Neuroanatomy of the Tube Feet and Tentacles in Holothuria Glaberrima (Holothuroidea, Echinodermata)
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Zoomorphology (2010) 129:33–43 DOI 10.1007/s00435-009-0098-4 ORIGINAL PAPER Neuroanatomy of the tube feet and tentacles in Holothuria glaberrima (Holothuroidea, Echinodermata) Carlos A. Díaz-Balzac · José E. Abreu-Arbelo · José E. García-Arrarás Received: 27 August 2008 / Revised: 19 June 2009 / Accepted: 15 September 2009 / Published online: 29 September 2009 © Springer-Verlag 2009 Abstract Echinoderms are a key group in understanding Introduction the evolution of the nervous system in the Metazoa. Remarkably, little is known about echinoderm neurobiol- Echinoderms comprise an important group of animals in ogy. The echinoderm podia, which are unique echinoderm the study of nervous system evolution. However, when modiWcations and comprise structures responsible for loco- compared to other deuterostomes and to some protostome motion and feeding, have been largely neglected in nervous groups, the echinoderm nervous system has been less stud- system studies. Here, we have applied immunohistological ied. Furthermore, most studies of echinoderm neurobiology approaches using diVerent neuronal markers to describe the focus on the radial nerves, the main structure of the echino- neuroanatomy of the holothurian podia and its relation to derm nervous system. In recent years, there has been a the muscular component. We show, using the sea cucumber growing interest in echinoderm neurobiology. We, and oth- Holothuria glaberrima (Selenka, 1867), the direct innerva- ers, have taken advantage of new markers, namely antibod- tion of the podia by the ectoneural component of the ner- ies against neuropeptides, to describe some of the vous system, as well as the existence of a connection components of the echinoderm nervous system (Moore and between the nervous system components in the main Thorndyke 1993; Birenheide et al. 1998; Inoue et al. 1999; nerves, the muscle, and the connective tissue. These Wnd- Nakajima et al. 2004; Burke et al. 2006a; Tamori et al. ings conWrm the ectoneural origin of the tube feet’s main 2007). Our group has focused on members of the Holothu- nervous system and demonstrate its neuroanatomic com- roidea (Díaz-Miranda et al. 1995, 1996). We have previ- plexity. We also show the presence of Wbers and neurons ously described the enteric nervous system (García-Arrarás within the tube feet mesothelium and connective tissue. et al. 1999, 2001) and, more recently, the nervous compo- The study of these simple structures will help us elucidate nent associated with the connective tissue (Díaz-Balzac the echinoderms’ neuromuscular circuit and their evolution- et al. 2007). Here, we focus on the nervous component of ary relationships. body wall structures associated with the water-vascular canal, namely, the tube feet and tentacles. We focus on Keywords Holothuria glaberrima determining the extent of the nervous system innervation (Holothuroidea, Echinodermata) · Tube feet · Tentacles · on the muscular component as well as within the connective Neuropeptides · Nervous system tissue, since the echinoderms are well known for having mutable connective tissues, which are under nervous sys- tem control (Motokawa 1984; Birenheide and Motokawa 1996). The tube feet are podia, which imply they are a type of body wall protrusion associated with the water-vascular C. A. Díaz-Balzac · J. E. Abreu-Arbelo · J. E. García-Arrarás (&) system. The podia vary greatly among the diVerent classes Department of Biology, University of Puerto Rico, of echinoderms in their number, localization, and function. Río Piedras Campus, Box 23360, Río Piedras, Puerto Rico 00931-3360 In holothurians, the tube feet are the main type of podia and e-mail: [email protected] are referred to as locomotor podia, in contrast to other types 123 34 Zoomorphology (2010) 129:33–43 of podia named non-locomotor or papillate podia (Hyman Tissue sections 1955). These are unique echinoderm modiWcations and rep- resent the main structures by which many holothurians Specimens were anesthetized in 0.2% 1,1,1-trichloro-2- attach to the substratum, achieve locomotion, and undergo methyl-2-propanol (Sigma, St. Louis, MO) for 10 min a large range of activities (Hyman 1955). Holothurian ten- and dissected by longitudinal section of the body wall. tacles are also associated with the water-vascular system, Samples from the anterior and ventral part of the body making them similar, in some respects, to the tube feet. In wall, where the tentacles and tube feet are located, respec- fact, some controversy exists on whether the tentacles are tively, were dissected and Wxed in 4% paraformaldehyde modiWed podia (Bouland et al. 1982; McKenzie 1987; at 4°C for approximately 24 h. Tissues were rinsed 3 Smiley 1994; Mashanov and Dolmatov 2000). times for 15 min with 0.1 M phosphate-buVered saline Almost all of the current knowledge of the podial and (PBS) and left in a 30% sucrose solution at 4°C for at least tentacular nervous system is based on classical histological 24 h before proceeding to embed them in Tissue-Tek descriptions done early during the last century (see Hyman (Sakura Finetek, Torrance, CA). Cryostat tissue sections 1955) or on more recent studies done with electron (8–20 m) were cut and mounted on Poly-L-lysine-coated microscopy (Wood and Cavey 1981; Cavey and Wood slides. 1981; Rieger and Lombardi 1987; Flammang and Jangoux 1992; Pentreath and Cobb 1972; Smiley 1994; Cavey Histology 2006). These studies reveal the detail of cells and Wbers, but the overall view of the circuitry and anatomical structures is Cryostat tissue sections were stained with Milligan’s still lacking. Thus, our current view of the podial and ten- Trichrome (Humason 1979). Shortly, the sections were tacular nervous system is that of a neural plexus located cleared in xylene for 10 min, hydrated in a decreasing alco- between the longitudinal muscle Wbers and the connective hol series (95–70%) and allowed to mordant in potassium tissue layer (Flammang and Jangoux 1992; Smiley 1994). dichromate solution (76.5 mM potassium dichromate-22% Here, we describe the innervation of podia and tenta- HCl-22% ethanol, 6 min). Sections were then stained using cles and their subdivisions into muscular and connective the following washes, dyes and times: water rinse, acid tissue components in a representative of the Holothuroi- fuchsin (1.2 mM, 7 min) (J. T. Baker Chemical, Phillipsburg, dea, the aspidochirote Holothuria glaberrima (Selenka, NJ), water rinse, 1% phosphomolybdic acid (3 min) 1867). We also provide a detailed characterization of the (Sigma, St. Louis, MO), orange G in 1% phosphomolybdic distribution and morphology of cells and Wbers within the acid (44.2 mM, 8 min) (Allied Chemicals, Gujarat, India), nervous system of the tube feet, and propose a model cir- 1% acetic acid (2 min), 2.4% aniline blue (8 min) (Sigma, cuitry. Using this new information, we compare it to St. Louis, MO), and 1% acetic acid (3 min). Once the available histological and neuroanatomical tube feet and protocol was completed, the sections were dehydrated tentacle data (Hyman 1955; Florey and Cahill 1977, 1982; through an increasing series of alcohol (from 70 to 95%), Moore and Thorndyke 1993) from other echinoderm cleared in xylene (10 min), and cover glass mounted using species that have been studied thoroughly, especially Piccolyte mounting media (Ward’s Natural Science Estab- Asteroidea and Echinoidea species. This cellular and his- lishment, Rochester, NY). The stained sections were observed tological description, together with the recent publication and photographs were taken on a Nikon Eclipse E600 of the purple sea urchin genome (Sea Urchin Genome microscope. Sequencing Consortium 2006) and the identiWcation of many conserved vertebrate neural genes (Burke et al. 2006b), Immunohistochemistry will surely enhance the growing interest in echinoderm neurobiology. The indirect immunoXuorescence method was followed (García-Arrarás 1993; Díaz-Balzac et al. 2007). In brief, tissues were incubated for 1 h in goat serum 1:50 (Invitro- Materials and methods gen, Carlsbad, CA), 15 min in 1% Triton X, and two other rinses in 0.1 M PBS, followed by the overnight incubation Animals at room temperature with the primary antibodies or FITC- labeled phalloidin (Sigma, St. Louis, MO) at a dilution of Adult Holothuria glaberrima (Selenka, 1867) (Holothuroi- 1:2,000. The primary antibodies used include the RN1 dea, Aspidochirotida) specimen (10–15 cm in length) were monoclonal antibody (Díaz-Balzac et al. 2007) raised collected from the rocky shores of the north coast of Puerto against a homogenate of the radial nerve of H. glaberrima Rico. The animals were kept in sea water aquaria at the and used at a dilution of 1:100,000; the rabbit antiserum University of Puerto Rico in Río Piedras. GFSKLYamide No. 23 2i2s (Second injection and second 123 Zoomorphology (2010) 129:33–43 35 bleeding) (Díaz-Miranda et al. 1995) prepared against a Classical dyes GFSKLYa synthetic peptide and used at a 1:1,000 dilution; the rabbit antiserum galanin-1 2i3s (Second injection and Tube feet were localized within the ventral body wall, sur- third bleeding) (Díaz-Miranda et al. 1996) prepared against rounded by the connective tissue of the dermis. Milligan’s galanin (Calbiochem Corp. San Diego, CA) and used at a Trichrome (MT) staining of longitudinal (Fig. 1a) and cross 1:1,000 dilution; the rat antiserum Sp-SynB: (Nakajima sections (Fig. 1b) of the tube feet stem clearly showed the et al. 2004; Burke et al. 2006a) prepared against the recom- diVerent tissue layers: the epidermis, connective tissue, binant protein made up of the amino acids 177-420 of the podial nerve, and mesothelium. The mesothelium can be predicted Sp-SynB protein and used at a 1:200 dilution; the divided into the muscle cells whose Wbers are oriented lon- monoclonal antibody -tubulin, (Sigma T-4026 Lot. gitudinally and the adluminal cells (sometimes referred to 024K4862) clone TUB 2.1 prepared against tubulin from as coelomic epithelial cells).