in the nervous system, consistent with the specific distribution of the vertebrate calbindins [13]. Thus far, reports showing the distribution of any other calbindin subfamily members in invertebrates are lacking. At present, the parvalbumin subfamily of EF-hand domain- containing CBP is one of the best-studied CBP subfamilies. These proteins have been well characterized at the molecular expression, and structural levels [18–22]. This subfamily is characterized by having two functional EF-hand motifs that bind calcium and is made up by two major isoforms, a and b (oncomodulin). In some organisms such as zebrafish, 9 isoforms, encoded by different genes, have been identified [19]. This protein is considered to be a vertebrate specific protein, as it has not been found in non- vertebrates. Interestingly, studies done with antibodies that recognize parvalbumin showed that there is parvalbumin-like immunoreactivity in the planarian nervous system [23]. Currently not much is known about the physiological function of CBP [24], but understanding their evolution may give us a good insight into their possible function. Thus identifying and studying CBP in basal deuterostomes, such as echinoderms, may be the key to unlocking this mystery. The phylum Echinodermata comprises a distinctive group of marine invertebrates characterized by radial pentameric symmetry originating in bilaterally symmetrical larvae. Echinoderms can be further subdivided into three subphylums, Asterozoa, Echinozoa, and Crinozoa. The subphylum Asterozoa includes the Asteroidea (starfishes) and Ophiuroidea (brittlestars); the subphylum Echinozoa includes the Echinoidea (sea urchins and sand dollars) and Holothuroidea (sea cucumbers); and the subphylum Crinozoa comprises the Crinoidea (sea lilies). These organisms are part of the branch of the deuterostome evolutionary tree that gave rise to chordates [25], thus, lie at a key evolutionary position. Therefore, studying CBP in echinoderms is vital in order to understand the evolution of these proteins in the chordates. In this study we tested three antibodies that recognize EF-hand domain containing CBP of the calbindin and parvalbumin subfamilies. We identified a subpopulation of cells and fibers of the nervous system of H. glaberrima that showed immunoreactivity to these antibodies. Characterization of the antigens recognized by these markers was done by bioinformatics analyses with S. purpuratus and H. glaberrima, and western blot experiments with H. glaberrima and L. variegatus. Additionally, we used these antibodies, together with the novel neuronal marker RN1, to further characterize the nervous system component of the connective tissues in H. glaberrima. Our findings provide new markers for a subpopulation of neurons and fibers in the echinoderm nervous system. The availability of these new markers make feasible the study of the evolutionary aspects of the calcium-binding proteins family, as has been done recently with other gene family, the retinal genes in S. purpuratus [26]. Furthermore, we identified the presence of calbindin-D32k in H. glaberrima and L. variegatus, supporting its presence in at least two classes of echinoderms Our data also supports the evolutionary conservation of the calbindin subfamily, which seems to have evolved from two primitive EF-hand domain-containing CBP. One of them gave rise to cabindin-D32k in invertebrates and to calbindin 2 and calbindin 1 in vertebrates, while the other gave rise to secretagogin in both vertebrates and invertebrates. Furthermore, these proteins are preferentially distributed to the nervous system in both vertebrates and invertebrates. Materials and Methods Animals Adult Holothuria glaberrima (Selenka, 1867) (Aspidochirotida, Holothuroidea) and adult Lytechinus variegatus (Lamarck, 1816) Calbindin-D32k in the Echinoderm Nervous System (Temnopleuroida, Echinoidea) specimens were collected from the shores of the north coast of Puerto Rico. The animals were kept in sea water aquaria at the University of Puerto Rico in R´ıo Piedras. Tissue sections H. glaberrima specimens were anesthetized in 0.2% 1,1,1- trichloro-2-methyl-2-propanol (Sigma, St. Louis, MO) for 10 min and dissected by longitudinal section of the body wall. Ventral and dorsal areas of the body wall were divided into anterior, middle and posterior portions, dissected and fixed in 4% paraformaldehyde at 4uC for approximately 1 h. Tissues were rinsed 3 times for 15 min with 0.1 M phosphate-buffered saline (PBS), and left in a 30% sucrose solution at 4uC. Once the tissues had been in 30% sucrose solution for at least 24 h, they were embedded in Tissue-Tek (Sakura Finetek, Torrance, CA). Cryostat tissue sections of 14 mm were cut and mounted on Poly-L-lysine-coated slides. Immunohistochemistry The indirect immunofluorescence method was followed [27,28]. In brief, tissues were rinsed for 5 min in 0.1 M PBS, followed by one rinse of 15 min in 1% Triton X, 1 h incubation with 0.1 M Glycine, and a 1 h incubation in goat serum 1:50 (Invitrogen, Carlsbad, CA). Subsequently, the primary antibodies were incubated overnight at room temperature (Table 1). All antibodies were diluted in RIA buffer (0.05 M PBS- pH 7.4, 0.15 M NaCl, 0.5% BSA, and 1.5 mM NaN3). The primary antibodies used include the RN1 monoclonal antibody [27,29] raised against a homogenate of the radial nerve of H. glaberrima and used at a dilution of 1:100,000 in RIA buffer; the monoclonal antibody anti- b-tubulin, (Sigma T-4026 Lot. 024K4862) clone TUB 2.1 prepared against tubulin from rat brain and used at a 1:500 dilution in RIA buffer; the rabbit antiserum anti-GFSKLYamide No. 23 2i2s [30] prepared against a GFSKLYa synthetic peptide and used at a 1:1,000 dilution in RIA buffer; the rabbit antiserum anti-galanin-1 2i3s [31] prepared against galanin (Calbiochem Corp. San Diego, CA) and used at a 1:1,000 dilution in RIA buffer; the rabbit polyclonal anti-calbindin 1 (Abcam ab11426 Lot. 378854) prepared against the calbindin 1 protein purified from rat kidney and used at a 1:500 dilution in RIA buffer; the rabbit polyclonal anti-parvalbumin (Affinity Bioreagents PA1-933 Lot. 762-116) prepared against purified parvalbumin from rat skeletal muscle and diluted in RIA buffer to a 1 mg/ml concentration; the rabbit polyclonal anti-calbindin 2 (Abcam ab702 Lot. 721984) prepared against full length calbindin 2 and used at a 1/100 dilution in RIA buffer. Negative controls were performed in all experiments by incubating the tissue sections with rabbit serum during the incubation period of the primary antibody and following the normal immunostaining protocol. The secondary FITC antibodies, Goat anti-mouse (Biosource, Camarillo, CA, #AMI0408 Lot. 3501) and Goat anti-rabbit (Biosource, Camarillo, CA, #ALI0408 Lot. 1502) were used at a 1:50 dilution in RIA buffer for double-labeling indirect immuno- histochemistry. Also, the Cy3-conjugated secondary antibodies, Goat anti-mouse (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, #115-165-068 Lot. 47814) and Goat anti-rabbit (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, #111-165-144 Lot. 50694), were used at a 1:2,000 dilution in RIA buffer for double-labeling indirect immunohistochemistry. Cell nuclei were stained with 2 mM DAPI (Sigma, St. Louis, MO) in the buffered glycerol solution used to mount the slides. In cases where double-labeling was performed, the two primary antibodies were applied simultaneously and later the two secondary antibodies were added together (see [28]). Tissues were PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32689