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A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells
Cell Differentiation and Development, 25 (1988) 47-56 Elsevier Scientific Publishers Ireland, Ltd.
41
CDF 00525
A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells Koichiro
Shiokawa
‘, Kosuke Tashiro I,*, Norihiko Nakakura I, Yuchang Fu ‘, Yasuo Atsuchi Sakiko Nakazato 2, Yoshinari Tsuzaki 3 and Kohji Ikenishi 3
‘,
’ Department oj Biology, Faculty ofScience 33, Kyushu University, Fukuoka 812, Japan; ’ Nippon Zeon K.K., Biological Science Institute, Kawasaki, Kawasaki 210, Japan; and ’ Department of Biology, Faculty of Science, Osaka City University, Sugimoto, Sumiyoshi, Osaka S58, Japan (Accepted
7 April 1988)
Embryos from a female of Xenopus hwvis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastnda stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest. Maternal-effect
mutant; RNA synthesis; Dissociated cells; Cell adhesion; Gastrula-arrest
Introduction In Mexican axolotl ( Ambystoma mexicanurn ), a considerable number of mutant strains have been reported, among which are ova-deficient ones that arrest development during gastrulation (Humphrey, 1966; Malacinski and Brothers, 1974; Briggs and Briggs, 1984; Malacinski and Barone, 1985). In such mutants, the defect has been attributed to the absence of a certain maternally in-
Correspondence address: Dr. of Biology, Faculty of Science 812, Japan. * Present address: Okinaka Research, Tokyo Toranomon 105, Japan. 0922-3371/88/$03.50
Koichiro Shiokawa, Department 33, Kyushu University, Fukuoka Memorial Hospital,
Institute Minatoku,
0 1988 Elsevier Scientific
for Medical Toranomon
Publishers
Ireland,
herited protein within the egg (Briggs and Justus, 1968; Brothers, 1976). Several mutants have been reported to occur also in Xenopus laeuis, such as anucleolate mutants (Elsdale et al., 1958; Miller and Gurdon, 1970) that contain only residual rDNA such as rDNA pseudogene (Tashiro et al., 1986a), periodic albinism (Hoperskaya, 1975) ‘ pale eggs’ and ‘ partial cleavage’ maternal-effect mutants (Droin and Fischberg, 1984) muscular response-mutant (unresponsioe) (Reinschmidt and Tompkins, 1984) and others (Krotoski et al., 1985). However, as in axolotl, the number of available mutants in X faeuis is relatively small, and biochemical studies of the mutants, especially those of maternal defect mutants, are scanty. Under these circumstances, Ikenishi and Tsuzaki (1988) happened to find in their laboraLtd.
48
tory population of X. laeuis a female (designated as no. 65) whose fertilized eggs are ova-deficient and arrest development at gastrulation regardless of the difference in males mated. Ikenishi and Tsuzaki (1988) analyzed nuclear structure of these defect eggs and found that nuclei are fragmented from the early stage of development. Also, these authors found that the ova-deficient eggs were devoid of an acidic protein (ca. 38 kDa) which normally occurs in wild-type eggs. Unfortunately, however, the genetic background of this female is unclear. Furthermore, absence of the acidic protein and defect in nuclear appearance (Ikenishi and Tsuzaki, 1988) have not been related to the defect in cellular function at or shortly before the start of gastrulation arrest. In the present experiments, in order to study cellular function of the embryos of the female no. 65, we dissociated these embryos and studied both reaggregation and RNA transcription at several stages. The results showed that RNA synthesis in the ova-deficient embryos starts to be greatly inhibited as the first detectable defect as early as the late blastula (stage 9.5) stage, when cellular reaggregating activity and DNA and protein synthetic activity are not greatly inhibited.
Materials and Methods Embryos Fertilized eggs of X. laevis were obtained from wild-type and no. 65 females by mating with different males as described previously (Kotani et al., 1973). Embryos were staged according to Nieuwkoop and Faber (1956). Embryos were dejellied with 1.5% cysteine-HCl solution (pH 8.0) (Ikenishi and Tsuzaki, 1988) and raised in l/10 Steinberg solution at 20-21” C. Dissociation of embryos and labeling of embryonic cells Embryos at various stages were dissociated and cultured for varying lengths of time in the Steams’ medium as described previously by Shiokawa and Yamana (1967). For RNA labeling, dissociated cells were administered with either [ 3H]uridine-5T
(25 Ci/mmol) or [8-3H]guanosine (6 Ci/mmol) (Shiokawa and Yamana, 1967). Labeled cells were washed with fresh Stearns’ medium by low speed centrifugation and kept frozen at -20°C until analyzed.
Extraction and fractionation of RNA Embryonic cells were homogenized in 0.1 M sodium acetate, pH 5.0, containing 0.5% sodium dodecyl sulfate (SDS) and 10 pg/ml bentonite. Homogenates were then treated once at 25°C with buffer-saturated phenol (pH 5.0) for l-2 h in a gyratory shaker (Shiokawa et al., 1986). RNAs were precipitated from the aqueous phase with 0.2 M NaCl and 75% ethanol. Volumes of the aqueous phase recovered after the phenol treatment were adjusted to be the same, in order to ensure direct comparison of the amounts of RNAs extracted within one experimental set. RNAs were electrophoresed on lo-cm gels of either 0.5% agarose-2.4% polyacrylamide or 8% polyacrylamide as described previously (Shiokawa et al., 1979). After the electrophoresis, distribution of UV-absorbing materials was determined in a Gilford densitometer at 260 nm. The 0.5% agarose-2.4% polyacrylamide gels were sliced at 2 mm width, unless otherwise stated. Gels of 8% polyacrylamide were frozen in solid CO,-acetone and then sliced at 1 mm width. The efficiency of the extraction of the labeled RNA was arqund 80% as determined by the recovery of the UV-absorbing materials (one embryo contains about 4 pg of RNA) (Shiokawa and Yamana, 1967).
Determination of the incorporation of precursors of DNA and protein into acid-insoluble fraction of cells Dissociated cells were prepared from five wildtype and five ova-deficient embryos and exposed for 4 h to 1 pCi/ml each of either [6-3H]thymidine (20 Ci/mmol) or [2,3-3H]alanine (40 Ci/mmol). After the labeling, the cells were collected, homogenized in distilled water under the ice-cold conditions and then precipitated with 7% trichloroacetic acid (TCA). The precipitates were collected on glass fiber filters, and radioactivity was counted in ACS II scintillation mixture in a scintillation spectrometer (Shiokawa et al., 1986).
49
Extraction and fractionation of DNA Embryos at the late blastula stage (stage 9.5) were homogenized in 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA, 300 mM NaCl, 1 mg/ml proteinase K and 2% SDS and incubated overnight at 37” C. The homogenate was treated with chloroform-phenol (1 : 1) and then with chloroform. DNA obtained by precipitation with ethanol was further purified by RNase treatment (Tashiro et al., 1986a). Five embryo-equivalents of DNA samples were electrophoresed on 0.7% agarose gels (Sigma type II) in 40 mM Tris-acetate buffer (pH 8.0), containing 2 mM EDTA. After electrophoresis, DNAs were stained by ethidium bromide and photographed on an UV lamp (Tashiro et al., 1986b).
Results Reaggregating activity of dissociated embryonic cells Ova-deficient and wild-type embryos were dissociated into cells at the early blastula stage (stage 8) (ca. 8 h after fertilization) and cultured in petri dishes separately. At 1 h. cells from both cultures started to reaggregate quite actively (Fig. lA, C). At this time-point, the outer appearance of the whole embryos (stage 8.5) was similar in wild-type and ova-deficient embryos (Fig. lB, D). When these dissociated cells were cultured for 6 h until wild-type embryos reached the late gastrula stage (stage 11) many aggregates of larger sizes were formed. Fig. 2 (A, D) shows the largest aggregates formed in the cultures of wild-type and
Embryos were dissociated at the early blastula stage Fig. 1. Outer appearance of embryos and their cells shortly after dissociation. and then cultured for 1 h. (A) Wild-type embryo cells; (B) wild-type embryo; (C) ova-deficient embryo cells; (D) ova-deficient embryo. Magnification is the same in the whole embryos (diameter ca. 1.2 mm) and dissociated cells in these and the following figures.
Fig. 2. Outer appearance of embryos and their dissociated and reaggregated cells. Embryos were dissociated at the early blastula stage and cultured for 7 h. (A) Aggregates of wild-type embryo cells; (B) a wild-type embryo, vegetal side; (C) a wild-type embryo, animal side; (D) aggregates of ova-deficient embryo cells; (E) an ova-deficient embryo, vegetal side; (F) an ova-deficient embryo, animal side.
ova-deficient embryo cells. These results show that cells from ova-deficient embryos have reaggregating activity which is comparable to that of wildtype embryo cells. When Ca2+ was eliminated by treatment with 0.02 M EDTA at this step, the aggregates were again completely dissociated. Therefore, the aggregation mechanism supporting the structures as in Fig. 2A and D is Ca2+-dependent. At this stage, the appearance of the whole embryo at the animal side was still not appreciably different in the wild-type and ova-deficient embryos (Fig. 2C, F). However, from the shape of
the blastopore lip, it is apparent that gastrulation was greatly retarded in the ova-deficient embryos (Fig. 2B, E). When wild-type embryos developed from stage 8 to the late neurula stage after culture for 25 h, ova-deficient embryos were not viable any more. Thus, the whole embryos appeared as round white mass composed of deteriorating cells. In the dissociated cell culture, most ova-deficient embryo cells stopped dividing, and large aggregates that had been formed were destroyed and dissociated into cells almost completely, and lysed and eventually
51
disappeared (data not shown), although wild-type embryo cells maintained large aggregates. Thus, it appears that cells of both dissociated and whole embryos of this ova-deficient type die at and after the gastrula stage.
2
0
b
Changes in the pattern of RNA synthesis Wild-type and ova-deficient embryos were dissociated at the early blastula stage (stage 8.0) as in Fig. 1, and cells were labeled with [3H]uridine or [3H]guanosine for 4 h until the whole embryos developed to the late blastula stage. When labeled RNAs were electrophoresed on 0.5% agarose-2.4% polyacrylamide gels and UV-absorbing material was scanned in a densitometer, there was no significant difference in the absorbancy profile between the wild-type and ova-deficient embryos (dotted lines in Fig. 3A, B). This shows that the ribosome content was much the same in the wildtype and ova-deficient eggs. In the cells from wild- type blastulae, incorporation of [ 3Hluridine occurred mainly in heterogeneous non-ribosomal RNA and 4 S RNA (Fig. 3A) as shown by Nakakura et al. (1987). When the RNA from ova-deficient embryo cells was analyzed, a quite similar pattern of labeling was obtained (Fig. 3B).
o
kw
m,,;
.... L
20
40
60 Distance
00 moved
,,,..,(.
\
I
I
I
,
20
40
60
80
I mm1
Fig. 3. Gel electrophoretic pattern of labeled high- and lowmolecular-weight RNAs of early blastula cells. Ten early blastulae were dissociated and labeled with 100 pCi of [3H]uridine for 4 h. RNAs were extracted and fractionated on 0.5% agarose-2.4% polyacrylamide gels. Dotted line: UV absorption at 260 nm expressed in an arbitrary unit. (A) Wild-type embryo cells; (B) ova-deficient embryo cells.
x
;
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c
20
40
60 Distance
80
20 moved
40
60
60
I mm)
Fig. 4. Gel electrophoretic patterns of labeled high- and lowmolecular-weight RNAs of late blastula cells. Ten embryos were dissociated at the late blastula (stage 9.5) stage and labeled with 100 PCi of [3H]uridine for 4 h. RNAs were extracted and fractionated as in Fig. 3. (A) Wild-type embryo cells; (B) ova-deficient embryo cells.
We obtained essentially similar results with [ 3H]guanosine-labeled RNAs (profiles omitted). Since radioactivity obtained with [ 3H]guanosine as a label was relatively large, we fractionated the [3H]guanosine-labeled RNAs further on 8% polyacrylamide gels. The profile obtained showed that the labeling of U3, U2 and Ul snRNAs, 5 S RNA, 4.5 S pre-tRNA and 4 S RNA was much the same between the wild-type and ova-deficient embryos (data omitted, but see Shiokawa et al. (1979) for the profile). These results show that the RNA synthetic activity is not appreciably impaired in ova-deficient embryos, at least at the early to midblastula stages. Dissociated cells were then obtained from embryos at the late blastula stage (stage 9.5), and they were similarly labeled with [3H]uridine for 4 h until the whole embryos reached the midgastrula stage. As shown in Fig. 4A, cells from wild-type embryos synthesized a measurable amount of 18 S and 28 S rRNAs (Shiokawa and Yamana, 1979; Shiokawa et al., 1981) as can be seen by the appearance of two radioactivity peaks at the optical density peaks of 18 S and 28 S rRNA (dotted lines). In the cells from ova-deficient embryos (Fig. 4B), however, the activity of RNA synthesis was greatly suppressed (over 90%) and only a
Distance
moved
(mm)
Fig. 5. Gel electrophoretic pattern of labeled high- and lowmolecular-weight RNA of cells at the early neurula stage. Ten embryos were dissociated at the late blastula stage and cultured for 5 h. The cells were then labeled with 100 pCi of [3H]uridine for 4 h until the control wild-type embryos developed to the early neurula stage. RNAs were extracted and fractionated as in Fig. 3. (A) Wild-type embryo cells; (B) ova-deficient embryo cells. The inset shows the results of counting of the gel sliced at 1 mm width.
small incorporation occurred in the 4 S RNA region (at about 20% level of the wild-type cells). The analysis of the two RNA preparations on 8% gel revealed that the inhibition occurred similarly in each of the major species of low-molecularweight RNAs (data omitted). It is noteworthy that the recovery of the UV-absorbing materials in the wild-type embryonic cells was not greatly different from that of the ova-deficient embryonic cells (Fig. 4, dotted line). This provides strong evidence that neither cell death nor poor recovery of RNA explains the observed difference in RNA labeling activity. We cultured the dissociated cells from late blastulae for 5 h in the medium without a label. Then, after changing the medium into a fresh one, we labeled the cells with [3H]uridine for 4 h. At the end of the labeling, control wild-type embryos developed to the early neurula stage (stage 13.5). When the RNA from wild-type embryo cells was analyzed, the activity of rRNA synthesis was found to be very high (Fig. 5A). In contrast, the RNA synthesis in the cells of ova-deficient embryos was again greatly inhibited (Fig. 5B). Furthermore, the amount of UV-absorbing materials recovered from
the ova-deficient embryo cells was no longer identical to that from the wild-type cells: only about one half of that from wild-type cells. These results show that there is already extensive cell death in the culture of ova-deficient embryo cells at this stage. We repeated the labeling experiment shown in Fig. 5B and carefully analyzed the label distribution in the high-molecular-weight RNA region by slicing the gel at 1 mm width. The result in the inset of Fig. 5B shows that in the RNAs labeled there are two small, but distinct, amounts of 28 S and 18 S rRNAs. Then, some of the ova-deficient embryo cells appear to survive a little longer and accumulate a small amount of rRNA even after extensive cell death started at the mid to late gastrula stage, although the number of such cells appears to be very small. Changes in DNA and protein synthesis Embryos were dissociated into cells at the pigment gastrula (stage 9.5) and late gastrula (stage 11) stages, and the incorporation of [ 3H]thymidine was measured by labeling the cells for 4 h at each stage. The incorporation of the label into the total acid-insoluble fraction of five embryo-equivalent cells at the pigment gastrula stage was 11900 cpm and 10600 cpm (average of two determinations) for the control and ova-deficient embryos, respectively. However, the incorporation was 31100 cpm and 2810 cpm at the late gastrula stage, respectively. Then, it is apparent that DNA synthesis in the ova-deficient embryo cells was nearly normal (inhibition is only 11%) at the late blastula (stage 9.5) stage, but was greatly inhibited (by as much as 91%) at the late gastrula stage. DNAs were then extracted from both wild-type and ova-deficient embryos at the pigment gastrula stage and were electrophoresed on an agarose gel. When stained by ethidium bromide, it was shown that amounts of both nuclear and mitochondrial DNAs were not greatly different in the wild-type and ova-deficient embryos (Fig. 6). These results are consistent with the incorporation data that DNA replication is not impaired appreciably until at the late blastula stage. For a study of protein synthesis, dissociated cells from embryos at the late blastula (stage 9.5) and late gastrula stages were labeled for 4 h with
53
Fig. 6. Agarose gel electrophoresis of DNA from wild-type and ova-deficient embryos. Embryos at the late blastula stage (stage 9.5) were homogenized and DNAs were extracted. DNAs were electrophoresed on 0.7% agarose gel stained with ethidium bromide and filmed with an UV-lamp. (A) DNA digested with Hind111 as size markers; (B) DNA from wild-type embryos; (C) DNA from ova-deficient embryos. M and Ch show the positions of mitochondrial and chromosomal DNAs.
[3H]alanine, and incorporation into the total acid-insoluble fraction of the cells was determined as above. The results showed that the inhibition in the incorporation in ova-deficient embryo cells was approximately 20% and 80% for the late blastula and late gastrula cells, respectively. Then, the synthesis of both DNA and protein appears to be nearly normal at the late blastula stage and starts to be inhibited only at the mid to late gastrula stage.
Discussion The female no. 65 used in the present experiment was found in a laboratory population of X. laevis, and its eggs were found to arrest development at the late gastrula stage. The eggs of female no. 65 have two unique features: (1) absence of an acidic protein (ca. 38 kDa), and (2) abnormal nuclear structure (Ikenishi and Tsuzaki, 1988). However, the physiological meaning of these unique features has not been clarified, although they are certainly assumed to be related to the gastrulation arrest.
In the present experiment, we studied the general features of the macromolecular synthesis with particular interest in the change in the RNA synthetic activity by using dissociated cells of both ova-deficient and wild-type embryos. It was found here that cells from ova-deficient embryos reaggregate quite actively at least until the late gastrula stage. The strong reaggregating activity observed in the dissociated cells of ova-deficient embryos provides evidence that the adhesion system may be normal in the ova-deficient embryos. Since the aggregates formed by the cells from the ova-deficient embryos were repeatedly dissociated and reaggregated by the treatment with EDTA-containing medium (data not shown), the reaggregation detected here must be due to the so-called Ca2+-dependent adhesion system. We have previously shown that the activity of early Xenopus Ca2+-dependent reaggregating activity is maintained by the continuous translation of maternal stock of mRNA for the adhesion molecules by the studies that utilized metabolic inhibitors (Shiokawa et al., 1983). Therefore, it appears that the ova-deficient eggs contain ample stock of such maternal mRNAs for Ca2+-dependent adhesion molecules, and their translational machinery is also normal. In the ova-deficient embryos, DNA and protein synthesis proceeded relatively normally until the late blastula stage, although these were severely damaged at the late gastrula stage. On the other hand, RNA synthesis was nearly normal until the early blastula stage, but was greatly inhibited at the late blastula stage. Thus, in the ova-deficient embryos, RNA synthesis appears to be the first that was damaged seriously prior to the gastrulation arrest. These results are comparable to the previous autoradiographic study in ova-deficient embryos of axolotl (A. mexicanum) (Carroll, 1974). It has been described that inhibition of RNA synthesis by cy-amanitin at the midblastula stage may lead to arrest at gastrulation even in wild-type embryos (Newport and Kirschner, 1982). Therefore, it appears that RNA synthesis that occurs between the midblastula and early gastrula stages is essential for the subsequent gastrulation movement. Occurrence of RNA species that is uniquely activated during these stages has been reported.
54
Thus, Krieg and Melton (1985) isolated a DNA clone (GS17) that is transcribed only during the early to mid gastrula stage and is then rapidly degraded after the gastrula stage. Therefore, it is interesting to test whether such unique RNA is activated in this ova-deficient mutant. Anyway, the large inhibition of RNA synthesis in our present ova-deficient embryos established by the late blastula stage could be the main cause for the observed arrest at the late gastrula stage. As noted above, Ikenishi and Tsuzaki (1988) showed that blastomeres of the ova-deficient embryos contain multiple small nuclei (karyomeres) and lack an acidic protein (ca. 38 kDa). Therefore, we assume that the inhibition of RNA synthesis observed may be a reflection of either the unusual nuclear conditions or the absence of the 38 kDa protein or both, although we still do not know how such unusual conditions could induce the observed inhibition in RNA synthesis.
Acknowledgements We thank Professor K. Yamana for his warm encouragement during the present study. The present work was supported, in part, by a Grant-inAid for Scientific Research (No. 61540523) to KS. from the Ministry of Education, Science and Culture of Japan and a grant from Takeda Science Foundation (1986) to K.S.
References Briggs, R. and F. Briggs: Discovery and initial characterization of a new conditional (temperature-sensitive) maternal effect mutation in the axolotl. Differentiation 26, 176-181 (1984). Briggs, R. and J.T. Justus: Partial characterization of the component from normal eggs which corrects the maternal effect of gene o in the Mexican axolotl (Ambystoma mexicanurn). J. Exp. Zool. 167, 105-116 (1968). Brothers, A.J.: Stable nuclear activation dependent on a protein synthesized during oogenesis. Nature 260, 112-115 (1976). Carroll, C.R.: Comparative study of the early embryonic cytology and nucleic acid synthesis of Ambysroma mexicanurn normal and o mutant embryos. J. Exp. Zool. 187,409-422 (1974). Droin, A. and M. Fischberg: Two recessive mutations with
maternal
effect upon colour and cleavage of Xenopus lueois Arch. Dev. Biol. 193, 86-89 (1984). Elsdale, T.R., M. Fischberg and S. Smith: A mutation that reduces nucleolar number in Xenopw laevis. Exp. Cell Res. 14,642-643 (1958). Hoperskaya, O.A.: The animals homozygous for a mutation causing periodic albinism (ap) in Xenopur loeuis. J. Embryol. Exp. Morphol. 34, 253-264 (1975). Humphrey, R.R.: A recessive factor (0, for ova deficient) determining a complex of abnormalities in the Mexican axolotl (Ambystoma mexicanurn). Dev. Biol. 13, 57-76 (1966). Ikenishi, K. and Y. Tsuzaki: A possible maternal-effect mutant of Xenopw laeuis. I. Cytological and biochemical analysis of the unfertilized eggs and embryos. Dev. Biol. 125, 458-461 (1988). Kotani, M., K. Ikenishi and K. Tanabe: Cortical granules remaining after fertilization in Xenopus heuis. Dev. Biol. 30, 228-232 (1973). Krieg, P.A. and D.A. Melton: Developmental regulation of a gastrula-specific gene injected into fertilized Xenopw eggs. EMBO J. 4, 3463-3471 (1985). Krotoski, D.M., D.C. Reinschmidt and R. Tompkins: Developmental mutants isolated from wild-caught Xenopus laeois by gynogenesis and inbreeding. J. Exp. Zool. 233, 443-449 (1985). Malacinski, G.M. and D. Barone: Towards understanding paternal extragenic contributions to early amphibian pattern specification: the axolotl rs-1 gene as a model system. J. Embryol. Exp. Morphol. 89, Suppl., 53-68 (1985). Malacinski, G.M. and A.J. Brothers: Mutant genes in the Mexican axolotl. Science 184, 1142-1147 (1974). Miller, L. and J.B. Gurdon: Mutations affecting the size of the nucleolus in Xenopur laeuis. Nature 227, 1108-1110 (1970). Nakakura, N., T. Miura, K. Yamana, A. Ito and K. Shiokawa: Synthesis of heterogeneous mRNA-like RNA and lowmolecular-weight RNA before the midblastula transition in embryos of Xenopus loeuis. Dev. Biol. 123, 421-429 (1987). Newport, J. and M. Kirschner: A major developmental transition in early XenopuF embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30, 675-686 (1982). Nieuwkoop, P.D. and J. Faber: Normal Table of Xenopur lueuis (Daudin). North-Holland, Amsterdam (1956). Reinschmidt, D.C. and R. Tompkins: Unresponsive, a new behavioral mutant in Xenopur lueuis. Differentiation 26, 189-193 (1984). Shiokawa, K. and K. Yamana: Pattern of RNA synthesis in isolated cells of Xenopus laeuis embryos. Dev. Biol. 16, 368-388 (1967). Shiokawa, K. and K. Yamana: Differential initiation of rRNA gene activity in progenies of different blastomeres of early Xenopus embryos: Evidence for regulated synthesis of rRNA. Dev. Growth Differ. 21, 501-507 (1979). Shiokawa, K., Y. Misumi, Y. Yasuda, Y. Nishio, S. Kurata, M. Sameshima and K. Yamana: Synthesis and transport of eggs. Roux’s
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various RNA species in developing embryos of Xenopus laeois. Dev. Biol. 68, 503-514 (1979). Shiokawa, K., Y. Misumi and K. Yamana: Demonstration of rRNA synthesis in pre-gastrular embryos of Xenopur loeuis. Dev. Growth Differ. 23, 579-587 (1981). Shiokawa, K., K. Tashiro, T. Oka and K. Yamana: Contribution of maternal mRNA for maintenance of Ca’+-dependent reaggregating activity in dissociated cells of Xenopw laevis embryos. Cell Differ. 13, 247-255 (1983). Shiokawa, K., Y. Kawazoe, H. Nomura, T. Miura, N. Nakakura, T. Horiuchi and K. Yamana: Ammonium ion as
a possible regulator of the commencement of rRNA synthesis in Xenopw laeuis embryogenesis. Dev. Biol. 115. 380-391 (1986). Tashiro, K., K. Shiokawa, K. Yamana and Y. Sakaki: Structural analysis of ribosomal DNA homologues in nucleolusless mutant of Xenopur laevis. Gene 44, 299-306 (1986a). Tashiro, K., M. Inoue, Y. Sakaki and K. Shiokawa: Preservation of Xenopus laeuis rDNA-containing plasmid, pXlrlOlA, injected into the fertilized egg of Xenopus laeuis. Cell Struct. Funct. 11, 109-114 (1986b).
41
CDF 00525
A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells Koichiro
Shiokawa
‘, Kosuke Tashiro I,*, Norihiko Nakakura I, Yuchang Fu ‘, Yasuo Atsuchi Sakiko Nakazato 2, Yoshinari Tsuzaki 3 and Kohji Ikenishi 3
‘,
’ Department oj Biology, Faculty ofScience 33, Kyushu University, Fukuoka 812, Japan; ’ Nippon Zeon K.K., Biological Science Institute, Kawasaki, Kawasaki 210, Japan; and ’ Department of Biology, Faculty of Science, Osaka City University, Sugimoto, Sumiyoshi, Osaka S58, Japan (Accepted
7 April 1988)
Embryos from a female of Xenopus hwvis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastnda stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest. Maternal-effect
mutant; RNA synthesis; Dissociated cells; Cell adhesion; Gastrula-arrest
Introduction In Mexican axolotl ( Ambystoma mexicanurn ), a considerable number of mutant strains have been reported, among which are ova-deficient ones that arrest development during gastrulation (Humphrey, 1966; Malacinski and Brothers, 1974; Briggs and Briggs, 1984; Malacinski and Barone, 1985). In such mutants, the defect has been attributed to the absence of a certain maternally in-
Correspondence address: Dr. of Biology, Faculty of Science 812, Japan. * Present address: Okinaka Research, Tokyo Toranomon 105, Japan. 0922-3371/88/$03.50
Koichiro Shiokawa, Department 33, Kyushu University, Fukuoka Memorial Hospital,
Institute Minatoku,
0 1988 Elsevier Scientific
for Medical Toranomon
Publishers
Ireland,
herited protein within the egg (Briggs and Justus, 1968; Brothers, 1976). Several mutants have been reported to occur also in Xenopus laeuis, such as anucleolate mutants (Elsdale et al., 1958; Miller and Gurdon, 1970) that contain only residual rDNA such as rDNA pseudogene (Tashiro et al., 1986a), periodic albinism (Hoperskaya, 1975) ‘ pale eggs’ and ‘ partial cleavage’ maternal-effect mutants (Droin and Fischberg, 1984) muscular response-mutant (unresponsioe) (Reinschmidt and Tompkins, 1984) and others (Krotoski et al., 1985). However, as in axolotl, the number of available mutants in X faeuis is relatively small, and biochemical studies of the mutants, especially those of maternal defect mutants, are scanty. Under these circumstances, Ikenishi and Tsuzaki (1988) happened to find in their laboraLtd.
48
tory population of X. laeuis a female (designated as no. 65) whose fertilized eggs are ova-deficient and arrest development at gastrulation regardless of the difference in males mated. Ikenishi and Tsuzaki (1988) analyzed nuclear structure of these defect eggs and found that nuclei are fragmented from the early stage of development. Also, these authors found that the ova-deficient eggs were devoid of an acidic protein (ca. 38 kDa) which normally occurs in wild-type eggs. Unfortunately, however, the genetic background of this female is unclear. Furthermore, absence of the acidic protein and defect in nuclear appearance (Ikenishi and Tsuzaki, 1988) have not been related to the defect in cellular function at or shortly before the start of gastrulation arrest. In the present experiments, in order to study cellular function of the embryos of the female no. 65, we dissociated these embryos and studied both reaggregation and RNA transcription at several stages. The results showed that RNA synthesis in the ova-deficient embryos starts to be greatly inhibited as the first detectable defect as early as the late blastula (stage 9.5) stage, when cellular reaggregating activity and DNA and protein synthetic activity are not greatly inhibited.
Materials and Methods Embryos Fertilized eggs of X. laevis were obtained from wild-type and no. 65 females by mating with different males as described previously (Kotani et al., 1973). Embryos were staged according to Nieuwkoop and Faber (1956). Embryos were dejellied with 1.5% cysteine-HCl solution (pH 8.0) (Ikenishi and Tsuzaki, 1988) and raised in l/10 Steinberg solution at 20-21” C. Dissociation of embryos and labeling of embryonic cells Embryos at various stages were dissociated and cultured for varying lengths of time in the Steams’ medium as described previously by Shiokawa and Yamana (1967). For RNA labeling, dissociated cells were administered with either [ 3H]uridine-5T
(25 Ci/mmol) or [8-3H]guanosine (6 Ci/mmol) (Shiokawa and Yamana, 1967). Labeled cells were washed with fresh Stearns’ medium by low speed centrifugation and kept frozen at -20°C until analyzed.
Extraction and fractionation of RNA Embryonic cells were homogenized in 0.1 M sodium acetate, pH 5.0, containing 0.5% sodium dodecyl sulfate (SDS) and 10 pg/ml bentonite. Homogenates were then treated once at 25°C with buffer-saturated phenol (pH 5.0) for l-2 h in a gyratory shaker (Shiokawa et al., 1986). RNAs were precipitated from the aqueous phase with 0.2 M NaCl and 75% ethanol. Volumes of the aqueous phase recovered after the phenol treatment were adjusted to be the same, in order to ensure direct comparison of the amounts of RNAs extracted within one experimental set. RNAs were electrophoresed on lo-cm gels of either 0.5% agarose-2.4% polyacrylamide or 8% polyacrylamide as described previously (Shiokawa et al., 1979). After the electrophoresis, distribution of UV-absorbing materials was determined in a Gilford densitometer at 260 nm. The 0.5% agarose-2.4% polyacrylamide gels were sliced at 2 mm width, unless otherwise stated. Gels of 8% polyacrylamide were frozen in solid CO,-acetone and then sliced at 1 mm width. The efficiency of the extraction of the labeled RNA was arqund 80% as determined by the recovery of the UV-absorbing materials (one embryo contains about 4 pg of RNA) (Shiokawa and Yamana, 1967).
Determination of the incorporation of precursors of DNA and protein into acid-insoluble fraction of cells Dissociated cells were prepared from five wildtype and five ova-deficient embryos and exposed for 4 h to 1 pCi/ml each of either [6-3H]thymidine (20 Ci/mmol) or [2,3-3H]alanine (40 Ci/mmol). After the labeling, the cells were collected, homogenized in distilled water under the ice-cold conditions and then precipitated with 7% trichloroacetic acid (TCA). The precipitates were collected on glass fiber filters, and radioactivity was counted in ACS II scintillation mixture in a scintillation spectrometer (Shiokawa et al., 1986).
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Extraction and fractionation of DNA Embryos at the late blastula stage (stage 9.5) were homogenized in 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA, 300 mM NaCl, 1 mg/ml proteinase K and 2% SDS and incubated overnight at 37” C. The homogenate was treated with chloroform-phenol (1 : 1) and then with chloroform. DNA obtained by precipitation with ethanol was further purified by RNase treatment (Tashiro et al., 1986a). Five embryo-equivalents of DNA samples were electrophoresed on 0.7% agarose gels (Sigma type II) in 40 mM Tris-acetate buffer (pH 8.0), containing 2 mM EDTA. After electrophoresis, DNAs were stained by ethidium bromide and photographed on an UV lamp (Tashiro et al., 1986b).
Results Reaggregating activity of dissociated embryonic cells Ova-deficient and wild-type embryos were dissociated into cells at the early blastula stage (stage 8) (ca. 8 h after fertilization) and cultured in petri dishes separately. At 1 h. cells from both cultures started to reaggregate quite actively (Fig. lA, C). At this time-point, the outer appearance of the whole embryos (stage 8.5) was similar in wild-type and ova-deficient embryos (Fig. lB, D). When these dissociated cells were cultured for 6 h until wild-type embryos reached the late gastrula stage (stage 11) many aggregates of larger sizes were formed. Fig. 2 (A, D) shows the largest aggregates formed in the cultures of wild-type and
Embryos were dissociated at the early blastula stage Fig. 1. Outer appearance of embryos and their cells shortly after dissociation. and then cultured for 1 h. (A) Wild-type embryo cells; (B) wild-type embryo; (C) ova-deficient embryo cells; (D) ova-deficient embryo. Magnification is the same in the whole embryos (diameter ca. 1.2 mm) and dissociated cells in these and the following figures.
Fig. 2. Outer appearance of embryos and their dissociated and reaggregated cells. Embryos were dissociated at the early blastula stage and cultured for 7 h. (A) Aggregates of wild-type embryo cells; (B) a wild-type embryo, vegetal side; (C) a wild-type embryo, animal side; (D) aggregates of ova-deficient embryo cells; (E) an ova-deficient embryo, vegetal side; (F) an ova-deficient embryo, animal side.
ova-deficient embryo cells. These results show that cells from ova-deficient embryos have reaggregating activity which is comparable to that of wildtype embryo cells. When Ca2+ was eliminated by treatment with 0.02 M EDTA at this step, the aggregates were again completely dissociated. Therefore, the aggregation mechanism supporting the structures as in Fig. 2A and D is Ca2+-dependent. At this stage, the appearance of the whole embryo at the animal side was still not appreciably different in the wild-type and ova-deficient embryos (Fig. 2C, F). However, from the shape of
the blastopore lip, it is apparent that gastrulation was greatly retarded in the ova-deficient embryos (Fig. 2B, E). When wild-type embryos developed from stage 8 to the late neurula stage after culture for 25 h, ova-deficient embryos were not viable any more. Thus, the whole embryos appeared as round white mass composed of deteriorating cells. In the dissociated cell culture, most ova-deficient embryo cells stopped dividing, and large aggregates that had been formed were destroyed and dissociated into cells almost completely, and lysed and eventually
51
disappeared (data not shown), although wild-type embryo cells maintained large aggregates. Thus, it appears that cells of both dissociated and whole embryos of this ova-deficient type die at and after the gastrula stage.
2
0
b
Changes in the pattern of RNA synthesis Wild-type and ova-deficient embryos were dissociated at the early blastula stage (stage 8.0) as in Fig. 1, and cells were labeled with [3H]uridine or [3H]guanosine for 4 h until the whole embryos developed to the late blastula stage. When labeled RNAs were electrophoresed on 0.5% agarose-2.4% polyacrylamide gels and UV-absorbing material was scanned in a densitometer, there was no significant difference in the absorbancy profile between the wild-type and ova-deficient embryos (dotted lines in Fig. 3A, B). This shows that the ribosome content was much the same in the wildtype and ova-deficient eggs. In the cells from wild- type blastulae, incorporation of [ 3Hluridine occurred mainly in heterogeneous non-ribosomal RNA and 4 S RNA (Fig. 3A) as shown by Nakakura et al. (1987). When the RNA from ova-deficient embryo cells was analyzed, a quite similar pattern of labeling was obtained (Fig. 3B).
o
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Fig. 3. Gel electrophoretic pattern of labeled high- and lowmolecular-weight RNAs of early blastula cells. Ten early blastulae were dissociated and labeled with 100 pCi of [3H]uridine for 4 h. RNAs were extracted and fractionated on 0.5% agarose-2.4% polyacrylamide gels. Dotted line: UV absorption at 260 nm expressed in an arbitrary unit. (A) Wild-type embryo cells; (B) ova-deficient embryo cells.
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Fig. 4. Gel electrophoretic patterns of labeled high- and lowmolecular-weight RNAs of late blastula cells. Ten embryos were dissociated at the late blastula (stage 9.5) stage and labeled with 100 PCi of [3H]uridine for 4 h. RNAs were extracted and fractionated as in Fig. 3. (A) Wild-type embryo cells; (B) ova-deficient embryo cells.
We obtained essentially similar results with [ 3H]guanosine-labeled RNAs (profiles omitted). Since radioactivity obtained with [ 3H]guanosine as a label was relatively large, we fractionated the [3H]guanosine-labeled RNAs further on 8% polyacrylamide gels. The profile obtained showed that the labeling of U3, U2 and Ul snRNAs, 5 S RNA, 4.5 S pre-tRNA and 4 S RNA was much the same between the wild-type and ova-deficient embryos (data omitted, but see Shiokawa et al. (1979) for the profile). These results show that the RNA synthetic activity is not appreciably impaired in ova-deficient embryos, at least at the early to midblastula stages. Dissociated cells were then obtained from embryos at the late blastula stage (stage 9.5), and they were similarly labeled with [3H]uridine for 4 h until the whole embryos reached the midgastrula stage. As shown in Fig. 4A, cells from wild-type embryos synthesized a measurable amount of 18 S and 28 S rRNAs (Shiokawa and Yamana, 1979; Shiokawa et al., 1981) as can be seen by the appearance of two radioactivity peaks at the optical density peaks of 18 S and 28 S rRNA (dotted lines). In the cells from ova-deficient embryos (Fig. 4B), however, the activity of RNA synthesis was greatly suppressed (over 90%) and only a
Distance
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Fig. 5. Gel electrophoretic pattern of labeled high- and lowmolecular-weight RNA of cells at the early neurula stage. Ten embryos were dissociated at the late blastula stage and cultured for 5 h. The cells were then labeled with 100 pCi of [3H]uridine for 4 h until the control wild-type embryos developed to the early neurula stage. RNAs were extracted and fractionated as in Fig. 3. (A) Wild-type embryo cells; (B) ova-deficient embryo cells. The inset shows the results of counting of the gel sliced at 1 mm width.
small incorporation occurred in the 4 S RNA region (at about 20% level of the wild-type cells). The analysis of the two RNA preparations on 8% gel revealed that the inhibition occurred similarly in each of the major species of low-molecularweight RNAs (data omitted). It is noteworthy that the recovery of the UV-absorbing materials in the wild-type embryonic cells was not greatly different from that of the ova-deficient embryonic cells (Fig. 4, dotted line). This provides strong evidence that neither cell death nor poor recovery of RNA explains the observed difference in RNA labeling activity. We cultured the dissociated cells from late blastulae for 5 h in the medium without a label. Then, after changing the medium into a fresh one, we labeled the cells with [3H]uridine for 4 h. At the end of the labeling, control wild-type embryos developed to the early neurula stage (stage 13.5). When the RNA from wild-type embryo cells was analyzed, the activity of rRNA synthesis was found to be very high (Fig. 5A). In contrast, the RNA synthesis in the cells of ova-deficient embryos was again greatly inhibited (Fig. 5B). Furthermore, the amount of UV-absorbing materials recovered from
the ova-deficient embryo cells was no longer identical to that from the wild-type cells: only about one half of that from wild-type cells. These results show that there is already extensive cell death in the culture of ova-deficient embryo cells at this stage. We repeated the labeling experiment shown in Fig. 5B and carefully analyzed the label distribution in the high-molecular-weight RNA region by slicing the gel at 1 mm width. The result in the inset of Fig. 5B shows that in the RNAs labeled there are two small, but distinct, amounts of 28 S and 18 S rRNAs. Then, some of the ova-deficient embryo cells appear to survive a little longer and accumulate a small amount of rRNA even after extensive cell death started at the mid to late gastrula stage, although the number of such cells appears to be very small. Changes in DNA and protein synthesis Embryos were dissociated into cells at the pigment gastrula (stage 9.5) and late gastrula (stage 11) stages, and the incorporation of [ 3H]thymidine was measured by labeling the cells for 4 h at each stage. The incorporation of the label into the total acid-insoluble fraction of five embryo-equivalent cells at the pigment gastrula stage was 11900 cpm and 10600 cpm (average of two determinations) for the control and ova-deficient embryos, respectively. However, the incorporation was 31100 cpm and 2810 cpm at the late gastrula stage, respectively. Then, it is apparent that DNA synthesis in the ova-deficient embryo cells was nearly normal (inhibition is only 11%) at the late blastula (stage 9.5) stage, but was greatly inhibited (by as much as 91%) at the late gastrula stage. DNAs were then extracted from both wild-type and ova-deficient embryos at the pigment gastrula stage and were electrophoresed on an agarose gel. When stained by ethidium bromide, it was shown that amounts of both nuclear and mitochondrial DNAs were not greatly different in the wild-type and ova-deficient embryos (Fig. 6). These results are consistent with the incorporation data that DNA replication is not impaired appreciably until at the late blastula stage. For a study of protein synthesis, dissociated cells from embryos at the late blastula (stage 9.5) and late gastrula stages were labeled for 4 h with
53
Fig. 6. Agarose gel electrophoresis of DNA from wild-type and ova-deficient embryos. Embryos at the late blastula stage (stage 9.5) were homogenized and DNAs were extracted. DNAs were electrophoresed on 0.7% agarose gel stained with ethidium bromide and filmed with an UV-lamp. (A) DNA digested with Hind111 as size markers; (B) DNA from wild-type embryos; (C) DNA from ova-deficient embryos. M and Ch show the positions of mitochondrial and chromosomal DNAs.
[3H]alanine, and incorporation into the total acid-insoluble fraction of the cells was determined as above. The results showed that the inhibition in the incorporation in ova-deficient embryo cells was approximately 20% and 80% for the late blastula and late gastrula cells, respectively. Then, the synthesis of both DNA and protein appears to be nearly normal at the late blastula stage and starts to be inhibited only at the mid to late gastrula stage.
Discussion The female no. 65 used in the present experiment was found in a laboratory population of X. laevis, and its eggs were found to arrest development at the late gastrula stage. The eggs of female no. 65 have two unique features: (1) absence of an acidic protein (ca. 38 kDa), and (2) abnormal nuclear structure (Ikenishi and Tsuzaki, 1988). However, the physiological meaning of these unique features has not been clarified, although they are certainly assumed to be related to the gastrulation arrest.
In the present experiment, we studied the general features of the macromolecular synthesis with particular interest in the change in the RNA synthetic activity by using dissociated cells of both ova-deficient and wild-type embryos. It was found here that cells from ova-deficient embryos reaggregate quite actively at least until the late gastrula stage. The strong reaggregating activity observed in the dissociated cells of ova-deficient embryos provides evidence that the adhesion system may be normal in the ova-deficient embryos. Since the aggregates formed by the cells from the ova-deficient embryos were repeatedly dissociated and reaggregated by the treatment with EDTA-containing medium (data not shown), the reaggregation detected here must be due to the so-called Ca2+-dependent adhesion system. We have previously shown that the activity of early Xenopus Ca2+-dependent reaggregating activity is maintained by the continuous translation of maternal stock of mRNA for the adhesion molecules by the studies that utilized metabolic inhibitors (Shiokawa et al., 1983). Therefore, it appears that the ova-deficient eggs contain ample stock of such maternal mRNAs for Ca2+-dependent adhesion molecules, and their translational machinery is also normal. In the ova-deficient embryos, DNA and protein synthesis proceeded relatively normally until the late blastula stage, although these were severely damaged at the late gastrula stage. On the other hand, RNA synthesis was nearly normal until the early blastula stage, but was greatly inhibited at the late blastula stage. Thus, in the ova-deficient embryos, RNA synthesis appears to be the first that was damaged seriously prior to the gastrulation arrest. These results are comparable to the previous autoradiographic study in ova-deficient embryos of axolotl (A. mexicanum) (Carroll, 1974). It has been described that inhibition of RNA synthesis by cy-amanitin at the midblastula stage may lead to arrest at gastrulation even in wild-type embryos (Newport and Kirschner, 1982). Therefore, it appears that RNA synthesis that occurs between the midblastula and early gastrula stages is essential for the subsequent gastrulation movement. Occurrence of RNA species that is uniquely activated during these stages has been reported.
54
Thus, Krieg and Melton (1985) isolated a DNA clone (GS17) that is transcribed only during the early to mid gastrula stage and is then rapidly degraded after the gastrula stage. Therefore, it is interesting to test whether such unique RNA is activated in this ova-deficient mutant. Anyway, the large inhibition of RNA synthesis in our present ova-deficient embryos established by the late blastula stage could be the main cause for the observed arrest at the late gastrula stage. As noted above, Ikenishi and Tsuzaki (1988) showed that blastomeres of the ova-deficient embryos contain multiple small nuclei (karyomeres) and lack an acidic protein (ca. 38 kDa). Therefore, we assume that the inhibition of RNA synthesis observed may be a reflection of either the unusual nuclear conditions or the absence of the 38 kDa protein or both, although we still do not know how such unusual conditions could induce the observed inhibition in RNA synthesis.
Acknowledgements We thank Professor K. Yamana for his warm encouragement during the present study. The present work was supported, in part, by a Grant-inAid for Scientific Research (No. 61540523) to KS. from the Ministry of Education, Science and Culture of Japan and a grant from Takeda Science Foundation (1986) to K.S.
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various RNA species in developing embryos of Xenopus laeois. Dev. Biol. 68, 503-514 (1979). Shiokawa, K., Y. Misumi and K. Yamana: Demonstration of rRNA synthesis in pre-gastrular embryos of Xenopur loeuis. Dev. Growth Differ. 23, 579-587 (1981). Shiokawa, K., K. Tashiro, T. Oka and K. Yamana: Contribution of maternal mRNA for maintenance of Ca’+-dependent reaggregating activity in dissociated cells of Xenopw laevis embryos. Cell Differ. 13, 247-255 (1983). Shiokawa, K., Y. Kawazoe, H. Nomura, T. Miura, N. Nakakura, T. Horiuchi and K. Yamana: Ammonium ion as
a possible regulator of the commencement of rRNA synthesis in Xenopw laeuis embryogenesis. Dev. Biol. 115. 380-391 (1986). Tashiro, K., K. Shiokawa, K. Yamana and Y. Sakaki: Structural analysis of ribosomal DNA homologues in nucleolusless mutant of Xenopur laevis. Gene 44, 299-306 (1986a). Tashiro, K., M. Inoue, Y. Sakaki and K. Shiokawa: Preservation of Xenopus laeuis rDNA-containing plasmid, pXlrlOlA, injected into the fertilized egg of Xenopus laeuis. Cell Struct. Funct. 11, 109-114 (1986b).