Expression of Pigment Cell-Specific Genes in the Ontogenesis of the Sea Urchin Strongylocentrotus intermedius

One of the polyketide compounds, the naphthoquinone pigment echinochrome, is synthesized in sea urchin pigment cells. We analyzed polyketide synthase (pks) and sulfotransferase (sult) gene expression in embryos and larvae of the sea urchin Strongylocentrotus intermedius from various stages of development and in specific tissues of the adults. We observed the highest level of expression of the pks and sult genes at the gastrula stage. In unfertilized eggs, only trace amounts of the pks and sult transcripts were detected, whereas no transcripts of these genes were observed in spermatozoids. The addition of shikimic acid, a precursor of naphthoquinone pigments, to zygotes and embryos increased the expression of the pks and sult genes. Our findings, including the development of specific conditions to promote pigment cell differentiation of embryonic sea urchin cells in culture, represent a definitive study on the molecular signaling pathways that are involved in the biosynthesis of pigments during sea urchin development.


Introduction
Polyketide compounds are a large group of structurally very diverse multifunctional proteins mainly found in bacteria, fungi, and plants. One of these polyketide compounds, the pigment echinochrome, is synthesized in sea urchin pigment cells in larvae and in adults [1,2]. These compounds from sea urchins, as well as many marine secondary metabolites, possess highly effective antioxidant, antibacterial, antifungal, antitumor, and psychotropic activities [3][4][5][6][7] and may play a role in immune defense [8].
Although great progress has been made in characterizing sea urchin quinone pigments [1,2,9], no definitive information is available on the molecular signaling pathways that are involved in pigment cell specification and the biosynthesis of pigments during sea urchin development. Three basic biosynthetic pathways, the polyketide pathway, the shikimic acid pathway, and the mevalonic acid pathway, are involved in the synthesis of quinones, including benzoquinones, naphthoquinones, anthraquinones, and upper quinones [10]. Different individual compounds are formed by modifications of the basic chemical structure. The bioactive secondary metabolite, echinochrome (2,3,5,6,8pentahydroxy-7-ethylnaphthoquinone), is in the chemical class of naphthoquinones (Figure 1(a)). It is generated after a series of enzymatic, oxidative, and photochemical reactions from shikimic acid, similar to the formation of chimaphilin through the mevalonic acid biosynthetic pathway, as shown in Figure 1(b).
The drug "histochrome" (registered trademark) was developed from the echinochrome base structure and has unique therapeutic properties [3,4]. There are three ways to produce echinochrome: aquaculture, chemical synthesis, and the in vitro production. The industrial-scale procurement of echinochrome may lead to the extinction of the organisms that produce this substance. Chemically synthesized echinochrome has some toxic effects. Cultured pigment cells of sea urchins could provide a source of pharmacologically important quinone pigments that would help reduce the impact on the adult sea urchin population. The in vitro production of biologically active substances is one promising way to solve this problem.  Figure 1: The structure of the naphthoquinone pigment echinochrome (a). One of the quinone biosynthesis pathways (the formation of chimaphilin from a shikimic acid through the mevalonic acid biosynthetic pathway) in accordance with [9] (b).
Pigment cells are the first type of secondary mesenchymal cells (SMCs) to be specified at the mesenchyme blastula stage in sea urchins [11]. These cells accumulate red-brown pigment granules in their cytoplasm [12] and become easily detectable from the late gastrula stage onwards. The cytoplasmic granules store carotenoids and naphthoquinone compounds [2,12,13], which have been suggested to function in body coloring and phototropism which aid in the defense of larval ectoderm [14,15]. Pigment cell precursors are released from the vegetal plate during the initial phase Evidence-Based Complementary and Alternative Medicine 3 of gastrulation, and they have the ability to migrate within the ectodermal layer of the larval epithelium [16]. The ability of phagocytosis exhibited by pigment cells suggests their participation in wound healing in larvae [17]. Changes in the normal sequence or rate of sea urchin embryo development affect echinochrome synthesis [18].
Studies have revealed that expression of genes involved in the regulation of embryogenesis and development of sea urchins is mediated by a complex and extended cisregulatory system [19]. The participation of the sea urchin gene regulatory networks in development has been characterized in detail [20]. The use of the whole mount in situ hybridization has revealed that the polyketide synthase (pks) gene cluster, three different members of the flavincontaining monooxygenase gene family, and a sulfotransferase gene (sult) are specifically expressed in pigment cells, suggesting that they are required for the biosynthesis of the pigment echinochrome [21]. Sea urchin embryos lacking Sppks (knock-down) develop pigment cells but appear unpigmented (albino phenotype) [21].
This study is focused on a detailed gene expression profile for two pigment cell-specific genes, Sipks and Sisult, during sea urchin embryo development and in specific adult tissues. The effect of a precursor of naphthoquinone pigments, shikimic acid, on the expression of pigment cellspecific genes and embryo development was investigated. In addition, specific conditions for promotion of pigment cell differentiation in sea urchin cell culture were developed. The present study is an attempt to increase our understanding of the intracellular mechanisms affecting echinochrome synthesis.

Animals.
Adult sea urchins of Strongylocentrotus intermedius were collected in the Sea of Japan (Amursky Bay or Vostok Bay) and kept in tubs filled with running, aerated seawater. The animals were rinsed free of any debris with UV-sterilized, filtered seawater and injected with 2-3 mL of 0.5 M KCl to chemically induce spawning. The embryonic material was obtained by artificial fertilization and then placed in tanks with UV-sterilized seawater (17 • C) throughout development until the mesenchymal blastula, gastrula, prism, or pluteus stages (14,24,34, and 72 hours after fertilization, hpf, resp.). After 48 hpf, the larvae were fed the microalga Isochrysis galbana (100 000 cells/mL) daily. The embryos and cell cultures were examined with an inverted microscope Axiovert 200 M (Carl Zeiss, Goettingen, Germany) with 10× and 20× dry objectives.

Real-Time Quantitative Polymerase Chain Reaction (Real-Time PCR)
. Quantitative real-time PCR was used to measure the relative amount of Sppks and Spsult transcripts during the course of development and in specific tissues of the adults. Using BLAST, we showed a high identity (98-99%) of the central part of the pks and sult genes in the sea urchin S. intermedius with that of the pks and sult genes in the closely related sea urchin S. purpuratus (GeneBank accession numbers XM 788471 and DQ176319 for the pks and sult genes, resp.). Then, we used the obtained nucleotide sequences from cDNA of S. intermedius to design the realtime PCR primers and probes.
Total RNA from spermatozoids, unfertilized eggs, coelomocytes, ambulacra, embryos, and larvae of the sea urchin S. intermedius at various stages of development was extracted with Yellow Solve reagent (Clonogen, St. Petersburg, Russia) and treated with DNase. The RNA pellet was washed with 1 mL of 75% ethanol. The sample was then centrifuged at 13,200 g at 4 • C for 10 min. Following centrifugation, the supernatant was removed, and the RNA pellet was airdried and stored at protein content in the supernatants was determined as described previously [23] and averaged 450-475 µg/mL. The cell cultures were maintained by changing the old medium with new medium at 3-5-day intervals for 20 days at 17 • C.

Statistical Analysis.
Statistical analysis was carried out using the Statistica 8.0 program. The results are represented as the mean ± standard error and were tested by paired Student's t-test. P < .05 was selected as the point of minimal statistical significance in all analyses.

Sipks and Sisult Expression Profiles in Sea Urchin Embryo Development and in Specific Adult
Tissues. In unfertilized eggs, only trace amounts of the pks and sult transcripts were detected, whereas no transcripts of these genes were observed in spermatozoids (Figure 2). We observed the highest level of expression of the pks gene at the gastrula stage (Figure 2(a)), which exceeded the expression level of this gene at the blastula, prism, and pluteus stages, and in coelomocytes, and ambulacra by 4.6-, 4.3-, 4.5-, 4.5-, and 1.9-fold, respectively. The gene expression profile for Sisult had a similar trend to that of Sipks. The onset of transcription for the sult gene began at the blastula stage, and then the level of the expression increased drastically through the start of gastrulation (approximately 24 hours) (Figure 2(b)). After that, the level of transcript decreased by more than 10 and 20 times at the prism and pluteus stages, respectively. In addition, sult gene expression was detected in coelomocytes and ambulacra, where the level of the sult gene expression was lower than that at the gastrula stage by 22.7-and 35.5fold, respectively.

Experiments with a Precursor of Naphthoquinone Pigments: Shikimic Acid (ShA). Sipks and
Sisult expression in embryo development was significantly increased after the incubation of sea urchin embryos with 0.1 mM-0.5 mM ShA (Figure 3), but not 2.0 mM ShA, which blocked the expression of the pigment genes (data not shown). No apparent effect on normal development (Figure 4) was detected after the addition of ShA (0.1 mM and 0.5 mM) to sea urchin zygotes, which developed into morphologically almost normal plutei (Figures 5(a)((1), (2))). In contrast, the addition of ShA (0.1 mM and 0.5 mM) to the blastula and gastrula embryos resulted in a marked slowdown of development (Figures 5(b) and 5(c)). In these cases, after 8 days of cultivation with ShA, the development of the sea urchin larvae was retarded in the prism stage, while the control embryos reached the pluteus stage. The addition of 2.0 mM ShA to the zygotes, blastula, and gastrula embryos 6 Evidence-Based Complementary and Alternative Medicine of pigment cells was dependent on the cell culture medium.
If the coelomic fluid of control sea urchins was used as the medium, all the pigment cells were well attached and spread ( Figure 6(b)). However, if the coelomic fluid of injured sea urchins was used as the culture medium, all the pigment cells were rounded and unspread ( Figure 6(c)). Following 20 days in culture, the pigment cells maintained their morphology; however, further cell division was not detected. Cell viability was 90-95% immediately after seeding and declined to 70-75% after 20-day cultivation.

Discussion
Marine organisms passed through the long path of evolution and adaptation, and this is reflected in the peculiarities of their biosynthesis and metabolism. It is known that the transcription factor glial cells missing (SpGCM) is required for the activation of transcription for pigment cell-specific differentiation genes; the onset of transcription of these genes occurs a few hours after the activation of Spgcm (12 hours) [20]. Phylogenomic studies have suggested that some animal genomes (sea urchins, birds, and fish) possess a previously unidentified group of pks genes in addition to fas genes used in fatty acid metabolism. These pks genes in the chicken, fish, and sea urchin genomes do not appear to be closely related to any other animal or fungal genes and instead are closely related to pks genes from the slime mold Dictyostelium and eubacteria [24]. Our results agree with the data of Calestani with colleagues that showed that the pks genes are expressed in sea urchin pigment cells beginning from the blastula stage and that this expression is maintained throughout the pluteus stage [21]. The level of pks transcripts has been found to be highest at the gastrula stage and then gradually decreases. The addition of shikimic acid (0.1 mM and 0.5 mM), a precursor of naphthoquinone pigments, to zygotes and embryos was shown to increase the expression of the pks and sult genes. The addition of lower concentrations of shikimic acid to sea urchin zygotes did not influence the larval developmental stages. However, the addition of 0.5 mM and 2.0 mM shikimic acid to the blastula and gastrula embryos resulted in a marked slowdown of normal development or in larval growth inhibition, respectively.
As shown by Kominami [25], pigment cells differentiate in embryos treated with aphidicolin, a specific inhibitor of DNA polymerase alpha although gastrulation and successive morphogenesis are blocked due to the absence of cell divisions and DNA synthesis. The number of pigment cells observed in aphidicolin-treated embryos increased as the treatment was initiated at later time points (from 9, 10, 12, 16, and 24 h of development) [25]. Pigment cells can be induced even from animal blastomeres at the 8-cell stage or mesomeres at the 16-cell stage, if the blastomeres are treated with LiCl [26,27]. These data indicate the possible existence of an inductive signal for the specification of the pigment cell lineage.
Using dissociated sea urchin embryos transfected with the yeast gene gal4, we have previously shown that the absorption spectrum of red-brown pigments extracted from the cultured cells coincides with that of echinochrome [28]. The number of cells containing the red-brown pigments in two-month-old cell culture reached 50-60%, while the number of naphthoquinone pigments in these cells, as calculated per one cell [29], increased 9-10-fold [28] compared to the cells of normal plutei in vivo [5]. Here, we continued the studies of the differentiation process of sea urchin pigment cells in culture and developed conditions for the promotion of pigment cell differentiation without transfection of sea urchin embryos with foreign genes. Many pigmented cells formed and showed spread morphology similar to pigment cells embedded in the embryonic or larval ectoderm [16,29]. However, there is no cell division in these cultures. Today, only cells of developmental anomalies in sea urchin embryos transformed by the yeast gal4 gene [30], and malignant mussel hemocytes [31] have been reported to be involved in active proliferation.
We have found the specific effect of the coelomic fluid of control and injured sea urchins on the morphology of cultivated pigment cells. The origin of this phenomenon is unclear. We failed to develop a potential permanent cell line; however, the results obtained allow us to assert that the culture conditions used promote pigment cell differentiation 8 Evidence-Based Complementary and Alternative Medicine and can be useful for studying sea urchin pigment cells. The technology of directed differentiation of marine invertebrate embryonic cells in vitro opens the pathway for solutionapplied tasks, including the generation of cell cultures that produce complex bioactive compounds with therapeutic potential.