Many marine invertebrate larvae depend upon physical and chemical cues to select a suitable location for settlement and metamorphosis. Chemosensation in developing echinoids (sea urchins and sand dollars) is inferred from the observations that exposure to soluble chemicals derived from a microbial film is sufficient to induce metamorphosis of larvae of the sea urchin Lytechinus pictus (Cameron and Hinegardner, 1974) and that ciliary beat reversals are induced upon exposure to such cues (Satterlie and Cameron, 1985). Metamorphosis of the sand dollar Dendraster excentricus larvae was induced by exposure to seawater conditioned with sand in which adults were present (Burke, 1984), supporting the possibility that either larvae or developing juveniles (or both) are capable of sensing dissolved chemicals from adults and transducing them into a developmental response. A larval organ or restricted set of cells responsible for these chemosensory responses has not been identified, supporting the proposition that larval chemosensation may not be spatially restricted beyond ciliated ectoderm (Satterlie and Cameron, 1985).
Bishop et al. (2001) reported that the gaseous signaling molecule nitric oxide (NO) and its downstream effector cyclic guanosine monophosphate (cGMP) operate as inhibitory signals during metamorphosis of L. pictus: inhibition of nitric oxide synthase (NOS) or soluble guanylate cyclase (sGC) induced metamorphosis of competent larvae. This study prompted us to initiate a survey of tissues that are capable of producing NO or cGMP to gain insight into the cellular context of this signaling pathway. Cells containing cGMP, NOS, or both, are candidates for involvement in the inhibitory regulation of metamorphosis. While physiological methods to assess chemosensory responses of tissues or cells are difficult to apply to small marine larvae, the responses to inductive agents after surgical deletion of parts of the larva can be informative (e.g., Burke, 1983; Pires and Hadfield, 1993; Degnan et al., 1997; Bishop et al., 2001, Bishop and Brandhorst, 2001; Leise and Hadfield, 2000).
Here we describe the development of larval nerve cells defined by their expression of NOS and identify cGMP-producing cells in developing larval and juvenile tissues in L. pictus. NOS-defined neurites project from a restricted region of the post-oral ciliary band. We provide experimental evidence that this section of the ciliary band receives settlement cues derived from biofilm.
NOS Expression in the Post-Oral Ciliary Band
We have shown previously that, prior to the formation of the juvenile rudiment, NOS is expressed in the apical ganglion, oral ganglion, endoderm, and arms of L. pictus larvae (Bishop and Brandhorst, 2001). As the juvenile rudiment begins to form, cells immunostained for NOS appear in a specific and striking pattern: a single cell in the central zone of the post-oral ciliary band (POCB) begins to express NOS (Fig. 1A, arrowhead). From this cell, two axon-like processes first project laterally along the POCB toward the post-oral arms (Fig. 1B,C) and then posteriorly toward the midgut. At the level of the midgut, these processes then change direction and project apically along each side of the mouth before extending across the upper lip of the mouth through the neuropile underlying the pre-oral ciliary band, henceforth referred to as the pre-oral neuropile (PON) (Fig. 1D,E arrowheads). Continuing to project across the PON, these processes of common cellular origin pass each other in opposite directions before descending on the opposite side of the mouth (Fig. 1F, arrowheads). We were not able to follow the trajectory of these processes beyond this point. As juvenile development proceeds, there is a gradual increase in the number of NOS-defined cell bodies that appear in the POCB and sequentially project processes to the PON, reiterating the projection pathway described above. In competent larvae, up to ten NOS-defined cell bodies were detected in the POCB.
In early larvae, the POCB forms an arc connecting the post-oral arms (Fig. 2A), but during larval development a pair of ectodermal lobes fringed by the POCB extends toward the mouth (Fig. 2B–F). The NOS-defined cells form in the central zone of the POCB, in the cleft between the two lobes.
In advanced larvae, several fibers in the PON were stained for NOS (Fig. 3A). NOS-defined neuron-like cells also express a neural marker detected by the monoclonal antibody 1E11 (Fig. 3B,D). 1E11 was raised against a lysate from radial nerves of the sea star Asterina pectinifera and recognizes many if not all elements of the larval and juvenile nervous system of several echinoderms and a hemichordate (Nakajima et al., 2004a,b; Hiroaki et al., 2006). It recognizes an epitope encoded by the neural specific synaptotagmin gene cloned from the sea urchin Strongylocentrotus purpuratus (Burke et al., 2006a). Thus, 1E11 serves here as both a counterstain for NOS staining and an indirect confirmation of the neural character of NOS-defined cells. Because NOS-defined cell bodies and projections from the POCB have a neural morphology and express an epitope recognized by 1E11, we will provisionally refer to these cells as NOS-defined neurons (NDNs).
After NDNs begin projecting processes, their soma migrate from a basal to an apical position with respect to the polarity of other 1E11 immunoreactive cells in the ciliary band (Fig. 3C, compare cells 1–4 to cell 5 and compare the position of cell bodies stained with anti-NOS to those stained with 1E11). Associated with this movement, cell bodies convert from a globose to an ovoid shape (Fig. 3E, compare cell 3 to cells 1 and 5 and to cells in Fig. 3C) and projections derived from the same cell body become closely apposed such that they can no longer be resolved (Fig. 3C, compare cell 2 to cell 4). The rate at which the ectodermal lobes in the POCB form relative to the appearance of NDNs is variable. The anatomical relationship of this NOS-defined neural tissue to the larval body is depicted in Figure 4.
We verified the specificity of the heterologous anti-NOS antibody using NADPH diaphorase (NADPHd) histochemistry. NOS is the only enzyme in the heme mono-oxygenase class known to retain catalytic activity after aldehyde fixation (Weinberg et al., 1996). NADPHd histochemistry is thus commonly used to provisionally indicate NOS expression and to corroborate other evidence of NOS expression. Consistent with the pattern observed from NOS immunostaining, NADPHd staining indicated NOS activity in cell bodies and their cilia in the POCB (Fig. 3F). These results are also consistent with shared patterns of NOS immunoreactivity and NADPHd histochemical staining reported previously (Bishop and Brandhorst, 2001), indicating that the anti-NOS antibody is specifically detecting NOS.
Because of the difficulty of visualizing neural projections through a whole mounted larval body, and juvenile rudiment, we verified the connectivity of the projections of NDNs from the POCB to the PON by excising ectoderm containing the portion of the POCB between the post-oral arms. Excision of this region of the ciliary band led to the gradual disappearance of NOS staining in the PON, presumably the result of degeneration of severed neurites, whereas 1E11 immunoreactivity remained in the PON after this operation (compare Fig. 5A,B), indicating that other nerves did not disappear. Remarkably, the excised tissue regenerated in 1–2 weeks along with NOS-defined cell bodies that projected processes to the PON (Fig. 5C). In other experiments described below, the same region of tissue was excised from more advanced larvae and regeneration was still observed. Therefore, long after the larval body plan is established, and even after metamorphic competence is acquired, L. pictus larvae retain the potential to regenerate the loss of specialized tissues.
A Role for the Post-Oral Ciliary Band in Responses to Biofilm
When exposed to biofilm, L. pictus larvae initiate ciliary beat reversals (Satterlie and Cameron, 1985) and display a stereotypic settling posture prior to an irreversible commitment to initiate metamorphosis (Cameron and Hinegardner, 1978). We analyzed this behavior by videomicroscopy and discovered an additional settlement posture. Upon contact with the substrate, larvae most frequently oriented with their dorsal surface in contact with the substrate (Fig. 6A). While in this orientation, the epaulettes, a densely ciliated transverse band of tissue, directed water flow over the larval body in intermittent bursts. During these bursts of ciliary activity, vortices of flow were created, directing water (and presumably compounds derived from biofilm) over the ventral surface of the larva and over the POCB in particular from an anterior-to-posterior direction (see Supplemental Video Material, which can be viewed at www.interscience.wiley.com/jpages/1058-8388/suppmat). In this first settlement posture (P1), neither the larval arms nor the larval body are flexed. During normal settlement, P1 ends either when larvae resumed swimming, or when larvae assume the second distinct settlement posture. Settlement posture 2 (P2) (Fig. 6B) is characterized by the flexing behavior previously reported by Cameron and Hinegardner (1974). During P2, the primary podia of the juvenile become very active and the juvenile partially everts from the vestibule while the larva re-orients its position such that the oral surface of the juvenile faces the substrate (Fig. 6C,D). In P2, one or more of the five primary podia attach to the substrate while the larval body dramatically flexes away from the vestibular opening. This flexure facilitates access of the podia to the substrate. After assuming this posture, metamorphosis is frequently initiated.
Based on their anatomical placement and formation during late larval development, we hypothesized that the NOS-defined cells residing in the cleft of the central POCB have a chemosensory role in detecting metamorphic cues. As an experimental test, we excised the POCB including these cells, predicting that settlement and initiation of metamorphosis would be disrupted. In preliminary experiments, we observed an intriguing response of competent larvae to the excision of the central POCB: many larvae immediately flexed, while swimming, and in the absence of biofilm. This response was neither observed when other tissues were excised, nor when the central POCB from pre-competent larvae was removed. Larvae from which this region was excised returned to a normal swimming posture within minutes and did not initiate metamorphosis. Using videomicroscopy, we analyzed the responses of larvae lacking the central POCB (POCB- larvae) during 10 min of exposure to biofilm. As controls, we used both intact larvae and larvae that had regenerated the excised POCB including the NDNs (Regen larvae). Larvae with a regenerated POCB served to verify that the absence of the POCB, rather than the surgery, was responsible for any behavioral differences that we observed. We allowed POCB- larvae a 24-hr recovery period prior to exposing them to biofilm.
POCB- larvae spent significantly more time swimming than either intact or Regen larvae (Fig. 6E), which spent most of the time in contact with the substrate. They also resumed swimming upon contact with the substrate more frequently than either of the controls (Fig. 6F). These observations suggest a difference in the level of “interest” in the substrate displayed by operated larvae compared to control larvae. However, the amount of time the operated and control larvae spent in P1 was not significantly different (Fig. 6G). Although juveniles occasionally extended their tube feet through the vestibular opening in POCB-larvae, none (0/10) of these larvae even partially everted during the 10-min observation period, in contrast to controls for which 5/10 or 6/10 everted and established this contact. The most obvious difference between POCB-larvae and controls was that in response to a biofilmed surface, the former always failed (0/10) to significantly flex either the larval arms or the body relative to the anterior–posterior axis, while 5/10 or 6/10 of the controls flexed. Accordingly, juveniles in POCB-larvae were never observed to contact the substrate in posture 2. However, mechanical stimulation of larvae by a water current from a Pasteur pipette generated a flexing response among all larvae tested. As part of a different series of experiments (to be reported elsewhere), we immersed larvae in 5 μM of the synthetic musk Traseolide. This compound induced a very robust flexing response within 15 min of application. POCB larvae also responded to this treatment, indicating that the excision of the POCB did not diminish the capacity to flex, but did interfere with the capacity of larvae to do so in response to biofilm. None of the POCB-larvae initiated metamorphosis within the 10-min observation period, whereas 4/10 and 2/10 did so among intact and Regen larvae, respectively.
Other POCB-larvae were exposed to biofilm beginning 1 hr after surgery and scored for initiation of metamorphosis. After 1 hr of exposure to biofilm, some POCB-larvae had initiated metamorphosis, but at significantly reduced frequency compared to intact larvae (Fig. 6H). After a 24-hr exposure to biofilm, however, there was no difference between intact controls and POCB-larvae in the percentage of metamorphosis, indicating that larvae lacking the NDNs are able to respond to biofilm, though more slowly than intact larvae. If larvae were allowed to recover for 24 hr after excision of the POCB, fewer responded to biofilm by initiating metamorphosis. This suggests that surgery potentiates the response to biofilm but that the operated larvae recover.
Inhibition of Metamorphosis by NO Operates Via cGMP Signaling
Previously, we reported that both NO and cGMP signals inhibit metamorphosis in L. pictus (Bishop and Brandhorst, 2001). Treatment of competent larvae with inhibitors of NOS (e.g., L-NAME) or sGC (e.g., ODQ) induced metamorphosis, while the NO donor SNAP abolished the inductive effects of L-NAME or exposure to biofilm, a natural inducer of metamorphosis. Thus, the inductive properties of biofilm appear to be mediated by enzymatically produced NO, which probably acts via its well-established stimulation of sGC activity (for a review, see Ignarro, 1990). Here we report that SNAP completely abolished the inductive properties of ODQ (Fig. 7A). This result indicates the presence in competent larvae of cGMP-producing cells that are both sensitive to NO and that, at least indirectly, play an inhibitory role in metamorphosis. A 2-hr treatment of competent larvae with ODQ caused a significant decrease in levels of cGMP (Fig. 7B); 48.3 ± 0.6% of larvae treated with ODQ settled and initiated metamorphosis, compared to 0% of the controls. These experiments indicate that some larval or juvenile cells constitutively produce cGMP and that a reduction in cGMP is associated with the induction of metamorphosis.
Spatial Distribution of cGMP Production in Larvae and Juveniles
In an effort to identify the cells that mediate the response to inhibition of metamorphosis by NO, we examined the distribution of cells capable of producing elevated levels of cGMP in developing larvae and juveniles using an anti-cGMP antibody. Original characterizations of this antibody failed to detect cross-reactivity with other cyclic nucleotides (de Vente et al., 1987). Early in larval life (50 hr post-fertilization at 16°C), cGMP immunoreactivity (cGMP-IR) was observed in two cells near the oral ganglion of the lower lip (Fig. 8A). These cGMP immunoreactive cells increase in number as the larva grows (Fig. 8B,C). Double-labeling experiments confirmed that the same oral ganglion cells also stained for NOS (Bishop, 2002), suggesting that NO and cGMP are operating as part of an intracellular transduction pathway in these cells. Around the time of juvenile rudiment formation, cGMP-IR became detectable prominently along the ciliary band (Fig. 8D) and as a punctate pattern in cells of the larval epithelium (Fig. 8J). When cGMP antiserum was pre-adsorbed with 10 mM cGMP before application to fixed specimens, the intensity of cGMP-IR was greatly reduced (compare Fig. 8D to E). To support this qualitative observation, we measured the intensity of cGMP-IR after treatment with ODQ to inhibit sGC. Treatment of larvae having small juvenile rudiments (about 3 weeks old) significantly decreased the staining intensity of the punctate bodies in the epithelium (Table 1). These results confirm that the anti-cGMP antibody detects an antigen in urchin larvae that is structurally comparable to cGMP and sensitive to ODQ treatment. Mesenchyme cells distributed throughout the larval body were weakly cGMP-IR under normal physiological conditions (Fig. 8F).
Table 1. Quantitation of cGMP-IR in Larval Epithelial Cells in Response to ODQ Treatment (see Fig. 8J)a
Mean pixel values (0–25)
Mean (± s.e.m) gray values were calculated for ten vesicular bodies per image. Brightness and contrast settings on the confocal microscope were identical for each image pair.
186.4 ± 14.78
139.7 ± 11.59
131.8 ± 12.36
161.4 ± 14.15
70.7 ± 3.88
57.6 ± 1.95
116.7 ± 8.13
32.5 ± 4.19
149.5 ± 15.47
99.9 ± 13.55
We also tested for the presence of larval or juvenile cells that have the capacity to produce cGMP, but due to the absence of sustained NO stimulation or to the presence of phosphodiesterase activity, were not detected by immunostaining for cGMP under normal physiological conditions. Larvae having advanced juvenile rudiments were treated with SNAP (a generator of NO), the non-specific phosphodiesterase inhibitor IBMX, or both. When treated with both SNAP and IBMX, a high level of cGMP-IR was observed in a ring of cells at the tip of the juvenile tube feet and mesenchyme cells distributed around the juvenile rudiment (Fig. 8H,I) in comparison with untreated controls (Fig. 8G). Interestingly, the larva in Figure 8I had developed twin juvenile rudiments (not uncommon in well-fed cultures) and cGMP-IR cells are present at the periphery of each vestibule. Cells in the tube feet were not cGMP-IR when treated with SNAP or IBMX alone, suggesting that these cells produce cGMP in response to NO, but phosphodiesterase activity may prevent its accumulation to detectable levels.
In summary, elevated cGMP in larvae and juveniles was distributed among four distinct cell types, three in a sustained fashion under normal conditions and one in response to SNAP/IBMX treatment. Larval mesenchyme cells that were cGMP-IR under normal conditions were also responsive to SNAP/IBMX treatment, suggesting that they are responsive to NO and regulate cGMP levels via phosphodiesterase activity.
Inhibitory NO signaling controls the timing of life history transitions among disparate organisms, including fungi, slime molds, and plants that lack nervous tissues, as well as animals having nervous systems (reviewed by Bishop and Brandhorst, 2003; Hodin, 2006). To properly assess the evolutionary significance of this widespread use of NO as a component of systems that regulate life history transitions, the cellular context in which it operates must be examined. Toward this end, we investigated the spatial context of NOS expression during larval and juvenile development of the sea urchin, and report here on a small population of NOS-defined cells that form late in larval ontogeny. We provisionally designate these NOS-defined cells as neurons because of their morphology, their projection of axon-like processes, and their expression of the epitope recognized by 1E11, an echinoderm neural-specific antibody. Physiological studies are required to confirm this designation, but NOS-defined cells in the POCB cannot be unambiguously identified in live tissue and ciliary beating generates excessive background noise when using fine recording electrodes (CB unpublished observations). The cell bodies of the NDN sequentially form and extend neurites from the POCB during juvenile rudiment development, but not prior to this stage. Larvae are capable of regenerating the POCB, complete with NDNs even after the completion of the larval developmental program. The pattern of neurite projection from the AL to the PON suggests that the latter is providing a neurotrophic signal, thus implicating long-range signaling in this aspect of L. pictus larval neurogenesis. Since the development of the NDNs and elaboration of the POCB is completed 3–4 weeks after larvae begin to feed, an essential function for the NDNs in feeding or locomotion is precluded.
A Chemosensory Role for the Post-Oral Ciliary Band
The following considerations support our contention that the central zone of the POCB including the NDNs has a chemosensory function:
1The folded structure of the POCB and surrounding ectoderm as well as the position of NOS-defined cell bodies therein are anatomically comparable to fish olfactory epithelia, where olfactory neurons are found in the valleys between ridges in the highly convoluted olfactory epithelium (Farbman, 1992). This arrangement is thought to generate vortices that increase the sampling interval of soluble factors (Bardach and Villars, 1974). Our behavioral analyses indicate that larvae direct water flow over the ventral surface using intermittent ciliary bursts from the epaulettes. These bursts may correspond to the ciliary beat reversals recorded from L. pictus larvae in response to biofilmed substrata (Satterlie and Cameron, 1985). It is notable that the ectodermal lobe on the opposite (dorsal) surface of the larva that comes into close contact with the biofilmed substrate lacks ciliary band tissue and is not exposed to this flow of seawater (Fig. 2G). Strathmann (1988) proposed that the position of ciliary band tissue on convoluted body ridges of echinoderm larvae (and thus the overall body plan) is directly related to functional requirements associated with filtering small particles of food from large volumes of seawater at low Reynolds numbers. In contrast, the shape of the mature POCB, the arrangement of NDNs therein, and the intermittent ciliary bursts during settlement may reflect a function in sampling soluble chemicals. Increasing the velocity of flow of fluid over structures decreases boundary layer thickness, allowing more rapid access of odorants to sensory cells. In contrast, thicker boundary layers retain odorants in the vicinity of sensory cells for longer periods, increasing sampling potential. This relationship between fluid flow and olfaction is at the root of all pulsatile chemosensory behavior, such as sniffing in mammals, and its functional equivalent, antennal flicking in crustaceans. We suggest that the cilia in epaulettes of L. pictus beat intermittently during settlement to generate pulses of water over the POCB and, as such, may represent another functional equivalent of sniffing.
2In vertebrates, olfactory neurons are unique among sensory neurons in that their maturation process involves an apical migration through the overlying epithelium (Farbman, 1992). Olfactory neurons are born from a mitotic division of globose stem cells in the basal olfactory epithelium whereupon they extend an axon and a dendrite, and form a dendritic cilium as they migrate apically and ultimately breach the apical surface of the epithelium at maturity (Farbman, 1994). All known olfactory neurons are bipolar, projecting a single unmyelinated axon to the olfactory bulb (Ronnet and Moon, 2002). The soma of the NDNs of the POCB in urchin larvae are initially globose and then become polarized, moving to the periphery of the ciliary band and extending a cilium to the exterior. Each NOS-defined cell body projects two axon-like processes toward the apical region of the larva, and marine invertebrate larvae are not known to have olfactory bulbs, two points of distinction.
3NO has a signaling function in olfactory circuits in animals as diverse as mollusks, insects, lampreys, and mammals (Breer and Shepherd, 1993; Gelperin, 1994; Elphick et al., 1995; Muller and Hildebrandt, 1995; Bicker, 2001; Hua et al., 2000; Ronnet and Moon, 2002). In insects, NOS is hypothesized to be an integrator of chemosensory stimuli (Muller and Hildebrandt, 1995). We suggest that NO, via NOS-defined cells in the POCB and PON, may have a similar function during L. pictus metamorphosis.
4Hypothesizing that NDNs residing in the central region of the POCB sense biofilm cues, we predicted that the removal of this tissue from competent larvae would disrupt larval responses to biofilm. Several aspects of the settlement process were disrupted among larvae lacking this section of the POCB but they were restored in larvae that regenerated the POCB. The most striking of these was the failure of larvae to initiate posture 2 by flexing the larval body and partially everting the juvenile. After contact with a biofilmed surface, larvae lacking the NDNs resumed swimming an order of magnitude more often than either intact controls. This suggests that these larvae were not receiving information about the substrate that would normally induce them to remain in contact with it and initiate the transition to posture 2. In contrast, the frequency that larvae adopted posture 1 while in contact with the substrate was not different among intact NDN- and Regen larvae, suggesting that mechanosensation is responsible for this orientation process.
We predicted that the removal of the central POCB would abolish the initiation of metamorphosis in response to biofilm cues. This manipulation significantly reduced the rate of, but did not abolish, initiation of metamorphosis. This suggests that the contribution of NDNs in the POCB may have a secondary role in chemosensation relevant to the induction of metamorphosis, with the juvenile perhaps having the primary role. Alternatively, the artificial nature of our bioassays (i.e., a protracted exposure of larvae to heavily biofilmed substrata in small volumes of stagnant water) may contribute to these observations. Juvenile tissues may have received higher concentrations of soluble biofilm cues for longer periods than would be experienced in the natural environment, thereby diminishing the importance of larval tissues during settlement. Indeed, during the more stringent 10-min video observation periods, 40% of the intact larvae metamorphosed in response to biofilm while none of those lacking the POCB did so.
With these considerations, we contend that the NDNs of the POCB of L. pictus larvae have a chemosensory role in regulating initiation of metamorphosis and that the associated ectoderm between the post-oral arms is morphologically specialized to support this function. Further evidence for this contention would include binding of inductive compounds purified from biofilm to cells in the POCB; comparative ultra-structural studies describing the structure and arrangement the NDNs; and presence of mRNA or proteins having known chemosensory function in the NDNs. Hundreds of chemosensory receptor genes have been predicted by bioinformatic analysis of the sequenced genome of S. purpuratus (Burke et al., 2006; Raible et al., 2006). In situ hybridization analysis of a small subset of these genes failed to indicate expression in the POCB of plutei (Raible et al., 2006); assessing more genes in competent plutei might be more informative.
Evidence for Control of Metamorphosis by a Paracrine NO/cGMP Signaling Pathway
cGMP, the most common effector of NO signaling, is produced by a stimulatory interaction between NO and the heme prosthetic group of sGC. Because of the capacity of NO to diffuse through cellular membranes, this interaction can occur in an autocrine, paracrine, or endocrine context (McDonald and Murad, 1996). We provide evidence that NO and cGMP are acting in concert by showing that the NO donor SNAP can suppress the inductive properties of the sGC inhibitor ODQ. As expected, ODQ treatment was found to reduce levels of cGMP detected by immunostaining or ELISA of extracts. These observations indicate that at least one NO-sensitive cell type in L. pictus inhibits metamorphosis via cGMP signaling and that the inductive properties of ODQ are associated with a decrease in cGMP. We sought to identify the candidate cells by immunostaining for cGMP.
In larvae that had initiated juvenile rudiment formation, elevated cGMP was detected in four distinct tissues. Cells in the oral ganglion, cells along the ciliary band, epithelial cells, and mesenchyme distributed around the vestibule produce detectable cGMP under normal physiological conditions. The complex of bodies staining for cGMP at the base of ectodermal cilia shown in Figure 8D and J are similar in appearance and position to the apical Golgi complexes in which the ciliary protein Spec3 accumulates in ectodermal epithelial cells of echinoid embryos (Eldon et al., 1990). The complex can sometimes be resolved into more than two distinct vesicular structures indicating that they are not the adjacent paired basal bodies of cilia. The intensity of staining of this complex was reduced by treatment of larvae with ODQ, indicating that cGMP is produced by sGC rather than the particulate form of the enzyme, though it is presumably membrane bound if it is indeed localized to the Golgi complex.
Cells near the tip of the tube feet of the developing juvenile are also capable of producing cGMP, but only when stimulated by SNAP in the presence of the phosphodiesterase inhibitor IBMX. The absence of detectable cGMP in these cells when larvae were incubated in either SNAP or IBMX alone suggests cGMP levels are normally low and regulated in these NO-responsive cells. Assuming that tissues playing an inhibitory role in metamorphosis via cGMP contain sustained and detectable levels of cGMP, the tube feet are excluded as candidates for a major inhibitory signaling function. Although the oral ganglion meets the criterion of this assumption, the hypothesis that the oral ganglion is playing a significant role in regulating metamorphosis was previously falsified (Bishop and Brandhorst, 2001). Moreover, NOS and cGMP are expressed in these cells very early in larval life, suggesting a role in feeding. Therefore, of the tissues observed to contain cGMP at elevated levels, the ciliary band, larval epithelium, and peri-vestibular mesenchyme are most likely to be participating in the regulation of metamorphosis with respect to inhibitory cGMP signaling. None of these tissues has been observed to express NOS.
The first overt sign of an irreversible commitment to metamorphosis is the protrusion of the larval skeleton through the arm tips as the epithelium retracts. This very rapid event is then followed by a complete retraction of the larval body over the course of about an hour (Cameron and Hinegardner, 1978). Cells of the ciliary band and larval epithelium that produce cGMP are thus candidates as mediators of the initial retraction event and subsequent resorption of the larval epithelium. One possible scenario is that chemosensory neurons in the POCB transduce biofilm cues via a decrease in the output of NO signaling. This decrease results in a behavioral response in which the juvenile everts from the vestibule and becomes exposed to the substrate (posture 2), with the response being propagated through the ciliary band and larval epithelium via a decrease in cGMP production. In concert with sensory input from the tube feet of the juvenile, this decrease in NO/cGMP signaling may mediate the collapse of the larval body and thus the irreversible initiation of metamorphosis.
Adults were purchased from Marinus Scientific (Garden Grove, CA) and held either at Simon Fraser University (Burnaby, BC, Canada) or at the University of Hawaii in recirculating seawater tanks until used. Larvae were cultured as described in Bishop and Brandhorst (2001) with the following modifications: larvae were raised exclusively on a mono-algal diet of Dunaliella tertiolecta (NEPCC strain 001 or a strain obtained from R. Strathmann, Friday Harbor Laboratories, WA; 5,000 cells/mL/day) and were incubated without stirring in the dark at temperatures ranging from 16–24° C. This temperature range reflects our attempts to find optimal growing conditions. Competent larvae were usually generated within four to five weeks.
All chemicals were purchased from Sigma-Aldrich Chemical Corp. L-nitroarginine-methyl-ester (L-NAME) was prepared as a 100-mM stock in distilled water. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was prepared as a 50-mM stock in DMSO. S-Nitroso-N-acetylpenicillamine (SNAP) was prepared as a 100-mM stock in DMSO. 1-Methyl-3-isobutylxanthine (IBMX) was prepared as a 2-mM stock solution in distilled water. Stock solutions were stored at −20°C and thawed and diluted with filtered seawater (FSW) immediately before use. DMSO (0.1% v:v FSW) was used as a control when experiments in which chemicals formulated in DMSO were conducted. This concentration of DMSO never induced metamorphosis.
Quantification of cGMP
cGMP was quantified using an enzyme-linked immunosorbent assay (ELISA) (Amersham Pharmacia Inc.). Four separate assays were conducted using larvae raised from different parents. To verify the effect of ODQ on cGMP levels, groups of ten competent larvae were treated with 50 μM ODQ until 50% metamorphosis was observed. This typically required a 2-hr exposure. Here we define metamorphosis as having begun when the skeletal rods of the arms protrude through the tips of the epidermis. In our experience, this is an invariant sign of an irreversible commitment to metamorphose. In three of the four experiments, larvae and newly metamorphosed juveniles were collected and processed immediately according the manufacturer's instructions. In the fourth experiment, larvae were collected and then frozen at −70°C until used. Larvae were homogenized in 100 μL of extraction buffer in 1.5-mL microfuge tubes using a plastic pestle mounted on a cordless drill and used immediately for the ELISA. Results from multiple wells of a single ELISA plate were pooled and treated as a single unit of replication. The Student's t-test was used to assess the significance of the difference in the mean cGMP level between untreated and ODQ treated larvae.
Immunohistochemistry and Histochemistry
All staining experiments were conducted as whole mount preparations. Universal anti-NOS polyclonal rabbit antibody (Affinity Bioreagents, Golden, CO) was used to detect NOS in fixed growing and mature larvae. Larvae were fixed for 1–2 hr at RT in 4% formaldehyde and then rinsed 3 times in phosphate buffered saline (PBS) pH 7.4. Fixed larvae were blocked with PBS containing 2.5–5% bovine serum albumin (BSA) and 0.1% Triton-X-100 (PBT) and then incubated in 1:100 anti-NOS overnight at 4°C. Larvae were incubated in secondary antibody (goat anti-rabbit-Alexa 568; Invitrogen/Molecular Probes, Eugene, OR) diluted 1:1,000 for 2 hr at RT or overnight at 4°C. The mouse monoclonal antibody 1E11 (Nakajima et al., 2004a) was used at a dilution of 1:10–50. The secondary antibody was goat-anti mouse-Alexa 488 (Molecular Probes) diluted 1:1,000. The cGMP antiserum, a gift from Dr. Jan de Vente (Universiteit Maastricht, Netherlands), was raised in sheep against a cGMP-formaldehyde-thyroglobulin conjugate (de Vente et al., 1987). cGMP staining was carried out essentially as described for NOS except that primary antibody concentrations were 1:1,000. An anti-sheep secondary conjugated to Alexa-488 (Molecular Probes) was used as a secondary antibody diluted 1:2,000. The specificity of the primary antibody was verified by pre-incubating it with 10 mM cGMP in blocking buffer for 1 hr at room temperature prior to the addition of specimens. After 1 hr of incubation in primary antibody (1:2,000) in the presence or absence of cGMP, specimens were incubated in secondary antibody for 1 hr at room temperature and then examined for qualitative differences in staining in ciliary band tissue. In all cases, omission of the primary antibody served as a control for non-specific binding of the secondary antibody.
To quantify differences in the intensity of cGMP immunoreactivity between larvae treated with 50 μM ODQ for 7 hr and control larvae, five pairs of images of both ODQ-treated and control specimens were collected. Using Northern Ecplise 6.0 image analysis software (Empix Inc., Toronto, Canada), the mean gray values for pixels contained within ten haphazard selections from each image were collected. Each selection contained a single stained body. Because brightness and contrast settings were specific for each image pair, mean pixel values could not be pooled. Differences in staining intensity for each image pair were, therefore, analyzed independently using a Student's t-test and then tabulated. For SNAP/IBMX-enhanced cGMP immunostaining, larvae were incubated in a solution of either 100 μM SNAP, 200 μM IBMX, 100 μM SNAP + 200 μM IBMX, or seawater for 2 hr in the dark at room temperature. These were then fixed and processed as above. All specimens were mounted in 70% glycerol in PBS and viewed using a Zeiss LSM 410 confocal microscope. Images were adjusted for brightness and contrast using Adobe Photoshop 6.0 and 8.0CS.
NADPH diaphorase (NADPHd) histochemical staining, as performed by Bishop and Brandhorst (2001), was used to verify the specificity of NOS immunostaining. The ectoderm including the PCOB stained using NADPHd histochemistry was excised prior to observation. All DIC images were captured with Northern Eclipse 6.0 image capturing software from a Sony DXC-950 3CCD camera mounted on an Olympus Vanox Microscope.
Surgical Operations and Experimental Setup
Surgical operations were performed using either fine-edged stainless steel surgical tools (Fine Science Tools, Vancouver, B.C.), or insect pins (Elephant no. 2 or 3) fused to a Pasteur pipette (as described by Bates and Jeffery, 1987). Both types of implement were honed using a Japanese water stone (Lee Valley Tools, Vancouver, BC). In all cases, when operations were performed for the first time, post-operative larvae were observed for 1 hr prior to further manipulation to ensure that the operations did not induce metamorphosis. None of the surgical manipulations caused mortality within the period of observation (up to 2 weeks in some experiments).
For regeneration experiments (presented in Fig. 5), the lobes hosting the central post-oral ciliary band (POCB) were removed from at least 100 larvae having small juvenile rudiments (approximately 3 weeks prior to competence). To determine the baseline counts for NOS-defined cell bodies in the POCB and to verify the efficacy of the operation in removing target tissues, some larvae lacking the central POCB and intact larvae were fixed immediately and processed for NOS staining. We observed no mortality from this operation but in some larvae (<5%) algae had populated the larva, presumably from having gained access via the wound in the ectoderm. As a result, for all subsequent experiments, post-operative larvae were placed in 500-mL plastic beakers without food for 24 hr. After this recovery period, larvae were cultured according to normal schedules. Control and experimental larvae were cultured at identical densities and food concentrations. To determine the state of NDNs after removal of the central POCB, larvae were allowed to regenerate for 5 and 12 days after surgery, at which time they were fixed and processed for NOS immunostaining. Throughout the regeneration period, the POCB zone was monitored microscopically. The mean number of cell bodies expressing NOS in the POCB of ten larvae was scored as a proximate measure of NDN regeneration.
For experiments that tested POCB function (presented in Fig. 7H), larvae were placed in contact with a heavily biofilmed dish either 1 or 24 hr after its removal. Biofilm for these experiments was generated by incubating Syracuse dishes in running seawater at Kewalo Marine Labs, Honolulu, Hawaii. Results were scored after 1- and 24-hr exposure to biofilm. In this experiment, replicates consisted of larvae derived from the same parents but cultured in separate vessels from the time of rudiment invagination until competence (about 1.5 weeks). That is, the vessel was the unit of replication. Intact larvae served as experimental controls. Experimental results were subjected to a one-way ANOVA and a Tukey post-hoc test for significance to detect the effect of the removal of the central PCOB on larval responses to biofilm.
Larvae were observed laterally with respect to the substrate using a Sony® Digital Video Camera mounted on one of the ocular lens tubes of a side-mounted Leica stereo microscope. Custom-manufactured microaquaria were used as observation chambers. After observing the behavior of several competent larvae in response to a biofilmed substrate, we generated five behavioral characters associated with settlement: time in contact with the substrate, time in posture 1, number of times larvae swam off the substrate after contact, number of larvae that flexed (assuming posture 2), and whether juvenile podia contacted the substrate. Larvae from which the central POCB had to be excised as described above were allowed to regenerate for 10–14 days prior to exposure to biofilm; each had been examined to insure that the ciliary band had been at least partially regenerated. Larvae were exposed to shards of glass coated with biofilm and responses were videotaped for 10-min periods. In addition, we used a suspension of either algae or azocarmine particles to visualize water flow over the larva during exposure to biofilm. Video images were imported and edited with Windows Movie Maker® using default settings. Each of the behavioral characters was scored in order to quantify differences in behaviors. Means of the data for “time in contact with the substrate” and “time in posture 1” were analyzed with a one-way ANOVA and a Tukey post-hoc test. Data for “number of times larvae swam off the substrate after contact” were heteroscedastic and, therefore, analyzed by the Kruskal-Wallis test. Raw scores for whether larvae flexed and whether the juvenile tube feet contacted the substrate were tabulated.
Our work has benefited from the generous gifts of the cGMP antibody from Jan de Vente (Universiteit Maastricht) and the 1E11 antibody developed by Yoko Nakajima in collaboration with Robert Burke (Dept. of Biology, University of Victoria). David McLachlan (Canadian Food Inspection Agency, Dartmouth, Canada) is acknowledged for his custom manufacture of microaquaria for videography of larvae. We thank Michael Hadfield (Kewalo Marine Laboratories) for providing the micro-video apparatus, culturing facilities, and some operating funds, and Joanna Bince from Athula Wikramanayake's lab (University of Hawaii) for providing young larvae. Brian Nedved conducted some statistical tests from the video analyses and critically commented on the manuscript. Discussions with E. Leise were timely and helpful. Greg Murray is thanked for confirming some cGMP immunostaining results. An anonymous reviewer provided helpful guidance on anatomical terminology. Our research was funded by a grant from the Natural Sciences and Engineering Research Council of Canada to B.P.B. and in part by an NSERC post-doctoral fellowship to C.D.B.