (A) anti-rat-liver arginase clone (92.13); (B) polyclonal anti-H1A serum; (C) monoclonal anti-'H5' (clone 106), secondary rabbit anti-mouse immunoglobulin serum 1:500 diluted; and (D) monoclonalanti-'H5' (clone 106), secondaryantiserum 1:8000 diluted.
use of a range of antibody dilutions in immunohistochemical studies. We have found, for instance, that antibodies that are able to detect a heterogeneity of carbamoyl-phosphate synthetase according to the vascular architecture in adult rat liver (Gaasbeek Janzen et al., 1984) fail to detect heterogeneity in neonatal livers at similar antibody concentrations, but do detect heterogeneity at higher antibody dilutions.
Discussion In X. laevis erythrocytes a lysine-rich histone occurs (denoted as H I ° / H 5 variant in this paper) that is immunologically related to chicken histone H5 (Moorman et al., 1984; Smith et al., 1984). Careful biochemical extractions and detailed elec-
trophoretic analyses have made it seem very likely that a protein indistinguishable from this variant, denoted as H1E, is present in liver, intestine and testis also (Risley and Eckhardt, 1981). The immunohistochemical procedure, as described in this paper, has permitted us to establish unambiguously the presence of this histone H I ° / H 5 variant in all somatic tissues that were examined and has excluded the possibility that the presence of this variant is due to erythrocyte contamination. The latter possibility had to be considered very seriously, as clearly illustrated in Fig. 1: most of the surface of the section is occupied by the large hepatocytes, but most of the nuclei are erythrocyte nuclei. It is noteworthy that erythrocyte nuclei in tissue sections react much better than smears of red blood cells (Moorman et al., 1984), apparently because the histone antigens are much better
115 accessible to immunoglobulin in sections than smears. In agreement with our previous observations on smears of red blood cells (Moorman et al., 1984), no heterogeneity of anti-H5 staining could be detected. Thus in Xenopus we have no indications for a heterogeneity in chromatin conformation as suggested for chicken erythrocyte nuclei (Mura and Stollar, 1981b). We consider the observed staining with the monoclonal 'H5' antibody as evidence for the presence of the H I ° / H 5 variant; first, because of the rigorously tested antibody specificity (Moorman et al., 1984) and second, because of the method specificity as summarized in the Results section. Essentially the same results were obtained with the fluorescence technique (data not shown). One of the advantages of an immunohistochemical approach is that it gives information on the cellular distribution of a given protein within a tissue. It is clear from the results presented that the nuclei of all cell types of the somatic tissues examined contain the histone H I ° / H 5 variant. Thus, a histone H5, defined as 'restricted to erythrocyte nuclei' seems not to occur in Xenopus. This raises the question of whether the Xenopus H I ° / H 5 variant has to be considered as functionally similar to mammalian H1 °. The immunological relationship between these histones (Smith et al., 1984) points to a common, conserved structure with a similar function. On the other hand, it is not immediately apparent from the observed cellular distribution in Xenopus that the Xenopus H I ° / H 5 variant is characteristic of tissues with low mitotic activity, as is the case for mammalian H1 °. This point deserves some consideration. First, in oocyte nuclei the H I ° / H 5 variant cannot be detected, whereas the surrounding tissues react with the anti-H5 serum, suggesting that this histone is not present in the amplified nucleoli at least, although oocytes are not very active in DNA synthesis or cell division. A trivial explanation is that the immunohistochemical procedure used did not allow the detection of histone 'H5' in oocyte nuclei. Another explanation might be that the H I ° / H 5 variant is absent in oocytes, because this cell is optimally prepared for the very rapid divisions just after fertilization. Also, the rapidly di-
viding, early stages of development do not contain the H I ° / H 5 variant, whereas H1A was present in all nuclei (Moorman et al., in preparation). Thus, if the H I ° / H 5 variant is involved in the cessation of DNA synthesis or mitotic activity, oocytes have solved this problem in another way. Previously, we showed that Xenopus oocytes have a large store of histone H1A (Van Dongen et al., 1983). Nevertheless, histone H1A could be detected only in the nucleoli located characteristically just beneath the nuclear membrane. Apparently, the way of storage prevents immunohistochemical detection with the procedures we have applied. The presence of histone HIA in the nucleoli at all stages of oocyte development, some of which are highly active in ribosomal RNA synthesis, is interesting in view of the discussions on the composition of active chromatin (see, e.g., Weisbrod, 1982) and dearly demonstrates that the absence of a lysine-rich histone is not a prerequisite for active chromatin. Second, the cells of the various stages of spermatogenesis undoubtedly react with the antiH5 serum. This reaction seems less intense compared with that of the surrounding connective tissue. This became apparent if higher antibody dilutions were used, suggesting that the H I ° / H 5 variant is present in lower amounts. Biochemical extractions have not revealed the presence of this variant in spermatogenic cells (Risley and Eckhardt, 1981), but if this variant represented approx. 1% of total histone content, its presence would easily have remained undetected. Thus, in testis the H I ° / H 5 variant seems more abundant in the somatic cells with low mitotic activity, analogous to mammalian H1 °. But both the nondividing spermatids and the dividing spermatogenic cells also contain the H I ° / H 5 variant. Third, whereas one can expect a difference in mitotic activity between stomach epithelium and the other stomach cells or skin germinative layer and the other skin cells, no difference in staining between the 'H5' and the H1A antiserum could be detected, suggesting no heterogeneity with respect to the distribution of the H I ° / H 5 variant. In conclusion, in somatic tissues from adult X. laeois the H I ° / H 5 variant seems a specialized subfraction of the lysine-rich histones that is not
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restricted to amitotic cells or cells with low mitotic activity and the specific function of which remains unknown. This condition is different from that in birds where H5 seems restricted to the nucleated erythrocytes and may be more like that in mammals. The presence of mammalian H1 ° has been suggested to be indirectly linked to cell proliferation and a role for H1 ° has been proposed in the termination of differentiation and/or maintenance of the differentiated state (Gjerset al., 1982). Our data indicate that the absence of the Xenopus HI°/H5 variant is not a prerequisite for mitotic activity, but whether this variant plays a similar role as proposed for mammalian H1 ° has to be established. The absence of this variant in early embryonic stages (Moorman and de Boer, in preparation) could point in this direction. A precise description of the appearance of the HI°/H5 variant during embryonic development is currently being carried out and may indicate a role for the HI°/H5 variant.
Acknowledgements We thank Mrs. M.Th. Linders and C.C. Verhoek-Pocock for expert technical assistance, Dr. W.H. Lamers and Prof. Dr. R. Charles for their continuous interest and many fruitful discussions, Dr. W. van Raamsdonk for much histochemical advice, Mrs. F. de Jong and C. Hersbach for help on microscopy and photography and Mrs. J. Husslage for secretarial help.
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