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Quantitative expression studies of aldolase A, B and C genes in developing embryos and adult tissues of Xenopus laevis
Mechanisms of Development 102 (2001) 283±287
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Gene expression pattern
Quantitative expression studies of aldolase A, B and C genes in developing embryos and adult tissues of Xenopus laevis Eri Kajita a, Junya Moriwaki a, Hitomi Yatsuki b, Katsuji Hori b, Kin-ichiro Miura c, Momoki Hirai d, Koichiro Shiokawa a,* a
Laboratory of Molecular Embryology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo ku, Tokyo 113-0033, Japan b Department of Biochemistry, Saga Medical School, 5-1-1 Nabeshima, Saga 849-0937, Japan c Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-0031, Japan d Department of Integrated Biosciences, Graduate School of Frontier Science, The University of Tokyo, Hongo 7-3-1, Bunkyo ku, Tokyo 113-0033, Japan Received 11 December 2000; received in revised form 6 February 2001; accepted 6 February 2001
Abstract We previously cloned cDNAs for all the members (A, B and C) of Xenopus aldolase gene family, and using in vitro transcribed RNAs as references, performed quantitative studies of the expression of three aldolase mRNAs in embryos and adult tissues. A Xenopus egg contains ca. 60 pg aldolase A mRNA and ca. 45 pg aldolase C mRNA, but contains only ca. 1.5 pg aldolase B mRNA. The percent composition of three aldolase mRNAs (A:B:C) changes from 56:1.5:42.5 (fertilized egg) to 54:10:36 (gastrula), to 71:14.5:14.5 (neurula) and to 73:20:7 (tadpole) during development. These results are compatible with the previous results of zymogram analysis that aldolases A and C are the major aldolases in early embryos, whose development proceeds depending on yolk as the only energy source. Aldolase B mRNA is expressed only late in development in tissues such as pronephros, liver rudiment and proctodeum which are necessary for the future dietary fructose metabolism, and the expression pattern is consistent to that in adult tissues. We also show that three aldolase genes are localized on different chromosomes as single copy genes. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aldolase A, B and C mRNAs; Absolute amounts; Embryo-type aldolase mRNA composition; Tissue speci®c expression; Chromosomal localization
Aldolase is a multilocus-type isozyme, whose functional form is either homo- or hetero-tetramer composed of two of the three different subunits (A, B and C) (Penhoet et al., 1967) coded for by different genes (Lebherz and Rutter, 1969; Horecker et al., 1972; Hori et al., 1987). Aldolase holoenzymes catalyze different reactions depending on the nature of the constituent subunits. Basically, aldolase A catalyses reversible cleavage of fructose-1, 6-bisphosphate in glycolysis (Horecker et al., 1972; Hers and Kusaka, 1953). Aldolase B catalyses cleavage of fructose-1-phosphate derived from dietary fructose (Hers and Kusaka, 1953; Munnich et al., 1985; Decaux et al., 1991; Rutter et al., 1968), and also catalyses gluconeogenesis (Decaux et al., 1991; Rutter et al., 1968). Aldolase C has the intermediate property, but is utilized mainly in the cleavage of fructose-1, 6-bisphosphate (Horecker et al., 1972). We report here the results of quantitative studies of the expression of
* Corresponding author. Tel./fax: 181-3-5841-4431. E-mail address: [email protected] (K. Shiokawa).
three aldolase mRNAs (A, B and C) in embryos and adult tissues.
1. Results and discussion We performed Northern blot analyses using different amounts of in vitro transcribed aldolase RNAs and RNAs extracted from embryos and adult tissues, and obtained calibration curves to correlate strengths of signals to amounts of aldolase RNAs (Fig. 1). We ®rst estimated the absolute amount of each of the three aldolase mRNAs in embryos at different stages (Fig. 2a). We found that a Xenopus fertilized egg contains approximately 60 pg of aldolase A mRNA, 1.5 pg of aldolase B mRNA and 45 pg of aldolase C mRNA. Thus, aldolase mRNAs constitute altogether ca. 0.0025% of the total egg RNA (ca. 4 mg of RNA), which is mostly rRNA (Brown and Littna, 1964; Shiokawa and Yamana, 1967). We then calculated the percent composition of three aldolase mRNAs in embryos at different stages (Fig. 2b). Aldolase A plus C mRNAs constituted 99% of the total
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00324-0
284
E. Kajita et al. / Mechanisms of Development 102 (2001) 283±287
bolism of dietary fructose which starts at the feeding tadpole stage. We examined the tissue-speci®c expression of three aldolase mRNAs using sectioned materials of tailbud embryos (Fig. 3). We detected the aldolase A mRNA signal very strongly in somites, and moderately strongly in heart anlage, pronephros, brain, and eye. Aldolase B mRNA signal occurred in pronephros, liver rudiment, proctodeum and epidermis. Aldolase C mRNA signal occurred in brain, retina, heart anlage, spinal cord and pronephros. Thus, already at the tailbud stage, aldolase A, B and C mRNAs are expressed in a dramatically tissue-speci®c manner. We then estimated absolute amounts of aldolase A, B and
Fig. 1. Calibration curves obtained from Northern blot analysis performed with in vitro transcribed aldolase RNAs and RNAs from embryos and adult tissues. In vitro transcribed aldolase A, B and C RNAs appeared as signals of ca. 1.5, 1.5 and 1.8 kb, respectively, and aldolase A, B and C mRNAs in embryos and adult tissues appeared as RNAs of similar sizes (insets). Strength of each signal was determined by denstometry in an image analyzer (BAS 2500). (a) A calibration curve for aldolase A mRNA. Five, 25, 50, 150 and 300 pg of in vitro transcribed aldolase A RNA, total RNA of one fertilized egg, and total ovary RNA (10 mg) were analyzed. (b) A calibration curve for aldolase B mRNA. One-half, 5, 50, 200, and 500 pg of in vitro transcribed aldolase B RNA, total RNA from one-half embryo at stage 35, and total liver RNA (10 mg) were analyzed. A stage 35 tadpole contained ca. 150 pg of aldolase B mRNA. (c) A calibration curve for aldolase C mRNA. Five, 25, 50, 150 and 300 pg of in vitro transcribed aldolase C RNA, total RNA of one fertilized egg, and total ovary RNA (10 mg) were analyzed.
aldolase mRNAs (A plus B plus C) from the cleavage to gastrula stage. The A plus C-type aldolase mRNA composition is probably necessary for the fructose metabolism in early embryos which develop solely depending on yolk. The accumulation of aldolase B mRNA takes place in post-gastrular embryos, and this is probably to prepare for the meta-
Fig. 2. Absolute amounts of aldolase A, B and C mRNAs in Xenopus laevis embryos during development. (a) The graph was drawn based on the results of Northern blot analyses of RNAs from embryos at different stages (data not shown, but see Kajita et al., 2000) and calibration curves obtained in Fig. 1. Two or three independent determinations were performed. From cleavage to the late gastrula stage the amount of aldolase A mRNA per embryo decreased from 60 to 15 pg, but it increased sharply thereafter to the level of 600 pg (at the tadpole stage). The period of this sharp increase corresponded to the period of somitegenesis. The amount of aldolase B mRNA (1.5 pg/egg) did not change until the midgastrula stage, but increased slightly at the late gastrula stage, then increased sharply at the tailbud stage to reach the level of 140 pg/embryo at the tadpole stage. The amount of aldolase C mRNA per embryo decreased from ca. 45 pg (egg) to ca. 3 pg (gastrula), and did not increase greatly in post-gastrular stages (50 pg/embryo even at the tadpole stage). (b) Circle graphs were drawn based on the data in (a). The difference in the size of the circle graph represents the difference in the total amount of the three aldolase mRNAs (A plus B plus C) at each stage.
E. Kajita et al. / Mechanisms of Development 102 (2001) 283±287
285
C mRNAs per unit amount (1 mg) of total RNA for each adult tissue (Fig. 4a). The total amount of three aldolase mRNAs is largest in muscles, and next largest in heart, which is reasonable in view of the high energy consumption in these tissues. We calculated here again the percent composition of three aldolase mRNAs (A, B and C) for each tissue (Fig. 4b). Aldolase A mRNA constituted 99% of the total aldolase mRNAs in muscles, whereas aldolase C mRNA constituted 70 and 60%, respectively, of total aldolase mRNAs in heart and brain. Thus, the percent composition of aldolase mRNAs in heart, a typical mesodermal tissue, is similar to that in brain, a typical neural tissue. Also, kidney, which is mesodermal in origin, has aldolase mRNA composition almost identical to that of liver (ca. 85% aldolase B), which is endodermal in origin. Thus, it is apparent here that each tissue has its characteristic aldolase mRNA composition, indifferently to its developmental origin. Finally, we prepared primary culture of Xenopus adult kidney cells and performed FISH to locate three aldolase genes on metaphase chromosomes. As shown in Fig. 5, all the three aldolase genes existed as a single copy gene on different chromosomes, a ®nding which seems to be reasonable in view of their completely different tissue-speci®c expression patterns in tailbud embryos and adult. 2. Materials and methods pXALDA, XALDB2, and pXALDC were linearized with XhoI and transcribed with T3 or T7 RNA polymerase to obtain aldolase A, B and C RNA, respectively (Kajita et al., 2000). RNAs were extracted from embryos and adult tissues of Xenopus laevis and subjected to Northern blot analysis (Sambrook et al., 1989) under high stringency conditions using speci®c probes (Kajita et al., 2000). Fig. 3. Spatial expression patterns of aldolase A, B and C mRNAs examined in the wild-type Xenopus embryo at the tailbud stage (stage 35) by wholemount in situ hybridization. Probes for aldolase A, B and C mRNA were, respectively, the HindIII fragment of XALDA (Hikasa et al., 1997), the A¯II-XhoI fragment of XALDB2 (Kajita et al., 2000), and the SacI-KpnI fragment of XALDC (Atsuchi et al., 1994), which did not cross-hybridize to each other (Kajita et al., 2000). All the signals were obtained with antisense probe, but not with the sense probe (data omitted). (a) An embryo hybridized with the XALDA antisense probe. Yellow lines indicate regions (b±d) where embryos were sectioned. h: heart anlage; p: pronephros, s: somites. In (b): b, brain; e, ear vasicle. In (c): s, somites; p, pronephros. In (d), somites are defferentially stained in dermatome (s (der)) and myotome (m (der)). (e) An embryo was ®xed, cut into halves, and only the anterior half was hybridized with the XALDA antisense probe. Note the absence of difference in the staining between dermatome and myotome, indicating that the weaker staining in myotome in (d) is simply due to the lesser accessibility of the probe to it. (f) An embryo hybridized with the XALDB antisense probe. Yellow lines indicate regions (g±j), where embryos were sectioned. In (f±j): lr, liver rudiment; p, pronephros; ep, epidermis; pr, proctodeum. (k) An embryo hybridized with the XALDC antisense probe. Yellow lines indicate regions (l±n) where embryos were sectioned. In (k±n): br, brain; h, heart; p, pronephros; sc, spinal cord; rt, retina.
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Fig. 4. Amounts of aldolase A, B and C mRNAs expressed in different Xenopus adult tissues. (a) The bar graph was drawn based on the results of Northern blot analysis of RNAs from various tissues (not shown, but see Kajita et al., 2000) and the calibration curves shown in Fig. 1. Note that keletal muscles expressed almost exclusively aldolase A mRNA, whereas cardiac muscles expressed much more aldolase C mRNA than aldolase A mRNA. Aldolase B mRNA constituted the major aldolase mRNA in liver and kidney, and also in stomach and intestine, which are necessary for dietary fructose metabolism (Munnich et al., 1985). (b) Circle graphs showing the percent composition and relative amount of three aldolase mRNAs in different tissues, which were drawn based on the data in (a). The difference in the size of each circle graph represents the difference in the total amount of three aldolase mRNAs (A plus B plus C) per unit amount (1 mg) of the total RNA, which is nearly equal to rRNA.
Whole-mount in situ hybridization was performed as in Kajita et al. (2000). Probes for aldolase A and C mRNAs were obtained as described (Kajita et al., 2000), and the probe for aldolase B mRNA was obtained from pXALDB2-3 0 (Kajita et al., 2000). Embryos were stained as in Hemmati-Brivanlou et al. (1990) Hemmato-Brivanlou et al. (1990), dehydrated, embedded in paraf®n, and sectioned at 8 mm. Fluorescence in situ hybridization (FISH) was performed as in Hirai et al. (1994). XALDA, XALDB2 and XALDC
Fig. 5. Chromosomal localization of Xenopus aldolase genes. We examined at least 30 metaphase cells with clear twin-spot hybridization signals for each cDNA clone. There were 36 chromosomes in each nucleus. Top (a): a metaphase plate showing doublet signals (arrows) of aldolase A gene on the telomeric region of the long arms of a pair of acrocentric chromosomes. Middle (c): a metaphase plate showing doublet signals (arrows) of aldolase C gene on the middle portion of the short arms of a pair of large submetacentric chromosomes. Bottom (b): a metaphase plate showing doublet signals (arrow) of aldolase B gene on the short arm of a large submetacentric chromosome, which was con®rmed by band pattern to be different from the chromosome which carries aldolase C gene. In the present data, we did not denote the number to each chromosome, since karyotype of Xenopus laevis has not yet been internationally standardized.
E. Kajita et al. / Mechanisms of Development 102 (2001) 283±287
(Kajita et al., 2000) were labeled with biotin-14-dATP by nick-translation and hybridized overnight to metaphase chromosomes in primarily-cultured Xenopus kidney cells. Signals were ampli®ed using rabbit antibiotin (Enzo, NY) and ¯uorescein-labeled goat anti-rabbit IgG (Enzo, NY). Chromosomes were counterstained with propidium iodide. References Atsuchi, Y., Yamana, K., Yatsuki, H., Hori, K., Ueda, S., Shiokawa, K., 1994. Cloning of a brain-type aldolase cDNA and changes in its mRNA level during oogenesis and early embryogenesis in Xenopus laevis. Biochem. Biophys. Acta 1218, 153±157. Brown, D.D., Littna, E., 1964. RNA synthesis during the development of Xenopus laevis, the south african clawed toad. J. Mol. Biol. 8, 669±687. Decaux, J.F., Marcillat, O., Pichard, A.L., Henry, J., Kahn, A., 1991. Glucose-dependent and -independent effect of insulin on gene expression. J. Biol. Chem. 266, 3432±3438. Hemmati-Brivanlou, A., Frank, D., Bolce, M.E., Brown, B.D., Sive, H.L., Harland, R.M., 1990. Localization of speci®c mRNAs in Xenopus embryos by whole-mount in situ hybridization. Development 110, 325±330. Hers, H.G., Kusaka, T., 1953. Le metabolisme du fructose 1-phosphate dans le foie. Biochem. Biophys. Acta 11, 427±430. Hikasa, H., Hori, K., Shiokawa, K., 1997. Structure of aldolase A (muscletype) cDNA and its regulated expression in oocytes, embryos and adult tissues of Xenopus laevis. Biochem. Biophys. Acta 1354, 189±203. Hirai, M., Suto, Y., Kanoh, M., 1994. A method for simultaneous detection
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of ¯uorescent G-bands and in situ hybridization signals. Cytogenet. Cell Genet. 66, 149±151. Horecker, B.L., Tsola, O., Lai, C.Y., 1972. Aldolases. In: Boyer, P.D. (Ed.). The Enzymes, Vol. 7. Academic Press, New York, pp. 213±258. Hori, K., Mukai, T., Joh, K., Arai, Y., Sakakibara, M., Yatsuki, H., 1987. Structure and expression of human and rat aldolase isozymes Genes: multiple mRNA species of aldolase A produced from a single gene, in Isozymes. Cur. Topics Biol. Med. Res. 14, 153±175. Kajita, E., Wakiyama, M., Miura, K., Mizumoto, K., Oka, T., Komuro, I., Miyata, T., Yatsuki, H., Hori, K., Shiokawa, K., 2000. Isolation and characterization of Xenopus laevis aldolase B cDNA and expression patterns of aldolase A, B and C genes in adult tissues, oocytes and embryos of Xenopus laevis. Biochem. Biophys. Acta 1493, 101±118. Lebherz, H.G., Rutter, W.J., 1969. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry 8, 109±121. Munnich, A., Besmond, C., Darguy, S., Reach, G., Valmont, S., Dreyfus, J.C., Kahn, A., 1985. Dietary and hormonal regulation of aldolase B gene expression. J. Clin. Invest. 75, 1045±1052. Penhoet, E.E., Kochman, M., Valentine, R., Rutter, W.J., 1967. The subunit structure of mammalian fructose diphosphate aldolase. Biochemistry 6, 2940±2949. Rutter, W.J., Rajkumar, T., Penhoet, E.E., Kochman, M., Valentine, R., 1968. Aldolase variants: structure and physiological signi®cance. Ann. N.Y. Acad. Sci. 151, 102±117. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Extraction, puri®cation, and analysis of messenger RNA from eucaryotic cells. In: Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, New York. Shiokawa, K., Yamana, K., 1967. Pattern of RNA synthesis in isolated cells of Xenopus laevis embryos. Dev. Biol. 16, 368±388.
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Gene expression pattern
Quantitative expression studies of aldolase A, B and C genes in developing embryos and adult tissues of Xenopus laevis Eri Kajita a, Junya Moriwaki a, Hitomi Yatsuki b, Katsuji Hori b, Kin-ichiro Miura c, Momoki Hirai d, Koichiro Shiokawa a,* a
Laboratory of Molecular Embryology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo ku, Tokyo 113-0033, Japan b Department of Biochemistry, Saga Medical School, 5-1-1 Nabeshima, Saga 849-0937, Japan c Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-0031, Japan d Department of Integrated Biosciences, Graduate School of Frontier Science, The University of Tokyo, Hongo 7-3-1, Bunkyo ku, Tokyo 113-0033, Japan Received 11 December 2000; received in revised form 6 February 2001; accepted 6 February 2001
Abstract We previously cloned cDNAs for all the members (A, B and C) of Xenopus aldolase gene family, and using in vitro transcribed RNAs as references, performed quantitative studies of the expression of three aldolase mRNAs in embryos and adult tissues. A Xenopus egg contains ca. 60 pg aldolase A mRNA and ca. 45 pg aldolase C mRNA, but contains only ca. 1.5 pg aldolase B mRNA. The percent composition of three aldolase mRNAs (A:B:C) changes from 56:1.5:42.5 (fertilized egg) to 54:10:36 (gastrula), to 71:14.5:14.5 (neurula) and to 73:20:7 (tadpole) during development. These results are compatible with the previous results of zymogram analysis that aldolases A and C are the major aldolases in early embryos, whose development proceeds depending on yolk as the only energy source. Aldolase B mRNA is expressed only late in development in tissues such as pronephros, liver rudiment and proctodeum which are necessary for the future dietary fructose metabolism, and the expression pattern is consistent to that in adult tissues. We also show that three aldolase genes are localized on different chromosomes as single copy genes. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aldolase A, B and C mRNAs; Absolute amounts; Embryo-type aldolase mRNA composition; Tissue speci®c expression; Chromosomal localization
Aldolase is a multilocus-type isozyme, whose functional form is either homo- or hetero-tetramer composed of two of the three different subunits (A, B and C) (Penhoet et al., 1967) coded for by different genes (Lebherz and Rutter, 1969; Horecker et al., 1972; Hori et al., 1987). Aldolase holoenzymes catalyze different reactions depending on the nature of the constituent subunits. Basically, aldolase A catalyses reversible cleavage of fructose-1, 6-bisphosphate in glycolysis (Horecker et al., 1972; Hers and Kusaka, 1953). Aldolase B catalyses cleavage of fructose-1-phosphate derived from dietary fructose (Hers and Kusaka, 1953; Munnich et al., 1985; Decaux et al., 1991; Rutter et al., 1968), and also catalyses gluconeogenesis (Decaux et al., 1991; Rutter et al., 1968). Aldolase C has the intermediate property, but is utilized mainly in the cleavage of fructose-1, 6-bisphosphate (Horecker et al., 1972). We report here the results of quantitative studies of the expression of
* Corresponding author. Tel./fax: 181-3-5841-4431. E-mail address: [email protected] (K. Shiokawa).
three aldolase mRNAs (A, B and C) in embryos and adult tissues.
1. Results and discussion We performed Northern blot analyses using different amounts of in vitro transcribed aldolase RNAs and RNAs extracted from embryos and adult tissues, and obtained calibration curves to correlate strengths of signals to amounts of aldolase RNAs (Fig. 1). We ®rst estimated the absolute amount of each of the three aldolase mRNAs in embryos at different stages (Fig. 2a). We found that a Xenopus fertilized egg contains approximately 60 pg of aldolase A mRNA, 1.5 pg of aldolase B mRNA and 45 pg of aldolase C mRNA. Thus, aldolase mRNAs constitute altogether ca. 0.0025% of the total egg RNA (ca. 4 mg of RNA), which is mostly rRNA (Brown and Littna, 1964; Shiokawa and Yamana, 1967). We then calculated the percent composition of three aldolase mRNAs in embryos at different stages (Fig. 2b). Aldolase A plus C mRNAs constituted 99% of the total
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00324-0
284
E. Kajita et al. / Mechanisms of Development 102 (2001) 283±287
bolism of dietary fructose which starts at the feeding tadpole stage. We examined the tissue-speci®c expression of three aldolase mRNAs using sectioned materials of tailbud embryos (Fig. 3). We detected the aldolase A mRNA signal very strongly in somites, and moderately strongly in heart anlage, pronephros, brain, and eye. Aldolase B mRNA signal occurred in pronephros, liver rudiment, proctodeum and epidermis. Aldolase C mRNA signal occurred in brain, retina, heart anlage, spinal cord and pronephros. Thus, already at the tailbud stage, aldolase A, B and C mRNAs are expressed in a dramatically tissue-speci®c manner. We then estimated absolute amounts of aldolase A, B and
Fig. 1. Calibration curves obtained from Northern blot analysis performed with in vitro transcribed aldolase RNAs and RNAs from embryos and adult tissues. In vitro transcribed aldolase A, B and C RNAs appeared as signals of ca. 1.5, 1.5 and 1.8 kb, respectively, and aldolase A, B and C mRNAs in embryos and adult tissues appeared as RNAs of similar sizes (insets). Strength of each signal was determined by denstometry in an image analyzer (BAS 2500). (a) A calibration curve for aldolase A mRNA. Five, 25, 50, 150 and 300 pg of in vitro transcribed aldolase A RNA, total RNA of one fertilized egg, and total ovary RNA (10 mg) were analyzed. (b) A calibration curve for aldolase B mRNA. One-half, 5, 50, 200, and 500 pg of in vitro transcribed aldolase B RNA, total RNA from one-half embryo at stage 35, and total liver RNA (10 mg) were analyzed. A stage 35 tadpole contained ca. 150 pg of aldolase B mRNA. (c) A calibration curve for aldolase C mRNA. Five, 25, 50, 150 and 300 pg of in vitro transcribed aldolase C RNA, total RNA of one fertilized egg, and total ovary RNA (10 mg) were analyzed.
aldolase mRNAs (A plus B plus C) from the cleavage to gastrula stage. The A plus C-type aldolase mRNA composition is probably necessary for the fructose metabolism in early embryos which develop solely depending on yolk. The accumulation of aldolase B mRNA takes place in post-gastrular embryos, and this is probably to prepare for the meta-
Fig. 2. Absolute amounts of aldolase A, B and C mRNAs in Xenopus laevis embryos during development. (a) The graph was drawn based on the results of Northern blot analyses of RNAs from embryos at different stages (data not shown, but see Kajita et al., 2000) and calibration curves obtained in Fig. 1. Two or three independent determinations were performed. From cleavage to the late gastrula stage the amount of aldolase A mRNA per embryo decreased from 60 to 15 pg, but it increased sharply thereafter to the level of 600 pg (at the tadpole stage). The period of this sharp increase corresponded to the period of somitegenesis. The amount of aldolase B mRNA (1.5 pg/egg) did not change until the midgastrula stage, but increased slightly at the late gastrula stage, then increased sharply at the tailbud stage to reach the level of 140 pg/embryo at the tadpole stage. The amount of aldolase C mRNA per embryo decreased from ca. 45 pg (egg) to ca. 3 pg (gastrula), and did not increase greatly in post-gastrular stages (50 pg/embryo even at the tadpole stage). (b) Circle graphs were drawn based on the data in (a). The difference in the size of the circle graph represents the difference in the total amount of the three aldolase mRNAs (A plus B plus C) at each stage.
E. Kajita et al. / Mechanisms of Development 102 (2001) 283±287
285
C mRNAs per unit amount (1 mg) of total RNA for each adult tissue (Fig. 4a). The total amount of three aldolase mRNAs is largest in muscles, and next largest in heart, which is reasonable in view of the high energy consumption in these tissues. We calculated here again the percent composition of three aldolase mRNAs (A, B and C) for each tissue (Fig. 4b). Aldolase A mRNA constituted 99% of the total aldolase mRNAs in muscles, whereas aldolase C mRNA constituted 70 and 60%, respectively, of total aldolase mRNAs in heart and brain. Thus, the percent composition of aldolase mRNAs in heart, a typical mesodermal tissue, is similar to that in brain, a typical neural tissue. Also, kidney, which is mesodermal in origin, has aldolase mRNA composition almost identical to that of liver (ca. 85% aldolase B), which is endodermal in origin. Thus, it is apparent here that each tissue has its characteristic aldolase mRNA composition, indifferently to its developmental origin. Finally, we prepared primary culture of Xenopus adult kidney cells and performed FISH to locate three aldolase genes on metaphase chromosomes. As shown in Fig. 5, all the three aldolase genes existed as a single copy gene on different chromosomes, a ®nding which seems to be reasonable in view of their completely different tissue-speci®c expression patterns in tailbud embryos and adult. 2. Materials and methods pXALDA, XALDB2, and pXALDC were linearized with XhoI and transcribed with T3 or T7 RNA polymerase to obtain aldolase A, B and C RNA, respectively (Kajita et al., 2000). RNAs were extracted from embryos and adult tissues of Xenopus laevis and subjected to Northern blot analysis (Sambrook et al., 1989) under high stringency conditions using speci®c probes (Kajita et al., 2000). Fig. 3. Spatial expression patterns of aldolase A, B and C mRNAs examined in the wild-type Xenopus embryo at the tailbud stage (stage 35) by wholemount in situ hybridization. Probes for aldolase A, B and C mRNA were, respectively, the HindIII fragment of XALDA (Hikasa et al., 1997), the A¯II-XhoI fragment of XALDB2 (Kajita et al., 2000), and the SacI-KpnI fragment of XALDC (Atsuchi et al., 1994), which did not cross-hybridize to each other (Kajita et al., 2000). All the signals were obtained with antisense probe, but not with the sense probe (data omitted). (a) An embryo hybridized with the XALDA antisense probe. Yellow lines indicate regions (b±d) where embryos were sectioned. h: heart anlage; p: pronephros, s: somites. In (b): b, brain; e, ear vasicle. In (c): s, somites; p, pronephros. In (d), somites are defferentially stained in dermatome (s (der)) and myotome (m (der)). (e) An embryo was ®xed, cut into halves, and only the anterior half was hybridized with the XALDA antisense probe. Note the absence of difference in the staining between dermatome and myotome, indicating that the weaker staining in myotome in (d) is simply due to the lesser accessibility of the probe to it. (f) An embryo hybridized with the XALDB antisense probe. Yellow lines indicate regions (g±j), where embryos were sectioned. In (f±j): lr, liver rudiment; p, pronephros; ep, epidermis; pr, proctodeum. (k) An embryo hybridized with the XALDC antisense probe. Yellow lines indicate regions (l±n) where embryos were sectioned. In (k±n): br, brain; h, heart; p, pronephros; sc, spinal cord; rt, retina.
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Fig. 4. Amounts of aldolase A, B and C mRNAs expressed in different Xenopus adult tissues. (a) The bar graph was drawn based on the results of Northern blot analysis of RNAs from various tissues (not shown, but see Kajita et al., 2000) and the calibration curves shown in Fig. 1. Note that keletal muscles expressed almost exclusively aldolase A mRNA, whereas cardiac muscles expressed much more aldolase C mRNA than aldolase A mRNA. Aldolase B mRNA constituted the major aldolase mRNA in liver and kidney, and also in stomach and intestine, which are necessary for dietary fructose metabolism (Munnich et al., 1985). (b) Circle graphs showing the percent composition and relative amount of three aldolase mRNAs in different tissues, which were drawn based on the data in (a). The difference in the size of each circle graph represents the difference in the total amount of three aldolase mRNAs (A plus B plus C) per unit amount (1 mg) of the total RNA, which is nearly equal to rRNA.
Whole-mount in situ hybridization was performed as in Kajita et al. (2000). Probes for aldolase A and C mRNAs were obtained as described (Kajita et al., 2000), and the probe for aldolase B mRNA was obtained from pXALDB2-3 0 (Kajita et al., 2000). Embryos were stained as in Hemmati-Brivanlou et al. (1990) Hemmato-Brivanlou et al. (1990), dehydrated, embedded in paraf®n, and sectioned at 8 mm. Fluorescence in situ hybridization (FISH) was performed as in Hirai et al. (1994). XALDA, XALDB2 and XALDC
Fig. 5. Chromosomal localization of Xenopus aldolase genes. We examined at least 30 metaphase cells with clear twin-spot hybridization signals for each cDNA clone. There were 36 chromosomes in each nucleus. Top (a): a metaphase plate showing doublet signals (arrows) of aldolase A gene on the telomeric region of the long arms of a pair of acrocentric chromosomes. Middle (c): a metaphase plate showing doublet signals (arrows) of aldolase C gene on the middle portion of the short arms of a pair of large submetacentric chromosomes. Bottom (b): a metaphase plate showing doublet signals (arrow) of aldolase B gene on the short arm of a large submetacentric chromosome, which was con®rmed by band pattern to be different from the chromosome which carries aldolase C gene. In the present data, we did not denote the number to each chromosome, since karyotype of Xenopus laevis has not yet been internationally standardized.
E. Kajita et al. / Mechanisms of Development 102 (2001) 283±287
(Kajita et al., 2000) were labeled with biotin-14-dATP by nick-translation and hybridized overnight to metaphase chromosomes in primarily-cultured Xenopus kidney cells. Signals were ampli®ed using rabbit antibiotin (Enzo, NY) and ¯uorescein-labeled goat anti-rabbit IgG (Enzo, NY). Chromosomes were counterstained with propidium iodide. References Atsuchi, Y., Yamana, K., Yatsuki, H., Hori, K., Ueda, S., Shiokawa, K., 1994. Cloning of a brain-type aldolase cDNA and changes in its mRNA level during oogenesis and early embryogenesis in Xenopus laevis. Biochem. Biophys. Acta 1218, 153±157. Brown, D.D., Littna, E., 1964. RNA synthesis during the development of Xenopus laevis, the south african clawed toad. J. Mol. Biol. 8, 669±687. Decaux, J.F., Marcillat, O., Pichard, A.L., Henry, J., Kahn, A., 1991. Glucose-dependent and -independent effect of insulin on gene expression. J. Biol. Chem. 266, 3432±3438. Hemmati-Brivanlou, A., Frank, D., Bolce, M.E., Brown, B.D., Sive, H.L., Harland, R.M., 1990. Localization of speci®c mRNAs in Xenopus embryos by whole-mount in situ hybridization. Development 110, 325±330. Hers, H.G., Kusaka, T., 1953. Le metabolisme du fructose 1-phosphate dans le foie. Biochem. Biophys. Acta 11, 427±430. Hikasa, H., Hori, K., Shiokawa, K., 1997. Structure of aldolase A (muscletype) cDNA and its regulated expression in oocytes, embryos and adult tissues of Xenopus laevis. Biochem. Biophys. Acta 1354, 189±203. Hirai, M., Suto, Y., Kanoh, M., 1994. A method for simultaneous detection
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