Five Pax-6 variants and their expression patterns in squid embryos and adult eye tissues

We performed a single 3′-RACE PCR for the pygmy squid Pax-6 gene (designated as IpPax-6) to investigate the splicing variants and multiple loci of Pax-6 in coleoid cephalopods. We found that there were no multiple loci in the pygmy squid, but we identified three Pax-6 variants of discrete lengths. Differences in the amino acid sequences among these Pax-6 variants were confined to limited regions. Hence, they were hypothesized to be the result of alternative splicing events of a single locus. We next validated the presence of splicing variants using RT-PCR and we finally obtained five types of splicing variants, including an apparent ortholog of authentic Pax-6 (Figure 1). The length and structure of authentic IpPax-6 were similar to those of the Pax-6 genes found in other squid species, Euprymna scolopes and Loligo pealei15,16. Authentic IpPax-6 (authentic form, 499 aa) comprises two independent DNA-binding domains, the PD and HD domains and a C-terminal P/S/T-rich domain (PST), which is the dedicated activator with a partner trans-activator protein, as shown in many animals (Figure 1). Both protein sequence similarity and the phylogenetic tree confirmed that IpPax-6 was an ortholog of fly ey and vertebrate Pax-4/6 (Supplementary figure 1). The four identified variants produced proteins with lengths differing from that of authentic IpPax-6 (Figure 1).

Figure 1 Diagrams of splicing variants found in the pygmy squid. The upper row shows an estimated exon-intron structure of the squid Pax-6 gene. The arrowhead shows an intron confirmed in squid species by genomic PCR analysis and in a previous study. The authentic form (499 aa) is the most abundant and is similar to the Pax-6 gene of other squid species. Variants 1 and 3 lack the exon 4-encoding the N-terminal half of the HD. Variants 2 and 3 have an additional exon, exon 6, in the PST domain. Variant 4 also shows an additional exon 3 encoding 20 amino acids in the linker region between the PD and HD domains. Full size image

To explore the stage-specific expression of the squid Pax-6 variants, we performed Q-PCR for various tissues and at various embryonic stages using primers designed to target the additional exons of IpPax-6 (Figure 2 & Figure S2). Squid eggs show epibolic gastrulation and direct development without typical molluscan larval stages17. Embryonic eyes appear from the external epiderm of the blastodisc and are differentiable after stage 18, with retinal pigmentation starting at stage 20. The lens appears as a transparent stick-like structure visible to the unaided eye at stage 25. We first performed Q-PCR utilizing primers targeting exon 2, which covers all five variants. The Q-PCR analysis showed that IpPax-6 was expressed at stage 16 prior to eye vesicle formation (Figure 2A). The expression intensity of IpPax-6 was gradually upregulated with the development of the squid embryo (Figure 2A), with the eyeball showing the highest expression intensities among the tissues tested. As observed in the other bilaterian animals, the authentic and variant forms of IpPax-6 were expressed at markedly depressed levels in the muscle tissue. We then utilized primers targeting variants lacking exon 4 (variants 1 and 3, Figure 2B). The primers detected variants 1 and 3 at low levels in the embryos at stage 16 and in the eyeball tissue. We also used primers targeting variants including exon 6 (variants 2 and 3, Figure 2C). The Q-PCR analysis showed that variants 2 and 3 were expressed in the eyeballs and optic lobes as well as in embryos at stages 16 and 25. As the formation of photoreceptor cells and the lens begins in embryos at stage 25, the variants including exon 6 may contribute to eye development. The results demonstrate that the expression patterns of the IpPax-6 variants differed significantly from that of authentic IpPax-6.

Figure 2 Expression of the pygmy squid Pax-6 variants. Expression levels of all IpPax-6 variants (A), variants without exon 4 (variants 1 and 3) (B) and variants including exon 6 (variants 2 and 3) (C) were quantified by real-time RT-PCR analysis. The expression level in each body part relative to stage16 (1.0) was calculated and subsequently normalized to the expression level of alpha-tubulin. The quantifications were performed twice on different cDNAs generated independently and geometric means were calculated. The y-axis is arbitrary. Error bars represent standard deviations. (D–G) Whole-mount in situ hybridization analyses with anti-sense RNA probes for IpPax-6 exon 2 (D, F) and IpPax-6 exon 4 (E, G). An RNA probe designed from exon 2 targeting all five variants showed the Pax-6 expression across the brain area of embryos at stage 22 (D) and at stage 25 (F). The RNA probe designed from exon 2 also indicates Pax-6 expression around the eyes (D', side view). An RNA probe designed from exon 4 targeting variants intrinsically as well as variant forms 2 and 4 showed similar expression patterns (E, G) to that of the probe targeting exon 2, except in the tissue around the eyes (E). This result suggests that the variants with an exon 4 deletion (variants 1 and 3) show specific localization in the tissue around the eyes compared with the other variants (arrowhead). Scale bars, 10 μm. Full size image

To distinguish which variants are present in each stage, we performed RT-PCR utilizing primer sets across exon boundaries. Variant 1 was considered to be expressed in all/some embryonic stages but not in the adult eyes (Supplementary figure 2). RT-PCR analysis also showed that variant 4 was strongly expressed in the adult eyes, particularly in the retina, but not in the lenses (Supplementary figure 2A). Variants 2 and 3 were expressed across all embryonic stages and also in adult tissues (Supplementary figure 2B).

To identify the tissue-specific expression of IpPax-6 variants, we performed in situ hybridization using RNA probes designed to bind specifically to each variant (Figure 2D–G). The RNA probe designed from exon 2 targets all five variants identified in this study. The RNA probe designed from exon 4 bound to the authentic form and to variants 2 and 4. IpPax-6 was found to be localized in the brain area, including the dorsal basal lobe, superior frontal lobe, peduncle/olfactory lobes and optic lobes (Figure 2D–G), as described in Hartmann et al.18 The tissue outside the retina (perhaps corresponding to the future iridophore layer) also clearly expressed IpPax-6 at stage 22 (Figures 2D and 2D'). IpPax-6 expression was observed in this layer until stage 25. The in situ hybridization utilizing the probe targeting exon 4 suggested that variants 2 and 4 had similar expression patterns in the brain but not in the eyes (Figure 2E). This finding suggests that variants 1 and 3 (lacking exon 4) are upregulated in the outer layer of the eyes. These consequences imply that each IpPax-6 variant is regulated independently across the processes of eye formation.

Exon-intron structure of Pax-6 in other cephalopods/molluscs

We investigated whether this type of alternative splicing was acquired only in coleoid cephalopods. Applying RT-PCR analysis to Japanese spear squid (Loligo bleekeri) embryonic RNAs, we found three types of mRNAs possibly derived from alternative splicing (exon 4 skipping, exon 3 insertion and exon 6 insertion) in the eyes (Figure 3A, B). The inserted exons 3 and 6 encoded 20 and 40 amino acids, respectively, whereas the skipped exon 4 encoded 51 amino acids. To survey the presence of similar alternative splicing in other molluscan genomes, we examined the exon-intron structures of Pax-6 in the owl limpet and pearl oyster. The complete genome sequence of the owl limpet (Lottia gigantea, obtained from JGI genome portal Lotgi v1.0, e_gw1.86.103.1)19 and of the pearl oyster (Pinctada fucata, obtained from the OIST Marine Genomics Unit genome browser P. fucata_ver1.0, transcript: pfu_aug1.0_8418.1_67856.t1, scaffold8418.1)20 showed that molluscan Pax-6 has five exons. Exon 4 in the squid was conserved across the molluscan species tested. However, exons 3 and 5 were not found in the pearl oyster Pax-6 gene. Thus, we found that variant forms 2 and 4 have been acquired in the coleoid cephalopod lineage (Figure 1).

Figure 3 Indels found in IpPax-6 variants and predicted 3D structures of the HD. Aligned nucleotide sequences of (A) exon 3 and (B) exon 6 of the pygmy squid and the Japanese spear squid, respectively. Alignment of translated amino acid sequences of the HD used in comparative modeling (C). Variants 1 and 3 of IpPax-6 lack a part of helix 1. The three-dimensional structure of the spliced HD obtained by homology modeling (D, D'). Green sticks indicate proteins of IpPax-6 and gray balls represent target DNA molecules. The dotted circle indicates the part of helix 1 lost by the deletion of exon 4. Full size image

To the best of our knowledge, our study is the first to report in-frame splicing variants of squid Pax-6 that were expressed differently according to the embryonic stage. Previous studies isolated discrete types of splicing variants that had lost the N-terminal half of the PD domain in other squid species15,18, but these variants did not show spatio-temporal differences in expression. Our study also suggested that the mechanisms underlying the acquisition of variations in Pax-6 transcripts by alternative splicing have been uniquely acquired in the coleoid cephalopod lineage, as the lower molluscs, such as bivalves, do not possess a corresponding exon-like fragment in their genomes.

Function of squid Pax-6 variants and their putative role in eye development

The addition and deletion of an amino acid fragment encoded in the alternatively used exons is expected to cause structural changes in the IpPax-6 protein variants, which may alter their function in the developmental process. Two of its variants (variants 1 and 3) lack a 153mer in the middle of authentic Pax-6 and half of the HD (Figure 1). To explore whether the deletion influences their functional properties, we performed three-dimensional (3D) structural predictions of the proteins based on comparative modeling. The putative 3D structures of the HDs of authentic IpPax-6 and the variant lacking the segment coded by exon 3 were constructed. The template structure was identified by DNA-bound form so that we could predict the structure of IpPax-6 and the variant in DNA-bound form. The putative 3D structure of the authentic form was reasonably well modeled; the core residues, namely, Phe on the loop before the first helix of the HD, Leu on the first helix, Leu on the second helix and Trp and Phe on the third helix of the model structure, were conserved and the three helices of the HD were apparently tightly packed into one another (Figure 3C). The residues important for DNA binding, namely, two Arg residues at the N-terminal arm and polar residues on the surface of the third helix, were located reasonably close to the DNA interface (Figure 3C, D). However, the putative 3D structure of the variant presented a number of problematic issues. In the modeled structure, the loss of the region encoded by exon 3, which encodes the N-terminal part of the first helix, was compensated by 15 residues encoded in exon 2. Thus, the amino acid sequences of the authentic and variant forms differed only in the region containing the 15 residues of the N-terminal side. This difference, however, significantly increased the structural energy of the variant and apparently destabilized the overall structure. This instability may result from a lack of the Phe on the loop before the first helix and of the Leu on the first helix. These components are evidently important for packing the three helices. In addition, two Arg residues at the N-terminal loop that bind to the DNA bases in the minor groove in the authentic form were missing in the variant. These problems in stability and DNA binding in the variant strongly suggest that the HD of the variant is unstable and that the domain has little binding affinity for DNA (Figures 3D and 3D'). The lack of a stable HD further suggests that variants 1 and 3 have different DNA target sites from that of authentic IpPax-6 in squid species.

Two variants (variants 2 and 3) also exhibited a 120 mer insertion within the PST domain (Figure 1). The inserted sequence was found to be specific to squid (Figure 2). This insertion might change the trans-activation activities of the PST domain. Variant 4 showed a unique insertion (57 mer) between the PD and HD. The Motif program (http://www.genome.jp/tools/motif/) found no known domains or signatures in the inserted sequence. This insertion elongates a linker between the PD and HD domains.