in SOC medium (Invitrogen, Calsbad, CA) for 1 h before plating. We analyzed for positive insertion using selective plating with ampicillin and Colony PCR, using pGEX specific primers. All DNA samples were analyzed in 1% agarose gels run at 150 V for 30 min in 10 mM Sodium Borate buffer, visualized with SafeView Classic (Applied Biological Materials, Richmond, BC) staining in a ChemiDoc XRS+ System, and Sanger sequencing was use to confirm the sequences. Expression of H. glaberrima calbindin-D32k Positive colonies were grown in LB medium with 50 mg/ml ampicillin to O.D.600 1.3, and induced with 1 mM IPTG (Sigma, St. Louis, MO). Cells were lysed using B-PER Reagent (Thermo Fisher Scientific, Rockford, IL) and protein extraction was carried out using TRIzol LS Reagent (Invitrogen, Calsbad, CA) according to the manufacturers protocol for bacterial protein extraction. The fragment was purified using a B-PER GST Fusion Protein Spin Purification Kit (Pierce, Rockford, IL), and fractions were collected after lysing, washing, and elution, for a total of seven fractions. As a negative control, another protein from a different bacterial strain containing the same vector but a different insert, translationally controlled tumor protein (TCTP), was purified along with our fragment. The fractions were then analyzed via SDS-PAGE and western blot. Results Neuronal specificity The radial nerve cord (RNC) is the most prominent neural structure of the echinoderm’s nervous system. It is subdivided anatomically into two major components, the ectoneural (ERN) and the hyponeural component (HRN) (Fig. 1A). The RNC is a ganglionated nerve, with cell bodies located in the periphery of the respective component. Nerves originating from the RNC inner- vate other body structures. The ectoneural peripheral nerve extends from the ERN into the dermis and tube feet, while the hyponeural peripheral nerve extends from the HRN into the body wall travelling between the coelomic epithelia and the circular muscle. We observed immunoreactivity to anti-calbindin 1, anti- parvalbumin, and anti-calbindin 2 within both components of the RNC (Fig. 1A–C). All three antibodies labeled cells in the RNC, but their numbers were not the same. Approximately 3064 (6%) (n = 7) anti-calbindin 1 immunopositive cells were observed per section [1964 (4%) in the ERN and 1164 (2%) in the HRN], as demonstrated by double-labeling with DAPI. A smaller number of cells were labeled with anti-parvalbumin [1464 (3%) (n = 4) cells per section- 963 (2%) in the ERN and 562 (1%) in the HRN] and with anti-calbindin 2 [461 (n = 4) (1%) cells per section- 461 (1%) in the ERN and 0 (0%) in the HRN] (all numbers are expressed as mean 6 SD). Anti-calbindin 1 immunoreactivity was observed in a large number of fibers equally distributed throughout both components of the RNC, contrary to fibers immunoreactive to anti- parvalbumin and anti-calbindin 2, which were mostly distributed to the distal region of the ERN and all throughout the HRN. Immunoreactivity to the three markers was also observed in the peripheral nerves extending from the ERN and the HRN. Individual immunoreactive fibers were best observed within the circular muscle, just distal to the hyponeural peripheral nerve. At this site, fibers immunoreactive to the anti-CBPs markers were present, but their number and morphology was different with each marker as observed in serial sections. The number of immuno- reactive fibers was larger with anti-calbindin 1 and lowest with anti-calbindin 2, leaving the amount of anti-parvalbumin Calbindin-D32k in the Echinoderm Nervous System immunopositive fibers in-between the first two. The thickness of immunoreactive fibers also differed in the same pattern. Anti- calbindin 1 labeled thick and thin fibers, while most of anti- calbindin 2 immunopositive fibers were thick fibers. Anti- parvalbumin immunopositive fibers were mostly thick fibers, though some thin fibers were observed. The localization of the fibers, as well as the pattern of direction in which the fibers run through was similar with all the three markers. We corroborated the neuronal specificity of the three markers by double-labeling immunohistochemistry using the CBP marker with the monoclonal antibodies RN1 or anti-b-tubulin, and by means of serial sections immunostained with markers that recognize the well-characterized neuropeptides GFSKLYamide and Galanin. Co-labeling of individual fibers with anti-calbindin 1 and RN1, anti-parvalbumin and RN1, and anti-calbindin 2 and RN1 was best observed in the circular muscle (CM) (Fig. 2). Although, most of the fibers were co-labeled, there were still fibers (especially those that had a smaller diameter) only labeled by RN1, but not by any of the CBPs markers. Connective tissue components The connective tissue component (CTC) of holothurians has been defined to be sandwiched between two epithelia and has been well described in the body wall and in the tube feet. This component is innervated by fibers that originate from the ectoneural nervous system. The CTC of the body wall can be divided into inner and outer dermis, according to the distribution of its fibers. All three markers labeled structures in the body wall CTC divisions, although more fibers were labeled in the outer dermis than in the inner dermis (Fig. 3). Both individual fibers and some small bundles were observed. Immunoreactive fibers in the inner dermis had a parallel orientation to the muscular fibers of the CM, while immunoreactive fibers in the outer dermis did not show a particular orientation (Fig. 3). In the inner dermis, fibers were oriented parallel to the circular muscle fibers that lie beneath. Cells immunoreactive to all three markers were also observed in the two dermal layers of the body wall. These cells were mostly isolated and had a bipolar or pyramidal morphology. Fibers could be observed extending from them through the dermis; though no association with a particular structure or localization could be identified. Although the tube feet are extensions of the body wall, their CTC is somewhat different from the body wall dermis. A larger number of fibers within the CTC of the tube feet were labeled by all three markers, compared to the outer dermis. Thicker fibers were also noted to be present in the CTC of the tube feet compared to the body wall (Fig. 4). The fibers present within the CTC of the tube feet were closely packed and presented an orthogonal orientation that clearly differed from those of the body wall. Thus, they outlined a cylindrical shape that distinctly delineated the boundary between the tube feet and the body wall dermis. Immunoreactive cells to all three markers were observed in CTC of the tube feet (Fig. 1D–F). Similar to those cells within the body wall, these had a bipolar or pyramidal morphology, and fibers from these could in some cases be observed extending to the podial nerve and the podial cylindrical fenestrated sheath of the tube feet. EF-Hand comain containing CBPs An alternative strategy to study the presence and relationship of EF-hand domain containing CBPs is to focus on the gene sequences identified in EST and genomic databanks. Sequences for EF-hand domain containing CBPs calbindin 1, calbindin 2, and parvalbumin in Echinodermata, were searched in the NCBI’s PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32689