Anatomy of the Hesse PhotoreceptorCell Axonal System in the Central

Nervous System of Amphioxus

ANTONIO CASTRO,1 MANUELA BECERRA,2 MARIA JESUS MANSO,1

NANCY M. SHERWOOD,3AND RAMON ANADON2*

1Department of Cell and Molecular Biology, Faculty of Sciences, University of A Coruna,15071-A Coruna, Spain

2Department of Cell Biology and Ecology, University of Santiago de Compostela, 15782-Santiago de Compostela, Spain

3Department of Biology, University of Victoria, Victoria,British Columbia V8W 3N5, Canada

ABSTRACTThe present study reports the organization of the Hesse cell axonal system in the central

nervous system of the amphioxus, with the use of a polyclonal antiserum raised againstlamprey gonadotropin-releasing hormone-I (GnRH-I). In the spinal cord, the rhabdomericphotoreceptor cells of the bicellular organs were well labeled with this antibody. These cellssent smooth, straight, lateral processes that bent and became beaded as they passed ven-trally and crossed to the contralateral side of the cord. There, the processes of several cellsaggregated to give rise to a longitudinal fiber bundle. Beaded collaterals of these processeswere directed to ventral neuropil and did not appear to contact giant Rohde cell axons. Thecrossed projections of the Hesse photoreceptors are compared with those of vertebrate retinalganglion cells. Other antisera raised against GnRH weakly labeled rhabdomeric photorecep-tors located dorsally in the brain, the Joseph cells. The finding that GnRH antibodies labelamphioxus photoreceptor cells and axons is not definitive proof that the photoreceptorscontain GnRH. Regardless of whether the antibody recognizes amphioxus GnRH, which hasnot yet been identified by structure, the antibody has revealed the processes of the Hessephotoreceptor cells. J. Comp. Neurol. 494:54–62, 2006. © 2005 Wiley-Liss, Inc.

Indexing terms: photoreceptor cells; brain; spinal cord; immunohistochemistry; lancelet;

Cephalochordates

Amphioxus (lancelets) and vertebrates share a numberof significant attributes, such as the presence of a noto-chord (at least in embryos), dorsal tubular central nervoussystem, segmented trunk musculature, paired lateral gillslits (at least in embryos), postanal tail, hepatic portalsystem, and endostyle (the thyroid gland homolog). Prob-ably amphioxus is the closest living invertebrate relativeof the vertebrates. The central nervous system (CNS) ofamphioxus consists of a short anterior region (the brain)and a long caudal region (spinal cord), all lying over thenotochord, which extends between the rostral and thecaudal ends of the body (Franz, 1924; Bone, 1960). Theorganization of the CNS in amphioxus is known from light(Retzius, 1891; Franz, 1924; Bone, 1959, 1960; Ekhart etal., 2003) and electron microscopic studies on differenttypes of cells in the brain and spinal cord (Eakin andWestphal, 1962; Meves, 1973; Obermuller-Wilen, 1976,

1979; Watanabe and Yoshida, 1986; Ruiz and Anadon,1989, 1991a–c; Lacalli et al., 1994; Lacalli, 1996, 2002a,b,2003, 2004; Lacalli and Kelly, 1999, 2000, 2003a,b).

A characteristic trait of amphioxus not observed in ver-tebrates is the presence of bicellular photoreceptor organs,the Hesse ocelli, distributed throughout most of the spinal

Grant sponsor: Spanish Education and Science Ministry; Grant number:BFU2004-05287/BFI; Grant sponsor: Natural Sciences and EngineeringResearch Council of Canada.

*Correspondence to: Ramon Anadon, Department of Cell Biology andEcology, University of Santiago de Compostela, 15782-Santiago de Com-postela, Spain. E-mail: bfanadon@usc.es

Received 13 December 2004; Revised 18 April 2005; Accepted 26 July2005

DOI 10.1002/cne.20783Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 494:54–62 (2006)

© 2005 WILEY-LISS, INC.

cord (Franz, 1924; Eakin and Westphal, 1962). Electronmicroscopic studies have revealed the rhabdomeric-likenature of Hesse photoreceptor cells, which are closelyassociated with a cup-shaped pigment cell (Eakin andWestphal, 1962; Nakao, 1964; Ruiz and Anadon, 1991a).Similar rhabdomeric photoreceptors, the Joseph cells, arefound in the dorsal region of the brain (Eakin and West-phal, 1962; Watanabe and Yoshida, 1986; Ruiz and Ana-don, 1991a). Recent evidence indicates that both rhabdo-meric receptors express amphioxus melanopsin and a Gqprotein, confirming their photoreceptive nature (Koyanagiet al., 2005). Putative amphioxus photoreceptors of ciliarytype (lamellate cells: Meves, 1973; Ruiz and Anadon,1991b; Lacalli et al., 1994; frontal eye receptor cells:Stokes and Holland, 1995; Lacalli, 1996) have also beenreported in the brain of amphioxus. The ultrastructure ofJoseph cells and Hesse cells reveals that they are moresimilar to rhabdomeric photoreceptors of invertebratesthan to those of the retina and pineal organ of vertebrates.However, the connections of amphioxus photoreceptorsare unknown, except for some connections of the firstHesse ocellus of the rostral brain of larval amphioxusrecently described by ultrastructural reconstruction fromserial ultrathin sections (Lacalli, 2002a).

The distribution of some neurotransmitters and neuropep-tides in the CNS of amphioxus is known from several immu-nohistochemical studies (for further references see Hollandand Holland, 1993; Uemura et al., 1994; Anadon et al., 1998;Candiani et al., 2001; Castro et al., 2003; Moret et al., 2004;Sherwood et al., 2005). The presence of gonadotropin-releasing hormone (GnRH) immunoreactivity has been re-ported in both brain and Hatschek’s pit (a preoral glandularorgan) of amphioxus with the use of antibodies against mam-malian GnRH (Chang et al., 1985). In vertebrates, the de-capeptide GnRH is key in the regulation of reproductivefunctions by the hypothalamohypophyseal-gonadal axis(Powell et al., 1996; Iwakoshi et al., 2002; Adams et al., 2003;Iwakoshi-Ukena et al., 2004). Almost all vertebrate speciessynthesize at least two isoforms of GnRH, a hypophysiotro-pic GnRH form (GnRH-I) and a second form (GnRH-II; forreview see Sherwood and Adams, 2005). Neurons expressingGnRH-II tend to project not to the hypophysis but to differ-ent brain regions and the spinal cord (Wright and Demski,1991; Yamamoto et al., 1998), indicating that this peptidemay be involved in basic neural functions. GnRH-III iso-

forms appear to have evolved in fishes (see Sherwood andAdams, 2005), whereas GnRH-IV includes only the twoforms of GnRH in lamprey (Gorbman and Sower, 2003).Nine different GnRHs have been found in tunicates, allsharing the residues 1–4 and 9 and 10 with the mammalianGnRH (Powell et al., 1996; Adams et al., 2003). A dodecapep-tide GnRH has been recently identified in octopus (Iwakoshiet al., 2002; Iwakoshi-Ukena et al., 2004), being the onlyGnRH characterized to date in a nonchordate species.

In the present study, we reasoned that amphioxus islikely, based on its phylogenetic position, to have someform of GnRH. We used a set of antisera generated againstdifferent GnRH peptides with the aim of revealing thedistribution of possible GnRH(s) in the nervous system ofadult amphioxus. One of the GnRH antibodies labeled theHesse photoreceptors, including their specialized fibersystem, providing evidence of the axonal projections of theHesse ocelli. These results are discussed in the context ofboth connections and possible functions, with the under-standing that the labeled molecule in the Hesse cellsmight not be GnRH.

MATERIALS AND METHODS

Animals

Sixteen adult amphioxus (Branchiostoma lanceolatumPallas), collected in the Rıa de A Coruna (Spain), wereused for this study. All specimens were anesthetized with0.1% 3-aminobenzoic acid ethyl ester methane sulfonatesalt (MS-222; Sigma, St. Louis, MO) in sea water and fixedby immersion in 4% paraformaldehyde in 0.1 M phosphatebuffer (PB; pH 7.4), during 24 hours at 4°C. Amphioxuswere cryoprotected in 30% sucrose in PB, frozen withliquid nitrogen, and cut on a cryostat at a thickness of12–20 �m in transverse and sagittal planes, mounted ongelatinized slides, and processed for immunocytochemis-try. All procedures for animal experimentation were ap-proved by the animal care and use committee of the Uni-versity of A Coruna and conformed to the guidelines of theEuropean Community.

Immunocytochemistry

Sections were processed for immunocytochemistry asfollows: they were 1) rinsed several times in 0.01 Mphosphate-buffered saline (PBS; pH 7.4); 2) treated with3% H2O2 in PBS for 30 minutes to block endogenousperoxidase activity; 3) rinsed thrice in PBS (5 minuteseach); 4) treated with normal goat serum (Dako, Glostrup,Denmark) diluted 1:10 in PBS for 1 hour; 5) incubatedwith one of the rabbit antisera raised against five differentGnRHs (see below; dilutions 1:1,000–10,000 in PBS) for20–48 hours at room temperature or at 4°C (48-hourincubations) in a moist chamber; 6) rinsed thrice in PBS (5minutes each); 7) incubated with goat anti-rabbit immu-noglobulin (Dako; diluted 1:100 in PBS) for 1 hour; 8)rinsed thrice in PBS (5 minutes each); 9) incubated withperoxidase-antiperoxidase complex (PAP; Dako; diluted1:200 in PBS) for 1 hour; and 10) rinsed twice in PBS,followed by a wash in 0.05 M pH 7.6 Tris-HCl buffer (5minutes each). Finally, the immune complex was visual-ized by incubation with 0.06% 3,3�-diaminobenzidine(DAB; Sigma) and 0.005% H2O2 in PBS for 5–10 minutes.Then, the sections were rinsed in Tris-HCl, dehydrated,and coverslipped.

Abbreviations

A atrial cavityCc central canalDf dorsal finDR dorsal nerve rootHP Hatschek’s pitLm large motoneuronsMf metapleural foldsMs muscle segmentmt muscle tailsN notochordOr oral regionPh pharynxRd dorsal Rohde axons (descending)Rv ventral Rohde axons (ascending)Sh notochord sheathSm small motoneuronsSmb somatomotor bundleT tail

55HESSE CELLS IN THE AMPHIOXUS CNS

GnRH antisera

The five antisera used in this study were raised inrabbits against different forms of GnRH: Bla-5 (anti-lamprey GnRH-I), 7CR-10 (anti-dogfish GnRH), FP-7(anti-salmon GnRH), 8CR-10 (anti-catfish GnRH), andJas-2 (anti-tunicate GnRH-I). These antisera were pre-pared in the Sherwood laboratory. Immunocytochemistrywith Bla-5 gave a clear staining of Hesse cell photorecep-tors, which were not recognized by the other antibodies.Amphioxus GnRH has not been characterized by struc-ture, so none of the antisera raised against GnRH could betested for cross-reactivity. Instead, in the present study,cross-reactivity for antiserum Bla-5 with 12 vertebrateGnRH peptides was tested, and relative cross-reactivitywas calculated with lamprey GnRH-I as the referencestandard. The Bla-5 antiserum was diluted 1:5,000 (v/v)final dilution with synthetic mammalian (m) mGnRH astracer. The results are summarized in Tables 1 and 2. Thisantiserum had been previously tested with six vertebrate(Somoza et al., 2002) and nine tunicate GnRH peptides(Adams et al., 2003). Antiserum 7CR-10 was tested forcross-reactivity against all 14 vertebrate (those indicatedin Tables 1 and 2) and two tunicate GnRH peptides; itcross-reacted with chicken GnRH-II (cGnRHII; 100%),salmon GnRH (sGnRH; 85%), dogfish GnRH (dfGnRH;25%), lamprey GnRH-III (13%), and lamprey GnRH-I (6%)but not with nine other vertebrate GnRHs nor with tuni-cate GnRH-I or -II (all less than 0.04%; Lescheid et al.,1997; and unpublished data). Antiserum FP-5 (an earlierbleed from the same rabbit as FP-7) was tested for cross-reactivity by using either the tunicate GnRH-V or -VItrace and cGnRH-II as the reference peptide. The relative

cross-reactivity for mGnRH or any of the nine tunicateGnRHs was less than 1.2% (unpublished data).

Immunostaining with Bla-5 was tested by replacementof the primary antiserum either with normal goat serumalone or with the primary antiserum previously incubatedfor 24–48 hours at 4°C with lamprey GnRH-I (20–40�M). These controls completely abolished the immunore-action in the amphioxus nervous system. Connective tis-sue and blood plasma were nonspecifically immunostainedboth with the primary antiserum and in controls omittingthe primary antiserum.

The Bla-5 antiserum has been previously shown to labelthe GnRH neurons that form a network around the dorsalstrand in tunicate Ciona intestinalis (Adams et al., 2003).Other antisera used here have been previously shown tolabel neurons with GnRH in several tunicate species. An-tiserum FP-5 (an earlier bleed from the same rabbit asFP-7) was used to label GnRH neurons in the tunicateCiona intestinalis (Adams et al., 2003). Catfish antiserum8CR-6, which is an earlier bleed from the same rabbit as8CR-10, also labeled GnRH in neurons around the dorsalstrand of Corella inflata (Mackie and Singla, 2004). Anti-serum Jas-2 labeled GnRH sensory cells in Corella inflata(Mackie and Wyeth, 2000; Mackie and Singla, 2004).Nonetheless, a protein labeled by GnRH antisera raised inother species might not be GnRH.

Photomicrography

Photomicrographs were taken with a digital camera(DP12; Olympus Co., Tokyo, Japan), converted to grayscale, and adjusted for contrast and brightness in Photo-Paint (Corel, Ottawa, Ontario, Canada).

RESULTS

We have used a battery of antibodies against GnRH ofdifferent chordate groups: tunicate (tGnRH), lamprey (la-GnRH), catfish (cfGnRH), salmon (sGnRH), and dogfish(dfGnRH; for details see Tables 1 and 2). In the brainand/or spinal cord of the adult amphioxus, most of theseantibodies produced labeling of some structures. The mostinteresting pattern of immunostaining was observed withthe laGnRH antibody, which labeled a specific system ofcells and fibers in the caudal brain and spinal cord at highantiserum dilutions (1:5,000–10,000). Moreover, this re-action was completely abolished by preabsorption of theprimary antibody with the specific peptide. Because am-phioxus GnRH(s) has not been sequenced, confirmation ofwhether the substance revealed in amphioxus is a GnRHmolecule must await further studies.

Immunolocalization of Hesse cells

In the nerve cord, the laGnRH antibody produced thelabeling of photoreceptor cells of Hesse eyecups (each con-sisting of a photoreceptor cell and a pigment cell; seeEakin and Westfall, 1962; Ruiz and Anadon, 1991a) withdifferent intensities, from faint to strong (Fig. 1A,C).When diluted appropriately (dilution 1:5,000–1:10,000),the antiserum clearly labeled the processes of these cells(Fig. 1). These cells did not label in controls in which theprimary antibody was either preabsorbed with lampreyGnRH-I or omitted (Fig. 1B), although some nonspecificlabeling was present in connective sheaths around the

TABLE 1. Relative Cross-Reactivity of Vertebrate GnRH Standards withAnti-Lamprey GnRH-I (Bla-5) and Mammalian GnRH Trace

Class GnRH peptide StructureCross-reactivity

(%)

IV Lamprey-I1 pQHYSLEWKPG-NH2 100.00III Salmon pQHWSYGWLPG-NH2 38.00I Seabream pQHWSYGLSPG-NH2 34.00I Whitefish pQHWSYGMNPG-NH2 33.00I Mammalian pQHWSYGLRPG-NH2 32.00I Chicken-I pQHWSYGLQPG-NH2 24.00I Pejerrey pQHWSFGLSPG-NH2 15.00I Catfish pQHWSHGLNPG-NH2 2.00I Herring pQHWSHGLSPG-NH2 1.00I Dogfish pQHWSHGWLPG-NH2 1.00II Chicken-II pQHWSHGWYPG-NH2 0.60IV Lamprey-III pQHWSHDWKPG-NH2 0.03I Guinea pig pQYWSYGVRPG-NH2 ntI Rana (frog) pQHWSYGLWPG-NH2 nt

1Reference peptide.nt, not tested.

TABLE 2. Relative Cross-Reactivity of Tunicate GnRH Standards withAnti-Lamprey GnRH-I (Bla-5) and Tunicate-3 GnRH Trace

GnRH peptide StructureCross-reactivity

(%)

Tunicate-1 pQHWSDYFKPG-NH2 �0.1Tunicate-2 pQHWSLCHAPG-NH2 0.5Tunicate-31 pQHWSYEFMPG-NH2 100.0Tunicate-4 pQHWSNQLTPG-NH2 �0.1Tunicate-5 pQHWSYEYMPG-NH2 117.0Tunicate-6 pQHWSKGYSPG-NH2 �0.1Tunicate-7 pQHWSYALSPG-NH2 1.5Tunicate-8 pQHWSLALSPG-NH2 2.2Tunicate-9 pQHWSNKLAPG-NH2 �0.1

1Reference peptide.

56 A. CASTRO ET AL.

Fig. 1. Transverse sections of the amphioxus spinal cord showingthe structures immunolabeled with an anti-lamprey GnRH-I antibody(Bla-5). A: Panoramic view showing distribution of immunostainedcells and fibers. Star, median giant Rohde axon; arrows, Hesse cellbundles; arrowhead, photoreceptor smooth process. B: Control sectionincubated without the primary antibody. C: Detail of the perikaryon(arrow) and smooth initial axonal process (arrowhead) of a Hessephotoreceptor cell. Star, pigment cell of the Hesse eyecup. D: Conti-nuity of the smooth (arrowheads) and beaded (arrows) parts of pro-cesses of photoreceptor cells. Star, pigment cell. E: Ventrally directed

initial process (arrowhead) of a Hesse photoreceptor located in themidline. Black star, median giant Rohde axon; arrow, Hesse photore-ceptor; white stars, pigment cells. F: Beaded Hesse photoreceptoraxon (arrow) crossing the ventral midline. Star, median giant Rohdeaxon. G,H: Detail of Hesse cell bundles (thick arrows) and axonalprocesses located near the lateral surface of the cord (wide arrows).Thin arrow, beaded fiber running toward the bundle. I: Detail ofbeaded axonal processes (arrows) coursing through the ventrolateralneuropil. Scale bars � 50 �m in A,B,F; 25 �m in C–E,G–I.

57HESSE CELLS IN THE AMPHIOXUS CNS

nerve cord and blood spaces and in faint clumps irregu-larly distributed in the nerve cord.

The Hesse photoreceptor perikaryon gave rise to a thin,straight, smooth process that coursed laterally from thecell for about a half of the thickness of the cord wall andthen turned abruptly in the ventral direction, changing itssmooth profile into a beaded morphology (Figs. 1A,C,D, 2).Sometimes, bundles of two or three straight axonal pro-cesses were observed, apparently following the direction ofa bundle of ependymal cell processes before changing theirdirection (Fig. 1C,D). The process rarely branches in theventral neuropil, always coursing near the ventral surfaceof the cord to the contralateral side (Fig. 1A,F), ascendingtoward a lateral fiber bundle that is present at a middleheight of the cord (Figs. 1A,G–I, 2). This bundle is herereferred to as the “Hesse cell bundle.” Processes of Hessephotoreceptor cells located at the left and right sides of thecord behave similarly, coursing in the contralateral side.Photoreceptor cell processes located below the central ca-nal sometimes course ventrally before crossing the mid-line (Fig. 1E). Upon entering the Hesse cell bundle, theseimmunostained axons appear to course longitudinally.The number of axons observed in transverse sections ofthis bundle varies along the cord; it was high at rostralspinal levels, where Hesse eyecups are more abundant(about two dozen axons in the bundle per section at rostral

pharyngeal levels), but was greatly reduced in centralregions and again increased in caudal regions. This indi-cates that axons from different Hesse photoreceptor cellsare overlapped at most for a few segments. The smallernumber observed in central regions correlates with thescarcity of Hesse eyecups at these regions, whereas theaxons and Hesse eyecups are abundant at both rostral andcaudal ends of the spinal cord. Immunostained beads aresometimes observed close to the lateral surface of the cordand lateral to the Hesse cell bundle (Figs. 1A,G,H, 2).

We have not observed any clear relationship betweenthe Hesse cell processes revealed by the laGnRH anti-serum and the giant axon system of amphioxus. Thissystem consists of a ventral midline (median) giant axonoriginating from the first Rohde cell, located at the entrylevel of the left nerve VI, and two groups of about sixthinner giant axons, dorsal and ventral, on each side of thenerve cord (Fig. 2). Smooth processes from midline Hessephotoreceptor cells sometimes pass close to the mediangiant axon, but they do not form apparent bouton-likestructures at this level. Also, the dorsal Rohde axons,originating from the other rostral Rohde cells, are not incontact with Hesse cell processes, and only the ventralRohde axons (originating from caudal Rohde cells) are inthe region traversed by beaded Hesse cell axons.

Fig. 2. A,B: Schematic representation of an amphioxus and atransverse section at the level of the pharynx (line in A), respectively.C: Schematic drawing showing the Hesse cell system in relation toother known structures of the amphioxus spinal cord. Hesse cells arelight gray (open arrow), axons in black. Arrowheads point to theinitial smooth part of the axon. Thin arrows point to the Hesse cell

bundles, and thick arrows to bouton-like processes near the cordsurface. Somatomotor neurons (large and small) are outlined at right,and several types of GABAergic cells are outlined at left and crossingthe central canal. Star, median giant Rohde axon. For abbreviationssee list.

58 A. CASTRO ET AL.

In the brain, the laGnRH antibody demonstrated the pres-ence of only some intensely stained cells in the ventral regionat intermediate and caudal levels (Fig. 3A,B). The cells la-beled with laGnRH antibody were small, and most showedlateral processes that arched ventrally and crossed the ven-tral midline to run in the contralateral side, similarly toHesse photoreceptor cell axons. The location of these cellswas lateral to the ventral part of the central canal, some-what dorsal to the Rohde nucleus as defined recently byEkhart et al. (2003). Most of these cells were observed caudalto the nerve IV entrance overlapping with the most rostralHesse cells.

Other GnRH antibodies

The immunolabeling obtained with the dfGnRH and cf-GnRH antibodies was rather different; they labeled only theJoseph cells with different intensities. These photoreceptorsare large cells located in the dorsal region of the brain (Fig.3C,D). As clearly shown with the dfGnRH antibody, thesecells extend from just caudal to the brain vesicle to the level

of the nerve V entrance; they are numerous at middle brainlevels, sometimes extending rather ventrally near the mid-line, and are more scarce at rostral and caudal brain levels.The antibodies labeled the cytoplasm but not the region ofthe rhabdome (Fig. 3D). No processes of Joseph cells werelabeled with GnRH immunocytochemistry.

The other two antibodies used here (anti-tGnRH-1and anti-sGnRH) apparently produced nonspecific la-beling of most neurons in the brain, including the Jo-seph cells and the cells of the nucleus of Rohde, whichwere faintly to moderately labeled. These antibodies didnot label the Hesse cells or their characteristic axons.

Hatschek’s pit

Hatschek’s pit is a glandular structure of the preoralregion of amphioxus consisting of a tall epithelium (seeNozaki and Gorbman, 1992). The GnRH antibodies usedhere did not produce any specific staining of glandularcells of this pit organ, as shown in the Figure 3E with theanti-lamprey GnRH-I antibody.

Fig. 3. Transverse sections of the brain (A–D) and Hatschek’s pit(E) showing immunohistochemical results obtained with Bla-5, anti-lamprey GnRH-I (A,B,E), and 7CR-10, anti-dogfish GnRH (C,D) an-tibodies. A: Section of the caudal brain at the level of the nucleus ofRohde (open arrow) showing a laGnRH immunostained cell (solidarrow). The arrowhead points to a fiber coursing near the notochord.B: Section through the caudal brain showing an laGnRH-immunostained cell perikaryon (arrow), fibers coursing transversely(single arrowheads) and fibers coursing longitudinally (double arrow-

head). C: Section through the rostral brain showing dfGnRH-immunostained Joseph cells (arrows) in the roof of the brain. D: Detailof Joseph cells showing that the rhabdomeric rim (arrows) is negative.E: Transverse section through Hatschek’s pit showing that the epi-thelium is GnRH negative. The arrows point to the apical surface ofthe epithelium. Note the nonspecific staining of connective sheaths.Star, funnel of the pit. Scale bars � 25 �m in A; 50 �m in B,C,E; 10�m in D.

59HESSE CELLS IN THE AMPHIOXUS CNS

DISCUSSION

Organization of the Hesse cell fiber system

Hesse eyecups are thought to be involved in motor re-sponses of amphioxus to light (Parker, 1908). The Hesseeyecups consist of a rhabdomeric photoreceptor cell asso-ciated with a cap-shaped pigment cell, forming a minute,directionally sensitive eye (Franz, 1924; Eakin and West-fall, 1962; Nakao, 1964; Ruiz and Anadon, 1991a). A short,straight, proximal part of the photoreceptor cell processwas foreseen in the classical study of amphioxus neuronsby Retzius (1891). Our results show for the first time thatthe axons of these cells project contralaterally, formingtogether a small lateral axonal bundle, the Hesse cellbundle. Moreover, our results show that, along theirtransverse trajectory, the commissural fibers are beaded,coursing in the ventrolateral neuropil, but they do notenter the region of the somatomotor bundle. In an 8-dayamphioxus larva, a portion of the axons of the two photo-receptor cells of the first ocellus, which are located in thebrain near the center of somite 5, was reconstructed byusing serial transmission electron microscopy (Lacalli,2002a). The two axons originating from this ocellus ap-pear to contact ipsilaterally the processes of motoneuronsof the dorsal compartment. This ipsilateral projection andclose relationship of photoreceptor axons with a specificportion of the locomotor system observed in early larvae isin contrast with the basically contralateral projection ob-served here. There is an absence of any obvious relation-ship between the Hesse cell fiber system and the somato-motor bundle and neuromuscular complexes revealed bycalretinin immunocytochemistry (Castro et al., 2004).Hesse cells of adult amphioxus appear responsible for thestartle locomotor responses evoked by light illumination ofthe cord (Parker, 1908; Guthrie, 1967, 1975). Contacts ofdifferent types of synaptic boutons on Rohde giant axonshave been observed with electron microscopy (Ruiz andAnadon, 1989), but, in light of our results, it appearsimprobable that Hesse cell axons actually form contactswith giant axons. However, both beaded Hesse cell axonsand motoneuron processes course through the ventral andventrolateral neuropil (Castro et al., 2004), making a re-lationship between the two systems appear possible.Whether this relationship is direct or indirect is notknown. In any case, connections of adult Hesse cells ap-pear to be very unlike from those reported in early larvae,which can be related to the major behavioral and locomo-tive differences observed between larvae and adults.

Our results pose another intriguing question. In bothvertebrates and amphioxus, the central projections of sen-sory nerves are basically ipsilateral. However, this doesnot apply to the vertebrate eye projections, which areprimarily crossed in fishes, although some tetrapodsevolved secondarily partial crossing of optic fibers in thechiasm. The present results indicate for the first time thatcrossed projections are the rule for amphioxus spinal eye-cups, which suggests that this is a shared ancient charac-teristic of visual projections. Although this finding is sug-gestive, any hypothesis about the origin of thecontralateral optic projections must take into accountdeep differences between the amphioxus spinal eyecupsystem and the vertebrate lateral eyes. On one hand,studies of amphioxus Hesse cells with proximity micro-electrodes have shown that brief, high-intensity flashes oflight evoke a local, slow potential wave, indicating the

presence of a depolarizing potential in the receptor (Guth-rie, 1975). This Hesse cell response appears of be a signcontrary to that reported in vertebrate photoreceptors andin some tunicates (salps and larval ascidians), which showa hyperpolarizing response after a brief flash of light (Gor-man et al., 1971). On the other hand, in vertebrates,photoreceptors are not projection neurons, if we exclude asmall proportion of ganglion cells of the retina expressingmelanopsin that are directly responsive to light (seeBelenky et al., 2003; Morin et al., 2003) and some putativephotoreceptors in the lamprey pineal that project to theoptic tectum (Pombal et al., 1999). However, it is conceiv-able that the indirect eye projections of vertebrate eyeswould have evolved from a simple crossed circuitry ofancestral rhabdomeric photoreceptors projecting directlyto their contralateral targets, as we found in adult am-phioxus. Melanopsin is closely related to the opsins ofinvertebrates (Bellingham et al., 2002), and a recent studyof phototransductive mechanisms provides molecular evi-dence of a rhabdomeric-like light-activated signaling cas-cade initiated by melanopsin within vertebrate cells(Isoldi et al., 2005; Melyan et al., 2005). In some respects,the photosensitive ganglion cells of vertebrates resembleHesse photoreceptors (Koyanagi et al., 2005). However,Hesse organs do not appear to express AmphiPax-6, thehomologue of the vertebrate Pax-6 that is expressed inmost retinal cells, including ganglion cells and photore-ceptors, as the ciliary photoreceptors of amphioxus possi-bly do (Glardon et al., 1998). Joseph cells and organs ofHesse apparently develop from a genetic program that isnot triggered by Pax-6, so they might be recent acquisi-tions of amphioxus (Glardon et al., 1998). Thus, the re-semblance of amphioxus photoreceptors with crossed cir-cuitry of vertebrate ganglion cells might be a result ofconvergence.

GnRH in rhabdomeric photoreceptors ofamphioxus?

The two main structures immunostained with thepresent set of GnRH antibodies in amphioxus, the Hessecells and the Joseph cells, are considered rhabdomericphotoreceptors. As far as we are aware, no vertebratephotoreceptor has been reported to be immunoreactivewith GnRH antibodies. However, increasing numbers ofstudies indicate that some hypothalamic neurons are op-sin immunoreactive (Garcıa-Fernandez and Foster, 1994;Yoshikawa et al., 1994, 1998; Alvarez-Viejo et al., 2003),and in lamprey large numbers of opsin (Garcia-Fernandezet al., 1997)- and GnRH (Nozaki et al., 2000; Sower et al.,2000)-immunoreactive neurons have been observed insimilar locations of the magnocellular preoptic nucleusand rostral hypothalamus. Thus, the possibility of collo-cation of these substances in deep brain photoreceptorsshould not be discarded without specific investigations.Because vertebrates have no spinal cord photoreceptors,and neither retinal nor pineal photoreceptors correspondto Hesse or Joseph cells, findings in vertebrates cannotconfirm or rule out the possibility that GnRH is actuallyexpressed in amphioxus rhabdomeric photoreceptors.

Our results showing that the Hesse photoreceptor cellsand the Joseph cells were immunolabeled by the laGnRHand by the cfGnRH and dfGnRH antibodies, respectively,indicates that these rhabdomeric photoreceptors of am-phioxus contain different molecular markers. Althoughthe neat immunolabeling of Hesse cells and controls per-

60 A. CASTRO ET AL.

formed with preabsorption of the anti-lamprey GnRH-Iantiserum with the corresponding peptide favors the viewthat the molecule(s) revealed in amphioxus by this anti-body is GnRH or a closely related molecule, we cannot ruleout the possibility that the antiserum was staining a dif-ferent protein. Thus, confirmation of the molecule re-vealed as GnRH must await sequencing of amphioxusGnRH(s) and in situ hybridization studies.

Some immunohistochemical studies in another am-phioxus (Branchiostoma belcheri) have reported the pres-ence of GnRH immunoreactivity in a variety of neurons inthe brain (Chang et al., 1985; Fang et al., 1999), which isunlike the present results showing GnRH immunoreactiv-ity in specific CNS systems. Moreover, whereas these ear-lier authors found GnRH-ir cells in Hatschek’s pit, specificGnRH immunoreactivity was not demonstrated in thispreoral glandular organ by the set of anti-GnRH antibod-ies presently used. The reasons for this notable discrep-ancy in the distribution of GnRH are not clear. Becausethe earlier authors used an antimammalian GnRH anti-body, one possibility is that immunolabeling obtained inthese studies was not specific, as suggested by the manyneuronal types described.

Whereas the main cells demonstrated in amphioxuswith GnRH antibodies are photoreceptors, in tunicates theimmunoreactive GnRH cells do not correspond to photo-receptors (Ohkuma et al., 2000), although immunoreac-tive GnRH is present in primary sensory cells of at leastone species of tunicate, Corella inflata (Mackie andWyeth, 2000; Mackie and Singla, 2004). Both labeledGnRH cells and labeled opsin (photoreceptive) neuronshave been reported in the cortex of the cerebral ganglion ofadult ascidians, although not in the same cell (Ohkuma etal., 2000).

CONCLUSIONS

Our results indicate for the first time that the photore-ceptive system of the amphioxus nerve cord forms well-defined crossed projections. Evidence favoring the pres-ence of GnRH in amphioxus Joseph cells appears to bevery weak, and their axonal system could not be demon-strated with the present set of GnRH antibodies. Ourimmunocytochemical results suggest a closer resemblanceof amphioxus Hesse cell material to lamprey GnRH-I thanto other GnRHs, although the primary amino acid se-quence(s) remains to be determined.

ACKNOWLEDGMENTS

We thank Dr. Santiago Parra (Spanish Institute ofOceanography, A Coruna) for collecting the amphioxusused in this study.

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