Calbindin-D32k Is Localized to a Subpopulation of Neurons in the Nervous System of the Sea Cucumber Holothuria glaberrima (Echinodermata)

Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates

The Adhesion Molecule KAL-1/anosmin-1 Regulates Neurite Branching through a SAX-7/L1CAM–EGL-15/FGFR Receptor Complex

Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system

Holothurian Nervous System Diversity Revealed by Neuroanatomical Analysis.

The Echinodermata comprise an interesting branch in the phylogenetic tree of deuterostomes. Their radial symmetry which is reflected in their nervous system anatomy makes them a target of interest in the study of nervous system evolution. Until recently, the study of the echinoderm nervous system has been hindered by a shortage of neuronal markers. However, in recent years several markers of neuronal and fiber subpopulations have been described. These have been used to identify subpopulations of neurons and fibers, but an integrative study of the anatomical relationship of these subpopulations is wanting. We have now used eight commercial antibodies, together with three antibodies produced by our group to provide a comprehensive and integrated description and new details of the echinoderm neuroanatomy using the holothurian Holothuria glaberrima (Selenka, 1867) as our model system. Immunoreactivity of the markers used showed: (1) specific labeling patterns by markers in the radial nerve cords, which suggest the presence of specific nerve tracts in holothurians. (2) Nerves directly innervate most muscle fibers in the longitudinal muscles. (3) Similar to other deuterostomes (mainly vertebrates), their enteric nervous system is composed of a large and diverse repertoire of neurons and fiber phenotypes. Our results provide a first blueprint of the anatomical organization of cells and fibers that form the holothurian neural circuitry, and highlight the fact that the echinoderm nervous system shows unexpected diversity in cell and fiber types and their distribution in both central and peripheral nervous components

Western blot of holothurian and echinoid radial nerve cords preparations using anti-calbindin 1 and anti-parvalbumin.

<p>(A) A 32 kDa immunoreactive band to anti-calbindin 1 was observed in <i>H. glaberrima</i> and <i>L. variegatus</i> radial nerve cords homogenates. A 28 kDa immunoreactive band corresponding to calbindin 1 was observed in the rat kidney positive control and no immunoreactive bands were observed in the synthetic peptide of an EF-hand domain-containing protein negative control. (B) A 32 kDa immunoreactive band to anti-calbindin 1 was observed in <i>H. glaberrima</i> and <i>L. variegatus</i> radial nerve cords homogenates. A 12 kDa major immunoreactive band corresponding to calbindin 1 was observed in the rat skeletal muscle positive control and no immunoreactive bands were observed in the synthetic peptide of an EF-hand domain-containing protein negative control.</p

Alignment of the peptide sequences and phylogenetic trees of members of the calbindin subfamily from different species.

<p>(A) Sequence alignment of rat calbindin 2 (NP_446440.1), <i>Drosophila</i> calbindin-D32k (NP_476838.1), <i>S. kowalevskii</i> calbindin-D32k (XP_002735965.1), and <i>S. purpuratus</i> calbindin-D32k proteins (XP_781517.2). Alignment of the proteins was made using the CLUSTALW multiple sequences alignment. Dots represent conserved residues and black lines below the residues represents the presence of an EF hand domain. (B) Phylogenetic tree of members of the calbindin subfamily from different species. Calb32_Dm <i>Drosophila melanogaster</i> (NP_476838.1), Calb32_Sk <i>S. kowalevskii</i> (XP_002735965.1), Calb32_Sp <i>S. purpuratus</i> (XP_781517.2), Calb32_Sm <i>S. mansoni</i> (XP_002574332.1), Calb2_Gg <i>G. gallus</i> (NP_990647.1), Calb2_Dr <i>D. rerio</i> (NP_957005.1), Calb2_Rn <i>R. norvegicus</i> (NP_446440.1), Calb2_Hs <i>H. sapiens</i> (NP_001731.2), Calb1_Xl <i>X. laevis</i> (NP_00108408.1), Calb1_Gg G. gallus (NP_990844.1), Calb1_Mm <i>M. musculus</i> (AAH16421.1), Calb1_Rn <i>R. norvegicus</i> (AAH81764.1), Calb1_Hs <i>H. sapiens</i> (NP_004920.1), SCGN_Sk <i>S. kowalevskii</i> (NP_001161653.1), SCGN_Sp <i>S. purpuratus</i> (XP_785060.2), SCGN_Dr <i>D. rerio</i> (NP_001005776.1), SCGN_Xl <i>X. laevis</i> (NP_001088097.1), SCGN_Rn <i>R. norvegicus</i> (NP_963855.1), SCGN_Mm <i>M. musculus</i> (NP_663374.1). The phylogenetic tree was constructed from an alignment created by using the unweighted pair group method with arithmetic mean. The scale bar represents 100 times the expected number of amino acid substitution.</p

Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the podial nervous system of <i>Holothuria glaberrima</i> (Holothuroidea).

<p>Longitudinal and transverse sections through the tube feet showing immunoreactivity to anti-calbindin 1 (A,D,G,M), anti-parvalbumin (B,E,I,N), and anti-calbindin 2 (C,F,K,O) in the different subdivisions of the podial nervous system. (A–F) Immunoreactivity was observed with all markers in the podial nerve (arrows) and in the podial cylindrical fenestrated sheath (arrowheads). (G–L) Co-labeling of anti-calbindin 1 (G) and RN1 (H), anti-parvalbumin (I) and RN1 (J), and anti-calbindin 2 (K) and RN1 (L) was observed in fibers and cells (arrows) of the connective tissue plexus. (M–O) Anti-calbindin 1 (M), anti-parvalbumin (N), and anti-calbindin 2 (O) immunoreactivity was also present in the nerve plate of the tube feet's disk. Al, ambulacral lumen; ctp, connective tissue plexus; icc, inner cluster of cells; me, mesothelium; np, nerve plate; occ, outer cluster of cells.</p

Identification of calbindin-D32k as the anti-calbindin 1 and anti-parvalbumin epitope in echinoderms.

<p>(A) Sequence alignment of <i>H. glaberrima</i> calbindin-D32k fragment (Calb32_Hg) and <i>S. purpuratus</i> calbindin-D32k isoform 1 (Calb32(1)_Sp) and isoform 2 (Calb32(2)_Sp), showing a high degree of homology. Alignment of the proteins was made using the CLUSTALW multiple sequences alignment. Dark gray represent conserved residues, light gray represent conservation of strong groups, and black lines below the residues represents the presence of an EF hand domain. (B) A 36 kDa immunoreactive band to anti-calbindin 1 and anti-parvalbumin was observed in the <i>H. glaberrima</i> GST-tagged calbindin-D32k fragment peptide (Calb32_Hg), and no immunoreactive band to anti-calbindin 1 and anti-parvalbumin was observed in the negative control, GST-tagged translationally controlled tumor protein (TCTP).</p

Schematic domain structure of members of the calbindin subfamily in rat (<i>R. norvegicus</i>), fly (<i>D. melanogaster</i>), and sea urchin (<i>S. purpuratus</i>).

<p>Domains 1, 3, 4, and 5 are present in all the proteins. The second domain doesn't bind calcium in the calbindin 1 protein, while it does in calbindin 2, and in <i>Drosophila and S. purpuratus'</i> calbindin-D32k. Domain 6 is present in <i>Drosophila</i> calbindin-D32k and <i>S. purpuratus</i> calbindin-D32k, but it is only able to bind calcium in the latter. Pairwise similarities of domains between proteins were calculated by aligning the domains as predicted by ScanProsite.</p

Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the peripheral nerve and circular muscle of <i>Holothuria glaberrima</i> (Holothuroidea).

<p>Longitudinal sections through the body wall showing colabeling of anti-calbindin 1 (A) and RN1 (D), anti-parvalbumin (B) and RN1 (E), and anti-calbindin 2 (C) and RN1 (F). Most of the immunoreactivity was observed in the peripheral nerves (arrowheads), while only a minor supopulations of fibers were co-labeled by the anti-CBP and RN1 (arrows).</p