Na, K-ATPase β2 isoform (atp1b2) expressed in the retina of Xenopus

Xenopus laevis was handled in accordance with animal welfare regulations of Institutional Animal Care and Use Committees (IACUC), Chonbuk National University Laboratory Animal Centre, South Korea. Xenopus laevis embryos were maintained according to standard protocols. All efforts were made to minimize the discomfort of animal used.

Total RNA was extracted and digested with DNase I and purified with RNeasy cleanup kit (Qiagen). First strand cDNA was synthesized from stage 35 embryos with RevertAid™ First Strand cDNA Synthesis Kit (Fermentas). RT-PCR was performed by using Maxim RT-PCR Premix Kit (iNtRON). The partial sequences of the X. laevis atp1b2, vsx1 and pax6 ORF were amplified using PCR from stage 30 cDNA using a set of primers designed against two conserved domain regions of each gene in several species. The resulting PCR products of 1,018 bp for atp1b2, 1,002 bp for vsx1 and 1,010 bp for pax6 were purified, subcloned into a pGEM-T Easy vector (Promega), and sequenced. Primers designed for cloning are shown in Table 1. The PCR conditions were 95°C for 30 seconds, 45°C for 30 seconds, 72°C for two minutes for 35 cycles, and final extension at 72°C for 15 minutes.

Xenopus laevis eggs were collected, fertilized, and embryos were cultured under standard procedure in amphibian [19]. Anti-sense Digoxigenin-labeled probe was transcribed as the standard procedures [20]. The atp1b2 plasmids were linearized with Spe I and transcribed with T7 polymerase. The retinal marker sox4, sox11 plasmids were linearized with Cla I and EcoR V, respectively, and transcribed with T7 polymerase. The other retinal marker vsx1 and pax6 plasmids were linearized with Sac II and transcribed with SP6 polymerase. Whole mount images were taken on an Olympus MV × 10 microscope.

Stage 27, 30, 35, 41 and 47 embryos were dehydrated with a series of ethanol, transferred to xylene, and then embedded in paraffin. Serial sections of 12 μm were cut with microtome (Thermo, MICROM, HM 325). Anti-sense Digoxigenin-labeled probe was transcribed for in situ hybridization on sections and counterstained with eosin. The images were acquired digitally using a Leica DM 2500 microscope.

Stage 47 embryos were dehydrated with a series of ethanol, transferred to Xylene, and then embedded in paraffin. Serial sections of 5 μm were cut with microtome and stained with hematoxylin and eosin.

The temporal expression patterns of Xenopus laevis atp1b2 (Fig. 1) were analyzed by RT-PCR using RNA of different developmental stages [21]. The atp1b2 expression, which was not observed during early cleavage stages (stage 1~9), was observed at the late gastrulation stage (stages 11.5) and continued to be present until stage 40.

Fig. 1. Temporal expression of atp1b2 during Xenopus development. Expression was analyzed by RT-PCR using RNA isolated from embryos at the indicated developmental stages. Elongation factor 1 alpha (Ef1α) was used as an internal control. Lane (-) shows a negative control in absence of RNA.

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Embryos of stage 15, 20, 30 (tailbud), 40 and stage 45 (tadpole) were used to examine the tissue distribution of Xenopus atp1b2. We were interested in establishing the onset of atp1b2 expression during Xenopus retinal development. The results of in situ hybridization analysis were shown in Fig. 2. Whole mount in situ hybridization showed that in early neurula stage embryos (stage 15) Xenopus atp1b2 was expressed in the whole neuroectoderm (Fig. 2A) and at late neurula stage (stage 20) it was expressed in the whole surface ectoderm (Fig. 2B). At tailbud stage (stage 30) atp1b2 was expressed in the eye and the developing brain with entire epidermal cells (Fig. 2C). Xenopus atp1b2 expression was observed in the developing gills in tadpoles (stage 40 and 45) along with other expression domain (Figs. 2D and 2E). Gene expression persisted at a high level at least until stage 45, the last stage examined in the present study.

Fig. 2. Developmental expression of atp1b2 by in-situ hybridization. Whole mount in situ hybridization results (A-E). Xenopus atp1b2 was expressed in the neuroectoderm at stage 15 (A), in the entire epidermal cell layer at stage 20 (B), in the surface ectoderm and in the eye and brain at stage 30 (C), and in the brain, eye, developing gills along with surface ectoderm at stage 40 and 45 respectively (D, E). F, G, H, I, J and, K are the results of in situ hybridization on serial sections at different stages. The atp1b2 was expressed in the epidermal cell layer and retina at stage 30 (F) and 35 (G), respectively. It was expressed in the pharyngeal epithelium at stage 35 (H) and in the epidermal cell layer, retina, pharyngeal epithelium along with a projections for developing gills at stage 41 (I, J). The expression of atp1b2 persisted in the brain and specifically in the outer nuclear layer of retina at stage 47 (K). Scale bar=200 μm.

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Whole mount in situ hybridization revealed that Xenopus atp1b2 was expressed in the eye at stages 30 to 45. To determine the specific layer in which atp1b2 was expressed, we performed in situ hybridization on the serial sections of stage 30, 35, 41 and 47 embryos. At tailbud stage (stage 30) Xenopus atp1b2 was expressed in the retina and distinguishingly in the epidermal cells (Fig. 2F). At stage 35 we found an exclusive expression domain that was pharyngeal epithelium (Figs. 2G and 2H) and its expression persisting as the same pattern at stage 41 including the lateral prominence for gills (Figs. 2I and 2J). In tadpole (stage 47), the expression of almost the same domains including neural tube and the future brain also persisted, but at this stage the retinal expression was more specific in the outer nuclear layer of retina (Fig. 2K).

We also performed in situ hybridization on sections with different proneural and panneural retinal cell markers. We used sox4 and sox11 to visualize ganglion cell layer, pax6 to visualize amacrine and ganglion cells, vsx1 to detect bipolar cells and compare the expression with atp1b2. After conducting in situ hybridization on sections, it was observed that atp1b2 was expressed in the retina at stage 27, in contrast to atp1b2, and all other marker genes were expressed in the retina at this stage (Figs. 3A-E). Xenopus atp1b2, vsx1, and pax6 were weakly expressed in the retina at stage 30 whereas sox4 and sox11 were strongly expressed (Figs. 3F-J). Since Xenopus atp1b2 was expressed in the retina we have sectioned later stage to see the specific layer of expression. At stage 35, atp1b2 was expressed in outer part of the retina and among the retinal marker genes sox4 and sox11 were expressed strongly in the ciliary marginal zone (CMZ) whereas pax6 was not expressed in the CMZ (Figs. 3K-O).

Fig. 3. Expression analysis of atp1b2 in the retinal development before cellular differentiation. Transverse sections were performed at three different stages along the anteroposterior axis. Each row shows sections of different stages embryos and each column shows expression of different marker genes for retinal development. Expression of atp1b2, sox4, sox11, vsx1 and pax6 at stage 27 (A-E), at stage 30 (F-J), at stage 35 (K-O) respectively. Scale bar=100 μm.

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The specific retinal layer was not clearly detectable at stage 41 but we found the different domain of expressions (Figs. 4A-E). At stage 47, Xenopus atp1b2 was specifically expressed in the outer nuclear layer of retina whereas sox4 and sox11 in the inner nuclear layer and ganglion cell layer, vsx1 in the inner nuclear layer, and pax6 were expressed both in the inner nuclear layer and ganglion cell layer (Figs. 4F-J).

Fig. 4. Expression analysis of atp1b2 in the retinal development after cellular differentiation. Expression of Xenopus atp1b2 in the retina at stage 41 (A-E) and at stag 47 (F-J), hematoxylin and eosin stained embryos (stage 47) showing different layer of retina (K), and diagram of eye showing different layer of retina (L). Scale bar=100 μm.

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