Sponge oscula are ciliated

Sponges are unusual in possessing both cilia and flagella (named for their differing beat patterns [17]) on somatic cells. These include ciliated epithelial cells of sponge larvae which are involved in locomotion and also photoresponses [18, 19], ciliated cells at the exit of the feeding choanocyte chambers [20, 21] and flagellated choanocytes involved in pumping water through the canal system (reviewed in [20]). In contrast, the epithelia of adult sponges are usually naked. We were therefore surprised to find cilia on all cells forming the epithelial lining of the osculum in the freshwater sponge Ephydatia muelleri, a demosponge that can be cultured in the laboratory (Figure 1a). The osculum is the most prominent feature of a sponge, and is the final exit of water filtered through the sponge body for food and oxygen.

Figure 1 Cilia are found on the epithelia lining the osculum . a. The sponge Ephydatia muelleri in the lake, and grown in the lab viewed from the side (upper inset) and from above (lower inset). The oscula (white arrows) extend upwards from the body. b, c, Scanning electron micrographs show cilia arise from the middle of each cell along the entire length of the inside of the osculum; b the lining of the osculum with cilia on each cell (inset shows an osculum removed from the sponge and sliced in half longitudinally); c, two cilia arise from each cell. d, e, Cilia in the oscula labeled with antibodies to acetylated α-tubulin (green), nuclei with Hoechst (blue, n), actin with phalloidin (red). f. A 3D surface rendering illustrates how the cilia arise just above the nucleus of the cell. Scale bars a 5 mm; inset 1 mm; b 20 μm; inset 100 μm c, 1 μm d, 20 μm e, f 5 μm. Full size image

In E. muelleri a pair of cilia, each 4–6 microns long, emerges above the nucleus of every epithelial cell (Figure 1b-f). A survey of 6 other demosponges showed that in each, the oscula are also lined by ciliated cells; in some species the cells have a single cilium, and others up to 4 cilia, all arising centrally above the cell nucleus (Additional file 1: Figure S1). Even glass sponges (class Hexactinellida), which are syncytial, have cilia at the lip of their large oscula. There is no data available so far for the other two classes, Calcarea and Homoscleromorpha, although the latter is known to have cilia throughout the canals, and therefore presumably also up to the osculum lip.

Serial sections through the base of the cilium in E. muelleri show basal bodies are simple, with no structures linking pairs of cilia in a cell (Figure 2a). In contrast to the flagella of choanocyte chambers, which have a central pair of microtubules, in cross section the oscula cilia have a 9 + 0 axonemal skeleton (Figure 2b), which is characteristic of sensory cilia in other organisms [3]. Both fluorescence and scanning electron microscopy show pairs of cilia in E. muelleri are oriented perpendicular to the water flow (Figure 2c), which may be important for sensing changes in flow. In live animals the cilia label with the vital dye FM 1–43, and high frequency time-lapse microscopy showed that they are non-motile and only vibrate in the flow that passes out of the osculum (Figure 2d, and Additional file 2: Movie S1).

Figure 2 Cilia are non-motile and are oriented perpendicular to the direction of water flow in the osculum. a. Serial longitudinal sections (86 nm apart) show each cilium arises just above the cell nucleus (n) from simple basal bodies (bb); no links between the bases of the ciliary pair were found. b. In cross-section the cilium lacks a central microtubule pair in contrast to the cross section of a flagellum from a choanocyte chamber. c. Cilia pairs are aligned parallel to the long axis of the cells in the osculum, and both the cilia pairs and the cells’ long axes lie perpendicular to the direction of water flow (shown by the blue arrow) at 345.12 ± 4.72° (mean ± SE) (rose diagram: H A :0°; V = 0.841; p < 0.001; n = 49). d. Still images from high-frequency time-lapse imaging of live cilia (arrows) labeled with FM1-43 (see Additional file 2: Movie S1). Scale bars: a, 500 nm b, 100 nm c, 10 μm d, 20 μm. Full size image

Cationic channel blockers inhibit sponge behaviour

In the last decade it has been recognized that most cells in the vertebrate body, and many in invertebrates, possess specialized sensory structures called ‘primary’ cilia, which function as sensory organelles as in kidney epithelial cells, chondrocytes, odontoblasts, embryonic endocardial cells, and ‘Kupffer’s vesicle’ [22]. Primary cilia, although similar to motile cilia in their basic structure, lack the radial spokes and dynein arms that enable motility. Instead they possess stretch-activated cationic channels that are part of the transient receptor potential (TRP) channel superfamily [23] including polycystin-1 (PC1) and polycystin-2 (PC2) [23] or their homologs, which allow them to function as sensory organelles [3, 22–24]. Remarkably, TRP channels are responsible for almost all forms of sensation experienced by eukaryotic cells, including movement, taste, smell, temperature, vision and osmolarity.

The function of TRP channel sensation is difficult to assess directly, and is therefore usually done by behavioral assay; for example inhibition of an avoidance reaction by the unicellular alga Chlamydomonas using TRP channel blockers has shown that TRP11 is involved in mechanosensation [2]. In multicellular organisms it is difficult to study the function of primary cilia in living tissues, except in cell culture. In contrast, freshwater sponges are small and transparent, and cilia can be viewed live. Furthermore, both of the freshwater sponges E. muelleri and S. lacustris can be triggered to inflate and then contract their whole body (a behaviour termed a ‘sneeze’ [14, 15]) in response to mechanical or chemical stimuli (Figure 3a). Because the osculum is the final channel through which water exits the sponge, we hypothesized that the cilia have a sensory role in controlling the canal diameter to optimize normal flow through the sponge filter, and in particular during the sneeze behaviour.

Figure 3 Cationic channel blockers reduce the ‘sneeze’ response. a. The sponge ‘sneeze’ behaviour involves contraction of the osculum (white arrows), inflation, then contraction of canals (black arrows) and recovery (bar shows canal diameter). b. Neomycin sulfate (red) and FM1-43 (blue) reduce the peak amplitude of the behaviour in E. muelleri (n = 8; p < 0.001). Gd3+ (solid green) eliminated all response (n = 3; p = 0.015), but after recovery for 24 h the sponge response was even greater than before (dotted green). c, d All three compounds caused lengthening of cilia relative to controls (left), but had no effect on choanocyte flagella (bottom right) in E. muelleri (*significance at p < <0.001; error bars show ± SE). Scale bars: a, 1,000 μm c, 10 μm. Full size image

Three commonly used chemicals–the antibiotic neomycin sulfate, styryl dye FM1-43, and cationic channel blocker Gadolinium (Gd3+)–have been shown to inhibit sensory ability of primary cilia in other organisms [25, 26]. These drugs are all thought to block TRPP2 (PC2) channels on the ciliary membrane. In sponges natural stimuli (sediment, vigorous mechanical agitation) as well as bath treatments of 75–90 μM L-glutamate trigger the inflation and contraction of the excurrent canals [14, 15]. Treatment of sponges with neomycin sulfate (300 μM) and FM 1–43 (35 μM) reduced the maximum amplitude of the inflation response by 60% (Figure 3b) in both cases, and treatment with Gd3+ (5 μM) eliminated the response; the effects were reversible (Figure 3b). After recovery, the Gd3+-treated sponges showed an enhanced response to L-Glu (Figure 3b). This knock-down and knockout of the sponge behaviour by drugs that are known to affect channels on ciliary membranes implicates the cilia in sensing stimuli and transducing them into behaviour. Further support for this idea comes from the direct effect the drugs had on ciliary length.

Lengthening of primary cilia in other organisms has been proposed to increase their sensitivity [27, 28]. Ciliary (and flagellar) length is determined by a dynamic process of intraflagellar transport (IFT) which continuously brings molecules, including tubulin, up and down the cilium [29]. Chemical or mechanical stimuli that interfere with Ca2+ influx have been shown to alter IFT, thereby changing cilium length [27, 28]. In E. muelleri cilia length increased 1.2-fold after only one hour of treatment in all three drugs (Figure 3c, d), and Gd3+ treated sponges recovered partially after a one-hour washout. These data suggest that the drugs interfere with IFT in the oscula cilia. Unlike cilia, the flagella in choanocyte chambers of E. muelleri did not change length (Figure 3d), implying that the effects of the drugs reported here are only on ciliated cells.

Although pharmacology is almost universally used to study the sensory roles of cilia and flagella in other organisms [2, 25–27, 30], neomycin sulfate, FM 1–43, and Gadolinium can also affect other calcium transport processes in tissues including smooth muscle contractility. We therefore tested whether another calcium channel blocker could equally affect the sponges. In contrast to neomycin sulfate which eliminates all response in the sponge, the L-type calcium channel blocker Verapamil (10 μM) had no effect on the amplitude of the sneeze reflex (Figure 4a). This finding is consistent with experiments on vertebrate primary cilia [25, 26]. We found that longer incubation in Neomycin sulfate (2 hr in S. lacustris compared to 10 min for E. muelleri) repressed the sneeze reflex for longer. FM 1–43 is fluorescent and was clearly localized primarily to the cilia (Additional file 2: Movie S1), but to determine where neomycin sulfate localized we incubated sponges in Texas Red-conjugates of neomycin sulfate. Cells in the sponge osculum labeled within 2 minutes of incubation in the dye, and the same cells co-labelled with YO-PRO1, which selectively labels hair cells in the lateral line of zebrafish (Danio rerio) [31, 32] (Figure 4b). Taken together, the effect of these treatments suggests that stretch-activated, nonselective cation channels are involved in the sponge behavior.

Figure 4 Cilia are specifically involved in the sponge behaviour. a. In contrast to Neomycin sulfate (solid red) which eliminates the ‘sneeze’ response (n = 3, p = 0.035), the calcium channel blocker Verapamil (dotted red) does not affect amplitude of the sneeze behaviour in S. lacustris (n = 5, p = 0.573). b. Texas-Red Neomycin sulfate conjugate (red) and YO-PRO1 (green) selectively label cells in the osculum. c. A 20 hr treatment in chloral hydrate eliminates the sneeze behaviour in S. lacustris (solid green; n = 5, p = 0.004), which does not return until more than 3 days after recovery (dotted green; n = 5, 24 hr washout p = 0.003, 72 hr washout p = 0.018, 120 hr washout p = 0.864)). d-f(SEM) d’-f’(fluorescence). Cilia are removed by chloral hydrate treatment; S. lacustris 0 hr (d, d’), 20 hr (e, e’), and 70 hr (f, f’) treatment in chloral hydrate. g. The sneeze behaviour in S. lacustris cannot be triggered when the osculum is removed (solid blue; n = 3, p = 0.010) until it has fully regrown (dotted blue; n = 3, p = 0.275). h. Ciliated cells on the surface of E. muelleri 8 hr post osculum removal and (i) in the newly formed osculum 24 hr post osculum removal. Ciliated cells do not become labeled with EdU until after the osculum has regrown suggesting they arise by migration of newly formed mesohyl cells which differentiate into ciliated pinacocytes. Cilia are labeled with acetylated α-tubulin (red), nuclei with Hoechst (blue), and newly synthesized DNA with EdU (green). Scale bars: b, 50 μm inset 10 μm d, e, 5 μm d’, e’, f, f’, h, i 10 μm. Full size image

While we cannot rule out the possibility that any of these drugs have other effects on the sponge in addition to working on the cilia, in our experience very few molecules cause the sponge to relax–most trigger contractions [15, 16]. However, to confirm that the cilia in the osculum, and the osculum itself, are indeed required for the sponge sneeze reflex we used both chloral hydrate to deciliate the sponge and removed the osculum, and tested the responsiveness of the sponge in each instance. Chloral hydrate is known to remove cilia from cells, causing a loss of behaviour in both metazoans [24] and unicellular eukaryotes [2, 4] after 20 hr exposure. It is thought to act by weakening the attachment of the cilium to the basal body, with full loss of cilia occurring after 68 hr in kidney epithelial cells [24]. We found that 20 hr exposure to 4 mM chloral hydrate eliminated the sneeze reflex and it took 120 hr for recovery of sensitivity (Figure 4c-e). As in kidney cells [24], it took 70 hr to remove all cilia from the epithelium of the osculum (Figure 4f).

We have found that when removed, a new osculum forms again after 8 hours. De-osculated sponges could not be triggered to sneeze (Figure 4g), and although the sponge continued to filter water at all times during repair of the osculum, it was only after the osculum had fully formed that the sneeze response returned. Together these results suggest that both the osculum and the cilia lining it are necessary for the sneeze reflex. To determine when ciliated cells first appear on newly formed oscula, we labeled sponges from which the osculum had been removed with the cell proliferation marker EdU and detected incorporation of uridine into new cells using Click-iT (Molecular Probes, Invitrogen). At 8 hr after the osculum was removed, cilia were found on cells in a few discrete places on the surface of the sponge (Figure 4h). Pinacocytes in the sponge surface are not usually ciliated, therefore we interpreted the differentiation of cilia on pinacocytes as an early marker of the location of a new osculum. Furthermore, although mesohyl cells were labeled within 6 hrs of incubation in EdU, cells of the new osculum were not labeled with EdU, and it was only 24 hr after the new osculum was formed that a few new ciliated cells labeled (Figure 4i). Although we were unable to trace the migration of cells in live animals, we interpret these data to suggest that cilia differentiate on cells in the surface of the sponge, thereby identifying the region as a potential osculum; then as the osculum grows to full height using cells already present in the sponge, new ciliated epithelial cells differentiate from newly formed mesohyl cells.

Sponges possess a repertoire of transient receptor potential channels

Considering the conserved role of TRP channels, and in particular PKD in sensory behaviour across eukaryotes [2], we searched the transcriptomes of 8 sponge species for homologs of both pkd1 and pkd2 and other TRP channels. A 700aa homolog of pkd2 (Type II TRP) was identified in Corticium candelabrum (Homoscleromorpha) and a 178aa sequence of a pkd2 (Type II TRP) gene was found in the freshwater Spongilla lacustris (Demospongiae) (Figure 5a, Additional file 1: Figures S2, S3). We found a 978aa sequence of a Type II TRP (ML) in Sycon coactum (Calcarea), and several sequences with similarity to various Type I TRP channels were found in all 4 Porifera classes (Figure 5a-c, Additional file 1: Figure S5). These candidates were included in an alignment containing more than 100 representatives for all the TRP families across bilaterians (Figure 5a; Additional file 1: Figures S2-S3). The ability to retrieve protein sequences depends on the quality of the transcriptome and the divergence of sequences in transcriptomes. Negative results do not imply conclusive absence. Our phylogenetic analysis grouped sponge pkd sequences with Type II TRP and specifically pkd2 channels genes from bilaterians with high support (91% bootstrap). Sponge pkd channel sequences showed similar domain architecture and proposed 3D protein folding to both mouse and Chlamydomonas sequences (Figure 5b), and other sponge sequences showed amino acids indicative of the TRP domain (Figure 5c; Additional file 1: Figure S5). Although the pharmacology of the sponge cilia is similar to that of cilia known to have pkd2 channels, several TRP channels from Chlamydomonas have also been found to transduce mechanical signals so we cannot rule out the possibility that other TRP channels are involved in flow sensing in sponges.