Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OlGC3, OlGC4, OlGC5, and OlGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OlGCS-α1, OlGCS-α2, and OlGCS-β1), neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OlGC3 and OlGC5 mRNAs were expressed in the proximal part and the OlGC4 and OlGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OlGCS-α2 and OlGCS-β1) of two soluble GC subunits (α2 and β1) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GC α1 subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (α2/β1 heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas.
In the photoreceptor cells of the vertebrate retina, cGMP plays an important role. The decrease of the cGMP level due to hydrolysis by phosphodiesterase (PDE) triggers the signal transduction from photoreception to neuronal signal output (Koch et al., 2002). The decreased cGMP level is restored to its initial level by the activation of guanylyl cyclases (GCs), which are enzymes that convert GTP to cGMP. GCs in living organisms are grouped into two major forms, those found membrane-bound in the plasma (membrane GCs) and those found in the cytoplasm (soluble GCs) (Lucas et al., 2000).
The membrane GCs responsible for phototransduction have been obtained from human (Shyjan et al., 1992; Lowe et al., 1995), rat (Yang et al., 1995), bovine (Goraczniak et al., 1994, 1997) and medaka fish retinas (Seimiya et al., 1997; Hisatomi et al., 1999). From a retinal cDNA library of the medaka fish Oryzias latipes, Seimiya et al. (1997) and Hisatomi et al. (1999) each independently isolated cDNA clones for a set of three membrane GCs, and named them OlGC3, OlGC4 and OlGC5, and OlGC-R1, OlGC-R2 and OlGC-C, respectively. A comparison of the nucleotide and deduced amino acid sequences of these six cDNAs indicates that OlGC-R1 and OlGC-C correspond to OlGC4 and OlGC5, respectively. Since OlGC-R2 is distinct from OlGC3, OlGC4 and OlGC5, it is assumed that four different membrane GCs, namely OlGC3, OlGC4 (OlGC-R1), OlGC5 (OlGC-C), and OlGC-R2, are expressed in the medaka fish retina (Kusakabe and Suzuki, 2000a).
Soluble GC is a heme-containing heterodimer composed of α and β subunits (Kamisaki et al., 1986). Soluble GC is activated primarily by nitric oxide (NO) generated from L-arginine by nitric oxide synthase (NOS) (Denninger and Marletta, 1999; Alderton, et al., 2001; Bellamy and Garthwaite, 2002), which is grouped into three subfamilies: neural NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) (Hall et al., 1994; Chartrain et al., 1994; Marsden et al., 1993). Two α (α1 and α2) and two β (β1 and β2) subunits of the soluble GC mRNAs are found in mammals (Wedel and Garbers, 1997; Lucas et al., 2000). In the medaka fish, mRNAs for soluble GC α1 and β1 subunits have been isolated and named OlGCS-α1 and OlGCS-β1, respectively (Mikami et al., 1998). The primary structure of the medaka fish α1 and β1 subunits is closely related to the structure of the respective mammalian subunits. Recently, we also obtained the α2 subunit gene (OlGCS-α2) of medaka fish soluble GC (Yao, Y., Yamamoto, T. and Suzuki, N., unpublished data). Several papers have demonstrated on the basis of immunohistochemistry and GC activation induced by NOS that some of the soluble GC subunits exist in the mammalian retina (Haberecht et al., 1998; Margulis et al., 1998; Sitaramayya, 2002). However, as far as we know, no identification of the type of the soluble GC subunit expressed in the retina has been performed yet. In addition, none of the papers dealing with the expression of the soluble GC subunit mRNA in the teleost retina have been published yet.
It has been reported that the NO/cGMP signaling pathway comprising NOS, soluble GC, cGMP-dependent protein kinase (cGK), and PDE plays an important role in smooth muscle contraction in the vascular system and in immune response (Lucas et al., 2000). In a study, we obtained a cDNA fragment of nNOS by RT-PCR from the whole body of a medaka fish (Yamamoto et al., 2003). Downstream of the cGMP signaling pathway, cGK functions similarly to cAMP-dependent protein kinase in the cAMP signaling pathway. Two distinct isoforms (cGK I and cGK II) of cGK have been identified in mammals (Wernet et al., 1989; Tamura et al., 1996; Ørstavik et al., 1996). Recently, we also obtained cDNA fragments of the medaka fish cGK I and II (Yamamoto et al., 2003). In the present study, we examined the localization of mRNAs for four membrane GCs, three soluble GC subunits, nNOS, and cGK I in the adult and embryonic retinas of the medaka fish Oryzias latipes by in situ hybridization (ISH). Here, we report that OlGC3 and OlGC5 are expressed in the proximal part of the adult and embryonic retinas and OlGC4 and OlGC-R2 are expressed in the outer nuclear layer of the adult and embryonic retinas, while OlGCS-α2 and OlGCS-β1 are expressed mainly in the inner nuclear layer and ganglion cell layer of the embryonic retina.
MATERIALS AND METHODS
Animals and embryos
Adult individuals and embryos of the orange-red variety of the medaka fish O. latipes were purchased from a dealer. They were kept in indoor tanks under artificial reproductive conditions (14 hr light, 10 hr dark, 27°C) and fed on Otohime B2 (Nisshin Seifun Group Inc., Tokyo, Japan). Naturally spawned and fertilized eggs were collected, and the embryos were cultured in distilled water containing 0.6 ppm methylene blue at 27°C. The developmental stage of the embryos was expressed in days, with the day of fertilization referred to as day 0.
The eyes of embryonic and adult medaka fish were fixed with 4% paraformaldehyde in 70 mM phosphate buffered saline (PBS), pH 7.3. After being rinsed in PBS, the samples were treated with 0.1% diethylenepyrocarbonate (DEPC) overnight at 4°C, and then left in DEPC-treated 30% sucrose-PBS at 4°C. The tissue was sliced into 15 μm-sections on a cryostat microtorm (LEICA, CM3000) and mounted on a glass slide coated with poly-L-lysin (Sigma).
Isolation of cDNA fragment encoding medaka fish nNOS and cGK-I
The medaka fish nNOS and cGK-I cDNA fragments were obtained by RT-PCR using degenerate primers designed using zebrafish and salmon sequences (Holmqvist et al., 2000; Øyan et al., 2000; Yamamoto et al., 2002). The primer pairs used for the first PCR were: nNOS, 5′-CCYGTBTTCCAYCAGGAGATG-3′ and 5′-RAAGGCRCARAASTGRGGGTA-3′. The primer pairs used for nested PCR were as follows: nNOS, 5′-CAGGAGATGCTCAACTATC-3′ and 5′-TCCAGTGCTCTCGAAGTTG-3′; cGK-I, 5′-ATCATCGACACCTTTGGAGTTGG-3′ and 5′-CACATTGTAAATGCTTTATCCAGAG-3′. The amplified PCR products of nNOS (502 bp) and cGK-I (1145 bp) were purified and subcloned into the plasmid vector pBluescript II KS (+) (Stratagene).
In situ hybridization (ISH)
The pBluescript II vector containing a cDNA fragment for the coding region of OlGC3 (nucleotides 431–1257), OlGC4 (nucleotides 5–1811), OlGC5 (nucleotides 283-1071), OlGC-R2 (nucleotides 1–1389), OlGCS-α1 (nucleotides 195–953), OlGCS-α2 (nucleotide 1356-2894), OlGCS-β1 (nucleotides 252-953), nNOS (nucleotides 1–502 ) or cGK I (nucleotides 1–1145) was used as a template to generate a digoxigenin-labeled cRNA probe using a DIG RNA Labeling Kit (Boehringer Mannheim) according to the manufacturer's protocol (Yamamoto et al., 2003).
ISH was carried out according to the protocol described by Yoshida et al. (1994) with the following modifications. The section was postfixed with 0.1 M phosphate buffer (PB, pH 7.4) containing 4% formalin for 20 min. The fixed section was treated with 10 μg/ml proteinase K (Boehringer Mannheim) in TE, and fixed again with PB containing 4% formalin. The acetylation of the section was carried out with 0.1 M triethanolamine containing 0.25% acetic acid. After being rinsed in PB, the section was treated with ethanol and chloroform. Prehybridization was carried out at 55°C for 1 hr in a hybridization buffer containing 50% formamide, 20 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 10% dextran sulfate, 0.2% sarcosyl, 0.02% salmon sperm DNA, and 1xDenhardt's solution. After an overnight hybridization with about 2 ng/μl antisense or sense probe in the hybridization buffer at 55°C, the section was washed with 4xSSC at 60°C for 20 min and then with a formamide buffer (50% formamide in 2xSSC) at 60°C for 30 min. The section was incubated in 1 μg/μl RNase A (Sigma) in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA and 500 mM NaCl at 37°C for 30 min. After the RNase A was rinsed out of the section using the same buffer, the section was treated with the formamide buffer again at 60°C for 30 min. The section was further rinsed with Buffer 1 containing 100 mM Tris-HCl (pH 7.5) and 150 mM NaCl, after which it was rinsed with a blocking buffer containing 1.5% blocking reagent (Boehringer Mannheim) in Buffer 1 for 1 hr at room temperature. The section was then incubated with alkaline phosphatase-conjugated anti-digoxigenin sheep antibody (1:500 dilution in the blocking buffer) overnight at room temperature and rinsed with the Buffer 1 and then Buffer 2 containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2. The hybridization signal on the section was visualized by a treatment with a solution containing 340 μg/ml nitroblue tetrazolium and 170 μg/ml 5-bromo-4-chloro-3-indolyl phosphate in Buffer 2 and 10% polyvinyl alcohol 2000 (Sigma). The section was then rinsed with TE to terminate the coloring reaction, mounted under coverslips with TE containing 50% glycerol and observed under Nomarski optics.
Expression of membrane GC genes in medaka fish embryonic retina
The most external layer of the medaka fish retina is a pigment epithelium containing a large number of melanin granules (Fig. 1). Photoreceptor cells consisting of rod and cone cells are arranged inside of the pigment epithelium. The cell bodies containing nuclei of the photoreceptor cells are located in one layer known as the outer nuclear layer. The outer layer of the outer nuclear layer in the photoreceptor cells can be further divided into two layers, the inner segment containing ellipsoid and myoid and the outer segment containing rod and cone cells. Under light-microscopic observations, the outer nuclear layer and the proximal part of the photoreceptor cells are delimited with an outer limiting membrane. The expression of four membrane GC genes, OlGC3, OlGC4, OlGC5 and OlGC-R2, in the adult medaka fish retina was examined by ISH, which demonstrated that the OlGC3 and OlGC5 genes are expressed in the proximal part of the photoreceptor cells and that the OlGC4 and OlGC-R2 genes are expressed in the outer nuclear layer and around the outer limiting membrane (Fig. 1). Unfortunately, the difference in the gene expression of the four GCs between the cone and rod cells could not be recognized clearly in this study. However, the present observations are almost consistent with the previous findings on the OlGC-C (OlGC5), OlGC-R1 (OlGC4) and OlGC-R2 expression in the medaka fish retina; OlGC-C is expressed in both the cones and myoids, while OlGC-R1 and OlGC-R2 are expressed in the cell bodies and myoids in the rod cells (Hisatomi et al., 1999). In addition to these findings, we showed that OlGC3 and OlGC5 are expressed in the proximal part of the photo-receptor cells (Fig. 1). The results obtained by ISH of the membrane GCs with the medaka fish embryonic retina are shown in Fig. 2. Although the expression of OlGC3, OlGC4, and OlGC5 was detected in the retinas of 5-day-old embryos, OlGC4 was expressed only in a part of the retinas of 4-day-old embryos. Moreover, the expression of OlGC4 gene was also detected even in the retinas of 3-day-old embryos. On the other hand, the expression of OlGC-R2 could not be detected in the retinas of 3-day- to 5-day-old embryos.
Expression of soluble GC subunit genes in medaka fish adult and embryonic retinas
The inner nuclear layer is a nuclear layer located between the outer nuclear layer and the ganglion cell layer of the photoreceptor cells. In the medaka fish adult and embryonic retinas, the expression of the soluble GC subunit genes (OlGCS-α1, OlGCS-α2, and OlGCS-β1) was examined with ISH (Fig. 3). A weak signal of OlGCS-α2 was detected in the inner nuclear layer of the adult retina, although OlGCS-α1 and OlGCS-β1 transcripts could not be detected in it. In the retinas of 5-day-old embryos, OlGCS-α2 and OlGCS-β1 were expressed extensively in the inner nuclear layer and the ganglion cell layer (Fig. 3). On the other hand, the expression of OlGCS-α1 could not be detected in the embryonic and adult retinas.
Expression of nNOS and cGK I genes in the medaka fish adult and embryonic retinas
In the adult and embryonic medaka fish retinas, the expression of nNOS and cGKI, both of which are present in the NO/cGMP signaling pathway, was examined by ISH. The transcript of nNOS was detected in the inner nuclear layer and the ganglion cell layer in the adult retina (Fig. 4). In contrast with the localization pattern of the OlGCS-α2 mRNA, the cells expressing the nNOS gene are scattered in both the ganglion cell layer and the inner layer of the inner nuclear layer of the adult retina. The expression of the nNOS gene was also detected in the retinas of 5-day-old embryos and adult fish with the same pattern. The identification of the cells expressing nNOS remains to be resolved. However, since it is known that amacrine cells are located in the innermost layer of the inner nuclear layer (Haverkamp et al., 2000), it is possible that the amacrine cells express the nNOS gene and synthesize nitric oxide. The ganglion cell layer contains cell bodies of midget and polysynaptic ganglion cells and astrocytes. The cell expressing the nNOS gene in the ganglion cell layer is not yet clear. The expression of the cGKI gene could not be detected in either the adult or the embryonic retina (Fig. 4).
It has been reported that four membrane GC genes, OlGC3, OlGC4 (OlGC-R1), OlGC5 (OlGC-C), and OlGC-R2, are expressed in the medaka fish adult and embryonic retina (Seimiya et al., 1997; Hisatomi et al., 1999; Kusakabe and Suzuki, 2000b, 2001). OlGC-R1 and OlGC-R2 are expressed around the nuclei of photoreceptor cells while OlGC-C is expressed proximal to the nuclei (Hisatomi et al., 1999). Our present results are consistent with the previous observations on the expression pattern of these membrane GCs in the adult medaka fish tissues. Furthermore, we showed in this study that the OlGC3 gene is expressed near the site where OlGC5 is expressed, indicating that the membrane GCs expressed in the medaka fish retina may be grouped into two groups: one comprising OlGC3 and OlGC5, which are expressed outside of the outer limiting membrane, and the other comprising OlGC4 and OlGC-R2, which are expressed in the cell bodies containing the nuclei of the photoreceptor cells. This classification based on the expression pattern of the retinal membrane GC genes coincides with the evolutionary distance of these membrane GCs as estimated by molecular phylogenetic analysis (Kusakabe and Suzuki, 2000a). However, the relationship between the functional difference between these membrane GCs and their localization is not clear at this point. In mammals, two retina-specific membrane GCs have been cloned in humans (RetGC-1 and Ret GC-2; Shyjan et al., 1992; Lowe et al., 1995), rats (GC-E and GC-F; Yang et al., 1995) and bovines (ROS-GC1 and ROS-GC2; Goraczniak et al., 1994, 1997). It has been reported that a GC-E-deficient mouse shows no abnormality in its apparent rod morphology and its behavior toward light, although its cones had disappeared completely within five weeks after birth (Yang et al., 1999), suggesting that the other retinal membrane GC (GC-F) compensates for the GC-E-deficient effects in the mouse. On the other hand, it is known that membrane GCs form homo-oligomers (Lowe, 1992; Chinkers and Wilson, 1992; Vaandrager et al., 1994). Yang and Garbers (1997) showed that the two retinal GCs, GC-E and GC-F, form homodimers preferentially, but a limited number of heterodimers are also formed. However, it remains to be solved whether the membrane GCs in the medaka fish retina compensate each other and form a homodimer and/or a heterodimer. In the present study, we demonstrated that OlGC4 is expressed in the retinas of 3-day- and 4-day-old embryos while OlGC3 and OlGC5 are expressed in the retinas of 5-day-old embryos, suggesting that OlGC3 and OlGC5 compensate for each other if necessary, and that OlGC4 may play a novel role in the developing retina.
The evidence showing that soluble GC exists in different types of retinal cells such as photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, and Müller cells has been presented, based on biochemical and immunohistochemical experiments (Sitaramayya, 2002). However, there has been no study dealing with the types of subunits expressing and functioning in these cells. In the present study, we showed the strong signals in the embryonic retina due to the mRNAs of OlGCS-α2 and OlGCS-β1, both of whose translation products form a NO-activating heterodimer, and the weak signals due to them in the adult retina. We could not detect any signal due to the α1 subunit gene in ither the adult or embryonic retina, which suggests that the soluble GC expressed in the medaka fish embryonic retina is a α2/β1 heterodimer. In previous papers, we demonstrated that the medaka fish soluble GC subunit genes, OlGCS-α1 and OlGCS-β1, are organized in tandem on the medaka fish genome and that their transcriptions seem to be coordinated (Mikami et al., 1999; Yamamoto and Suzuki, 2002). Recently, we showed that the transcripts of OlGCS-α1 and OlGCS-β1 are colocalized in the embryonic whole brain and kidney (Yamamoto et al., 2003). The genes of the human soluble GC α1 and β1 subunits are mapped on the same chromosome 4q32 (Giuili et al., 1993; Lucas et al., 2000), while the human soluble GC α2 gene is located on chromosome 11 (Yu et al., 1996). It has been reported that the human soluble GC α2 subunit forms an enzymatically active heterodimer with the β1 subunit when both subunit genes are co-expressed in the cultured cells (Yu et al., 1996), and the physiological existence of the α2/β1 heterodimer has been found in the human placenta (Russwurm et al., 1998) and rat brain synaptosomes (Russwurm et al., 2001). Moreover, it has been reported that the α2/β1 heterodimer interacts with postsynaptic density-95 protein (PSD-95), which is a synaptic protein containing three PDZ protein motifs that also interact with nNOS and NMDA receptor and which contributes to anchoring the heterodimer to the plasma membrane (Brenman et al., 1996; Russwurm et al., 2001; Russwurm and Koesling, 2002). At the present time, we do not know whether the transcription of the OlGCS-α2 gene is coordinated with that of the OlGCS-β1 gene in the medaka fish. However, we demonstrated that the expression site of the OlGCS-α2 and OlGCS-β1 genes is rather restricted in the embryonic retina (Fig. 3). Recently, we demonstrated that the knock-down of the soluble GC α2 subunit gene using morpholino antisense oligonucleotide results in abnormal development of the eye in the medaka fish (Yamamoto et al., 2003). In this regard, it should be mentioned that the diminishment of the soluble GC activity due to the mutation in the α subunit gene causes defects in the development of the Drosophila visual system leading to defects in the visual system function and in visually mediated behavior (Gibbs et al., 2001). Taking account of these reported facts and our results presented here, we presume that a soluble GC consisting of α2 and β1 subunits plays a novel role in the retinal development in medaka fish embryos, although the downstream of the cGMP signaling pathway in the medaka fish retina after soluble GC remains to be determined.
There have been many papers demonstrating that by using immunohistochemical and NADPH diaphorase histochemical methods, NOS, not being specified either as nNOS, eNOS or iNOS, is detected in amacrine cells in the inner nuclear layer and ganglion cell layer of rat, guinea pig, and turtle retinas (Chun et al., 1999; Oh et al., 1999; Haverkamp et al., 2000), in amacrine and ganglion cells as well as horizontal and photoreceptor cells in zebrafish retinas (Devadas et al., 2001), and in amacrine and ganglion cells as well as Mülar cells in goldfish and catfish retinas (Liepe et al., 1994). As shown in Fig. 4, we showed that nNOS is expressed in the inner nuclear layer and ganglion cell layer in both the adult and embryonic retinas as a series of spots, suggesting that in the medaka fish, nNOS is also expressed in amacrine cells in the inner nuclear layer as well as in ganglion cells in the adult and embryonic retinas. It is known that the major target molecule of NO generated by NOS is soluble GC (α1/β1 and/or α2/β1 heterodimer) (Russwurm and Koesling, 2002), a key enzyme in the NO/cGMP signaling pathway (Denninger and Marletta, 1999; Kusakabe and Suzuki, 2000a). Several investigators reported that activation of soluble GCs or cGMP elevation by NO or NO donors occurs in bipolar cells but not in amacrine cells in the mammalian retina (Shiells and Falk, 1992; Koistinaho et al., 1993). On the other hand, there have been several papers reporting that NO is involved in many optic systems such as the control of retinal flow (Deussen et al., 1993; Toda et al., 1994; Wiencke et al., 1994) and the induction of neurotoxic effects leading to retinal destruction (Lipton and Rosemberg, 1994; Cui and Harvey, 1995; Goureau et al., 1995), although the relationship with the cGMP signaling pathway is unknown. Inconsistent with the above papers, it has been reported that NO exhibits a neuroprotective effect in retinal development, and its effect is partially mediated by soluble GC (Guimaraes et al., 2001). Moreover, the NO in the retina is also reported to have cGMP-independent effects on the phototransduction and synaptic output from photoreceptor cells (Kurenny et al., 1994). Although there are several apparent inconsistencies and points of confusion among the reported results and explanations of experimentally obtained facts regarding the NO/cGMP signaling pathway, the most plausible explanation is that NO and soluble GC are required to regulate the appropriate development of the retina (Ernst et al., 1998; Guimarases et al., 2001; Gibbs et al., 2001). In this sense, we postulate here that in the medaka fish, NO itself or NO with a soluble GC (α2/β1 heterodimer) plays a role as a regulator of retinal development.
We would like to thank the staff members of the Central Laboratory for Research and Education of Asahikawa Medical College for the use of their laboratory facilities. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (no. 11236202) and by the National Project on Protein Structural and Functional Analyses.