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Nutrient Uptake by Marine Invertebrates: Cloning and Functional Analysis of Amino Acid Transporter Genes in Developing Sea Urchins (Strongylocentrotus purpuratus)

Eli Meyer^* and Donal T. Manahan

Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0371

To whom correspondence should be addressed. E-mail: manahan{at}usc.edu

	Abstract

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Transport of amino acids from low concentrations in seawater by marine invertebrates has been extensively studied, but few of the genes involved in this physiological process have been identified. We have characterized three amino acid transporter genes cloned from embryos of the sea urchin Strongylocentrotus purpuratus. These genes show phylogenetic proximity to classical amino acid transport systems, including Gly and B0+, and the inebriated gene (INE).

Heterologous expression of these genes in frog oocytes induced a 40-fold increase in alanine transport above endogenous levels, demonstrating that these genes mediate alanine transport. Antibodies specific to one of these genes (Sp-AT1) inhibited alanine transport, confirming the physiological activity of this gene in larvae. Whole-mount antibody staining of larvae revealed expression of Sp-AT1 in the ectodermal tissues associated with amino acid transport, as independently demonstrated by autoradiographic localization of radioactive alanine. Maximum rates of alanine transport increased 6-fold during early development, from embryonic to larval stages. Analysis of gene expression during this developmental period revealed that Sp-AT1 transcript abundance remained nearly constant, while that of another transporter gene (Sp-AT2) increased 11-fold. The functional characterization of these genes establishes a molecular biological basis for amino acid transport by developmental stages of marine invertebrates.

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion Literature Cited

 
The transport of amino acids into animal cells is important for many metabolic processes including nutrition, osmoregulation, and protein synthesis (Christensen, 1990). Concentrative transport of amino acids is driven by the energy stored in electrochemical gradients, typically through co-transport with sodium ions (Saier, 2000). Physiologists have described a number of sodium-dependent neutral amino acid transport systems in mammals, including systems A (Christensen et al., 1965), ASC (Christensen et al., 1967), and B (Stevens et al., 1984). Many transport systems take up a broad range of amino acid substrates (Stevens et al., 1982), with certain systems transporting overlapping sets of substrates (Palacin et al., 1998). These studies have shown that cells can transport a given amino acid substrate (e.g., alanine) through multiple transport systems (e.g., systems A, ASC, and B).

The genes that underlie amino acid transport systems like the well-described mammalian systems have been cloned and characterized in a wide range of organisms (e.g., Malandro and Kilberg, 1996; Nelissen et al., 1997; Jack et al., 2000). Cloning these transporter genes has made it possible to obtain experimental proof of function using expression systems including oocytes of the frog Xenopus laevis (Gurdon and Wickens, 1983). Molecular cloning of transporter genes has provided tools for investigating the regulation of transporter gene expression (Santalucia et al., 1992; Lescale-Matys et al., 1993; Corey et al., 1994; Hirsch et al., 1996). Comparisons of protein sequences and physiological functions have made it possible to characterize the phylogenetic relationships among amino acid transporter genes (Saier, 2000; Boudko et al., 2005).

In contrast to the more detailed understanding of genes that regulate amino acid transport systems in model systems, far less is known about the corresponding genes involved in amino acid transport by marine invertebrates. The uptake of dissolved organic material from seawater by marine animals has been investigated for nearly a century (Putter, 1909; Krogh, 1931; Stephens and Schinske, 1961; Jorgensen, 1976; Wright, 1988a; Manahan, 1990; Gomme, 2001). These previous studies have shown that the uptake of dissolved organic material from dilute concentrations in seawater (sub-micromolar) is a physiological process that is broadly distributed among many marine animals, representing most marine phyla. The process is thought to fill osmoregulatory and nutritional roles, although the quantitative extent of these contributions remains uncertain (Gomme, 2001). Little is known about the specific molecular biological mechanisms involved in these transport processes because very few of the genes associated with amino acid transport by marine animals have been identified (Hosoi, 2005; Toyohara et al., 2005).

Sea urchin embryos and larvae have been used for decades in studies of evolutionary, developmental, and molecular biology (Davidson, 1986; Raff, 1996). Recently, the genome of the sea urchin Strongylocentrotus purpuratus (Stimpson 1857) was sequenced (Sea Urchin Genome Sequencing Consortium et al., 2006), providing a unique evolutionary context for the study of transport processes, since the sea urchin is the first non-chordate deuterostome with a fully sequenced genome (Davidson, 2006). The uptake of dissolved amino acids from dilute concentrations in seawater by developing sea urchins has been characterized in detail (Tyler et al., 1966; Gross and Fry, 1966; Epel, 1972; Manahan et al., 1989). In S. purpuratus, high-affinity amino acid transport systems are active throughout embryogenesis and larval development (Epel, 1972; Manahan et al., 1989). These transport systems function in a sodium-dependent manner (Epel, 1972; Davis et al., 1985) similar to the amino acid transport systems described in mammals (Malandro and Kilberg, 1996). Transport systems with broad substrate specificity (i.e., multiple amino acids transported through a single transport system) have been described in sea urchin embryos (Tyler et al., 1966). These amino acid transport systems in echinoderms are comparable in substrate specificity to some mammalian transport systems—for example, ATB0+ (Epel, 1972; Allemand et al., 1984, 1985). Echinoderm larvae can transport amino acids from dilute solution in seawater at rates sufficient to contribute substantially to the energy requirements of development (Manahan et al., 1983; Shilling and Manahan, 1994). Despite the many detailed studies of amino acid transport systems in developing echinoderms, the genes responsible for regulating flux rates have yet to be identified.

The current study presents (i) the first functional characterization of amino acid transporter genes in a marine invertebrate larval form, (ii) a classification of these genes as members of an evolutionarily conserved gene family, and (iii) a quantitative analysis of the expression of these genes during early sea urchin development. The findings presented assign physiological functions to previously uncharacterized genes that were sequenced during the recently completed sea urchin genome project (Sea Urchin Genome Sequencing Consortium et al., 2006). Additionally, this study highlights the evolutionary significance of the fact that a single transporter gene family can produce diverse physiological functions depending upon context (e.g., expression in different tissues, species, or developmental stages).

	Materials and Methods

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Cloning and sequencing of amino acid transporter genes
Putative transporter genes were isolated from a cDNA library, representing more than one million clones, that was constructed using purified mRNA from 4-cell-stage embryos of the sea urchin Strongylocentrotus purpuratus (4-h-old). Appropriate clones were selected using ³²P-radiolabeled degenerate probes based on conserved regions within previously characterized mammalian transporter genes. The consensus amino acid sequences of these conserved regions were (1) FPYLCYKNGGGAFLIPYFI and (2) GKVVYFTATFP. Nucleotide sequences corresponding to these regions were deduced from codon preference tables for S. purpuratus (Wada et al., 1991), resulting in the following degenerate oligonucleotide probes: (1) TCCCITACCTITGCTACAARAACGGHGGIGGHGCHTTCCTIATCCCITACTTCAT; and (2) GGGAAIGTDGCDGTGAAGTAIACIACYTTDCC (degenerate nucleotide codes: I = inosine; D = guanine, adenine, or thymine; R = adenine or guanine; and Y = cytosine or thymine). The initial screening of the cDNA library with Probe 1 (corresponding to conserved region 1, above) identified several dozen clones. Further screening with Probe 2 (corresponding to conserved region 2, above), with endonuclease restriction analysis of selected clones, narrowed the range of possibilities to three putative transporter genes, designated as Sp-AT1, Sp-AT2, and Sp-AT3 (S. purpuratus amino acid transporter). Nucleotide sequences for these clones were initially obtained for both DNA strands, using transposon-facilitated insertion of sequencing primers throughout the full length of each cDNA (Strathmann et al., 1991); and cDNA sequences were subsequently confirmed by capillary electrophoresis (Beckman CEQ: Beckman-Coulter, Fullerton, CA). The nucleotide sequences for Sp-AT1, Sp-AT2, and Sp-AT3 have been deposited in the GenBank database under accession numbers EF538763, EF538764, and EF538765, respectively.

DNA sequence analysis
Structural features of the Sp-AT1, Sp-AT2, and Sp-AT3 transporter proteins were predicted on the basis of the deduced amino acid sequences of the above cDNA clones. Open reading frames (ORFs) of each clone were determined using the Vector NTI software package, ver. 10.3.0 (Informax, Inc., Bethesda, MD). Transmembrane topology models were predicted on the basis of the classical Kyte-Doolittle method (Kyte and Doolittle, 1982) and the "consensus-based" ConPredII method (Arai et al., 2004). Putative O- and N-glycosylation sites were identified using the Center for Biological Sequence Analysis servers (Gupta et al., 2004; Julenius et al., 2005).

The nucleotide sequences of cDNA clones for Sp-AT1, Sp-AT2, and Sp-AT3 were queried against GenBank using TBLASTN (Altschul et al., 1997). These comparisons confirmed that each cDNA clone matches a distinct region in the sea urchin genome. These searches also revealed sequence similarities between the sea urchin genes and previously characterized genes from the SLC6 gene family. Based on these similarities, a phylogenetic analysis was conducted to characterize the position of these sea urchin genes within this well-studied gene family. The protein sequences for a selection of 34 SLC6 genes from human, Caenorhabdites elegans, and Drosophila melanogaster were obtained from National Center for Biotechnology Information (accession numbers are shown in legend for Fig. 3). These genes were aligned with the deduced amino acid sequences of Sp-AT1, Sp-AT2, and Sp-AT3 using ClustalW ver. 1.83 (Thompson et al., 1994). Alignments were minimally adjusted by hand to reduce the number of gaps (Baldauf, 2003), and positions containing gaps were excluded from further analyses using the BioEdit software package, ver. 7.0.9.0 (Hall, 1999). The alignment was bootstrapped (n = 1000) and a consensus neighbor-joining tree based on protein distance was generated using the Phylip sequence analysis package, ver. 3.67 (Felsenstein, 1993). The resulting tree was used to assign each Sp-AT gene to a particular sub-family within the SLC6 gene family.

View larger version (26K): [in this window] [in a new window]   Figure 3. Protein distance analysis of sea urchin transporter genes Sp-AT1, Sp-AT2, and Sp-AT3. Thirty-four different SLC6 genes, from human (Hs), Caenorhabdites elegans (Ce), and Drosophila melanogaster (Dm) were included in this analysis. The neighbor-joining tree shown is based on a ClustalW multiple sequence alignment. Scale bar (0.1) represents units of protein distance calculated by the Jones-Taylor-Thornton model in the PHYLIP software package (Felsenstein, 1993). Bootstrap support values (n = 1000) for all nodes with 50% or higher support are shown as percentages. Accession numbers: Ce_BGT1, AAZ23105; Ce_DAT, AAC83661; Ce_MOD5, AAK84832; Ce_SNF2, NP_492396; Ce_SNF5, NP_496735; Ce_SNF6, NP_497416; Ce_SNF9, NP_503077; Dm_CG10804, NP_726868; Dm_CG15088, NP_788408; Dm_CG15279, NP_001014484; Dm_CG1698, NP_610517; Dm_CG1732, NP_651930; Dm_CG3252, NP_572219; Dm_CG4476, NP_648293; Dm_CG8850, NP_610755; Dm_DAT, NP_523763; Dm_INE, NP_477012; Dm_SERT, NP_523846; Hs_ATB0+, NP_009162; Hs_BGT1, NP_003035; Hs_CT1, NP_005620; Hs_DAT1, NP_001035; Hs_GAT3, NP_055044; Hs_GLYT1, NP_001020016; Hs_GLYT2, NP_004202; Hs_NET1, NP_001034; Hs_NTT4, NP_001010898; Hs_NTT73, NP_877499; Hs_PROT, NP_055043; Hs_SERT, NP_001036; Hs_SLC6A19, NP_001003841; Hs_TAUT, NP_003034; Hs_XTRP2, NP_872438; Hs_XTRP3, NP_064593.

In vitro transcription of the linearized cDNA constructs described above was achieved using the T7 mMessage mMachine RNA transcription kit (Ambion, Austin, TX). This resulted in the production of different cRNAs, each containing the ORF of either Sp-AT1, Sp-AT2, or Sp-AT3. Prior to injection into frog oocytes, each cRNA preparation was analyzed on standard denaturing formaldehyde-MOPS agarose gels to confirm molecular weight and integrity (Ausubel et al., 1994).

The cRNA prepared from each of the Sp-AT genes was used for heterologous expression in oocytes of X. laevis according to standard procedures (Quick and Lester, 1994). Individual oocytes were injected with 25 ng (50 nl) of cRNA prepared as described above, or with 50 nl of nuclease-free water as controls. Injected oocytes were incubated for 3–5 days on a rotary shaker (70 rpm) at 18 °C in ND96+ medium (96 mmol l^–1 NaCl, 2 mmol l^–1 KCl, 1 mmol l^–1 MgCl₂, 1.8 mmol l^–1 CaCl₂, 5 mmol l^–1 HEPES, 50 µg ml^–1 gentamycin, pH 7.5). During this culture period, oocytes were examined daily, unhealthy oocytes removed, and the remaining oocytes transferred into fresh medium.

Amino acid transport rates of cRNA-injected oocytes were measured using [¹⁴C]alanine as a tracer (Perkin-Elmer, Wellesley, MA) after 3–5 days of incubation with injected cRNA. Transport rates were measured by incubating oocytes in ND96 containing 100 µmol l^–1 alanine (specific activity, 0.37 kBq nmol^-1; total activity per 200 µl assay = 187 kBq). Preliminary experiments showed that transport rates of [¹⁴C]alanine at a concentration of 100 µmol l^–1 were linear for over 1 h, so in subsequent experiments oocytes were incubated with [¹⁴C]alanine for 40 min. Transport assays were terminated by rinsing each oocyte four times with 10 ml of Na⁺-free ND96 (96 mmol l^–1 choline-Cl, 2 mmol l^–1 KCl, 1 mmol l^–1 MgCl₂, 1.8 mmol l^–1 CaCl₂, 5 mmol l^–1 HEPES, pH 7.5). Oocytes were dissolved with 0.5 ml of 2.5% sodium dodecyl sulfate for 24 h, and the total radioactivity in each sample measured with quench-corrected, liquid scintillation counting. Transport rates were calculated on the basis of the total radioactivity in each oocyte, the specific activity of [¹⁴C]alanine in the transport medium, and the elapsed time (pmol Ala oocyte^-1 h^–1). In parallel with the above assays, separate experiments in which Na⁺-free ND96 was substituted for standard ND96 medium (with Na⁺) were conducted to evaluate the sodium dependency of the induced amino acid transport activity in frog oocytes from injected sea urchin genes.

Localization of Sp-AT1 expression in larval tissues
To visualize the tissue-specific expression of one of these transporter genes in larvae, antibodies were raised against synthetic peptides designed to match a 20 amino acid residue region of high antigenic index (Jameson and Wolf, 1988) within a predicted extracellular domain of Sp-AT1. This synthetic peptide antigen (RPSEEYWEENVLRQSQSMND) was used to raise rabbit-anti-Sp-AT1 polyclonal antibodies (Bio-Synthesis, Lewisville, TX). Polyclonal antibodies were purified from crude serum by affinity chromatography using the same peptide antigen (Bio-Synthesis, Lewisville, TX).

The specificity of protein binding for this polyclonal antibody preparation was verified by immunoblots of protein (Western blots) extracted from embryos (4-cell-stage) and larvae (4-d-old) of S. purpuratus according to standard methods (Ausubel et al., 1994). Briefly, 10 µg of total protein from each developmental time point was electrophoresed (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose membranes (Amersham Pharmacia, San Francisco, CA). Membranes were blocked with bovine serum albumin (BSA) and incubated with the anti-Sp-AT1 primary antibody at 0.5 µg ml^-1. Membranes were washed and incubated with a 1:10,000 dilution of alkaline phosphatase (AP)-conjugated secondary antibody AP-goat-anti-rabbit-IgG (Sigma, St. Louis, MO), and then washed and visualized with Western-Blue AP substrate solution (Promega, Madison, WI).

The tissue-specific expression of the Sp-AT1 amino acid transporter in larvae was assessed by whole-mount antibody staining of pluteus-stage larvae. Larvae were preserved with buffered formalin (4%, pH 7.5). Larvae preserved using the above procedure were rinsed with filtered seawater and incubated in TBST/BSA (20 mmol l^–1 Tris-HCl, 150 mmol l^–1 NaCl, 0.05% Tween-20, 0.5% BSA, pH 7.5) for 1 h to block nonspecific antibody binding. Preserved larvae were incubated with the anti-Sp-AT1 primary antibody at 100 µg ml^-1 for 1 h. Larvae were then rinsed in TBST and incubated for 1 h in TBST/BSA containing the alkaline phosphatase (AP)-conjugated secondary antibody AP-goat-anti-rabbit-IgG (1:10,000 dilution). Larvae were incubated in Western-Blue AP substrate solution (Promega, Madison, WI), and the sites of antibody binding visualized by light microscopy.

Autoradiographic analysis of amino acid transport
Autoradiography was used to identify the tissues involved in transport of dissolved amino acids from seawater by sea urchin larvae. Details of the methods used to expose larvae to radioactive amino acids and prepare sections of larvae for autoradiographic analysis are given in Manahan and Crisp (1983). Briefly, for the current studies pluteus-stage larvae were exposed for 100 min to [³H]alanine added to seawater at a concentration of 1 µmol l^–1. Following fixation and dehydration through a graded series of alcohol washes (final being 100% ethanol), larvae were embedded in Spurr's epoxy resin and serially sectioned (1 µm thickness). Alternate sections were either stained with Richardson's stain for light microscopy or exposed to autoradiographic emulsion (Kodak Nuclear Track Emulsion) to visualize tissues containing [³H]alanine.

Measurement of alanine transport rates during sea urchin development
Maximum alanine transport capacities were measured throughout early development according to previously published methods (Manahan et al., 1989). Transport rates were measured by suspending embryos or larvae at 1000 individuals ml^-1 in 0.2-µm (pore size) filtered seawater (FSW) containing 100 µmol l^–1 [¹⁴C]alanine (specific activity, 14 kBq µmol^-1; total activity, 19 kBq per 10-ml assay). Samples were collected at 2-min intervals and washed on 8.0-µm (pore size) polycarbonate filters using FSW and vacuum filtration. Tissue was solubilized by overnight digestion in 0.5 ml Scintigest (Fisher Scientific, Hampton, NH), and the total activity of each sample was measured by quench-corrected liquid scintillation counting. Transport rates were calculated on the basis of the slopes of regressions of [¹⁴C]alanine content per larva against elapsed time. Preliminary measurements using embryos (4-cell-stage) and larvae (4-d-old) from four independent cultures confirmed that transport rates at 100 µmol l^–1 [¹⁴C]alanine were equivalent to those at 200 µmol l^–1 concentrations. This comparison supports the use of 100 µmol l^–1 for measuring maximal transport capacities. Alanine transport rates were subsequently measured at this saturating substrate concentration at multiple developmental stages (0–4 d) in an additional four independent cultures of sea urchins.

A separate set of transport rate measurements was conducted to confirm the previously reported broad substrate specificity of amino acid transport systems in developing sea urchins (e.g., Epel, 1972; Allemand et al., 1984) in our experimental material. Pluteus-stage larvae of S. purpuratus (7-d-old) were suspended in FSW containing 50 µmol l^–1 [¹⁴C]alanine alone, or the same concentration of [¹⁴C]alanine with either 500 µmol l^–1 glycine or 500 µmol l^–1 serine. Transport assays in the presence or absence of these competing amino acid substrates were conducted essentially as described above.

A separate series of experiments was undertaken to confirm the physiological activity of the Sp-AT1 gene in larvae. The hypothesis tested was that the antibody specific to Sp-AT1 should selectively inhibit alanine transport but not the transport of other non-amino acid substrates by sea urchin larvae (glucose and uridine). These non-amino-acid substrates were selected on the basis that they are known to be transported by different transport systems than amino acids. For these inhibition assays, pluteus-stage larvae (6-d-old) were incubated with and without anti-Sp-AT1 antibody (100 µg ml^-1 in FSW). The rates of transport of alanine, glucose, and uridine in the presence and absence (control) of antibody were conducted as described above. Larvae were enumerated and suspended at 1000 larvae ml^-1 in FSW with one of three different substrates: [¹⁴C]alanine at 5.7 or 19 µmol l^–1; [¹⁴C]glucose at 3.1 µmol l^–1; or [³H]uridine at 0.42 µmol l^–1. Transport rates were calculated from the resulting regressions. To assess the effects of anti-Sp-AT1 antibodies on transport rates of alanine, glucose, and uridine, the slopes of regressions obtained from these treatments (presence and absence of antibody) were compared by analysis of variance (ANOVA).

Analysis of Sp-AT1 and Sp-AT2 gene expression during development
Embryos and larvae collected throughout early development (0–4 d) for RNA analysis were homogenized in denaturing solution (4 mol l^–1 guanidinium thiocyanate, 25 mmol l^–1 sodium citrate pH 7.0, 0.1 mol l^–1 β-mercaptoethanol, and 0.5% sodium sarcosyl) using a rotor-stator homogenizer (Tissue-Tearor, Biospec Products, Bartlesville, OK). Homogenates were immediately frozen by immersion in liquid nitrogen and stored at –80 °C. RNA was extracted using the RNEasy kit (Qiagen) according to the manufacturer's instructions. RNA was eluted and stored in 1x MOPS/RNasin (40 mmol l^–1 3-[n-morpholino]-propanesulfonic acid, 10 mmol l^–1 sodium acetate, 1 mmol l^–1 EDTA, 100 U ml^-1 RNasin, pH 7.0).

Quantified RNA samples from each developmental stage sampled were electrophoresed under denaturing conditions (1% agarose, 6% formaldehyde, 1x MOPS buffer; Ausubel et al., 1994). Preliminary RNA blot analyses of embryos and larvae revealed that Sp-AT1 transcripts were more abundant than Sp-AT2, so 5 µg of total RNA per sample was used for analysis of Sp-AT1 and 10 µg for Sp-AT2. Gels were stained with ethidium bromide to verify RNA integrity and equal loading. Intensity of ethidium bromide-stained 18S rRNA bands was quantified with the software ImageJ ver. 1.38x (National Institutes of Health) and used to normalize subsequent analyses of ³²P-labeled band-density in RNA blots (hybridization of ³²P-labeled DNA probes, details below). RNA was transferred onto nylon membranes (Brightstar-Plus, Ambion, Austin, TX) according to standard downward capillary-transfer methods (Ausubel et al., 1994). Gels and membranes were visualized under UV light to verify complete transfer of RNA. RNA was crosslinked to membranes by exposure to UV light (Stratalinker, Stratagene, La Jolla, CA), and membranes were stored at –80 °C.

Radiolabeled probes for RNA blot analysis (Northern blots) of Sp-AT1 and Sp-AT2 transcript abundance were prepared from cDNA clones. DNA templates for radiolabeling of probes were obtained by restriction digests. The enzymes chosen for Sp-AT1 were AatII and PstI, resulting in a 889-bp fragment corresponding to a region within the ORF. For Sp-AT2, the enzyme used was NcoI, resulting in a 939-bp fragment corresponding to a region within the ORF. Following restriction digestion, templates were gel purified using the Qiaquick gel extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Random-primed radiolabeled DNA probes were then synthesized using the Prime-A-Gene kit (Promega, Madison, WI). To radiolabel each probe, 1.85 MBq of ³²P-dCTP was used per 50-µl reaction volume (Perkin-Elmer, Wellesley, MA). A molecular-weight marker template (Millennium Marker Template, Ambion, Austin, TX) was also used to generate probes that would hybridize with markers of known molecular weight in each gel. Following synthesis and purification of probes according to the manufacturer's instructions, the radioactivity of each probe was quantified by liquid scintillation counting.

RNA blots were pre-hybridized at 42 °C for 1 h in Ultrahyb hybridization solution (Ambion, Austin, TX) according to the manufacturer's instructions. Probes for Sp-AT1 and Sp-AT2 were added to achieve final activities of 17 kBq ml^-1. Molecular-weight marker probes were added to a final activity of 0.8 kBq ml^–1. Hybridizations were conducted overnight at 42 °C in a rotary hybridization oven (Bambino, Midwest Scientific, St. Louis, MO). Blots were washed, wrapped in plastic, and exposed to PhosphorImager imaging plates (Amersham Biosciences, Piscataway, NJ) from which digital images were obtained with an FX Molecular Imager (Bio-Rad, Hercules, CA). Measurements of radioactivity (dpm) with PhosphorImager systems are linear over a wide dynamic range compared to X-ray film (see manufacturer's instructions). Band densities (as pixels) were quantified using ImageJ (NIH) and then normalized to the amount of 18S rRNA to correct for any minor differences in RNA loading among different samples. These mRNA analyses of the expression of Sp-AT1 and Sp-AT2 were performed in duplicate for each developmental stage sampled.

	Results

TOP Abstract Introduction Materials and Methods Results Discussion Literature Cited

 
DNA sequence analysis of putative amino acid transporters
Open reading frames (ORFs) were identified within the nucleotide sequences of sea urchin genes Sp-AT1, Sp-AT2, and Sp-AT3. The deduced amino acid sequences are shown in Figure 1. The gene Sp-AT1 contains 164 bp of 5' untranslated region (UTR), a 149-bp 3' UTR, and a 1329-bp ORF encoding a protein calculated to be 48.8 kDa in molecular weight. Sp-AT2 contains a 407-bp 5' UTR, a 447-bp 3' UTR, and an 1857-bp ORF encoding a 69.6-kDa protein. Sp-AT3 contains a 130-bp 5' UTR, an 899-bp 3' UTR, and an 1821-bp ORF encoding a 67.5-kDa protein. The topology models predicted for the Sp-AT genes, based on the classical Kyte-Doolittle hydrophobicity plot (Kyte and Doolittle, 1982) and the consensus-based ConpredII method (Arai et al., 2004), contain multiple transmembrane domains (n = 9–13), intracellular N-termini, extracellular C-termini, and large (68–110 amino acid residues) extracellular domains between transmembrane helices 3 and 4 for all three clones (Fig. 2). A number of putative glycosylation sites were identified on the basis of amino acid sequences (four sites per gene, on average), and the majority of these were located between transmembrane helices 3 and 4. These analyses reveal similarities in sequence and structure between the Sp-AT genes and amino acid transporter genes in other organisms (Saier, 2000).

View larger version (82K): [in this window] [in a new window]   Figure 1. Multiple sequence alignment comparing amino acid sequences of the sea urchin (Strongylocentrotus purpuratus) transporter genes Sp-AT1, Sp-AT2, and Sp-AT3 with the human glycine transporter gene (hs_GLYT2; AAD27892 ). Amino acids are represented using single letter codes. Gaps in the alignment are indicated by dashes. Black boxes denote identical amino acids, and grey boxes denote similar amino acids (conservative substitution of a similar amino acid group). Solid horizontal lines indicate conserved regions of the protein from which oligonucleotide probes were designed for selection of clones from a cDNA library prepared from early stage embryos.

View larger version (42K): [in this window] [in a new window]   Figure 2. Hydrophobicity plots of sea urchin transporter genes Sp-AT1, Sp-AT2, and Sp-AT3. Hydropathy was calculated with a window size of 19 residues. Vertical grey bars overlaid on the hydropathy plot indicate the transmembrane helices predicted by the consensus-based ConPredII method.

The above sequence comparisons support the annotation of Sp-AT1, Sp-AT2, and Sp-AT3 as members of the SNF gene family. To resolve their position within this family, a phylogenetic analysis was conducted in which these sea urchin genes were compared with 34 other members of the SNF gene family (Fig. 3). This analysis revealed that Sp-AT1 is most similar to the amino acid transporter subfamily, a group that includes the human GLYT2 and ATB0+ amino acid transporter genes (accession numbers: NP_004202 and NP_009162, respectively). Bootstrap support for this clade was reasonably high (78%). Sp-AT2 clustered with the C. elegans SNF6 gene (accession number: NP_497416) with weak (50%) bootstrap support. Sp-AT3 was most similar to the D. melanogaster inebriated gene (accession number: NP_477012) with 100% bootstrap support for this node. These analyses confirm the close relationship between the Sp-AT genes and the SNF gene family that was suggested by BLAST searches, and highlight the sequence diversity among the genes identified in the current study.

Testing of physiological function by heterologous gene expression
Significant differences in alanine transport rates were observed between oocytes of Xenopus laevis injected with cRNA encoding either Sp-AT1, Sp-AT2, or Sp-AT3, and control oocytes injected with nuclease-free water (Fig. 4). Oocytes injected with Sp-AT1, Sp-AT2, and Sp-AT3 showed alanine transport rates (given as values plus or minus the standard error) of 570 ± 23, 603 ± 28, and 642 ± 23 pmol alanine oocyte^–1 h^–1, respectively, while the corresponding nuclease-free-water-injected controls showed transport rates of only 14 ± 1.5 pmol alanine oocyte^–1 h^–1. These results demonstrate a greater than 40-fold increase in alanine uptake rates of cRNA-injected oocytes relative to those measured in controls (Student's one-tailed t-test; P < 0.001 for each comparison between cRNA-injected and control oocytes). Oocytes injected with a partial Sp-AT2 construct (deletion of a 51-bp region from the 5' end of the ORF) transported alanine at rates that were not significantly different from those of water-injected controls (t-test; P > 0.05), supporting the conclusion that the transport activity induced by injection of full-length cDNA constructs is a specific property of the genes injected.

View larger version (14K): [in this window] [in a new window]   Figure 4. Alanine transport rates measured in oocytes of Xenopus laevis injected with nuclease-free water (control: H2O) or cRNA encoding sea urchin genes Sp-AT1, Sp-AT2, and Sp-AT3. Values shown represent means of replicate measurements each made on individual oocytes (n = 10) ±s.e.m.

Localization of Sp-AT1 expression in larval tissues
The major morphological features of the larval stage studied can be seen in the light photomicrograph of a whole-mount pluteus-stage larva (Fig. 5A). In the larval section shown in Figure 5B, the general tissue staining reagent (Richardson's) allows visualization of the same morphological features in a sectioned larva. An autoradiogram of the location of [³H]alanine transported from seawater by larvae is shown in Figure 5C. In this autoradiogram both oral and aboral ectoderm show heavy labeling, visualized as white silver grains under dark-field illumination. Exposure of larvae to the anti-Sp-AT1 antibody added to seawater revealed specific binding of this antibody to oral and aboral ectoderm (Fig. 5D, E). Figure 5D shows the control measurement of larvae that were exposed to only the secondary antibody and visualized by the presence of alkaline phosphatase. As expected, the endogenous alkaline phosphatase in the digestive system of larvae yielded a positive reaction (dark purple staining in stomach, ‘st', seen as darkened region in black-and-white photomicrograph) to the substrate used to visualize the secondary antibody. Figure 5E shows larvae that were exposed to the primary antibody (anti-Sp-AT1) and subsequently reacted with the secondary antibody. The location of the amino acid transporter proteins are seen as the appearance of the dark purple staining of alkaline phosphatase in oral and aboral ectoderm of the whole larvae (Fig. 5E, seen as darkened region in black-and-white photomicrograph). These antibody labeling experiments show that Sp-AT1 gene product is located in the ectoderm of sea urchin larvae, which autoradiography data indicate are the same tissues involved in transport.

View larger version (110K): [in this window] [in a new window]   Figure 5. Sites of amino acid uptake and localization of Sp-AT1 expression in larvae of Strongylocentrotus purpuratus. Key to labels: a, arms; ae, aboral ectoderm; e, esophagus; m, mouth; oe, oral ectoderm; sk, skeletal rods; st, stomach. (A) Photomicrograph of intact pluteus-stage larva viewed by dark-field light microscopy, illustrating relevant morphological features. Scale bar is 200 µm. (B) Section (1 µm) of larva viewed by light microscopy. (C) Autoradiogram of larva that had been incubated with [3H]alanine (1-µm section shown). Tissues containing [3H]alanine visualized as white silver grains under dark-field illumination. (D) Whole-mount of larvae incubated with only the secondary antibody (anti-rabbit-IgG), but without the primary antibody (control). Purple regions (dark in black-and-white image) in the stomach (st) and digestive tract result from activity of endogenous alkaline phosphatase associated with the secondary antibody assay. An original uncropped photomicrograph is shown, where two larvae are seen in their entirety and a third is partially visible near the top left corner of ‘D’. (E) Whole-mount of larvae incubated with both the primary antibody (anti-Sp-AT1) and the secondary antibody. Purple regions (dark in black-and-white image) indicate the localization of the transporter protein (Sp-AT1) in ectoderm, as detected by primary and secondary antibody binding. An original photomicrograph is shown: two larvae are seen in their entirety, and a third is partially visible near the top right corner of ‘E’.

View larger version (22K): [in this window] [in a new window]   Figure 6. (A) Specific inhibition of in vivo alanine transport rates in pluteus-stage larvae of Strongylocentrotus purpuratus by anti-Sp-AT1 antibody. Values shown represent the amount of [14C]alanine transported in the presence and absence of the Sp-AT1 antibody. Open circles represent larvae assayed in the presence of anti-Sp-AT1 antibody (100 µg ml–1); filled circles represent control larvae assayed in the absence of this antibody. (B) Immunoblots of 10-µg samples of protein from embryos (5 h) and larvae (4 d) using the same anti-Sp-AT1 antibody. The single band at 66 kDa was visualized with a colorimetric alkaline phosphatase substrate.

View larger version (12K): [in this window] [in a new window]   Figure 7. Alanine transport during development of Strongylocentrotus purpuratus. (A) Competitive inhibition of [14C]alanine transport by larvae of S. purpuratus in the presence of molar excess glycine or serine. Values shown represent alanine transport rates measured with either 50 µmol l–1 [14C]alanine alone, white bar; 50 µmol l–1 [14C]alanine with 500 µmol l–1 unlabeled glycine (‘+Gly’); or 50 µmol l–1 [14C]alanine with 500 µmol l–1 unlabeled serine (‘+Ser’). (B) Comparison of maximum alanine transport rates (Jmax) measured at different substrate concentrations (white bars = 100 µmol l–1 alanine; shaded bars = 200 µmol l–1 alanine). Values (±s.e.m.) shown represent mean transport rates for developmental stages from three cultures (gametes from different adults). (C) Maximum alanine transport rates during development of embryos and larvae. Transport of 14C-alanine at 100 µmol l–1 (Jmax) was measured throughout early development for four independent cultures (each type of symbol represents a different culture; n = 16 different sets of transport assays). Values shown represent transport rates and standard errors calculated from regressions of accumulated alanine against elapsed time (n = 7–9 time points per assay; see Materials and Methods). Filled symbols (black squares) represent transport rate measurements conducted on the same culture of animals for which transcript abundances of Sp-AT genes were quantified (Figs. 8, 9); other open symbols represent different cultures.

Having shown that 100 µmol l^–1 alanine was sufficient to produce maximum transport rates, a more detailed analysis was undertaken to define changes in maximum transport capacities at 100 µmol l^–1 throughout embryonic and larval development (Fig. 7C). This developmental increase in transport capacity was described by the equation: transport rate (pmol alanine individual^–1 h^–1) = 2.08*Age + 1.61 (ANOVA of linear regression: R² = 0.96, P < 0.001). The slope of this regression provides a quantitative measure of the increase in maximum alanine transport rates during development of 2.08 ± 0.11 pmol alanine individual^–1 h^–1 per day of development. As is evident from Figure 7C, maximum transport rates obtained from this series of measurements are highly reproducible between the seven independent cultures tested and represent maximum alanine transport rates for early developmental stages of S. purpuratus.

Ontogenetic changes in Sp-AT1 and Sp-AT2 expression
For comparison of the developmental change in physiological transport rates and levels of transporter gene expression, RNA was extracted from the same cultures of embryos and larvae for which the transport rates are shown as filled symbols (black squares) in Figure 7B. RNA blot analysis (Northern blot) of these samples revealed that the ³²P-labeled probe for Sp-AT1 hybridized specifically to a single transcript of 5.3 kb in molecular weight (Fig. 8A). The density of this band decreased during embryonic development to the pluteus-stage larvae (0–4 d). Band densities were quantified and normalized to the amount of 18S rRNA (Fig. 8B). These analyses revealed that maximal expression was found in unfertilized eggs, followed by a rapid decline to 57% of this initial abundance within the first 5 h of development (Fig. 8C). The abundance of Sp-AT1 transcript remained relatively constant throughout the remaining period of early development to the 4-d-old larval stage.

View larger version (33K): [in this window] [in a new window]   Figure 8. Changes in Sp-AT1 transcript abundance during early development (up to 4 d) of the sea urchin Strongylocentrotus purpuratus, as measured with RNA blots. (A) Numbers at top of figure represent the age of embryos and larvae (days post-fertilization) from which RNA was extracted. Digital image of an RNA blot produced by hybridization with a [32P]-labeled DNA probe for Sp-AT1 and exposure to a PhosphorImager plate. Band shown is 5.3 kb in molecular weight. (B) Ethidium-bromide-stained 18S rRNA band obtained from the RNA gel used for the blot shown in A. (C) Quantified Sp-AT1 band densities from duplicate RNA blots, normalized to the amount of 18S rRNA in each lane.

View larger version (32K): [in this window] [in a new window]   Figure 9. Changes in Sp-AT2 transcript abundance during early development (up to 4 d) of the sea urchin Strongylocentrotus purpuratus, as measured with RNA blots. (A) Numbers at top of figure represent the age of embryos and larvae (days post-fertilization) from which RNA was extracted. Digital image of an RNA blot produced by hybridization with a [32P]-labeled DNA probe for Sp-AT2 and exposure to a PhosphorImager plate. Band shown is 8.9 kb in molecular weight. (B) Ethidium-bromide-stained 18S rRNA band obtained from the RNA gel used for the blot shown in A. (C) Quantified Sp-AT2 band densities from duplicate RNA blots, normalized to the amount of 18S rRNA in each lane.

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion Literature Cited

Sequence analyses revealed similarities between the cDNA clones characterized in this study (Sp-AT1, Sp-AT2, and Sp-AT3) and transporter genes previously described in other species. Public database searches revealed similarities with the sodium-dependent neurotransmitter transporter gene family SNF (also known as Solute Carrier Family 6, or SLC6). Phylogenetic analyses have revealed at least six subfamilies within this gene family, including clades specific for -aminobutyric acid, monoamine neurotransmitters, or amino acids, as well as other clades specific to certain taxa (e.g., insects or C. elegans) (Chen et al., 2004; Boudko et al., 2005; Thimgan et al., 2006). Protein distance analyses comparing Sp-AT1, Sp-AT2, and Sp-AT3 with a representative sampling of SNF genes reveals the close relationship between these sea urchin genes and those previously characterized SLC6 sequences (Fig. 3). These sea urchin genes each clustered with a different subfamily in this analysis: Sp-AT1 with the amino acid transporter subfamily that includes GLYT1 and ATB0+ (78% bootstrap support), Sp-AT3 with the arthropod inebriated gene (100% bootstrap support), and Sp-AT2 with the C. elegans gene SNF6, although bootstrap support for this last grouping was equivocal (50%).

The finding of an inebriated ortholog (Sp-AT3; Fig. 3) among these cDNA clones was unexpected since these genes have previously been identified only in arthropods (Boudko et al., 2005). This SNF gene is associated with osmoregulation and ion transport, and is expressed in the central nervous system of insects (Chiu et al., 2000) although its substrate remains unknown and a transport function has not been demonstrated in insects. For the sea urchin inebriated homolog Sp-AT3, our findings demonstrate that this gene is capable of mediating alanine transport when expressed in Xenopus oocytes (Fig. 4). This result does not imply that alanine is the preferred substrate for the gene in vivo, but the finding of a transport function for an inebriated homolog remains noteworthy and suggests that further investigation into echinoderm inebriated genes might help to elucidate the physiological roles of these genes across taxa.

The recent publication of the genome sequence for S. purpuratus (Sea Urchin Genome Sequencing Consortium et al., 2006) prompted a further comparison of the genes characterized in the present study (cDNA) with the coding sequences predicted by computational analysis of the sea urchin genome. The Sp-AT1, Sp-AT2, and Sp-AT3 cDNA clones were each compared with the corresponding genomic region (accession numbers: NW_001347949, NW_001354582, and NW_001469296, respectively). For each genomic region, the Sp-AT gene protein sequence was compared with the coding sequence predicted by each of two methods: Gnomon, the gene prediction method used by the National Center for Biotechnology Information; and the FGENESH+ gene prediction model (Softberry Inc., Mount Kisco, NY). The Sp-AT clones matched FGENESH+ predictions closely, to within 24 amino acid residues. Larger discrepancies were found between the Sp-AT clones and Gnomon predictions. The Gnomon-predicted protein XP_001179122 is 271 residues longer than Sp-AT1, XP_001177914 is 81 residues longer than Sp-AT2, and XP_001196519 is 24 residues longer than Sp-AT3. These discrepancies, in the context of the functional data presented for the cDNA clones described here, emphasize the need for more accurate prediction and experimental analysis of genes that contribute to physiological processes.

The open reading frames (ORF) predicted for the Sp-AT1, Sp-AT2, and Sp-AT3 cDNA clones ranged from 442 to 618 amino acid residues, similar to the range found across the eight families of amino acid transporter genes expressed in animals (300–710 residues for these families; Saier, 2000). The Sp-AT1 ORF is slightly smaller than the 600–700 residues characteristic for the SNF gene family (Saier, 2000). The predicted transmembrane topologies for Sp-AT1, Sp-AT2, and Sp-AT3 contain 9, 11, and 13 transmembrane helices, respectively (Fig. 2). These predicted topologies differ markedly from the well-conserved pattern of 12 transmembrane helices characteristic of the SNF gene family, although they are consistent with the topologies reported among the eight amino acid transporter gene families expressed across animal phyla (6–15 transmembrane helices: Saier, 2000). Despite these unusual features, the cDNA clones characterized in this study are thought to represent complete, functional ORFs for these genes, because they induce alanine transport when expressed in a null system (Fig. 4) and contain characteristic structural features such as the large extracellular loop between transmembrane helices 3 and 4 (Fig. 2) that is well conserved in this gene family (Chiu et al., 2000).

Gene sequences are frequently used to infer putative functions, but direct tests of these assumptions are needed for understanding the molecular biological bases of specific physiological processes. In this study, different DNA constructs, each containing one of the three sea urchin genes cloned from a cDNA library prepared from embryos of S. purpuratus, were used to test physiological function. This was achieved by heterologous expression of sea urchin transporter genes in frog oocytes. These experiments showed that Sp-AT1, Sp-AT2, and Sp-AT3 mediate alanine transport (Fig. 4). Antibody staining revealed that the transporter protein encoded by Sp-AT1 is expressed in both oral and aboral ectoderm of larvae (Fig. 5E), a finding that matches well with the results of autoradiographic analyses which showed that alanine transported from seawater accumulated in these same ectodermal tissues (Fig. 5C). A further testing of transporter function in larvae was undertaken using the anti-Sp-AT1 antibody to inhibit alanine transport; in the presence of antibody, transport was inhibited by 50% (Fig. 6). Since the other transporter genes identified in this study (Sp-AT2, Sp-AT3) are also expressed in larvae at this developmental stage, the residual transport activity (i.e., the remaining 50%) from these inhibition experiments could be explained in part by their activity. Further experiments with these antibodies might allow estimation of the quantitative contribution of these different genes to the overall activity. The inhibition data shown here confirm the activity of the Sp-AT1 transporter protein in larvae. The sodium dependency of these sea urchin genes is consistent with the extensive literature showing that integumental amino acid transport systems in echinoids and marine invertebrates, in general, are sodium-dependent (Epel, 1972; Davis et al., 1985; Wright, 1988b; Preston, 1993; Gomme, 2001). The amino acid substrate used in our transport assays was alanine, but this does not imply that alanine is the preferred substrate for these transporter genes in vivo. Alanine was chosen because it is a substrate that is taken up by the well-characterized neutral amino acid transport systems of developing echinoderms (Tyler, 1966) and is commonly used to measure the activity of neutral amino acid transport systems in marine invertebrates (Gomme, 2001). The results presented here do not describe the full range of substrates taken up by these transporters, but demonstrate that the Sp-AT genes can transport the "model" substrate alanine in a sodium-dependent fashion. These findings support the conclusion that Sp-AT1, Sp-AT2, and Sp-AT3 represent components of the sodium-dependent neutral amino acid transport system(s) of developing sea urchins described in previous reports (Epel, 1972; Manahan et al., 1983, 1989).

Three different amino acid transporter genes are expressed during development of the sea urchin S. purpuratus (Fig. 1), highlighting the complexity of the relationship between changes in gene expression and physiological transport rates. Of the three genes cloned from the cDNA library screen, genes Sp-AT1 (Fig. 8) and Sp-AT2 (Fig. 9) were both expressed at levels detectable by analyses of RNA blots. Similar blots for the third amino acid transporter gene (Sp-AT3) cloned showed that this gene is present early in development, but nonspecific hybridization on blots prevented quantitative analysis of changes in its expression. There were differences in the levels of expression of Sp-AT1 and Sp-AT2 during development. Preliminary analyses revealed that a larger amount of total RNA was required to produce a detectable signal for Sp-AT1 than for Sp-AT2. The signal intensity obtained for Sp-AT2 from analysis of 10 µg of RNA was about 17-fold lower than the signal obtained from half that amount of RNA for Sp-AT1, suggesting that Sp-AT1 transcripts are about 30 times more abundant in unfertilized eggs. Sp-AT2 increased in expression during development and by the larval stage (4-d-old) had increased to half the level of Sp-AT1.

The increases in maximal transport rates (J_max) measured in the present study (Fig. 7) are in close agreement with those previously reported for early embryos (Epel, 1972) and larvae (Manahan et al., 1989) of S. purpuratus. For example, Manahan et al. (1989) reported a J_max value for alanine transport by 4-d-old larvae of 12.3 pmol larva^–1 h^–1, similar to the value reported in the present study of 10.2 pmol larva^–1 h^–1 (Fig. 7). Previous studies also reported developmental changes in transporter affinities (K_t) for kinetics of amino acid transport. A high-affinity (K_t = 1.4 µmol l^–1) system is activated shortly after fertilization (Epel, 1972). At the larval stage, a biphasic kinetic transport system is present that contains both high- and low-affinity transporters for amino acids (K_t values of 1 and 132 µmol l^–1: Manahan et al., 1989). In this context, it is noteworthy that Sp-AT1 and Sp-AT2 are differentially expressed during development. It is possible that these expression profiles are linked to developmental changes in the kinetics of amino acid transport.

Previous studies in our laboratory on changes in ion transport (sodium pump) activities during development of S. purpuratus showed that increases in activities of Na⁺, K⁺-ATPase (Leong and Manahan, 1997) lagged behind developmental increases in gene expression (amount of mRNA: Marsh et al., 2000). In the current study, amino acid transport rates similarly increased during development, and multiple genes capable of mediating alanine transport were expressed. During the developmental period investigated in the current study (unfertilized egg to 4-d-old larval stage: Figs. 8, 9), there was a 6-fold increase in alanine transport capacity. This change in physiological transport contrasts with the changes measured in expression of the amino acid transporter genes, where Sp-AT1 decreased by 55% (Fig. 8) and Sp-AT2 increased 11-fold (Fig. 9). This contrast between mRNA abundance and physiological rates of an organic-substrate transporter has been observed in other species. For example, ovine intestinal transport of glucose by the Na⁺-dependant transporter SGLT1 increased by 60–90-fold upon infusion with glucose, while mRNA levels for this gene increased by only about 2-fold in this same treatment (Lescale-Matys et al., 1993). This comparison suggests that multiple mechanisms at the molecular biological level (e.g., transcriptional, translational, and post-translational regulation) are involved in regulating transport rates. Additionally, our observation that gene expression for amino acid transport changes during sea urchin development (Figs. 8, 9) is consistent with other reports of transport physiology in mammalian embryos (Van Winkle, 2001). During mammalian development, changes in the utilization of metabolic substrates are thought to result from the energetic demands of cell division (e.g., macromolecular synthesis; Martin, 2000). Changes in the expression of other transporter genes for amino acids and sugars also occur during mammalian development (Ito and Groudine, 1997; Pantaleon et al., 2001). Mammalian glucose transporters provide another example, where GLUT1 is expressed at high levels during fetal stages of mouse development, while expression of a second glucose transporter gene (GLUT4) is initiated post-natally (Santalucia et al., 1992).

The finding that multiple amino acid transporter genes are expressed during development of S. purpuratus is supported by preliminary experiments we have undertaken with other species of sea urchins. Partial clones of putative amino acid transporter genes have been obtained from developmental stages of two other urchin species, based on a polymerase chain reaction analysis using oligonucleotide primers designed from the same conserved regions as those used for probe design in the present study (Fig. 1). For embryos of the temperate urchin Lytechinus pictus, three distinct clones were obtained on the basis of deduced amino acid sequences (ranging from 52% to 56% similarity in amino acid sequence between these clones). Comparison of the deduced amino acid sequences of the three cDNA clones from L. pictus with the amino acid transporter genes from S. purpuratus revealed a high sequence similarity for one clone (99% similarity between Sp-AT2 and a clone from L. pictus). In another species, the Antarctic sea urchin Sterechinus neumayeri, two distinct clones were present in embryos (with 45% amino acid sequence similarity between these clones). These preliminary results suggest that expression of multiple amino acid transporter genes during development may be widespread among different sea urchin species. Further experiments using cloned genes should provide novel insights into the evolutionary relationships of amino acid transporters in sea urchins, and the physiological roles of transporter proteins during development in diverse environments.

This identification of amino acid transporter genes in S. purpuratus represents a substantial increase in our understanding of how developmental stages of marine invertebrates transport amino acids from seawater. These findings demonstrate that amino acid transporter genes are expressed in developing sea urchins and that the genes involved are closely related to genes associated with synaptic neurotransmitter transport (SNF family). This relationship with gene families involved in neuronal signaling is worth noting, since Sp-AT1, Sp-AT2, and Sp-AT3 were cloned from early embryonic stages lacking a nervous system (i.e., 4-cell-stage embryos). The SNF gene family serves a number of well-characterized physiological roles in diverse organs and tissues, including the nervous, renal, and digestive systems (Malandro and Kilberg, 1996; Palacin et al., 1998; Saier, 2000). In contrast, the expression of these genes in the ectoderm of developing sea urchins (e.g., Sp-AT1 in Fig. 5D, E) represents a context in which the physiological roles of these genes have not been characterized. In sea urchin larvae, the transporter proteins are located in ectodermal tissues that are in direct contact with seawater, facilitating the transport of organic nutrients from the environment. In these developmental stages of sea urchins, amino acid transport can play an important nutritional role (Manahan, 1990). Osmoregulatory roles for members of this gene family have been shown to be crucial for survival of mammalian embryos (Van Winkle, 2001; Steeves et al., 2003), suggesting this as another possible physiological role during sea urchin development—that is, maintenance of high intracellular concentrations of organic osmolytes (Wright and Secomb, 1986; Gomme, 2001). As a neurotransmitter transporter gene family, SNF genes also play a major role in neural signaling and sensory physiology. Small molecular weight organic compounds dissolved in seawater are known to serve as chemical cues for fertilization, for aspects of larval behavior, and as inducers of metamorphosis (Zimmer-Faust and Tamburri, 1994; Riffel et al., 2002; Hadfield and Koehl, 2004). Some of these chemical cues are amino acids or their derivatives. The expression of SNF transporter genes in sea urchin embryos and larvae, along with receptors yet to be identified, suggests a possible role for the SNF gene family in chemosensory processes in marine organisms.

In conclusion, the findings reported here demonstrate that the sea urchin genes Sp-AT1, Sp-AT2, and Sp-AT3 encode functional amino acid transporters and are expressed during embryonic and larval stages of development. Ontogenetic increases in maximum alanine transport rates correspond to changes in the expression of Sp-AT genes, suggesting that these genes play a role in regulating transport physiology during development. The results presented here constitute, to our knowledge, the first molecular biological and functional characterization of amino acid transporter genes in a larval form of marine invertebrates. The identification of these genes will facilitate further studies into the mechanisms underlying the activation of transport systems at fertilization (Epel, 1972) and known ontogenetic changes in transport kinetics during later larval development (Manahan et al., 1989). These findings constitute an essential step toward understanding the mechanisms underlying the physiological role and regulation of amino acid transport processes during development of marine invertebrates.

	Acknowledgments

 
We are especially grateful to Dr. Eric H. Davidson for providing cDNA libraries and to EHD and Dr. Andy Cameron for their expert guidance in the early stages of this work. Dr. Adam G. Marsh assisted with the antibody staining experiments and some of the transport assays. D. Dimster-Denk assisted with autoradiographic analyses. Amanda Haag helped to produce the gene expression constructs, Dr. Patricia von Dippe and Andrew Nosal assisted with preliminary analysis of transporter genes in other sea urchin species. Dr. Michael Quick provided oocytes and helpful advice with many aspects of the heterologous expression experiments. This research was supported by the National Science Foundation under grant numbers 0130398 and 0412696 to DTM.

	Footnotes

 
Received 9 October 2007; accepted 22 April 2009.

^* Current address: Department of Integrative Biology, University of Texas at Austin, Austin, TX 78712.

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