Evolutionary origins of sensation in metazoans: functional evidence for a new sensory organ in sponges

Evolutionary origins of sensation in metazoans: functional evidence for a new sensory organ in sponges
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  Evolutionary srcins of sensation in metazoans:functional evidence for a new sensory organin sponges Ludeman  et al. Ludeman  et al. BMC Evolutionary Biology   2014,  14 :3  RESEARCH ARTICLE Open Access Evolutionary srcins of sensation in metazoans:functional evidence for a new sensory organin sponges Danielle A Ludeman 1 , Nathan Farrar 1 , Ana Riesgo 2 , Jordi Paps 3 and Sally P Leys 1* Abstract Background:  One of the hallmarks of multicellular organisms is the ability of their cells to trigger responses to theenvironment in a coordinated manner. In recent years primary cilia have been shown to be present as  ‘ antennae ’ on almost all animal cells, and are involved in cell-to-cell signaling in development and tissue homeostasis; how thissophisticated sensory system arose has been little-studied and its evolution is key to understanding how sensationarose in the Animal Kingdom. Sponges (Porifera), one of the earliest evolving phyla, lack conventional muscles andnerves and yet sense and respond to changes in their fluid environment. Here we demonstrate the presence of non-motile cilia in sponges and studied their role as flow sensors. Results:  Demosponges excrete wastes from their body with a stereotypic series of whole-body contractions usinga structure called the osculum to regulate the water-flow through the body. In this study we show that short cilialine the inner epithelium of the sponge osculum. Ultrastructure of the cilia shows an absence of a central pair of microtubules and high speed imaging shows they are non-motile, suggesting they are not involved in generatingflow. In other animals non-motile,  ‘ primary ’ , cilia are involved in sensation. Here we show that molecules known toblock cationic ion channels in primary cilia and which inhibit sensory function in other organisms reduce or eliminatesponge contractions. Removal of the cilia using chloral hydrate, or removal of the whole osculum, also stops thecontractions; in all instances the effect is reversible, suggesting that the cilia are involved in sensation. An analysisof sponge transcriptomes shows the presence of several transient receptor potential (TRP) channels including PKDchannels known to be involved in sensing changes in flow in other animals. Together these data suggest that ciliain sponge oscula are involved in flow sensation and coordination of simple behaviour. Conclusions:  This is the first evidence of arrays of non-motile cilia in sponge oscula. Our findings provide supportfor the hypothesis that the cilia are sensory, and if true, the osculum may be considered a sensory organ that isused to coordinate whole animal responses in sponges. Arrays of primary cilia like these could represent the firststep in the evolution of sensory and coordination systems in metazoans. Keywords:  Porifera, Primary cilia, Evolution of nervous systems, Sensory systems, PKD Background Sensory systems use specialized cells or organelles toreceive signals that are conducted through the body electrically or chemically [1]. Signal transduction in many unicellular eukaryotes occurs via cilia, which often haveboth motile and sensory roles [2-4]. The evolution of multi- cellularity necessarily involved the ability to transducesignals over longer distances, which in animals is now doneby nerves [5] to allow rapid coordinated movements of thewhole organism [6]. Although cilia play an importantrole in sensing the environment in both unicellular andmulticellular animals, the evolutionary relationship of sensory cilia in unicellular eukaryotes, fungi and meta-zoans is unclear. Studies of sensory systems in the earliestevolving metazoans could shed light on shared commonmechanisms of sensation.Sponges lack a nervous system and while they are usually considered representatives of the first multicellular animals * Correspondence: 1 Department of Biological Sciences, University of Alberta, CW 405 BiologicalSciences Building, Edmonton, Alberta T6G 2E9, CanadaFull list of author information is available at the end of the article © 2014 Ludeman et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the CreativeCommons Attribution License (, which permits unrestricted use, distribution, andreproduction in any medium, provided the srcinal work is properly cited. Ludeman  et al. BMC Evolutionary Biology   2014,  14 :3  [7-10], some recent phylogenomic analyses place cteno- phores more basally [11,12] calling into question our un- derstanding of the evolution of nerves and the ancestralmetazoan state. Analysis of sponge genomes and tran-scriptomes has revealed a complex assortment of signalingmolecules and proteins necessary for a post-synapticscaffold [13,14]. Together with physiological evidence that glutamatergic signaling occurs in sponges [15,16] this suggests that a signaling system similar to that seen inother metazoans may be used to coordinate sponge behav-ior. Whereas sensory organs are well-known from cteno-phores, in sponges the mechanism for transducing sensory information from the environment has as yet remainedunknown.Here we provide experimental data which suggest thatan array of non-motile cilia in the sponge osculum – thechimney-like structure through which water exits fromthe sponge – functions as a sensory system to detect changesin flow and control whole animal responses. We used anemergent model system, the freshwater sponge, to investi-gate the ultrastructure and physiology of the cilia. We alsostudied the molecular evolution of sensory channels of theTransient Receptor Potential family in Porifera. Regardlessof whether sponges as we know them today were orwere not the earliest multicellular animals to evolve, itis intriguing to consider that an array of sensory cilialike this in sponge oscula could have given rise to morecomplex signalling cells, such as nerves and sensory sensilla, in the early evolution of animals. Results and discussion Sponge oscula are ciliated Sponges are unusual in possessing both cilia and flagella(named for their differing beat patterns [17]) on somaticcells. These include ciliated epithelial cells of sponge larvaewhich 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 allcells forming the epithelial lining of the osculum in thefreshwater sponge  Ephydatia muelleri , a demosponge thatcan be cultured in the laboratory (Figure 1a). The osculumis the most prominent feature of a sponge, and is the finalexit of water filtered through the sponge body for foodand oxygen.In  E. muelleri  a pair of cilia, each 4 – 6 micronslong, emerges above the nucleus of every epithelial cell(Figure 1b-f). A survey of 6 other demosponges showedthat in each, the oscula are also lined by ciliated cells;in some species the cells have a single cilium, and othersup to 4 cilia, all arising centrally above the cell nucleus(Additional file 1: Figure S1). Even glass sponges (classHexactinellida), which are syncytial, have cilia at the lipof their large oscula. There is no data available so far forthe other two classes, Calcarea and Homoscleromorpha,although the latter is known to have cilia throughoutthe canals, and therefore presumably also up to theosculum lip.Serial sections through the base of the cilium in  E.muelleri  show basal bodies are simple, with no structureslinking pairs of cilia in a cell (Figure 2a). In contrastto the flagella of choanocyte chambers, which have acentral pair of microtubules, in cross section the osculacilia have a 9+0 axonemal skeleton (Figure 2b), which ischaracteristic of sensory cilia in other organisms [3].Both fluorescence and scanning electron microscopy show pairs of cilia in  E. muelleri  are oriented perpendicular tothe water flow (Figure 2c), which may be important forsensing changes in flow. In live animals the cilia label withthe vital dye FM 1 – 43, and high frequency time-lapsemicroscopy 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). Cationic channel blockers inhibit sponge behaviour In the last decade it has been recognized that most cells inthe vertebrate body, and many in invertebrates, possessspecialized sensory structures called  ‘ primary  ’  cilia, whichfunction as sensory organelles as in kidney epithelial cells,chondrocytes, odontoblasts, embryonic endocardial cells,and  ‘ Kupffer ’ s vesicle ’  [22]. Primary cilia, although similarto motile cilia in their basic structure, lack the radialspokes 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 assensory organelles [3,22-24]. Remarkably, TRP channels are responsible for almost all forms of sensation experiencedby eukaryotic cells, including movement, taste, smell,temperature, vision and osmolarity.The function of TRP channel sensation is difficult toassess directly, and is therefore usually done by behavioralassay; for example inhibition of an avoidance reaction by the unicellular alga  Chlamydomonas  using TRP channelblockers has shown that TRP11 is involved in mechano-sensation [2]. In multicellular organisms it is difficult tostudy the function of primary cilia in living tissues, exceptin cell culture. In contrast, freshwater sponges are smalland 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 wholebody (a behaviour termed a  ‘ sneeze ’  [14,15]) in response to mechanical or chemical stimuli (Figure 3a). Because theosculum is the final channel through which water exitsthe sponge, we hypothesized that the cilia have a sensory  Ludeman  et al. BMC Evolutionary Biology   2014,  14 :3 Page 2 of 10  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 pairwere found.  b . In cross-section the cilium lacks a central microtubule pair in contrast to the cross section of a flagellum from a choanocytechamber.  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 perpendicularto 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 imagesfrom 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  μ m  d , 20  μ m. 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 fromthe side (upper inset) and from above (lower inset). The oscula (white arrows) extend upwards from the body.  b ,  c , Scanning electron micrographsshow 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 labeledwith antibodies to acetylated  α -tubulin (green), nuclei with Hoechst (blue, n), actin with phalloidin (red).  f  . A 3D surface rendering illustrates how thecilia arise just above the nucleus of the cell. Scale bars  a  5 mm; inset 1 mm;  b  20  μ m; inset 100  μ m  c , 1  μ m  d , 20  μ m  e ,  f   5  μ m. Ludeman  et al. BMC Evolutionary Biology   2014,  14 :3 Page 3 of 10  role in controlling the canal diameter to optimize normalflow through the sponge filter, and in particular during thesneeze behaviour.Three commonly used chemicals – the antibiotic neomy-cin sulfate, styryl dye FM1-43, and cationic channel blockerGadolinium (Gd 3+ ) – 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 onthe ciliary membrane. In sponges natural stimuli (sediment, vigorous mechanical agitation) as well as bath treatmentsof 75 – 90  μ M L-glutamate trigger the inflation and con-traction of the excurrent canals [14,15]. Treatment of  sponges with neomycin sulfate (300  μ M) and FM 1 – 43(35  μ M) reduced the maximum amplitude of the inflationresponse by 60% (Figure 3b) in both cases, and treatmentwith Gd 3+ (5  μ M) eliminated the response; the effectswere reversible (Figure 3b). After recovery, the Gd 3+ -treatedsponges showed an enhanced response to L-Glu (Figure 3b).This knock-down and knockout of the sponge behaviourby drugs that are known to affect channels on ciliary membranes implicates the cilia in sensing stimuli andtransducing them into behaviour. Further support forthis idea comes from the direct effect the drugs had onciliary length.Lengthening of primary cilia in other organisms hasbeen proposed to increase their sensitivity [27,28]. Ciliary  (and flagellar) length is determined by a dynamic processof intraflagellar transport (IFT) which continuously bringsmolecules, including tubulin, up and down the cilium [29].Chemical or mechanical stimuli that interfere with Ca 2+ influx have been shown to alter IFT, thereby changingcilium length [27,28]. In  E. muelleri  cilia length increased -50050100150    C   h  a  n  g  e   i  n  e  x  c  u  r  r  e  n   t  c  a  n  a   l   d   i  a  m  e   t  e  r   (   %   ) Time (s) Control300µM Neomycin35µM FM1-43 -10001002000 600 1200 1800 2400 3000 Control3µM Gd 3+ Washout Cilia Flagella   Washout  G d   3 +   F  M 1 - 4  3  N e o m  y c  i n C o n  t r o  l 0102030    )   l  o  r   t  n  o  c   f  o   %   (   h   t  g  n  e   l  n   i  e  g  n  a   h   C Gd 3+ 01020300102030 0 s 750 s 1600 s 3590 sControl Neomycin FM1-43 Gd 3+ ab cd * * * n>200n>200n>200n>200n=48n=126 * n=10n=13 * 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). Gd 3+ (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  μ m  c , 10  μ m. Ludeman  et al. BMC Evolutionary Biology   2014,  14 :3 Page 4 of 10


Apr 28, 2018


Apr 28, 2018
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