Developmental Expression of Otoconin-22 in the Bullfrog Endolymphatic Sac and Inner Ear
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka, Japan
Correspondence to: Dr. Shigeyasu Tanaka, Dept. of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan. E-mail: sbstana{at}ipc.shizuoka.ac.jp
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Summary |
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Key Words: otoconin-22 endolymphatic sac inner ear developmental expression in situ hybridization immunohistochemistry bullfrog (Rana catesbeiana)
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Introduction |
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The inner ear emerges embryologically as the otic placode at both sides of the head by thickening of the epithelial ectoderm. This placode grows to form a pair of sacs and then divides into two parts, the utriculus and the sacculus.
In this study we examined expression of otoconin-22 during the development of the endolymphatic sac and inner ear by using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques.
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Materials and Methods |
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RT-PCR of Developing Embryos
The developmental expression of otoconin-22 mRNA was analyzed by RT-PCR. Using TRIZOL RNA extract reagent (Life Technologies; Rockville, MD), total RNA was prepared from each of 10 embryos at Shumway stage 18 to the early phase of stage 25. After treatment of 20 µg of total RNA with DNase (4 U; Takara, Kyoto, Japan), a 10-µg aliquot of the total RNA was reverse-transcribed in 20 µl of reaction buffer containing a 1-mM concentration of each dNTP, 9.9 U of RNase inhibitor (Toyobo; Osaka, Japan), and 7.5 mM oligo-dT(19) primer (Life Technologies) at 42C for 1 hr and then at 52C for 30 min. PCR was then performed basically by the same method as described previously (Yaoi et al. 2003a), using the following homologous primers: otoconin-22 sense, 5'-CTGTGACTCAGACATACCGCTTCTA-3' (247271 b); otoconin-22 antisense, 5'-CATCGCACTCACAGACCATCTT-3' (324345 b). Bullfrog ß-actin was used as an internal standard during detection of otoconin-22 mRNA expression. The ß-actin cDNA was amplified by using a set of primers designed to amplify a ß-actin fragment of 96 bp (Yaoi et al. 2003a
). The RT-PCR products were analyzed on a 2% agarose gel containing ethidium bromide (EtBr; 0.5 µg/ml) with Marker 6 (
/Sty1 digest; Wako Pure Chemicals, Osaka, Japan) molecular weight markers. The gels were transferred onto a nylon membrane (Roche Molecular Biochemicals; Meylan, France) and subjected to Southern blotting analysis using bullfrog otoconin-22 cDNA as the probe.
Antibody
The antibody used in this study was raised in rabbits and characterized as described previously: anti-bullfrog otoconin-22 (previously termed secretory phospholipase A2-like protein) against a synthetic peptide corresponding to N-terminal amino acids 113 (ST-135: TPAQFDEMIKVTT) of bullfrog otoconin-22 (Yaoi et al. 2001).
Dual mRNA and Protein Staining
DIG-labeled antisense and sense cRNA probes were prepared from the full coding region of otoconin-22 cDNA by in vitro transcription, as described previously (Saito et al. 2002). Bullfrog embryos from Shumway stage 19 to stage 25 (ca. 3 and 5 mm in head length) were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4, for 16 hr at 4C. After fixation, the embryos were dehydrated through a graded alcohol series, cleared in methyl benzoatecelloidin, and embedded in Paraplast. Sections were cut at 4-µm thickness and mounted on silane-coated slides. ISH was carried out according to a method described previously (Saito et al. 2002
). Briefly, deparaffinized sections were digested with 5 µg/ml proteinase K for 20 min, fixed in 4% PFA for 20 min, and then incubated with the DIG-labeled cRNA at 65C for 16 hr. After hybridization, the sections were treated with 1 µg/ml RNase solution for 30 min at 37C, washed with 0.2 x SSC at 50C for 2 hr, and then incubated with alkaline phosphatase-conjugated sheep anti-DIG Fab antibody (Roche) for 16 hr The label was detected with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP; Roche).
After the mRNA had been stained as described above, the sections were washed with PBS and incubated with rabbit anti-otoconin-22 serum (1:2000) for 16 hr, followed by Cy3-labeled donkey anti-rabbit IgG (1:400; Jackson Immunoresearch, West Grove, PA) for 2 hr. The sections were washed with PBS, then mounted in PermaFluor (Immunon; Pittsburgh, PA) and examined under an Olympus BX50 microscope equipped with a BX-epifluorescence attachment (Olympus Optical; Tokyo, Japan).
Immunofluorescence
The embryos [Shumway stage 25 (late phase)], tadpoles (TK stages IX, XXIII, and XXV), and young adults (1 month after metamorphosis) were fixed by immersion in BouinHollande solution for 2 days. After the tissues had been treated with 10% EDTA in water at 4C for 3 days to demineralize the calcium carbonate crystals, they were dehydrated through an ethanol series and embedded in Paraplast. Sections were cut at 4 µm and mounted on gelatin-coated slides. Immunohistochemistry was performed by the indirect immunofluorescence method. Deparaffinized sections were incubated sequentially at room temperature with the following reagents: 5% normal goat serum for 2 hr, rabbit anti-bullfrog otoconin-22 serum (1:2000) for 16 hr, and FITC-labeled donkey anti-rabbit IgG (1:400; Jackson) for 2 hr. The sections were finally washed with PBS and then mounted in PermaFluor (Immunon). The specificity of the immunostaining was also examined by a preabsorption test. The diluted antiserum was mixed with the antigen peptide at a final concentration of 10 µg/ml and preabsorbed for 12 hr at 4C before use in the specificity test.
Whole-mount ISH
The embryos of Shumway stage 25 (early phase, mid-phase, late phase, and last phase) were fixed with 4% PFA in 0.1 M phosphate buffer, pH 7.4, at 4C for 16 hr and dehydrated with a methanol series. After infiltration with absolute methanol for 16 hr, the specimens were hydrated through a decreased methanol series and then bleached with 6% H2O2 for 16 hr. They were incubated with 10 µg/ml proteinase K for 1 hr, fixed in 4% PFA for 20 min, and then incubated with the DIG-labeled cRNA at 65C for 16 hr. They were washed with solution 1 containing 50% formamide, 0.2% SSC, and 0.1% Tween-20 for 1 hr at 65C, treated with 20 µg/ml RNase for 30 min at 37C, washed again with solution 1 for 1 hr at 65C, and then incubated with alkaline phosphatase-conjugated sheep anti-DIG Fab antibody (Roche) for 16 hr. The label was detected with NBT/BCIP (Roche).
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Results |
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Discussion |
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The present RT-PCR analysis of the embryos from Shumway 18 to the early phase of stage 25 demonstrated that otoconin-22 mRNA began to be expressed from stage 20 and continued, peaking in the early phase of stage 25. However, we did not observe otoconin-22 mRNA in any cells of the embryos from stage 20 to stage 21 with ISH. Because RT-PCR allows one molecule of RNA to be amplified a billionfold, a tiny amount of otoconin-22 mRNA that is not detectable by ISH may be observed. Constant expression of otoconin-22 mRNA and transcription of the protein were seen in the embryos from stage 24. This is consistent with the time when the larvae begin to swim and eat. During the intake of nutrient from the yolk, it is not necessary to store calcium in the endolymphatic sac. It is believed that the sac stores calcium ingested in food that is eaten. We should also consider the other important role of otoconin-22 protein: the formation of otoconia in the inner ear. In the present study we showed that otoconia formed after the expression of otoconin-22. In embryos before stage 25, otoconin-22 was expressed only in the endolymphatic sac. However, whole-mount ISH analysis detected otoconin-22 mRNA throughout the inner ear after the mid-phase of stage 25. At the late phase of stage 25, when the otoconia have formed in the inner ear, an otoconin-22 mRNA-positive reaction was seen in the periphery of the otoconia. These results indicate that otoconin-22 mRNA, involved in the formation of otoconia in the inner ear, started to be expressed from the mid-phase of stage 25. Furthermore, in the present immunoflorescence study an otoconin-22-immunopositive reaction was observed in the epithelial cells of the inner ear. Therefore, otoconin-22 mRNA in the mid-phase of stage 25 is transcribed into the protein, thereby forming the otoconia in the inner ear.
Pote et al. (1993) identified otoconin-22, the major protein of aragonitic otoconia in the saccule of the inner ear of Xenopus. The otoconin-22 is considered to play a crucial role in depositing calcium carbonate. In amphibians, calcite is present in the utricle and aragonite in the saccule (Pote and Ross 1993
; Kido and Takahashi 1997
; Oukda et al. 1999
). In the present study we detected otoconin-22 protein in both the utricle and the saccule, which suggests that aragonite is also made in the utricle, although it is generally accepted that the utricle contains calcite. Identifying amphibian otoconin-90 protein, which is considered to be involved in calcite formation, would clarify the molecular mechanism of calcite formation.
The calcium carbonate crystals in the endolymphatic sac and paravertebrate lime sac are considered to be stores of calcium to be used in bone formation and in bone injury (Guardabassi 1960; Robertson 1969a
,b
). This means that there should be a decrease in the quantity of calcium carbonate crystals and degeneration of the endolymphatic sac during the bone formation that occurs during metamorphosis, but we did not detect this. The climactic stages are consistent with the time when ossification of cartilage begins and the tadpoles do not eat. In this period we did not observe any significant decrease in quantity of calcium carbonate crystals or degeneration of the endolymphatic sac. Observation indicated that the epithelial cells in the endolymphatic sac of bullfrog adults have more numerous secretory granules bearing otoconin-22 than those of tadpoles (data not shown). Therefore, it is conceivable that bullfrogs secrete larger amounts of otoconin-22 protein during growth, thereby forming calcium carbonate crystals in the lumen of the sac. Ultimobranchialectomy decreases the secretory activity of the epithelial cells and the quantity of crystals in the endolymphatic sac (Robertson, 1969a
,b
) and calcitonin increases the quantity of calcium carbonate crystals in anuran amphibians (Swarup and Krishna 1979
; Oguro et al. 1984
; Srivastav and Rani 1989
). Therefore, the endolymphatic sac, including the paravertebral lime sac. is important for maintaining calcium homeostasis.
Taken together, the findings of the present study demonstrated that otoconin-22 is expressed as metamorphosis progresses and that the expression pattern differs between the endolymphatic sac and the inner ear.
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Acknowledgments |
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Footnotes |
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Received for publication December 22, 2003; accepted January 16, 2004
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