Developmental Expression of Otoconin-22 in the Bullfrog Endolymphatic Sac and Inner Ear

Abstract

In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog (Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear.

Keywords

otoconin-22 endolymphatic sac inner ear developmental expression in situ hybridization immunohistochemistry

Organisms produce hard tissues in a process called biomineralization. Hard tissue includes bones, teeth, calculus, and otoliths, which are present in many vertebrates, in scales in fish, and in eggshell in birds. In invertebrates, examples of hard tissue are the shells of mollusks and diatoms, the ossicles of sea urchins, the exoskeletons of crustaceans, and the coccoliths of coccolithophriods. Recently, several cDNAs encoding proteins involved in the formation of ear stones have been cloned and sequenced, otoconin-90 (also called otoconin-95) in mammals (Wang et al. 1998; Verpy et al. 1999; Thalmann et al. 2001) and otolith matrix protein-1 (Murayama et al. 2000) and otolin-1 (Murayama et al. 2002) in teleosts. Analysis of the molecular mechanism of the ear stone formation has advanced rapidly. In most vertebrates the endolymphatic sac, which arises as the endolymphatic duct from the junction of the utriculus and the sacculus, terminates in a small blind-ending vesicle within the braincase, whereas the amphibian endolymphatic sac not only enlarges to form processes around the brain but also extends caudally along the vertebral canal and protrudes between the vertebrae, where it is referred to as the paravertebral lime sac. The lumen of these sacs contains many tiny crystals consisting of calcium carbonate, which are formed by otoconin-22 protein. In a previous study, we found that otoconin-22 protein is present in the endolymphatic sac surrounding the pituitary gland and the paravertebral lime sac in the bullfrog (Yaoi et al. 2001). Later, we cloned and sequenced a cDNA encoding bullfrog otoconin-22 and found that calcitonin regulates the expression of otoconin-22 mRNA in the endolymphatic sac, thereby stimulating the formation of calcium crystals in the lumen of the endolymphatic sac (Yaoi et al. 2003b). Furthermore, we noted that the inner ear expressed otoconin-22 mRNA but not calcitonin receptor mRNA, although the receptor mRNA was expressed in the endolymphatic sac. Consequently, otoconin-22 mRNA in the endolymphatic sacs is believed to be regulated by calcitonin via the calcitonin receptor. The regulatory mechanism of otoconin-22 in the inner ear may be different.

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.

Materials and Methods

Animal

Bullfrog (Rana catesbeiana) tadpoles at various developmental stages were purchased from Ouchi (Misato, Japan). The embryos and larvae were staged according to Shumway (1940) for embryonic stages and according to Taylor and Korllos (1946) for metamorphotic stages. The embryos of Shumway stage 25 were further divided into four phases according to head length: early phase (ca. 5 mm), mid-phase (ca. 7 mm), late phase (ca. 10 mm) and last phase (ca. 12 mm). The tadpoles were decapitated and prepared for RT-PCR, ISH, and immunofluorescence microscopy.

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′ (247–271 b); otoconin-22 antisense, 5′-CATCGCACTCACAGACCATCTT-3′ (324–345 b). Bullfrog β-actin was used as an internal standard during detection of otoconin-22 mRNA expression. The β-actin cDNA was amplified by using a set of primers designed to amplify a β-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 (λ/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 1–13 (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 × 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 Bouin-Hollande 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).

Results

Developmental Time Course of Otoconin-22 mRNA Expression Identified by RT-PCR

To investigate the developmental time course of otoconin-22 mRNA expression, we performed RT-PCR using total RNA from the embryos at Shumway stage 18 to 25 (early stage). A 99-bp band indicating the presence of otoconin-22 mRNA was detected in the embryos at Shumway stage 20 to stage 25. The otoconin-22 mRNA levels increased as development progressed (Figure 1). The RT-PCR result was confirmed by Southern blotting analysis (data not shown).

Figure 1

RT-PCR of otoconin-22 mRNA in the embryos from Shumway stage 18 to 25 (early stage). RT-PCR products using primers as described in Materials and Methods were separated on a 2% aga-rose gel and stained with ethidium bromide.

Expression of Otoconin-22 mRNA and Protein

To evaluate the relationship between mRNA and protein expression, we applied immunofluorescence staining with anti-otoconin-22 after ISH histochemistry using DIG-labeled otoconin-22 cRNA as the probe. No positive staining was obtained in the embryos from stage 19 to 21 (Figure 2a), although a positive reaction was detected at stages 20 and 21 by the RT-PCR method. In stage 22 embryos, the first otoconin-22 mRNA-positive reaction was observed at the basal side of the placode of the endolymphatic sac differentiated from the upper side of the utriculus (Figure 2b). The endolymphatic sac has not yet formed vesicular structures at stage 22. Otoconin-22 protein was not detected at the same stage but was first observed in the epithelial cells of the endolymphatic sac at stage 24 (Figure 2c). In stage 25 (early stage) embryos with a head length of 3 and 5 mm, the endolymphatic sac displayed follicular structures consisting of a single epithelium, which reacted strongly with the DIG-labeled cRNA probe and anti-otoconin-22 serum (Figures 2d and 2e). A positive reaction was observed in the apical cytoplasm of the epithelial cells, which is consistent with the results obtained in the adult (Yaoi et al. 2001).

Figure 2

Expression sites of otoconin-22 mRNA in bullfrog embryos. (a) Shumway stage 19; (b) stage 22; (c) stage 24; (d) stage 25 (early stage). Number “1” indicates detection of otoconin-22 mRNA by ISH, “2” indicates higher magnification of the corresponding figures, and “3” indicates expression of otoconin-22 protein, which has immunofluorescence images in the same section as “2.” Otoconin-22 mRNA is detected in the embryos from stage 22 and the otoconin-22 mRNA expression continues afterward (arrows), whereas otoconin-22 protein is observed at the embryos from stage 24 (arrowheads). OV, otic vesicle; ELS, endolymphatic sac. Bars: 1 = 100 μm; 2,3 = 10 μm.

Because otoconin-22 mRNA was not detected in the inner ear of the embryos at stage 22 to the early phase of stage 25 by the usual ISH, we decided to use whole-mount ISH, which is considered to have a higher sensitivity. This method demonstrated the presence of otoconin-22 mRNA in the inner ear. No positive reaction was seen in the early phase of stage 25 (Figure 3a). In the mid-phase of stage 25, otoconin-22 mRNA was detected in the inner ear (Figure 3b). By the late phase of stage 25, when ear stones had formed, otoconin-22 mRNA was detected in the inner ear (Figure 3c). Intense expression of otoconin-22 mRNA was observed in both the inner ear and the paravertebral lime sac in the last phase of stage 25, when the endolymphatic sac had enveloped the brain and had enlarged at the vertebrae (Figure 3d). No positive reaction was obtained with the cRNA sense probe (data not shown).

Figure 3

Expression sites of otoconin-22 mRNA during Shumway stage 25 detected by whole-mount ISH. Micrographs indicate a part of head region. (a) No positive reaction was seen in the embryos in the early phase of stage 25. (b) At mid-phase of stage 25, otoconin-22 mRNA was detected in the inner ear. (c) The late phase of stage 25 when otoconia form in the inner ear. (d) The last phase of stage 25: otoconin-22 mRNA is visible in the inner ear and in the endolymphatic sac, which can be seen along the vertebrae. Bars = 500 μm.

When expression of otoconin-22 protein was examined in the embryos after the late phase of stage 25 by the immunofluoresence technique, positive reactions were seen in the endolymphatic sac and the inner ear (data not shown). In the tadpoles at TK stages IX to XXV and metamorphosed frogs (ca. 1 month), expression of otoconin-22 protein was limited to the endolymphatic sac and inner ear (Figures 4a and 4b). The inner ear was composed of the utriculus, sacculus, and three semicircular canals. Positive reaction for otoconin-22 protein was observed in the epithelial cells of the utriculus and sacculus (Figure 4b1) and in the supporting cells composing the crista ampullaris, which connects the semicircular canals and utriculus (Figure 4b2). The epithelial cells of the paravertebral lime sac, which protrudes from both sides of the vertebrae in metamorphosed frogs, were also stained with anti-otoconin-22 (Figures 4c and 4d). There was no positive reaction with the preabsorbed antiserum (data not shown).

Figure 4

Expression of otoconin-22 protein in tadpoles at TK stage IX and young adult 1 month after complete of metamorphosis. The drawing of the tadpole and the frog show the sites of the sections in a and c. (a1) Immunofluorescence images in the vicinity of the spinal cord in the tadpole at TK Stage IX. An otoconin-22-immunopositive reaction is visible in the endolymphatic sac. (a2) Higher magnification of the boxed area (ELS) in a1. Arrows indicate otoconin-22-immunopositive reaction. (b1) Immunofluorescence images in the vicinity of the inner ear in the cross-section of the head. (b2) Higher magnification of the boxed area (ELS) in b1. An otoconin-22-immunopositive reaction (arrows) is observed in the supporting cells of the crista ampullaris. (c) Hematoxylin-eosin staining image in the vicinity of the spinal cord in a cross-section of the ventral part of the body. (d1) Immunofluorescence image of otoconin-22 protein corresponding to c. (d2) Higher magnification of the boxed area (PVLS) in d1. Arrows indicate otoconin-22-immunopositive reaction. (d3) Higher magnification of the boxed area (PVLS) in d1. An otoconin-22-immunopositive reaction is visible in the epithelial cells of the paravertebrate lime sac (arrows) and in otoconia present in the lumen (arrowheads). ELS, endolymphatic sac; PVLS, paravertebral lime sac. Bars: a1,b1,c,d1 = 500 μm; a2,b2,d2,d3 = 10 μm.

Discussion

Bullfrog embryos absorb nutrition from the yolks stored at the ventral side of the body until Shumway stage 24, and begin to swim actively at stage 25. Bullfrog larvae remain in stage 25 for a long time, growing from ∼1 cm to between 3 and 6 cm in body length without undergoing metamorphosis. Individual differences in growth during stage 25 are due to the time of birth and the environment. Larvae born later, and those that have delayed growth, spend the winter in the early phase of stage 25 or TK stages I-XIX. During the climactic stages, the larvae do not eat because remodeling of the gut is taking place.

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 billion-fold, 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.

Footnotes

Acknowledgements

Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (ST).

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