Restricted expression of karyopherin alpha mRNA in the sea urchin suggests a role in neurogenesis

Gene Expression Patterns 16 (2014) 51–60

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Gene Expression Patterns j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e p

Restricted expression of karyopherin alpha mRNA in the sea urchin suggests a role in neurogenesis Christine A. Byrum *, Jason Smith, Marietta R. Easterling, M. Catherine Bridges Department of Biology, College of Charleston, Rita Liddy Hollings Science Center, 58 Coming Street, Room 214, Charleston, SC, USA

A R T I C L E

I N F O

Article history: Received 7 October 2013 Received in revised form 11 June 2014 Accepted 25 June 2014 Available online 10 September 2014 Keywords: Karyopherin Importin Sea urchin Lytechinus Nuclear transport Embryogenesis

A B S T R A C T

Karyopherin alpha (KAP-α) proteins are critical for the transport of many molecules into the nucleus. In this study, we identified three members of the KAP-α family in the sea urchin Lytechinus variegatus and described the developmental expression of these proteins. Although many importins are assumed to have ubiquitous expression, we found that all three genes were differentially expressed. Both LvKPNA1/5/6 and LvKPNA3/4 accumulated at high levels during cleavage, exhibiting cyclic expression as cells divided. By the blastula and gastrula stages expression decreased, remaining highest in the vegetal plate and archenteron, and by the prism/pluteus stages expression was restricted to the oral surface and gut. Expression of a third KAP-α gene, LvKPNA2/7, was examined in embryos from the mesenchyme blastula to pluteus stages. LvKPNA2/7 mRNA is present in vegetal cells of the mesenchyme blastula and, during gastrulation, it is localized to the archenteron and appears in additional groups of ectodermal cells. Prism/ pluteus stage embryos expressed LvKPNA2/7 in the gut and scattered distribution of transcripts in the ciliary band resembled expression patterns of neural cells. We hypothesize that LvKPNA2/7 maintains pluripotency in the neural precursors prior to activation of neural differentiation and believe that this study is an important first step in an effort to better understand the roles of importins during embryogenesis. © 2014 Elsevier B.V. All rights reserved.

Nuclear transport is a process critical to the survival of any eukaryote. Even organisms as simple as yeasts produce proteins specialized to transfer molecules across the nuclear envelope. To do this, cargo often pass through a cylindrical aqueous channel called the nuclear pore complex (NPC). Highly conserved among eukaryotes (DeGrasse et al., 2009), the NPC is a massive macromolecular structure. For example, in yeast a single NPC is composed of 456 individual proteins that can be categorized into 30 distinct protein types called nucleoporins (Alber et al., 2007). Although molecules smaller than 40 kDa can freely diffuse through the NPC, translocation of larger macromolecules (e.g. RNA and proteins) requires active transport mediated by the karyopherins (Alber et al., 2007). Karyopherins attach to cargo and form low affinity but highly specific bonds with the nucleoporins. Repeated phenylalanine residues in the nucleoporins insert into crevices between alpha helices of

Abbreviations: ASW, artificial seawater; ESC, embryonic stem cell; IMP, importin; KAP-α, karyopherin alpha; KAP-β, karyopherin beta; KPNA, karyopherin alpha; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; RanBP1, Ran binding protein 1; SAF, spindle assembly factor. * Corresponding author. Address: Department of Biology, College of Charleston, Rita Liddy Hollings Science Center, 58 Coming Street, Room 214, Charleston, SC 29401, USA. Tel.: +1-843-953-5504; fax: +1-843-953-5453. E-mail address: [email protected] (C.A. Byrum). http://dx.doi.org/10.1016/j.gep.2014.06.005 1567-133X/© 2014 Elsevier B.V. All rights reserved.

the karyopherin, allowing the cargo–karyopherin complex to quickly transfer across the nuclear membrane (Aitchison and Rout, 2012). Karyopherins are a diverse group of proteins and each is specialized to transport a specific cargo type. While some karyopherins (importins) typically transport cargo from the cytoplasm to the nucleus, others are most likely to transfer cargo from the nucleus to the cytoplasm (exportins) or to shuttle cargo in both directions (transportins). Further descriptions of the specific cargo of each karyopherin type are reviewed in Mosammaparast and Pemberton (2004) as well as Chook and Süel (2011). To detect a specific cargo type, karyopherins bind to signal sequences in the cargo. During nuclear import, karyopherins bind to the nuclear localization signal (NLS). This signal sequence is often strongly hydrophilic, but can vary widely in composition, again allowing the karyopherins to functionally specialize in the types of cargo transported (Poon and Jans, 2005). Similarly, during nuclear export, cargo molecules bind to the karyopherin via a nuclear export signal (NES) (Görlich and Kutay, 1999). By preferentially masking the NLS and/or NES signals (e.g. by phosphorylation or binding of a heterologous protein to these sites), cells can regulate transport of cargo across the NPC (Kauffman et al., 1998; Li et al., 1998; reviewed in Poon and Jans, 2005). There are two structurally distinct families of karyopherin proteins: the karyopherin alpha (KAP-α) family and the karyopherin beta (KAP-β) family. While many of the KAP-β importins interact

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Cytoplasm

Nuclear membrane

Karyopherin alpha (KPNA)

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Karyopherin beta (KPNB) KPNA RanGTP

CAS

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RanGTP

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RanGDP

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CAS

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Fig. 1. Summary of molecular interactions in karyopherin alpha mediated transport. (A) Karyopherin alpha forms a ternary complex, binding to a karyopherin beta importin as well as the NLS site in the cargo molecule. (B) After the cargo is transported through the nuclear pore complex, RanGTP binds to KPNB, causing the KPNA and the cargo to dissociate from KPNB. (C) The RanGTP bound KPNB is transported through the NPC and both CAS and RanGTP bind to the KPNA. (D) RanGTP is converted to RanGDP and KPNB is released in the cytoplasm. Also, the KPNA/CAS/RanGTP complex moves through the NPC. (E) Ran GTP is converted to RanGDP and KPNA and CAS dissociate. CAS and RanGDP move back through the NPC and RanGDP is converted to RanGTP.

directly with the NPC to transport cargo into the nucleus, nuclear import of other cargo requires the assistance of an additional protein, a member of the KAP-α family (also known as the importin subunit α or importin-α family). In this process (reviewed by Pemberton and Paschal, 2005) KAP-α acts as an intermediary, binding to both the cargo molecule via the NLS and to the KAP-β molecule, KPNB1, to form a ternary complex (Fig. 1A). This complex then docks at the NPC and is transported through the central channel to the nucleus. Inside the nucleus, the small GTPase RanGTP binds to KPNB1, causing a conformational change in the molecule that allows it to dissociate from the complex (Fig. 1B). The KPNB1/RanGTP complex is then translocated through the NPC to the cytoplasm where it binds to Ran binding protein 1 (RanBP1) (Fig. 1C). This interaction with RanBP1 converts RanGTP into an intermediate that can be acted on by another cytoplasmic protein, RanGAP. When RanGAP encounters this intermediate, it stimulates GTPase activity by Ran (in RanGTP), causing conversion of RanGTP into RanGDP and resulting in the release of KPNB1 (Fig. 1D). In the nucleus, binding of Nup50/Npap60 to the KAP-α importin allows release of the cargo (Matsuura and Stewart, 2005). After this, the KAP-α importin is transported back to the cytoplasm by the exportin CAS (also known as XPO-2). CAS traverses the NPC with the KAP-α cargo by binding to

RanGTP (Fig. 1C, D). Dissociation of the complex in the cytoplasm occurs in a manner similar to that described for KPNB1 (Fig. 1E). When importins were first discovered, many investigators assumed that these gene products would be uniformly distributed throughout the organism. This, however, is not always true and subsequent studies have identified karyopherins that are both spatially and/or temporally restricted (Song and Wessel, 2007; Umegaki et al., 2007; Whiley et al., 2012; Yasuhara et al., 2007). Because each karyopherin specializes in the transport of particular cargo types, investigators have hypothesized that differential expression of these nuclear transport proteins could influence the availability of transcription factors in the nucleus and thereby affect cell differentiation (Okada et al., 2008; Poon and Jans, 2005; Yasuda et al., 2012). Studies examining functional roles of the KAP-α importins have supported this hypothesis and it is clear that the KAP-α family members are sequentially expressed in mammals during differentiation of sperm (Hogarth et al., 2006; Whiley et al., 2012), macrophages (Köhler et al., 2002), muscle (Hall et al., 2011) and neurons (Yasuhara et al., 2007, 2013). The work of Yasuhara et al. (2007, 2013) provides some of the strongest support for this hypothesis. They showed that expression of the KAP-α subtypes switches from KPNA2 to KPNA1 during

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neural differentiation of mouse embryonic stem cells (ESCs). They found that the ESCs are maintained in an undifferentiated state when KPNA2 levels are high because KPNA2 facilitates nuclear import of Oct3/4, a Class V POU transcription factor needed to maintain pluripotency. KPNA2 also prevents nuclear transport of two Class III POU transcription factors that induce neural differentiation, Brn2 and Oct6. It accomplishes this by binding to these proteins in the cytoplasm and preventing them from interacting with other importins, thus creating a dominant-negative effect. Only after KPNA2 expression decreases do levels of KPNA1 rise. After the loss of KPNA2, the transcription factors Brn2 and Oct6 are free to bind to KPNA1, which will transfer them into the nucleus where they activate neural differentiation. To learn more about potential roles of the KAP-α gene family in early developmental processes, we examined the spatial distribution of KAP-α mRNAs at different developmental stages (fertilization to 36hpf at 23 °C) in embryos of the sea urchin model Lytechinus variegatus. Sea urchins are excellent organisms for these sorts of studies. They have simple larval stages with few cell types, develop quickly, produce many offspring (>1000), and are nearly transparent during embryogenesis, making it easier to observe cellular interactions. One of the greatest strengths of the sea urchin embryo is that detailed systems analysis (http://sugp.caltech.edu/endomes/ #EndomesNetwork) has vastly improved our understanding of the gene regulatory processes acting during early developmental events. This information provides a valuable foundation to build upon. Also, among deuterostomes, members of the Echinodermata are basal to the phylum Chordata and evolved prior to gene duplication events that occurred in the vertebrate line. Consequently, molecular interactions in the sea urchin tend to be similar, but not as complex as those in vertebrates, making the study of molecular processes easier. In recent years, investigators have examined the process of neurogenesis in developing sea urchins (e.g. Burke et al., 2006a;

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Bradham et al., 2009; Yaguchi et al., 2010; Angerer et al., 2011; Yaguchi et al., 2012; Range et al., 2013); however, much remains to be learned and the potential roles of KAP-α family members in these processes need to be evaluated. Only two previous investigations (Song and Wessel, 2007; Tu et al., 2012) have examined the expression of karyopherins in the sea urchin. In the Song and Wessel study, spatial and temporal distribution of genes that act in the production and function of small regulatory RNAs was examined. One gene evaluated was the KAP-β family member exportin-5. Song and Wessel found that although exportin-5 transcripts were present throughout development, expression was highest in the oocyte and it decreased substantially by the 32-cell stage (Song and Wessel, 2007). In a second study, Tu et al. (2012) published a temporal analysis of the Strongylocentrotus purpuratus transcriptome that can be accessed in the “Quantitative Developmental Transcriptomes of S. purpuratus” as a resource of SpBase (Cameron et al., 2009) (http://www.spbase.org:3838/ quantdev/). This study annotated more than 7000 transcripts including members of the KAP-α and KAP-β families. Their database reports expression profiles of three KAP-α family members (a summary of the temporal profiles of these genes based on the results of the study by Tu et al. (2012) is presented in Fig. 2). Two of these, SpImp_1 and SpKPNA3, are present at highest levels during cleavage, but are expressed at lower levels during other stages of development. Expression of the other KAP-α gene, SpKPNA2, increases at the hatched blastula stage and peaks during the late prism and pluteus stages. Our study examines the expression of the KAP-α gene family during early developmental processes in embryos of a related sea urchin species, L. variegatus. In this paper, we identified three KAP-α genes. Two of these karyopherins, LvKPNA1/5/6 and LvKPNA3/4, are highly expressed during cleavage, but are also present in the gut and oral territories during later developmental stages. The third

Fig. 2. Summary of the expression of KAP-α gene products in Strongylocentrotus purpuratus as reported in Tu et al. (2012). Both SpImp_1 and SpKPNA3 are present at high levels during cleavage, whereas expression of SpKPNA2 first increases during the hatched blastula stage (18 hpf), but rises to highest levels during late development (64– 72 hpf). Note that S. purpuratus developmental times correspond to the following stages when incubated at 14 °C: unfertilized egg (0 hpf), cleavage (10 hpf), hatched blastula (18 hpf), mesenchyme blastula (24 hpf), early gastrula (30 hpf), mid gastrula (40 hpf), late gastrula (48 hpf), prism (56 h), late prism (64 hpf), and pluteus (72 hpf).

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importin, LvKPNA2/7, is found in the endoderm and appears in groups of ectodermal cells that eventually form portions of the ciliary band. These results are exciting for a few reasons. First, they provide further evidence that the KAP-α family is differentially expressed during development. Also, our findings suggest that LvKPNA2/7 may act in the process of neural differentiation. This karyopherin is expressed in portions of the embryo that undergo neurogenesis and it displays an ectodermal expression pattern similar to that seen in other genes associated with neural differentiation in mammals. We propose that KPNA2/7 maintains pluripotency in neural precursors prior to neural differentiation. Finally, this study is the first detailed report of the spatial distribution of KAP-α gene products in an echinoderm and it provides important preliminary data for future functional studies that will allow us to better understand potential roles of KAP-α genes in early developmental events. 1. Results and discussion 1.1. Characterization of the karyopherin alpha family in L. variegatus In BLAST searches of the pre- and postgastrula embryonic transcriptomes of the NCBI Sequence Read Archive we identified three distinct L. variegatus KAP-α sequences. Using reverse transcriptase PCR, KAP-α fragments ranging from 800 to 1200 bp in length were isolated and sequenced three to eight times. Using the consensus sequences, phylogenetic analysis (Fig. 3) was performed to confirm that the expressed LvKAP-α sequences were orthologous to human and S. purpuratus (the purple sea urchin) KPNAs. Although there are seven different KAP-α sequences in humans, only three were identified in L. variegatus. This is not surprising. Among invertebrates, most groups have three KAP-α sequences (Mason et al., 2009) (although genomes of different Drosophila species were recently shown to have four to five KAP-α forms due to gene duplications and losses) (Phadnis et al., 2012). Genome duplication events during evolution of the vertebrate line later led to expansion of the KAP-α family. Because of this, investigators categorize the KAP-α genes into three different clades: KAP-α1 (includes human KPNA1, KPNA5, and KPNA6), KAP-α2 (includes human KPNA2 and KPNA7), or KAP-α3 (includes human KPNA3 and KPNA4) (Goldfarb et al., 2004; Köhler et al., 1997; Malik et al., 1997; Mason et al., 2009; Tsuji et al., 1997).

Fig. 3. Phylogenetic analysis of the karyopherin alpha sequences found in Lytechinus variegatus. Neighbor-joining analysis showing the phylogenetic positions of L. variegatus KAP-α sequences (Lv, burgundy squares) relative to orthologous human (Hs) and purple sea urchin sequences (Strongylocentrotus purpuratus; Sp, blue squares). Numbers at each node indicate the bootstrap value.

Our phylogenetic analysis also included three S. purpuratus sequences: SpKPNA2, SpKPNA3, and SpImp_1 (equivalent to KPNA1/ 5/6). Gene models for each are presented in Echinobase (http:// www.echinobase.org/Echinobase/), a portion of the SpBase (http:// spbase.org) (Cameron et al., 2009; Tu et al., 2012) database for echinoderm genomic and transcriptomic data. Sea urchin and human KAP-α sequences that were used in this phylogeny appear in Supplementary Table S1. This table also identifies accession numbers of equivalent sequences in other databases. Because some gene models in these databases are more accurate than others, we have reviewed differences between the sequences in the Supplementary material. Based on investigations in other organisms, we know that the stereotypical KAP-α protein contains a single N-terminal importin β binding domain (IBB) followed by a series of 10 tandem ARM repeats that comprise the NLS-cargo binding domain (Conti et al., 1998; Fontes et al., 2000; Goldfarb et al., 2004; Herold et al., 1998). The importin β binding domain (IBB domain) is used to attach to the KAP-β protein during nuclear transport and, in the absence of a KAP-β protein, it allows dimerization with other KAP-α proteins, reducing the ability of cargo to bind to the KAP-α form. The other domains, the ARM repeats, bind to cargo and help facilitate transport by providing a grooved surface that the nucleoporins interact with as they move the protein through the NPC. Also, the 10th ARM repeat usually contains a binding site necessary for export of the KPNA from the nucleus to the cytoplasm. In our LvKAP-α sequences, domain structure was quite similar to that observed in other organisms. Although only eight to nine ARM repeats were detected using domain prediction software, this is within the normal range of variability for the KAP-α sequences.

1.2. LvKPNA1/5/6 and LvKPNA3/4 are both expressed during cleavage To assess spatial distribution of LvKPNA1/5/6 and LvKPNA3/4 at different developmental stages, we performed whole-mount in situ hybridization (WMISH). All reactions performed with sense probes were clear (see Supplementary Figs. S1–S3) and reactions performed with antisense probes produced the following results. Expression of LvKPNA1/5/6 and LvKPNA3/4 was evident during cleavage (1-cell to 60-cell stages) (Figs. 4A–L, 5A–L); however, staining varied quite a bit from embryo to embryo. In some cases, KPNA1/ 5/6 or KPNA3/4 mRNA was present throughout (e.g. Figs. 4A, C–E, G, 5A, E, G, J) while in others staining was less obvious or absent (Figs. 4B, F, H, K, 5A, C, F, L). There were even instances in which LvKPNA1/5/6 or LvKPNA3/4 staining was restricted to a subset of the cells. In these embryos, the cell population that was stained varied from embryo to embryo and no clear pattern was evident (Fig. 5D, H, I, K). This variation may reflect an extremely fast turnover rate of these transcripts during cleavage, or, more likely, is due to variation in amplification of the WMISH signal within the same sample. During later developmental stages, expression of LvKPNA1/5/6 and LvKPNA3/4 was also observed but staining was often faint. At 8 hours postfertilization (hpf) and in the mesenchyme blastula stages (Figs. 4M, N, 5M, N) these karyopherins, when detected, were typically found throughout the embryo and the presence of LvKPNA3/4 was observed more frequently than LvKPNA1/5/6. In the 16 hpf gastrula (Figs. 4O, 5O), staining remained faint with slightly higher levels observed near the vegetal plate and in the archenteron, and by the late gastrula stage it became quite difficult to detect these two karyopherins (Figs. 4P, 5P). Distribution of LvKPNA1/5/6 and LvKPNA3/4 in prism and pluteus stages (Figs. 4Q–T, 5Q–T) was restricted to the endoderm and oral regions, particularly the ciliary band, but was absent in aboral cells.

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Fig. 4. LvKPNA1/5/6 expression is highest in cleaving cells during early embryogenesis, but is also present during later developmental stages. LvKPNA1/5/6 mRNA is present throughout the embryo at the (A) fertilized egg, (C) 2-cell, (D) 4-cell, (E) 8-cell, and (G) 16-cell stages; however, expression levels appear to fluctuate as the cell cycle progresses such that expression may also decrease to very low levels during each of these embryonic stages (as in B, F, H). At the 32-cell stage (I, J) and 4 hours postfertilization (hpf) (K, L) embryos have high (J, L) or low levels of expression (I, K) and this expression is not observed in the micromere progeny (arrowheads). Lower levels of the transcript are present throughout the blastula (M) and mesenchyme blastula (N) and distribution of LvKPNA1/5/6 gradually increases more at the vegetal pole. By the mid gastrula (O) to late gastrula (P) stage, highest expression is observed at the vegetal pole and in the archenteron. Expression may also be present in some of the mesenchymal cells, but this varies greatly from one embryo to another. This is also true at the prism (Q, P), 28 hpf pluteus (S), and 36 hpf pluteus (T) stages and it is noteworthy that expression is high in the oral half of the embryo, but absent in aboral cells (R, T). Expression appears to be high in the ciliary band as well. In all photos the animal pole is oriented to the top and frontal views are shown with the following exceptions: (D, T) viewed from vegetal pole, (I) viewed from animal pole, (R) right lateral view. or = oral, ab = aboral.

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Fig. 5. Spatial distribution of LvKPNA3/4 mRNA is similar to that of LvKPNA1/5/6 mRNA. Faint to strong expression is observed at the 1-cell stage (A) and LvKPNA3/4 is found throughout the embryo (B, E, G, J), in varying cells (D, H, I, K), or at low levels (C, F, L) from the 2-cell stage until 4 hpf. Similar dynamic patterns were observed for LvKPNA1/5/6. By the blastula stage (M) expression of LvKPNA3/4 has decreased but remains present throughout the embryo. In the mesenchyme blastula (N) and early gastrula stages (O), highest levels of the transcript are found at the vegetal pole and in the forming archenteron. In late gastrula (P), prism (Q, R), and pluteus (S, T) stage embryos, LvKPNA3/4 is present at low levels in the oral surface and gut, but is absent in aboral cells. In all photos the animal pole is oriented to the top and frontal views are shown with the following exceptions: (C, F, T) viewed from vegetal pole, (E) oblique view, (R) view from animal pole, (S) right lateral view. or = oral, ab = aboral.

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The temporal expression of homologous genes in S. purpuratus (developing at 14 °C) reported by Tu et al. (2012) (Fig. 2) is consistent with the spatial distributions of these gene products in L. variegatus. It is clear that both KPNA1/5/6 and KPNA3/4 are both expressed early in development and that they persist, although likely at lower concentrations (based on the Tu et al. (2012) temporal analysis), throughout embryogenesis. Tu et al. (2012) found that SpImp_1, the S. purpuratus KPNA1/5/6 homolog, was present at low levels in unfertilized eggs, was highest during cleavage (10 hpf), and was expressed at lower concentrations in the hatched blastula to pluteus stages (18–72 hpf). Temporal expression of SpKPNA3 was similar to that for SpImp_1, but was much higher. Again, transcripts were present in the unfertilized egg and the number of transcripts per embryo increased during cleavage, but then decreased in the hatched blastula to pluteus stages. Although temporal expression patterns of SpKPNA3 and SpImp_1 were quite similar, SpKPNA3 levels were proportionately higher (relative to expression at other stages) at the hatched blastula stage. The observation that LvKPNA1/5/6 and LvKPNA3/4 are both present during cleavage is not surprising. Several studies have reported effects of the KAP-α proteins on cell proliferation although the effects were not necessarily the same in each study. For example, Hall et al. (2011) found that knockdown of KPNA1 in mouse myoblasts resulted in higher rates of cell proliferation in the forming muscle. Interestingly, knockdown of KPNA2 had the opposite effect, reducing proliferation of the myoblasts. Also, the work of Quensel et al. (2004) in human HeLa cells linked activity of the KPNAs to cell proliferation. Unlike Hall, these investigators found that HsKPNAs 1, 3, and 6 were necessary for HeLa cell proliferation and that knockdown of these KPNAs reduced cell numbers by 25–80%. They also reported that knockdown of HsKPNA2 had little effect. It is noteworthy that KPNAs have been implicated as regulators of mitotic spindle assembly. In dividing cells, the importin beta/ KPNA complex can bind to spindle assembly factors (SAFs) preventing them from assembling microtubules during mitosis. This occurs in all areas of the cell except those where the mitotic chromosomes are located. Since Ran GTP is made in areas near the chromosomes, presence of the protein causes dissociation of the KPNA and KPNB complex from the SAFs, allowing them to participate in formation of the spindle (Ciciarello et al., 2004; Gruss et al., 2001; Nachury et al., 2001; Trieselmann et al., 2003; Wiese et al., 2001). In sea urchin KAP-α forms, LvKPNA1/5/6 and LvKPNA3/4 may be functionally redundant in this process and expression of these KAP-α forms during mitosis may be related, in part, to critical roles of these proteins in the regulation of the spindle assembly factors during cell cycles. Distribution during the later developmental stages, may again reflect roles of these genes in cell proliferation. Following gastrulation, cells in the oral ectoderm continue to divide as neurogenesis takes place and as the mouth forms, but cell divisions cease in the aboral portions as this territory differentiates to form squamous epithelium (Coffman et al., 2004). The presence of the LvKPNA1/5/6 and LvKPNA3/4 transcripts in oral cells may reflect the fact that these karyopherins regulate cell proliferation. In these later stages the presence of LvKPNA1/5/6 may also be related to roles of the gene product in neural differentiation. As mentioned before, Yasuhara et al. (2007) found that both KPNA1 and KPNA4 were upregulated during neural differentiation of mouse embryonic stem cells (ESCs). Although overexpression or knockdown of KPNA4 had no effect on murine neural cell differentiation, they found that upregulation of KPNA1 induced formation of neural cells. Also, even if the ESCs were exposed to conditions favorable for neural differentiation, cells experiencing KPNA1 knockdown were unable to differentiate into neural cells and continued to express markers of pluripotency. Thus, in mouse ESCs, KPNA1 is critical for neural cell differentiation. Based on these findings, we expected that expression of KPNA1/5/6 would be robust during neurogenesis in

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pluteus stage embryos. Although faint staining of both KPNA1/5/6 and KPNA3/4 was detected in oral cells and the ciliary band in prism to pluteus stage embryos, portions of the sea urchin embryo known to produce neural cells (Angerer et al., 2011), further investigation will be necessary to determine whether this change is sufficient to influence neurogenesis. Tu et al. (2012) also found that S. purpuratus KPNA1/5/6 expression was lower during late development than during cleavage. It may be that levels of KPNA1/5/6 do not need to change much to influence neurogenesis or perhaps expression of KPNA1/5/6 is more robust in concentrated regions of the ciliary band later in development and we are only observing early portions of the process. Our investigation has focused on the 0–36 hpf period, but neural differentiation continues during later developmental stages. These questions will be addressed further in future studies. 1.3. LvKPNA2/7 is expressed in endoderm and patches of ectodermal/ ciliary band cells Using wholemount in situ hybridization, spatial expression of LvKPNA2/7 mRNA was examined in L. variegatus embryos between 12 hpf (mesenchyme blastula) and 36 hpf (pluteus). In mesenchyme blastulae, LvKPNA2/7 occurred in isolated ectodermal patches (Fig. 6A) and faint staining was often also detected in the vegetal cells (not shown). As gastrulation progressed, LvKPNA2/7 mRNA became localized to the archenteron and had spread to additional patches of ectodermal cells (Fig. 6B–D). In many later stage embryos, distribution of these patches appeared to be almost bilaterally symmetrical, but it is difficult to verify this, since expression of LvKPNA2/7 was quite faint in some areas. Ectodermal and endodermal expression in the prism stage (Fig. 6E, F) was similar to that seen during gastrulation; however, it was clear that LvKPNA2/7 expression was absent in the aboral cells by this stage (Fig. 6F). In plutei, LvKPNA2/7 was primarily found in clusters of cells within the ciliary band (Fig. 6G, I–K, M) and in the gut (Fig. 6G, H, K, M). Groups of cells expressing LvKPNA2/7 in the ciliary band were often located close to the apical plate (Fig. 6G, I, K–M), but also occurred in other regions of the ciliary band along the sides of the embryo (Fig. 6M), in the arms (Fig. 6J, M), and around the mouth (Fig. 6I, K, M). Although LvKPNA2/7 was present in oral ectoderm (Fig. 6G–I, K, L), it was not expressed in aboral ectoderm. Many of our observations are consistent with temporal expression patterns of the homologous gene, SpKPNA2, reported in the related sea urchin S. purpuratus (Tu et al., 2012) (Fig. 2). Tu et al. found that SpKPNA2 mRNA was quite low in the unfertilized egg, and that it remained fairly low in the cleaving embryo (10 hpf). By the hatched blastula stage they noted that SpKPNA2 levels were much higher (18 h). In our study, the spatial distribution of LvKPNA2/7 was evaluated from the mesenchyme blastula stage until the pluteus stage. In both S. purpuratus and L. variegatus, expression of KPNA2/7 is present at the mesenchyme blastula stage and, as in L. variegatus, transcription of this product is maintained through the prism stage and appears to increase in the pluteus. The expression pattern described for LvKPNA2/7 is not commonly encountered in the sea urchin; however, two genes related to neural cell fate specification (Synaptotagmin B and Brn1/2/4) have somewhat similar spatial patterns. The first gene, Synaptotagmin B, is commonly used as a marker for differentiated neural cells. This protein binds to calcium in the synaptic vesicle-associated fusion complex and is the antigen recognized by the sea urchin neural antibody 1E11 (Burke et al., 2006b). Like LvKPNA2/7, this gene is expressed in patches of cells within the ciliary band and in neural fibers around the mouth. It is not found in the endoderm. The other gene with similar expression to LvKPNA2/7 is SpBrn1/2/4. SpBrn1/ 2/4 is found in the foregut, in ectoderm of the stomodeum, and in patches of cells throughout the ciliary band (Cole and Arnone, 2009). Although examination of the roles of SpBrn1/2/4 in the sea urchin

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Fig. 6. LvKPNA2/7 is expressed in ciliary band cells and endoderm. LvKPNA2/7 transcripts were detected at the mesenchyme blastula stage (A) in patches of ectodermal cells (indicated by dots). In some embryos faint expression was also observed in the vegetal ectoderm, cells that will later form the endoderm. By the mid gastrula (B) and late gastrula (C, D) stages, LvKPNA2/7 is evident in the endoderm as well as patches of cells in the ectoderm. This pattern persists in the prism (E, F) and pluteus stages (G–M) and staining is also observed in cilated cells surrounding the mouth (I, K, M). In each photo the animal pole is oriented toward the top and a frontal view is shown with the following exceptions: (H) vegetal view, (F, L) right lateral view, (J) view of a single arm with the animal pole to the right, and (M) view of the oral surface. (I) Inset showing the oral region in a frontal view with the apical plate toward the top.

have primarily focused on its function in formation of the midgut and the regulation of SpEndo16 (Yuh et al., 2005), the work of Yasuhara et al. (2007, 2013) in mouse embryonic stem cell (ESC) lines noted that Brn2 is a key transcription factor activating neural cell differentiation. In mouse ESCs, Brn2 distribution is regulated by KPNA2. Yasuhara et al. (2007) showed that undifferentiated mouse ESCs initially express high levels of KPNA2 prior to neural differentiation. If expression of KPNA2 is blocked (using a KPNA RNAi vector) KPNA1 and Brn2 expression increase while expression of Oct3/4 (a transcription factor needed to maintain pluripotency) decreases. In another experiment, Yasuhara et al. (2007) observed that expression of the neural markers Nestin and Tuj1 is robust in ESCs where KPNA1 has been overexpressed and KPNA2 has been depleted (Yasuhara et al., 2007). Finally, they found that overexpressing KPNA2 prevented neural differentiation even under conditions that normally stimulate neural differentiation. These cells also exhibit increased expression of Oct3/4. Based on these experiments and several other findings, Yasuhara et al. (2013) have proposed that KPNA2 is an important factor

necessary for maintaining pluripotency in neural precursor cells. Although Brn2 and another neural transcription factor, Oct6, are both present in the undifferentiated ESCs, they remain restricted to the cytoplasm at this stage because they bind to a novel C-terminal NLS domain in KPNA2 (Yasuhara et al., 2013). This interaction produces a dominant-negative effect preventing movement of these transcription factors into the nucleus. Unlike Brn2 and Oct 6, the transcription factor Oct3/4, continues to be imported into the nucleus in the undifferentiated ESC because it binds to a different region of KPNA2 (the major NLS domain), allowing KPNA2 to interact with the cargo and transfer it to the nucleus. Based on the Yasuhara et al. model and the distribution of LvKPNA2/7 in L. variegatus embryos, we hypothesize that an important role of LvKPNA2/7 in the sea urchin embryo is the maintenance of pluripotency in the neural precursors located in the ciliary band and around the mouth just prior to neural differentiation. In the future, we plan to test whether overexpression of LvKPNA2/7 during late embryogenesis interferes with neural differentiation (as was observed in ESCs by Yasuhara et al., 2007), whether LvKPNA2/7 expression decreases as neurogenesis

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proceeds, and to determine whether KPNA2/7 expression directly overlaps with that for LvBrn1/2/4. 2. Experimental procedures 2.1. Methods 2.1.1. Culture of L. variegatus adults and embryos Adult L. variegatus were obtained from the Florida Keys (KP Aquatics LLC, Tavernier, FL, USA) and maintained in aquaria at room temperature. To induce spawning, adults were injected with 1 mL of 5M KCl. Eggs were collected in filtered artificial seawater (ASW) and sperm were collected dry. Approximately 100 μL of sperm diluted 2:500 in ASW were used to fertilize eggs in 200 mL of ASW. After fertilization, additional seawater was added, bringing the total volume to 1 L. Embryos were incubated in a large finger bowl or beaker at 23 °C. 2.1.2. Characterization of KPNA sequences Using predicted KPNA sequences from the S. purpuratus genome and confirmed human KPNA sequences, NCBI Sequence Read Archive BLAST searches were performed to identify homologous transcriptomic sequences in L. variegatus. Short reads were aligned and the consensus sequences obtained were used to design primers for PCR and amplify 800–1200 bp reverse transcriptase PCR products using L. variegatus cDNA from 0 to 32 hpf embryos (see Supplementary Table S2 for a summary of the primer sequences used). Each L. variegatus KPNA described in this paper was sequenced three to eight times and representative sequences have been deposited into GenBank: KM_233700 (LvKPNA1/5/6), KM_233701 (LvKPNA2/7), and KM_233702 (LvKPNA3/4). Domain searches were performed using EMBL’s Interpro site. Phylogenetic analysis was performed using MEGA 5 (Tamura et al., 2011). Sea urchin sequences selected for phylogenetic analysis have at least 50% coverage when compared to the most similar human KPNA. Multiple sequence alignments were performed in Muscle and trimmed to a 549 bp segment to minimize non-overlapping regions. An unrooted neighbor-joining tree was produced (Saitou and Nei, 1987) followed by bootstrap analysis of the data (500 replicates). The maximum composite likelihood method (Tamura et al., 2007) was utilized to compute evolutionary distances (measured as the number of base substitutions per site). Accession numbers of all human and predicted S. purpuratus KPNA sequences used to produce this tree are summarized in Supplementary Table S2. 2.1.3. Whole-mount in situ hybridization (WMISH) Digoxigenin-labeled antisense probes were synthesized from T7tailed PCR products using a DIG RNA labeling kit (Roche, Indianapolis, IN, USA) and labeled sense probes were produced in a similar manner from reverse complementary SP6-tailed PCR products. Primer sets used to obtain these products are presented in Supplementary Table S1. Embryos examined for the presence of KPNA1/5/6 or KPNA3/4 mRNA were collected at the 1-cell, 2-cell, 4-cell, 8-cell, 16cell, and 32-cell stages as well as every 4 hours until 36 hpf. Those tested for the presence of KPNA2/7 mRNA were tested from 12 hpf to 36 hpf. In each case, embryos were fixed in filtered artificial seawater containing 4% paraformaldehyde and 10 mM EPPs for 1–1.5 hours at room temperature. This was followed by postfixation in cold MeOH (three washes) and storage at −20 °C. In cases where unhatched embryos were to be collected, 0.5 mM 3-amino-1,2,4triazole was added at fertilization to prevent hardening of the fertilization envelope. Shortly after fertilization, these embryos were also exposed to pH 5.0 seawater for 1 minute and 20 s followed by the addition of 14 drops of Tris base. This removes the jelly coat and embryos were then passed through 100 μm Nitex mesh to manually remove fertilization envelopes. Whole-mount in situ

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hybridizations were performed using standard approaches previously described by Croce et al. (2003) and Walton et al. (2009). DIC images of the embryos were collected using a Zeiss AxioImager equipped with a high-resolution digital camera (AxioCam MRc5) and AxioVision 4.6.3 software. For each developmental stage, 10– 30 stained embryos were photographed. When necessary, additional image processing was performed using Adobe Photoshop. 2.2. Conclusions In this study, we have identified three members of the KAP-α family of nuclear transport proteins and examined their spatial distribution throughout early development in embryos of the sea urchin L. variegatus. It is noteworthy that all three KAP-α sequences were differentially expressed. Although many previously assumed that nuclear transport proteins would be generally distributed, it is clear that the KAP-α family is a dynamic group of proteins likely to affect early developmental events. Two of these karyopherins, LvKPNA1/ 5/6 and LvKPNA3/4, have overlapping expression patterns. They are both expressed during cleavage, but by the mesenchyme blastula stage staining is less obvious and primarily found in the vegetal pole (although low levels continue to be produced throughout the embryo). During gastrulation LvKPNA1/5/6 and LvKPNA3/4 are present in the archenteron and, by the prism and pluteus stages, expression is restricted to the gut and oral territories. Our finding that LvKPNA1/5/6 and LvKPNA3/4 expression were present during cleavage and additional evidence that orthologous genes regulate cell proliferation (Ciciarello et al., 2004; Gruss et al., 2001; Hall et al., 2011; Nachury et al., 2001; Quensel et al., 2004; Trieselmann et al., 2003; Wiese et al., 2001) suggests that LvKPNA1/5/6 and LvKPNA3/4 may influence mitotic events, possibly acting in conjunction with KPNB1 to inhibit spindle assembly. In addition, LvKPNA1/5/6 may act in later stages of development to influence neurogenesis. This will be further examined in future studies. Expression of a third KAP-α gene, LvKPNA2/7, was examined in embryos from the mesenchyme blastula to pluteus stages. LvKPNA2/7 mRNA is present in vegetal cells of the mesenchyme blastula and is later observed in the endoderm of the archenteron and gut. It is also expressed in patches of cells within the ectoderm, ultimately ending up in groups of cells within the ciliary band and around the mouth. Distribution of LvKPNA2/7 is similar to the spatial distribution of SpBrn1/ 2/4 and SpSynaptotagmin B, two genes associated with the specification and differentiation of neural cells. These findings and the reported roles of KPNAs in neural cell fate specification of mouse embryonic stem cells (Yasuhara et al., 2007) suggest that LvKPNA2/7 may influence the specification of nerves associated with the ciliary band and the mouth. We hypothesize that KPNA2/7 maintains pluripotency in the neural precursors prior to activation of neural differentiation and look forward to further examining roles of these karyopherins in future studies. Acknowledgements Funding for this study was generously provided by a College of Charleston Biology Department Research and Development grant and class funding for students of the Genomics class that participated in this research. Portions of the paper were composed by CAB during a writing workshop sponsored by the College of Charleston. We are also grateful to Zachary Schwarz, Shalika Kumburegama, Cyndi Bradham, and Agnes Southgate for discussions about this project and technical advice. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.gep.2014.06.005.

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